Int. J. BioRes. 1(2): 23-28 February, 2010 Rahman et al.

Full Length Research Paper

PREVALENCE OF SUBCLINICAL MASTITIS IN DAIRY COWS REARED IN SYLHET

DISTRICT OF BANGLADESH
M M Rahman1, M R Islam1, *M B Uddin1, M Aktaruzzaman2
1
Department of Medicine and Surgery, 2Department of Physiology and Pharmacology, Faculty of Veterinary and Animal
Science, Sylhet Agricultural University, Sylhet – 3100, Bangladesh
*Correspondence: bashir_vet@yahoo.com
Accepted on 27 February 2010

ABSTRACT

The study was carried out to estimate the prevalence of sub clinical mastitis (SCM) in dairy cows kept by Sylhet Govt.
Dairy Farm (SGDF) and different local dairy farms at Sylhet (LFS) of Bangladesh. Sub clinical mastitis was assessed using
the modified white side test (MWST) and revealed negative, trace (+), distinct (++), strong (+++), and doubtful (±) etc.
reactions according to the severity of mastitis. Bacteriological examination of milk samples of 158 milch cows (632
quarters) revealed that 81 cows (51.3%) had suffering from SCM with different bacterial infection, of which 27 (17.1%)
cows had mono-bacterial infection and 17 (10.8%) cows had mixed bacterial infections. Statistical analysis of the result of
single and mixed bacterial infection in the milk of apparently healthy milch cows revealed that the single infection was
significantly (P < 0.05) higher than mixed bacterial infection. The 17 (10.8%) cows had mixed infection, of which 4 (3.13%
had Staphylococcus spp. + Streptococcus spp., 7 (7.5%) had E. coli + Staphylococcus spp. 6 (3.8%) had Staphylococcus
spp. + Bacillus spp. and no one had Corynebacterium spp. + Staphylococcal infection of the 158 milch cows examined, 33
(20.9%) had Staphylococcal infection was recorded in cows of SGDF (30.0%) than LFS (18.8%), Sylhet. The overall
prevalence of SCM was found to be 51.3%, of which 43.3% at the SGDF, Sylhet and 53.1% at the LFS of the 81 bacterial
isolates, 27 (17.1%) had mono bacterial infection and 17 (10.8%) had mixed bacterial infection.

Key words: SCM, Prevalence, SGDF, LFS, Sylhet

INTRODUCTION

Bovine mastitis is a single most common disease syndrome in adult dairy cows, accounting for about 38% of morbidity
(Smith, 1996). Mastitis is also associated with number of zoonotic diseases in which milk acts as a vehicle of infection
(Jenkins, 1982). Recently, about 20,582 mini dairy farms each with five or more high yielding cross-bred cows have been
established in private sector in Bangladesh (Faruq, 1996) and most of these dairy farms are confronted with problems of
clinical and subclinical mastitis (Rahman et al., 1997). The clinical mastitis can be diagnosed on history and clinical findings
but laboratory examinations are required to ascertain the subclinical mastitis. The prevalence of SCM has been shown to be 15
to 40 times more than the clinical mastitis (Philpot, 1984). The prevalence of bovine mastitis is higher in farms with larger
herd sizes than in those with lower herd sizes (Radostits et al., 2000). The prevalence is also higher in cows with lesions and
tick infestations on the skin, udders and teats than cows without these factors, in early lactation stage than in the mid-lactation
stage (Biffa et al., 2005).
Epidemiological studies on mastitis revealed that mastitogenic organisms are widespread on different body sites of the cows,
milker’s hands, milking cans and in the milk samples. Teat apices have been reported to be the most common site (Itagaki et
al, 1999), from where these organisms have been isolated (Malhotra and Kapur, 1982; Prabhakar et al., 1990). The mastitis
causing organism, Staphylococci, the chief udder pathogen, has been isolated from almost all the body site examined and
environment but Streptococci from fewer body sites, whereas the prevalence of Escherichia coli has been reported to be
widespread (Probhakar et al., 1990; Joshi et al., 1998).
The prevalence of subclinical mastitis in milch cow have been reported to be 16.5% with Whiteside Test (WST) and 15.8%
with California Mastitis Test (CMT) from Baghabarighat, Sirajgonj district by Prodhan et al. (1996). Staphylococci are the
major etiological agents, followed by Streptococci and E. coli causing subclinical mastitis in cows in India (Singh and Baxi,
1982) but only Staphylococci have been isolated from subclinical mastitis cases of cows from Bangladesh (Rahman et al.,
1968). However, Mahbub-E-Elahi et al. (1996) isolated and identified Staphylococci (49.3%), Streptococci (14.0%),
Corynebacterium (8.0%), E. coli (6.0%), Bacillus sp. (4.7%) and 18% unidentified bacteria from clinically affected mastitis
cows of Manikgonj Milkshed area, Bangladesh Agricultural University Dairy Farm (BAUDF) and BAU Veterinary Clinic.
Very limited research works on mastitis have been carried out in Bangladesh (Chowdhury et al., 1967; Choudhury and Ali,
1975; Mahbub-E-Elahi et al., 1996; Sen et al., 1996; Rahman et al., 1997). Considering the potential of the area and the
economic significance of dairy production to the local community there have been repeated efforts by governmental and non-

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governmental aid organizations to improve the dairy productivity Conversely, bovine mastitis was reported to be one of the
most prevalent dairy health problems in most parts of Bangladesh where dairy activities are practiced. Yet, the information on
the prevalence of sub clinical mastitis in the area is lacking and what available is fragments of information from cases of
clinical mastitis that has been presented to veterinary clinic for the treatment. This investigation was proposed to determine
the prevalence of sub clinical mastitis and to isolate the dominant bacteria from those cases in the study area.

MATERIALS AND METHODS

Selection of study animals and site and duration

In this study, a total of 158 dairy cows were sampled during the period from April 2008 to May 2009 from Sylhet Govt. Dairy
Farm (SGDF) and different local dairy farms (LDF) at Sylhet district of Bangladesh.
Questionnaires
A structured questionnaire was developed and clinically selected mastitis suspected patients owners or farmers were asked and
the pre-tested questionnaire was filled. Each asking time about 10–15 min. The questionnaires contained information
regarding Sex, age, breed, health and vaccination status and management. Open-ended questions were collected and recorded.
Sample collection
A total number of thirty (30) apparently healthy milch cows of the SGDF and hundred twenty eight (128) milch cows of LFS
were randomly selected for this study. Before taking the milk sample, a teatful of milk was milked into a tube and later
discarded. This was to flush out any bacteria that might be present in the teat canal. The teat and teat tips were disinfected
with a swab soaked in 70% alcohol. Milk samples were collected aseptically from the udder at the time of morning milking.
All the milk collected vials were labeled with the identification number of cow (and others symbol).
Detection of sub clinical mastitis
Immediately after collection the samples were subjected to Modified Whiteside test (MWST) described by Dunn et al., (1942)
and Murphy et al., (1941). Briefly, in the original technique, 10 ml of milk are mixed with 2 ml of 4% sodium hydroxide
solution in a test tube, while in the modified technique 5 drops of milk are mixed with two drops of sodium hydroxide solution
on a microscope slide. If the milk is normal, and has homogenous turbidity develops within 20-30 seconds then it is detected
as negative .If, milk is flakes form and mixer become stringy then is detected as positive.
Grading of sub clinical mastitis milk
In this study, the results of modified Whiteside test were graded (Fig. 1) as strong (+++), distinct (++), trace (+), doubtful (±)
and negative (normal) on the basis of two factors such as (1) amount and degree of distinction of viscous mass (gel)
formation, of fiber like material or flakes or shred formation and entirely free of precipitant; (2) time of reaction whether it is
immediate, moderate or slow reaction or without any reaction.
Pre-incubated and incubated milk samples
Each pre-incubated milk sample and incubated (at 370 C for 24 hours) milk sample was stained with Gram’s Method and was
examined under microscope as described by Samad (1996). The findings were recorded and then the samples which were
found positive for bacteria used for the isolation and identification of bacteria.
Bacterial culture
From all milk samples a standard of 0.01 ml of milk sediment was removed and cultured on Blood agar medium, Nutrient
agar medium, EMB agar, MacConkey’s agar, Salmonella Shigella agar etc. Bacterial growth was identified and recorded after
24 and 48 hour of incubation. Identification of bacteria was made on the basis of standard features and procedures (Carter and
Chengappa, 1991)
Data analysis
Percentages were used to express the prevalence and the proportion of the isolates of the different general of bacteria that
causing mastitis in the area.

RESULTS AND DISCUSSIONS

Prevalence of subclinical mastitis

Milk samples from 30 milch cows of SGDF (120 quarters), Sylhet and from 128 milch cows of different local dairy farms
(512 quarters), Sylhet were randomly collected aseptically and examined bacterilogically, of which 13 (43.3%) and 68

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Int. J. BioRes. 1(2): 23-28 February, 2010 Rahman et al.

(53.1%) showed positive SCM, respectively (Table 1). A significantly (P > 0.05) higher prevalence of SCM was recorded in
milch cows of LFS (53.1%), than SGDF (43.3%), Sylhet, (Table 1).
Table 1: Percent (%) prevalence of Sub Clinical Mastitis.
Sources Total No. of cases No of positive cases No of Negative cases % of SCM in Herd
Sylhet Govt. Dairy Farm 30 13 17 43.3
Local Farms in Sylhet 128 68 60 53.1
Total 158 81 77 51.3
Legends: %= percentage; SCM= Sub Clinical Mastitis
Milk samples from seventy seven (77) cows out of 158 were negative to modified Whiteside test, while milk samples of
eighty one (81) cows showed various degrees of reactions. Milk samples of forty six (46) cows showed +1(29.1%), milk
samples of nineteen (19) cows showed +2 (12.0%) and milk samples of thirteen (13) cows showed +3 (8.2%) reaction.
Besides this only milk samples from seven (7) cows exhibited doubtful (1.9%) reaction. The reaction of the milk samples
which varied according to the sources of the collected samples are presented in Table 1.
The total 51.3% of the cows were found positive to modified Whiteside test (Fig.1). These findings are in support with the
observation of Singh and Baxi (1982) who reported 54.0% incidence of SCM in cows on bacteriological examination.
Karimuribo et al. (2008) reported the percent prevalence subclinical mastitis were 75·9 per cent when assessed by the CMT
and 43·8 per cent when assessed by culture. Also higher incidence of SCM (75.3%) has been reported from India by Devi et
al. (1997). Argaw and Tolosa (2008) reported the prevalence of sub clinical mastitis 89.54 % in cow and 63.1 % quarters were
recorded and the resulting quarter infection rate was 56.70 % in Ethiopia which is higher than others. Comparison among
other areas of Bangladesh, higher rate of bacterial infection was recorded in milk samples of the Local Farms in Sylhet
(53.1%) than Sylhet Govt. Dairy Farm (43.3%), Sylhet.
Rate of Sub clinical Mastitis
48.73

29.11 +(Trace)

++(Distinct)
77
+++(Strong)
12.03
46
(Doubtful)
1.90
8.23 -(Negative)
19 03
13

No of samples reacted Percentage

Figure 1: Rate of sub clinical mastitis

However, wide variation in the incidence of SCM has been reported since beginning which is largely due to changing
managemental conditions, and use of different diagnostic tests used. In Bangladesh, Prodhan et al. (1996) reported 16.5% and
15.8% incidence of SCM with Whiteside test (WST) and California Mastitis Test (CMT), respectively. Sen et al. (1996)
reported 14.4% and 11.5% incidence of SCM with CMT and WST, respectively. Nooruddin et al. (1997) and Rahman et al.
(1997) reported 21.2% and 18.5% incidence of SCM, respectively by using modified WST. In India, Bhatnagar and Melhotra
(1969) reported 10 to 24%, Misra et al. (1973) reported 25%, Singh and Baxi (1982) reported 17%, Pal et al. (1989) reported
23.1% and Parai et al. (1992) reported 19.3% and Dhote et al. (1999) reported 20.7% incidence of SCM with indirect tests but
higher incidence of SCM have also been reported by Verma (1978) who reported 36.1 to 61%, Singh and Baxi (1980)
reported 54.0%, Motice et al. (1985) reported 54 to 81.5% and Ramachandraih et al. (1990) reported 63.7 Jha et al. (1993)
reported 18.8% SCM in cows from Nepal.
Causative organisms of SCM
Bacteriological examination of milk samples of 158 milch cows (632 quarters) revealed that 81 cows (51.3%) had suffering
from SCM with different bacterial infection, of which 27 (17.1%) cows had mono-bacterial infection and 17 (10.8%) cows
had mixed bacterial infections (Table 2). However, Shike et al. (1998) found 7 (31.8%) pure and 15 (68.2%) mixed cultures
from the SCM and clinical mastitis in cross-bred cows. Statistical analysis of the result of single and mixed bacterial infection
in the milk of apparently healthy milch cows revealed that the single infection was significantly (P < 0.05) higher than mixed
bacterial infection (Table 2). Of the 27 mono-bacterial isolates, of which 37 (31.0%) isolates were Staphylococci, 17 (11.3%)
isolates were Escherichia coli 6 (4.7%) isolated were Bacillus spp. and 4 (3.1%) were Streptococcus spp. (Table 2). The 17

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(10.8%) cows had mixed infection, of which 4 (3.13%) had Staphylococcus spp. + Streptococcus spp., 7 (7.5%) had E. coli +
Staphylococcus spp. 6 (3.8%) had Staphylococcus spp. + Bacillus spp. and no one had Corynebacterium spp. +
Staphylococcal infection (Table 2). Of the 158 milch cows examined, 33 (20.9%) had Staphylococcal infection was recorded
in cows of SGDF (30.0%), than LFS (18.8%), Sylhet, (Table 2). Of the 68 positive cases of SCM of the LFS, Sylhet, 24
(18.7%) cows had Staphylococcus spp. 17 (11.3%) cows had E. coli, 6 (4.7%) cows had Bacillus spp., 4 (3.1%) cows had
Streptococcus spp., 4 (3.1%) cows had Staphylococcus spp. + Streptococcus spp., 7 (7.5%) cows had E. coli + Staphylococcus
spp. (Table 2), whereas of the 13 positive cases of SCM of the SGDF, Sylhet of which 09 (30.0%) cows had Staphylococcus
spp. and 04 (13.3%) cows had mixed infection with Staphylococcus spp. + Bacillus spp. (Table 2).
Table 2. Prevalence and effects of sub clinical mastitis on milk production in dairy cows
Sl Organisms isolated Farms No. of Positive Mean milk yield (L/cow/day)
No. cows No. % Pre-testing Record at Difference
tested highest records testing time
1. Stapylococcus spp. SGDF 30 09 30.0 4.5 4.1 -0.4
LFS 128 24 18.8 10.8 9.8 -1.0
Single 158 33 20.9 7.6 7.0 -0.7
2. Escherichia coli SGDF 30 00 00 00.0 00.0 -2.9
LFS 128 17 11.3 12.6 9.6
3. Bacillus spp. SGDF 30 00 00 00.0 00.0 -1.2
LAS 128 06 4.7 09.3 08.1
4. Streptococcus spp. SGDF 30 00 00 00.0 00.0 -1.7
LFS 128 04 3.1 09.7 08.7
Single 158 27 17.1 10.5 8.8 -1.7
5. Staph. + Strep. SGDF 30 00 00 00.0 00.0 -0.7
LFS 128 04 3.1 09.7 09.0
6. E. coli + Staph SGDF 30 00 00 00.0 00.0 -0.7
LFS 128 07 7.5 08.0 07.4
7. Staph + Bacillus SGDF 30 04 13.3 5.5 4.4 -1.1
LFS 128 02 01.6 8.8 7.5 -1.3
Mixed 158 17 10.8 8.0 7.0 -0.9
Total SGDF 30 13 43.3 5.0 4.2 -0.8
LFS 128 68 53.1 9.9 8.5 -1.3
Overall 158 81 51.3 7.4 6.4 -1.1
SGDF = Sylhet Govt. Dairy Farm; LFS = Local Farms of Sylhet; Cory = Corynebacterium.
The percentage of the single causative organism Staphylococci (20.9%) was significantly higher, followed in order by E. coli
(11.3%), Bacillus sp. (4.69%), Streptococcus sp. (3.1%) in pure culture. These findings support the report of Mahbub-E-Elahi
et al. (1996) who isolated and identified Staphylococcus aureus (31.33%), Staph. epidermidis (18.0%), Streptococcus sp.
(14.0%), Corynebacterium pyogenes (8.0%), E. coli (6.0%), Bacillus sp. (4.7%), and 18.0% unidentified bacteria from the
cases of bovine mastitis. However, Rahman et al. (1968) isolated and identified the distribution of different strains of
Staphylococci from mastitic and apparently normal bovine mammary gland. These findings also correspond well with the
Indian reports made by Sen et al. (1981), Singh and Baxi (1982), Shike et al. (1998) and Umakanthan (1998) who reported
highest rate of Staphylococci, followed in order by E. coli, Streptococci and Corynebacteria. Misra et al. (1973), Rahman and
Boro (1990), Gupta et al. (1992) and Jha et al. (1993), Shike et al. (1998) and Chanda et al. (1998) who reported
Staphylococci as the chief pathogens of mastitis. The increased incidence of E. coli (11.1%) recorded in this study in
comparison to the earlier finding (6.0%) of Mahbub-E-Elahi et al. (1996) may be attributed to the indiscriminate use of
penicillin which might have led to the elimination of gram positive organisms in certain cases.

CONCLUSIONS

The study was carried out to find out the prevalence of subclinical mastitis (SCM) in the cows of the Sylhet region,
Bangladesh. Milk samples of 158 milch cows were examined bacterilogically, of which 81 (51.3%) had bacterial infection.
The overall prevalence of SCM was found to be 51.3%, of which 43.3% at the SGDF, Sylhet and 53.1% at the LFS of the 81
bacterial isolates, 27 (17.1%) had mono bacterial infection and 17 (10.8%) had mixed bacterial infection.
The present study showed high prevalence of sub clinical mastitis in cow in the study area. The isolated mastitis causing
pathogens were many of which Staphylococcus, Streptococcus, Escherichia coli, and Bacillus are the major one. Further
studies are required to identify risk factors for sub clinical mastitis and antibiotic sensitivity tests as this would help to devise
treatment and control of sub clinical mastitis in dairy cow in the area.
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