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Subclinical Bovine Mastitis in Rural, Peri-Urban and Suburban Regions of Jaipur
District of Rajasthan, India
Biswadeep Jena1*, Nilesh Kumar Pagrut2, Abhishek Sahoo3 and Abrar Ahmed1
1Department of Teaching Veterinary Clinical Complex, MJF College of Veterinary and Animal Science,
Chomu, Jaipur, Rajasthan, INDIA
2Department of Veterinary Pathology, MJF College Of Veterinary And Animal Science, Chomu, Jaipur, Rajasthan, INDIA
3Department of Animal Nutrition, Arawali Veterinary College, Sikar, Rajasthan, INDIA
*Corresponding author: B Jena; Email- biswadeep44@gmail.com
Received: 18 December, 2014 Accepted: 02 February, 2015
ABSTRACT
A cross section study was carried out from June 2013 to May 2014 on a total of 110 lactating cows of rural, peri-urban and
suburban regions of Jaipur District of state of Rajasthan, for sub clinical mastitis by using California Mastitis Test (CMT),
White side test (WST), Surf Field Mastitis Test (SFMT) and Somatic cell count (SCC). Prevalence of subclinical bovine
mastitis in animal level was recorded as 67.27, 64.55, 63.64 and 74.55 % by CMT, WST, SFMT and SCC, respectively whereas
39.55, 38.86, 37.95 and 45.23 % by CMT, WST, SFMT and SCC, respectively in the level of quarters. Staphylococcus species
(46.3%) occupied the prime position among the bacterial isolates followed by Streptococcus species (9.76%), Escherichia coli
(6.1%), mixed growth (32.96%) and sterile growth (4.88%). Antibiotic susceptibility test revealed highest sensitivity towards
Enrooxacin. However, antibiotics showing higher rate of resistance patterns were Streptomycin, Penicillin G, Ampicillin,
Cloxacillin, Amoxicillin, Kanamycin and Lincomycin. This reects the poor quality of milk available to the consumers, lack of
adequate hygienic practices, pre-emptive prophylactic regimen and indiscriminate use of antimicrobials.
Keywords: Subclinical mastitis, bovine, CMT, SFMT, WST, SCC, bacterial isolates, antibiotic sensitivity test
Mastitis is an inammation of the mammary gland (Suojala
et al., 2011) The incidences of mastitis highly affects the
economics of dairy industry, due to sudden drop in milk
yield, increase in treatment costs, recovery time and
nally culling of the affected cows (Bar et al., 2008; Hertl
et al., 2011). Mastitis compromises animal welfare as
well as its treatment, associated with human health hazard
(Fogsgaard et al., 2011; Rasmussen et al., 2011). Bovine
mastitis is categorized as one of the most problematic
diseases affecting the dairy industry throughout the world
(Bachaya et al., 2011). It was the most prevalent and
cost sparing diseases of dairy animals worldwide, with
an annual economic loss of over 1.7 billion dollars in the
USA (Sahoo et al., 2012) and 526 million dollars in India
(Varshney and Naresh, 2004). It is characterized by heat,
redness, swelling, hardness and pain with abnormalities in
milk like increased somatic cells, especially leukocytes,
in the milk and by pathological changes in the mammary
tissue (Ranjan et al., 2010). Various forms of clinical and
subclinical mastitis occur in bovines. In clinical mastitis all
the ve cardinal signs of udder inammation (redness, heat,
swelling, pain and loss of milk production) are recorded,
while in the sub-clinical form no obvious manifestations of
inammation are found. Subclinical mastitis is 3-40 times
more common than clinical mastitis that causes greater
overall loss in most dairy herds (Bachaya et al., 2011).
In 2004, Varshney and Naresh reported 70% economic
losses due to subclinical mastitis. The situation has been
compounded by the continued indiscriminate use of
antibiotics without culture and sensitivity testing of milk.
This may be attributed to callous approach of the dairy
farmers, who instead of consulting qualied veterinarian,
prefer to take over the counter supply of medicine by
the drug retailers. Veterinarians who do not capitalize
Journal of Animal Research: v.5 n.1, p. 175-182. April. 2015
DOI Number: 10.5958/2277-940X.2015.00028.5
176 Journal of Animal Research: v.5 n.1. April 2015
Jena et al.
on the available diagnostic tests are no less responsible
for increase in the incidence of mastitis (Ranjan et al.,
2010). However, the detection of mastitis is difcult.
Clinical mastitis is conrmed by observation of clinical
signs by the farmer (direct detection) (Hokmabad et al.,
2011). Subclinical mastitis can be recognized by indirect
detection: the somatic cell count in milk (Hokmabad et
al., 2011) or by animal-side milk tests (Bachaya et al.,
2011), but most of the farmers in Jaipur (rural), Rajasthan
are not acquainted with these practices. Therefore it is
indispensable to recognize and enumerate the causative
organisms to review the sufciency of the therapeutic
armory, evade auxiliary complications and acclimatize
management practices for the efcient control of mastitis.
The purpose of this investigation was to elucidate the
prevalence of subclinical mastitis in apparently healthy
dairy cows in rural, peri-urban and suburban regions of
Jaipur district of state of Rajasthan, to nd out the major
causative agents causing subclinical mastitis and to study
their drug sensitivity. The study was conducted on 110
cows in the Jaipur rural region of state of Rajasthan, India.
MATERIALS AND METHODS
Location
The study was conducted on randomly selected apparently
healthy cows who visited Teaching Veterinary Clinical
Complex, M.J.F. College of Veterinary and Animal
Science, Chomu, Jaipur during a period of 11 months
from June 2013 to May 2014. Cows which were tested
under this study mostly belong to rural, peri-urban and
suburban regions of Jaipur district of state of Rajasthan.
The study area was found at and around 26.9260° N
longitude, 75.8235° E latitude with an altitude range of
431 m above sea level. Jaipur has a semiarid subtropical
climate, receiving over 650 millimetres (26 in) of rainfall
annually only during the monsoon. The average daily
temperature is around 30°C during summer and 15-
18°C during winter.
Animals
In the present study, a total number of 110 (440 quarters)
apparently healthy cows without any clinical signs of
mastitis were screened for SCM in and around Jaipur
District during the period of 11 months from June 2013
to May 2014. Animals were managed under extensive
and semi-intensive production system. The traditional
extensive production system consists of indigenous
breeds graze for feed with minor supplementations. On the
other hand, in the semi-intensive production system, the
animals are mainly belongs to crossbred cattle. They are
reared indoors with occasional grazing in the eld. They
are supplemented with concentrates in addition to the
natural pasture, crop by products, straw. This type of dairy
husbandry system is booming and becoming an important
source of milk supplies to households, nearby Dairy plants
and a means of income generation in rural and peri-urban
Table 1 : Animal wise and quarter wise prevalence of subclinical mastitis detected by four screening tests.
Tests used Types Sample
tested
Positive
cases
Prevalence
(in %)
95%
Condence
Intervals
Chi-square
value P-value
California mastitis test
(Score > 1+)
Animal wise 110 74 67.27 55.97-76.75 13.127 0.000*
Quarter wise 440 174 39.55 34.25-44.89 19.236 0.000*
White side test
(Score > 1+)
Animal wise 110 71 64.55 53.16-74.31 9.309 0.000*
Quarter wise 440 171 38.86 33.59-44.20 21.827 0.000*
Surf eld mastitis test
(Score > 1+)
Animal wise 110 70 63.64 52.23-73.49 8.182 0.000*
Quarter wise 440 167 37.95 32.71-43.28 25.536 0.000*
Somatic cell count
(SCC > 5 × 105)
Animal wise 110 82 74.55 63.65-83.07 26.509 0.000*
Quarter wise 440 199 45.23 39.784-50.615 4.009 0.000*
Subclinical Bovine Mastitis in Rajasthan
Journal of Animal Research: v.5 n.1. April 2015 177
Table 2.: Bacteria isolated from 82 numbers of cases of SCM
in cows.
Bacteria
Number of
isolates
Percentage (%)
Staphylococcus Spp. 38 46.3
Streptococcus Spp. 8 9.76
E. coli 5 6.1
Staphylococcus spp.+
Streptococcus spp. 10 12.2
Staphylococcus spp.+ E.
coli 911
Streptococcus spp. + E. coli 5 6.1
Staphylococcus spp.+
Streptococcus spp. + E. coli 3 3.66
Negative growth 4 4.88
areas of Jaipur District, Rajasthan, India. Manure removal
is generally made on a daily basis. Although milking is
done by hand, pre- and post-milking hygienic protocols,
such as washing of udder and subsequent drying are not
followed. All the tested animals were apparently healthy
during preceding lactations. Full hand method of milking
was performed twice a day (6 and 18 h). Among the 110
animals 64, 25, 13 and 8 were crossbred Holstein Friesian,
Haryana, Rathi and Non-descript breeds, respectively.
White side test (WST)
One drop of 4 per cent sodium hydroxide and ve drops of
milk from each quarter were placed on the glass slide and
mixed with a glass rod (Doxey, 1985). Results were read
after 20 sec, according to the change in viscosity of milk
as negative, 1+, 2+ and 3+. Samples scoring 1+, 2+ or, 3+
considered as positive case for subclinical mastitis.
Somatic cell count (SCC)
It was done as described by Schalm et al. (1971). Milk was
mixed thoroughly before testing. Ten microliter of milk
from each quarter was spread over 1 cm2 marked square
area on a glass slide. The milk lm was left undisturbed
at room temperature until it dried, and then the smear
was xed in Xylol for 5 min and stained with Loffer’s
methylene blue reagent. Cell counting was made under
oil immersion as per the procedure described by Dhakal
(2006). Animal sample, showing somatic cell count more
than 5 × 105 of somatic cells, is considered as positive case
for subclinical mastitis as per criteria cited by International
Dairy Federation (IDF) and Hegde et al. (2013).
Table 3. Antibiotic sensitivity pattern in term of high to
moderate and mild to resistant antibiotic sensitivity for selection
of antibiotics for therapeutic use.
List of the antibiotics with its
MIC (µg)
Bacterial Isolates
H, Mo
Mi, R
No.
%
No.
%
Enrooxacin (Ex, 10) 76 92.7 6 7.3
Ciprooxacin (Cf, 10) 73 89 9 11
Amikacin (Ak,30) 70 85.4 12 14.6
Ceftriaxone (Ci, 30) 66 80.5 16 19.5
Chloramphenicol (C, 30) 60 73.2 22 26.8
Cephotaxime (Ce, 30) 56 68.3 26 31.7
Gentamicin (G, 10) 47 57.3 35 42.7
Peoxacin (Pf, 5) 40 48.8 42 51.2
Cephalexin (Cp, 30) 37 45.1 45 54.9
Neomycin (N, 30) 31 37.8 51 62.2
Lincomycin (L, 10) 25 30.5 57 69.5
Kanamycin(K, 30) 23 28 59 72
Amoxycillin (Am, 10) 22 26.8 60 73.2
Cloxacillin(Cx, 10) 21 25.6 62 74.4
Ampicillin(A, 10) 18 22 64 78
Penicillin G (PG, 10) 13 15.9 69 84.1
Streptomycin(S, 10) 10 12.2 72 87.8
Surf Field Mastitis Test (SFMT)
This test was performed and scored following the method
described by Muhammad et. al. (2010) in brief, about 2
ml milk from each quarter was drawn from bottle into test
cup and an estimated 2 ml of 3% solution of household
detergent (Surf Excel®, Uniliver, India Ltd.,). Mixing
was accomplished by gentle circular motion of the paddle
in a horizontal plane for few seconds. The reaction
developed almost immediately with milk containing a
high concentration of somatic cells. The peak of reaction
was obtained within 30 seconds and immediately scored
as 1+, 2+ and 3+.
178 Journal of Animal Research: v.5 n.1. April 2015
Jena et al.
California mastitis test (CMT)
The California mastitis test was carried out as screening
test for selections of samples for culture following the
method described by Schalm et al. (1971) and Quinn et al.
(1994). A squirt of milk, about 2 ml from each quarter was
placed in each of four shallow cups in the CMT paddle.
An equal amount of the commercial reagent was added
to each cup. A gentle circular motion was applied to the
mixtures, in a horizontal plane for 15 sec. The reaction
was interpreted according to Schalm et al. (1971), Quinn
et al. (1994) and David et al. (2005). The results of visible
reactions were classied into 5 scores: (0) = negative, (±)
= trace, (+1) = weak positive, (+2) = distinct positive, and
(+3) = strong positive. In this study, CMT score of 1+ and
above was considered positive for mastitis and of trace
and negative (± and 0) together was considered negative
for subclinical mastitis.
Milk Sampling
Quarters that scored negative and trace were assumed
healthy and the quarters with different positive scores
through any screening tests were assumed infected.
A cow with at least one affected quarter at the time of
examination was considered positive for subclinical
mastitis. Similar criteria were used to characterize a cow
positive for SCM (Sharma et al., 2007). All the animals
found positive in SCC were selected for further analysis
under bacteriological prevalence and antibiotic sensitivity
test. Only one of the infected quarters from each cow was
selected for milk sampling except when the cow had four
severely infected quarters so one more milk sample was
collected. Then the teat end of the selected quarter was
swabbed with cotton soaked in 70% ethyl alcohol and 10
milliliters of milk was approximately collected into sterile
containers. The rst 3-4 streams of milk were discarded.
The collecting vial was as near horizontal as possible and
by turning the teat to a near horizontal position, 15 ml of
milk was collected into the vial. Samples were transported
to the laboratory in a special box with ice at 4°C for
bacteriological investigation (Quinn et al., 1994).
Bacteriological culture and identication of
microorganisms
The samples were subjected to bacteriological study in
the laboratory by inoculating approximately 0.01 ml of
milk sample on to blood agar, nutrient agar, MacConkey’s
agar, Sabourads dextrose agar and Eosine Methylene
Blue agar plates and the plates were incubated “under
aerobic conditions” at 37° C for 24 to 48 hours. The
staining and cellular morphological features of organisms
were ascertained by microscopic examination of Gram
stained smears. The bacteria isolated were identied on
the basis of their cultural, morphological and biochemical
characteristics as per the method of (Cruickshank et al.,
1975).
Antibiotic susceptibility test
All the isolates were subjected to in vitro drug sensitivity
test as per method described by Bauer et al. (1966). The
antimicrobials (µg) available commercially in market
like cloxacillin (10), amoxycillin(10), streptomycin (10),
penicillin G (10), and neomycin (30) were tested for their
in vitro effectiveness against various bacterial isolates.
In addition, old and new generation antimicrobials (µg)
like chloramphenicol (30), lincomycin (10), gentamicin
(10), ciprooxacin (10), cephalexin (30), enrooxacin
(10), amikacin (30), cefotaxime (30), ceftriaxone (30),
peoxacin (5), kanamycin (30) and ampicillin (10) were
also tested. The antibiotic discs (Hi-Media®, Mumbai,
India) were impregnated on the surface of an agar plate
previously inoculated with a standard amount of the
organism under scrutiny. The plates were incubated
at a temperature of 37°C for duration of 18 - 24 hours.
Subsequently, the plates were examined for the zone
of inhibition developed around the discs, followed by
the diameter of the zone of inhibition was measured
in milimeter and compared with the values listed in
standard chart provided by the manufacturer, on the
basis of which the isolates were categorized as resistant
(R), mildly sensitive (Mi), moderately sensitive (Mo) or
highly sensitive (H) to the antimicrobial contained in that
particular disc (Ranjan et al., 2010).
Statistical analysis
All collected data were entered in Microsoft Ofce®
2007 excel sheet and analyzed by SYSTAT® version 12
computer package program. In the present study chi-
square test and condence intervals were calculated.
Subclinical Bovine Mastitis in Rajasthan
Journal of Animal Research: v.5 n.1. April 2015 179
RESULTS AND DISCUSSION
Prevalence of subclinical bovine mastitis in animal level
was recorded as 67.27, 64.55, 63.64 and 74.55 % by
CMT, WST, SFMT and SCC, respectively whereas 39.55,
38.86, 37.95 and 45.23 % by CMT, WST, SFMT and
SCC, respectively in the level of quarters, as summarized
inTable 1. The gures on both animal wise and quarter
wise prevalence of SCM based on individual tests closely
proximated with the observations of Sharma et al. (2004
and 2007). However, both lower (Sharma and Sindhu,
2007; Sharma and Maiti, 2010; Supriya et al., 2010;
Bachaya et al., 2011; Hegde et al., 2013) and higher
(Muhammad et al., 2010) prevalence rates of SCM have
been reported in the literature. This large variability of
prevalence of SCM found around Jaipur could be due to
prevalence of risk factors e.g., a large proportion of cow
conned to zero grazing production system, differences in
management practices and poor hygienic standards of the
dairy environment cum milking conditions. Other factors
that could inuence the prevalence of SCM could be due to
immune responses, genetic variation in disease resistance
amongst the breeds, some heritable characteristics
such as milk production capacity, teat structure, udder
conformation, use of different methods of diagnosing of
subclinical mastitis and the denition of infection, which
is variable according to Mdegela et al. (2005). According
to IDF criteria, 15.38 % quarters of cows were suffering
from sub clinical mastitis on account of having somatic
cell count (SCC) more than 5,00,000 per ml of milk and
culturally positive. The prevalence rate of SCM on IDF
criteria was lower than cultural examination or SCC alone.
These ndings are in agreement with the observations of
Tuteja (1993) and Sharma and Kapur (2000), Supriya et
al. (2010) and Hegde et al. (2013).
Among all the four indirect tests, SCC showed highest
efcacy of 74.55 % with respect to diagnosis of SCM. The
efcacy of SCC is followed by CMT, WST and SFMT
which in accordance to Sharma et al. (2008). Hence all the
animals found positive in SCC were selected for further
analysis under bacteriological prevalence and antibiotic
sensitivity test. The data summarized in Table 2 indicates
the relative occurrence of various bacteria isolated from
cows. The pathogens isolated from 82 milk samples,
found positive under SCC, were Staphylococcus spp.
38 (46.3%), followed by Streptococcus spp. 8 (9.76%),
Escherichia coli 5 (6.1%), mixed growth 27 (32.96%) and
no growth were found in 4 (4.88%) milk samples. Among
the mixed growth, prevalence of Staph. spp. and Strep.
spp. was the most predominant combination with 12.2%
of prevalence. This was followed by Staph. spp. + E. coli
combination, Strep. spp. + E. coli combination and Staph.
spp. + Strep. spp. + E. coli combination with 11%, 6.1%
and 3.66% prevalence, respectively.
On cultural examination the Staph. spp. was found to be
the chief etiological agent causing SCM. This nding is in
agreement with the earlier reports of Sharma and Sindhu
(2007); Sumathi et al. (2008); Sharma and Maiti (2010);
Harini and sumathi (2011) and Ranjan et al. (2011).The
highest incidence of Staph. spp. are closely associated with
hygiene. It becomes pathogenic whenever the hygienic
conditions of the animal or environment become meager.
Moreover, the existence of high concentration of Staph.
spp. in milk also indicates the relatively poor quality of
milk, related with unhygienic milking practices as this
pathogen is mainly spread during milking via milkers’
hands [Bradley 2002]. This also might be due to harboring
of the organism in the skin, udder and milk of the infected
gland which acts as reservoir (Olmsted and Norcross,
1992). Davidson (1961) have shown that the ability of
Staph. spp. to bind to epithelial cells of the ductile and
alveoli in mammary gland is also an important virulence
factor.
Strep. spp. was the second largest mastitogen group
of isolates recovered from our experiment. This was
in accordance with reports of Sahoo et al., (2009) and
Sharma and Maiti (2010). Strep. spp. which is an obligate
parasite of the epithelium and tissue of mammary gland,
multiplies in the milk and on the mammary epithelial
surfaces, generally causing a subacute or chronic
inammatory reaction with periodic acute are-ups. The
affected tissue eventually is destroyed resulting in reduced
milk production (Sharma et al., 2012).
The E. coli isolates in the present study accounted for 6.1%
share and third most prevalent organism among different
isolates of mastitis milk, which is very low with respect to
previous literature (Ranjan et al., 2011). Despite E. coli is
the environmental pathogen, but low or sporadic incidence
of E. coli has also been reported by various workers (Shukla
et al., 1998) as observerd in this study. Opsonization of
bacteria by IgM with subsequent phagocytosis and killing
by neutrophil are some of the factors, which prevent
180 Journal of Animal Research: v.5 n.1. April 2015
Jena et al.
establishment of E. coli mastitis (Gyles and thoen, 1993).
These inherent properties of udder defense against E. coli
infection might be responsible for reduced incidence of E.
coli mastitis in the present study. Prevalence of E.coli is an
indication of poor hygienic practices in dairy environment,
as these organisms originate from the cow’s environment
and infect the udder through the teat canal. Contamination
of end of the teat is a major predisposing factor in the
development of environmental mastitis (Bradley, 2002;
Sumathi et al., 2008; Sharma et al., 2012).
Different combinations of the mastitogenic organisms
were detected in mixed infection. Most predominant
combination of the isolates was Staph. spp. and Strep.
spp. followed by Staph. spp. and E. coli similar to ndings
of Srinivasan et al. (2013). Therefore, it is of principal
magnitude that a particular dairy herd/organized farm
should be screened for the disease routinely, espouse
an efcient treatment protocol and sound prophylactic
measures, that would prevent serious economic losses in
terms of decreased milk production, cost of treatment and
culling of animals.
The failure of some pathogens to grow in vitro may
be due to the fact that certain microorganisms require
specic culture media (Ranjan et al. 2010). It could also
be explained by the possible premedication of the animals
with antibiotics (Hawari and Al-Dabbas, 2008) because
the withdrawal time may not have been respected.
The data summarized in Table-3 indicates the zone of
inhibition and antibiotic sensitivity of the isolates. Out
of 82 isolates obtained from cases of SCM from cows
tested for their antibiogram, revealed percent of isolates
were most sensitive to Enrooxacin (92.7%), followed
by Ciprooxacin (89%), Amikacin (85.4%), Ceftriaxone
(80.5%), Chloramphenicol (73.2%), Cephotaxime
(68.3%), Gentamicin (47%), Peoxacin (40%),
Cephalexin (37%) and Neomycin (31%). On the contrary,
antibiotics showing higher rate of resistance patterns
were Streptomycin, Penicillin G, Ampicillin, Cloxacillin,
Amoxicillin, Kanamycin and Lincomycin showing 87.8%,
84.1%, 78%, 74.4%, 73.2%, 72% and 69.5% resistance,
respectively.
The emergence of drug resistant organisms causing
mastitis due to indiscriminate use of antibiotics is well
known. Moreover, due to lack of prophylactic agents,
chemotherapy continues to play a major role in the
therapeutic management of the disease. For success
of the treatment, sensitivity testing plays a pivotal role.
Recently newer antibiotics have been introduced for the
treatment of both sub clinical and clinical mastitis. Thus,
it has become imperative to control this dreaded disease
with most effective antibiotic therapy. Hence the present
study was also designed to prove into in-vitro sensitivity
of isolated bacterial strains from cases of SCM against a
range of traditional as well as newly introduced antibiotics
potentially useful for the treatment and control programme
(Sharma et al., 2007).
Highest sensitivity of bacteria towards Enrooxacin is in
agreement with Sahoo et al., (2009); Ranjan et al., (2010)
and Sharma et al. (2012). Isolates in the present study
showed moderate sensitivity or resistance to Streptomycin
and Penicillin – G. Indiscriminate and frequent use
of these antibiotics in animals could be the reason for
their ineffectiveness against bacterial isolates. Similar
observations of resistance towards Penicillin were also
observed by Ranjan et al. (2010) and Harini and sumathi
(2011).
The distressing level of resistance of organisms to a
particular drug might be due to the indiscriminate use
of the respective drugs. In view of the diversied gamut
of pathogens resulting in mastitis, its control needs
the selection of apt antimicrobial by establishing an
antibiogram. Hence, the control of mastitis should be
directed by administration of distinct regime of antibiotics
and holistic advance to the disease management.
Hence the present study concluded that prevalence of
subclinical mastitis in rural, peri-urban and suburban
provinces of Jaipur was found to be highly contagious.
The rationale behind this high prevalence is multifaceted,
but there are a few points need scrupulous elucidation
such as lack of regular screening for early detection and
prophylactic treatment, inadequate housing with improper
sanitation of environment, udder and milker’s hand, zero
grazing , late stage of lactation, high parity; all seem to
augment the risk of getting SCM. Although we did not
perform any statistics on it, the results of this study
provide new information and will hopefully contribute to
a possibly lower prevalence of SCM in the future.
Subclinical Bovine Mastitis in Rajasthan
Journal of Animal Research: v.5 n.1. April 2015 181
ACKNOWLEDGEMENTS
The authors are thankful to the Dean, M.J.F. College of
Veterinary and Animal Science, Chomu, Jaipur, Rajasthan,
India for his support and cooperation in carrying out the
study. The fund for the study was provided by Department
of Teaching Veterinary Clinical Complex, M.J.F. College
of Veterinary and animal science, Chomu, Jaipur,
Rajasthan, India
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