Mastitis is recognised worldwide as one of the most costly diseases of dairy
animals because it incurs great financial loss due to reduced milk yield, increased
culling rates and treatment costs as well as lowering nutritive value of milk
(Radostits et al., 2007). In contrast to visible
changes in the acute form of mastitis there is absence of gross abnormalities
in the milk or udder in case of subclinical mastitis which leads to a greater
loss to the dairy industry. In India, Singh and Singh (1994)
and Dua (2001) reported the economic loss due to SCM
was higher (483.10 crores and 1723.32 crores) than the acute mastitis (234.59
and 696.29 crores) in buffaloes. Apart from causing huge economic losses, this
disease also possesses the risk for the transmission of zoonotic diseases to
human beings (Radostits et al., 2007).
Namakkal, a district of Tamil Nadu having buffalo population of 1,18,202 with
milk production of 118.675 tons and contributing 57.76% of total milk production
i.e., 205.448 tones (Census, 2004). However, limited
information regarding the prevalence and etiology of SCM in clinically normal
buffaloes in this region was available. Keeping the above facts in view, the
present investigation was undertaken to find out the prevalence and etiology
of SCM in buffaloes of marginal farmers maintained under prevailing field conditions
by indirect chemical tests, somatic cell count and bacteriological examination.
MATERIALS AND METHODS
In the present study, a total number of 206 (824 teats) apparently healthy
buffaloes without any clinical signs of mastitis were screened for SCM in and
around Namakkal district during a period of one year from March 2007 to February
2008. All the screened buffaloes were housed in stables during rainy days and
allowed to move freely in spring and summer. All the tested animals were apparently
healthy during the preceding lactations. Full hand method of milking was performed
twice a day (6 and 18 h). Of the 206 animals 68, 50 and 88 were graded Murrah,
graded Surti and Non descript breeds, respectively. Information regarding, stage
of lactation, parity, milking methods and previous incidence of mastitis were
collected. The animals were divided into three groups on the basis of stage
of lactation viz., early (up to 120 days), mid (121- 240 days) and late (above
240 days) stage. The parity of the buffaloes were grouped into 1, 2, 3, 4, 5,
6 and above 6.
Collection of milk samples: A total of 824 quarter milk samples were collected from 206 apparently healthy lactating buffaloes. Teat end were cleaned with chlorhexidine solution. After discarding first few streams of fore milk, 5 mL of milk from each quarter was collected into sterile screw caped vials for laboratory examination. Each vial was labeled as Left Front (LF), Left Hind (LH), Right Front (RF) and Right Hind (RH). Individual quarter milk samples was subjected to California Mastitis Test (CMT), White Side Test (WST) and SCC, where as the composite or pooled milk from each buffaloes were utilized for bacteriological examination. The WST and CMT were done in the spot i.e., milking byre itself and for confirmatory tests, milk samples were taken to laboratory in a thermal flask containing crushed ice.
White side test: The WST was done according to Doxey
(1985). One drop of 4 percent sodium hydroxide and five drops of milk were
placed on the glass and mixed with a glass rod. The results were read after
20 sec, according to the change in viscosity of milk as negative, 1+, 2+ and
California mastitis test: The test was carried out according to the
method of Schalm and Noorlander (1957). Equal volume
of milk and CMT reagent (obtained from IVPM, Ranipet, Tamil Nadu) were mixed
on a test plate. The formation of viscous gel was evaluated by rotating the
plate gently. According to the changes of colour and formation of gel, the result
was interpreted as negative, 1+, 2+ and 3+ as described by Schalm
et al. (1971). Quarters with CMT score of 1+ and above were judged
as positive. Buffaloes were considered positive for SCM, when at least one quarter
turned out to be positive for CMT.
Somatic cell count: It was done as described by Schalm
et al. (1971). Milk was mixed thoroughly before testing. Ten microliter
(One loopful) of milk from each quarter was spread over 1 cm2 marked
square area on a glass slide. The milk film was left undisturbed at room temperature
until it dried, then the smear was fixed in Xylol for 5 min and stained with
Loffers methylene blue reagent. Cell counting was made under oil immersion
as per the procedure described by Dhakal (2006).
Microbiological examination: The CMT and WST positive samples were held
in ice box until transported to the laboratory, where the samples were kept
at room temperature for 30 min before streaking on the culture plates. One hundred
microliter of each buffalo pooled milk from indirect test positive sample were
streaked on the Mac Conkeys and blood agar plates for bacterial culture
and isolation. Pure colonies were identified on the basis of colony characters,
Grams reaction, morphological findings and biochemical tests as described
by Barrow and Feltham (1993).
The bacteriological examination was reported to be most reliable method for diagnosis of subclinical mastitis. Hence an animal was considered positive for SCM irrespective of the number of quarters affected if the milk samples showed increased SCC along with bacteriological growth.
Statistical analysis: The data were subjected to Chi-square test of
analysis and completely randomized block design as per Snedecor
and Cochran (1994).
In the present study, total number of animals affected with SCM were 54 (26.21 percent) out of 206 buffaloes. Among the different breeds Graded Murrah (GM), graded Suruti (GS) and Non-descript (ND) showed 15.33, 5.83 and 4.85% positivity to SCM, respectively on SCC and bacteriological examination (Table 1). The breed wise analysis showed no significant difference in subclinical mastitis among different breeds.
The prevalence of SCM in different stages of lactation has been shown in Table
2. The prevalence of SCM was 17.44, 19.29 and 44.44% in early, mid and late
stages of lactation, respectively. Among the different breeds also the prevalence
was high during late stage of lactation.
|| Breed wise prevalence of subclinical mastitis in buffaloes
|1: Indicate number of animals positive for SCM, 2: Indicate
percent of positivity to total number of animals screened (i.e., 206)
|| Prevalence SCM in different stages of lactation
|*Number of animals positive and figures in parenthesis indicate
percent of positivity in different breeds
|| Prevalence of SCM in different parities of buffaloes
The Chi-square analysis of SCM at different stages of lactation revealed a
significant difference (p>0.05) among different stages of lactation with
regard to percentage of occurrence of mastitis. However, there is no significant
difference between different breeds studied.
The percentage of SCM prevalence in different parities has been shown in Table 3. The prevalence of SCM was observed to increase along with parity. The percent of prevalence being 17.94, 20.58, 23.07, 32.50, 30.55 and 38.89 in first, second, third, fourth, fifth and above sixth parity, respectively. The overall incidence was highest during above 6th lactation and it was found to increasing after 4th lactation. However, there is no significant difference among different parities.
Quarter (s) involvement in buffaloes showed that maximum number of positive animals (51.85%) was having infection in single quarter. The percentage involvement of two, three and four quarter (s) was 33.33, 9.26 and 5.56, respectively. The prevalence of SCM in LF, LH, RF and RH quarters were 12 (11.11%), 50 (46.30%), 06 (5.5%) and 40 (37.04%), respectively. There was higher prevalence in hind quarters and among which left one (46.30%) was found to be more susceptible. In case of fore quarters, left quarter (11.11%) was more susceptible.
The indirect tests were compared with SCC and bacteriological examination in order to know their efficacy. The percentage of agreement of indirect tests i.e., WST and CMT with SCC and bacteriological examination were 88.52 and 93.10 percent, respectively. Based on the above observation, SCC and bacteriologically positive samples were considered as SCM positive animals. Hence, in the present study, total number of animals affected with SCM were 54 (26.21 percent) out of 206 animals.
The mean values of milk SCC in different scores of CMT are given in Table
4. Out of 206 animals tested, 58 showed CMT positive reaction and the score
varied from Negative to 3+. The mean SCC in Negative, +, ++ and +++ were 5.25±0.002,
5.93±0.005, 6.56±0.002 and 7.02±0.000, respectively and
they differed highly significantly (p<0.01).
|| CMT and Mean SCCx105/mL of milk in SCC affected
|Means bearing different superscript differ significantly (p<0.01)
|| Microorganisms isolated from cases of SCM in buffaloes
In the negative samples, the SCC ranged from 0.72 to 2.80x105.
Two samples which showed positive reaction (+) in CMT revealed SCC with in the
negative range values (2.08 and 2.64x105).
The pooled milk samples of 58 and 61 buffaloes which showed positive reaction for SCM by CMT and WST respectively were tested bacteriologically, of which 54 revealed different bacterial infections (Table 5). Among them 47 (87.04%) showed monobacterial infection and 7 (12.96%) revealed mixed bacterial infection. The major pathogen isolated from milk samples were Staphylococcus sp. 25 (46.30 %) followed by Streptococcus sp. 11 (20.37%), E.coli 6 (11.11%), Bacillus sp. 3 (5.56%) and Klebsiella sp. 2 (3.70%) in pure culture. Staphylococcus sp. and Streptococcus sp. were also the chief etiological agents associated with mixed infections. In this study 73.79% of the samples did not yield any bacteria.
Infections of mammary gland by pathogenic bacteria result in decreased milk
production and compositional changes that vary with the intensity and duration
of the infection. Subclinical mastitis are those for which no visible changes
occur in the appearance of milk or the udder but milk production decreases,
bacteria are present in the secretion and composition is altered (Harmon,
1993). It is very difficult to detect SCM in the udder without the help
of laboratory diagnostics. Among the various tests, isolation and identification
of pathogenic organisms from milk is most reliable and direct indicator for
diagnosis of SCM, but it is cumbersome and expensive for regular testing. Hence
indirect tests such as WST and CMT were employed to screen this form of mastitis
and the results were compared with bacteriological examination.
In the present study, total number animals affected with SCM were 54 (26.21%)
out of 206 buffaloes. Sigh and Baxi (1980) and Dhakal
(2006) were reported 23.86 and 21.7% of SCM respectively which might be
comparable with the present findings.
Among the different breeds, graded Murrah (15.53%) showed higher incidence.
The significant difference between the different breeds may be due to their
milk production potentials. Radostits et al. (2007)
stated that high yielding animals are more susceptible to mastitis than low
The prevalence of SCM was higher in later stage of lactation (44.44%). The
finding of the present study was contrary to earlier observation (Palanivel
et al., 2005), in which higher incidence during early (70.80%) and
low in late (36.8%) stage of lactation were reported. Comparison of the available
information on the incidence of clinical and subclinical infections of udder
indicates that incidence of clinical mastitis may be more during early and mid
lactation (Bendixen et al., 1988). The incidence
of SCM may show the opposite trend (Trisanram et al.,
Comparison of the incidence of SCM in different parity groups showed in general
the incidence of infection increased as the lactation number increased. The
incidence was minimum during first (17.94%) and maximum during above 6th (38.89%)
lactation which was in agreement with Palanivel et al.
(2005), who found that the risk of SCM increases significantly with advancing
the age of animals which approximate to the parity number. The most likely cause
of the higher prevalence of the SCM in multiparous animals might be due to prolonged
period of risk.
In most of the animals it was observed that only one quarter (51.85%) was affected
which was in agreement with Saini et al. (1994).
The possible reason could be that the pathogenic organisms might not have entered
all the quarters at the same time. The predisposing factors like injury, defective
sphincter and prevalence of pathogens and immune status of different quarters
also play a role in the development of the disease (Saini
et al., 1994).
Quarter wise presence of SCM was found to be higher in hind quarters (83.34%)
than fore quarters (16.36%). Naiknaware et al. (1998)
and Chisty et al. (2007) also observed higher
rate of hind quarter affections. The higher prevalence in hind quarters may
be due to their frequent exposure to dung and urine, while milking they are
pulled forward and sideways which may lead to undue stress on them, the left
side quarter get first chance of contamination by milkers, being milking approach
side in buffaloes and comparatively more milk yield from hind quarters increases
their susceptibility to mastitis.
The bacteriological test was reported to be most reliable method for diagnosis
of mastitis. Keeping this in view the indirect tests were compared with them
in order to know their efficacy. The percent agreements of WST and CMT with
SCC and bacteriological examination were 88.52 and 93.10%, respectively. The
present finding was close agreement with Saravanan et
al. (2008). This indicates that any one of the test can be safely resorted
to detect the incidence of SCM in buffaloes under field condition.
In the present study, SCC increased with the increasing severity of SCM as
determined by CMT. The SCC in SCM cases were above 3,28,000 with an average
of 10.04x105/mL of milk. Our results are in conformity with Gosh
et al. (2004), who also recorded higher SCC in SCM buffaloes compared
to normal non infected buffaloes. Increase in SCC in SCM might be due to increased
number of leukocytes and epithelial cells in milk. The great variation in SCC
in SCM (3.28 to 22.80x105/mL) might be due to management, breed,
age, stage and parity of lactation and infectious status (Harmon,
Staphylococcal (46.30%) and Streptococcal sp. (20.37%) were predominant
subclinical mastitis pathogens in buffaloes in the present study. These findings
were in close agreement with earlier workers (Palanivel
et al., 2005; Piepers et al., 2007).
The higher incidence of Staphylococcal infection might be due to their ubiquitous
nature and its well adaptation to survive in the udder and establish a mild
subclinical infection of long duration from which it is shed in milk, facilitate
transmission to healthy animals during milking procedure and the emergence of
drug resistant strains which destroy penicillin through the production of enzyme
pencillinase. Moreover, these organisms survive better in the environment and
are widely distributed at different body sites of lactating animal leads to
easy infection. Of the 54 buffaloes infected with SCM, 47 (87.03) infected with
monobacterial and 7 (12.97%) with concurrent bacterial infection which confirming
the earlier results of Naiknaware et al. (1998).
In conclusion, SCM was observed in a considerable percentage of buffaloes in
Namakkal area of Tamil Nadu. Hence, a study on the risk factors associated with
this problem will help in control and reduce the possible economic losses.
The authors are thankful to the Dean, Veterinary College and Research Institute, Namakkal, Tamil Nadu Veterinary and Animal Sciences University (TANUVAS) for providing necessary facilities to carry out the work.