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Co-infection of methicillin-resistant Staphylococcusepidermidis, methicillin-resistant Staphylococcusaureus and extended spectrum β-lactamaseproducing Escherichia coli in bovine mastitis – threecases reported from IndiaSamiran Bandyopadhyaya, Indranil Samantab, Debaraj Bhattacharyyaa, Pramod KumarNandaa, Debasish Kara, Jayanta Chowdhuryc, Premanshu Dandapata, Arun Kumar Dasa,Nayan Batuld, Bimalendu Mondala, Tapan Kumar Duttae, Gunjan Dase, Bikash ChandraDasa, Syamal Naskara, Uttam Kumar Bandyopadhyaya, Suresh Chandra Dasa & SubhasishBandyopadhyaya
a Indian Veterinary Research Institute, Eastern Regional Station, 37 Belgachia Road,Kolkata - 700 037, Indiab Department of Veterinary Microbiology, West Bengal University of Animal and FisherySciences, Kolkata, Indiac State Livestock Farm, Kalyani, Indiad Department of Veterinary Medicine, West Bengal University of Animal and FisherySciences, Kolkata, Indiae College of Veterinary Sciences, Central Agricultural University, Aizwal, IndiaPublished online: 02 Dec 2014.
To cite this article: Samiran Bandyopadhyay, Indranil Samanta, Debaraj Bhattacharyya, Pramod Kumar Nanda,Debasish Kar, Jayanta Chowdhury, Premanshu Dandapat, Arun Kumar Das, Nayan Batul, Bimalendu Mondal, TapanKumar Dutta, Gunjan Das, Bikash Chandra Das, Syamal Naskar, Uttam Kumar Bandyopadhyay, Suresh Chandra Das &Subhasish Bandyopadhyay (2014): Co-infection of methicillin-resistant Staphylococcus epidermidis, methicillin-resistantStaphylococcus aureus and extended spectrum β-lactamase producing Escherichia coli in bovine mastitis – three casesreported from India, Veterinary Quarterly, DOI: 10.1080/01652176.2014.984365
To link to this article: http://dx.doi.org/10.1080/01652176.2014.984365
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CASE SERIES
Co-infection of methicillin-resistant Staphylococcus epidermidis, methicillin-resistant
Staphylococcus aureus and extended spectrum b-lactamase producing Escherichia coli
in bovine mastitis � three cases reported from India
Samiran Bandyopadhyaya*, Indranil Samantab, Debaraj Bhattacharyyaa, Pramod Kumar Nandaa, Debasish Kara, Jayanta
Chowdhuryc, Premanshu Dandapata, Arun Kumar Dasa, Nayan Batuld, Bimalendu Mondala, Tapan Kumar Duttae,
Gunjan Dase, Bikash Chandra Dasa, Syamal Naskara, Uttam Kumar Bandyopadhyaya, Suresh Chandra Dasa
and Subhasish Bandyopadhyaya
aIndian Veterinary Research Institute, Eastern Regional Station, 37 Belgachia Road, Kolkata - 700 037, India; bDepartment ofVeterinary Microbiology, West Bengal University of Animal and Fishery Sciences, Kolkata, India; cState Livestock Farm, Kalyani,India; dDepartment of Veterinary Medicine, West Bengal University of Animal and Fishery Sciences, Kolkata, India; eCollege of
Veterinary Sciences, Central Agricultural University, Aizwal, India
(Received 17 April 2014; accepted 2 November 2014)
Emergence of antimicrobial resistance among bovine mastitis pathogens is the major cause of frequent therapeutic failureand a cause of concern for veterinary practitioners. This study describes intra-mammary infection of methicillin-resistantStaphylococcus epidermidis (MRSE), methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrumb-lactamase (ESBL) producing Escherichia coli in two Holstein Friesian crossbred cows with subclinical mastitis and onenon-descript cow with clinical mastitis in two different districts of West Bengal, India. In total, three MRSE, one MRSAand three ESBL producing E. coli were isolated from these cases. Both the crossbreds were detected with MRSE (HFSE1and HFSE2) and ESBL producing E. coli (HFEC1 and HFEC2), whereas, simultaneous infection of three pathogens viz.MRSA (NDSA1), MRSE (NDSE1) and ESBL producing E. coli (NDEC1) was found in the non-descript cow. Themethicillin-resistant isolates possessed mecA gene and exhibited resistance to various antibiotics such as amikacin,tetracycline and glycopeptides. The ESBL producers were positive for blaCTX-M and blaTEM genes; in addition, HFEC1and HFEC2 were positive for blaSHV and possessed the genes for class I integron (int1), sulphonamide resistance (sul1),quinolone resistance (qnrS) and other virulence factors (papC, iucD and ESTA1). All the ESBL producers exhibitedresistance to a variety of antibiotics tested including third- and fourth-generation cephalosporins and were alsointermediately resistant to carbapenems. This is the first ever report on simultaneous occurrence of MRSE, MRSA andESBL producing E. coli in bovine mastitis indicating a major concern for dairy industry and public health as well.
Keywords: bovine; cow; India; ESBL; mastitis; MRSA; MRSE
Introduction
Two Holstein Friesian (HF) crossbred cows (HFC1 and
HFC2) and one non-descript cow (NDC, without any
progeny record) from Nadia and Kolkata districts of West
Bengal, India, respectively, were reported with abnormal
udder secretion and drop in milk yield (Table 1). In all
three cows (HFC1, HFC2 and NDC), appearance of flakes
was noticed in milk. Besides, NDC was detected off-fed
for three days with mild rise of temperature (39.4 �C), hotpainful mildly swollen udder besides appearance of blood
clots in milk (Figure 1).
Milk samples, collected from all the animals, were
subjected to White Slide Test (WST), California Mastitis
Test (CMT) and Somatic Cell Count (SCC) following
standard procedure (Radostits et al. 2000). Diluted milk
(10¡2 to 10¡3 dilution) was incubated at 37 �C for 24 h
for determination of total viable bacterial count (TVBC)
and expressed in cfu/ml. The blood collected via jugular
vein and the smear prepared from ear vein puncture were
checked for routine haematological examination and
blood protozoa, respectively.
For direct examination of etiological bacteria, the smear
prepared from the individual milk samples was examined
microscopically following Gram’s staining. In the stained
smear, both Gram-positive cocci in bunches and Gram-neg-
ative bacilli were detected in all samples. For isolation of
the bacteria, milk samples were inoculated separately into
the nutrient broth (HiMedia, Mumbai, India) and incubated
overnight at 37 �C. The growth in nutrient broth was fur-
ther transferred into Baird Parker Agar (HiMedia, Mumbai,
India) with egg yolk emulsion. All the distinct black-col-
oured colonies with or without opalescence were further
subjected to standard biochemical tests, such as catalase,
coagulase and oxidase tests, glucose and mannitol fermen-
tation and lecithinase reactions (Quinn et al. 1994). For iso-
lation of Gram-negative bacilli, the growths in nutrient
broth were inoculated into Mac-Conkey agar (HiMedia,
Mumbai, India) and further into Eosin Methylene Blue
agar (HiMedia, Mumbai, India). The colonies with charac-
teristic metallic sheen were further subjected to standard
biochemical tests, such as catalase, oxidase, indole, methyl-
red, VP, citrate and urease (Quinn et al. 1994).
*Corresponding author. Email: [email protected], [email protected]
� 2014 Taylor & Francis
Veterinary Quarterly, 2014
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All the biochemically confirmed Staphylococcus iso-
lates (11) were examined for their sensitivity to methicil-
lin (Met, 5 mg), oxacillin (Ox, 1 mg), cefoxitin (Cx,
30 mg), ciprofloxacin (Cf, 30 mg), amoxicillin and clavu-
lanic acid (AMC, 30 mg), amikacin (AK, 30 mg), tetracy-
cline (TE, 30 mg), vancomycin (VA, 30 mg), teicoplanin
(TEI, 30 mg) and tigecycline (TGC, 15 mg) by standard
disc diffusion assay (CLSI 2014). Minimum inhibitory
concentration (MIC) for the drugs oxacillin, cefoxitin and
vancomycin was measured using Ezy-MIC-strips (HiMedia,
Mumbai, India) according to the manufacturer’s instruc-
tions. Methicillin resistance was also confirmed by the
growth of the Staphylococcus isolates in oxacillin and
cefoxitin supplemented media (Hichrome MeRSA Agar,
HiMedia, Mumbai, India). For determination of vancomy-
cin resistance, isolates with zone diameters � 15 mm in
disc diffusion assay and for which vancomycin MICs
were �16 mg/mL (for Staphylococcus aureus)/�32 mg/
mL (for S. epidermidis) were classified as vancomycin-
resistant, whereas the isolates with zone diameters �15 mm and for which vancomycin MICs were 4�8 mg/
mL (for S. aureus)/ 8�16 mg/mL (for S. epidermidis)
were classified as vancomycin-intermediate (http://www.
cdc.gov). For determination of tigecycline susceptibility,
interpretation of zone diameters of all the Staphylococcus
isolates was done using the US FDA (Food and Drug
Administration) tigecycline susceptible breakpoints listed
for S. aureus. Isolates with zone diameter of �19 mm
only were classified as susceptible (Behera et al. 2009).
The isolates exhibiting resistance to methicillin, cefoxitin
and oxacillin were further screened for the presence of
methicillin resistance (mecA), coagulase (coa) and ther-
monuclease (nuc) genes by PCR as described earlier
(Wong et al. 2010).
All the seven E. coli isolates recovered from three
cows were further assessed for their sensitivity/resistance
to ceftazidime (CAZ, 30 mg), cefotaxime (CTX, 30 mg),
ceftriaxone (CTR, 30 mg), cefepime (CPM, 30 mg),
amoxicillin and clavulanic acid (AMC, 30/10 mg), tetra-
cycline (TE, 30 mg), gentamicin (G, 10 mg), amikacin
(AK, 30 mg), co-trimoxazole (Co, 25 mg), ciprofloxacin
(Cf, 30 mg), chloramphenicol (C, 30 mg), imipenem
(IMP, 10 mg), ertapenem (ETP, 10 mg) and meropenem
Figure 1. Mildly swollen udder of non-descript cattle.
Table1.
Clinico-pathologicalprofileofthreecasesofbovinemastitis.
Laboratory
findings
Bacteriology
Patients
District
Disease
Clinicalfeatures
WST�
CMT�
SCC� /ml
(Log10)
Haematology
Totalviable
bacterialcount
(cfu/m
l)
Bacteriaisolated
(number
ofisolatesin
parenthesis)
Holstein
Friesian
cross
1(H
FC1)
Nadia
Subclinical
mastitis
Notsignificant,
occasionalflakes
inmilkfrom
all
thequarters
CC
5.93
Nosignificant
change
0.05£
106
Staphylococcus
epidermidis(3),
Escherichia
coli(2)
Holstein
Friesian
cross
2(H
FC2)
Nadia
Subclinical
mastitis
Notsignificant,
occasionalflakes
inmilkfrom
all
thequarters
CC
5.85
Nosignificant
change
0.05£
106
S.epidermidis(2),
E.coli(2)
Non-descript
cow
(NDC)
Kolkata
Clinical
mastitis
Hotpainfulmildly
swollen
udder,
fever,off-fed
for
threedays,blood
clotandflakes
inmilk
CCC
C6.28
Neutrophilia
(36%
DLC)
2.1£
106
S.aureus(3),
S.epidermidis(3),
E.coli(3)
� WST:WhiteSlideTest;CMT:CaliforniaMastitisTest;SCC:SomaticCellCount.
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(MRP, 10 mg). MIC values of all isolates were determined
for ceftazidime, cefepime, cefotaxime, and ceftriaxone
using Hi-comb MIC test (HiMedia, Mumbai, India). The
isolates with zone of inhibition beyond the Clinical and
Laboratory Standards Institute (CLSI) recommended cut-
off values or with a breakpoint of MIC > 2 mg/ml for the
third- or fourth-generation cephalosporins were further
subjected to ESBL phenotypic determination by com-
bined disc method and ESBL E-test (CLSI 2014). The
combined disc method was performed using cefotaxime
(30 mg), ceftazidime (30 mg) and cefepime (30 mg) disc
alone or with clavulinic acid (10 mg) and an increase of
5 mm or more in the zone of inhibition in the presence of
clavulinic acid was considered positive for ESBL produc-
tion. ESBL E-test was performed using triple ESBL Ezy-
MIC-strip (HiMedia, Mumbai, India) and eightfold
increase in MIC in presence of b-lactamase inhibitors was
confirmed for ESBL production. All the three confirmed
ESBL producing E. coli isolates were subjected to modi-
fied hodge test (MHT) [http://www.cdc.gov] and disc dif-
fusion test with carbapenemase inhibitor [ethylene
diamine tetra-acetic acid (EDTA) and phenylboronic acid
(PBA)] impregnated agar as described by Birgy et al.
(2012). Furthermore, these ESBL producers were
screened for major b-lactamase-gene families blaCTX-M,
blaSHV, blaTEM, blaNDM OXA1 and OXA2 (Bhattacharjee
et al. 2007; Sarma and Ahmed 2010; Manchanda et al.
2011), plasmid‑mediated quinolone resistance (PMQR)
determinants (qnrA, qnrB, qnrS and aac(6 0)-ib-cr (Cie-
sielczuk et al. 2013), class I integron (int1), sulphonamide
resistance (sul1) genes (Lindstedt et al. 2003), cardinal
virulence markers for Shiga toxin producing E. coli (stx1and stx2), necrotoxigenic E. coliand extra-intestinal patho-
genic E. coli (ExPEC) by PCR as describer earlier (Paton
and Paton 1998; Van Bost et al. 2003).
Of the three suspected cases investigated, the cross-
breds (HFC1 and HFC2) were confirmed for subclinical
and the NDC for clinical mastitis by the WST, CMT and
SCC tests (Table 1). The haematogical parameters did not
reveal any significant change except neutrophilia in the
NDC.
The TVBC in milk was higher in the NDC than in the
crossbreds. In total, eight S. epidermidis, three S. aureus
and seven E. coli were isolated from these three cases of
bovine mastitis (Table 1). From HFC1, three S. epidermi-
dis were isolated of which one was methicillin resistant
(HFSE1) as revealed by disc diffusion assay and their
growth in Hichrome MeRSA agar. Of the two S. epidermi-
dis isolates from HFC2, one was methicillin resistant
(HFSE2). From the NDC with clinical mastitis, three S.
epidermidis and three S. aureus were isolated. Of them,
each of S. aureus (NDSA1) and S. epidermidis (NDSE1)
were methicillin resistant (Table 2).
The MRSE isolates (HFSE1, HFSE2 and NDSE1)
recovered in this study were phenotypically resistant to
methicillin, oxacillin, amoxicillin and clavulanic acid,
amikacin, tetracycline, vancomycin and not susceptible to
tigecycline (Table 2). On the other hand, MRSA isolate
(NDSA1) from NDC was phenotypically resistant to
methicillin, oxacillin, cefoxitin, amikacin, tetracycline,
vancomycin but was susceptible to tigecycline. In PCR,
all the four methicillin-resistant isolates harboured the
mecA gene and the S. aureus isolate (NDSA1) also carried
nuc and coa genes (Table 2).
Of the seven E. coli isolated in this study, only three
isolates (HFEC1, HFEC2 and NDEC1) were phenotypi-
cally confirmed as ESBL producers both by E-test and
combined disc method. All these three ESBL producers
exhibited resistance to the third- and fourth-generation
cephalosporins like ceftazidime, cefotaxime, ceftriaxone,
cefepime, along with amoxicillin and clavulanic acid, tet-
racycline, gentamicin, amikacin, co-trimoxazole and
chloramphenicol (Table 2). Furthermore, the isolates were
intermediately resistant to carbapenems (imipenem, mero-
penem and etrapenem). However, the isolates were found
negative in MHT and inhibitor based (EDTA and PBA)
phenotypic tests for carbapenemase or metallo-beta-lacta-
mase production. In contrast, all the ESBL producers and
methicillin-resistant Staphylococcus spp. isolated in this
study were sensitive or intermediately resistant to cipro-
floxacin except the MRSE isolate (NDSE1) obtained from
NDC.
Again, all the three ESBL producers (HFEC1, HFEC2
and NDEC1) possessed two major genes of b-lactamase
family (blaCTX-M, blaTEM) and the b-lactamase gene
blaSHV was detected in two isolates from HFEC1 and
HFEC2. Besides, the latter also possessed the genes for
class I integron (int1) and sulphonamide resistance (sul1).
However, none of the ESBL producers possessed studied
PMQR genes except HFEC2, which harboured qnrS.
Among other studied virulence factors, the papC,iucD
and ESTA1were detected in HFEC1 and HFEC2. In con-
trast, none of the isolates possessed the genes for Shiga
toxins (stx1, stx2) and cytotoxic necrotizing factor (cnf1,
cnf2) or other resistant traits investigated (OXA, NDM
and aac(60)-ib-cr.).Bovine mastitis continues to be a major challenge the
dairy industry is facing despite implementation of several
control programmes. The condition is further complicated
with the colonization and infection of drug-resistant path-
ogen(s) increasing the cost of treatment of sick animals.
Detection of such resistant pathogens is a major public
health concern due to the possibility of infection via con-
taminated unpasteurized milk and milk products intended
for human consumption. Although individual cases of
methicillin-resistant Staphylococcus and extended spec-
trum b-lactamase (ESBL) producing Enterobacteriaceae
have been reported in bovine mastitis in recent years
(Geser et al. 2012; Dahmen et al. 2013; Gindonis et al.
2013), there is no report apparently available on mixed
infection of three different kind of pathogens in bovine
mastitis.
This report describes co-infection of either or both
methicillin-resistant S. epidermidis (MRSE), methicillin-
resistant S. aureus (MRSA), and ESBL producing E. coli
harbouring virulence genes such as papC, ESTA1, and
iucD in three such cases of bovine mastitis in two cross-
breds and one NDC from West Bengal, India. The find-
ings revealed more association of MRSE than MRSA
with the bovine mastitis. Reports from other countries like
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Table2.
CharacterizationofMRSA,MRSEandESBLproducingE.coliisolatesfrom
bovinemastitis.
1.Methicillin-resistantStaphylococcusepidermidisandmethicillin-resistantStaphylococcusaureus
Genes
Antibioticresistance
profile
Isolates
mecA
nuc
coa
Met
Ox
Cx
Cf
AMC
AK
TE
VAa
TEI
TGCb
HFSE1
C¡
¡R
RI
IR
RR
RR
NS
HFSE2
C¡
¡R
RR
IR
RR
II
NS
NDSA1
CC
CR
RR
SI
RR
RI
S
NDSE1
C¡
¡R
RI
RR
RR
RR
NS
2.Extended
spectrum
b-lactamaseproducingEscherichia
coli
Antibioticresistance
profile
Isolates
CAZ
CTX
CPM
CTR
AMC
TE
GAK
Co
Cf
CIM
PMRP
ETP
HFEC1
RI
RI
RR
RR
RI
RI
II
HFEC2
RR
RR
RR
RR
RS
RI
II
NDEC1
RR
RR
RR
RR
RS
RI
SS
Resistance
andvirulence
genotypes
Phenotypicconfirm
ation
ofESBLproduction
Isolates
bla
CTX-M
bla
TEM
bla
SHV
sul1
int1
OXA1/2
NDM
qnrA
qnrB
qnrS
aac(60 )-ib-cr
CNF-1/2
stx1/2
Others
DDDT
E-Test
HFEC1
CC
CC
C¡
¡¡
¡¡
¡¡
¡ESTA1
CC
HFEC2
CC
CC
C¡
¡¡
¡C
¡¡
¡iucD
,papC,ESTA1
CC
NDEC1
CC
¡¡
¡¡
¡¡
¡¡
¡¡
¡¡
CC
Note:R:Resistant;S:Susceptible;NS:Not-susceptible;I:Interm
ediate.
Ceftazidim
e(CAZ,30mg),cefotaxim
e(CTX,30mg),ceftriaxone(CTR,30mg),cefepim
e(CPM,30mg),ciprofloxacin
(Cf,30mg),co-trimoxazole(Co,25mg),chloramphenicol(C,30mg),am
oxicillinandclavulanicacid
(AMC,30/10mg),tetracycline(TE,30mg),gentamicin
(G,10mg),am
ikacin
(AK,30mg),im
ipenem
(IMP,10mg),ertapenem
(ETP,10mg)andmeropenem
(MRP,10mg).
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the Netherlands, Finland and USA also indicate that
MRSE is an emerging etiological factor to cause bovine
mastitis (Sawant et al. 2009; Sampimon et al. 2011; Gin-
donis et al. 2013). Even the presence of MRSE alone with-
out any MRSA was also recorded from bovine subclinical
and clinical mastitis cases (Kot et al. 2012).
The ESBL-producing E. coli possessing blaCTX-M var-
iants were reported previously from mastitic cattle in
France, Japan and United Kingdom (Geser et al. 2012;
Dahmen et al. 2013; Ohnishi et al. 2013). Again, drug-
resistant Enterobacteriaceae possessing b-lactamase
encoding genes, such as blaTEM, blaSHV, blaCTX-M, blaOXAand other drug resistant determinants like qnr and class I
integron, were reported from bovine mastitis in Egypt
(Ahmed and Shimamoto 2011). However, there is hardly
any report of simultaneous infection of MRSA, MRSE
and ESBL-producing E. coli in the same mastitic cow
available to compare with the present findings. Further-
more, all the three ESBL producers from crossbreds were
found to carry virulence markers for ExPEC like papC,
iucD and ESTA1 which is rarely reported among the
ESBL producers from bovine mastitis, although such viru-
lence factors were reported among non-ESBL E. coli iso-
lates from clinical bovine mastitis in recent past (Suojala
et al. 2011). Detection of virulence genes required for
establishment of extra-intestinal infection in mastitic E.
coli isolates further makes the condition worse because
they can transmit the genes in commensal human E. coli
and convert them into extra-intestinal pathogenic E. coli
such as uropathogenic E. coli (Samanta et al. 2014.).
One of the ESBL producers (HFEC2) despite carrying
the plasmid-mediated (fluoro)quinolone resistance gene
(qnrS), was still sensitive to ciprofloxacin. This could be
due to the fact that qnr proteins are considered to confer
only low level of quinolone resistance by protecting the
bacterial DNA gyrase from (fluoro)quinolones. However,
in few instances, these can also facilitate higher resistance
during presence of (fluoro)quinolones at therapeutic con-
centration (Robicsek et al. 2006).
Similar to this finding, MRSE strains isolated from
mastitis milk samples of cattle also exhibited resistance to
tetracycline and different macrolides as reported by Kot
et al. (2012). Likewise, S. aureus isolated from goat milk
was also resistant to (oxy)tetracycline (Chu et al. 2012). It
is worrying that MRSA and MRSE isolates recovered in
this study also exhibited resistance or reduced susceptibil-
ity to glycopeptides or glycylcycline antibiotics like van-
comycin, teicoplanin and tigecycline, although these
drugs are never recommended in veterinary practice in
India. The glycopeptide resistance among S. aureus iso-
lates of animal origin is rarely found even though it is
widely detected in human (Thati et al. 2011). This could
be a grave public health concern as glycopeptides or gly-
cylcycline antibiotics are the choice of treatment in
MRSA infection in human beings. The ESBL producing
E. coli isolated in this study exhibited resistance to antibi-
otics belonging to multiple antimicrobial classes. Previ-
ously, ESBL-producing E. coli isolated from mastitic
milk showed resistance against chloramphenicol, genta-
micin and tetracycline in Switzerland (Geser et al. 2012).
However, the phenotypic expression of drug resistance in
ESBL producers is highly variable depending upon the
type of b-lactamase enzyme present and other resistance
traits they harbour in the plasmid, although multi-drug
resistance is commonly found among ESBL-producing E.
coli (Rakotonirina et al. 2013). Despite their intermediate
resistance to carbapenems, none of the ESBL producers
showed any evidence of MBL or carbapenemase produc-
tion either by phenotypic screening test or for blaNDMgene in PCR. Such resistance may be either due to high
expression of ESBL or in combination with reduced
expression of porin channels as suggested previously
(Birgy et al. 2012).
In conclusion, present investigation is an unique report
of simultaneous occurrence of MRSE, MRSA and ESBL-
producing E. coli in bovine mastitis. However, in absence
of any previous report or precise information of earlier
disease conditions/treatment of these cases, it is very diffi-
cult to comment, or conclude with regard to frequency of
such cases. Even though it seems to be an isolated case of
its own kind, such type of bovine intra-mammary infec-
tion is a cause of concern for public health.
Acknowledgements
The authors are thankful to the Director, IVRI, Izatnagar (UP)for providing necessary research facilities to carry out the pres-ent work.
Funding
The financial support received from DBT, Government of Indiais acknowledged.
Disclosure statement
The authors have no conflict of interest.
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