Incidence rate of Staphylococcus aureus and Streptococcus agalactiae in subclinical mastitis at smallholder dairy cattle farms in Hawassa , Ethiopia

A longitudinal study was undertaken from December 2009 to February 2010 at Hawassa town, Ethiopia, in smallholder dairy farms to identify the role of Staphylococcus aureus and Streptococcus agalactiae in causation of subclinical mastitis and also to assess the role of selected risk factors in the transmission of these pathogens. A total of seven farms were selected. The farms were first screened for subclinical mastitis by California Mastitis Test (CMT) and those free of the disease were monitored. Each farm was visited at intervals of two weeks and during each visit CMT was conducted. A milk sample was aseptically collected from quarters that were CMT positive (a CMT score of greater than or equal to one). Milk samples were cultured, and S. aureus and S. agalactiae were isolated. A cow found positive in CMT in the first test and had S. aureus and S. agalactiae was then excluded and was not subsequently tested and sampled. The average subclinical mastitis incidence rate due to S. aureus and S. agalactiae was found to be 21.84 ± 0.06 Sd per 100 cowmonths at risk. Out of 165 CMT positive milk samples cultured for isolation of S. aureus and S. agalactiae, 88 (53.32%) yielded S. aureus and 30 (18.17%) had S. agalactiae. Co-infection by S. aureus and S. agalactiae was found in 14 (8.48%) of CMT positive milk samples. Generally, 104 CMT positive milk samples (63.03%) were due to S. aureus and S. agalactiae. Out of the 12 questions to the milking practice and other contagious mastitis control measures, only two were practiced by all farms: milking mastitic cows last and treating all cases of clinical mastitis. This study reveals that S. aureus and S. agalactiae were the major causes of subclinical mastitis and mastitis control strategies in those farms, and possibly other local dairies which have to focus on these pathogens.


INTRODUCTION
In most Western countries it is now possible to reduce the incidence of subclincal mastitis using udder health monitoring programs by setting a regulatory limit for bulk milk somatic cell count (BMSCC) (Barkema et al., 1998).
Most smallholder dairy farmers in Africa including Ethiopia are generally aware of clinical mastitis (Almaw et al., 2008;Kivaria et al., 2006) because of the signs exhibited by the cow.However, farmers' awareness of *Corresponding author.E-mail: tiwawyedegera@yahoo.com.Tel: +251113380895/96/97.Fax: +251113380220.subclinical mastitis is very low.According to Almaw et al. (2008) none of the interviewed Ethiopian farmers knew and screened their cows for subclinical mastitis except seeking veterinarian assistance whenever their cows got sick.Generally, smallholder dairy production in Northwestern Ethiopia is characterized by hand milking and poor sanitary milking practices (Tassew and Seifu, 2009).This practice could facilitate the spread of contagious mastitis pathogens.Several studies have been conducted in Ethiopia and elsewhere to isolate pathogens from subclinical mastitis (Getahun et al., 2008;GIanneechini et al., 2002;Moret-Stalder et al., 2009).Contagious mastitis tends to be sub-clinical in nature.The focus on mastitis prevention and control programs has to differ between regions and should be farm specific based on the existing situations.The new infection trend of S. aureus and S. agalactiae in the absence of adequate control measure has not been studied in Ethiopia.The implementation of effective specific control program could result in the eradication of S. agalactiae and substantial reduction in the incidence of S. aureus subclinical mastitis.This study was undertaken to estimate the incidence rate of S. aureus and S. agalactiae in subclinical mastitis under the smallholder dairy production system where milking is almost always by hand.

Farm selection
The study was conducted in Hawassa town, the capital city of Southern Nations Nationalities and People Regional State of Ethiopia from December 2009 to February 2010.A total of seven smallholder dairy farms were selected; one government owned and six private.The farms were coded as A, B, C, D, E, F and G dairy farms.The herd size ranged from 5 to 19 lactating dairy cows.

Study animals
All animals included in the study were cows producing milk at various stages of lactation.The majority were crossbreds (Holstein-Zebu) of different blood level.Only two farms had pure local breeds (Zebu).Farm D had one and F had five local zebu breeds.All the cows were hand milked and milked two times a day in the morning and evening.The animals were kept indoor, the whole day and fed roughage ad-libtum with nug cake supplement.Milk yield was between 4 to 20 L per day in high grade exotic breeds and 1.5 to 5 L per day in both local and low grade cross breeds.

Study design and sampling
A longitudinal study was conducted to determine the incidence of subclinical bovine mastitis due to S. aureus and S. agalactiae.The farms were first screened for subclinical mastitis using California Mastitis test (CMT).The CMT was scored 0, T, 1, 2, and 3 and was interpreted as negative, trace, weak positive, distinct positive and strong positive in that order.For the purpose of this study a score of greater than or equal to one was taken as positive.CMT was conducted according to Quinn et al. (1999).From CMT positive quarters milk sample was collected aseptically according to the National Mastitis Council (NMC, 1990) for isolation of S. aureus and S. agalactiae.Those free of the disease were monitored at intervals of two weeks for a total of six visits.This was based on the assumption that subclinically infected cows remain so at least for two weeks so that there will not be missing of cows that became sick and recovered during the interval period.A cow with a positive CMT test and had S. aureus or S. agalactiae was excluded in the next visit and was not tested and sampled for the second time to avoid persistent infection.However, cows with CMT positive but culture negative were reexamined for both CMT and bacteriology until positive for S. aureus or S. agalactiae.A cow found positive for S. aureus or S. agalactiae was considered at risk for S. agalactiae or vice versa and was monitored until found S. agalactiae or S. aureus positive or end of the study period.

Data collection
Factors that were thought to have potential association with contagious mastitis pathogens (S. aureus and S. agalactiae) and udder health problems were recorded.Twelve check lists were prepared to collect data on milking practice and other contagious mastitis control measures.The check list itmes were hand wash before milking, udder wash before milking, towel usage, milking mastitic cows last, dry cow therapy, pre and post milking teat dipping and culling of chronic mastitic cows.

California mastitis test
The California Mastitis Test was carried out as screening test to detect subclinical mastitis.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 horizontal plane for 5 s and then the reaction was interpreted as described in study and sampling part of the paper.

Milk sample collection
Milk sample collection and storage was carried out following procedures recommended by NMC (1990) and Quinn et al. (1999).Quarter milk was collected from CMT positive cows only.The teat orifice was cleaned using cotton soaked in 70% ethyl alcohol and 5 to 10 ml of milk was collected in to sterile test tubes for bacteriological examination.During collection the test tube was held nearly horizontal to prevent contamination by dirt droppings.The sample was transported immediately to Hawassa University, Faculty of Veterinary Medicine, Microbiology Laboratory using ice box.Samples were processed immediately without storage.

Staphylococcus aureus and Streptococcus agalactiae isolation
Isolation of S. aureus and S. agalactiae from CMT positive milk samples was performed following standard procedures described by NMC (1990) and Quinn et al. (1999).One loopful from each milk sample was inoculated on to blood agar base enriched with 7% sheep blood.Blood agar plates were incubated at 37°C for 24 to 48 h.Each plate was examined for growth, morphology and hemolytic characteristics, Gram stain reaction and catalase tests.Staphylococci were identified based on catalase test, growth

Data analysis
Chi square analysis was used to compare the incidence rate of subclincal mastitis between farms.The incidence rate (IR) of subclinical mastitis due to S. aureus and S. agalactiae (combined) at cow level was calculated according to the formula given in Thrusfield (2005).

Incidence rate of subclinical mastitis caused by Staphylococcus aureus and Streptococcus agalactiae
In seven farms, a total of 69 lactating cows were monitored for the period of three months for the incidence rate of subclinical mastitis.At cow level out of 69 lactating cows 39 had subclinical mastitis caused by S. aureus and S. agalactiae.The average incidence rate was 21.84 ± 0.06 Sd per 100 cow-months.The highest subclinical incidence rate was observed on farm A (30 per 100 cowmonths at risk) and the lowest was at farm B (11.8 per 100 cow-months at risk) (Table 1) and the difference was statistically significant (χ 2 (1) = 7.4667 Pr = 0.006).Regarding the infection rate of S. aureus and S. agalactiae, there was no a continuous increase or decrease during visit period (Table 2).S. aureus was more prevalent than S. agalactiae.In farm A new infection by S. agalactiae ranging from 6.7 to 23% throughout all checking times was seen.And in all farms except D (S. agalactiae positive in the final check up) and E, which were positive for S. agalactiae in the previous check up, new infection by S. agalactiae, was seen at least in the next check up.

Bacteriology
A total of 165 CMT positive milk samples were cultured for isolation of S. aureus and S. agalactiae.Of these 88 (53.32%) yield S .aureusand 30 (18.17%) S. agalactiae (Table 3).Growth due to other bacteria was observed in 46 (27.90%)CMT positive samples but these were not further isolated.Out of 165 CMT positive milk samples, 104 (63.03%) were due to the contagious pathogens of S. aureus and S. agalactiae.Co-infection by S. aureus and S. agalactiae was seen in 14 (8.48%) samples (Table 3).

Milking practice and other contagious mastitis control measures
Of the 12 check lists which are considered important in contagious mastitis control, most of them were not in use by the farms studied (Table 4).All dairy workers used tap water to clean milking equipment, wash their hands and cows udder.All farms practiced hand milking where none of the milkers in all farms use soap to wash their hands (Table 4).The association of these milking practices with the occurrence of S. aureus and S. agalactiae were not tested statistically.

DISCUSSION
This study reveals that the majority of subclinical cases

𝐼𝑅 =
Number of new cases of disease that occur in a population during a particular period of time The sum of overall individuals of the length of time at risk of developing disease  (63.02% of CMT positive samples) were due to contagious pathogens (S. aureus and S. agalactiae) and the dominant pathogen was S. aureus (53.32%).This might be related to poor milking and contagious mastitis control practice seen in the studied farms.In the absence of hygienic milking practice contagious mastitis pathogens either from infected cow or milkers hand can easily spread.In cross-sectional studies of subclinical mastitis various isolation rates of S. aureus have been reported (Abera et al., 2010;Gianneechini et al., 2002;Moret-stalder et al., 2009).However, studies indicating the transmission rates are not common.In Germany, Sommerhäuser and his colleagues (2003) evaluated the spread of S. aureus in a six herds after implementing six point control measure including strip cup testing, udder cleaning before milking using individual paper towel, post-milking teat disinfection, proper milking technique, culling and dry cow therapy.At the beginning of their study the intramammary infection rate for S. aureus was 24.2 to 27.1% in three herds and 4.2 to 11.9% in the other three herds.At the end of the study (Sommerhäuser et al., 2003) there was no new infection and persistent infection was observed only in one herd (1.2% of the cows) suggesting the control measures were effectively controlling the transmission of contagious mastitis.However, their study also indicated that there was dynamicity in the occurrence of S. aureus infection as herds which were negative in the previous check up showed 9.1% S. aureus new infection in the subsequent check up, in agreement with the present study.Unlike the German study, in the present study there was not a control measure in place except treating clinical cases and culling and hence the rate of new infection was bound to increase.The chance of a cow getting new S. aureus and S. agalactiae infection in a month per 100 lactating cows was found to be 21.84%.In Tanzania intervention trials were studied for a period of one year in smallholder dairy farming involving a total of 160 smallholder dairy farms with 247 lactating cows (Karimuribo et al., 2006).These studies were aimed to evaluate the effectiveness of two mastitis control practices: a single antibiotic infusion during lactation, and hypochlorite post-milking teat dipping.The result was intramammary antibiotics significantly reduced the proportion of bacteriologically positive quarters in the short-term (14 days post-infusion) but teat dipping had no detectable effect on bacteriological infection and CMT positive quarters.In the present study 30 of 165 CMT positive milk samples were having S. agalactiae.S. agalactiae an obligate parasite of the bovine mammary gland and which is susceptible to treatment with a variety of antibiotics and can be eradicated from a herd.Keefe (1997) in his review on S. agalactiae concluded that protocols for therapy of all infected animals in a herd were generally successful in eradicating the pathogen from the herd, especially if they are followed up with good udder hygiene techniques.
In conclusion the major causes of sub-clinical mastitis in smallholder dairy farms at Hawassa, Ethiopia were S. aureus and S. agalactiae; S. aureus being the dominant pathogen.This may be related to the absence of a lack of control measures for contagious mastitis pathogens observed in this study.

Table 1 .
Incidence rate of subclinical bovine mastitis at cow level in smallholder farms at Hawassa, Ethiopia from December 2009 to February 2010.
a S. aureus and/or S. agalactiae; b sd: standard deviation.characteristics on Manitol salt agar and coagulase test.Coagulase positive Staphylococcus species (that is, S. aureus, S. intermedius and S. hyicus) were identified on the basis of acetoin production from glucose (Voges Proskauer test).Staphylococcus aureus is acetoin positive where as S. intermidius and S. hycus do not produce acetoin.In addition S. hyicus does not produce haemolysis on sheep blood agar.Isolates presumptively identified as Streptococci were characterized according to CAMP reaction, catalase test, and hydrolysis of esculin.A CAMP test positive S.agalactiae was differentiated from S. uberis which is also CAMP positive by production of dark brown colony on esculin blood agar (Edwards's medium) indicating esculin hydrolysis.

Table 2 .
Percentage of newly infected cows with S. aureus or S. agalactiae pathogens at the time of herd check-up.
a S. aureus.

Table 3 .
Frequency of S. aureus and S. agalactiae isolates in CMT positive sample in subclinical bovine mastitis in smallholder dairy farms at Hawassa, Ethiopia.

Table 4 .
Milking practices and other contagious mastitis control measures in selected farms at Hawassa, Ethiopia.