VA - Pakistan Research Repository

EPIDEMIOLOGICAL, HAEMATOLOGICAL AND BIOCHEMICAL

RISK FACTORS OF PARTURIENT HAEMOGLOBINURIA IN

BUFFALOES

by

ALTAF MAHMOOD

2007 - VA - 442

THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE

REQUIREMENT FOR THE DEGREE

OF

DOCTOR OF PHILOSOPHY

IN

EPIDEMIOLOGY AND PUBLIC HEALTH

FACULTY OF VETERINARY SCIENCE

UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES,

LAHORE

(2011)

To

The Controller of Examinations,

University of Veterinary and Animal Sciences,

Lahore.

We, the Supervisory Committee, certify that the contents and form of thesis

submitted by Mr. Altaf Mahmood, have been found satisfactory and recommend that it be

processed for evaluation by the External Examiner(s) for award of the degree.

Chairman _____________________________

Prof. Dr. Muhammad Athar Khan

Member ______________________________

Prof. Dr. Muhammad Arif Khan

Member ______________________________

Prof. Dr. Zafar Iqbal Chaudhry

DEDICATION

This humble effort is dedicated to my favorite and ideal

personality Muhammad (PBUH) who is ever a beacon of

knowledge and guidance for humanity, my Parents - who always

raised their hands for my success.

AACCKKNNOOWWLLEEDDGGEEMMEENNTTSS

I have no words to express my deepest sense of gratitude and innumerous

thanks to ALMIGHTY ALLAH, the compassionate, the merciful, the only creator of

universe, whose countless blessings enabled me to complete this work. I offer my

humblest thanks to the HOLY PROPHET MUHAMMAD (PEACE BE UPON HIM) who

is ever a beacon of guidance and knowledge for humanity.

I express my deep sense of gratitude, to my reverend and learned Supervisor,

Prof. Dr.Mohammad Athar Khan, Chairman, Department of Epidemiology & Public

Health, University of Veterinary and Animal Sciences, Lahore for his sympathetic

attitude, skillful and expert guidance which enabled me to present this endeavour in

the present form.

I express my appreciations to members of the supervisory committee viz, Dr.

Muhammad Arif Khan, Professor of SURGERY, University of Veterinary and Animal

Sciences, Lahore and Prof. Dr. Zafar Iqbal Ch., Ex-Dean, Faculty of Veterinary

Science, University of Veterinary and Animal Sciences, Lahore, for their positive

criticism and assistance in my research project.

I pay my cordial gratitude to DR. ATTA-UR-REHMAN, ex-Chairman Higher

Education Commission, Pakistan, for initiating such a merit oriented program of PhD

fellowship and DR. JAVED LEGHARI for continuing it instead of political

impediments.

I affectionally like to revive and appreciate the sincerity, help and

encouragement of my professional colleagues, and all of my other friends.

Last but not least, I must acknowledge my indebtedness to my loving family

members for the motivation to take up this program of studies, and their hands in

prayers for my success, and great patience and goodwill throughout the fairly long

period of training at this institution.

ALTAF MAHMOOD

CONTENTS

DEDICATIONS_________________________________________________________ (i)

ACKNOWLDGEMETNS_________________________________________________ (ii)

TABLE OF CONTENTS__________________________________________________ (iii)

LIST OF FIGURES______________________________________________________ (iv)

LIST OF TABLES _______________________________________________________ (v)

S. No. Chapter Page No.

01 Introduction 1

02 Review of Literature 6

03 Materials and Methods 50

04 Results 72

05 Discussion 133

06 Summary 161

07 Literature cited 164

TABLE OF CONTENTS

Sr. # CHAPTER PAGE

#

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 6

2.1 Distribution and Frequency 6

2.2 Determinants 14

2.3 Haematological Studies 29

2.4 Biochemical Analysis 33

2.5 Urinalysis 39

2.6 Treatment 41

2.7 Gossypol Toxicity 47

3. MATERIALS AND METHODS 50

3.1 Study Area 50

3.2 Active Surveillance 51

3.3 Case-Control Study 54

3.4 Clinical Trial 57

3.5 Cross-Sectional Study 60

3.6 Experimental Study 70

4. RESULTS 72

4.1

Active Surveillance. 72


4.3 Clinical Trial 101



Sr.# CHAPTER Page#

5. DISCUSSION 133

5.1 Active Surveillance 133


5.3 Clinical Trial

141



5.6 Recommendations 159

6. SUMMARY 161

7. LITERATURE CITED 164

LIST OF FIGURES

Fig. # Title Page #

1 Mortality rates of buffalo diseases in eight selected villages of

district Chakwal.

75

2 Proportional mortality rates of buffalo diseases in eight selected

villages of district Chakwal.

76

3 Distribution of buffalo diseases occurred during one year period

(April 2010 – March 2011) in eight selected villages of district

Chakwal.

81

4 Group wise mortality rates of buffalo diseases occurred during one

year period (April 2010 – March 2011) in eight selected villages

of district Chakwal.

82

5 Proportional mortality rates of buffalo diseases occurred during

one year period (April 2010 – March 2011) in eight selected

villages of district Chakwal.

83

6 Number of exposed and unexposed animals in case - control

groups with respect to age.

86


groups with respect to lactation number.

87


groups with respect to stage of pregnancy.

88

9

10

Number of exposed and unexposed animals in case - control

groups with respect to post-partum period.

Number of exposed and unexposed animals in case - control

groups with respect to daily milk yield.

89

90

11 Monthly distribution of parturient haemoglobinuria cases (n =

180) occurred in district Chakwal from April 2009 – January

2011.

95

12 Distribution of parturient haemoglobinuria cases (n = 180)

occurred in district Chakwal from April 2009 – January 2011 with

respect to lactation number.

96



respect to stage of pregnancy.

97

Fig. # Title Page #



respect to post-partum period.

98



respect to previous history of disease (repeat effectees).

99

16 Number of parturient haemoglobinuria effected (n = 180)

recovered and died animals in district Chakwal recorded during

April 2009 – January 2011

100


groups with respect to RBCs. 110


groups with respect to haemoglobin concentration.

111


groups with respect to haematocrit.

112


groups with respect to mean corpuscular volume (MCV).

113


groups with respect to mean corpuscular haemoglobin (MCH).

114


groups with respect to erythrocyte sedimentation rate (ESR).

115


groups with respect to serum glucose.

116


groups with respect to serum cholesterol.

117


groups with respect to serum alkaline phosphatase.

118


groups with respect to serum billirubin conjugated.

119


groups with respect to serum billirubin unconjugated.

120

Fig. # Title Page #


groups with respect to serum total billirubin.

121


groups with respect to serum inorganic phosphorous.

122


groups with respect to serum copper.

123


groups with respect to serum molybdenum.

124


groups with respect to serum selenium.

125

LIST OF TABLES

Table # Title Page #

1 Epidemiological rates of buffalo diseases occurred during one year

period (April 2010 – March 2011) in eight selected villages of district

Chakwal

74

2 Distribution of buffalo diseases occurred during one year period (April

2010 – March 2011) in eight selected villages of district Chakwal

77

3 Monthly distribution of buffalo diseases occurred during one year

period (April 2010 – March 2011) in eight selected villages of district

Chakwal

78

4 Distribution of buffalo diseases occurred during one year period (April


80

5 Results of case – control study for parturient haemoglobinuria

conducted on 180 case control pairs in district Chakwal from April

2009 – January 2011

85

6 Monthly distribution of parturient haemoglobinuria cases (n = 180)

occurred in district Chakwal from April 2009 – January 2011 93

7 Season wise distribution of parturient haemoglobinuria cases (n = 180)

occurred in district Chakwal from April 2009 – January 2011

93

8 Distribution of parturient haemoglobinuria cases (n = 180) occurred in

district Chakwal from April 2009 – January 2011 with respect to

lactation number

93


district Chakwal from April 2009 – January 2011 with respect to stage

of pregnancy

94


district Chakwal from April 2009 – January 2011 with respect to post-

partum period

94


district Chakwal from April 2009 – January 2011 with respect to

previous history of disease (repeat effectees)

94

12 Recovery rates of different treatment packages for parturient

haemoglobinuria in buffaloes (n = 30)

102

Table # Title Page #

13 Odds ratios with 95% confidence intervals and P-values for

haematological and serum biochemical variables of haemoglobinuric

buffaloes with control group I

105



buffaloes with control group II

106



buffaloes with control group I and II combined

109

16 Haematological values of haemoglobinuric and healthy buffaloes

128

17 Serum biochemical values of haemoglobinuric and healthy buffaloes

129

18 Haematological and biochemical values of rabbits after gossypol

treatment

132

LIST OF APPENDICES

Appendix # Title Page #

Appendix-1 Meta-analysis of disease (parturient haemoglobinuria) distribution with

respect to various descriptive characteristics

178

Appendix-2 Tests of between-subjects effects in cases and controls for different

variables in case control study 179

Appendix-3 Tests of between-subjects effects in cases and controls for

haematological variables

180

Appendix-4 Tests of between-subjects effects in cases and controls for biochemical

variables

181

Appendix 5 Analysis of variance for haematological parameters of cases and

controls in cross-sectional study

182

Appendix 6 Analysis of variance for serum biochemical parameters of cases and


184

CHAPTER - 1

INTRODUCTION

Buffalo is the main dairy animal in Indo-Pakistan subcontinent contributing almost 70%

of the total milk. Out of total 166 millions world buffalo population, 96% is inhabited in

Asia where buffaloes are almost exclusively owned by small landless farmers owing 2 –

3 animals only. According to an estimate, Pakistan has third largest buffalo herd in the

world comprising of 22 million heads after 92 millions and 23.5 millions in India and

China respectively. Majority (64%) of the total buffalo population of Pakistan is

inhabited in Punjab followed by Sind (28%), KPK (07%) and Baluchistan (01%). Buffalo

milk is preferred in Pakistan due to its delicious taste and more butter fat (5 – 9%) as

compared to the milk of cattle. Buffalo population of Pakistan is an important national

asset comprising of the best dairy breeds of the world i.e. Nili Ravi and Kundi. Male

buffaloes are also an important source of beef in Pakistan. Despite decades of neglect in

terms of research of diseases and genetic make up, buffalo herd of Pakistan has shown an

increasing trend in the population growth which has been estimated 2.6% for 1987 –

1997 as compared to 2% and 1.3% of India and China respectively. An average annual

growth rate of 5.7% was also recorded in milk production in Pakistan during 1987 – 1997

which was also the highest worldwide (Anonymous, 2009, Khan, 2009, Panezai, 2007,

Dalir-Naghadeh et al, 2005, Akhtar et al, 2006, Anonymous, 1998).

Inspite of great production potential, buffalo is exposed to various raging and fatal

diseases and the farmers therefore have to bear heavy economic losses. These facts

indicate that the great production potential of buffalo may be utilized to improve the

INTRODUCTION

2

agriculture based rural economy of Pakistan by placing more emphasis on health status of

this specie.

According to recommendations of 2nd

world buffalo congress, (1988), New Delhi, India,

“Field investigations related to disease surveillance, monitoring and forecasting should be

given top priority in the list of basic research on buffalo diseases”.

Among the major disease problems of buffaloes, parturient haemoglobinuria is a potent

threat to the breeding age buffaloes of India and Pakistan affecting a considerable number

of buffaloes every year (Gahlawat et al., 2007, Akhtar, 2006). According to a survey

conducted during 1996, the estimated annual economic losses due to parturient

haemoglobinuria were Rs. 490.2 millions in Punjab, Pakistan (Anonymous, 1996).

It is a non infectious hemolytic syndrome of buffaloes characterized by intravascular

haemolysis, haemoglobinaemia, haemoglobinuria and anaemia (Akhtar et al, 2007, Rana

and Bhardwaj, 1988). No etiological pathogen has so far been isolated from the affected

animals (Pirzada et al., 1989, Cheema et al., 1980). The incidence rate is much higher in

buffaloes as compared to cattle (Dalir-Naghadeh et al, 2005, Pirzada et al, 1989). The

buffaloes are highly susceptible either in advanced pregnancy and / or in early lactation

usually close to parturition with majority of animals in their third to sixth lactations

(Dalir-Naghadeh et al, 2005, Heuer and Bode, 1998, Chugh et al., 1996, Goel et al,

1988). The exact pathogenesis is not known however a variety of causal factors have

been reported to be associated with this disease in different parts of the continent (Akhtar

et al., 2008, khan et al, 2007). Determinants include dietary phosphorus deficiency,

cruciferous plants, saponin from berseem, mineral deficiency and competition of mineral

absorption (Brechbuhl et al, 2008, Radostits et al 2007, Khan et al, 2007, Neto et al,

INTRODUCTION

3

2007, Mac. William et al., 1982, Pirzada et al., 1989, Mohamed et al, 1988). Excess of

molybdenum in soil and fodder reduces phosphorous contents of the body by interfering

with its absorption from gastro intestinal tract and increasing its elimination through urine

(Dhilon et al, 1972). Hypermolybdenosis also induces copper deficiency which is

considered another important etiological factor of parturient haemoglobinuria (Radostits

et al, 2007, Kahn and Line, 2005, Dua, 2003). Copper deficiency reduces the activity of

the copper containing enzyme, superoxide dismutase because it is a part of the

erythrocyte protection mechanism against oxidative stress (Smith et al., 1975).

This process helps in rendering them vulnerable to “Heinz body formation” as a result of

feeding on poisonous (toxic) plants (Jain, 1993) like sugar beet leaves, alfalfa hay,

brassica and berseem. S.methyl cystein sulfoxide in brassica, saponin in sugar beets,

alfalfa and berseem are primary haemolytic factors (Radostits et al 2007, Dalir-

Naghadeh et al, 2005, Pirzada and Hussain, 1998, Mohamed and El-bagoury, 1990,

Mohamed et al, 1988, Smith et al, 1974). Heinz bodies are most commonly associated

with hemolytic anaemias caused by toxic agents causing damage to erythrocytes leading

to erythrolysis or intravascular haemolysis (Feldman et al, 2000, Gardner et al, 1976,

Coles, 1986). According to Hutchison, 1977, if a practitioner detects Heinz bodies on a

bovine blood smear, the differential diagnosis may rule in the feeding of poisonous

plants.

The mammalian red blood cells depend on glucose metabolism for main source of

energy for viable function and structure. This makes them susceptible to the factors

inhibitory to glycolytic pathways (Radostits et al., 2007). Low serum phosphorus level is

also associated with reduced erythrocyte glycolytic activity which results in less ATP

INTRODUCTION

4

production. As a matter of fact, ATP is required to maintain RBC shape and plasticity. If

levels fall below a critical point, the cells loose their deformability (normally erythrocyte

is deformable), become spherocytic and rigid resulting in their rapid removal from

circulation (Radostits et al, 2007, Akhtar et al, 2007, Elison et al., 1986). Glucose-6-

phosphate dehydrogenase enzyme is part of intraerythrocytic metabolic chain which

protects haemoglobin from oxidative denaturation in mild oxidant attacks. An excess

oxidant attack will deplete the erythrocytes of glucose 6-phosphate dehydrogenase and its

dependent enzymes, allowing the denaturation of the globin with its resulting

precipitation and formation of Heinz bodies (Ahmad, 2008, Khan et al, 2007, Valli,

1976). Mohamed et al., 1988, observed that in many apparently healthy buffaloes there

was a highly significant drop in serum phosphorus level, even more than

haemoglobinuric animals. This therefore suggested that hypophosphataemia may be a

predisposing cause of the disease and some other hemolytic agents in the fodder may be

the causal factors associated with haemoglobinuria.

Parturient haemoglobinuria cases may be treated with sodium acid phosphate,

antifibrinolytic drugs or combination of both as antifibrinolytic drugs are compatible with

sodium acid phosphate. Some recovered animals develop severe respiratory distress due

to very low level of haemoglobin and die of anaemic anoxia. These animals may be

treated with combination of inosine (a hypoxantaine derivative) and sodium acid

phosphate because these therapeutic agents act as oxygen releasers and are effective in

combating anaemic anoxia (Goel et al., 1988, Chugh et al., 1988, Bhardwaj and Chugh,

1988, Shah et al, 1988). According to Chugh et al., 1988, the results of treatment with

antifibrinolytic drugs strongly suggested that increased fibrinolysis or proteolysis could

INTRODUCTION

5

be precipitating factor in parturient haemoglobinuria. The prevention of disease includes

supplementation with dicalcium phosphate (Pirzada et al., 1989, Pirzada and Ali, 1990).

Information on epidemiological, hematological, serum biochemical, preventive and

therapeutic aspects of parturient haemoglobinuria in the buffalo rearing countries

particularly in Pakistan is quite scanty. Further more these are important for planning a

control strategy of this disease. The present study is therefore designed to investigate the

aforementioned multidimensional aspects of disease for the achievement of following

objectives:

Burden of parturient haemoglobinuria and its distribution through active

surveillance system.

Quantification of epidemiological risk factors associated with disease through a

case - control study.

Recovery rates of different treatment packages for parturient haemoglobinuria

through randomized controlled trial.

Quantification of haematological and biochemical risk factors associated with

parturient haemoglobinuria through a cross-sectional study.

Determination of toxic effects of gossypol on hematological and biochemical

parameters by its experimental induction in rabbits.

CHAPTER - 2

REVIEW OF LITERATURE

2.1: DISTRIBUTION AND FREQUENCY

Durrani et al (2010) recorded prevalence of post parturient haemoglobinuria in buffaloes

(n=1000) grouped according to varying stages of lactation and pregnancy. 1%, 12%,

26%, 29%, 35%, 30% and 18% prevalence was recorded for 1st, 2

nd, 3

rd, 4

th, 5

th, 6

th and

7th

lactations respectively. Prevalence according to different stages of pregnancy and

parturition / lactation was 8%, 12%, 4%, 28%, 4% and 78% for 3 months pregnant

animals, 3 - 4 months pregnant animals, 4 – 7 months pregnant animals, 7 – 10 months

pregnant animals, lactating non-pregnant animals and animals within 4 weeks postpartum

respectively.

Dua (2009) reported that highest incidence of nutritional haemoglobinuria in many parts

of Punjab, India was recorded in spring followed by winter and summer.

Durrani et al (2009) recorded prevalence of post parturient haemoglobinuria in one

thousand buffaloes. These buffaloes were grouped according to varying stages of

lactation and pregnancy. Highest (60%) prevalence of disease was recorded within four

weeks postpartum. Majority of haemoglobinuric animals were in their 5th

lactation.

Radostits et al (2007) stated that post parturient haemoglobinuria effects only adult cows

usually 2 – 4 weeks after calving mostly in their 3rd

to 6th

lactations. High producing

animals are more susceptible where as beef cattle are not commonly affected. The disease


7

is distributed in many countries with relatively low incidence. Case fatality rate is 50%.

They also described two distinct forms of disease in Newzealand and North America

In Newzealand, young cattle of almost two years age are affected with subclinical

Heinz body anaemia and without hypophosphataemia.

In North America, disease effects mature, high producing cows with

hypophosphataemia in diseased animals as well as in healthy herd mates.

Gahlawat et al (2007) recorded epidemiological features of 12 haemoglobinuric

buffaloes. It was observed that disease effected buffaloes within 60 days of parturition

during 2nd

to 6th

lactation.

Khan and Akhtar (2007) recorded epidemiological observations of 60 haemoglobinuric

buffaloes selected from field cases in three districts of Punjab, Pakistan. Highest

incidence of disease (41.7%) was recorded in winter followed by summer (28.3%), spring

(23.3%) and autumn (6.7%). The disease lasted from 3 – 9 days with 15% case fatality

rate. 18.3% (11) of the diseased buffaloes were repeat effectees and among these (11)

repeat effectees 8.3% (5) and 10% (6) had suffered during 1 and 2 previous lactations

respectively. Significantly (P˂ 0.001) higher milk production was recorded before disease

(7.54 ± 3.40 liters/ day) than milk production after disease (4.50 ± 2.68 liters/ day).

Highest incidence was recorded in buffaloes yielding 10 liters or more milk per day. 25%

of the effected buffaloes were in fourth lactation. 45% (27) of haemoglobinuric buffaloes

suffered during post calving period and majority of these (59.3%) were within 23 days of

calving. 55% (33) of the diseased buffaloes were pregnant and 18 (54.6%) among these

were in third trimester. Berseem (Trifolium alexandrinum) was major constituent of


8

fodder of 65% (39) haemoglobinuric buffaloes. Maize (Zea mays), Sorghum (sorghum

vulgare), sugarcane (saccharam officinarum), wheat straw (triticum destivum), lucerne

(medicago sativa) and sarson (brassica compestris) were other fodders offered to diseased

animals.

Radwan and Rateb (2007) stated that parturient haemoglobinuria is a sporadic disease

which mostly affects buffaloes. Higher incidence of disease is reported from May to

January which coincides with late pregnancy and early parturition as most of the

buffaloes are either in advanced pregnancy or early postpartum phase during this period.

Akhtar et al (2006) studied clinical and epidemiological aspects of parturient

haemoglobinuria in buffaloes. For this purpose haemoglobinuric (n=60) and healthy

(n=60) buffaloes were randomly selected from Faisalabad, Toba tek singh and Jhang

districts of Punjab, Pakistan. A significantly higher occurrence (41.8%) of disease was

recorded in winter season. Most of the diseased buffaloes were in 4th

lactation. 45% (27)

buffaloes developed haemoglobinuria in post calving period and majority of them

(59.3%) was within 23 days of calving. Total 33 (55%) haemoglobinuric buffaloes were

pregnant out of which 18 (54.6%) were in 3rd

trimester. The highest number of diseased

buffaloes (42.5%) was yielding 10 liters or more milk per day. Case fatality rate was

15%.

Akhtar (2006) conducted a study on haemoglobinuric buffaloes (n=60) randomly selected

from Faisalabad, Toba tek singh and Jhang districts of Punjab, Pakistan. Majority of

haemoglobinuria cases were recorded during winter months. Most of the buffaloes were

affected in advanced pregnancy or within three weeks of parturition. Comparatively


9

higher incidence of disease was recorded in 3rd

and 4th

lactations. High milk producing

animals were more susceptible. Case fatality rate was 15%.

Stockdale et al (2005) recorded cases of haemoglobinuria during the conduct of an

experiment designed to examine the nutritional management of 72 dairy cows in late

pregnancy. The cows developed haemoglobinuria two to four weeks after calving and

incidence rate was 5.55%.

Kahn and Line (2005) described that post parturient haemoglobinuria (PPH) is distributed

worldwide with low incidence but high case fatality rate (10 – 30%). Most of the animals

are affected within 2 - 5 weeks of lactation and disease rarely effects beef and non

lactating cattle. It is sporadic in North America and effects high producing dairy cows

where as herd outbreaks occur in Newzealand effecting cows of all ages.

Dalir-Naghadeh et al (2005) described haemoglobinuria in 12 Iranian river buffaloes. The

age of affected buffaloes ranged from 5 – 10 years and all said animals had been recently

calved. The disease occurred within 12 – 52 days after calving and incidence varied from

second to seventh parturition. Milk yield of affected animals ranged from 2 – 7 liters per

day before the onset of clinical signs. Wheat straw, barley straw, and alfa alfa hay were

major constituents of feed offered to the diseased animals. No seasonal pattern of disease

was observed in this study because incidence was recorded in all seasons. From the

present study, it was suggested that the term post-parturient haemoglobinuria is

appropriate for this buffalo syndrome.

Iqbal et al (2005) reported that parturient haemoglobinuria (an acute disease) is one of the

most important diseases of high yielding buffaloes in Pakistan.


10

Bhikane et al (2004) recorded 91 clinical cases of parturient haemoglobinuria in

buffaloes during 1994 – 2000 and observed that,

Most of the cases occurred during January.

Age of effected animals ranged from 6 – 12 years.

Most of the haemoglobinuric animals were yielding more than four liters of milk.

Dua (2003) reported that incidence of nutritional haemoglobinuria is higher in buffaloes

as compared to cattle in Punjab, India where clinical and subclinical hypophosphataemia

is an important nutritional disorder of dairy animals and only 13.7% buffaloes have

normal mineral status.

Rahman, (2002) conducted epidemiological study of various livestock diseases and cost - benefit

ratio at farmer level in tehsil DG Khan. The incidence rate, mortality rate and case fatality rate

due to parturient haemoglobinuria were 3.09%, 0.48% and 16.54% respectively in buffaloes

while in cattle these findings were 1.17%, 0.19% and 16.32% respectively.

Khan, (2001) studied descriptive epidemiology, active surveillance and economic

importance of various livestock diseases in district Malakand. The incidence, mortality

and case fatality rate in buffaloes due to haemoglobinuria were 2.64%, 0.22% and 8.3%

respectively while in cattle these findings were 0.86%, 0.15% and 18.18% respectively.

Muhammad et al (2000) recorded epidemiological features of 116 (111 buffaloes and 5

cows) cases of parturient haemoglobinuria in Punjab province of Pakistan for a period of

two years. Eighty (68.96%), 19 (16.38%), 10 (8.62%) and 7(6.03%) cases were recorded

during November to February (winter), March to April (spring), May to August (summer)

and September to October (fall) respectively. 21 (18%) haemoglobinuric animals were in


11

prepartum phase where as 95 (81.9%) haemoglobinuric animals were in postpartum

period. Most of the postpartum cases occurred within first two months of parturition. 21

(18.1%) haemoglobinuric animals were repeat effectees. Among these repeat effectees 6

(5.17%) and 15 (12.93%) suffered from haemoglobinuria during the same and previous

lactations / gestations respectively. Berseem (trifolium alexandrinum) and brassica napus

were major constituents of fodders of 88 (75.86%) and 45 (38.79%) cases respectively.

Thompson and Badger (1999) described outbreak of post parturient haemoglobinuria in a

dairy herd. It was observed that out of 300 newly autumn calved cows, 50 developed

signs of haemoglobinuria at about 3 weeks postpartum. Incidence, mortality and case

fatality rates were 16.66%, 28% and 4.66% respectively.

Pirzada and Hussain (1998) stated that parturient haemoglobinuria is disease of economic

importance in buffalo rearing areas, particularly India, Pakistan and Egypt. In Indian

subcontinent the disease was first reported in buffaloes in 1939 from Faisalabad

(Lyallpur) district of Punjab, Pakistan. The disease mostly affects high yielding buffaloes

particularly in Rawalpindi, Jhelum, Attock and Faisalabad districts of Punjab, Pakistan.

Occurrence is reported throughout the year with relatively higher incidence in winter and

stall fed animals are mostly affected.

Heuer and Bode (1998) recorded epidemiological observations of 39 haemoglobinuric

buffaloes during December 1984 to March 1985 in Punjab province of Pakistan. Most of

the animals were affected in early lactation stage. The disease occurred seasonally from

December to March and no case was recorded between May and October.


12

Chugh et al (1996) recorded epidemiological features of 131 haemoglobinuric buffaloes.

The highest occurrence of disease was observed in September (25.2%) with the lowest in

February and March (0.8%). 88% of effected buffaloes were in 3rd

to 6th

lactations. Out

of 41 pregnant buffaloes, 32 were six or more months pregnant. 81(66.4%) of effected

buffaloes developed haemoglobinuria during first four weeks postpartum. 78 (75%) of

104 haemoglobinuric buffaloes were producing more than 10 liters of milk in their

previous lactations. Out of total 51 buffaloes with full records, 14(27.5%) developed

haemoglobinuria during 1 (12), 2(1) or 3(1) previous years.

Jain (1993) described that post parturient haemoglobinuria (PPH) mostly occurs within 2

– 3 weeks after calving but may also occur up to 42 days postpartum. The disease may be

sporadic or a herd problem. It also affects water buffaloes, sheep and goats.

Pervez (1992) studied epidemiological aspects of parturient haemoglobinuria in buffaloes

and cattle in Vehari district of Punjab, Pakistan. Incidence rate was higher in buffaloes

(3.12%) than cattle (0.5%) and the disease mostly occurred within 2 - 4 weeks of

parturition. Incidence was higher in winter as compared to summer and high milk

producing animals were more susceptible. 48.25% and 38.46% case fatality rates were

recorded for buffaloes and cattle respectively whereas 47.43% was overall case fatality

rate for buffaloes and cattle. Higher incidence of disease was recorded where

management was poor. Economic losses due to this disease were 13% based on total

losses due to common livestock diseases of cattle and buffaloes in district Vehari.

Ali (1991) recorded epidemiological features of 75 haemoglobinuric buffaloes from 8

districts of Punjab, Pakistan. He observed that:


13

Most of the animals were effected before or after parturition

Majority (36%) of diseased animals were in third lactation

Highest number of cases (44.46%) were recorded during January

Majority (88%) of effected animals were in post partum phase.

Mohamed and El-Bagoury (1990) stated that haemoglobinuria effects Egyptian buffaloes

in advanced pregnancy and puerperal period as well as heifers every year during the

green season i.e. from November to May. The degree of severity varies from year to year

according to health status of animals, stage of pregnancy and number of calvings.

Akram et al (1990) analyzed data of 131 haemoglobinuric buffaloes brought to veterinary

services for treatment. Occurrence of disease/ haemoglobinuria in 110 (83.93%) animals

was recorded during December to March. 111 (84.73%) of affected animals were in post

parturient phase. Out of these 111 cases 67 ( 51.14%) and 44 (33.59% ) animals

developed disease during 0 to 3 months and more than 3 months postpartum respectively.

Yamin (1989) studied epidemiology and economic importance of various livestock

diseases in district Lahore. Out of total 667 cattle only 2 were affected and died. The

morbidity and mortality rates were therefore 0.30% and case fatality rate was 100%.

Mohamed et al (1988) studies haemoglobinuria in 103 effected buffaloes from Monofia

province of Egypt. All the said buffaloes were high producing animals in their 3rd

to 6th

lactations and were kept on berseem without any supplement throughout the season i.e.

from Nov to May. The disease lasted for 5 - 6 days and mortality rate ranged from 2 – 5

% according to condition of the effected animals. The disease often reappeared in

subsequent lactations.


14

Bhardwaj and Chugh (1988) reported that parturient haemoglobinuria in buffaloes is an

epidemic problem in India.

Shah et al (1988) conducted a questionnaire based retrospective survey on 1987

buffaloes in Lahore city to access the incidence of parturient haemoglobinuria. Out of

1987, 130 animals (6.5%) suffered form disease during the period of last 10 years.

Incidence rates of 45.95%, 23.42%, 17.12%, 9.91% and 3.6% were recorded for 3rd

, 4th

,

2nd

, 5th

and 1st lactations respectively. Majority (69.38%) of affected animals developed

the disease within 16 to 30 days after parturition. 18.92%, 16.22 %, 13.51%, 13.50% and

9.91% cases occurred in August, June, April, May and July respectively.

Macwilliams et al (1982) described bovine post parturient haemoglobinuria (PPH) in

North America and Newzealand with different features. In North America multiparous

high producing dairy cattle are affected in their third to sixth lactations 2 – 4 weeks after

calving during the winter season. The disease is sporadic with a case fatality rate of 10 –

50 %. In Newzealand post parturient haemoglobinuria is a herd problem which affects

younger cows during their first and second lactations 2 – 4 weeks after calving. The

incidence rate is up to 40% with occasional mortality.

2.2: DETERMINANTS

Dua (2009) described that nutritional haemoglobinuria in buffaloes and cattle is

associated with molybdenum induced hypophosphataemia and hypocupraemia which

results from exclusive and prolonged feeding of berseem.

Brechbuhl et al (2008) discussed the report of a post parturient haemoglobinuria (PPH)

case in cow for awareness of veterinarians about risk factors of the disease. The said case


15

was referred to the clinic for ruminants, Vetsuisse, Faculty of Berne, Switzerland during

the winter of 2005 - 2006, when several other cases with serious haemoglobinuria were

also referred to the clinic. Anaemia, hypophosphataemia and haemoglobinuria were

characteristic findings. Analysis of feed revealed that only 60% of the required

phosphorous ration was offered to these animals during the dry and start periods. It was

recommended that further risk factors like feed containing hemolytic agents and lack of

energy in the ration should also be investigated.

Akhtar et al (2007) analyzed blood and serum samples of haemoglobinuric (n=60) and

healthy (n=60) buffaloes and concluded that anaemia, hypophosphataemia, decreased

glucose 6 phosphate dehydrogenase ( G6PD), hypocupraemia and hypermolybdenaemia

are factors which increase vulnerability of erythrocytes to haemolysis leading to

haemoglobinuria.

Khan and Akhtar (2007) investigated parturient haemoglobinuria in 60 buffaloes from

three districts of Punjab, Pakistan. They suggested that disease is strongly associated with

berseem feeding throughout the winter season.

Radwan and Rateb (2007) described that recent parturition, inadequate dietary

phosphorous intake, high calcium ratio, copper deficiency, drinking of cold water and

exposure to extreme cold weather are predisposing factors associated with parturient

haemoglobinuria in buffaloes.

Neto et al (2007) reported that post parturient haemoglobinuria in buffaloes is a

complication of phosphorous deficiency.


16

Akhtar et al (2007) studied serum mineral profile of 60 haemoglobinuric and 60 healthy

buffaloes along with mineral analysis of soil (n=50) and fodder (n=50) from disease

prone areas to determine their interrelationship. There was non significant (P>0.05)

difference in the levels of calcium and potassium between upper and lower soil surface.

Significantly (P<0.05) higher mean values of potassium, copper, iron, selenium and

molybdenum were observed in upper soil surface than in lower soil surface. The fodders

offered to haemoglobinuric animals were deficient in phosphorous and copper and were

having excess of potassium, iron and selenium. Lucerne, berseem, sarson and sorghum

were not deficient in calcium where as maize, sugarcane and wheat straw had no required

levels for dairy animals. All fodders had adequate molybdenum contents for dietary

requirements of the dairy animals. Significantly (P<0.05) decreased serum phosphorous,

copper and selenium where as increased (P<0.05) potassium, iron and molybdenum were

recorded in haemoglobinuric buffaloes as compared to healthy controls. It was concluded

that phosphorous deficiency which is a predisposing factor for haemoglobinuria is

transferred from soil to plants and ultimately animals leading to hypophosphataemia.

Radostits et al (2007) described that following predisposing factors are associated with

post parturient haemoglobinuria,

Dietary phosphorous deficiency.

Cruciferous plants in diet of the effected animals.

Copper and selenium deficiency.

Drinking of cold water or exposure to extreme cold weather.


17

Gahlawat et al (2007) investigated erythrocytic oxidative stress by comparing

malondialdehyde and reduced glutathione levels in RBCs of 12 haemoglobinuric

buffaloes with matching healthy controls. They suggested that enhanced

malondialdehyde and low reduced glutathione levels in erythrocytes of effected buffaloes

indicate the involvement of oxidative stress in this disease.

Akhtar et al (2007) investigated haemoglobinuria in buffaloes from three districts of

Punjab, Pakistan. They concluded that hypophosphataemia and decreased erythrocytic

glucose-6 Phosphate dehydrogenase (G6PD) predispose erythrocytes to hemolysis

leading to haemoglobinuria in buffaloes.

Akhtar et al (2006) studied epidemiological aspects of parturient haemoglobinuria in

buffaloes. Berseem was observed as major component (65%) of fodders of the

haemoglobinuric buffaloes. It was concluded that high milk production, 3rd

to 5th

lactation / gestation, early lactation, advanced pregnancy, berseem feeding and winter

season are risk factors of parturient haemoglobinuria in buffaloes.

Akhtar (2006) analyzed serum samples of haemoglobinuric (n=60) and healthy (n=60)

buffaloes along with soil and fodder samples from Faisalabad, Toba tek singh and Jhang

districts of Punjab, Pakistan. The fodders offered to haemoglobinuric animals were

deficient in phosphorous, and copper but having excess of potassium, iron and selenium.

Berseem, lucern, sarson and sorghum had adequate calcium concentration where as

maize, sugarcane and wheat straw did not contain required levels for dairy animals. All

analyzed fodders had adequate molybdenum contents. It was concluded that phosphorous


18

deficiency is transferred from soil to plants and ultimately animals leading to

hypophosphataemia and haemoglobinuria.

Stockdale et al (2005) designed an experiment to examine the nutritional management of

72 dairy cows (36 thin & 36 fat cows) in late pregnancy. It was observed that four fat

cows developed acute haemoglobinuria two to four weeks after calving. Significantly

(P<0.05) decreased plasma phosphorous concentration (0.87mmole/L) was recorded in

fat cows as compared to thin cows (1.12mmole/L). Plasma inorganic phosphorous

concentration of four fat haemoglobinuric cows was less than 0.3mmole/L. However

similar declined level of plasma inorganic phosphorous concentration was recorded in

another 12 cows without developing any sign of haemoglobinuria. Marginally decreased

phosphorous concentration was recorded in the feed offered to cows. It was concluded

that inadequate dietary phosphorous is predisposing factor in post parturient

haemoglobinuria.

Kahn and Line (2005) described that exact cause of post parturient haemoglobinuria

(PPH) is unknown and predisposing factors associated with this disease are,

Phosphorous deficiency (increases osmotic fragility of erythrocytes).

Copper deficiency (makes erythrocytes susceptible to oxidative injury).

Hemolytic or oxidative plant toxins (brassica specie, sugar beets, green forage).

Selenium deficiency and

Ketoacidosis


19

Dalir-Naghadeh et al (2005) recorded epidemiological features of 12 haemoglobinuric

buffalos in west Azerbaijan, Iran. They hypothesized that disease is strongly associated

with hypophosphataemia.

Iqbal et al (2005) described that dietary phosphorous deficiency is major predisposing

factor of parturient haemoglobinuria.

Habib et al (2004) investigated disorders associated with phosphorous deficiency in

buffaloes in Buner area of NWFP, Pakistan. Excessive amount of copper (28.50 µg/ g)

and low level of phosphorous (0.010 g/ 100g) was reported in range gross hay of the

study area. They described that excessive copper intake may cause haemoglobinuria in

phosphorous deficient buffaloes.

Dua (2003) stated that molybdenosis induced hypocuprosis (which is endemic in dairy

animals of many parts of Punjab, India) results in nutritional haemoglobinuria.

Muhammad et al (2000) analyzed 116 (111 buffaloes and 5 cows) cases of parturient

haemoglobinuria in Punjab province of Pakistan for a period of two years and concluded

that disease is associated with berseem and brassica napus.

Chugh et al (2000) studied lipid peroxidation in RBCs of haemoglobinuric buffaloes. For

this purpose, level of malondialdehyde was observed in RBCs of haemoglobinuric (n =

20) and healthy (n = 20) buffaloes. Level of malondialdehyde in RBCs of

haemoglobinuric buffaloes was significantly (P<0.05) higher (493.88 ± 38.17nmoles/ml)

as compared to healthy controls (237.17 ± 17.01nmoles/ml). The enhanced lipid

peroxidation in RBCs of haemoglobinuric buffaloes indicates the role of oxidative

damage to erythrocytes in this disease. These results were further validated by


20

antioxidant therapy. Vitamin E was orally administered to haemoglobinuric buffaloes at

the rate of 12 grams followed by 6 grams after every 12 hours up to maximum six doses

and 65% efficacy was recorded as 13 of 20 buffaloes recovered. Malondialdehyde level

in RBCs of four buffaloes which had responded to vitamin E therapy were 498.38 ±

64.55, 364.57 ± 37.22 and 257.20 ± 22.99 n moles/ml at 0, 72 and 120 hours after

treatment. Malondialdehyde levels in three buffaloes which had not responded to vitamin

E therapy were 646.87 ± 46.75 and 536.84 ± 30.58 n moles/ml at 0 and 72 hours after

treatment. The enhanced lipid peroxidation and its decreasing trend with antioxidant

(vitamin E) therapy validated the role of oxidative damage to RBCs in this disease.

Thompson and Badger (1999) described factors associated with post parturient

haemoglobinuria in a dairy herd in Newzealand where 50 cows developed

haemoglobinuria. A combination of grass, barley, and a mix of fruit, vegetable canary

waste and silage were fed to the effected cows. A marked decrease of phosphorous

concentration was observed in silage. The disease was therefore associated with

phosphorous deficient feed.

Chugh et al (1998) assessed the antioxidant status of erythrocytes by measuring

tocopherol (vitamin E) and reduced glutathione in haemoglobinuric (n = 20) and healthy

(n = 20) buffaloes. Significantly decreased concentration of tocopherol (1.76 ± 0.11

micro g/ml) and reduced glutathione (23.74 ± 2.86mg%) were recorded in erythrocytes of

diseased buffaloes as compared to healthy controls which had 2.45 ± 0.14 micro g/ml and

73.71 ± 3.87 mg% of tocopherol and reduced glutathione respectively. Severe

hypophosphataemia was also observed in haemoglobinuric buffaloes. It was concluded


21

that insufficient antioxidant potential of erythrocytes in this disease is associated with

hypophosphataemia.

Heuer and Bode (1998) analyzed epidemiological data of haemoglobinuric (n=39) and

healthy (n=24) buffaloes from Punjab province of Pakistan for quantification of risk

factors associated with disease. Crude odds ratios and their 95% confidence intervals for

low serum inorganic phosphorous, age (up to 7 years or above), postpartum period (up to

60 days or above) and stage of pregnancy (up to 6 months or above) were 20.9 (5.6 –

77.8), 1.9 (0.5 – 6.3) and 1.4 (0.4 – 5.1) respectively. They suggested that dietary and

seasonal factors play an important role in the pathogenesis of this disease as the onset of

disease (in December) coincides with the change of diet (from maize to berseem). They

also investigated the association between serum inorganic phosphorous, season and host

factors by analyzing serum samples of 426 adult female buffaloes from 139 farms.

Lowest serum inorganic phosphorous was recorded from December to March which was

interpreted as soil borne and related to feed changes i.e. from maize to berseem.

Pirzada and Hussain (1998) described that:

Mineral deficient soils in buffalo rearing areas of Punjab, Pakistan and depletion

of phosphorous reserves from soil due to intensive cropping

Acute shortage of green fodder in non-irrigated areas of Pakistan

Feeding of berseem (responsible for development of hypophosphataemia due to

its low phosphorous content 0.04%) throughout the green season i.e. from

November to May and brassica compestris


22

Saponin (a haemolytic agent) present in sugar beet leaves, alfa alfa hay and

berseem

Other haemolytic agents in cruciferous plants

Ingestion of cabbage and turnip leaves

Oxidative stress on erythrocytes

Hypophosphataemia induced decreased red cell glycolysis and ATP synthesis

leading to loss of deformability, increased fragility and vulnerability to

haemolysis and

Significantly wider calcium / phosphorous ratio of blood from 7th

month of

pregnancy than earlier stages

are predisposing factors associated with parturient haemoglobinuria in buffaloes.

Chugh et al (1996) hypothesized following risk factors on the basis of epidemiological

features of 131 haemoglobinuric buffaloes.

Lactation number: it was observed that most of the effected buffaloes were in

their 3rd

to 6th

lactations.

Pregnancy: majority (32 of 41) of pregnant buffaloes had six or more month’s

pregnancy.

Postpartum period: 66.4% (81 of 122) buffaloes developed disease within first

four weeks postpartum.

Milk yield: 75% (78 of 104) buffaloes were producing more than 10 liters of milk

during their previous lactations.


23

Previous history of haemoglobinuria: 27.5% (14 of 51) buffaloes with full records

had history of haemoglobinuria during 1 (12), 2 (1) & 3(1) previous years.

Fodder: berseem was major constituent of fodder of 37.3% (22of 59) buffaloes.

Ration: 46.8% (20 of 62) effected buffaloes were maintained on cottonseed cake

or cottonseed cake and wheat or wheat flour alone or with pearl millet, gram,

mustard cake and cluster bean were feeds of 42% of haemoglobinuric buffaloes.

Jain (1993) described that exact pathogenesis of post parturient haemoglobinuria

(PPH) is not known however phosphorous and copper deficiencies are suspected as

predisposing factors associated with this disease. He further explained that

erythrocytes of phosphorous deficient cattle have subnormal ATP and GSH

concentrations where as increased methemoglobin and enhanced osmotic fragility.

These metabolically compromised erythrocytes are hemolysed when exposed to plant

hemolysins (such as saponin from sugar beets or alfa alfa hay) or an oxidant.

Similarly copper deficiency compromises the ability of erythrocytes to withstand

oxidative damage by decreasing the superoxide dismutase level, rendering them

vulnerable to Heinz body formation and hemolysis as a result of feeding on poisonous

plants.

Ali (1991) studied epidemiological aspects of haemoglobinuric (n=75) and healthy

(n=25) buffaloes from Punjab province of Pakistan. He suggested that green fodders

offered to the effected animals during winter seasons (turnips, berseem and sarson) were

low in phosphorus contents and considered to be the predisposing factors of the disease.


24

Jub et al (1990) described post parturient haemoglobinuria (PPH) in 11 Holstein –

Friesian cows with wide range of ages, body condition and production levels. The

effected cows were selected from eight dairy herds in East Gippsland, Victoria.

Hypophosphataemia, Heinz bodies and strong ketoneuria were observed in seven of nine,

four of eight and six of ten cows respectively. Disease persisted in four cows despite

phosphorous therapy. Most of the herd mates having similar calving dates had normal

serum phosphorous levels and none of them had Heinz bodies. There was no evidence of

following predisposing factors.

Dietary phosphorous deficiency

Hypocuperosis

Excessive phosphorous drain due to high production.

Cruciferous plants in the diet.

It was suggested that hypophosphataemia occurs following recovery from pre parturient

ketoacidosis which is caused by undernutrition in late pregnancy and then becoming

nutritionally replete in early postpartum period. Circulating oxidants cause erythrocyte

damage due to hypophosphataemia being a predisposing factor.

Mohamed and El-Bagoury (1990) described that saponin (present in berseem, sugar beet

leaves and alfa alfa hay), cabbage and turnip leaves, a haemolytic factor in brassica plant

and low serum inorganic phosphorous concentration are predisposing factors associated

with haemoglobinuria in buffaloes. They also described the presence of haemoglobinuria

in buffaloes having normal serum inorganic phosphorous concentration who were fed

cabbage and turnip leaves as vegetable market wastes. They designed an experiment to


25

study the haemolytic effect of saponin on erythrocytes of haemoglobinuric and healthy

buffaloes in vitro. Eighty buffaloes were selected for investigation which were kept on

Trifolium alexandrium (Berseem) throughout the green seasons from 1988 to 1990. Out

of these eighty buffaloes forty were divided into clinical cases of haemoglobinuria

(n=20), sub-clinical cases (n=10) and healthy controls (n=10) according to their clinical

conditions. Samples of berseem offered to these buffaloes were also collected from

different stages of cutting for extraction of saponin. Significantly higher level of saponin

was observed in serum and plasma of haemoglobinuric buffaloes as compared to

subclinical cases and healthy controls. It was also observed that saponin extracted from

berseem haemolysed the erythrocytes of the diseased (n=20) and healthy (n=20)

buffaloes in vitro. From the present study it was suggested that saponin is an important

haemolytic agent which causes hemolysis of erythrocytes and consequently

haemoglobinuria in buffaloes.

Akram et al (1990) investigated 131 haemoglobinuric buffaloes for associated risk

factors. They concluded that disease is associated with hypophosphataemia which is

apparently due to low phosphorous content (0.04%) of berseem being used as fodder

during the green season i.e. from December to May. They further described that buffalo

rearing areas of Punjab, Pakistan have phosphorous deficient soils and buffaloes from

these areas have low blood phosphorous concentration.

Singari et al (1989) studied the effect of decreased plasma inorganic phosphorous on

erythrocyte metabolism by estimation of red cell glutathione content. Lower GSH values

(28.53 ± 1.59/100ml RBCs) were recorded in haemoglobinuric buffaloes with low

plasma inorganic phosphorous content (2.68 ± 0.16/100ml) as compared to healthy


26

controls (102.33 ± 6.37mg/100mlRBCs). It was concluded that phosphorous deficiency

adversely effects pentose phosphate pathway of erythrocytes predisposing them to

hemolysis.

Bhardwaj and Chugh (1988) stated that studies on erythrocyte metabolism, ATP, and

membrane total phospholipids in parturient haemoglobinuria affected buffaloes indicate

the involvement of oxidative stress on erythrocytes in this disease.

Mohamed et al (1988) investigated haemoglobinuric (n=103) and healthy (n=30)

buffaloes in Monofia province of Egypt and suggested that disease is associated with:

Hypophosphataemia

Haemolytic agents in the fodder

Berseem (Trifolium alexandrinum) feeding throughout the season without any

supplement

On the basis of biochemical analysis and erythrocytic fragility tests, they attributed the

lowering of serum inorganic phosphorus and cholesterol levels of affected buffaloes to

low phosphorous content of berseem and cholesterol binding activity of saponin in

berseem respectively. According to them, these two may be the contributory factors for

increased fragility of erythrocytes in affected buffaloes.

Rana and Bhardwaj (1988) estimated intraerythrocytic ATP and total membrane

phospholipids in RBCs of haemoglobinuric (n=13) and healthy (n=10) buffaloes to

determine their possible relationship with hypophosphataemia, keeping in view the key

role of phosphorus in red cell metabolism, membrane structure and stability.

Nonsignificant decrease of ATP and significant decrease of total membrane


27

phospholipids were recorded in RBCs of haemoglobinuric buffaloes as compared to

healthy controls. Scanning electron microscopy revealed spheroechinocytosis of RBCs in

haemoglobinuric buffaloes which indicate changes in membrane permeability and

integrity. They suggested that hypophosphataemia results in decreased ATP and

membrane total phospholipids leading to reduced membrane deformability and ultimately

intravascular haemolysis.

Bhardwaj et al (1988) estimated reduced glutathione (GSH), glutathione peroxidase

(GPX), glutathione reductase (GR), red cell catalase and plasma inorganic phosphorus in

haemoglobinuric buffaloes, healthy buffaloes, from endemic area and healthy buffaloes

from organized farms with no report of parturient haemoglobinuria to investigate the

phenomenon of intravascular haemolysis in this disease. Significantly (P˂0.05) decreased

levels of reduced glutathione, glutathione peroxidase, plasma inorganic phosphorus,

increased activity of glutathione reductase and decreased activity of red cell catalase in

haemoglobinuric buffaloes in this study indicated the involvement of:

Oxidant stress to erythrocytes

Initiation of protective mechanism against oxidant stress

Predisposition of erythrocyte membrane to peroxidative damage and vulnerability

to haemolysis.

Higher levels of plasma inorganic phosphorus in healthy buffaloes from organized farms

with no report of parturient haemoglobinuria indicated its protective role against above

predisposing factors in this disease.


28

Wang et al (1985) studied the effect of hypophosphataemia on red cell glycolysis and

ATP synthesis. For this purpose washed red blood cells of healthy cows were incubated

as 10% and 20% suspensions in media containing 0, 2.5 and 25mM phosphate. It was

observed that rate of glycolysis was dependent on the inorganic phosphate concentration.

Consumption of glucose and production of 2, 3 diphosphoglycerate and adenosine 5-

triphosphate (ATP) were reduced in the absence of phosphate. In another experiment,

moderate hypophosphataemia was induced in two calves. It was observed that washed

red blood cells of these calves showed a depletion of ATP when compared with red cells

from a control calf. The results indicate that red cell glycolysis and ATP synthesis is

reduced in hypophosphataemia. Subnormal ATP concentration is a predisposing factor

which alters the normal deformability of red blood cells leading to increased fragility and

haemolysis.

Macwilliams et al (1982) described bovine post parturient haemoglobinuria with different

etiological factors in North America and New Zealand. Recent parturition, heavy milk

production, dietary phosphorous deficiency and ingestion of cruciferous plants (contain

low phosphorous content i.e. 0.4% dry matter, wider calcium to phosphorous ratio i.e. ˃

2:1 and haemolytic substances e.g. saponin) are predisposing factors associated with

disease in North America. In New Zealand the disease is reported to be associated with

molybdate fertilizer induced hypocupraemia (molybdenum interferes with copper

absorption from the gut). High concentration of molybdenum and low levels of copper

during highest incidence of disease (July, August, September and October) in pasture

samples validated the hypothesized etiological factors.


29

Cecvre et al (1979) reported a case of post parturient haemoglobinuria (PPH) in Quebec

dairy herd. Anaemia and hypophosphataemia were recorded in many animals without any

clinical sign of PPH. The other animals were showing severe signs of PPH.

Gardner et al (1976) suggested that PPH is associated with oxidative denaturation of

haemoglobin.

2.3: HAEMATOLOGICAL STUDIES

Durrani et al (2010) investigated haematological parameters of haemoglobinuric

buffaloes in Pakistan. Lower mean erythrocyte count (2.9 ± 1.5 × 1012

/ L), haemoglobin

concentration (6.5 ± 1.5 gram/ dl) and haematocrit (19.4 ± 3.2%) whereas higher

erythrocyte sedimentation rate (110 ± 35.9 mm/ h) and total leukocyte count (11± 2.1 ×

109 / L) were recorded in affected buffaloes as compared to healthy controls.

Akhtar et al (2007) studied haematological alterations in haemoglobinuric buffaloes. For

this purpose blood samples were collected from 60 haemoglobinuric and 60 healthy

buffaloes and analyzed for haematological parameters. There was significant (P<0.05)

decrease in mean erythrocyte count (3.6 ± 1.0 × 1012

), haemoglobin concentration (5.8 ±

1.4g /dl), haematocrit (16.9 ± 2.8%) and lymphocytes (48.7 ± 2.9%) where as significant

(P<0.05) increase in erythrocyte sedimentation rate (104 ± 36.2mm/hour) and neutrophils

(43 ± 4.5%) in haemoglobinuric buffaloes as compared to healthy controls.

Radostits et al (2007) described that low RBC count, low PCV, reduced haemoglobin

concentration and presence of Heinz bodies in erythrocytes are consistent findings in post

parturient haemoglobinuria (PPH).


30

Khan and Akhtar (2007) recorded haematological observations of haemoglobinuric

(n=60) and healthy (n=60) buffaloes from three districts of Punjab, Pakistan. Significant

(P˂0.001) decrease in total erythrocyte count, haemoglobin concentration and

haematocrit whereas significant (P˂0.01) increase in erythrocyte sedimentation rate and

neutrophils was recorded in haemoglobinuric buffaloes compared to healthy controls.

Blood smears of haemoglobinuric buffaloes exhibited macrocytes and spherocytes.

Difference in total leukocyte count, monocytes and eosinophils between the two groups

was non significant.

Radwan and Rateb (2007) investigated haemotological parameters of haemoglobinuric

(n=20) and healthy buffaloes. Marked decrease in total erythrocyte count, haemoglobin

concentration and haematocrit were recorded in haemoglobinuric buffaloes compared to

healthy controls.

Akhtar et al (2007) analyzed blood samples of haemoglobinuric (n=60) and healthy

(n=60) buffaloes selected from Toba Tek Singh, Faisalabad and Jhang districts of Punjab,

Pakistan to investigate haematological alterations. Significantly (P˂0.001) lower mean

erythrocyte count (3.6 ± 1.0× 1012

/ l), haemoglobin concentration (5.8 ± 1.4 gm/ dl) and

haematocrit (16.9 ± 2.8%) were observed in haemoglobinuric buffaloes compared to

healthy controls. erythrocytes sedimentation rate (104 ± 36.2 mm/ hr) and neutrophils (43

± 4.5%) were significantly (P˂0.001) higher in haemoglobinuric buffaloes. Total

leukocyte count (10 ± 1.9×109/ l), monocytes (5.1 ± 1%) and eosinophils (3.2 ± 0.9%) did

not differ significantly.


31

Dalir- Naghadeh et al (2005) recorded haemotological features of 12 haemoglobinuric

buffalos in west Azerbaijan, Iran. Low packed cell volume (PCV), anisocytosis and

spherocytosis were characteristic haemotological alterations in effected buffaloes.

Bhikane et al (2004) studied 91 clinical cases of haemoglobinuria in buffaloes during

1994 – 2000. Macrocytic hypochromic anaemia and neutrophilia were haematological

findings of effected animals.

Thompson and Badger (1999) studied factors associated with postparturient

haemoglobinuria in a dairy herd. Haematological examination revealed a profound

regenerative anaemia.

Akhtar (2006) analyzed blood samples of haemoglobinuric (n=60) and apparently healthy

(n=60) buffaloes randomly selected from Faisalabad, Toba tek singh and Jhang districts

of Punjab, Pakistan for haematological parameters. Significantly (P<0.05) decreased

total erythrocyte count, haemoglobin concentration, haematocrit and lymphocyte count

where as significantly (P<0.05) increased erythrocyte sedimentation rate and neutrophil

count was recorded in diseased buffaloes as compared to healthy controls.

Jain (1993) reported that regenerative macrocytic hypochromic anaemia and Hienz

bodies in erythrocytes of effected animals are prominent haematological findings in post


Shah et al (1988) reported haemogram values of 111 haemoglobinuric buffaloes affected

during last 10 years in Lahore city. Lower RBC count (3.68 millions/cubic mm),

haemoglobin concentrations (5.8 g/dl) and PCV (19%) values were recorded in


32

haemoglobinuric buffaloes. The decrease in these haemogram values was 45.24%,

43.69% and 44.12% for RBC count, haemoglobin and PCV respectively.

Goel et al (1988) recorded haemogram values of 9 haermoglobinuric buffaloes. 3.63 ±

0.06 and 13.61 ± 0.71 were mean values for haemoglobin (gram/ %) and PCV (%)

respectively.

Bhardwaj and Rana (1988) reported scanning electron microscopic appearance of

buffaloes, human and cattle erythrocytes. Most of the buffalo erythrocytes were

biconcave discs with relatively deeper concavity as compared to human and cattle

erythrocytes and their diameter ranged from 4 to 5.6 µm with an average of 4.8 µm.

Extensive poikilocytosis was also seen. The different shapes detected were discocyte,

stomatocyte, spherocyte and kinzocyte. A drapano-echinocyte was alos detected. The

buffalo erythrocyte indicated poor survivability (majority of the cells became spherical)

as compared to cattle and human erythrocytes when stored in normal saline at 4ºC for 48

hours.

Rana and Bhardwaj (1988) investigated cytomorphological changes in erythrocytes of

haemoglobinuric buffaloes through scanning electron microscopy. Heparinized blood

was collected from effected and healthy buffaloes. Spheroechinocytosis of RBCs was

observed in haemoglobinuric buffaloes and most of the discocytes had irregular margins.

In healthy buffaloes pokilocytosis was observed. They also clearly ruled out the

possibility of Heinz bodies and involvement of compliment system activation in this

disease.


33

Elison et al (1986) described haematological features of 16 cases of post parturient

haemoglobinuria from 15 farms in Newzealand. Marked regenerative anaemia,

spherocytosis and Heinz body formation were characteristic findings. Widespread

anaemia in clinically healthy herd mates was recorded on two of eight farms. In one of

these two farms, anaemia was associated with large number of Heinz bodies.

Macwilliams et al (1982) reported decreased packed cell volume (PCV), icteric plasma,

intensified erythrogenesis, polychromasia, anisocytosis, macrocytosis, basophilic

stippling, reticulocytosis, presence of echinocytes, neutrophilic leukocytosis and

increased osmotic fragility of erythrocytes as characteristic haematological features of

bovine post parturient haemoglobinuria (PPH). Heinz bodies have been consistently

observed in erythrocytes of PPH affected New Zealand cattle. They also stated that in

PPH affected New Zealand herds, presence of Heinz body anaemia is greater than the

incidence of clinical haemoglobinuria.

Gardner et al (1976) reported in Northland that Heinz body anaemia is a consistent

finding in cattle with post parturient haemoglobinuria (PPH). It was observed that in PPH

affected herds, the prevalence of Heinz body anaemia was greater than the prevalence of

clinical haemoglobinuria.

2.4: BIOCHEMICAL ANALYSIS

Durrani et al (2010) studied biochemical profile of haemoglobinuric buffaloes in

Pakistan. Decreased serum phosphorous (1.8 ± 0.4 mg/ dl) and copper (63.4 ± 5.8 µg/ dl)

whereas increased molybdenum (185.2 ± 53.9 µg / dl), urea (50.5 ± 8.2mg / dl) and


34

creatinine (2.8 ± 0.8 mg / dl) levels were recorded in diseased buffaloes as compared to

healthy controls.

Akhtar et al (2008) assessed the role of glucose, billirubin, cholesterol and alkaline

phosphatase in haemoglobinuric buffaloes. Significantly (P˂0.05) increased blood

glucose, serum total billirubin and serum alkaline phosphatase concentrations were

recorded in effected buffaloes as compared to healthy controls.

Akhtar et al (2007) analyzed serum samples of haemoglobinuric (n=60) and healthy

(n=60) buffaloes for biochemical parameters. Significantly (P˂0.001) lowered values of

phosphorous (1.9 ± 0.6 mg/ dl), copper (65.4 ± 0.6µgm/ dl) and erythrocytic G6PD (92.3

± 13.2 mu/ 109 TECs) whereas significantly (P ˂ 0.001) higher values of molybdenum

(171.5 ± 56.7 µgm/ dl), urea (49.7 ± 7.8 mg/ dl) and creatinine (2.1 ± 0.4 mg/ dl) were

recorded in haemoglobinuric buffaloes as compared to healthy controls. Difference in

serum calcium concentration between the two groups was non significant (P= 0.907).

Khan and Akhtar (2007) recorded biochemical observations of haemoglobinuric (n=60)

and healthy (n=60) buffaloes from three districts of Punjab, Pakistan. Significantly

(P˂0.001) decreased serum phosphorous, copper, selenium, erythrocytic glucose-6

phosphate dehydrogenase (G6PD) whereas significantly (P˂0.001) increased potassium,

iron and molybdenum were recorded in haemoglobinuric buffaloes compared to healthy

controls.

Radwan and Rateb (2007) analyzed serum samples of haemoglobinuric (n=20) and

healthy buffaloes. Highly significant drop in serum inorganic phosphorous, calcium and

magnesium was recorded in diseased buffaloes compared to healthy controls.


35

Akhtar et al (2007) analyzed serum samples of haemoglobinuric (n=60) and healthy

(n=60) buffaloes for biochemical parameters. Significant (P<0.05) increase in urea (49.7

± 7.8mg/dl), createnin (2.1 ± 0.4mg/dl) and molybdenum 171.5 ± 56.7 mug/dl) whereas

significant decrease (P<0.05) in glucose-6 phosphate dehydrogenase (92.3 ± 13.2mu/109

TECs), phosphorous (1.9 ± 0.6 mg/dl) and copper (65.4 ± 6mug/dl) was observed in

haemoglobinuric buffaloes as compared to healthy controls.

Radostits et al (2007) described that marginally decreased serum inorganic phosphorous

and low copper status in blood and liver of effected animals are main biochemical

alterations in this disease.

Gahlawat et al (2007) analyzed blood samples of haemoglobinuric (n=12) and healthy

(n=12) buffaloes for malondialdehyde, reduced glutathione and plasma inorganic

phosphorus. Significantly (P˂0.01) increased malondialdehyde whereas decreased

reduced glutathione and plasma inorganic phosphorus levels were recorded in

haemoglobinuric buffaloes compared to healthy controls.

Akhtar et al (2006) recorded biochemical profile of haemoglobinuric and healthy

buffaloes. Significantly (P<0.05) decreased serum phosphorous, copper and selenium

where as increased potassium, iron and molybdenum were recorded in haemoglobinuric

buffaloes as compared to healthy controls.

Akhtar (2006) analyzed serum samples of haemoglobinuric (n=60) and healthy (n=60)

buffaloes randomly selected from Faisalabad, Toba tek singh and Jhang districts of

Punjab, Pakistan for biochemical parameters. Significant (P<0.05) increase in glucose,

urea, total billirubin, createnin, alkaline phosphatase, potassium, iron and molybdenum


36

where as significant (P<0.05) decrease in erythrocytic glucose 6 phosphate

dehydrogenase (G6PD), inorganic phosphorous, copper and selenium was observed in

haemoglobinuric buffaloes as compared to healthy controls.

Dalir-Naghadeh et al (2005) studied haemoglobinuria in 12 river buffaloes in West

Azerbaijan, Iran. In all 12 cases serum inorganic phosphorus level was below 0.65mmol/l

which was four times lower when compared with healthy buffalos.

Iqbal et al (2005) stated that low blood phosphorous level is characteristic biochemical

alteration in parturient haemoglobinuria.

Bhikane et al (2004) studied 91 haemoglobinuric buffaloes during 1994 - 2000.

Biochemical analysis revealed severe hypophosphataemia, wide calcium: phosphorous

ratio, hyperbillirubinaemia, increased serum urea, nitrogen and createnin values.

Dua (2003) stated that in dairy animals of Punjab, India significantly high level of

molybdenum is consistently recorded in clinical cases of hypophosphataemia.

Fa-qing et al (2002) studied phospholipid constituents of erythrocyte membrane in three

groups of cows: the haemoglobinuria group (HN), the hypophosphataemia group (HP)

and the control group (CK). The contents of the phospholipid constituents in HN and HP

groups were changed. There was significant decrease in phosphatidylethaanolamine (PE)

content in HN group as compared to HP and CK groups where as significant increase was

observed in sphingomyline (SM) and phosphatidylcholine (PC) + phosphatidylserine

(PS) content in HN group than two other groups. There was significant positive and

negative correlation between serum phosphorous and PE content, serum phosphorous and

SM content respectively.


37

Thompson and Badger (1999) studied an outbreak of post parturient haemoglobinuria in a

dairy herd. Marked decrease in serum inorganic phosphorous of effected animals was

recorded on biochemical analysis.

Pirzada and Hussain (1998) stated hypophosphataemia and decreased erythrocytic

glutathione (GSH) level as prominent biochemical alterations in haemoglobinuric

buffaloes.

Jain (1993) reported that decreased plasma inorganic phosphorous (2.68 ± 0.16 mg/dl

compared to normal 5.40 ± 0.24 mg/dl), erythrocyte membrane phospholipids (1.83 ±

0.42 mg/ml compared to healthy animals 2.86 ± 0.39mg/ml) and increased glutathione

reductase levels are prominent biochemical findings recorded in haemoglobinuric water

buffaloes.

Ali (1991) studied haemoglobinuria in buffaloes from 8 districts of Punjab, Pakistan.

Biochemical analysis of clinically effected buffaloes revealed 1.89% increase in serum

calcium level whereas 44.01% and 43.67% decrease in serum inorganic phosphorus and

copper levels respectively.

Mohamed and El-Bagoury (1990) determined serum inorganic phosphorous levels in

clinical cases (n=20) of haemoglobinuria, subclinical cases (n=100 and apparently

healthy buffaloes (n=10). Significantly (P˂0.001) decreased serum inorganic

phosphorous was recorded in subclinical cases (0.66 ± 0.15 mg/ 100ml) as compared to

clinical cases (1.83 ± 0.21 mg/ 100ml) and healthy controls (5.20 ± 0.41 mg/ 100 ml). It

was also observed that serum inorganic phosphorous level of some subclinical cases was

0.1mg/ 100 ml without development of disease / haemoglobinuria.


38

Akram et al (1990) analyzed serum samples of haemoglobinuric (n=38) and healthy

(n=24) buffaloes for determination of inorganic phosphorous concentration. Below 1mg

%, below 2mg % and below 3mg % inorganic phosphorous levels were recorded in 19

(50%), 8 (21%) and 6 (16%) haemoglobinuric buffaloes respectively with a mean of 1.6

mg %. Out of 24 healthy animals 4 (17%) and 1 (4%) exhibited below 3mg % and below

2mg % serum inorganic phosphorous concentrations respectively with an average value

of 3.9mg %.

Mohamed et al (1988) analyzed non heparinized blood samples of haemoglobinuric

(n=103) and apparently healthy (n=30) buffaloes for calcium, inorganic phosphorus,

magnesium, sodium, potassium, chlorides and cholesterol. The affected buffaloes

exhibited non significant (P˃0.01) variation in serum calcium, significant (P˂0.01)

increase in magnesium whereas significant (P˂0.001) decrease in inorganic phosphorous,

cholesterol, sodium, potassium and chloride levels.

Bhardwaj and Chugh (1988) stated that hypophosphataemia, hypocupraemia and

hypermolybdenaemia are main biochemical alterations in haemoglobinuric buffaloes.

Bhardwaj et al (1988) estimated plasma inorganic phosphorous in haemoglobinuric

buffaloes (n=42), healthy buffaloes from endemic area (n=15) and healthy buffaloes from

organized farms with no report of parturient haemoglobinuria (n=17). Significantly

(P˂0.05) decreased level was recorded in haemoglobinuric buffaloes (2.68 ± 0.16 mg/100

ml) as compared to healthy buffaloes from endemic area (4.7 ± 0.40 mg/ 100 ml) and

healthy buffaloes from organized farms with no report of this disease (6.02 ± 0.24 mg/

100 ml).


39

Elison et al (1986) described biochemical features of 16 post parturient haemoglobinuria

cases from 15 farms in Newzealand. Subnormal serum inorganic phosphorous

concentration was recorded in nine of eleven examined cases. There was widespread

hypophosphataemia in four of ten herds where as individual cows were

hypophosphataemic in all ten. There was low selenium status in six of eight herds.

Hypomagnesaemia and hypocupraemia were not consistent. It was concluded that two

distinct entities of post parturient haemoglobinuria exist in the region.

Young cow was effected with subclinical widespread Heinz body anaemia in herd

mates. Normal serum inorganic phosphorous was recorded.

High producing animals were effected with low serum inorganic phosphorous.

Macwilliams et al (1982) described two distinct entities of bovine post parturient

haemoglobinuria (PPH) in North America and New Zealand. Low level of serum

inorganic phosphorous (0.4 – 1.5 mg/ dl) is characteristic biochemical finding in PPH

affected cattle of North America. Moderate hypophosphataemia (2 – 3 mg/ dl) is also

reported in lactating but clinically normal cows of PPH affected herds whereas majority

of non lactating cows have normal serum inorganic phosphorous concentrations. In New

Zealand low levels of copper in serum and liver of PPH affected animals is prominent

biochemical alteration whereas hypophosphataemia is not a consistent finding.

2.5: URINALYSIS

Akhtar et al (2008) analyzed urine samples of haemoglobinuric and healthy buffaloes

selected from Faisalabad, Toba Tek Singh and Jhang districts of Punjab, Pakistan. They

reported that urine of clinically effected buffaloes ranged from red, dark red to coffee


40

color with strongly alkaline PH. It was positive for haemoglobin (100%) and albumin

(95%) but negative for sugar and ketone bodies. No intact erythrocytes were observed on

microscopic examination.

Radwan and Rateb (2007) analyzed urine samples of 20 haemoglobinuric buffaloes and

reported that urine was dark red brown to black in color. After centrifugation, the

sediment retained uniform clear red color without clotted blood or puss cells.

Akhtar (2006) analyzed urine samples of haemoglobinuric (n=60) and healthy (n=60)

buffaloes randomly selected from Faisalabad, Toba tek singh and Jhang districts of

Punjab, Pakistan for biochemical parameters. Urine of haemoglobinuric buffaloes was

positive for haemoglobin (100%) and albumin (95%) where as it was negative for sugar

and ketone bodies. Microscopic examination revealed no intact erythrocytes.

Significantly (P<0.05) higher PH was recorded in diseased buffaloes as compared to

healthy controls.

Dalir-Naghadeh et al (2005) studied haemoglobinuria in 12 Iranian river buffaloes. It was

observed that urine samples of effected buffaloes retained a uniform clear red colour

when allowed to stand for a specific period.

Ali (1991) studied 75 haemoglobinuric buffaloes from Punjab province of Pakistan. He

reported that urine of effected buffaloes was light brown to coffee coloured and it was

positive for haemoglobin.

Macwilliams et al (1982) reported the presence of billirubin, protein and ketones in urine

of post parturient haemoglobinuria (PPH) affected cattle.


41

2.6: TREATMENT

Durrani et al (2010) conducted therapeutic trial on 120 haemoglobinuric buffaloes

divided in four groups, A, B, C and D comprising 30 animals each. Sodium acid

phosphate (60 gm in 300 ml water i/v, subcut and oral after every 12 hours), Toldimfos

sodium (Fosfan injection 100mg in 50ml packing – Samu chemical industries @ 25ml

per animal) and tea leaves (orally @ 250 gm boiled in 2 liters of water) were

administered to each animal of group A, B and C respectively whereas, group D was used

as untreated control. Discoloration of urine was taken as index of recovery. 85% efficacy

rate was recorded for Toldimfos sodium followed by tea leaves (56%) and sodium acid

phosphate (18%).

Durrani et al (2009) conducted therapeutic trial of post parturient haemoglobinuria in

buffaloes with sodium acid phosphate, tolfimfos sodium and tea leaves. 85%, 56% and

18% efficacy was recorded for tolfimfos sodium, tea leaves and sodium acid phosphate

respectively.

Brechbuhl et al (2008) reported a post parturient haemoglobinuria (PPH) case of

hospitalized cow in Switzerland. The effected cow responded well when treated with

intraveinous and oral administration of phosphorous along with blood transfusion.

Radostits et al (2007) recommended whole blood transfusion, fluid therapy, oral,

subcutaneous and intravenous administration of sodium acid phosphate (60 grams in 300

ml distilled water), bone meal or DCP, and hematinics for treatment of post parturient

haemoglobinuria (PPH).


42

Gahlawat et al (2007) investigated antioxidant effect of sodium acid phosphate in 12

haemoglobinuric buffaloes. Sodium acid phosphate was administered to 10 effected

buffaloes @ 80 grams as 20% solution in double distilled water intraveinously followed

by similar oral dose once daily till clinical recovery. The remaining two clinical cases did

not receive any treatment and were used as untreated controls. All treated cases recovered

with average 3-5 intraveinous and oral doses of sodium acid phosphate whereas 2

untreated controls died within 5 - 6 days of illness. It was observed that sodium acid

phosphate therapy declined malondialdehyde, increased reduced glutathione and restored

the plasma inorganic phosphorus levels which revealed its antioxidant potential in

haemoglobinuric buffaloes.

Radwan and Rateb (2007) conducted a therapeutic trial on 20 haemoglobinuric buffaloes

divided into three groups. 1st, 2

nd and 3

rd groups were treated with di-basic sodium

phosphate (El-Gomhouria Co.60g dissolved in 300ml normal saline i/v and similar dose

subcut for 1st three days followed by oral dose of 60gm for 7 days), 20% tonophosphan

(Intervet Egypt Co. 50ml of 20% solution i/v, 25ml i/m and 25ml subcut for 5 days) and

monobasic sodium phosphate (El- Gomhouria Co. 60g in 300 ml normal saline i/v and

similar dose subcut for 5 days followed by 60g oral dose for 7 days) respectively. The

clinical signs of haemoglobinuria disappeared within two days (1st group), 4 – 5 days (2

nd

group) and after 6 days (3rd

group) as a result of respective treatment.

Kahn and Line (2005) suggested whole blood transfusion, crystalloid fluids, sodium acid

phosphate (60 grams in 300 ml sterile water oral, subcut., and intravenous) and copper

glycinate (120 mg available copper) for treatment of post parturient haemoglobinuria.


43

Iqbal et al (2005) suggested phosphorous supplements for control of parturient

haemoglobinuria.

Bhikane et al (2004) studied 91 haemoglobinuric buffaloes during 1994 – 2000. The

cases were divided into two treatment groups i.e. group A and group B. Group A was

treated with sodium acid phosphate where as group B was treated with organic

phosphorous. On comparative analysis, almost equal therapeutic response was observed

in both groups but the percent increase in serum inorganic phosphorous was more

pronounced in group A as compared to group B.

Dalir-Naghadeh et al (2005) suggested sodium acid phosphate and blood transfusion for

treatment of post- parturient haemoglobinuria.

Chugh et al (2000) conducted a therapeutic trial on 20 haemoglobinuric buffaloes with

vitamin E. twelve grams of vitamin E was orally administered followed by six grams at

every 12 hours interval till clinical recovery or up to maximum six doses in case of no

response. 65% efficacy was recorded as 13 of 20 buffaloes recovered. For clinical

recovery, average number of required doses was 8.30 ± 0.45 in an average period of 4.15

days.

Heuer and Bode (1998) suggested phosphorous supplementation, oral laxatives and

rumen stimulants for treatment of parturient haemoglobinuria.

Thompson and Badger (1999) investigated an outbreak of post parturient

haemoglobinuria in a dairy herd in Newzealand. Out of 300 newly autumn calved cows,

50 developed haemoglobinuria. The said cases were treated with phosphorous

supplementation and supportive therapy and most eventually recovered.


44

Pirzada and Hussain (1998) suggested sodium acid phosphate (20% solution

intraveinous, subcut and oral), inosine (oxygen releaser to combat anaemic anoxia),

antifibrinolytic drugs (botrophase a blood coagulant prepared from snake venom

Bothrops jararaca), blood transfusion and phosphorous supplementation for treatment of

parturient haemoglobinuria in buffaloes. Dicalcium phosphate and bone meal are

recommended in advanced stages of pregnancy for prevention.

Jain (1993) suggested sodium acid phosphate and copper glycerate for treatment of post


Pervez (1992) conducted treatment trials for parturient haemoglobinuria in buffaloes in

Vehari district of Punjab, Pakistan with sodium acid phosphate (60 gms per animal), Cal-

D-Mag (300ml per animal) and 5% saline dextrose (1000ml per animal) in three groups

comprising 20 animals each. 85%, 35% and 0% efficacy rates were recorded for sodium

acid phosphate (Merck), Cal-D-Mag (Pfizer) and saline dextrose (Medi Pak) respectively.

Shah et al (1988) reported treatment of 111 haemoglobinuric buffaloes on the basis of

retrospective survey of Lahore city. They divided the affected buffaloes in three groups,

A (45 animals), B (45 animals) and C (21 animals) on the basis of treatment provided.

Sodium acid phosphate with oxytetracycline, sodium acid phosphate with

streptopenciline injection and sodium acid phosphate alone were administered to group

A, B and C respectively. All the three groups exhibited equal (almost 100%) response to

the respected treatment packages in an average period of 3 (group A), 3.6 (group B) and

6.6 days.


45

Goel et al (1988) investigated therapeutic role of oxygen releases to combat anaemic

anoxia. For this purpose they conducted a clinical trial on 9 buffaloes which had

recovered from parturient haemoglobinuria but still had respiratory distress due to severe

anaemia. The said buffaloes were treated with inosine (a hypoxantaine derivative) as

oxygen releaser. 500 mg of inosine was dissolved in 5 ml dilute HCl and intraveinously

administered to each buffalo once daily for 3 consecutive days after mixing with 540 ml

of 5% dextrose saline. The treatment significantly (P˂0.01) stabilized the respiration rate

from 24 ± 1.69 to 16.33 ± 0.41 per minute within 4 hours. All the affected buffaloes tided

over the situation of severe anaemia and survived.

Goel et al (1988) treated 28 haemoglobinuric buffaloes having low haemoglobin with

combination of inosine, sodium acid phosphate and para amino methyl benzoic acid

(PAMBA); a potent antifibrinolytic drug. 10 grams of sodium acid phosphate and 30 ml

para amino methyl benzoic acid were intraveinously administered to each effected

buffalo for 2 - 3 days after mixing with 540 ml of 5% dextrose saline. Eight, twelve and

seven treated buffaloes recovered after six, two and three injections respectively whereas

the remaining one buffalo responded to treatment but died.

Goel et al (1988) treated 10 haemoglobinuric buffaloes with combined therapy of sodium

acid phosphate and para amino methyl benzoic acid (PAMBA). Two, six and one

buffaloes survived after one, two and three injections respectively whereas one did not

respond and died.


46

Bhardwaj and Chugh (1988) suggested sodium acid phosphate, epsilon amino caproic

acid (EACA), para amino methyl benzoic acid (PAMBA) and inosine (as oxygen releaser

to combat anaemic stress) for treatment of parturient haemoglobinuria in buffaloes.

Chugh et al (1988) estimated therapeutic efficacy of antifibrinolytic drugs: epsilon amino

caproic acid (EACA), para amino methyl benzoic acid (PAMBA) and botropase for

parturient haemoglobinuria in buffaloes. EACA powder (sigma chemical.Co. St.Louis,

USA) 20gms in 540ml of 5% dextrose saline, unipamba injection (Unichem Lab.Ltd.,

Bombay, India) 300mg in 540ml 5% dextrose saline and botropase injection (Juggat

Pharma Pvt. Ltd, Banglore, India) 10ml in 20ml normal saline were intraveinously

administered once daily into each of 30, 13 and 18 haemoglobinuric buffaloes

respectively till clinical recovery or death. 90%, 92.30% and 94.40% efficacy was

recorded for EACA, PAMBA and botropase after average number of 3.07, 3.25 and 3

injections respectively.

Chugh et al (1988) treated 15 haemoglobinuric buffaloes with combination of sodium

acid phosphate (SAP) and antifibrinolytic drug: epsilon amino caproic acid (EACA).

EACA (10grams) and SAP (80 grams) were mixed in 540ml of 5% dextrose saline and

administered to each effected animal as slow intraveinous injection twice daily till

clinical recovery or death. 86.6% efficacy was recorded after an average number of 3.30

± 0.31 injections as 13 buffaloes recovered and remaining 2 responded to treatment but

died.

Chugh et al (1988) treated 15 and 12 haemoglobinuric buffaloes (which had already been

given sodium acid phosphate in the field for 2 or 3 occasions and had not recovered) with


47

epsilon amino caproic acid (EACA 20gms in 540ml 5% dextrose saline) and para amino

methyl benzoic acid (PAMBA 300 mg in 540ml 5% dextrose saline) respectively @ one

slow intraveinous injection daily for 2 – 3 days. Efficacy for EACA and PAMBA was

80% and 83.3% after an average number of 4.18 and 3.90 injections respectively.

Macwilliams et al (1982) suggested two different treatment packages for bovine post

parturient haemoglobinuria (PPH) in North America and New Zealand being two distinct

entities with respect to etiological factors of disease (PPH). Intraveinous administration

of sodium acid phosphate (60 gm in 300 ml distilled water), transfusion of fresh blood,

intraveinous fluid therapy and bone meal (100 gm orally twice daily) are recommended

for treatment of PPH in North America whereas parenteral administration of copper

(120mg per animal) is suggested for treatment of PPH affected New Zealand cattle. They

reported that pre-calving use of copper in cows significantly (P˂0.01) decreased the

incidence (5.18% compared to 25.51%) of PPH in New Zealand. They further reported

that increase in blood, liver and pasture copper concentrations followed by marked

decrease in incidence of PPH were recorded when top dressing of copper sulphate was

applied to pastures four months prior to calving.

2.7: GOSSYPOL TOXICITY

Prasad et al (2009) fed three different diets to 15 buffalo calves for a period of 120 days.

The calves were divided into three groups, group I /control group (n = 5), group II (n = 5)

and group III (n = 5). Group I was fed conventional diet whereas group II and III were

fed urea treated cotton stem with legume fodder and untreated cotton stem with legume

fodder respectively. Significantly increased levels of haemoglobin and packed cell


48

volume (PCV) were recorded in group II and III as compared to control group.

Insignificant difference was recorded between the groups with respect to total erythrocyte

count (TEC), total leukocyte count (TLC), mean corpuscular volume (MCV) and mean

corpuscular haemoglobin concentration (MCHC) whereas significantly increased level of

mean corpuscular haemoglobin (MCH) was recorded in group II as compared to group I.

The MCH level of group III was also higher as compared to group I but the difference

was not significant.

Kahn and Line (2005) described that excessive intake of free gossypol for long periods

can produce toxic effects in high producing dairy animals as well as other mature

ruminants. They described that decreased red blood cells, haemoglobin and packed cell

volume, decreased oxygen release from oxyhaemoglobin, decreased oxygen carrying

capacity of blood due to reduced haemoglobin and PCV values (resulting from

complexing of iron by gossypol) and increased erythrocyte fragility are main

haematological effects of gossypol toxicity.

Dabrowski et al (2000) recorded declined haemoglobin and haematocrit values of

rainbow trout due to increased gossypol concentration in the feed.

Brocas et al (1997) and Randel et al (1996) suggested that gossypol cytotoxicity effects

are associated with decreased haemoglobin, decreased packed cell volume and increased

erythrocyte fragility.

Lindsey (1980) evaluated the potential of gossypol toxicity in dairy cows by feeding

large amounts of cottonseed meal during early lactation. Decreased haemoglobin

concentration and increased erythrocyte fragility was recorded whereas packed cell


49

volume and plasma copper concentration were not effected. He suggested that long term

excessive feeding of cottonseed meal with high free gossypol content can cause toxicity

in mature ruminants.

Herman (1970) recorded almost 50% decrease in haemoglobin concentration and

haematocrit of rainbow trout fed a diet supplemented with 1000ppm free gossypol for a

period of 14 months.

CHAPTER – 3

MATERIALS AND METHODS

3.1: STUDY AREA

District Chakwal was selected from arid / barani zone of Punjab, Pakistan for present

study. Its area is 6524 square kilometers which is composed of plain as well as hilly land.

Weather is hot in summer whereas dry and cold in winter with a temperature of 42ºc and

8ºc in summer and winter respectively. Most of the parts of district are deficient in

subsoil / underground water except the towns of Choa Saiden Shah and Kallar kahar. The

district is administratively divided in four tehsils including Chakwal, Talagang, Choa

saiden shah and Kallar kahar. Wheat, sorghum, millet, maize, rape / mustard seed, gram

and groundnut are main crops of the district whereas brassica, berseem, lucerne, wheat

straw, sorghum, maize and millet are different fodders offered to the dairy animals. Total

buffalo population of the district is 145000 (Anonymous, 2009) out of which 73660 are

breeding age buffaloes (Anonymous, 2006) which constituted the study population for

present project. The study was conducted from April, 2009 to August, 2011 under

following five titles:

1. Active surveillance

2. Case control study

3. Clinical trial


51

4. Cross-sectional study

5. Experimental study

3.2: ACTIVE SURVEILLANCE

Descriptive epidemiological study was conducted in district Chakwal from April, 2010 to

March, 2011 through active surveillance system to see the distribution of parturient

haemoglobinuria and assess its burden (magnitude of problem) following recommended

procedures (Anonymous, 1999) as described below.

3.2.1: CASE DEFINITION AND DIAGNOSIS

Parturient haemoglobinuria is a non infectious hemolytic syndrome of buffaloes

characterized by hypophosphataemia, intravascular haemolysis, haemoglobinaemia,

haemoglobinuria and anaemia. Its clinical diagnosis was made on the basis of specific

signs (red/coffee colored urine & constipation/straining during defecation) whereas; other

similar disease conditions including haemoparasites and leptospirosis were ruled out

through standard laboratory techniques (Cole et al., 1973; Anwar et al., 2005).

3.2.2: TARGET POPULATION

All breeding age buffaloes of Punjab province constitute the target population for the

present study.

3.2.3: STUDY POPULATION

From the target population, all breeding age buffaloes of district Chakwal were selected

as study population.


52

3.2.4: SAMPLING PLAN

Sample was selected from study population by simple random sampling according to

following procedure.

3.2.4.1: FIRST STAGE SAMPLING

A total of 461 villages of district Chakwal constituted the sampling frame whereas one

village /town was taken as sampling unit.

3.2.4.2: SECOND STAGE SAMPLING

In second stage sampling, all breeding age buffaloes of selected villages constituted the

sampling frame whereas one breeding age buffalo was taken as sampling unit.

3.2.5: SAMPLE SIZE

Sample size was calculated according to recommendations of Thrusfield (2005) using the

following model:

n = 1.962

Pexpected (1 – Pexpected)

d2

n = required sample size

Pexpected = expected prevalence

d = desired absolute precision

By specifying the values of expected prevalence and desired absolute precision as 4%

and 5% respectively, the desired sample size was n = 59.


53

3.2.6: RANDOMIZATION

A total of eight villages including Buchal kalan, Wallana, Dandi, Saghar, Dhiryala

kahoon, Dalwal, Mureed and Karyala were randomly selected from all villages and towns

of district Chakwal for collection of required information.

3.2.7: DATA COLLECTION

Required information about all breeding age buffaloes from each household of eight

selected villages was collected on monthly basis from April, 2010 to March, 2011 by

directly interviewing the owners of respective animals and recorded in performa /

questionnaire designed for the present study. One performa / questionnaire was filled for

each (one) household. The contents of questionnaire include,

Date of interview

Name of village

Tehsil

Total number of breeding age buffaloes (denominator information)

Morbidity and mortality of breeding age buffaloes due to parturient

haemoglobinuria during the month

Morbidity and mortality of breeding age buffaloes due to diseases other than

parturient haemoglobinuria during the month

3.2.8: STATISTICAL ANALYSIS


54

The recorded information was finally pooled in data entry sheet and analyzed for

determination of incidence rate, mortality rate, case fatality rate and proportional

mortality rate of buffalo diseases following the recommendations of Thrusfield (2005).

3.3: CASE CONTROL STUDY

Population based case - control study of parturient haemoglobinuria was conducted in

district Chakwal from April, 2009 to March, 2011 for quantification of epidemiological

risk factors associated with disease according to recommended procedures (Beaglehole et

al, 2006 and Mir, 2001).

3.3.1: CASE DEFINITION



haemoglobinuria and anaemia.

3.3.2: TARGET POPULATION

All breeding age buffaloes of Punjab province constituted the target population for

present project.


All breeding age buffaloes of district Chakwal constituted the study population for

present project.

3.3.4: SAMPLE SIZE


55

Sample size was estimated according to the model recommended by Schlesselman (1982)

for two sided tests of significance. As parturient haemoglobinuria mostly effects the

buffaloes before or after parturition according to majority of previous reports, stage of

pregnancy and postpartum period were therefore considered as hypothesized risk factors

for estimation of sample size. By specifying the values of α (type I error probability), ᵦ

(type II error probability), R (the hypothesized relative risk of sufficient biologic

importance) and p0 (expected rate of exposure among controls) as 0.05, 0.10, 6 and 0.02

respectively, the desired sample size was n = 158 per group (cases and controls).

3.3.5: SELECTION OF CASES

Buffaloes suffering from parturient haemoglobinuria were selected from study

population. The disease was diagnosed on the basis of characteristic clinical signs

(haemoglobinuria & straining during defecation) and epidemiological features (advanced

pregnancy or recent parturition). The possibility of other diseases causing red urine

(haemoparasites, leptospirosis & bacillary haemoglobinuria) was ruled out through

standard laboratory methods (Cole et al., 1973; Anwar et al., 2005).

3.3.6: SELECTION OF CONTROLS

One healthy control was selected against each case of parturient haemoglobinuria.

3.3.7: MATCHING


56

Each control was individually matched to its respective case with respect to various

descriptive and demographic characteristics including age, lactation number, pregnancy

status and area/village.

3.3.8: INFORMED CONSENT

Informed consent was obtained from owner of each animal (case and control)

individually before the start of investigation.

3.3.9: MINIMIZING BIAS

Only new (incident) cases were selected to minimize the chances of bias (selection bias,

recall bias).

3.3.10: DATA COLLECTION

Data were collected about each case and control individually by interviewing the owners

of respective animals (cases and controls) from April, 2009 to March, 2011 and recorded

in a performa/ questionnaire already designed for the study. One performa/ questionnaire

was filled for each animal (case or control). The contencts of the questionnaire include;

date of interview, village, tehsil, date of start of illness i.e. haemoglobinuria (only for

cases), age of the animal, lactation number, stage of pregnancy, postpartum period, milk

yield, previous history of haemoglobinuria, previous history of other diseases, fodder,

feed/ ration, use of mineral mixture, grazing, use of drugs, use of oxytocine injection and

vaccination. The data recorded in respective columns of questionnaires were finally

pooled in a data entry sheet for analysis and comparison.



57

The data were analyzed for calculation of odds ratios, their 95% confidence intervals and

P-values using SPSS 17 software for following hypothesized risk factors.

1) Age ≥ 7 years

2) Lactation number ≥ 3

3) Stage of pregnancy ≥ 7 months

4) Postpartum period ≤ 60 days

5) Milk yield ≥ liters per day

6) Previous history of haemoglobinuria

7) Previous history of other diseases

8) Cruciferous / toxic plants

9) Feed / ration (cotton seed cake)

10) No use of mineral mixture

11) Use of drugs

12) Use of oxytocine injection

13) Vaccination

14) Constipation

15) Grazing

The data of cases were also analyzed with respect to month of disease occurrence, season

of disease occurrence, lactation number, stage of pregnancy, post-partum period and

previous disease history (parturient haemoglobinuria) of effected animals by applying chi

square test for proportion at 5% significance level using SPSS 17 software.

3.4: CLINICAL TRIAL


58

A randomized controlled trial was conducted during December 2010 – January 2011

in district Chakwal to compare and assess the therapeutic efficacy of three different

treatment packages for parturient haemoglobinuria in buffaloes following the

recommended procedures (Beaglehole et al, 2006).






Haemoglobinuric buffaloes of district Chakwal constituted the study population for

this project.

3.4.3: SELECTION OF STUDY SUBJECTS

Study subjects were selected from the study population according to following inclusion

and exclusion criteria.

3.4.4: INCLUSION AND EXCLUSION CRITERIA

Animals suffering from other diseases causing red urine (haemoparasites, leptospirosis &

bacillary haemoglobinuria) were excluded from the study after confirmation of diagnosis

through standard laboratory methods. The haemoglobinuric animals who received any

prior treatment for this disease were also excluded from the study whereas remaining

animals were selected as potential participants for the present study.


59

3.4.5: INFORMED CONSENT

Owners of potential participants (haemoglobinuric animals) were invited to participate in

the study and their informed consent was obtained before the start of investigation. A

total of 30 animals were finally selected for the study as a result of informed consent.

3.4.6: ETHICAL CONSIDERTATIONS

All the study subjects were exposed to medication and no placebo group was established

to address the ethical issues.

3.4.7: RANDOMIZATION

A total of 30 haemoglobinuric animals selected for present study were randomly divided

into three groups each comprising of 10 animals for allocation of respective treatment

packages.

3.4.8: DETAIL OF TREATMENT

3.4.8.1: PACKAGE I

Each animal of first group (regimen I) was treated by administering sodium acid

phosphate (Merck) @ 80g as 20 % solution in distilled water intraveinously as well as 80

g orally once daily till clinical recovery or death. Fresh blood was also administered @ 3

bags (500ml × 3) per animal on first day of treatment

3.4.8.2: PACKAGE II.

Ten animals of the second group (regimen II) were treated with a combination of

antifibrinolytic drug tranexamic acid (Transamine injection 250mg × 10) and sodium acid


60

pohosphate. 80g sodium acid phosphate and 2.5g tranexamic acid (Transamine injection

250mg × 10) were mixed with 500 ml of 5% dextrose saline and intraveinously

administered to each animal once daily till clinical recovery or death. A similar daily

dose of sodium acid phosphate (80g) was also administered to each animal orally once

daily till clinical recovery or death.

3.4.8.3: PACKAGE III

The remaining ten animals of third group (regimen III) were exposed to combined

therapy of Tranexamic acid and Novacoc forte injection. 2.5g tranexamic acid

(Transamine injection 250mg × 10) in 500 ml of 5% dextrose saline was intraveinously

administered to each animal once daily till clinical recovery or death. 500ml Novacoc

forte injection (Waseem Impex) was also intraveinously administered to each animal only

for one day i.e. first day of treatment.

In all three groups, the absence of haemoglobin in urine (after verification by the

benzidine test) was taken as index of recovery.


The treatment packages were compared with respect to their recovery rates and average

number of doses utilized by applying Chi-square test for proportion and Kruskal Wallis H

test respectively at 5% significance level using SPSS 17 software.

3.5: CROSS-SECTIONAL STUDY


61

Cross-sectional epidemiological study was conducted in district Chakwal during 2010 -

2011 for quantification of haematological and biochemical risk factors associated with

parturient haemoglobinuria in buffaloes.





3.5.2: SELECTION OF CASES

Thirty buffaloes suffering from parturient haemoglobinuria were randomly selected from

field cases during 2010 – 2011. Clinical diagnosis of disease was made on the basis of

characteristic signs (haemoglobinuria, straining during defication) and epidemiological

features (advanced pregnancy or early lactation) which was further validated by ruling

out other diseases causing red urine (haemoparasites, leptospirosis and bacillary

haemoglobinuria) through standard laboratory techniques. Blood and urine samples of

haemoglobinuric buffaloes were also analyzed for antimicrobial susceptibility testing to

rule out the chances of bacterial infection.

3.5.3: SELECTION OF CONTROLS

Thirty matched healthy buffaloes from the same areas maintained under similar

conditions like that of cases were selected as control group I. Another control group II of

30 buffaloes maintained under similar conditions and from the same areas but having

lactation number ± 2 as compared to control group I were also selected for analysis and


62

comparison. Each case was therefore compared with two controls to increase the strength

and precision of results.

3.5.4: COLLECTION OF SAMPLES

Blood and urine samples were collected from haemoglobinuric and healthy animals

according to following procedures.

3.5.4.1: BLOOD SAMPLES

Blood samples were collected from jugular vein of each animal (cases and controls) with

the help of sterilized disposable syringe into 3ml sterile vacuum tubes containing ethyl

diamine tetra acetic acid (EDTA) @ 1mg / ml for haematological studies. Blood samples

were also collected from each animal into 4ml sterile vacuum tubes without

anticoagulant, serum was separated and stored at -20ºc until biochemical analysis.

3.5.4.2: URINE SAMPLES

Urine samples were collected from each animal with sterilized catheter into dry sterilized

glass bottles for gross, microscopic and biochemical examination.

3.5.5: LABORATORY ANALYSIS

Blood, serum and urine samples were processed for laboratory analysis according to

following procedures.

3.5.5.1: HAEMATOLOGICAL STUDIES

Blood samples collected with anticoagulant were analyzed for following haematological

parameters:


63

3.5.5.1.1: ERYTHROCYTE MORPHOLOGY

Blood smears were prepared from fresh blood of each animal (case and control), air

dried, fixed with methanol, stained with Giemsa’s stain, washed, dried and examined

under microscope using oil immersion lens for erythrocyte morphology and

haemoparasites.

3.5.5.1.2: RETICULOCYTE COUNT

Staining solution for reticulocytes was prepared by dissolving 1g of new methylene blue

in 100ml of iso-osmotic phosphate buffer (PH 7.4). The staining solution and EDTA

blood were mixed in a ratio of 1: 4 in 75 × 10mm glass tube with the help of a pasteur

pipette. The mixture was allowed to stand at 37ºc for 20 minutes, erythrocytes

resuspended by gentle mixing, films prepared on glass slides in usual way, dried and

examined under microscope for determination of reticulocyte percentage according to the

following formula (Dacie and Lewis, 1991).

x

Reticulocyte percentage = × 100 %

n × y

Where,

x = Number of reticulocytes in the field

y = average number of cells per field

n × y = total number of cells in n fields

3.5.5.1.3: DEMONSTRATION OF HEINZ BODIES


64

Staining solution for Heinz bodies was prepared by dissolving 0.5 gm of methyl violet

stain in 100ml of 9g/l (9%) NaCl. The solution was filtered. 1ml of EDTA blood was

added to 4ml staining solution (1:4) and suspension was allowed to stand for 10 minutes

at 20º C. Then blood films were prepared on glass slides in usual way, dried and

examined under microscope for demonstration of Hienz bodies (Dacie & Lewis, 1991).

3.5.5.1.4: ERYTHROCYTE SEDIMENTATION RATE

Erythrocyte sedimentation rate (ESR) was determined by Westergren’s method (Khan &

Aslam 2001). Blood collected with anticoagulant was aspirated into the Westergren’s

tube (300mm length, 2mm diameter, and graduated from 0 – 200 at 1mm intervals) up to

the mark zero by dipping its bottom in the blood. ESR was recorded in mm after one

hour.

3.5.5.1.5: COMPLETE BLOOD COUNT

The blood samples collected with anticoagulant (EDTA) were processed through

automated haemotology analyzer Celltac a Mek 6420K ( Nihon Kohden Japan) according

to manufacturer’s instructions for various haematological parameters including white cell

count (WBC), red cell count (RBC), haemoglobin (HGB), haematocrit (HCT), mean

corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular

haemoglobin concentration (MCHC), platelets (PLT), plateletcrit (PCT), lymphocytes

(LY), monocytes (MO), granulocytes (GR), red cell distribution width (RDW), mean

platelet volume (MPV) and platelet distribution width (PDW).

3.5.5.2: SERUM BIOCHEMICAL STUDIES


65

Serum samples of cases (haemoglobinuric buffaloes) and controls (healthy buffaloes)

were analyzed for determination of glucose, cholesterol, alkaline phosphatase, bilirubin

(total & direct), calcium, phosphorous, copper, molybdenum and selenium according to

the following procedures.

3.5.5.2.1: GLUCOSE

Glucose was determined through spectrophotometer by using diagnostic kit according to

manufacturer’s instructions. Glucose oxidizes in the presence of glucose oxidase to form

gluconic acid and hydrogen peroxide.

Glucose oxidase

Glucose + oxygen + Water Gluconic acid + hydrogen peroxide

Hydrogen peroxide reacts with phenol and 4-aminophenazone in the presence of

peroxidase forming a red violet quinoneimine dye as indicator.

Peroxidase

Hydrogen peroxide + 4-aminophenazone + phenol Quinoneimine + water

3.5.5.2.2: CHOLESTEROL

Serum cholesterol was determined with the help of spectrophotometer using diagnostic

kit following manufacturer’s instructions. The test is based on enzymatic determination

of cholesterol with the help of cholesterol esterase and cholesterol oxidase. Cholesterol

esters react with water in the presence of cholesterol esterase to form free cholesterol and

fatty acids.

Cholesterol esterase


66

Cholesterol esters + water Cholesterol + fatty acids

Cholesterol is then converted to cholesten-3-on and hydrogen peroxide by reacting with

oxygen in the presence of cholesterol oxidase.

Cholesterol oxidase

Cholesterol + oxygen cholesten-3-on + hydrogen peroxide

The formed hydrogen peroxide reacts with phenol and 4-aminoantipyrine in the presence

of peroxidase to form quinoeimine dye and water.

Peroxidase

Hydrogen peroxide + phenol quinoeimine dye + water

+ 4-aminoantipyrine

3.5.5.2.3: ALKALINE PHOSPHATASE

Serum alkaline phosphatase was determined by spectrophotometer using the diagnostic

kit following manufacturer’s instructions. Test is based on kinetic determination of

alkaline phosphatase (ALP) activity as per DGKC recommendations. P- nitrophenyl

phosphate is converted to P-nitrophenol and phosphate in the presence of alkaline

phosphatase.

Alkaline phosphatase

P-nitrophenyl phosphate P-nitrophenol + phosphate

3.5.5.2.4: BILIRUBIN (TOTAL & DIRECT)


67

Billirubin (total & direct) was determined by spectrophotometer using diagnostic kit

following Jendrassik Grof method as per manufacturer’s recommendations. Total

billirubin combines with sulfanilic acid in the presence of caffeine accelerator forming a

red azobilirubin dye whose color intensity is proportional to bilirubin concentration.

HCl

Sulfanilic acid + sodium nitrite Diazotized sulfanic acid

HCl

Bilirubin + diazotized sulfanic acid Azobilirubin

The concentration of direct bilirubin was determined without caffeine additive.

3.5.5.2.5: CALCIUM

Serum calcium concentration was determined photometrically using diagnostic kit

following manufacturer’s instructions. The test is based on the reaction of calcium with

o-cresolphthalein complexone in alkaline solution resulting in formation of calcium-o-

cresolphthalein complex (purple complex) whose color intensity is directly proportional

to the calcium concentration.

Alkaline solution

Calcium + o-cresolphthalein complexone Calcium-o-cresolphthalein-complex

3.5.5.2.6: PHOSPHOROUS

Serum phosphorous was estimated with the help of spectrophotometer using reagent kit

according to the recommendations of manufacturer. Inorganic phosphorous forms


68

phosphomolybdic complex by reacting with molybdic acid. Its subsequent reduction in

alkaline medium results in creation of a blue molybdenum colour the intensity of which is

directly proportional to the phosphorous concentration in the sample.

3.5.5.2.7: ANALYSIS OF MICROMINERALS

Serum copper, molybdenum and selenium were determined by atomic absorption

spectrophotometer after preparation of samples following wet digestion method. 0.5ml of

serum sample was digested with 10ml concentrated nitric acid in a digestion flask for

almost 20 minutes at low temperature till the clearance of contents and then with 5ml

perchloric acid for about 15 minutes. The solution was vigorously heated until about 3ml

colourless material was left which was then cooled and diluted with redistilled water in a

volumetric flask upto the level of 20 ml. Mineral concentrations in the diluted samples

were measured and final quantities were determined by comparing sample reading with

standard curves.

3.5.5.3: URINALYSIS

Fresh urine samples collected from haemoglobinuric and healthy animals were processed

for gross, microscopic and biochemical analysis. Appearance, Colour, PH (PH meter),

specific gravity (urinometer), protein (Robert test), Glucose (Benedict’s test), nitrite

(commercial dipstick), blood (Benzidine test), ketones (Ross test) and bile (Gemelin test)

were determined following the procedures of Anwar et al (2005) and Khan and Aslam

(2001). For microscopic examination each urine sample was centrifuged at 1000 rpm for

3 minutes and all supernatent fluid was removed. A drop of the sediment was placed on a

clean glass slide, covered with a cover slip and examined under microscope for


69

identification of RBCs, WBCs (puss cells), epithelial cells, casts, crystals and bacteria

(Anwar et al, 2005).


Data were analyzed for calculation of odds ratios, their 95% confidence intervals and P-

values by comparing cases (n = 30) with control group I (n = 30), control group II (n =

30) and control group I and II combine (n = 60) for following hypothesized

haematological and biochemical risk factors using SPSS 17 software.

Red blood cells ˂ 5 × 1012

/µl

Haemoglobin ˂ 8 g /dl

Haematocrit ˂ 25%

Mean corpuscular volume ˃ 50 fl

Mean corpuscular haemoglobin ˃ 20 pg

Mean corpuscular haemoglobin concentration ≥ 30 g /dl

Erythrocyte sedimentation rate ˃ 80mm /1st hour

Serum glucose ˃ 60 mg /dl

Cholesterol ˃ 90 mg /dl

Alkaline phosphatase ˃ 100 u /l

Bilirubin (conjugated) ˃ 0.2 mg /dl

Bilirubin (unconjugated) ˃ 0.2 mg /dl

Bilirubin (total) ˃ 0.3 mg /dl

Calcium ≥ 2 mmoles /l

Phosphorous ˂ 2.5 mg /dl


70

Copper ˂ 95 µg /dl

Molybdenum ˃ 70 µg /dl

Selenium ˂ 15 µg /dl

Cases (n =30) and controls (n = 60) were also compared with respect to haematological

and biochemical parameters by applying independent sample t-test at 5% significance

level using SPSS 17 software.

3.6: EXPERIMENTAL STUDY

Experiment was conducted on a group of commercial female rabbits (n = 8) of 6 months

age purchased form local market and maintained at University of Veterinary & Animal

Sciences, Lahore from February 2011 to April 2011 under optimum conditions. The

cotton seed cake was then purchased from local market and analyzed for determination of

free gossypol contents following American Oil Chemist’s Society official method Ba – 7

– 58 (Irfan, 2007). The cotton seed cake containing free gossypol contents of 0.25% was

fed to rabbits @ 4 grams per kg per day in addition to their routine diet including good

quality fresh vegetables (cucumbers, spinach, cabbage & carrots) and clean water ad-

libitum. Blood samples were collected from each rabbit by ear vein puncture with the

help of sterilized needles into the vaccutainers with and without anticoagulant (EDTA)

for haematological and serum biochemical studies respectively on day 0 (before the start

of experiment), day 15, day 30, day 45 and day 60. The samples were analyzed for

complete blood count, RBC morphology, Heinz bodies, serum calcium and phosphorous

following the same procedures as described under the heading 3.4.4 (laboratory analysis).



71

The data was analyzed by applying repeated measurement analysis of variance at 5%

significance level using SPSS 17 software.

CHAPTER - 4

RESULTS


A total of eight villages including Buchal kalan, Wallana, Dalwal, Diryala kahoon,

Dandi, Saghar, Mureed and Karyala were randomly selected from district Chakwal for

active disease surveillance. Information collected from each household of these selected

villages about all diseases of adult milking buffaloes occurred during one year period was

analyzed for various epidemiological parameters and is described in detail as under.

4.1.1: INCIDENCE RATE

The highest incidence rate of 22.6% was recorded for worm infestation followed by foot

and mouth disease (15.01%), pyrexia of unknown origin (11.71%), mastitis (10.42%),

impaction (9.9%), tick infestation (8.61%), pneumonia (4.79%), parturient

haemoglobinuria (3.97%), tympany (3.81%), endometritis (3.66%), idiopathic diarrhea

(3.56%), anoestrous (2.27%), mange (1.85%), milk fever (1.49%), retention of placenta

(1.44%), uterine prolapse (1.18%), haemorrhagic septicaemia (1.08%), haemoparasites

(0.92%), dystokia (0.87%), repeat breeding (0.72%), aspiration pneumonia (0.61%),

carbohydrate engorgement (0.51%), abortion (0.51%), cystic ovaries (0.51%), partial

paralysis (0.46%), uterine torsion (0.41%) and traumatic pericarditis (0.36%). These

results along with village wise information of cases are summarized in table 1.

RESULTS

73

4.1.2: MORTALITY RATE

The highest mortality rate (1.03%) was recorded for parturient haemoglobinuria. The

mortality rates recorded for other buffalo diseases in descending order of ranking were

0.72%, 0.51%, 0.41%, 0.36%, 0.36%, 0.36%, 0.36%,0.25%, 0.25%, 0.15%, 0.1%, 0.1,

0.1, and 0.05% for foot and mouth disease, uterine prolapse, carbohydrate engorgement,

haemorrhagic septicaemia, partial paralysis, tympany, traumatic pericarditis, aspiration

pneumonia, pneumonia, impaction, haemoparasites, idiopathic diarrhea, uterine torsion

and dystokia respectively. The results are summarized in table 1.

4.1.3: CASE FATALITY RATE

The case fatality rate of traumatic pericarditis was 100% followed by carbohydrate

engorgement (80%), partial paralysis (77.77%), uterine prolapse (43.47%), aspiration

pneumonia (41.66%), haemorrhagic septicaemia (33.33%), parturient haemoglobinuria

(25.97%), uterine torsion (25%), haemoparasites (11.11%), tympany (9.45%), dystokia

(5.88%), pneumonia (5.37%), foot and mouth disease (4.81%), idiopathic diarrhea

(2.89%) and impaction (1.56%). The results are summarized in table 1.

4.1.4: PROPORTIONAL MORTALITY RATE

The proportional mortality rate of parturient haemoglobinuria out of all buffalo diseases

was 20% followed by foot and mouth disease (14%), uterine prolapse (10%),

carbohydrate engorgement (8%), haemorrhagic septicaemia (7%), partial paralysis (7%),

tympany (7%), traumatic pericarditis (7%), aspiration pneumonia (5%), pneumonia (5%),

impaction (3%), haemoparasites (2%), idiopathic diarrhea (2%), uterine torsion (2%) and

dystokia (1%). The results are summarized in table 1.

RESULTS

74

Table 1: Epidemiological rates of buffalo diseases occurred during one year period

(April 2010 – March 2011) in eight selected villages of district Chakwal (total number

of adult/milking buffaloes in eight selected villages i.e. denominator = 1938).

DISEASES

Total

cases

Total

Deaths

Incidence

Rate %

Mortality

Rate %

Case Fatality

Rate %

Proportional

MortalityRate

%

Idiopathic diarrhoea 69 2 3.56 0.1 2.89 2

Worm infestation 438 0 22.6 0 0 0

Impaction 192 3 9.9 0.15 1.56 3

Tympany 74 7 3.81 0.36 9.45 7

Carbohydrate

engorgement

10

8

0.51

0.41

80

8

Aspiration pneumonia

12

5

0.61

0.25

41.66

5

Pneumonia 93 5 4.79 0.25 5.37 5

Abortion 10 0 0.51 0 0 0

Uterine prolapse

23

10

1.18

0.51

43.47

10

Uterine torsion 8 2 0.41 0.1 25 2

Dystokia 17 1 0.87 0.05 5.88 1

Endometritis 71 0 3.66 0 0 0

Anoestrous 44 0 2.27 0 0 0

Repeat breeding

14

0

0.72

0

0

0

Retension of placenta

(ROP)

28

0

1.44

0

0

0

Cystic ovaries 10 0 0.51 0 0 0

Tick infestation 167 0 8.61 0 0 0

Mange 36 0 1.85 0 0 0

Parturient

haemoglobinuria

(PHU)

77

20

3.97

1.03

25.97

20

Milk Fever 29 0 1.49 0 0 0

Partial paralysis 9 7 0.46 0.36 77.77 7

Foot and Mouth

disease (FMD)

291

14

15.01

0.72

4.81

14

Haemorrhagic

septicaemia (HS)

21

7

1.08

0.36

33.33

7

Pyrexia of unknown

origin (PUO)

227

0

11.71

0

0

0

Haemoparasites 18 2 0.92 0.1 11.11 2

Mastitis 202 0 10.42 0 0 0

Traumatic pericarditis

7

7

0.36

0.36

100

7

RESULTS

75

Figure 1: Mortality rates of buffalo diseases in eight selected villages of district

Chakwal.

AP: Aspiration pneumonia

CRB. ENGORG: Carbohydrate engorgement

FMD: Foot & mouth disease

HS: Haemorrhagic septicaemia

PHU: Parturient haemoglobinuria

RESULTS

76

Proportional mortality%

Name of disease

uterine torsion

Uterine prolapse

Tympany

Trauma.pericarditis

Pneumonia

Part.haemoglobinU

ria

Partial paralysis

Impection

Idiopathic diarrhoea

Haem

orrhagic septica

Haem

oparasites

Foot and Mouth disea

Dystokia

Carbohydrate engorge

Aspiration pneumonia

Fre

qu

en

cy (

%)

30

20

10

0 2

10

77

5

20

7

32

7

2

14

8

5

Figure 2: Proportional mortality rates of buffalo diseases in eight selected villages of

district Chakwal.

RESULTS

77

Table 2: Distribution of buffalo diseases occurred during one year period (April


Name of villages with denominator (number of adult / milking buffaloes)

DISEASES

Buchal

kalan

(224)

Wallana

(250)

Diryiala

kahoon

(82)

Dalwal

(252)

Saghar

(64)

Dandi

(281)

Mureed

(631)

Karyala

(154)

Total

cases

Idiopathic diarrhea

4

9

4

6

4

19

8

15

69

Worm infestation

5

116

12

103

12

103

76

11

438

Impaction 4 65 7 72 7 9 13 15 192

Tympany 1 4 2 3 2 9 4 49 74

Carbohydrate

engorgement

0

1

2

1

1

0

4

1

10

Aspiration

pneumonia

0

2

0

0

1

4

4

1

12

Pneumonia 22 11 7 9 5 9 17 13 93

Abortion 0 1 1 1 1 3 3 0 10

Uterine prolapse

0

2

2

2

2

5

4

6

23

Uterine torsion 0 1 1 0 1 1 2 2 8

Dystokia 1 1 0 1 0 2 10 2 17

Endometritis 0 9 4 8 4 9 24 13 71

Anoestrous 1 9 3 5 3 7 16 0 44

Repeat breeding

0

3

2

2

2

1

4

0

14

Retension of placenta

(ROP)

5

4

4

4

4

0

4

3

28

Cystic ovaries 0 1 1 1 1 2 4 0 10

Tick infestation 3 46 9 34 9 34 32 0 167

Mange 2 6 4 5 4 6 9 0 36

Parturient

haemoglobinuria

3

5

5

7

3

12

35

7

77

Milk Fever 0 4 0 5 0 7 7 6 29

Partial paralysis 1 2 0 2 0 2 2 0 9

Foot and Mouth

disease (FMD)

0

0

26

40

5

55

117

48

291

Haemorrhagic

septicaemia (HS)

0

0

1

0

0

0

0

8

21

Pyrexia of unknown

origin (

13

40

14

40

12

15

93

0

227

Haemoparasites 0 3 0 3 1 6 5 0 18

Mastitis 39 15 22 25 9 25 40 27 202

Traumaticpericarditis. 1 1 1 1 0 0 2 1 7

RESULTS

78

Table 3: Monthly distribution of buffalo diseases occurred during one year period

(April 2010 – March 2011) in eight selected villages of district Chakwal.

Diseases

Apr

May

Jun

Jul

Aug

Sep

Oct

Nov

Dec

Jan

Feb

Mar

Total

Idiopathic

diarrhea

0

9

5

3

15

0

18

7

3

1

5

3

69

Worm infestation 44 47 43 32 43 34 33 16 63 27 33 23 438

Impection 25 7 20 4 20 15 24 19 22 19 14 3 192

Tympany 11 1 6 14 1 10 4 14 5 5 0 3 74

Carb. Engorge. 8 0 1 0 0 1 0 0 0 0 0 0 10

Aspirration

pneumonia

1

1

3

2

1

0

1

1

0

1

0

1

12

Pneumonia 6 7 5 7 4 3 8 10 16 14 13 0 93

Abortion 0 1 2 1 0 3 0 0 0 0 1 2 10

Uterine prolapse 2 2 1 0 4 2 3 3 2 1 2 1 23

Uterine torsion 0 0 1 1 0 2 3 0 0 0 1 0 8

Dystokia 0 0 0 0 4 2 5 1 2 0 1 2 17

Endometritis 0 5 5 18 11 6 7 4 4 0 1 10 71

Anoestrous 3 7 4 4 2 7 4 5 0 1 4 3 44

Repeat breeding 0 1 1 2 0 2 2 2 4 0 0 0 14

Retension of

placenta

4

1

0

7

0

2

7

1

1

5

0

0

28

Cystic ovaries 2 0 1 0 1 0 1 0 0 3 2 0 10

Tick infestation 15 22 22 29 48 18 0 0 2 0 2 9 167

Mange 5 0 0 6 2 7 2 2 0 9 3 0 36

Parturient

haemoglobinuria

6

0

0

1

0

0

3

9

22

18

12

6

77

Milk Fever 2 2 2 2 2 0 5 4 2 0 3 5 29

Partial paralysis 0 0 4 0 0 0 0 1 2 0 0 2 9

Foot and Mouth

disease (FMD)

118

65

0

0

22

13

0

0

0

13

25

35

291

Haemorrhagic

septicaemia (HS)

0

0

6

7

7

1

0

0

0

0

21

Pyrexia of

unknown origin

9

17

15

21

27

28

19

17

14

18

16

26

227

Haemoparasites 1 2 3 5 7 0 0 0 0 0 0 0 18

Mastitis 24 21 16 20 11 24 16 18 10 9 14 17 201

Traumatic

pericarditis

2

0

1

1

1

0

2

0

0

0

0

0

7

RESULTS

79

4.1.5: CLASSIFICATION / GROUP WISE ANALYSIS OF BUFFALO DISEASES

All the diseases of adult milking buffaloes occurred during one year study period in eight

selected villages of district Chakwal were grouped into various categories for further

analysis and comparison including gastro-intestinal disorders (idiopathic diarrhea, worm

infestation, impaction, tympany & carbohydrate engorgement), respiratory problems

(aspiration pneumonia & pneumonia), reproductive ailments (abortion, uterine prolapse,

uterine torsion, dystokia, endometritis, anoestrous, repeat breeding, retention of placenta

& cystic ovaries), skin abnormalities (tick infestation & mange), metabolic / deficiency

diseases (parturient haemoglobinuria, milk fever & partial paralysis), generalized

conditions (foot and mouth disease, haemorrhagic septicaemia, pyrexia of unknown

origin & haemoparasites), managemental disorders (mastitis) and miscellaneous

conditions (traumatic pericarditis).

The highest incidence rate (40.38%) was recorded for gastro-intestinal disorders followed

by generalized conditions (28.72%), reproductive ailments (11.5%), skin abnormalities

(10.46%), managemental disorders (10.42%), metabolic/deficiency diseases (5.92%) and

respiratory problems (5.40%). The mortality rate of metabolic/deficiency diseases was

highest (1.39%) among all groups. Mortality rates of other diseases in descending order

of ranking were 1.18%, 1.02%, 0.66%, 0.5% and 0.36% for generalized conditions,

gastro-intestinal disorders, reproductive ailments, respiratory problems and miscellaneous

conditions respectively. Proportional mortality rate of metabolic /deficiency diseases (out

of all buffalo problems) was 27% followed by generalized conditions (23%), gastro-

intestinal disorders (20%), reproductive ailments (13%), respiratory problems (10%) and

miscellaneous conditions (7%). These results are summarized in table 4 and figures 3,4,5.

RESULTS

80

Table 4: Distribution of buffalo diseases occurred during one year period (April


Diseases

Total

Gast

ro-

Inte

stin

al

dis

ord

ers

Res

pir

ato

ry

pro

ble

ms

Rep

rod

uct

ive

Ail

men

ts

Sk

in

Ab

norm

ali

ties

Met

ab

oli

c/

Def

icie

ncy

dis

ease

Gen

erali

zed

con

dit

ion

s

Man

agem

enta

l

dis

ord

er

Mis

cell

an

eou

s

Con

dit

ion

s

Vil

lag

es

Buchal 14 22 7 5 4 13 39 1 105

Dalwal 185 9 24 39 14 83 25 1 380

Dandi 140 13 30 40 21 76 25 0 345

Diryiala 27 7 18 13 5 41 22 1 134

Karyiala 91 14 26 0 13 56 27 1 228

Mureed 105 21 71 41 44 215 40 2 539

Saghar 26 6 18 13 3 18 9 0 93

Wallana 195 13 31 52 11 43 15 1 361

Total 783 105 225 203 115 545 202 7 2185

RESULTS

81

Diseases

Mics. C

onditions

Managm

ental disorder

Generalized conditio

Metabolic/D

eficiency

Skin Abnorm

alities

Reproductive Ailm

ent

Respiratory problem

GIT disorders

Fre

qu

en

cy (

%)

100

90

80

70

60

50

40

30

20

10

0

Villages

Wallana

Saghar

Mureed

Karyiala

Diryiala

Dandi

Dalw al

Buchal kalan

14781026141225

6

86

29203938

20

3220

13

14

13

101112

1312

14

11

8

6

8

7

12

1418

20

13

1218

1412

1512

19

11

924

141921

Figure 3: Distribution of buffalo diseases occurred during one year period (April 2010 –

March 2011) in eight selected villages of district Chakwal.

RESULTS

82

Mortality (%)

Disease

Mics. C

onditions

Generalized conditio

Metabolic/D

eficiency

Reproductive Ailm

ent

Respiratory problem

GIT disorders

Fre

qu

en

cy (

%)

1.6

1.4

1.2

1.0

.8

.6

.4

.2

0.0

.4

1.2

1.4

.7

.5

1.0

Figure 4: Group wise mortality rates of buffalo diseases occurred during one year

period (April 2010 – March 2011) in eight selected villages of district Chakwal.

RESULTS

83

Figure 5: Group wise proportional mortality rates of buffalo diseases occurred during

one year period (April 2010 – March 2011) in eight selected villages of district

Chakwal.

RESULTS

84

4.2: CASE CONTROL STUDY

Case-control study was conducted on 180 case-control pairs for quantification of

epidemiological risk factors associated with parturient haemoglobinuria of buffaloes.

Odds ratio of 15.80 was recorded for ≥ 7 months pregnancy. The odds ratios for other

variables in descending order were 6.39 for ≥ 3 lactation number, 6.23 for ≤ 60 days

postpartum period, 5.56 for ≥ 7 years age, 3.41 for previous history of haemoglobinuria

(repeat effectees), 3.34 for constipation, 2.51 for cruciferous / toxic plants (brassica,

berseem & turnips), 1.32 for use of oxytocine injection, 1.19 for previous history of

other diseases (metabolic, infectious), 1.07 for ≥ 8 liters milk yield, 1.07 for use of drugs,

1 for cotton seed cake, 1 for vaccination (HS & FMD), 0.912 for no grazing and 0.818 for

no use of mineral mixture. The odds ratios of age, lactation number, stage of pregnancy,

postpartum period, milk yield, previous history of haemoglobinuria and ingestion of

cruciferous /toxic plants were significant whereas those of cottonseed cake, no use of

mineral mixture, use of drugs, use of oxytocine injection, grazing and previous history of

diseases other than haemoglobinuria were insignificant (P ˃ 0.05) All these odds ratios

along with their 95% confidence intervals, P values and detail of exposures are

summarized in table 5 and figures 6, 7, 8, 9 and 10.

RESULTS

85

Table 5: Results of case – control study for parturient haemoglobinuria conducted

on 180 case control pairs in district Chakwal from April 2009 – January 2011

Variables

Cut off

Points

Study Groups

OR 95 % C.I P-value Cases

n = 180

Controls

n = 180

Age ≥ 7 years 151 87 5.56 3.39 – 9.11 0.000

Lactation number ≥ 3 163 108 6.39 3.57 – 11.43 0.000

Month of Pregnancy ≥7 months 56 5 15.80 6.15 – 40.59 0.000

Postpartum Period ≤ 60 days 62 14 6.23 3.31 – 11.65 0.000

Milk Yield ≥ 8 litters 101 98 1.07 0.706 – 1.62 0.000

Previous history of

Haemoglobinuria Yes 60 23 3.41 1.99 – 5.83 0.000

Previous history of

Other diseases Yes 38 33 1.19 0.70 – 2.006 0.508

Cruciferous / Toxic

plants Yes 86 48 2.51 1.61 – 3.91 0.000

Cotton seed cake Yes 160 160 1 0.518 – 1.93 1

Use of Mineral Mix Yes 88 97 0.818 0.54 – 1.23 0.343

Use of drugs Yes 64 61 1.07 0.69 – 1.6 0.740

Use of Oxytocine Inj Yes 13 10 1.32 0.56 – 3.120 0.518

Vaccination (HS&FMD) Yes 136 136 1 0.618 – 1.61 1

Constipation Yes 103 0 3.34 2.76 – 4.02 0.000

Grazing Yes 41 44 0.912 0.5 – 1.48 0.71

RESULTS

86

Figure 6: Number of exposed and unexposed animals in case - control groups with

respect to age. Seven years or more age is taken as risk factor in this study.

GROUPS

ControlCases

Co

un

t160

140

120

100

80

60

40

20

0

Age Groups (years)

7 or More

< 7

93

29

87

151

RESULTS

87


respect to lactation number. Lactation number ≥ 3 is taken as risk factor in this study.

GROUPS

ControlCases

Co

un

t200

100

0

Lactation number

3 or More

< 3

72

17

108

163

RESULTS

88

GROUPS

ControlCases

Fre

qu

en

cy

200

100

0

Stage of Pregnancy

7th Months

< 7 Months

175

124

56


respect to stage of pregnancy. Seven months or above pregnancy is taken as risk factor in

this study.

RESULTS

89

GROUPS

ControlCases

Co

un

t

200

100

0

Postpartum period

60 days and below

More than 60 days

166

118

14

62


respect to post-partum period. Post-partum period of ≤ 60 days is taken as risk factor in

this study.

RESULTS

90


respect to daily milk yield. Ten liters or more milk yield is taken as risk factor in this

study.

GROUPS

ControlCases

Co

un

t110

100

90

80

70

Milk yield (liters)

< 8

8 or More

82

79

98

101

RESULTS

91

4.2.1: DISTRIBUTION OF PARTURIENT HAEMOGLOBINURIA WITH RESPECT

TO VARIOUS DESCRIPTIVE CHARACTERISTICS

Parturient haemoglobinuria cases (n = 180) were analyzed with respect to month, season,

lactation number, stage of pregnancy, post-partum period and previous history of disease

(parturient haemoglobinuria) by applying chi square test for proportion at 5%

significance level. Detailed description of result with respect to each variable is as under.

4.2.1.1: MONTH OF DISEASE OCCURRENCE

Significantly (P ˂ 0.05) increased number of parturient haemoglobinuria cases (n = 42 /

23.3%) occurred in December followed by January (n = 34 / 18.9%), August (n = 28 /

15.6%), November n = 18 / 10%), October (n = 16 / 8.9%), March (n = 14 / 7.8%), April

(n = 8 / 4.4%), September (n = 8 / 4.4%), July (n = 6 / 3.3%), June (n = 4 / 2.2%) and

May (n = 2 / 1.1%).

4.2.1.2: SEASON OF DISEASE OCCURRENCE

Significantly (P ˂ 0.05) higher occurrence of parturient haemoglobinuria (n = 108 / 60%)

was recorded in winter season i.e. from November to March as compared to summer (n

= 72 / 40%) i.e. from April to October.

4.2.1.3: LACTATION NUBER OF EFFECTED ANIMALS

Frequency of parturient haemoglobinuria was significantly (P ˂ 0.05) higher (n = 56 /

31.1%) in 4th

lactation followed by 3rd

(n = 47 /26.1%), 5th

(n = 34 / 18.9%), 6th

(n = 18 /

10%), 2nd

(n = 17 / 9.4%), 8th

(n = 6 / 3.3%) and 7th

(n = 2 / 1.1%) lactations.

RESULTS

92

4.2.1.4: PREGNANCY STATUS OF HAEMOGLOBINURIC ANIMALS

Out of total pregnant haemoglobinuria cases (n = 90 / 50%), significantly (P ˂ 0.05)

increased number of animals (n = 22 / 24.44%) had 10th month of pregnancy whereas (n

= 16 / 17.77%), (n = 12 / 13.33%), (n = 12 / 13.33%), (n = 10 / 11.11%), (n = 8 / 8.88%),

(n =4 / 4.44%), (n = 4 / 4.44%) and (n = 2 / 2.22%) haemoglobinuric pregnant animals

had 8th

, 6th

, 4th

, 7th

, 9th

, 2nd

, 3rd

and 5th

months of pregnancy respectively. Out of 90

pregnant haemoglobinuria cases, 56 (62.22%) had ≥ 7 months pregnancy. The remaining

90 (50%) haemoglobinuric animals were not pregnant.

4.2.1.5: POST-PARTUM PERIOD OF HAEMOGLOBINURIC ANIMALS

A significantly (P < 0.05) increased proportion of haemoglobinuric animals (n = 118 /

65.6%) developed the disease after 60 days of parturition. Out of total 90 (50%) non

pregnant animals, 62 (68.88%) were effected within 60 days after parturition.

4.2.1.6: PREVIOUS HISTORY OF HAEMOGLOBINURIA

A significantly decreased proportion (n = 60 / 33.3%) of effected animals had history of

haemoglobinuria during their previous lactations (repeat effectees).

Out of total 180 cases 68 (37.77%) died and 112 (62.22%) recovered. These results in

relation to aforementioned descriptive characteristics are summarized in tables 6, 7, 8, 9,

10, 11 and figures 11, 12, 13, 14, 15 and 16.

RESULTS

93

Table 6: Monthly distribution of parturient haemoglobinuria cases (n = 180)


Month Cases Percent

Jan 34 18.9

Marc

h 14 7.8

April 8 4.4

May 2 1.1

June 4 2.2

July 6 3.3

Aug 28 15.6

Sep 8 4.4

Oct 16 8.9

Nov 18 10.0

Dec 42 23.3

Total 180 100.0

Table 7: Season wise distribution of parturient haemoglobinuria cases (n = 180)


Season Cases Percent

Nov-

March 108 60.0

April –

Oct 72 40.0

Total 180 100.0

Table 8: Distribution of parturient haemoglobinuria cases (n = 180) occurred in

district Chakwal from April 2009 – January 2011 with respect to lactation number

Lactation

number Cases Percent

2 17 9.4

3 47 26.1

4 56 31.1

5 34 18.9

6 18 10.0

7 2 1.1

8 6 3.3

Total 180 100.0

RESULTS

94


district Chakwal from April 2009 – January 2011 with respect to stage of pregnancy

Month of

pregnancy Cases Percent

0 90 50.0

2 4 2.2

3 4 2.2

4 12 6.7

5 2 1.1

6 12 6.7

7 10 5.6

8 16 8.9

9 8 4.4

10 22 12.2

Total 180 100.0


district Chakwal from April 2009 – January 2011 with respect to post-partum

period


district Chakwal from April 2009 – January 2011 with respect to previous history of

disease (repeat effectees)

Previous

history of

haemoglobin

uria Cases Percent

Yes 60 33.3

No 120 66.7

Total 180 100.0

Post-partum period Cases Percent

60 days

and below 62 34.4

More than

60 days 118 65.6

Total 180 100.0

RESULTS

95

Figure 11: Monthly distribution of parturient haemoglobinuria cases (n = 180) occurred

in district Chakwal from April 2009 – January 2011.

Months

DecNovOctSepAugJulyJuneMayAprilMarchJan

Fre

qu

en

cy

50

40

30

20

10

0

42

1816

8

28

64

8

14

34

RESULTS

96

Figure 12: Distribution of parturient haemoglobinuria cases (n = 180) occurred in

district Chakwal from April 2009 – January 2011 with respect to lactation number.

Lactation number

8765432

Fre

qu

en

cy

60

50

40

30

20

10

0

6

18

34

56

47

17

RESULTS

97

Figure 13: Distribution of parturient haemoglobinuria cases (n = 180) occurred in district

Chakwal from April 2009 – January 2011 with respect to stage of pregnancy.

Stage of pregnancy(month)

10th9th

8th7th

6th5th

4th3rd

2nd

Not P

regnant

Fre

qu

en

cy

100

80

60

40

20

0

22

8

16

101212

90

RESULTS

98

Figure 14: Distribution of parturient haemoglobinuria cases (n = 180) occurred in

district Chakwal from April 2009 – January 2011 with respect to post-partum period.

65.6%

34.4%

More than 60 days

60 days and below

RESULTS

99

Figure 15: Distribution of parturient haemoglobinuria cases (n = 180) occurred in district

Chakwal from April 2009 – January 2011 with respect to previous history of disease

(repeat effectees).

66.7%

33.3%

No

Yes

RESULTS

100

GROUPS

ControlCases

Co

un

t

200

180

160

140

120

100

80

60

40

Result

Died

Recovered

Not Applicable

180

112

68

Figure 16: Number of parturient haemoglobinuria effected (n = 180) recovered and died

animals in district Chakwal recorded during April 2009 – January 2011

RESULTS

101

4.3: CLINICAL TRIAL

The experiment was conducted on 30 haemoglobinuric buffaloes randomly divided into

three groups regimen I, regimen II and regimen III each having 10 animals. Each group

was treated with its respective treatment package till clinical recovery (absence of

haemoglobin in urine) or death.

Out of total 10 treated animals of regimen I, 7 and 3 animals recovered after 3 and 4 days

respectively. The recovery rate of respective treatment package (package I) was 100%

and average number of doses used was 3.3 ± 0.48.

Four and three animals of regimen II recovered after three and four days respectively

whereas remaining three animals did not respond to treatment and died after four (one

animal) and five (two animals) days. Recovery rate of respective treatment package

(package II) was 70% and average number of doses used was 3.8 ± 0.79.

Two and three animals of regimen III recovered after three and four days respectively.

The remaining five animals of this group did not respond to treatment and died after four

(two animals) and five (three animals) days. The recovery rate of respective treatment

package (package III) was therefore 50% and average number of doses used was 4.1 ±

0.74. These results are summarized in table 12.


Average number of doses used and recovery rates were compared by applying Kruskal

Wallis H test and Chi-square test for proportion respectively at 5% significance level

using SPSS 17 software.

RESULTS

102

Table 12: Recovery rates of different treatment packages for parturient

haemoglobinuria in buffaloes (n = 30)

Treatment Packages Package I Package II Package III

Dose & Route

Sodium acid

phosphate 80

grams as 20%

solution in distilled

water i/v and 80

grams orally once

daily.

Blood transfusion

(3 bags × 500ml)

for day I only

Sodium acid

phosphate 80 grams

and tranexamic acid

2.5 grams

(Transamine

injection 250mg ×

10) in 500 ml of 5%

dextrose saline i/v

with 80 grams of

sodium acid

phosphate as 20%

solution in distilled

water orally once

daily.

Tranexamic acid 2.5

grams (Transamine

injection 250mg ×

10) in 500 ml of 5%

dextrose saline by

slow i/v injection

once daily. Novacoc

injection 500ml i/v

for day 1only.

Cases treated 10 10 10

Cases recovered 10 7 5

Died 0 3 5

*Average number of

doses used

3.3 ± 0.48 3.8 ± 0.79 4.1 ± 0.74

**Recovery rate % 100 70 50

*P = 0.048 (significant) Kruskal Wallis H test

**P = 0.000 (significant) Chi-square test for proportion

RESULTS

103

4.4: CROSS SECTIONAL STUDY

A cross sectional epidemiological study was conducted on haemoglobinuric (n = 30) and

healthy (n = 60) buffaloes in district Chakwal during 2010 – 2011 for quantification of

haematological and biochemical risk factors associated with parturient haemoglobinuria

of buffaloes. The healthy buffaloes (n = 60) were divided into two groups (control group

I and control group II) each having 30 animals. The cases (haemoglobinuric buffaloes)

were then compared with control group I, control group II and group I & II combine (n =

60) with respect to various haematological and serum biochemical parameters. Results

are described in detail as under.

4.4.1: CASES VERSUS CONTROL GROUP I

The odds ratios recorded for various haematological parameters were significant (P ˂

0.05) including 26 (for red blood cells ˂ 5 × 106 /µl), 16 (for haemoglobin ˂ 8g /dl), 26

(for haematocrit ˂ 25%), 9.33 (for mean corpuscular volume ˃ 50fL), 11 (for mean

corpuscular haemoglobin ˃ 20pg) and 32.67 (for erythrocyte sedimentation rate ˃ 80mm

/1st hour).

The significant (P ˂ 0.05) odds ratios recorded for serum biochemical parameters were

40.88 (for billirubin unconjugated ˃ 0.2mg /dl), 40.88 (for billirubin total ˃ 0.3mg /dl), 1

(for calcium ≥ 2mmoles /L), 9.75 (for phosphorous ˂ 2.5mg /dl), 8 (for molybdenum ˃

70µg /dl) and 5.5 (for selenium ˂ 15 µg /dl). The insignificant odds ratios of serum

biochemical parameters include 3.25 (for glucose ˃ 60mg /dl), 3.5 (for cholesterol ˃

90mg /dl), 1 (for alkaline phosphatase ˃ 100 U /L) and 0.762 (for copper ˂ 95µg /dl).

RESULTS

104

These odds ratios along with their 95% confidence intervals and P-values are summarized

in table 13.

RESULTS

105

Table 13: Odds ratios with 95% confidence intervals and P-values for

haematological and serum biochemical variables of haemoglobinuric buffaloes with

control group I

Parameters

Cut off

points

Study Groups

OR 95 % C.I P-value

Cases (n=30) Control (n=30)

Red Blood Cells (106/µl) < 5 26 6 26 6.53-103.49 0.000

Haemoglobin g/dl < 8 24 6 16 4.51-56.69 0.000

Haematrocrit (%) < 25 26 6 26 6.53-103.49 0.000

MCV fL > 50 28 18 9.33 1.86 – 46.68 0.002

MCH pg > 20 22 6 11 3.29-36.75 0.000

ESR mm/1st hour > 80 28 9 32.67 6.37-167.27 0.000

Serum Glucose mg/dl > 60 26 20 3.25 0.88-11.89 0.060

Cholesterol mg/dL > 90 28 24 3.5 0.645 – 18.98 0.129

Alkaline Phosphatase u/l > 100 20 20 1 0.342-2.92 1

Bilirubin (unconj)mg/dl > 0.2 30 18 40.88 2.28- 732.44 0.001

Bilirubin (Total) mg/dl > 0.30 30 18 40.88 2.28- 732.44 0.001

Calcium mmole/L ≥ 2 30 30 1 1.35 – 2.80 0.000

Phosphorous mg/dl < 2.5 18 4 9.75 2.70 - 35.11 0.000

Copper µg/dl < 95 12 14 0.762 0.274 – 2.12 0.602

Molybdenum µg/dl > 70 24 10 8 2.47 – 25.86 0.000

Selenium µg/dl < 15 20 8 5.5 1.81 - 16.68 0.002

RESULTS

106

4.4.2: CASES VERSUS CONTROL GROUP II

The odds ratios of 26 (for red blood cells ˂ 5 × 106 /µl), 20 (for haemoglobin ˂ 8g /dl),

32.5 (for haematocrit ˂ 25%), 56 (for mean corpuscular volume ˃ 50fL), 13.75 (for mean

corpuscular haemoglobin ˃ 20pg) and 21 (for erythrocyte sedimentation rate ˃ 80mm /1st

hour) were significant (P ˂ 0.05).

Odds ratios of serum biochemical parameters including 0.098 (for glucose ˃ 60mg / dl), 3

(for alkaline phosphatase ˃ 100 U /L), 16.18 (for billirubin unconjugated ˃ 0.2mg / dl),

16.18 (for billirubin total ˃ 0.3mg /dl), 1 (for calcium ≥ 2mmoles /L), 6 (for phosphorous

˂ 2.5mg /dl), 16 (for molybdenum ˃ 70µg / dl) and 8 (for selenium ˂ 15 µg /dl) were

significant whereas odds ratios of 0.186 (for cholesterol ˃ 90mg /dl) and 1 (for copper ˂

95µg /dl) were insignificant (P ˃ 0.05). These results are summarized in table 14.

RESULTS

107



control group II

Parameters

Cut off

points

Study Groups

OR 95 % C.I P-value


Red Blood Cells (106/µl) < 5 26 6 26 6.53-103.49 0.000

Haemoglobin g/dl < 8 24 5 20 5.38-74.29 0.000

Haematrocrit (%) < 25 26 5 32.5 7.81-135.10 0.000

MCV fL > 50 28 6 56 10.32-303.68 0.000

MCH pg > 20 22 5 13.75 3.91 – 48.26 0.000

ESR mm/1st hour > 80 28 12 21 4.19-105.03 0.000

Serum Glucose mg/dl > 60 26 30 0.098 0.005 - 0.90 0.038

Cholesterol mg/dL > 90 28 30 0.186 0.009 - 4.06 0.129

Alkaline Phosphatase u/l > 100 20 12 3 1.04-8.60 0.038

Bilirubin (unconj)mg/dl > 0.2 30 24 16.18 0.083 – 0.30 0.010

Bilirubin (Total) mg/dl > 0.30 30 24 16.18 0.083 – 0.30 0.010

Calcium mmole/L > 2 30 30 1 1.35 - 2.80 0.000

Phosphorous mg/dl < 2.5 18 6 6 1.89-19.04 0.002

Copper µg/dl < 95 12 12 1 0.356-2.80 1

Molybdenum µg/dl > 70 24 6 16 4.5-56 0.000

Selenium µg/dl < 15 20 6 8 2.47-25.86 0.000

RESULTS

108

4.4.3: CASES VERSUS CONTROL GROUP I AND II (COMBINED)

The odds ratios of 26, 17.81, 28.95, 21, 12.25 and 26 recorded for red blood cells ˂ 5 ×

106 /µl, haemoglobin ˂ 8g / dl, haematocrit ˂ 25%, mean corpuscular volume ˃ 50fL,

mean corpuscular haemoglobin ˃ 20pg and erythrocyte sedimentation rate ˃ 80mm / 1st

hour respectively were significant (P ˂ 0.05) whereas odds ratio of 0.50 recorded for

mean corpuscular haemoglobin concentration ≥ 30g/dl was insignificant.

The significant odds ratios for serum biochemical parameters were 26.55, 26.55, 0.50,

7.50, 11 and 6.57 recorded for billirubin unconjugated ˃ 0.2mg /dl, billirubin total ˃

0.3mg /dl, calcium ≥ 2mmoles /L, phosphorous ˂ 2.5mg /dl, molybdenum ˃ 70µg /dl

and selenium ˂ 15 µg /dl respectively whereas odds ratios of 1.30, 1.55, 1.75 and 0.872

recorded for glucose ˃ 60mg /dl, cholesterol ˃ 90mg /dl, alkaline phosphatase ˃ 100 U

/L and copper ˂ 95µg /dl respectively were insignificant (P ˃ 0.05). The results

including odds ratios, their 95% confidence intervals, P-values and number of exposed

and unexposed animals among cases and controls are summarized in table 15 and figures

17 – 32.

RESULTS

109



control group I and II combined

Parameters

Cut off

points

Study Groups

OR 95 % C.I P-value


Red Blood Cells (106/µl) < 5 26 12 26 7.61 – 88.78 0.000

Haemoglobin g/dl < 8 24 11 17.81 5.88 – 53.96 0.000

Haematrocrit (%) < 25 26 11 28.95 8.38 – 99.98 0.000

MCV Fl > 50 28 24 21 4.55 – 96.46 0.000

MCH pg > 20 22 11 12.25 2.32 – 34.67 0.000

MCHC g/dl > 30 30 60 0.50 0.009 - 26.42 0.511

ESR mm/1st hour > 80 28 21 26 5.63 – 120.01 0.000

Serum Glucose mg/dl > 60 26 50 1.30 0.371 – 4.54 0.681

Cholesterol mg/dl > 90 28 54 1.55 0.29 – 8.21 0.600

Alkaline Phosphatase u/l > 100 20 32 1.75 0.72 – 4.36 0.227

Bilirubin (unconj)mg/dl > 0.2 30 42 26.55 1.54 - 457.80 0.001

Bilirubin (total) mg/dl > 0.30 30 42 26.55 1.54 - 457.80 0.001

Calcium mmole/L > 2 30 60 0.50 0.009 - 0.26 0.000

Phosphorous mg/dl < 2.5 18 10 7.50 2.76 – 20.33 0.000

Copper µg/dl < 95 12 26 0.872 0.358 – 2.12 0.763

Molybdenum µg/dl > 70 24 16 11 3.80-31.81 0.000

Selenium µg/dl < 15 20 14 6.57 2.5-17.27 0.000

RESULTS

110

Study groups

ControlsCases

Fre

qu

en

cy

60

50

40

30

20

10

0

Red blood cells

<5

5 or more

48

4

12

26


respect to RBCs. Red blood cells ˂ 5 × 106 /µl is taken as risk factor in this study.

RESULTS

111

Study groups

ControlsCases

Fre

qu

en

cy

60

50

40

30

20

10

0

Haemoglobin g/dl

<8

8 or more

49

6

11

24


respect to haemoglobin concentration. Haemoglobin ˂ 8g /dl is taken as risk factor in this

study.

RESULTS

112


respect to haematocrit. Haematocrit ˂ 25% is taken as risk factor in this study.

RESULTS

113

Study groups

ControlsCases

Fre

qu

en

cy

40

30

20

10

0

MCV(fL)

>50

50 or less

36

2

24

28


respect to mean corpuscular volume (MCV). Mean corpuscular volume of ˃ 50fL is taken

as risk factor in this study.

RESULTS

114

Study groups

ControlsCases

Fre

qu

en

cy

60

50

40

30

20

10

0

MCH (pg)

> 20

20 or less

49

8

11

22


respect to mean corpuscular haemoglobin (MCH). Mean corpuscular haemoglobin ˃

20pg is taken as risk factor in this study.

RESULTS

115

Study groups

ControlsCases

Fre

qu

en

cy

50

40

30

20

10

0

ESR (mm/1st hour)

> 80

80 or less

39

21

28


respect to erythrocyte sedimentation rate (ESR). Erythrocyte sedimentation rate of ˃

80mm /1st hour is taken as risk factor in this study.

RESULTS

116


respect to serum glucose. Serum glucose level of ˃ 60mg /dl is taken as risk factor in this

study.

RESULTS

117


respect to serum cholesterol. Serum cholesterol level of ˃ 90mg /dl is taken as risk factor

in this study.

RESULTS

118


respect to alkaline phosphatase. Serum alkaline phosphatase level of ˃ 100 U /L is taken


RESULTS

119

Study groups

ControlsCases

Fre

qu

en

cy

70

60

50

40

30

20

10

0

Billirubin (conj.)

> 0.2

0.2 or less

60

8

22


respect to billirubin conjugated. Serum billirubin conjugated level of ˃ 0.2mg /dl is taken


RESULTS

120

Study groups

ControlsCases

Fre

qu

en

cy

50

40

30

20

10

Billirubin (unconj.)

> 0.2

0.2 or less

18

42

30


respect to billirubin unconjugated. Serum billirubin unconjugated level of ˃ 0.2mg /dl is

taken as risk factor in this study.

RESULTS

121

Study groups

ControlsCases

Fre

qu

en

cy

50

40

30

20

10

Billirubin (total)

> 0.3

0.3 or less

18

42

30


respect to total billirubin. Serum total billirubin level of ˃ 0.3mg /dl is taken as risk factor

in this study.

RESULTS

122


respect to serum inorganic phosphorous. Serum inorganic phosphorous level of ˂ 2.5mg

/dl is taken as risk factor in this study.

RESULTS

123


respect to serum copper. Serum copper level of ˂ 95µg /dl is taken as risk factor in this

study.

RESULTS

124


respect to serum molybdenum. Serum molybdenum level of ˃ 70µg /dl is taken as risk

factor in this study.

RESULTS

125


respect to serum selenium. Serum selenium level of ˂ 15µg /dl is taken as risk factor in

this study.

RESULTS

126

4.4.4: HAEMATOLOGICAL AND SERUM BIOCHEMICAL PROFILES OF

HAEMOGLOBINURIC AND HEALTHY BUFFALOES

Haematological and serum biochemical profiles of haemoglobinuric and healthy

buffaloes were also compared by applying independent sample t-test at 5% significance

level. Results are described in detail as under.

Significant (P ˂ 0.05) decrease in mean erythrocyte count (3.58 ± 1.03 × 106 /µl),

haemoglobin concentration (6.90 ± 1.39g /dl), haematocrit (19.26 ± 4.28 %), mean

corpuscular haemoglobin (21.46 ± 2.52 g/dl), relative lymphocyte count (30.67 ± 8.57

%) and absolute lymphocyte count (3.63 ± 1.07 × 103 /µl) whereas significant (P ˂ 0.05)

increase in mean corpuscular volume (60.72 ± 8.49 fL), , red cell distribution width

(16.80 ± 1.81 %), reticulocytes (1.25 ± 0.66 % ), erythrocyte sedimentation rate (129.20

± 21.15 mm /1st hour), total leukocyte count (11.55 ± 5.31 × 10

3 /µl), monocytes (17.06

± 15.04 %), platelets (208.13 ± 85.26 × 103 /µl), plateletcrit (0.09 ± 0.04 %) and mean

platelet volume (4.99 ± 1.85 fL) was recorded in haemoglobinuric buffaloes as compared

to healthy controls. Insignificant (P ˃ 0.05) difference was recorded between

haemoglobinuric and healthy animals with respect to mean corpuscular haemoglobin

concentration, granulocytes % and platelet distribution width (table 16).

Blood smears of haemoglobinuric buffaloes exhibited macrocytosis and spherocytosis.

Heinz bodies were not observed on blood smears of haemoglobinuric as well as healthy

buffaloes.

Significantly (P ˂ 0.05) decreased levels of serum calcium (2.12 ± 0.11 mmoles /l),

phosphorous (2.67 ± 0.79 mg /dl) and selenium (15.77 ± 4.95 µg /dl) whereas

RESULTS

127

significantly (P ˂ 0.05) increased levels of serum alkaline phosphatase (164.20 ± 88.95 u

/l), billirubin conjugated (0.40 ± 0.17 mg /dl), billirubin unconjugated (0.67 ± 0.22 mg

/dl), billirubin total (1.07 ± 0.39 mg /dl), and molybdenum (115.33 ± 30.08 µg /dl) were

recorded in haemoglobinuric buffaloes as compared to healthy controls. There was

insignificant (P ˃ 0.05) difference between cases and controls with respect to serum

glucose, cholesterol and copper levels (table 17).

RESULTS

128

Table 16: Haematological values of haemoglobinuric and healthy buffaloes

Parameters Mean ± S.D.

P-value

Cases (n = 30) Controls (n = 60)

Red blood cells (106/µl) 3.58 ± 1.03 6.73 ± 1.28 0.000

Haemoglobin (g /dl) 6.90 ± 1.39 11.20 ± 2.06 0.000

Haematocrit % 19.26 ± 4.28 31.69 ± 5.39 0.000

Mean corpuscular volume (fL) 60.72 ± 8.49 47.97 ± 4.03 0.000

Mean corpuscular haemoglobin

(pg)

21.46 ± 2.52 17.39 ± 1.73 0.000

Mean corpuscular haemoglobin

concentration (g /dl)

35.82 ± 2.15 36.02 ± 0.97 0.545

Red cell distribution width % 16.80 ± 1.81 16.08 ± 0.70 0.000

Reticulocytes % 1.25 ± 0.66 0.21 ± 0.16 0.000

Erythrocyte sedimentation rate

( mm /1st hour)

129.20 ± 21.25 66.35 ± 37.88 0.000

White blood cells (103 /µl) 11.55 ± 5.31 9.20 ± 1.67 0.002

Lymphocytes % 30.67 ± 8.57 46.33 ± 11.32 0.000

Lymphocytes (103 /µl) 3.63 ± 1.07 4.31 ± 1.50 0.030

Monocytes % 17.06 ± 15.04 12.13 ± 2.81 0.016

Granulocytes % 42.89 ± 20.93 41.53 ± 10.55 0.684

Platelets (103 /µl) 208.13 ± 85.26 169.68 ± 70.01 0.001

Plateletcrit % 0.09 ± 0.04 0.07 ± 0.03 0.025

Mean platelet volume (fL) 4.99 ± 1.85 4.45 ± 0.65 0.049

Platelet distribution width % 15.94 ± 2.47 16.15 ± 0.81 0.560

RESULTS

129

Table 17: Serum biochemical values of haemoglobinuric and healthy buffaloes

Parameters Mean ± S.D.

P-value

Cases (n = 30) Controls (n = 60)

Serum Glucose mg/dl 80.20 ± 18.02 84.83 ± 18.39 0.260

Cholesterol mg/dl 123.60 ± 17.98 119.13 ± 19.74 0.300

Alkaline Phosphatase u/l 164.20 ± 88.95 120.20 ± 76.99 0.017

Billirubin (conj) mg/dl 0.40 ± 0.17 0.10 ± 0.02 0.000

Billirubin (unconj)mg/dl 0.67 ± 0.22 0.28 ± 0.06 0.000

Billirubin (total) mg/dl 1.07 ± 0.39 0.38 ± 0.08 0.000

Calcium mmole/L 2.12 ± 0.11 2.20 ± 0.08 0.000

Phosphorous mg/dl 2.67 ± 0.79 4.01 ± 1.12 0.000

Copper µg/dl 100.80 ± 13.27 97.50 ± 6.38 0.114

Molybdenum µg/dl 115.33 ± 30.08 68.43 ± 13.11 0.000

Selenium µg/dl 15.77 ± 4.95 24.37 ± 6.70 0.000

RESULTS

130

4.4.5: URINALYSIS

Urine colour of haemoglobinuric buffaloes ranged from red (n = 12 / 40%) to coffee

colour (n= 18 / 60%) with smokey turbid (n = 21 / 70%), smokey (n = 9 / 30%)

appearance and alkaline PH (n = 30 / 100%). The urine samples were positive for

haemoglobin (n = 30 / 100%), protein (n = 25 / 83.33%), epithelial cells (n = 25 /

83.33%), WBCs /pus cells (n = 30 / 100%) and amorphous phosphate (n = 21 / 70%)

whereas negative for ketones, bilirubin, glucose, nitrite, RBCs, casts, crystals and

bacteria. The average range of WBCs /pus cells and epithelial cells was 5 – 7/HPF and 1

– 2/HPF respectively. The average specific gravity of all urine samples was 1.006.

The urine colour of healthy buffaloes was light yellow with clear appearance and alkaline

PH. The average specific gravity of urine samples was 1.005. These samples were

positive for WBCs /pus cells and epithelial cells with an average range of 2 – 3 /HPF and

occasional /HPF respectively whereas negative for haemoglobin, protein, nitrite, ketones,

bilirubin, glucose, RBCs, casts, crystals and bacteria.

RESULTS

131


Significant decrease was recorded in total erythrocyte count (6.69 ± 0.48 × 106/µL to 5.37

± 0.11× 106/µL), haemoglobin concentration (11.7±1.29g/dL to 9.41±0.12g/dL),

haematocrit (36.28±4.14% to 28.42±0.95%), mean corpuscular volume (54±2.72 fL to

48.75±0.89 fL) and serum inorganic phosphorous (3.82±0.04 mg/dL to 3.7±0.07 mg/dL)

whereas significant increase was recorded in mean corpuscular haemoglobin

(17.45±0.89pg to 18.25±0.22pg), mean corpuscular haemoglobin concentration

(32.27±1.46 g/dl to 36.17±0.73 g/dl), red cell distribution width (17.32±0.78% to

20±0.77%), total leukocyte count (3.42±1.52 × 103/µL to 4.68±0.47 × 10

3/µL),

lymphocytes (2.69±1.05 × 103/µl to 4.44±0.43 × 10

3/µl) and monocytes (0.02±0.013 ×

109/L to 0.04±0.01 × 10

9/L) from 0 – 60

th day with the passage of time. Non significant

(P˃0.05) difference was recorded with respect to granulocytes and serum calcium

concentration. The results are presented in table 18.

Blood smear examination of rabbits revealed microcytosis and absence of Heinz bodies.

RESULTS

132

Table 18: Haematological and biochemical values of rabbits after gossypol

treatment

Parameters Mean ± S.D

Pvalue Day 0 Day 15 Day 30 Day 45 Day 60

Red blood cells

106/µL

6.69 ± 0.48 6.29 ± 0.296 5.81 + 0.18 5.77 +0.09 5.37 ± 0.11 0.0000

Haemoglobin g/dL 11.7±1.29 11.07±0.27 10.38±0.46 9.78±0.38 9.41±0.12 0.000

Haematocrit % 36.28±4.14 33.03±1.06 30.57±1.92 28.85±0.84 28.42±0.95 0.0000

Mean corpuscular

volume fL 54±2.72 52±1.07 50.5±1.19 49.5±1.19 48.75±0.89 0.000

Mean corpuscular

haemoglobin pg 17.45±0.89 17.65±0.63 17.9±0.4 18.1±0.39 18.25±0.22 0.033

Mean corpuscular

haemoglobin

concentration g/dl

32.27±1.46 33.55±0.82 34.65±0.89 35.28±0.95 36.17±0.73 0.000

Red cell

distribution width

%

17.32±0.78 18±1.15 18.7±1.087 19.57±0.87 20±0.77 0.000

White blood cells

103/µl

3.42±1.52 4.44±0.41 4.57±0.51 4.66±0.47 4.68±0.47 0.008

Lymphocytes

103/µl

2.69±1.05 3.70±0.85 3.9675±0.52 4.29±0.42 4.44±0.43 0.000

Monocytes 109/L 0.02±0.013 0.03±0.01 0.04±0.01 0.04±0.01 0.04±0.01 0.002

Granulocytes

103/µl

0.7±0.58 0.7025±0.96 0.705±0.95 0.70±0.96 0.70±0.97 0.967

Calcium mmole /L 3.445±0.06 3.4175±0.05 3.43±0.04 3.41±0.05 3.4175±0.07 0.866

Phosphorous

mg/dL 3.82±0.04 3.7125±0.058 3.715±0.07 3.705±0.07 3.7±0.07 0.012

CHAPTER - 5

DISCUSSION


characterized by intravascular haemolysis, haemoglobinaemia, haemoglobinuria and

anaemia. Most of the animals are affected during advanced pregnancy or early lactation

usually close to parturition. The exact pathogenesis is not known, however diversified

risk factors have been reported to be associated with this disease in different parts of the

continent. Present study was therefore conducted for assessment of disease (parturient

haemoglobinuria) burden and its distribution as well as quantification of associated

epidemiological, haematological and biochemical risk factors.


Active surveillance was conducted in district Chakwal for a period of one year from April

2010 to March 2011 to assess disease burden (parturient haemoglobinuria in buffaloes)

and its distribution in order to suggest future research priorities by dissemination of

findings to livestock farmers, field veterinarians, researchers and higher ups through

newspapers and journals. Out of total 461 villages of district Chakwal, eight villages

were randomly selected for collection of required information. All breeding age buffaloes

of eight selected villages (1938) being population at risk for parturient haemoglobinuria

were taken as denominator. Required data about diseases of each breeding age buffalo of

selected villages were collected and recorded in performa/questionnaire.

DISCUSSION

134

For the present study, desired sample size estimated according to recommendations of

Thrusfield (2005) was n = 59. Results will be hopefully more precise due to the bigger

sample (77 parturient haemoglobinuria cases) than estimated value i.e. n = 59.

Disease burden/magnitude of problem with respect to parturient haemoglobinuria was

assessed by calculating epidemiological rates (incidence rate, mortality rate, case fatality

rate and proportional mortality rate) and comparing them with other disease problems of

breeding age buffaloes whereas the distribution of disease (parturient haemoglobinuria)

was determined by analyzing the data of haemoglobinuric buffaloes with respect to

various descriptive characteristics including month of disease occurrence, season of

disease occurrence, lactation number, pregnancy status, postpartum period and previous

disease history of effected buffaloes.

A total of 77 parturient haemoglobinuria cases were recorded during the study period i.e.

from April 2010 to March 2011 out of which 20 effected animals died and remaining 57

recovered. The incidence, mortality and case fatality rates of parturient haemoglobinuria

were therefore 3.97%, 1.03% and 25.97% respectively whereas its proportional mortality

rate out of all buffalo diseases was 20%.

In the present study, parturient haemoglobinuria appeared as number one disease among

all disease problems of breeding age buffaloes with respect to mortality rate (1.03%) and

proportional mortality rate (20%) whereas it appeared at 8th

and 7th

position with respect

to incidence rate (3.97%) and case fatality rate (25.97%) respectively. The highest

mortality and proportional mortality rates of parturient haemoglobinuria indicate that it is

most important disease of buffaloes causing heavy losses every year. Although the

DISCUSSION

135

incidence rate is relatively low but the proportional mortality rate of 20% indicates that

5th

part of the total deaths of breeding age buffaloes is due to parturient haemoglobinuria.

Pirzada and Hussain (1998) stated that parturient haemoglobinuria mostly affects high

yielding buffaloes particularly in Rawalpindi, Jhelum, Attock and Faisalabad districts of

Punjab, Pakistan. Kahn and Line (2005) described that parturient haemoglobinuria is

distributed worldwide with low incidence but high case fatality rate (10 – 30%). Iqbal et

al (2005) reported that parturient haemoglobinuria is one of the most important diseases

of high yielding buffaloes in Pakistan. Rahman (2002) reported incidence, mortality and

case fatality rates of 3.09%, 0.48% and 16.54% respectively for parturient

haemoglobinuria in buffaloes in tehsil DG Khan. Khan (2001) reported 2.64%, 0.22%

and 8.3% incidence, mortality and case fatality rates for parturient haemoglobinuria in

buffaloes in district Malakand. Pervez (1992) reported incidence and case fatality rates of

3.12% and 48.25% respectively for parturient haemoglobinuria in buffaloes in district

Vehari. Shah et al (1988) reported 6.5% incidence rate for parturient haemoglobinuria in

buffaloes on the basis of retrospective survey in Lahore city.

Findings of the present study with respect to disease burden are nearly close to these

previous reports with some variation which is probably due to different sample sizes and

geo-climatic conditions.

The distribution of parturient haemoglobinuria was determined with respect to various

descriptive characteristics. For this purpose, data of 77 parturient haemoglobinuria cases

from eight selected villages and data of 103 parturient haemoglobinuria cases collected

from the same district i.e. district Chakwal during April 2009 to March 2010 were pooled

DISCUSSION

136

and analyzed with respect to month of disease occurrence, season of disease occurrence,

lactation number, pregnancy status, postpartum period and previous disease history of

affected animals. Relatively higher proportion of total (180) parturient haemoglobinuria

cases was recorded in month of December, during winter season i.e. from November to

March, during 10th

month of pregnancy and after 60 days of parturition. Out of total 180

cases, 60 (33.3%) were repeat effectees.

Durrani et al (2010) analyzed data of 1000 haemoglobinuric buffaloes with respect to

lactation number, stage of pregnancy and postpartum period. They reported highest

occurrence of disease in 5th

lactation, during 7th

to 10th

month of pregnancy and within 60

days postpartum. Gahlawat et al (2007) reported the occurrence of parturient

haemoglobinuria within 60 days postpartum and during 2nd

to 6th

lactations. Khan and

Akhtar (2007) recorded epidemiological data of 60 haemoglobinuric buffaloes from three

districts of Punjab, Pakistan and reported highest occurrence of parturient

haemoglobinuria in winter followed by summer spring and autumn. 45% buffaloes were

affected during post-calving period and majority of them were within 23 days of calving.

18.3% of the effected buffaloes were repeat effectees. Radwan and Rateb (2007) reported

highest incidence of parturient haemoglobinuria from May to January effecting majority

of buffaloes during advanced pregnancy or early postpartum phase. Bhikane et al (2004)

recorded 91 clinical cases of parturient haemoglobinuria in buffaloes and reported that

most of the cases occurred during January and majority of effected buffaloes were

yielding more than 4 liters milk. Muhammad et al (2000) analyzed epidemiological data

of 111 haemoglobinuric buffaloes from Punjab province of Pakistan and reported highest

occurrence of disease during winter (November to February) followed by spring (March

DISCUSSION

137

to April), summer (May to August) and fall (September to October). 18% of the affected

animals were in prepartum whereas 82% were in postpartum phase and most of the

postpartum cases occurred within first two months of parturition. 18.1% haemoglobinuric

animals were repeat effectees. Chugh et al (1996) analyzed data of 131 haemoglobinuric

buffaloes and reported highest occurrence of disease in September and lowest in February

to March. 88% of the haemoglobinuric animals were affected during 3rd

to 6th

lactations.

Majority (32 of 41) of the pregnant buffaloes were affected during or after 6th

month of

pregnancy. Majority of non pregnant buffaloes developed haemoglobinuria during first

four weeks postpartum. Out of total 51 buffaloes with full records, 14 (27.5%) were

repeat effectees. Pervez (1992) studied epidemiological aspects of parturient

haemoglobinuria in buffaloes in Vehari district of Punjab, Pakistan and reported higher

incidence of disease in winter as compared to summer and most of the animals are

affected within 2 – 4 weeks of parturition. Ali (1991) reported epidemiological features

of 75 haemoglobinuric buffaloes from eight district of Punjab, Pakistan and reported that

majority of animals were affected after parturition in their 3rd

to 6th

lactations during the

month of January. Akram et al (1990) analyzed data of 131 haemoglobinuric buffaloes

and reported that 111 animals were affected in postpartum phase and out of these 111

cases, 67 (51.14%) and 44(33.59%) animals developed disease during 0 – 3 months and

more than 3 months postpartum respectively. Mohamed et al (1988) studied parturient

haemoglobinuria in 103 affected buffaloes from Monofia province of Egypt and reported

that disease often appeared in subsequent lactations.

Findings of the present study are almost close to this previously reported distribution

pattern of parturient haemoglobinuria except some variations but the overall distribution

DISCUSSION

138

pattern is almost same (appendix 1). These few variations may be due to different sample

sizes and geo-climatic conditions. The findings of present study are contrary to the

previous report of Heuer and Bode (1998) with respect to seasonal pattern of disease.

They recorded no case of parturient haemoglobinuria between May and October.

Findings of the present study are also contrary to majority of previous reports with

respect to occurrence of disease within 60 days postpartum. In the present study, majority

(65.6%) of the animals were affected after 60 days of parturition.

5.2: CASE-CONTROL STUDY

The investigation was conducted on 180 case-control pairs for quantification of risk

factors associated with disease. The desired sample size estimated as per

recommendations of Schlesselman (1982) was n = 154 for the present study. It is hoped

that results are more precise and accurate due to the larger sample size (n = 180) than

desired (n = 154). Risk factors of parturient haemoglobinuria were quantified in terms of

odds ratios. Age, lactation number, stage of pregnancy, postpartum period, previous

history of haemoglobinuria and ingestion of cruciferous plants /toxic plants (berseem,

brassica and turnips) appeared as risk factors whereas use of mineral mixture and grazing

were recorded as protective factors. No association of parturient haemoglobinuria was

recorded with high milk yield, previous disease history of effected animals other than

parturient haemoglobinuria, cottonseed cake, use of drugs, use of oxytocine injection and

vaccination.

DISCUSSION

139

Significant (P ˂ 0.05) odds ratio of 5.56 was recorded for ≥ 7 years age which means that

buffaloes of ≥ 7 years age have 5.56 times more risk of parturient haemoglobinuria as

compared to the buffaloes of ˂ 7 years age.

Significant (P ˂ 0.05) odds ratio of 6.39 recorded for ≥ 3 lactation number indicates that

risk of parturient haemoglobinuria increases 6.39 times during and after 3rd

lactation as

compared to 1st and 2

nd lactations.

Highest and significant (P ˂ 0.05) odds ratio of 15.80 recorded for ≥ 7 months pregnancy

indicates 15.80 times increase in risk of parturient haemoglobinuria during and after 7th

month pregnancy whereas significant (P ˂ 0.05) odds ratio of 6.23 recorded for ≤ 60 days

postpartum period indicates 6.23 times increase in risk of parturient haemoglobinuria

during early lactation (1 – 60 days).

Significant odds ratios of 3.41 and 2.51 recorded for previous history of haemoglobinuria

and ingestion of cruciferous plants respectively indicate 3.41 and 2.51 times increased

risk for buffaloes with respective attributes.

Khan and Akhtar (2007) recorded epidemiological observations of 60 haemoglobinuric

buffaloes and reported that 11 (18.3%) were repeat effectees. Mahammad et al (2000)

recorded epidemiological features of 111 buffaloes and reported that 21 (18.1%) were

repeat effectees. Arif (1997) reported that out of 60 haemoglobinuric buffaloes, 11

(18.33%) were repeat effectees. Chugh et al (1996) reported that out of 51

haemoglobinuric buffaloes with full records, 14 (27.5%) were repeat effectees. In the

present study, 60 (33.33%) cases and 23 (12.77%) controls were repeat effectees.

DISCUSSION

140

Chugh et al (1996) recorded epidemiological features of 131 haemoglobinuric buffaloes

and hypothesized 3rd

to 6th

lactations, ˃ 6 months pregnancy, 1 – 28 days postpartum

period, ˃ 10 liters daily milk yield, previous history of haemoglobinuria, berseem and

cottonseed cake as risk factors of parturient haemoglobinuria. Results of the present study

are in agreement with this report except high milk yield which is not associated with

disease because of its significant odds ratio of 1.07.

Results of present study with respect to age, lactation number, stage of pregnancy,

postpartum period, previous history of haemoglobinuria and ingestion of cruciferous

/toxic plants being putative risk factors of parturient haemoglobinuria are in accord with

previous reports of Khan and Akhtar (2007), Radostits et al (2007), Akhtar et al (2006),

Muhammad et al (2000), Pirzada and Hussain (1998), Arif (1997), Samad (1997), Chugh

et al (1996), Pervez (1993), Ali (1991), Mohamed and El-Bagoury (1990), Akram et al

(1990), Mohamed et al (1988) and Mac Williams et al (1982). In these reports,

epidemiological risk factors of parturient haemoglobinuria are not quantified in terms of

odds ratios except Samad (1997) who analyzed six years data of haemoglobinuric

buffaloes and recorded odds ratio of 3.77 for pregnancy and late stage of gestation. He

described that parturition is sparing factor whereas pregnancy and late stage of gestation

are risk factors of parturient haemoglobinuria. Results of present study are contrary to the

previous reports with respect to high milk yield because significant (P˂ 0.05) odds ratio

of 1.07 recorded for ≥ 8 liters daily milk yield indicates that parturient haemoglobinuria

is not associated with high milk yield.

Results of the present study are also contrary to the previous report of Heuer and Bode

(1998). They described that ˃ 7 years age and early lactation (1 – 60 days) are not

DISCUSSION

141

associated with parturient haemoglobinuria. This contradiction is probably because of too

small sample size (39 cases and 24 controls) in said investigation.

Buffaloes are at the level of peak production during 3rd

to 6th

lactations resulting in stress

on mineral balance (Akhtar, 2006). This stress is further intensified by pregnancy and late

stage of gestation which coincides with ingestion of cruciferous plants in winter season

resulting in development of parturient haemoglobinuria (Heuer and Bode, 1998).

No quantitative analysis is available in previous reports with respect to previous disease

history of effected animals other than parturient haemoglobinuria, use of mineral mixture,

use of drugs, use of oxytocine injection, vaccination and grazing to compare the results of

present study.

5.3: CLINICAL TRIAL

Experiment was conducted on 30 haemoglobinuric buffaloes to compare and assess the

recovery rates of three treatment packages against parturient haemoglobinuria in

buffaloes. The effected buffaloes were therefore randomly divided into three groups with

10 animals in each group for allocation of respective treatment packages.

Highest recovery rate was recorded for package I (100%) followed by package II (70%)

and package III (50%). Findings of the present study are in close agreement with the

previous reports of Brechbuhl et al (2008), Gahlawat et al (2007), Radwan and Rateb

(2007), Bhikane et al (2004), Thompson and Badger (1999), Pirzada and Hussain (1998),

Pervez (1992), Shah et al (1988), Goel et al (1988) and Chugh et al (1988).

DISCUSSION

142

Durrani et al (2010) and Durrani et al (2009) reported 18% efficacy rate for sodium acid

phosphate which do not support the findings of present study. This difference is probably

due to the reason that in present study sodium acid phosphate therapy is supported by

blood transfusion and antifibrinolytic drug in group I and group II respectively.

Chugh et al (1988) estimated therapeutic efficacy of three antifibrinolytic drugs including

epsilon amino caproic acid (EACA), para amino methyl benzoic acid (PAMBA) and

botropase for parturient haemoglobinuria in buffaloes. They reported 90%, 92.30% and

94.40% efficacy for EACA, PAMBA and botropase after average number of 3.07, 3.25

and 3 injections respectively. In the present study, 50% recovery rate was recorded for

antifibrinolytic drug (tranexamic acid) after average number of 4.1 ± 0.74 injections. This

difference is probably due to different types of antifibrinolytic drugs in both experiments.

In conclusion, it is suggested that sodium acid phosphate along with whole blood

transfusion is best treatment for parturient haemoglobinuria in buffaloes followed by

combined therapy of sodium acid phosphate and antifibrinolytic drugs.

Hypophosphataemia decreases glucose utilization rate and ATP production in

erythrocytes of haemoglobinuric buffaloes leading to decreased synthesis as well as

reduction of glutathione. This process predisposes the erythrocytes to harmful effects of

oxidants because reduced glutathione is required to protect erythrocyte membrane against

lipid peroxidation and haemoglobin against oxidative denaturation. Its deficiency

therefore leads to lipid peroxidation of red cell membrane and ultimately intravascular

haemolysis. Sodium acid phosphate reduces lipid peroxidation by acting through

antioxidant mechanism. Hence, sodium acid phosphate restores the plasma inorganic

DISCUSSION

143

phosphorous level as well as the antioxidant potential of erythrocytes leading to increased

red cell vitality (Gahlawat et al, 2007). Transfusion of whole blood for treatment of

parturient haemoglobinuria is also recommended by various previous authors including

Radostits et al (2007), Kahn and Line (2005) and Dalir-Naghadeh et al (2005). Whole

blood transfusion helps in reducing anaemic hypoxia by improving haematological

indices and ultimately the chances of recovery.

5.4: CROSS-SECTIONAL STUDY

Cross-sectional epidemiological study was conducted for quantification of

haematological and biochemical risk factors associated with parturient haemoglobinuria.

Odds ratios for hypothesized risk factors were calculated by comparing cases (n = 30)

with control group I (n = 30), cases (n =30) with control group II (n = 30) and cases (n =

30) with control group I and II combined (n = 60). Low red cell count (≤ 5× 106/ µL),

haemoglobin concentration (˂ 8mg/ dL) and haematocrit (˂ 25%) whereas increased

mean corpuscular volume (˃50fL), mean corpuscular haemoglobin (˃20pg) and

erythrocyte sedimentation rate (˃ 80mm/ 1st hour) were recorded as significant (P˂0.05)

haematological risk factors associated with parturient haemoglobinuria in all above three

comparisons. Mean corpuscular haemoglobin concentration (≥30g/dL) appeared as

protective factor due to insignificant (P ˃ 0.05) odds ratio of 0.504.

Significant and higher odds ratios recorded for decreased red cell count, haemoglobin

concentration and haematocrit whereas increased mean corpuscular volume, mean

corpuscular haemoglobin and erythrocyte sedimentation rate indicate their strong

association with parturient haemoglobinuria. Low red cell count, haemoglobin

DISCUSSION

144

concentration and haematocrit indicate severe anaemia which is attributed to

intravascular haemolysis in this disease. Akhtar et al (2007) recorded significant (P˂0.05)

decrease in mean erythrocyte count, haemoglobin concentration and haematocrit whereas

significant (P˂0.05) increase in erythrocyte sedimentation rate of haemoglobinuric

buffaloes (n =60) as compared to healthy controls (n = 60). Results of the present study

with respect to red cell count, haemoglobin concentration, haematocrit and erythrocyte

sedimentation rate are in accord with the findings of Durrani et al (2010), Khan and

Akhtar (2007), Radwan and Rateeb (2007), Akhtar (2006), Dalir- Naghadeh et al

(2005), Shah et al (1988) and Goel et al (1988).

The absolute values or red cell indices including mean corpuscular volume, mean

corpuscular haemoglobin, mean corpuscular haemoglobin concentration and red cell

distribution width are used for morphological classification of anaemia. Significantly

(P˂0.05) increased values of mean corpuscular volume and red cell distribution width,

significantly decreased mean corpuscular haemoglobin whereas insignificantly changed

mean corpuscular haemoglobin concentration indicate macrocytic anaemia (Anwar et al,

2005, Dacie and Lewis, 1991). Increased mean corpuscular volume is also an indication

of erythriod regenerative response resulting in release of immature cells of increased size

and this erythriod regenerative response is associated with blood loss or haemolysis

(Feldman et al, 2000). These cytomorphological changes of erythrocytes are attributed to

decreased ATP production due to impaired glycolytic activity of erythrocytes in this

disease. ATP is required to maintain RBC shape and plasticity. Due to its decreased

production, RBCs loose their normal deformability and become spherocytic and rigid

(Radostits et al, 2007, Akhtar et al, 2007).

DISCUSSION

145

Khan and Akhtar (2007) reported that blood smears of haemoglobinuric buffaloes

exhibited macrocytes and sphereocytes. Dalir- Naghadeh et al (2005) recorded

anisocytosis and spherocytosis in haemoglobinuric buffaloes. Bhikane et al (2004)

reported Macrocytic hypochromic anaemia in 91 haemoglobinuric buffaloes during 1994

– 2000. Jain (1993) described regenerative macrocytic hypochromic anaemia in

haemoglobinuric animals. Rana and Bhardwaj (1988) reported spheroechinocytosis of red

blood cells in haemoglobinuric buffaloes as observed by scanning electron microscopy.

Elison et al (1986) reported spherocytosis in 16 haemoglobinuric animals. Mac Williams

et al (1982) also described polychromasia, anisocytosis and macrocytosis as characteristic

haematological features of parturient haemoglobinuria. In present study, significantly

(P˂0.05) increased mean corpuscular volume and red cell distribution width indicate

macrocytosis and anisocytosis. Findings of the present study are therefore in accord with

these previous reports with respect to absolute values of erythrocytes i.e. mean

corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean

corpuscular haemoglobin concentration (MCHC).

Increased bilirubin unconjugated (˃0.2mg/dL), bilirubin total (˃0.3mg/dL) and

molybdenum (˃70µg/dL) whereas decreased inorganic phosphorous (˂2.5mg/dL) and

selenium (˂15µg/dL) appeared as significant (P˂0.05) biochemical risk factors associated

with parturient haemoglobinuria in all three mentioned comparisons (cases versus control

group I, cases versus control group II& cases versus control group I & II combined).

Calcium (≥2mmoles/L) was not associated with disease when cases were compared

separately with both control groups (control group I & control group II) whereas it

appeared as significant (P˂0.05) protective factor when cases were compared with

DISCUSSION

146

control group I and II combined (n = 60). Copper (˂95µg/dL) appeared as insignificant

(P˃0.05) protective factor in first (cases versus control group I) and third comparison

(cases versus control group I & II combined) whereas it was not associated with

parturient haemoglobinuria in second comparison (cases versus control group II) due to

insignificant odds ratio of 1.

Serum glucose (˃60mg/dL) and cholesterol (˃90mg/dL) appeared as insignificant

(P˃0.05) risk factors when cases were compared with control group I whereas

insignificant (P˃0.05) protective factors when cases were compared with control group

II. No association of parturient haemoglobinuria was recorded with serum glucose and

cholesterol on comparing cases with control group I and II combined due to insignificant

(P˃0.05) odds ratios of 1.30 and 1.55 respectively. Alkaline phosphatase (˃100U/L)

appeared as significant (P˂0.05) risk factor in second comparison with odds ratio of 3

whereas it was not associated with disease in first and third comparisons due to

insignificant (P˃0.05) odds ratios of 1 and 1.75 respectively.

The adjusted odds ratios were calculated for mean corpuscular haemoglobin

concentration, bilirubin unconjugated, bilirubin total and calcium by adding 0.5 in each

cell.

Significantly (P˂0.05) increased level of serum alkaline phosphatase in haemoglobinuric

buffaloes may be attributed to generalized hypoxia as a result of decreased haemoglobin

level due to intravascular haemolysis. The consequently developed anoxic conditions

cause damage to liver, heart and kidney cells resulting in leakage of alkaline phosphatase

and thus increasing its concentration in general circulation (Cornelius, 1980).

DISCUSSION

147

Significantly (P˂0.05) increased concentration of unconjugated (indirect) bilirubin in

present study may be attributed to intravascular haemolysis whereas significantly

increased conjugated (direct) bilirubin may be attributed to hepato-cellular damage,

anorexia and dehydration. The hypoxic conditions developed as a result of severe

intravascular haemolysis effect the structure and function of liver and therefore decrease

its capacity for bilirubin conjugation and secretion (Anwar et al, 2005, Latimer et al,

2003, Banjamin, 1978). No previous report was available on serum bilirubin conjugated

and unconjugated levels of haemoglobinuric buffaloes for comparison with present study.

This is first report in this connection which will advance the prevailing knowledge with

reference to parturient haemoglobinuria in buffaloes.

Significantly (P˂0.05) decreased serum inorganic phosphorous concentration of

haemoglobinuric buffaloes in the present study may be due to phosphorous deficient diets

including cruciferous/toxic plants. Heavy drainage of phosphorous through milk also

leads to phosphorous deficiency in high producing animals maintained on low

phosphorous diets / rations (Radostits et al 2007, Mac Williams et al, 1982). Akhtar

(2006) described that low phosphorous rations with high calcium content cause

hypophosphataemia by decreasing phosphorous absorption from the gastro-intestinal tract

due to wider calcium phosphorous ratio. The soils of dry tropical countries like Pakistan

are deficient in phosphorous. This deficiency is therefore transferred from soil to plants

and ultimately to animals due to prolonged feeding on these phosphorous deficient plants

particularly berseem (Akhtar et al, 2006, Akram et al, 1990). Habib et al (2004) reported

low level of phosphorous in range grass hay of Buner district, KPK province of Pakistan.

Iqbal et al (2004) described that dietary phosphorous deficiency is major predisposing

DISCUSSION

148

factor of parturient haemoglobinuria. Dua (2009) and Dua (2003) also described that

parturient haemoglobinuria of buffaloes is associated with molybdenum induced

hypophosphataemia which results from excessive and prolonged feeding of berseem. The

excess of molybdenum decreases phosphorous content by interfering with its absorption

and increasing its elimination through urine leading to hypophosphataemia and

haemoglobinuria (Khan and Akhtar, 2007).

Serum molybdenum concentration was significantly (P˂0.05) increased (115.33 ±

5.49µg/dL) in haemoglobinuric buffaloes. Akhtar (2006) reported significantly (P˂0.05)

increased molybdenum (171.53 ± 56.69µg/dL) in haemoglobinuric buffaloes (n = 60).

Digraskar et al (1996) also reported significantly increased molybdenum 229.34 ± 4.18)

in haemoglobinuric buffaloes.

Significantly (P˂0.05) decreased serum selenium level (15.77 ± 0.90µg/dL) of

haemoglobinuric buffaloes in the present study is in agreement with the findings of

Akhtar (2006) who reported mean selenium level of 12.27 ± 3.16µg/dL in

haemoglobinuric buffaloes (n = 60). Elison et al (1986) also reported low selenium level

in six of eight herds affected with parturient haemoglobinuria. This decreased selenium

level of haemoglobinuric buffaloes may be attributed to decreased feed intake due to

progressive loss of appetite (Sarwar and Hassan, 2001).

Results of the present study with respect to increased bilirubin, alkaline phosphatase and

molybdenum whereas decreased phosphorous and selenium levels of haemoglobinuric

animals are in agreement with the previous reports of Durrani et al (2010), Akhtar et al

(2008), Akhtar et al (2007a), Khan and Akhtar (2007), Akhtar et al (2007b), Gahlawat et

DISCUSSION

149

al (2007), Akhtar et al (2006), Dalir-Naghadeh et al (2005), Bhikane et al (2004) and

Bhardwaj and Chugh (1988).

Non significant (P˂0.05) difference in serum copper concentration of haemoglobinuric

and healthy buffaloes in the present study is contrary to the previous reports of Durrani et

al (2010), Akhtar et al (2007), Khan and Akhtar (2007), Akhtar et al (2006), Akhtar

(2006), Ali (1991) and Bhardwaj and Chugh (1988). Majority of these previous

investigators attributed decreased serum copper level of haemoglobinuric animals to

increased molybdenum because of three way interaction between copper, molybdenum

and sulfur (Mc Donald et al, 2002) as a result of which thiomolybdates are produced in

the rumen (Mc Dowell, 1992, Spears, 2003). Sulfides produced by the rumen

microorganisms due to reduction of sulphate and degradation of sulfur amino acids react

with molybdate to form thiomolybdates. These thiomolybdates bind with copper forming

highly insoluble complex which do not release copper rendering it unavailable to the

animal for utilization leading to copper deficiency (Sarwar and Hassan, 2001). This

phenomenon is probably not involved in the present study because the serum

molybdenum level of haemoglobinuric animals (115.33 ± 5.49µg/dL) although

significantly higher than healthy animals but it is lower as compared to the previous

reports of Akhtar (2006) and Digraskar et al (1996) which were 171.53 ± 56.69µg/dL and

229.34 ± 4.18µg/dL respectively.

Mac Williams et al (1982) described two distinct entities of bovine post-parturient

haemoglobinuria (PPH) in North America and Newzealand. Low level of serum

inorganic phosphorous was characteristic biochemical finding in PPH affected cattle of

North America whereas in Newzealand, low levels of copper in serum and liver of PPH

DISCUSSION

150

affected animals was prominent biochemical alteration but hypophosphataemia was not a

consistent finding. This report supports the results of present study with respect to

occurrence of disease in North America where hypophosphataemia without hypocuprosis

is a prominent biochemical alteration associated with post-parturient haemoglobinuria.

Significantly (P˂0.05) decreased serum calcium concentration of haemoglobinuric

animals (n = 30) as compared to healthy controls (n = 60) in the present study is in

accordance with the findings of Radwan and Rateb (2007). They reported highly

significant (P˂0.01) drop in serum calcium level of haemoglobinuric buffaloes (n = 20)

as compared to healthy controls. The findings of present study are contrary to the

previous reports of Ghanam and El-Deeb (2010), Ali (1991), Akhtar et al (2007) and

Mohamed et al (1988). Ghanam and El-Deeb (2010) reported non significant difference

in blood calcium level of haemoglobinuric (n = 5) and healthy buffaloes (n = 5). Ali

(1991) investigated parturient haemoglobinuria in buffaloes from eight districts of

Punjab, Pakistan and reported 1.89% increase in clinically effected buffaloes. Akhtar et al

(2007) reported non significant difference in serum calcium concentration of

haemoglobinuric (n = 60) and healthy buffaloes (n = 60). Mohamed et al (1988) also

reported non significant variation in serum calcium concentration of haemoglobinuric (n

= 103) and apparently healthy buffaloes (n = 30).

Non significant (P˃0.05) difference in serum glucose level of haemoglobinuric (n = 30)

and healthy (n = 60) buffaloes in the present study is contrary to the previous reports of

Akhtar et al (2008) and Akhtar (2006). They reported significantly (P˂0.05) increased

serum glucose level in haemoglobinuric buffaloes (n = 60) as compared to healthy

controls (n = 60) and attributed it to phenomenon of glycogenolysis which occurs as a

DISCUSSION

151

result of anorexia. Ruminants depend on volatile fatty acids for main source of energy.

Progressive loss of appetite in parturient haemoglobinuria leads to non availability of

volatile fatty acids in sufficient quantity. Consequently the effected animals have to

depend on glucose oxidation metabolism for their energy requirements leading to

development of glycogenolysis and ultimately hyperglycaemia. Glucocorticoids released

as a result of stress of digestive disorders in parturient haemoglobinuria may also

stimulate phenomenon of glycogenolysis and gluconeogenesis resulting in development

of hyperglycaemia (Latimer et al, 2003, Singh et al, 1989, Banjamin, 1978).

The contradiction of these previous reports with the findings of present study with respect

to serum glucose level of haemoglobinuric animals may be due to difference in time at

which the effected animals were sampled. In parturient haemoglobinuria, anorexia

develops progressively and the previous investigators probably sampled the majority of

parturient haemoglobinuria affected animals few days after the onset of clinical signs

when anorexia and resultant phenomenon of glycogenolysis and gluconeogenesis had

been developed.

Insignificant (P˃0.05) difference in serum cholesterol level of haemoglobinuric (n = 30)

and healthy (n = 60) buffaloes in the present study is in agreement with the previous

reports of Akhtar et al (2008) and Akhtar (2006) whereas contrary to the previous reports

of Ghanem and El-Deeb (2010) and Mohamed et al (1988). Ghanem and El-Deeb (2010)

reported significant (P˂0.001) decrease in total cholesterol of haemoglobinuric buffaloes

(n = 5) as compared to healthy controls (n = 5). Mohamed et al (1988) analyzed the blood

samples of haemoglobinuric (n = 103) and healthy (n = 30) buffaloes and reported

DISCUSSION

152

significant (P˂0.001) decrease in serum cholesterol concentration of haemoglobinuric

buffaloes.

Significantly (P˂0.05) increased total leukocyte count in 30 haemoglobinuric buffaloes

(11.55 ± 0.97 × 103/µL) as compared to 60 controls (9.20 ± 0.22 × 10

3/µL) is close to the

values reported by Durrani et al (2010) and Durrani et al (2009). They reported total

leukocyte count of (11 ± 2.1 × 109/L) in haemoglobinuric buffaloes as compared to (9.5 ±

2.2 ×109/L) of healthy buffaloes. These findings are contrary to the previous reports of

Khan and Akhtar (2007), Akhtar et al (2007) and Akhtar (2006). They reported non

significant difference in total leukocyte count of haemoglobinuric and healthy buffaloes.

Significant (P˂0.05) decrease in lymphocytes of haemoglobinuric buffaloes (n = 30) as

compared to healthy controls (n = 60) is in agreement with the previous reports of Akhtar

et al (2007) and Akhtar (2006). This decrease in lymphocytes may be due to endogenous

release of corticosteroids as a result of oxidative stress on erythrocytes in this disease

(Singari et al, 1989, Bhardwaj et al, 1988). Corticosteroids cause lymphopenia by lysing

the lymphocytes in all tissues and declining lymphoid mitosis in lymph nodes (Latimer et

al, 2003).

In the present study, significantly (P˂0.05) increased monocytes were recorded in

haemoglobinuric buffaloes (n = 30) as compared to healthy controls (n = 60). These

findings are contrary to the previous reports of Khan and Akhtar (2007), Akhtar et al

(2007) and Akhtar (2006). They reported non significant difference in monocytes of

haemoglobinuric and healthy buffaloes.

DISCUSSION

153

Non significant (P˃0.05) difference was recorded between haemoglobinuric (n = 30) and

healthy (n = 60) buffaloes with respect to granulocytes. Khan and Akhtar (2007), Akhtar

et al (2007) and Akhtar (2006) reported significant (P˂0.05) increase in neutrophil

members of granulocytes whereas non significant difference in eosinophil members of

granulocytes between haemoglobinuric and healthy buffaloes. Bhikane et al (2004) also

reported neutrophilia in haemoglobinuric buffaloes.

Significantly (P˂0.05) increased values of platelets, plateletcrit and mean platelet volume

were recorded in haemoglobinuric buffaloes (n = 30) as compared to healthy controls (n

= 60) in the present study whereas non significant (P˃0.05) difference was recorded

between haemoglobinuric and healthy buffaloes with respect to platelet distribution

width.

No previous report is available on platelet parameters of haemoglobinuric buffaloes to

compare the results of present study. This is therefore first report with reference to

platelet parameters of haemoglobinuric buffaloes which will advance the prevailing

knowledge about parturient haemoglobinuria in buffaloes.

Significantly (P˂0.05) increased number of reticulocytes in haemoglobinuric animals (n

= 30) as compared to healthy controls (n= 60) indicate erythropoietic activity /

regenerative response (Anwar et al, 2005). These findings of present study are in

agreement with the previous reports of Thompson and Badger (1999), Jain (1993), Elison

et al (1986) and Mac Williams et al (1982). They reported the presence of regenerative

anaemia in haemoglobinuric animals.

DISCUSSION

154

In the present study, Heinz bodies were not observed on blood smears of

haemoglobinuric (n = 30) and healthy (n = 60) buffaloes. These findings are in agreement

with the previous report of Rana and Bhardwaj (1988). They investigated

cytomorphological changes in erythrocytes of haemoglobinuric buffaloes through

scanning electron microscopy and clearly ruled out the possibility of Heinz bodies and

involvement of compliment system activation in this disease.

The findings of present study with respect to Heinz body formation in erythrocytes of

parturient haemoglobinuria affected buffaloes are contrary to the previous reports of

Radostits et al (2007), Jain (1993), Elison et al (1986), Mac Williams et al (1982) and

Gardner et al (1976). These previous investigators reported the formation of Heinz bodies

as characteristic haematological alteration in erythrocytes of haemoglobinuric animals.

Mac Williams et al (1982) described two distinct entities of bovine post-parturient

haemoglobinuria in North America and Newzealand. Decreased serum inorganic

phosphorous level was characteristic finding in post-parturient haemoglobinuria affected

cattle of North America. In Newzealand, low levels of copper in serum and liver with

Heinz bodies in erythrocytes were characteristic findings in post-parturient

haemoglobinuria affected cattle whereas hypophosphataemia was not a consistent

finding.

Results of the present study are in close agreement with previously described North

American entity of post-parturient haemoglobinuria where disease developed with

hypophosphataemia but without hypocuprosis and Heinz bodies.

DISCUSSION

155

Hypocuprosis results in decreased activity of copper containing enzyme superoxide

dismutase which is part of erythrocyte protection mechanism against oxidative stress

/damage. This process helps in rendering the erythrocytes vulnerable to Heinz body

formation as a result of feeding on cruciferous /toxic plants (Jain, 1993, Smith et al,

1975).

These previous reports indicate that Heinz body formation in erythrocytes of

haemoglobinuric animals is associated with hypocoprosis. In the present study,

hypocuprosis is not recorded in haemoglobinuric buffaloes which might be the reason for

absence of Heinz bodies in erythrocytes of effected buffaloes.

Findings of the present study are therefore contrary to the previously described post-

parturient haemoglobinuria (PPH) entity of Newzealand.

Urine colour of haemoglobinuric buffaloes ranged from red to coffee colour with smokey

turbid and smokey appearance depending on severity and duration of disease. The urine

of affected buffaloes was positive for haemoglobin, protein, epithelial cells, WBCs/pus

cells and amorphous phosphate. These changes in urine may be associated with kidney

damage resulting from anaemic hypoxia due to excessive haemolysis (Latimer et al,

2003, Benjamin, 1978). These findings are in agreement with the previous reports of

Akhtar et al (2008), Radwan and Rateb (2007), Akhtar (2006), Dalir-Naghadeh et al

(2005), Arif (1997) and Ali (1991) whereas contrary to the previous report of Mac

Williams et al (1982) who reported the presence of bilirubin and ketones in urine of

haemoglobinuric animals.

DISCUSSION

156


Cotton is one of the major crops produced in Indo-Pakistan subcontinent. Its by-products

such as cotton seed cake are therefore widely used as protein source in livestock rations.

Instead of being a good protein source, its use is restricted due to the presence of

gossypol (C30H30O8), a polyphenolic compound naturally present in the cotton plant that

protects it from insect damage. Gossypol is toxic in free form whereas it is not toxic in

bound form. Ruminants can detoxify gossypol because it binds with microbial protein in

the rumen and is therefore not absorbed. Hence buffaloes are not affected at normal level

of feeding but this ability is reduced at higher levels of cottonseed cake feeding (Prasad et

al, 2009).

Glucose 6- phosphate dehydrogenase enzyme is required to maintain the integrity of

erythrocytes because it is part of intraerythrocytic metabolic chain which protects

haemoglobin from oxidative denaturation in mild oxidant attacks (Khan et al., 2007). As

gossypol permeates cells and also acts as an inhibitor for dehydrogenase enzymes

(Heinstein et al., 1970), it can increase erythrocyte fragility.

It is hypothesized that gossypol at higher levels of cotton seed cake feeding may

predispose the buffaloes to parturient haemoglobinuria by effecting their normal

haematological indices. Present study was therefore designed to investigate the effects of

gossypol on haematological parameters of experimental animals i.e. rabbits.

Eight adult female rabbits of six months age were maintained on cotton seed cake in

addition to their routine diet and their blood samples were collected from 0 – 60th

day

after every 15 days interval. Significantly (P˂0.05) decreased total erythrocyte count,

DISCUSSION

157

haemoglobin concentration, haematocrit and packed cell volume were recorded with the

passage of time. This decreasing trend in haemogram values of rabbits after gossypol

treatment indicate that gossypol can cause anaemia which is due to its iron binding

capacity. Gossypol binds with iron and thus reduces its availability for erythropoiesis

(Kahn and Line, 2005, Bressani, 1980). These findings are in agreement with the

previous reports of Rinchard et al (2003), Blom et al (2001), Dabrowski et al (2000),

Brocas et al (1997), Randel et al (1996), Lindsey (1980) and Herman (1970) whereas

contrary to the previous report of Prasad et al (2009). Prasad et al (2009) reported non

significant change in total erythrocyte count, total leukocyte count, mean corpuscular

volume and mean corpuscular haemoglobin concentration in gossypol treated buffalo

calves. They attributed this non significant difference of haemogram values to

neutralization of gossypol by ruminal microbial proteins.

Significantly (P˂0.05) decreased mean corpuscular volume whereas significantly

increased red cell distribution width of experimental animals with the passage of time

after gossypol treatment indicate the presence of microcytosis and anisocytosis which

may be associated with decreased iron availability for erythropoiesis due to iron binding

capacity of gossypol. Akingbemi and Aire (1994) reported anosocytosis and marked

poikilocytosis in gossypol treated rats.

Gossypol treatment caused leukocytosis in experimental animals by significantly

(P˂0.05) increasing the agranulocyte members of leukocytes i.e. lymphocytes and

monocytes. However, non significant (P˃0.05) change was recorded with respect to

granulocytes and the number of granulocytes remained almost same throughout the

experimental period. Significantly increased values of total leukocyte count, monocytes

DISCUSSION

158

and lymphocytes may be due to the reason that gossypol is a terpenoid aldehyde and it

probably acts like other lipid antigens. The number of naive lymphocytes specific for

any antigen is very small. On repeated stimulation activated CD4+ helper T lymphocytes

proliferate and differentiate into effector cells whose function is mediated by different

cytokines. In early response CD4+ helper T cells secrete interleukin-2 (IL-2) which

stimulates the clonal expansion of lymphocytes (Abbas, et al., 2007). These findings of

the present study are contrary to the previous report of Akingbemi and Aire (1994). They

reported leucopenia in gossypol treated rats but the rats were malnourished. They

described that lymphopenia in some gossypol treated rats indicate that gossypol may

affect immuno-competence in the presence of protein malnutrition. However, they also

suggested that further clarification is required for this speculation because the circulating

immunoglobulins of the gossypol treated rats were compared favourably with those of the

controls.

In the present study, significant (P˂0.05) decrease was recorded in serum inorganic

phosphorous whereas non significant (P˃0.05) change was recorded in serum calcium

level. Lee et al. (2006) reported significant (P˂0.01) decrease in coefficient of total tract

apparent digestibility of phosphorous in rainbow trout as a result of increasing dietary

cottonseed meal supplementation. Akingbemi and Aire (1994) investigated the effects of

protein malnutrition in gossypol treated rats and reported significantly (P˂0.01) increased

concentrations of calcium and magnesium whereas decreased potassium as a result of

gossypol treatment. They reported non significant difference with respect to phosphorous.

They attributed the increased calcium and magnesium concentrations to increased urinary

loss of these electrolytes.

DISCUSSION

159

From the present study, it is concluded that parturient haemoglobinuria is top most

disease among all problems of breeding age buffaloes with respect to mortality and it

affects majority of buffaloes during 4th

, 3rd

, 5th

, 6th and 2nd

lactations. The disease is

strongly associated with ≥7 years age, ≥3 lactation number, ≥7months pregnancy, ≤60

days postpartum period, previous disease history of effected animals, ingestion of

cruciferous/toxic plants, decreased red cell count, haemoglobin concentration,

haematocrit, serum inorganic phosphorous and selenium whereas increased mean

corpuscular volume, mean corpuscular haemoglobin and serum molybdenum. The

affected animals may be treated with combined therapy of sodium acid phosphate and

blood transfusion or sodium acid phosphate and antifibrinolytic drug tranexamic acid.

5.6: RECOMMENDATIONS

Keeping in view the findings of present study, it is suggested that investigation along

following lines would be helpful for clearer understanding of etiology, prevention and

control of parturient haemoglobinuria in buffaloes.

A cohort study with large sample size should be conducted to investigate the levels of

macro and micro minerals before the onset of clinical signs and their relationship with

host and environmental factors.

Investigation of seasonal changes in mineral status of soil, fodder and buffaloes in

different agro-ecological zones of Punjab thereby suggesting fertilizers, feed supplements

and mineral mixtures accordingly would help in prevention and control of parturient

haemoglobinuria.

DISCUSSION

160

Distribution of parturient haemoglobinuria should be determined in different buffalo

breeds i.e. Nili, Ravi, Kundi and other (non descript) and if association of disease is

proved with some specific breed, it might be excluded from further breeding programs to

prevent the disease.

Free gossypol is neutralized by microbial proteins in the rumen but this ability of

ruminants is reduced at higher levels of cottonseed cake feeding. Investigations to

determine the amount of free gossypol that can be safely neutralized by ruminal

microbial proteins in milking buffaloes thereby suggesting the optimum level of cotton

seed cake feeding would minimize the chances of gossypol toxicity effects on blood

components and would consequently help in prevention of parturient haemoglobinuria.

CHAPTER – 6

SUMMARY

Parturient haemoglobinuria is disease of economic importance which affects a

considerable number of buffaloes every year in India, Pakistan and Egypt. It is a non

infectious hemolytic syndrome characterized by intravascular haemolysis,

hypophosphataemia, haemoglobinaemia, haemoglobinuria and anaemia. The exact

pathogenesis is not known and diversified etiological factors have been associated with

this disease in different parts of the continent. Information on multidimensional

etiological aspects of this buffalo syndrome is quite scanty. The present study was

therefore carried out in district Chakwal for assessment of disease burden (parturient

haemoglobinuria), its distribution and quantification of associated epidemiological,

haematological and biochemical risk factors in order to suggest control measures and

future research priorities.

Active surveillance was conducted in eight randomly selected villages of district

Chakwal from April 2010 – March 2011. All breeding age buffaloes (1938) of these

selected villages were taken as sampling frame whereas one breeding age buffalo was

taken as sampling unit. Parturient haemoglobinuria appeared as number one disease

among all problems of breeding age buffaloes with respect to mortality rate (1.03%) and

proportional mortality rate (20%) whereas it appeared as 8th

and 7th

disease respectively

with respect to incidence (3.97%) and case fatality (25.97%) rates.

Case-Control study was conducted for quantification of epidemiological risk factors

associated with disease by analyzing the data of 180 case-control pairs for various

SUMMARY

162

hypothesized risk factors. ≥ 7 months pregnancy, ≥ 3 lactation number, ≤ 60 days

postpartum period, ≥ 7 years age, previous history of haemoglobinuria and ingestion of

cruciferous plants were recorded as significant (P ˂ 0.05) risk factors with odds ratios of

15.80, 6.39, 6.23, 5.56, 3.41 and 2.51 respectively.

Clinical trial was conducted on 30 haemoglobinuric buffaloes randomly divided into

three groups with 10 animals in each group to compare and assess the recovery rates of

three different treatment packages against parturient haemoglobinuria. The highest

recovery rate (100%) was recorded for combined therapy of sodium acid phosphate and

blood transfusion followed by sodium acid phosphate with antifibrinolytic drug

tranexamic acid (70%) and tranexamic acid with Novacoc injection (50%).

Cross-sectional epidemiological study was conducted on haemoglobinuric (n = 30) and

healthy (n = 60) buffaloes for quantification of haematological and biochemical risk

factors associated with parturient haemoglobinuria. Red cell count (˂ 5 × 106 /µl),

haemoglobin (˂ 8g / dl), haematocrit ˂ 25%, mean corpuscular volume (˃ 50fL), mean

corpuscular haemoglobin (˃ 20pg) and erythrocyte sedimentation rate( ˃ 80mm / 1st

hour) were recorded as significant (P ˂ 0.05) haematological risk factors with odds ratios

of 26, 17.81, 28.95, 21, 12.25 and 26 respectively whereas billirubin unconjugated (˃

0.2mg /dl), billirubin total ( ˃ 0.3mg /dl), phosphorous (˂ 2.5mg /dl), molybdenum (˃

70µg /dl) and selenium (˂ 15 µg /dl) were recorded as significant (P˂0.05) biochemical

risk factors with odds ratios of 26.55, 26.55, , 7.50, 11 respectively.

Experimental study was conducted to determine the effect of orally administered

gossypol on haematological and biochemical parameters of eight female rabbits of six

SUMMARY

163

months age purchased from local market and maintained at university of veterinary and

animal sciences from February 2011 – April 2011 under optimum conditions. The cotton

seed cake containing free gossypol contents of 0.25% was fed to rabbits @ 4 grams per

kg per day in addition to their routine diet including good quality fresh vegetables

(cucumbers, spinach, cabbage & carrots) and clean water ad-libitum. Blood samples of

each rabbit were collected after every 15 days interval and analyzed for haematological

and serum biochemical parameters. Significant (P˂0.05) decrease was recorded in total

erythrocyte count, haemoglobin concentration, haematocrit, mean corpuscular volume

and serum inorganic phosphorous whereas significant increase was recorded in mean

corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell

distribution width, total leukocyte count, lymphocytes and monocytes from 0 – 60th

day

with the passage of time whereas non significant (P˃0.05) difference was recorded with

respect to granulocytes and serum calcium concentration.

CHAPTER - 7

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APPENDICES

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Appendix 1: Meta-analysis of disease (parturient haemoglobinuria) distribution

with respect to various descriptive characteristics

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dis

ease

occ

urr

en

ce

Rep

eat

effe

ctee

s

Reference Samp

le

Size

≤ 2 ˃ 2 ≤ 6 ˃ 6 ≤ 60 ˃ 60 ≤ 9 ˃ 9 Nov. -

March

April

-

Octo

ber

Yes No

Khan and

Akhtar

(2007)

60 ---- ---- 15 18 16 11 ---- ---- 39 21 11 49

Akhtar

(2006)

60 13 47 15 18 19 8 23 17 39 21 ---- ----

Akhtar et al

(2006)

60 --- --- 15 18 16 11 35 25 ----- ----- 11 49

Dalir-

Naghadeh et

al (2005)

12 2 10 00 00 12 00 12 00 8 4 ---- ----

Muhammad

et al (2000)

116 --- ---- 15 6 95 00 ---- ----- 99 17 21 95

Chugh et al

(1996)

131 15 116 9 32 81 9 53 78 ------ ----- 14 37

Pervez

(1992)

156 4 152 00 38 118 00 71 85 130 26 ---- -----

Ali (1991) 75 29 46 00 9 66 00 ---- ---- 75 00 ---- -----

Akram et al

(1990)

131 --- ---- 00 00 67 64 ---- ---- 110 21 ---- ----

Total 801 63 371 69 139 490 103 194 205 500 110 57 230

Per

cen

t

63

/43

4=

14.5

1

37

1/4

34=

85.4

8

69

/80

1=

8.6

1

13

9/8

01=

17.3

5

49

0/8

01=

61.1

7

10

3/8

01=

12.8

5

19

4/3

99=

48.6

2

20

5/3

87=

52.9

7

50

0/6

10=

81.9

6

11

0/6

10=

18.0

3

57

/28

6=

19.9

3

23

0/2

86=

80.4

1

Akhtar (2006): Sample size is 60 but the data of 40 parturient haemoglobinuria cases is

given with respect to milk production.

Chugh et al (1996): Sample size is 131 but the data of 51 buffaloes is given with respect

to previous history of parturient haemoglobinuria.

APPENDICES

179

Appendix 2: Tests of between-subjects effects in cases and controls for different

variables in case control study

Source Dependent Variable

Type III

Sum of

Squares df Mean Square F Sig.

Corrected Model Age of animal

(years) 83.618(a) 1 83.618 20.432 .000

Lactation number 27.528(b) 1 27.528 13.111 .000

Stage of pregnancy 100.876(c) 1 100.876 9.430 .002

Postpartum period

(days) 696.090(d) 1 696.090 .042 .837

Milk yield (liters) 49.640(e) 1 49.640 3.896 .050

Intercept Age of animal

(years) 10792.112 1 10792.112 2637.084 .000

Lactation number 2476.944 1 2476.944 1179.727 .000

Stage of pregnancy 1438.404 1 1438.404 134.465 .000

Postpartum period

(days)

4989658.44

9 1 4989658.449 303.767 .000

Milk yield (liters) 8471.820 1 8471.820 664.889 .000

Cases vs. controls Age of animal

(years) 83.618 1 83.618 20.432 .000

Lactation number 27.528 1 27.528 13.111 .000

Stage of pregnancy 100.876 1 100.876 9.430 .002

Postpartum period

(days) 696.090 1 696.090 .042 .837

Milk yield (liters) 49.640 1 49.640 3.896 .050

APPENDICES

180

Appendix 3: Tests of between-subjects effects in cases and controls for

haematological variables

Dependent Variable

Type III

Sum of


Red blood cells

(RBC) 10/µl 206.146 2 103.073 74.005 .000

Haemoglobin

(HGB) g/Dl 369.510 2 184.755 52.546 .000

Haematocrit (HCT)

% 3091.793 2 1545.897 59.938 .000

Mean corpuscular

volume (MCV) fL 3307.704 2 1653.852 48.078 .000

Mean corpuscular

haemoglobin (MCH)

pg

367.732 2 183.866 49.314 .000

Mean corpuscular

haemoglobin

concentration

(MCHC) g/Dl

7.736 2 3.868 1.834 .166

Red cell distribution

width (RDW) % 16.958 2 8.479 6.253 .003

White blood cells

(WBC) 10/µL 118.832 2 59.416 5.302 .007

Lymphocytes (LY)

% 8830.451 2 4415.225 66.632 .000

Lymphocytes (LY)

10/µl 61.052 2 30.526 23.165 .000

Monocytes (MO) % 485.484 2 242.742 3.006 .055

MOnocytes (MO)

10/µl 19.219 2 9.609 7.243 .001

Granulocytes (GR)

% 3986.201 2 1993.100 11.311 .000

Granulocytes (GR)

10/µl 103.590 2 51.795 7.353 .001

Platelets (PLT)

10/µl 33718.067 2 16859.033 2.958 .057

Plateletcrit (PCT) % .006 2 .003 2.391 .098

Mean platelet

volume (MPV) fL 6.195 2 3.097 2.170 .120

Platelet distribution

width (PDW) % 7.574 2 3.787 1.573 .213

Erythrocyte

sedimentation rate

(ESR) mm/Ist hour

79279.800 2 39639.900 35.380 .000

Reticulocytes % 21.647 2 10.823 67.550 .000

APPENDICES

181

Appendix 4: Tests of between-subjects effects in cases and controls for biochemical

variables

Dependent Variable

Type III

Sum of


Serum Glucose

mg/dL 5844.356 2 2922.178 10.613 .000

Cholesterol mg/dL 4112.089 2 2056.044 6.245 .003

Alkaline

phosphatase u/L 39105.067 2 19552.533 2.939 .058

Billirubin

(conjugated) mg/dL 1.761 2 .880 85.236 .000

Billirubin

(unconjugated)

mg/dL

3.148 2 1.574 85.290 .000

Billirubin (total)

mg/dL 9.614 2 4.807 89.465 .000

Calcium mmole/L .128 2 .064 7.648 .001

Phosphorous mg/dL 36.401 2 18.200 17.285 .000

Copper µg/dL 592.800 2 296.400 3.615 .031

Molybdenum 44813.600 2 22406.800 54.817 .000

Selenium 1639.267 2 819.633 22.317 .000

APPENDICES

182

Appendix 5: Analysis of variance for haematological parameters of cases and


Sum of

Squares df

Mean

Square F Sig.

Red blood cells

(RBC) 10/µl

Between Groups 206.146 2 103.073 74.005 .000

Within Groups 121.172 87 1.393

Total 327.317 89

Haemoglobin

(HGB) g/Dl

Between Groups 369.510 2 184.755 52.546 .000


Total 675.406 89

Haematocrit

(HCT) %

Between Groups 3091.793 2 1545.897 59.938 .000

Within Groups 2243.874 87 25.792

Total 5335.667 89

Mean corpuscular

volume (MCV) fL

Between Groups 3307.704 2 1653.852 48.078 .000

Within Groups 2992.760 87 34.400

Total 6300.464 89

Mean corpuscular

haemoglobin

(MCH) pg

Between Groups 367.732 2 183.866 49.314 .000


Total 692.109 89

Mean corpuscular

haemoglobin

concentration

(MCHC) g/Dl

Between Groups 7.736 2 3.868 1.834 .166


Total 191.184 89

Red cell

distribution width

(RDW) %

Between Groups 16.958 2 8.479 6.253 .003


Total 134.921 89

White blood cells

(WBC) 10/µL

Between Groups 118.832 2 59.416 5.302 .007

Within Groups 975.016 87 11.207

Total 1093.847 89

Lymphocytes (LY)

%

Between Groups 8830.451 2 4415.225 66.632 .000

Within Groups 5764.826 87 66.262

Total 14595.27

7 89

Lymphocytes (LY)

10/µl

Between Groups 61.052 2 30.526 23.165 .000


Total 175.698 89

Monocytes (MO)

%

Between Groups 485.484 2 242.742 3.006 .055

Within Groups 7024.663 87 80.743

Total 7510.146 89

MOnocytes (MO)

10/µl

Between Groups 19.219 2 9.609 7.243 .001


Total 134.644 89

Granulocytes (GR)

%

Between Groups 3986.201 2 1993.100 11.311 .000

Within Groups 15330.11 87 176.208

APPENDICES

183

7

Total 19316.31

8 89

Granulocytes (GR)

10/µl

Between Groups 103.590 2 51.795 7.353 .001


Total 716.445 89

Platelets (PLT)

10/µl

Between Groups 33718.06

7 2 16859.033 2.958 .057

Within Groups 495846.4

33 87 5699.384

Total 529564.5

00 89

Plateletcrit (PCT)

%

Between Groups .006 2 .003 2.391 .098

Within Groups .106 87 .001

Total .112 89

Mean platelet

volume (MPV) fL

Between Groups 6.195 2 3.097 2.170 .120


Total 130.361 89

Platelet

distribution width

(PDW) %

Between Groups 7.574 2 3.787 1.573 .213


Total 217.041 89

Erythrocyte

sedimentation rate

(ESR) mm/Ist hour

Between Groups 79279.80

0 2 39639.900 35.380 .000


0 87 1120.403

Total 176754.9

00 89

Reticulocytes % Between Groups 21.647 2 10.823 67.550 .000

Within Groups 13.940 87 .160

Total 35.587 89

APPENDICES

184

Appendix 6: Analysis of variance for serum biochemical parameters of cases and


Serum Glucose mg/dL Between Groups 5844.356 2 2922.178 10.613 .000

Within Groups 23954.133 87 275.335

Total 29798.489 89

Cholesterol mg/dL Between Groups 4112.089 2 2056.044 6.245 .003

Within Groups 28645.067 87 329.254

Total 32757.156 89

Alkaline phosphatase

u/L

Between Groups 39105.067 2 19552.533 2.939 .058


3 87 6652.245

Total 617850.40

0 89

Billirubin (conjugated)

mg/dL

Between Groups 1.761 2 .880 85.236 .000


Total 2.660 89

Billirubin

(unconjugated) mg/dL

Between Groups 3.148 2 1.574 85.290 .000


Total 4.753 89

Billirubin (total)

mg/dL

Between Groups 9.614 2 4.807 89.465 .000


Total 14.289 89

Calcium mmole/L Between Groups .128 2 .064 7.648 .001


Total .856 89

Phosphorous mg/dL Between Groups 36.401 2 18.200 17.285 .000


Total 128.006 89

Copper µg/dL Between Groups 592.800 2 296.400 3.615 .031

Within Groups 7132.800 87 81.986

Total 7725.600 89

Molybdenum Between Groups 44813.600 2 22406.800 54.817 .000

Within Groups 35562.000 87 408.759

Total 80375.600 89

Selenium Between Groups 1639.267 2 819.633 22.317 .000

Within Groups 3195.233 87 36.727

Total 4834.500 89

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