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International Journal of Scientific Research and Innovative Technology ISSN: 2313-3759 Vol. 3 No. 6; June 2016

137

Prevalence of Bovine Subclinical Mastitis and its Association with Bacteria

and Risk Factors in Milking Cows of Batticaloa District in Sri Lanka

Sanotharan, N1., Pagthinathan, M.

1*. and Nafees, M.S.M

1

1 Department of Animal Science, Faculty of Agriculture, Eastern University, Sri Lanka, Vantharumoolai, Sri

Lanka

Abstract

The present study was carried out to investigate prevalence of subclinical mastitis (SCM) in milking cows and

risk factors associated with sub-clinical mastitis. A total of 152 lactating cows were randomly selected to

identify the SCM from 15 veterinary ranges of Batticaloa District and cow information was collected from the

farmers’ thought interview. Milk samples were collected aseptically from California mastitis test (CMT)

positive cows and dispatched to laboratory for microbiological and biochemical analysis. Result showed that

66 lactating cows (43%) and 116 (19.1%) quarters were positive to CMT. While, 93.9% of CMT positive

quarters showed a bacterial growth after the culturing. Among the pathogens isolated Staphylococcus spp.

(90.5%) was most frequent bacteria positive for SCM. The prevalence of SCM was (p < 0.05) significantly

high in European crossbreds (60.7%) compared to sahiwal (55.5%) and local cattle (0%). The highest

prevalence found in the age group of more than 8 years and the highest prevalence in parity more than 5 year

was 75%. The prevalence was significantly (p < 0.05) higher in cows with late lactation (71.1%) and the

higher prevalence was found in the daily milk yield of 3-5 Lit. The prevalence of SCM was comparatively

higher in intensive system (66.7%) and close housing system (58.6%) other system adopted respectively.

Keywords: California Mastitis Test, Prevalence, Subclinical mastitis, Bacteria, lactating cows

1. Introduction

Livestock sector is an important part in the Agriculture sector of Sri Lanka. Livestock sector contribute 0.8 %

to total GDP (Central Bank Report, 2014). Livestock sector’s contribution towards the GDP is higher in 2014

than the previous years; it might due to increase in milk production and egg production in Sri Lanka (Central

Bank Report, 2014). Therefore, milk production has played an important role in livestock production of the

country. At present, milking cows’ population and average milk production are 299,440 and 716,790 liters per

day, respectively (Central Bank Report, 2014). The dry zone of Sri Lanka contains large amount of cattle

population which are main source of income to cattle farmers (Gamage, 2009). Batticaloa District located in

agro ecological zone of low country dry zone of Sri Lanka which has total cattle and milking cow population

are 75,570 and 24,350, respectively (Central Bank Report, 2014) and daily cow milk production is recorded as


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31,140 liters (Central Bank Report, 2014). However, dairy farmers are facing many challenges to improve the

dairy production in this district even in Sri Lanka. One of the main challenges is a disease caused by infectious

and non-infectious agents. Mastitis is one of major disease in milking cow which can drastically change the

milk production and its quality in cow. In Sri Lanka, mastitis has a significant role in reduction in milk

production and its lost has been estimated as 4.3 million Sri Lankan rupees per year (Chandrasri, 2009).

In subclinical mastitis, there are no obvious clinical signs such as abnormal milk, udder swelling or

tenderness, or systemic signs such as fever, depression. Instead there is an in-crease in somatic cell counts of

the milk (Kayesh et al., 2014; Radostis et al. 2007). Subclinical mastitis causes more than three times losses as

compared to clinical mastitis (Kayesh et al., 2014; Singh and Singh 1994). The subclinical mastitis is more

serious and causes much greater loss to the dairy industry (Abrahmsen et al., 2014)

Cows with subclinical mastitis maintain a reservoir of infection within the dairy herd and increase the

potential exposure of uninfected cows to contagious pathogens. The causative organisms of mastitis are

categorized as contagious pathogens including Staphylococcus aureus, Streptococcus agalactiae, and

Mycoplasma bovis or as environmental pathogens such as streptococci (e.g., Streptococcus dysagalactiae and

Streptococcus uberis), and the enterobacteriaceae (Shahid et al., 2011). California Mastitis Test (CMT) has

been recognized as a highly sensitive test to detect bovine subclinical mastitis (Kayesh et al., 2014; Madut et

al. 2009). In Sri Lanka, total of 11,264 mastitis cases have been recorded in government veterinary offices.

Among these, 2,095 cases have been recorded in Eastern province (Department of animal production and

health, 2014). So, it is important to study the prevalence of subclinical mastitis in Batticaloa District.

Therefore, the study was undertaken to determine the prevalence of subclinical mastitis in lactating cows and

to isolate and identify the bacterial agents and risk factors associated with sub-clinical mastitis in the study

area.

2. Materials and Methods

2.1.Study area

This study was conducted in Batticaloa District, Eastern Province of Sri Lanka from March 2015 to December

2015 which located at 7°43ʹN latitude 81°45ʹE longitude and the elevation is 7.65 m above sea level. Land

area of Batticaloa District is 2,854 km2 and it comes under agro ecological zone of low country dry zone.

Mean temperature and annual rainfall and relative humidity were 28.4°C, 1,973.7 mm and 89%, respectively.

2.2. Sample size and sampling method

In this study, 152 milking cows were randomly selected from the 15 veterinary ranges in Batticaloa district.

Pre-tested questionnaire was used to collect the information on farmers to find out the associated factors, in

relation to subclinical mastitis in this area.

2.3. Information from farmers

Data were collected from farmers regarding management aspects such as herd sizes, housing systems, rearing

system, feeding system, milking system and frequency milking. Cow parameters such as breed, age, parity,

stage of lactation, infected quarters, milk production and health condition were collected from the farmers and

records

2.4. Detection of subclinical mastitis

The CMT test was conducted using score from 1 to 5 according to the Scandinavian scoring system, where 1

is negative result (no gel formation), 2 is traceable (possible infection), and 3 or above indicates a positive


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result, where 4 and 5 has the most gel formation. All quarters with CMT ≥3 were milk sampled during milking

for further bacterial examination (Klastrup et al., 1975).

2.5. Aseptic milk sample collection

Milk samples were collected from 152 milking animal. Just before collect the milk, udder and milker’s hand

were washed with clean water and teats were swabbed with cotton by using 75% alcohol. Then, milk samples

were collected aseptically from the teats at the time of milking into sterilized sample bottles after discarding

the first 3 milking streams. After collection, the milk samples were labeled and immediately transported in an

ice-cooled box and later transferred into a fridge at 4 °C. Milk samples were processed within 24 h after the

collection as described by Biru (1989).

2.6. Isolation and identification of bacterial agents

All laboratory works were performed at Department of Animal Science laboratory. Milk samples were

examined for the presence of bacteria according to the procedure described by Quinn et al. (1999). According

to Kayesh et al. (2014), Abrahmsen et al. (2014), Doherr et al. (2007) and (Mekibib et al. (2010), quarters

with a positive CMT (≥ 3) reaction were considered as subclinically inflamed and accordingly milk samples of

these quarters were subjected to bacteriological analysis as follows: After thorough mixing, 10 µl aliquots

were taken from each milk sample and were streaked on 7% sheep blood agar, MacConkey agar and nutrient

agar. Following that, these media were incubated under aerobic conditions at 37°C and examined for bacterial

growth after 16 to 48 hours of incubation. The bacterial isolates were identified by their cultural,

morphological and biochemical characters. Identification of the bacterial agents from the pure culture were

done based on their colony characteristics, Gram staining reaction, hemolysis pattern and biochemical test as

described by Kayesh et al. (2014), Castaneda et al. (2013) and Cheesbrough (1985).

2.7. Data analysis

Analyses of the frequency of positive cows (at least one quarter with CMT ≥3) were first performed.

Prevalence was calculated as the number of positive cases of subclinical mastitis out of 152 cows tested. The

association between the prevalence of subclinical mastitis and risk factors such as farming system, housing

system, milking place, hygiene practices, age, breed, parity, milking with calf suckling, stage of lactation, and

daily milk production with the CMT result was statistically analyzed in logistic regression model using

statistics package for social science (SPSS version 20.0) software. All the risk factors were explained in odds

ratio (OR) value which mean an odds ratio is a measure of association between an exposure and an outcome. P

values less than 0.05 were considered the level of significance of the result.

3. Results and Discussions

3.1. Prevalence of bovine subclinical mastitis

The study was interpreting by California Mastitis Test to identify the subclinical mastitis. The present study

was conducted using a total of 152 milking cows, out of 66 (43.4%) lactating cows were positive to California

Mastitis Test (CMT) Table 1. A total of 116 (19.1%) quarters from 608 quarters were positive to CMT. There

was a variation of CMT score even in different quarters of individual lactating cow. Among the 66 CMT

positive lactating cows, 62 (93.9%) of cows showed bacterial growth after the culturing of milk sample in the

blood agar plate.

3.2. Isolation and identification of bacterial species

Milk samples obtained from 116 CMT positive quarters were examined for the isolation and identification of

bacterial agents. Out of 116 milk samples, only three bacterial agents were isolated namely; Staphylococcus


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spp (90.5%), Escherichia coli (6.0%) and Streptococcus spp (3.5%). Staphylococcus aureus spp is contagious

bacterial agents and it could live on skin of the teats and in human skin (Wattiaux, 2011). The finding of this

study is closely similar with the findings of Kayesh et al. (2014) who reported 73.3% prevalence of

Staphylococcus spp. in the both clinical and subclinical mastitis which was followed by Streptococcus spp.

(33.33%) and Escherichia coli (6.67%). The finding of this study is also closely similar with the findings of

Deressa et al. (2013), Bitew et al. (2010) and Sharma et al. (2010) who reported 72.2% prevalence of

Staphylococcus spp. in the both mastitis.

3.3. Factors associated with subclinical mastitis detected by CMT

Out of the eleven variables, only ten variables significantly (P< 0.05) associated with CMT positive in binary

logistic regression analysis. Prevalence was relatively higher in European crosses as explained in odds ratio

(OR) value 21.34 based on the local breeds as a reference (Table 2). Further, risk factors such as age, parity,

stage of lactation, farming system, housing system, calf sucking after milking, milking place, hygiene,

isolation of infected cows and milk production were analyzed for relationship with subclinical mastitis

detected by CMT test (Table 2).

3.3. Breed of cow

Among the 84 European crosses, 51 (60.7%) were positive to CMT and among the 27 Sahiwal breeds, 15

(55.5%) cows were positive to CMT. None of the local breeds showed the CMT positive (Figure 1). A total of

94 quarters (28%) of European cross were CMT positive. Similarly, 22 quarters (20%) of Sahiwal breeds were

positive to CMT. European crosses and Sahiwal breeds had more chances for CMT positive than local breeds

in terms of OR value. The OR value for Sahiwal and European cross breed were 9.4 and 21.3 times more than

local breeds (Table 1). This is could be explained as European cross breeds are high yielding breeds and they

are more susceptible to mastitis diseases and anatomical structure also favors to cause the SCM in European

breeds. But, local breed which has more resistance to mastitis and they are low milk producer than cross breed

cows (Alemu et al., 2013; Sudhan et al., 2010).

3.4. Age of animal

Prevalence between age and CMT result for European cross breeds is shown in Figure 2. The prevalence of

different age groups was recorded ranges of bellow 5 years (45%), 5-8 years (62.5%) and above 8 years

(75%). In Sahiwal breeds, the prevalence of CMT positive in different age groups was found to be bellow

5years (50%), 5-8years (53.8%) and above 8years (66.7%). The highest prevalence of CMT positive in both

breeds were found in the age group of above 8 years and the lower prevalence were found in the age group of

below 5 years. The age group 5 – 8 years and above 8 years cows had more chances for CMT positive

in term of OR value, which expressed for 5 – 8 years and above 8 years cows were 2.9 and 4.5 times more

than below 5 years cows (Table 4.2). This might be higher chances for SCM infection in older animal than

younger and its teat canal more dilated and partially or permanently opened. Therefore, high chances for

entering of environmental and skin pathogens into teat canal (Shittu et al., 2012). The increase in subclinical

mastitis with age is consistent with other studies (Kader et al. 2003, Ghosh et al., 2004).

3.5. Parity

The prevalence of CMT positive showed that increasing tendency with the increase in number of parity

(Figure 3). The highest prevalence of CMT positive was observed in >5 parity number (75%), whereas in 1-2

and 3-5 parity the prevalence was 28.4 % and 53.3%, respectively. Among 3-5 and above 5 parity number of

cows had more chances for CMT positive in terms of OR value compare to below 2 parity number of cows

(Table 2). The lactation stage was categorized into early stage, mid stage and late stage based on lactation


141

period. Result is shown in Figure 4 that, the highest prevalence of CMT positive was recorded in a later stage

of lactation (71.1%) and the lowest prevalence was found in early stage of lactation (27.7%). Mid and late

stage of lactation of cows had more chances for CMT positive in terms of OR value, which was expressed for

mid and late stage of lactation of cows were 1.4 and 6.4 times, respectively compare to early stage of lactation

of cows (Table 2). The results are not in conformity with Kayesh et al. (2014) who recorded lower prevalence

of subclinical mastitis in stages of lactation above five months. However, Kayesh et al. (2014) and Rahman et

al. (1997) reported higher prevalence (34.00%) of subclinical mastitis during the mid of lactation

3.6. Dairy milk production

In European cross breeds, prevalence of CMT positive was recorded based on daily milk yield. Prevalence of

CMT positive for the daily milk production of 0-3 Lit, 3-6 Lit, 6-9 Lit and 9-12 Lit, were 60%, 72.5%, 45.4%

and 16.7%, respectively. In Sahiwal breeds, prevalence of CMT positive for the daily milk production for 0-3

Lit, 3-6 Lit, 6-9 Lit and 9-12 Lit were 50%, 70.5%, 25% and 0%, respectively (Figure 5). Milk yield of 0-3

Lit, 3-6 Lit and 6-9 Lit had more chances for CMT positive in terms of OR value for milk yield compared to

9-12 Lit cow with daily milk (Table 2).

3.7. Farming system

Prevalence of CMT positive recorded in intensive system (66.7%), semi intensive system (44.3%) and

extensive system (16.3%). High prevalence was observed in the intensive system and the lowest prevalence

was found in the extensive system (Figure 6). Semi intensive and intensive had more chances for CMT

positive in terms of OR value for semi intensive and intensive system were 4.1 and 10.3 times more than

extensive farming system (Table 2). These differences of prevalence rates of subclinical mastitis might be due

to difference of breeds of animals, management practices and the tests used for screening of the milk samples.

3.8. Housing system

The result revealed in Figure 7 that cows in closed housing system had higher prevalence (58.6%) of CMT

positive than, housing night only (45%) and open housing system (13.3%). Closed housing and housing night

only had more chances for CMT positive in terms of OR value compared to open housing system (Table 2).

Closed housing system increases the risk of mastitis because the confinement of the animals and contagious of

microorganisms in the various litters chance to form the mastitis (Sudhan et al., 2010).

3.9. Calf suckling practices

Highest prevalence of CMT positive (89.7%) was found in calf not suckling after milking and the lowest

prevalence of CMT positive (32.5%) was found in calf suckling after milking. Calf not suckling after milking

had more chances for CMT positive in terms of OR value than calf suckling after milking (Table 2). If calf not

suckling after milking, higher chances for pathogens development due to the residual milk could be remained

in teat canal to facilitating the development of mastitis (Sedano et al., 2010).

3.10. Isolation of infected cows and hygiene practices

The highest prevalence of CMT positive (80%) was recorded in rearing of infected cows with other animals

with same shed. Lowest prevalence (36.2%) was recorded in rearing of infected cows with isolated shed.

Rearing of all animals together with infected milking cows had more chances (7.0 times) for CMT positive in

terms of OR value compared to isolated shed for infected cows (Table 2). The pathogens present in animal can

be easily spread into herd to other animals, it might be a reason for higher prevalence (Duval, 1995). The

highest prevalence of CMT positive was recorded in poor hygiene (75.4%) and lowest was recorded in good

hygiene (19.5%). Poor hygienic practices had more chances (12.6 times) for CMT positive in term of OR


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value than good hygienic practices (Table 2). In general, poor hygienic practices directly influence to any

infection. This might leads to higher prevalence of mastitis in poor hygienic cow (Shittu et al.,2012).

3.11. Estimation of economic losses from SCM in Batticaloa District

In lactating cows, average milk yield/ quarter/ day of European cross and Sahiwal breeds was 1.62 and 1.46

Lit, respectively. The average lactation period of cow is 240 days. Economic losses were estimated for CMT

positive infected cows as described by Biru et al. (2013). Table 3 shows that milk yield of one quarter per day

based on CMT score.

In this study, the economical lost was estimated based on the milk yield loss of lactation and quarter-wise

level from 66 CMT positive cows out of 152 lactating cows. This estimation was attempted to determine milk

production with or without CMT positive cows. The difference between the milk yield based on the CMT

score of 1, 2 , 3 and 4 constituted the milk loss at the corresponding quarters. Milk production losses were

estimated to account for 69.6 liters/ quarter/ lactation in European cross breeds and 60 liters/ quarter/ lactation

in Sahiwal breeds. Economical loss was LKR Rs. 392,544 per lactation estimated from 94 CMT positive

quarters in European cross breeds. In Sahiwal breeds, economical loss was LKR Rs. 79,200 per lactation

estimated from 22 CMT positive quarters from 15 cows. This indicated indirect losses due to mastitis,

particularly the subclinical form, are not well recognized by many farmers. It is generally accepted that

subclinical mastitis accounts for the majority of economic costs of mastitis (Petrovski et al., 2006). The

economic loss incurred by mastitis is also an essential part of other management decisions, such as treating

infected udder quarters, culling mastitis cows, and discarding milk with high SCC in order to obtain a higher

milk price.

4. Conclusions

Overall prevalence of subclinical mastitis in lactating cows in Batticaloa District, Sri Lanka is 43%. Moreover,

the prevalence of subclinical mastitis in European crossbred cattle is more than those of sahiwal and local

breeds. The Staphylococcus spp. was most frequent bacterial agents associated with subclinical mastitis in

cows in the study area. From this study, SCM associated several risk factors including age, breed, parity; stage

of lactation, milk production, farming system, housing system, calf suckling after milking, isolation of

infected cows and hygienic practices may have been responsible for higher prevalence of subclinical mastitis

in lactating cows. The highest prevalence of SCM was found in old age group (>8 years) animal, late lactating

animal and daily milk yield of 3 -6 Lit animal. Good management practices might involve reduction of

prevalence of subclinical mastitis while close housing and intensive system were increased prevalence of

subclinical mastitis in cattle. The SCM drastically reduce the milk production of cows and, which was

significantly contribute the economic losses among the farmers.


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Table 1: Cattle and quarter-wise prevalence of subclinical mastitis in lactating cows in Batticaloa

district detected by CMT

Table 2: Binary logistic regression analysis of potential risk factors for subclinical mastitis in lactating cows

95.0% C.I. for OR Sig. OR Lower Upper

Breed .027

Local Reference

Sahiwal .008 9.412 5.852 32.235

European cross .005 21.34 3.133 27.051

Types

Sample tested Positive cases Prevalence

Cattle 152 66 43.4%

Quarters 608 116 19.1%


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Age .006

< 5 Years Reference

5 - 8 Years .006 2.923 1.595 12.965

> 8 Years .005 4.547 1.353 6.316

Parity .002

< 2 Reference

3-5 .004 2.807 1.399 46.449

> 5 .010 8.842 1.683 15.632

Stage of lactation .000

Early stage Reference

Mid stage .041 1.614 1.408 5.232

Late stage .000 6.438 2.597 15.958

Production .000

9-12 liters Reference

<3 liters .039 .933 .834 5.608

3-6 liters .028 6.944 1.236 39.017

6-9 liters .009 1.250 1.005 9.615

Farming system .000

Extensive Reference

Semi intensive .004 4.084 1.573 10.606

Intensive .000 10.286 3.755 28.178

Housing system

Open housing Reference

Housing night only .008 5.318 1.553 18.209

Closed housing .000 9.208 3.527 24.039

Calf suckling practice

Yes Reference

No .000 17.983 5.136 62.970

Milking space

Separated shed Reference

Out door .528 .449 0.270 35.326

International Journal of Scientific Research

Rearing shed .365

Separation of infected cow

Separated Referen

All together .000

Hygiene practice

Good hygiene Referen

Poor hygiene .000

Table 3: Average milk production fo

CMT Score A

Europ

1 2 3

4

Figure 1: Association betw

60.7%

39.3%

European cross

CM

ch and Innovative Technology ISSN: 2313-3759

147

65 3.086 0.037 5.404

rence

00 7.043 2.478 20.02

rence

00 12.61 5.814 27.35

for Subclinical mastitis.

Average milk yield (L) / quarter/ day

opean cross breeds Sahiwal breeds

1.62 1.46 1.58 1.42 1.29 1.20

1.13 1.02

etween breeds and subclinical mastitis detected

55.5%

0%

44.5%

100%

Sahiwal Local

MT positive CMT negative

Vol. 3 No. 6; June 2016

04

02

5

ed by CMT

100%


Figure 2: Association

Figure 3: Association between par

45%

62.5%

55%

37.5%

<5 Years 5-8 Years

28.4%

71.6%

<2 Number

European cros


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on between age subclinical mastitis detected by

arity and subclinical mastitis detected by CMT

75%

50%

53.8%

66.7%

25%

50%46.2%

>8 Years <5Years 5-8 Years >8

CMT positive CMT

52.3%

75%

46.7%

25%

3-5 Number >5 Numbe

CMT positive CMT neg

ross Sahiwal

Vol. 3 No. 6; June 2016

y CM

T Stage of lactation

66.7%

33.3%

>8 Years

T negative

25%

ber

negative


Figure 4: Association between stage of

Figure 5: Association between milk pr

27.7%

72.3%

Early stage

60%

72.5%

45.4%40%

27.5%

54.6%

< 3 L 3-6 L 6-9 European cros


149

of lactation and subclinical mastitis detected by

production and subclinical mastitis detected by

35%

71.1%

65%

28.9%

Mid stage Late stage

CMT positive CMT ne

45.4%

16.7%

50%

70.5%

25%

54.6%

83.3%

50%

29.5%

75%

9 L 9-12 L < 3 L 3-6 L 6-9 L

CMT positive CMT ne

cross Sahiwal

Vol. 3 No. 6; June 2016

by CMT

by CMT

28.9%

ge

negative

0% 0%

9-12 L

negative


Figure 6: Association between farming

Figure 7: Association between housing

66.7%

33.3%

Intensive

58.6%

41.4%

Closed housing


150

ing system and subclinical mastitis detected by

ng system and subclinical mastitis detected by

44.3%

16.3%

55.7%

83.7%

Semi intensive Extensive

CMT positive CMT ne

45%

13.3%

55%

86.7%

Housing night only Open housi

CMT positive CMT ne

Vol. 3 No. 6; June 2016

y CMT

y CMT

83.7%

ive

negative

86.7%

n housing

negative

prevalence of bovine subclinical mastitis and its association with ... · prevalence of bovine...

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