Lesion scoring technique for assessing the virulence and ...

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259ISSN 0372-5480Printed in Croatia

VETERINARSKI ARHIV 81 (2), 259-271, 2011

Lesion scoring technique for assessing the virulence and Lesion scoring technique for assessing the virulence and pathogenicity of Indian fi eld isolates of avian pathogenicity of Indian fi eld isolates of avian EimeriaEimeria species species

Muthusamy RamanMuthusamy Raman11*, Samsudeen S. Banu*, Samsudeen S. Banu11,, Sankaralingam GomathinayagamSankaralingam Gomathinayagam11, , and Gopal D. Rajand Gopal D. Raj22

11Department of Veterinary Parasitology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Department of Veterinary Parasitology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Chennai, IndiaSciences University (TANUVAS), Chennai, India

22Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu, Veterinary and Animal Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu, Veterinary and Animal Sciences University (TANUVAS), Chennai, IndiaSciences University (TANUVAS), Chennai, India

RAMAN, M., S. S. BANU, S. GOMATHINAYAGAM, G. D. RAJ: Lesion scoring technique for assessing the virulence and pathogenicity of Indian fi eld isolates of avian Eimeria species.. Vet. arhiv 81, 259-271, 2011.Vet. arhiv 81, 259-271, 2011.

ABSTRACTABSTRACTChicken coccidiosis caused by the genus Chicken coccidiosis caused by the genus EimeriaEimeria is a major health impediment causing high morbidity and is a major health impediment causing high morbidity and

mortality in commercial poultry. Assessment of the virulence and pathogenicity of mortality in commercial poultry. Assessment of the virulence and pathogenicity of EimeriaEimeria species are the vital species are the vital factors for formulating effective control strategies. The gross lesion score was used in this study for assessing factors for formulating effective control strategies. The gross lesion score was used in this study for assessing the virulence and pathogenicity of indigenous six the virulence and pathogenicity of indigenous six Eimeria Eimeria sp. in a controlled experimental trial using six groups sp. in a controlled experimental trial using six groups of one hundred and twenty chicks of three weeks of age, inoculated with 2 × 10of one hundred and twenty chicks of three weeks of age, inoculated with 2 × 10 2 2 to 2 × 10 to 2 × 103 3 numbers of oocysts numbers of oocysts of six species of of six species of Eimeria.Eimeria. While an extended prepatent period (2 - 6 hrs) was confi rmed in the majority of the While an extended prepatent period (2 - 6 hrs) was confi rmed in the majority of the species, the gross lesion scores were mostly within the low score of 1-2 compared to the international reference species, the gross lesion scores were mostly within the low score of 1-2 compared to the international reference strains such as Houghton, so indicating possible natural attenuation of the fi eld isolates. The gross lesion scores strains such as Houghton, so indicating possible natural attenuation of the fi eld isolates. The gross lesion scores (GLS) were assessed ranging from a scale of 0 (no gross lesion) to 4 (most severe lesion). This study seems to (GLS) were assessed ranging from a scale of 0 (no gross lesion) to 4 (most severe lesion). This study seems to be the fi rst such attempt to standardize a lesion scoring technique for assessing the virulence and pathogenicity be the fi rst such attempt to standardize a lesion scoring technique for assessing the virulence and pathogenicity of indigenous isolates of of indigenous isolates of EimeriaEimeria in the Indian sub-continent. in the Indian sub-continent.

Key words: Key words: chickens,chickens, EimeriaEimeria, assessment, virulence, prepatent period, gross lesion score, assessment, virulence, prepatent period, gross lesion score

IntroductionIntroductionThe poultry industry in India has grown in leaps and bounds from a mere backyard The poultry industry in India has grown in leaps and bounds from a mere backyard

to a mammoth Industry with a staggering 220 million layers and 80 million broilers to a mammoth Industry with a staggering 220 million layers and 80 million broilers ((ANONYM., 2004ANONYM., 2004). Of the infectious diseases, chicken coccidiosis caused by obligatory ). Of the infectious diseases, chicken coccidiosis caused by obligatory intracellular apicomplexan protozoa viz. intracellular apicomplexan protozoa viz. EimeriaEimeria, is one of the economically important , is one of the economically important

*Corresponding author:Dr. Muthusamy Raman, M.V.Sc., Ph.D., Principal Investigator and Associate Professor, Department of Veterinary Parasitology, Dr. Muthusamy Raman, M.V.Sc., Ph.D., Principal Investigator and Associate Professor, Department of Veterinary Parasitology, Madras Veterinary College, TANUVAS, Chennai 600 007, India, Phone: +91 44 2538 1506; Fax : +91 44 2536 2787; Madras Veterinary College, TANUVAS, Chennai 600 007, India, Phone: +91 44 2538 1506; Fax : +91 44 2536 2787; E-mail: [email protected]: [email protected]

260 Vet. arhiv 81 (2), 259-271, 2011

M. Raman et al.: Lesion scoring technique for assessing the virulence and pathogenicity of avian M. Raman et al.: Lesion scoring technique for assessing the virulence and pathogenicity of avian Eimeria Eimeria speciesspecies

enteric-protozoan parasites affecting millions of commercial poultry. Assessment of the enteric-protozoan parasites affecting millions of commercial poultry. Assessment of the virulence and pathogenicity of various fi eld isolates of virulence and pathogenicity of various fi eld isolates of Eimeria Eimeria is an important component is an important component for control of chicken coccidiosis (for control of chicken coccidiosis (LONG et al., 1976LONG et al., 1976). As the authors as a team had ). As the authors as a team had been recently given the task of developing the fi rst indigenous vaccine against chicken been recently given the task of developing the fi rst indigenous vaccine against chicken coccidiosis, it was felt that assessing of the virulence and pathogenicity of fi eld isolates of coccidiosis, it was felt that assessing of the virulence and pathogenicity of fi eld isolates of EimeriaEimeria species collected from commercial broiler and layer birds would be a critical step species collected from commercial broiler and layer birds would be a critical step before inclusion of the above isolates in the vaccinal strain (before inclusion of the above isolates in the vaccinal strain (CHAPMAN, 2004CHAPMAN, 2004). ).

As the biological features of the Eimerial parasite are highly specifi c, such as the As the biological features of the Eimerial parasite are highly specifi c, such as the prepatent period, development within the intestine, type of lesion (prepatent period, development within the intestine, type of lesion (METTIELLO et al., METTIELLO et al., 20002000) and the morphological appearance of various endogenous stages in the mucosa of ) and the morphological appearance of various endogenous stages in the mucosa of the intestine, the lesion scoring technique (LST) gained more signifi cance in confi rmation the intestine, the lesion scoring technique (LST) gained more signifi cance in confi rmation ((SCHNITZLER et al., 1998SCHNITZLER et al., 1998) and accurate diagnosis of chicken coccidiosis () and accurate diagnosis of chicken coccidiosis (CONWAY and CONWAY and McKENZIE, 1991McKENZIE, 1991). ).

Based on the above, the LST is considered suitable for detecting the site specifi c Based on the above, the LST is considered suitable for detecting the site specifi c relative gross lesions in various parts of intestines affected with the relative gross lesions in various parts of intestines affected with the EimeriaEimeria species. species. The currently available lesion scoring technique, standardized for inbred or pure lines of The currently available lesion scoring technique, standardized for inbred or pure lines of chicken in European countries cannot be taken as a reference for Indian Eimerian isolates. chicken in European countries cannot be taken as a reference for Indian Eimerian isolates. This is due to the fact that chicken of different genetic makeup are reared under different This is due to the fact that chicken of different genetic makeup are reared under different selection pressures and management practices in India. Hence an attempt was made to selection pressures and management practices in India. Hence an attempt was made to standardize one such LST for Indian isolates of avian standardize one such LST for Indian isolates of avian Eimeria Eimeria to assess their virulence to assess their virulence and pathogenicity. This work was carried out as part of the Department of Biotechnology, and pathogenicity. This work was carried out as part of the Department of Biotechnology, Govt.of India funded project to develop a multivalent oral vaccine against chicken Govt.of India funded project to develop a multivalent oral vaccine against chicken coccidiosis, using local isolates. coccidiosis, using local isolates.

Materials and methodsMaterials and methodsMaintenance of chicks.Maintenance of chicks. One hundred and twenty day-old broiler chicks obtained from One hundred and twenty day-old broiler chicks obtained from

the Poultry Research Station, Tamil Nadu Veterinary and Animal Sciences University, the Poultry Research Station, Tamil Nadu Veterinary and Animal Sciences University, Chennai, were maintained in coccidia free elevated cages. They were separated into six Chennai, were maintained in coccidia free elevated cages. They were separated into six groups of 20 chicks in each group, representing the six species of groups of 20 chicks in each group, representing the six species of Eimeria Eimeria sp., and were sp., and were maintained until 4-6 weeks of age. The chicks were fed with coccidiostat- free- broiler maintained until 4-6 weeks of age. The chicks were fed with coccidiostat- free- broiler mash with all other ingredients and were given watermash with all other ingredients and were given water ad libitum ad libitum throughout the whole throughout the whole experiment. experiment.

Inoculation of oocysts.Inoculation of oocysts. EimeriaEimeria oocysts collected from different locations of Southern oocysts collected from different locations of Southern India were sporulated and enumerated (Table 1). In each group, fi ve chicks were inoculated India were sporulated and enumerated (Table 1). In each group, fi ve chicks were inoculated with 2 × 10with 2 × 10 2 2 to 2 × 10 to 2 × 103 3 oocysts, thus four different fi eld isolates of a single oocysts, thus four different fi eld isolates of a single Eimeria Eimeria species species were inoculated with six were inoculated with six EimeriaEimeria species on days 18 and 36 (Table 2) as per species on days 18 and 36 (Table 2) as per ECKERT et ECKERT et al. (1995)al. (1995), and were examined for the prepatent period and gross post mortem lesions at , and were examined for the prepatent period and gross post mortem lesions at regular intervals. Three chicks in each group were sacrifi ced randomly when the birds regular intervals. Three chicks in each group were sacrifi ced randomly when the birds

261Vet. arhiv 81 (2), 259-271, 2011


started showing clinical signs, from 72 hours up to 120 hours in some species of started showing clinical signs, from 72 hours up to 120 hours in some species of EimeriaEimeria, , to assess the status of infection and virulence by lesion scoring technique. to assess the status of infection and virulence by lesion scoring technique.

Table 1. Field isolates of avian Table 1. Field isolates of avian EimeriaEimeria from Southern India used for the study from Southern India used for the study

Sl. No. Name of the species Location State1 E. acervulina Namakkal Tamil Nadu

Udumalpet Tamil NaduV. Kota Andhra PradeshNellore Andhra Pradesh

2 E. brunetti Namakkal Tamil NaduTirupur Tamil NaduNellore Andhra Pradesh

Cuddalore Tamil Nadu3 E. maxima Chittoor Andhra Pradesh

Bangalore KarnatakaNamakkal Tamil Nadu

V. Kota Andhra Pradesh4 E. necatrix Madurai Tamil Nadu

Namakkal Tamil NaduNellore Andhra Pradesh

Udumalpet Tamil Nadu5 E. mitis Bangalore Karnataka

Namakkal Tamil NaduV. Kota Andhra PradeshNellore Andhra Pradesh

6 E. tenella Coimbatore Tamil Nadu Bangalore Karnataka

V. Kota Andhra PradeshNellore Andhra Pradesh

Assessment of gross lesion score. Assessment of gross lesion score. The prepatent period was assessed for various The prepatent period was assessed for various species of species of EimeriaEimeria sp. based on the day of excretion of the oocyst after inoculation. sp. based on the day of excretion of the oocyst after inoculation. The gross lesion scores (GLS) were assessed using the criteria of The gross lesion scores (GLS) were assessed using the criteria of JOHNSON and REID JOHNSON and REID (1970)(1970) with the scores ranging from a scale of 0 (no gross lesion) to 4 (most severe gross with the scores ranging from a scale of 0 (no gross lesion) to 4 (most severe gross lesion). Various portions of the intestines (anterior, middle, posterior, including caecal lesion). Various portions of the intestines (anterior, middle, posterior, including caecal loop) were collected, slit open and preserved in 10% buffered formalin. The organ loop) were collected, slit open and preserved in 10% buffered formalin. The organ segments were routinely processed in paraffi n, sectioned parallel to the cut edge and segments were routinely processed in paraffi n, sectioned parallel to the cut edge and stained with haematoxylin and eosin stain for the histological examination (stained with haematoxylin and eosin stain for the histological examination (CONWAY CONWAY and McKENZIE, 1991and McKENZIE, 1991). The deep scrapings of the affected intestinal serosa and mucosa, ). The deep scrapings of the affected intestinal serosa and mucosa,

262 Vet. arhiv 81 (2), 259-271, 2011


as well as the intestinal contents, were examined by light microscope (Leica research as well as the intestinal contents, were examined by light microscope (Leica research binocular microscope, Germany) at the sites of petechiae, plaques and some other lesions binocular microscope, Germany) at the sites of petechiae, plaques and some other lesions ((LONG et al., 1976LONG et al., 1976).).

Table 2. Inoculum dose and prepatent period of variousTable 2. Inoculum dose and prepatent period of various Eimeria species Eimeria species in experimental chickenin experimental chicken

S. No. Name of the species

Dose of oocysts per chick

Prepatent period (in hrs) (Present

study)

Prepatent period (in hrs) (Eckert

et al., 1995)Site of

development

1 Eimeria acervulina 2.0 × 103 96 89 Small intestine

(duodenum)

2 Eimeria brunetti 2.0 × 103 122 120 Small intestine, caeca and rectum

3 Eimeria maxima 2.5 × 102 120 120 Small intestine(middle part)

4 Eimeria mitis 2.0 × 103 96 91 Small intestine(middle part)

5 Eimeria necatrix 2.0 × 103 142 138 Small intestine6 Eimeria tenella 5.0 × 103 138 132 Caeca

In the current study, the lesion scoring of In the current study, the lesion scoring of JOHNSON and REID (1970)JOHNSON and REID (1970) was made as a was made as a baseline for assessing the gut lesions from the anterior, middle, posterior and caecal loops baseline for assessing the gut lesions from the anterior, middle, posterior and caecal loops of chickens infected with various species of of chickens infected with various species of EimeriaEimeria. Thus, the following scoring system . Thus, the following scoring system was standardized for a scale of 0 to 4 for chicks infected with was standardized for a scale of 0 to 4 for chicks infected with EE. . tenella tenella ((DUFFY et al., DUFFY et al., 20052005). ).

A score of “0” was given for intestines without any gross lesions;A score of “0” was given for intestines without any gross lesions;+1 Few petechiae in the cecal wall with the presence of normal contents+1 Few petechiae in the cecal wall with the presence of normal contents+2 Mild ballooning with thickened cecal wall and bloody contents at the proximal end +2 Mild ballooning with thickened cecal wall and bloody contents at the proximal end

(Fig.1.1.)(Fig.1.1.)+3 Moderate ballooning of caecal loops with caecal core (Fig.1.2.)+3 Moderate ballooning of caecal loops with caecal core (Fig.1.2.)+4 Complete ballooning and distension of caecum packed with caseous cores (Fig.1.3.)+4 Complete ballooning and distension of caecum packed with caseous cores (Fig.1.3.)

The following scoring system was standardized for chicks infected with The following scoring system was standardized for chicks infected with EE. . necatrixnecatrix..+1 Scattered petechiae and white spots observed on the serosa of intestine;+1 Scattered petechiae and white spots observed on the serosa of intestine;+2 Ballooning of mid gut portion (Fig. 2.1.); +2 Ballooning of mid gut portion (Fig. 2.1.); +3 Pin point haemorrhages in the serosa with ballooning up to the lower part of the small +3 Pin point haemorrhages in the serosa with ballooning up to the lower part of the small

intestine (Fig. 2.2.);intestine (Fig. 2.2.);+4 Massive haemorrhage with brownish mucus contents; complete ballooning of the mid +4 Massive haemorrhage with brownish mucus contents; complete ballooning of the mid

intestineintestine..

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The scoring system for chicks infected with The scoring system for chicks infected with EE. . acervulinaacervulina standardized for a scale of standardized for a scale of 0 to 4 was: 0 to 4 was:

+1 Scattered petechiae in the anterior intestine (duodenum) with lesions extending along +1 Scattered petechiae in the anterior intestine (duodenum) with lesions extending along the axis of the intestinal wall (Fig. 3.1.);the axis of the intestinal wall (Fig. 3.1.);

+2 Vast lesions extending up to end of the mid intestine beyond duodenum (Fig. 3.2.);+2 Vast lesions extending up to end of the mid intestine beyond duodenum (Fig. 3.2.);+3 Intestine has grayish coated appearance with thickened wall containing watery +3 Intestine has grayish coated appearance with thickened wall containing watery

contentscontents +4 the entire mucosa may be bright red with typical ladder like lesions in the mid intestine. +4 the entire mucosa may be bright red with typical ladder like lesions in the mid intestine.

The intestine is fi lled with creamy contents.The intestine is fi lled with creamy contents.

Similarly, the following scoring system for chicks infected with Similarly, the following scoring system for chicks infected with EE. . maximamaxima was was standardized as follows: standardized as follows:

+1 Small amount of orange slimy mucus present (Fig. 4.1.);+1 Small amount of orange slimy mucus present (Fig. 4.1.);+2 Intestine fi lled with orange mucus and the wall of the intestines thickened without +2 Intestine fi lled with orange mucus and the wall of the intestines thickened without

ballooning (Fig. 4.2.);ballooning (Fig. 4.2.);+3 Thickening of intestinal wall with mucus;+3 Thickening of intestinal wall with mucus;+4 Ballooning of whole intestine with putrid odour of contents. +4 Ballooning of whole intestine with putrid odour of contents.

Likewise, the following scoring system was standardized for a scale of 0 to 4 for Likewise, the following scoring system was standardized for a scale of 0 to 4 for chicks infected with chicks infected with EE. . brunettibrunetti::

+1 Stagnation of faecal contents with mild ballooning of intestine (Fig. 5.1.);+1 Stagnation of faecal contents with mild ballooning of intestine (Fig. 5.1.);+2 Slightly greyish intestinal wall, thickening of lower intestine with fl ecks of salmon +2 Slightly greyish intestinal wall, thickening of lower intestine with fl ecks of salmon

colour material sloughed off the intestine;colour material sloughed off the intestine;+3 Moderate thickening of intestinal wall with blood tinged catarrhal exudates; Transverse +3 Moderate thickening of intestinal wall with blood tinged catarrhal exudates; Transverse

red. Streaks markedly visible in lower rectum (Fig. 5.2.);red. Streaks markedly visible in lower rectum (Fig. 5.2.);+4 Severe lesions extending up to the middle intestine.+4 Severe lesions extending up to the middle intestine.

Results Results The evaluationThe evaluation of the severity of gross lesions is usually the easiest method of of the severity of gross lesions is usually the easiest method of

assessment of the status of the infection in experimental chickens, because of the known assessment of the status of the infection in experimental chickens, because of the known factors, such as the oocyst dose etc. In the present study, it was obvious that the most factors, such as the oocyst dose etc. In the present study, it was obvious that the most species of species of EimeriaEimeria produced observable gross changes in the intestine with the confi rmed produced observable gross changes in the intestine with the confi rmed presence of various developmental stages in the mucosa of the intestine. presence of various developmental stages in the mucosa of the intestine.

In the present study, the birds exposed to 2 × 1In the present study, the birds exposed to 2 × 102 E. tenellaE. tenella oocyst had gross lesions oocyst had gross lesions even when sacrifi ced on day 6, which provided a true representation of the severity of the even when sacrifi ced on day 6, which provided a true representation of the severity of the infection as normally found in broiler fl ocks. infection as normally found in broiler fl ocks.

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Fig. 1. Descriptive lesions in Fig. 1. Descriptive lesions in Eimeria tenellaEimeria tenella. Fig. 1.1. Mild ballooning with thickened cecal wall . Fig. 1.1. Mild ballooning with thickened cecal wall and bloody contents at the proximal end (+2); Fig.1.2. Moderate ballooning of caecal loops with and bloody contents at the proximal end (+2); Fig.1.2. Moderate ballooning of caecal loops with caecal core (+3); Fig. 1.3. Complete ballooning and distension of caecum packed with caseous caecal core (+3); Fig. 1.3. Complete ballooning and distension of caecum packed with caseous

cores ( +4).cores ( +4).

1.1. 1.1

1.3.

1.2.

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Fig. 2. Descriptive lesions in Fig. 2. Descriptive lesions in Eimeria necatrixEimeria necatrix. 2.1. Ballooning of mid intestine as seen through . 2.1. Ballooning of mid intestine as seen through serosa, 2.2. Pin point haemorrhages in the serosa. serosa, 2.2. Pin point haemorrhages in the serosa.

The lesions for The lesions for E. necatrixE. necatrix were invariably found to be mild to moderate in spite were invariably found to be mild to moderate in spite of the varying number of oocysts in most birds sacrifi ced during the different phases of of the varying number of oocysts in most birds sacrifi ced during the different phases of patency. This could be attributed to the possibility of the more natural attenuation of the patency. This could be attributed to the possibility of the more natural attenuation of the species as it occurs in egg laying layers and backyard poultry (species as it occurs in egg laying layers and backyard poultry (SHIRLEY and MILLARD, SHIRLEY and MILLARD, 19861986). ).

It was observed in the investigation thatIt was observed in the investigation that E. acervulina E. acervulina,, E. maxima E. maxima and and E. brunettiE. brunetti occupied a much greater area beyond the typical restricted sites of their development, occupied a much greater area beyond the typical restricted sites of their development, which could be explained by the passive migration of merozoites due to the peristaltic which could be explained by the passive migration of merozoites due to the peristaltic waves of the intestine beyond the infected areas in heavy infection, as reported by waves of the intestine beyond the infected areas in heavy infection, as reported by WILLIAMS (2001)WILLIAMS (2001). .

2.1.

2.2.

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Fig. 3. Descriptive lesions in Fig. 3. Descriptive lesions in Eimeria acervulinaEimeria acervulina. 3.1. Scattered petechiae and ballooning of the . 3.1. Scattered petechiae and ballooning of the anterior intestine as seen through serosa; Lesions extending up to end of mid intestine beyond anterior intestine as seen through serosa; Lesions extending up to end of mid intestine beyond

duodenum. duodenum.

3.1.

3.2.

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Fig. 4. Descriptive lesions in Fig. 4. Descriptive lesions in Eimeria maximaEimeria maxima. 4.1. Intestine fi lled with orange mucous and . 4.1. Intestine fi lled with orange mucous and thickening of wall; 4.2. Thickening of mid-intestine wall without ballooning. thickening of wall; 4.2. Thickening of mid-intestine wall without ballooning.

4.1.

4.2.

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Discussion Discussion CONWAY et al. (1990)CONWAY et al. (1990) demonstrated that lesion scores provided a measure of parasite demonstrated that lesion scores provided a measure of parasite

infection and associated gross lesions. They added that lesion scores did not measure infection and associated gross lesions. They added that lesion scores did not measure nor could they possibly show the pathophysiological changes related to weight gain. nor could they possibly show the pathophysiological changes related to weight gain. However, the histopathological evaluation of the caecal segments done at 60 hrs after However, the histopathological evaluation of the caecal segments done at 60 hrs after pi revealed 30% or more diffuse or moderate infections in spite of the lack of gross pi revealed 30% or more diffuse or moderate infections in spite of the lack of gross lesions. lesions. GOODWIN (1994)GOODWIN (1994) emphasized the need for the superior diagnostic testing in emphasized the need for the superior diagnostic testing in the absence of gross lesions in gastrointestinal tracts, outlining the different diagnostic the absence of gross lesions in gastrointestinal tracts, outlining the different diagnostic protocols in pallor chicks, such as histopathology apart from culling, cultures and other protocols in pallor chicks, such as histopathology apart from culling, cultures and other laboratory investigations. In the present study, lesion scores observed in most of the local laboratory investigations. In the present study, lesion scores observed in most of the local strains of chicken were usually low (+2) with overlapping lesions in the anterior, middle strains of chicken were usually low (+2) with overlapping lesions in the anterior, middle and posterior parts of the intestines. Hence, it was very diffi cult to assess virulence and and posterior parts of the intestines. Hence, it was very diffi cult to assess virulence and

Fig. 5. Descriptive lesions in Fig. 5. Descriptive lesions in Eimeria brunettiEimeria brunetti. 5.1. Stagnation of faecal contents with mild . 5.1. Stagnation of faecal contents with mild ballooning of intestine; 5.2. Transverse red streaks markedly visible in lower rectum.ballooning of intestine; 5.2. Transverse red streaks markedly visible in lower rectum.

5.1.

5.2.

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pathogenicity using standard lesion score parameters in the above groups of chicken pathogenicity using standard lesion score parameters in the above groups of chicken ((GARI et al., 2008GARI et al., 2008). ).

McDOUGALD et al. (1987)McDOUGALD et al. (1987), during a study of the sensitivity of drugs to coccidia in , during a study of the sensitivity of drugs to coccidia in Brazil, reported that even though most of the isolates of Brazil, reported that even though most of the isolates of E. acervulina, E. maxima, E. E. acervulina, E. maxima, E. tenella, E. brunetti tenella, E. brunetti and and E. mitis E. mitis produced gross lesions very close to the description by produced gross lesions very close to the description by JOHNSON and REID (1970)JOHNSON and REID (1970), some of the isolates of the different species caused indistinct , some of the isolates of the different species caused indistinct lesions, for which speciation was confi rmed by microscopic examination of the oocysts lesions, for which speciation was confi rmed by microscopic examination of the oocysts and other endogenous stages. The prepatent period of most and other endogenous stages. The prepatent period of most EimeriaEimeria species, as assessed species, as assessed in the present study, was very similar to in the present study, was very similar to ECKERT et al. (1995)ECKERT et al. (1995) with minor deviations, with minor deviations, which could be due to variations characteristic or typical to the local isolates (which could be due to variations characteristic or typical to the local isolates (RAMAN RAMAN et al., 2005et al., 2005). The extended prepatent period in most of the species could be attributed to ). The extended prepatent period in most of the species could be attributed to the wild nature of the fi eld isolates (the wild nature of the fi eld isolates (McDOUGALD et al., 1987McDOUGALD et al., 1987). This investigation further ). This investigation further confi rmed the signifi cance of LST as an assessment tool to evaluate the virulence and confi rmed the signifi cance of LST as an assessment tool to evaluate the virulence and pathogenicity of fi eld isolates of the pathogenicity of fi eld isolates of the EimeriaEimeria species ( species (CONWAY et al., 1990CONWAY et al., 1990). ).

Defi nitive identifi cation of the Defi nitive identifi cation of the Eimeria Eimeria species in chicken requires a combination of species in chicken requires a combination of different methods (different methods (SHIRLEY and MILLARD, 1986SHIRLEY and MILLARD, 1986). In this study, we used lesion scoring ). In this study, we used lesion scoring technique along with the prepatent period and other clinical signs. As the main scope of technique along with the prepatent period and other clinical signs. As the main scope of this study was to investigate the virulence and pathogenicity of the various fi eld isolates in this study was to investigate the virulence and pathogenicity of the various fi eld isolates in Tamil Nadu and the neighbouring south Indian states, to assess their potential as vaccinal Tamil Nadu and the neighbouring south Indian states, to assess their potential as vaccinal strains, the LST was used as a tool. The scoring system standardized in the present study strains, the LST was used as a tool. The scoring system standardized in the present study would be very useful for a comparative analysis of the local strains for their virulence and would be very useful for a comparative analysis of the local strains for their virulence and pathogenicity. pathogenicity.

_______AcknowledgementsThe senior author is grateful to Department of Biotechnology, Ministry of Science and Technology, Government The senior author is grateful to Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi (Scheme code No.BT/PR7275/AAQ/01/278/2006) for their fi nancial assistance and the of India, New Delhi (Scheme code No.BT/PR7275/AAQ/01/278/2006) for their fi nancial assistance and the Dean, Madras Veterinary College, Chennai for the facilities provided.Dean, Madras Veterinary College, Chennai for the facilities provided.

ReferencesReferencesANONYMOUS (2004): All India Poultry Business Directory (year book) 2003-04 special ANONYMOUS (2004): All India Poultry Business Directory (year book) 2003-04 special

millennium issue, Sadana Publishers, pp. 59-388. millennium issue, Sadana Publishers, pp. 59-388. CHAPMAN, H. D. (2004): Review article: Walter T. Johnson (1892 to 1937): pioneer of coccidiosis CHAPMAN, H. D. (2004): Review article: Walter T. Johnson (1892 to 1937): pioneer of coccidiosis

research in the fowl. Avian Pathol. 33, 107-116.research in the fowl. Avian Pathol. 33, 107-116.CONWAY, D. P., M. E. McKENZIE (1991): Examination of lesions and lesion scoring. In: Poultry CONWAY, D. P., M. E. McKENZIE (1991): Examination of lesions and lesion scoring. In: Poultry

Coccidiosis - Diagnostic and Testing Procedures, 2Coccidiosis - Diagnostic and Testing Procedures, 2ndnd ed. Pfi zer Inc., New York. pp.17-36. ed. Pfi zer Inc., New York. pp.17-36.CONWAY, D. P., E. M. McKENZIE, A. D. DAYTON (1990): Relationship of coccidial lesion CONWAY, D. P., E. M. McKENZIE, A. D. DAYTON (1990): Relationship of coccidial lesion

scores and weight gain in infections of scores and weight gain in infections of EimeriaEimeria. Avian Pathol. 19, 489-496.. Avian Pathol. 19, 489-496.

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DUFFY, C. F., G. F. MATHIS, R. F. POWER (2005): Effects of NatusatDUFFY, C. F., G. F. MATHIS, R. F. POWER (2005): Effects of NatusatTMTM supplementation on supplementation on performance, feed effi ciency and intestinal lesion scores in broiler chickens challenged with performance, feed effi ciency and intestinal lesion scores in broiler chickens challenged with Eimeria acervulinaEimeria acervulina, , E. maximaE. maxima and and E. tenellaE. tenella. Vet. Parasitol. 130, 185-190.. Vet. Parasitol. 130, 185-190.

ECKERT, J., R. BRAUN, M. W. SHIRLEY, P. COUDERTECKERT, J., R. BRAUN, M. W. SHIRLEY, P. COUDERT (1995): COST 89/820 Biotechnology: (1995): COST 89/820 Biotechnology: Guidelines on techniques in coccidiosis research, European commission, Agriculture Guidelines on techniques in coccidiosis research, European commission, Agriculture Biotechnology, Luxemburg L 2920, pp. 1-190. Biotechnology, Luxemburg L 2920, pp. 1-190.

GARI, G., G. TILAHUN, GARI, G., G. TILAHUN, Ph. Ph. DORCHIESDORCHIES (2008): Study on poultry coccidiosis in Tiyo district, (2008): Study on poultry coccidiosis in Tiyo district, Arsi Zone, Ethiopia. Int.Arsi Zone, Ethiopia. Int. J.J. PoultPoult Sci.7, 251-256.Sci.7, 251-256.

GOODWINGOODWIN, M. A. (1994): Differential diagnosis of gastrointestinal disease in broilers. Poult. , M. A. (1994): Differential diagnosis of gastrointestinal disease in broilers. Poult. Digest. 8-19.Digest. 8-19.

IDRIS, A. B., D. I. BOUNOUS, M. A. GOODWIN, J. BROWN, E. A. KRUSHINSKIE (1997): IDRIS, A. B., D. I. BOUNOUS, M. A. GOODWIN, J. BROWN, E. A. KRUSHINSKIE (1997): Lack of correlation between microscopic lesion scores and gross lesion scores in commercially Lack of correlation between microscopic lesion scores and gross lesion scores in commercially grown broilers examined for small intestinal grown broilers examined for small intestinal Eimeria Eimeria sp. Coccidiosis. Avian Dis. 41, 388-sp. Coccidiosis. Avian Dis. 41, 388-391.391.

JOHNSON, J., W. M. REIDJOHNSON, J., W. M. REID (1970): Anticoccidial Drugs: Lesion scoring techniques in battery and (1970): Anticoccidial Drugs: Lesion scoring techniques in battery and fl oor-pen experiments with chicken. Exp. Parasitol. 28, 30-36.fl oor-pen experiments with chicken. Exp. Parasitol. 28, 30-36.

LONG, P. L., B. J. MILLARD, L. P. JOYNER, C. C. NORTON (1976): A guide to the laboratory LONG, P. L., B. J. MILLARD, L. P. JOYNER, C. C. NORTON (1976): A guide to the laboratory techniques used in the study and diagnosis of avian coccidiosis. Folia Vet. Lat. 6, 201-217.techniques used in the study and diagnosis of avian coccidiosis. Folia Vet. Lat. 6, 201-217.

McDOUGALD, McDOUGALD, L. R.L. R., , J. M.J. M. DA SILVA, S. J. BRAGA (1987): A survey of sensitivity to anticoccidial DA SILVA, S. J. BRAGA (1987): A survey of sensitivity to anticoccidial drugs in 60 isolates of coccidia from broiler chickens in brazil and Argentina. Avian Dis. 31, drugs in 60 isolates of coccidia from broiler chickens in brazil and Argentina. Avian Dis. 31, 287-292.287-292.

METTIELLO, R., J. D. BOVIEZ, L. R. McDOUGALD (2000): METTIELLO, R., J. D. BOVIEZ, L. R. McDOUGALD (2000): EimeriaEimeria brunettibrunetti and and E. necatrixE. necatrix in in chickens of Argentina and confi rmation of seven species of chickens of Argentina and confi rmation of seven species of EimeriaEimeria. Avian Dis. 44, 711-714. . Avian Dis. 44, 711-714.

RAMAN, M., V. GOWTHAMAN, R. MAHAPRABHU, T. ANNA, T. J. HARIKRISHNAN (2005): RAMAN, M., V. GOWTHAMAN, R. MAHAPRABHU, T. ANNA, T. J. HARIKRISHNAN (2005): Avian chorio-allantoic membrane-an Avian chorio-allantoic membrane-an inin--ovoovo model for detection anticoccidial drug resistance, model for detection anticoccidial drug resistance, Indian. J. Anim. Sci. 75, 5-7. Indian. J. Anim. Sci. 75, 5-7.

SCHNITZLERSCHNITZLER, B. E., P. L.THEBO, J. G. MATTSSON, F. M. TOMLEY, M. W. SHIRLEY, A. , B. E., P. L.THEBO, J. G. MATTSSON, F. M. TOMLEY, M. W. SHIRLEY, A. UGGLA (1998): Diagnosis of poultry UGGLA (1998): Diagnosis of poultry Eimeria:Eimeria: from Lesion scoring to nucleotide sequences. from Lesion scoring to nucleotide sequences. Parasitol. Int. 47, 71-95. Parasitol. Int. 47, 71-95.

SHIRLEY, M. W., B. J. MILLARD SHIRLEY, M. W., B. J. MILLARD (1986): Studies on the immunogenicity of seven attenuated (1986): Studies on the immunogenicity of seven attenuated lines of lines of EimeriaEimeria given as a mixture to chickens. Avian Pathol. 15, 629-638. given as a mixture to chickens. Avian Pathol. 15, 629-638.

WILLIAMSWILLIAMS, R. B. (2001): Quantifi cation of the crowding effect during infections with the seven , R. B. (2001): Quantifi cation of the crowding effect during infections with the seven EimeriaEimeria species of the domesticated fowl: its importance for experimental designs and the species of the domesticated fowl: its importance for experimental designs and the production of oocyst stocks. Int. J. Parasitol. 31, 1056-1069.production of oocyst stocks. Int. J. Parasitol. 31, 1056-1069.

Received: 16 September 2009Accepted: 21 December 2010

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RAMAN, M., S. S. BANU, S. GOMATHINAYAGAM, G. D. RAJ: Bodovanje jačine RAMAN, M., S. S. BANU, S. GOMATHINAYAGAM, G. D. RAJ: Bodovanje jačine patoanatomskih promjena za procjenu virulencije i patogenosti indijskih terenskih patoanatomskih promjena za procjenu virulencije i patogenosti indijskih terenskih izolata ptičjih vrsta rodaizolata ptičjih vrsta roda Eimeria. Eimeria. Vet. arhiv 81, 259-271, 2011.Vet. arhiv 81, 259-271, 2011.

SAŽETAKSAŽETAKKokcidioza uzrokovana vrstama roda Kokcidioza uzrokovana vrstama roda EimeriaEimeria jedna je od glavnih prijetnji za zdravlje pilića, jer može jedna je od glavnih prijetnji za zdravlje pilića, jer može

uzrokovati njihov veliki pobol i pomor. Procjena virulencije i patogenosti vrsta roda uzrokovati njihov veliki pobol i pomor. Procjena virulencije i patogenosti vrsta roda EimeriaEimeria od velike je od velike je važnosti za donošenje učinkovitih kontrolnih mjera. Bodovanje jačine patoanatomskih promjena rabljeno je za važnosti za donošenje učinkovitih kontrolnih mjera. Bodovanje jačine patoanatomskih promjena rabljeno je za procjenu virulencije i patogenosti šest udomaćenih vrsta roda procjenu virulencije i patogenosti šest udomaćenih vrsta roda EimeriaEimeria u kontroliranim pokusima na šest skupina u kontroliranim pokusima na šest skupina pilića. U svakoj skupini bilo je 120 pokusnih pilića u dobi od tri tjedna. Pilići su bili zaraženi s 2 ×pilića. U svakoj skupini bilo je 120 pokusnih pilića u dobi od tri tjedna. Pilići su bili zaraženi s 2 ×102 do 2 ×o 2 ×103 oocista šest vrsta oocista šest vrsta EimeriaEimeria. Potvrđen je produženi prepatentni period (2 - 6 sati) za većinu vrsta, a patoanatomske . Potvrđen je produženi prepatentni period (2 - 6 sati) za većinu vrsta, a patoanatomske promjene procijenjene su većinom s malim brojem bodova i to 1 - 2 u usporedbi s međunarodnim referentnim promjene procijenjene su većinom s malim brojem bodova i to 1 - 2 u usporedbi s međunarodnim referentnim sojevima poput soja Houghton. To upozorava na moguću prirodnu oslabljelost terenskih izolata. Patoanatomske sojevima poput soja Houghton. To upozorava na moguću prirodnu oslabljelost terenskih izolata. Patoanatomske promjene bile su procijenjene na skali od 0 (bez promjena) do 4 (jako teške promjene). Ovo je istraživanje promjene bile su procijenjene na skali od 0 (bez promjena) do 4 (jako teške promjene). Ovo je istraživanje prvi pokušaj standardizacije bodovanja jačine patoanatomskih promjena za procjenu virulencije i patogenosti prvi pokušaj standardizacije bodovanja jačine patoanatomskih promjena za procjenu virulencije i patogenosti izvornih izolata vrsta roda izvornih izolata vrsta roda EimeriaEimeria na Indijskom potkontinentu. na Indijskom potkontinentu.

Ključne riječi:Ključne riječi: pilići, pilići, EimeriaEimeria spp., virulencija, prepatentni period, bodovanje, jačina patoanatomskih spp., virulencija, prepatentni period, bodovanje, jačina patoanatomskih promjena promjena

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