Many infectious agents have been implicated as causes of mastitis in cattle (Owen et al. 1997; Radostits 2001), with over 135 different microorganisms having been isolated (Hawari & Fowzi 2008). The agents can be categorised into host-adapted pathogens, the most common organisms being Streptococcus agalactiae and Staphylococcus aureus (CSA 2004; Lim et al. 2007), and environmental pathogens, primarily coliforms and environmental Streptococci that are frequently found in the cow’s environment (Quinn et al. 2002).

The frequencies and distributions of the various microbial causes of udder infections on any given farm will determine the severity of their costs to farmers (Gill et al. 1990). In Kenya, the most common organisms reported to cause udder infections are Streptococcus, Staphylococcus, Escherichia coli, Trueperella and Pseudomonas species (Gitau et al. 2011; Gitau et al. 2012). However, because these Kenyan results were from laboratory submissions, it is unclear how often these organisms are currently infecting cows epidemiologically. Knowledge of the most common organisms and their antibiotic sensitivity is also needed so that one can determine the potential antibiotic regimen to use even before the laboratory results on culture and sensitivity tests are available (Godden et al. 2007).

Mastitis is considered to be one of the major reasons for antibiotic use in dairy animals (Hogan & Smith 2003). Identification of the mastitis causing pathogens and their antibiotic sensitivity patterns is needed not only to treat and control mastitis effectively (Ruegg 2004; Siamak et al. 2001), but also to support public health concerns on judicious use of antibiotics in developed and developing countries (Dhakal et al. 2007). Costly treatment failure in mastitis is often due to indiscriminate use of antibiotics without testing in vitro sensitivity, and can result in development of resistance to antimicrobial drugs (Health Canada 2003; Silva et al. 2005).

The objectives of this study were to investigate the prevalence and distribution of mastitis in dairy cattle in Mukurwe-ini and Nakuru Districts, Kenya, and to determine the antibacterial sensitivity of the organisms causing bovine mastitis in these districts to aid better decision-making in mastitis treatment and control efforts in Kenya and other similar dairy production areas. Knowledge of the prevalence of mastitogenic organisms and their antibiotic sensitivities could improve treatment efficacy and cow longevity and aid in a pro-active approach towards mastitis prevention in Kenya and possibly elsewhere in sub-Saharan Africa.

Nakuru County is one of the 14 counties of the Rift Valley Province. The county covers an area of about 7230 km² and is located between longitudes 35°28′E and 35°36′E and latitudes 0°12′S and 1°10′S (Ministry of Planning and National Development 2010b). Dairy farmers in the area practise both zero-grazing and semi-zero-grazing, where cattle are housed but allowed to graze at certain times (Ministry of Livestock Development 2008b).

In Nakuru, simple random selection was employed at farm level using a sampling frame of the dairy farms provided by the District Livestock Production Officer. In Mukurwe-ini, a convenient sampling method was used for logistical reasons, as the research was conducted alongside another project (Dohoo et al. 2012, 2013). The other project included 32 farms with biogas digesters recently constructed on the farm by a non-governmental organisation and a reference group of 32 randomly selected farms without biogas digesters in the region, matched on age of the participant, family size, and number of cows. Due to the similarity of farming practices across farms in the district, the sample was considered fairly representative of the populations in the district. The study farms were visited twice during the study period to increase the sample size and determine the consistency of the results, as the researchers were on the farms for other reasons.

Within the herds, cows were eligible for the study if they were lactating. All cows were identified with an ear tag (cows without an ear tag were given one on the first visit). For farms with less than five lactating cows, all the cows were selected to participate. For the farms that had more than five lactating cows, the lactating cows were systematically randomly selected by examining alternate cows in the crush, starting with the first one. Fewer than 10 cows were examined on each farm. Of the 128 farms, 68 farms had only one lactating cow, whilst four farms had six or more cows. There were 33, 13, 10 and zero farms having two, three, four and five cows, respectively. There were 113 and 128 cows sampled in Mukurwe-ini and Nakuru Districts respectively.

From quarters that were CMT-positive (> 1 CMT), milk samples were collected for culture after routine teat cleaning and disinfection using 70% alcohol, as described by the National Mastitis Council (1999). If more than one quarter was CMT-positive in the same cow, a composite milk sample was taken from all CMT-positive quarters. The composite samples taken from different quarters were comprised of one milk stream from each quarter, which was considered to be nearly the same volume. The first stream of milk from each quarter was discarded prior to sampling for both CMT and cultures. The milk samples were refrigerated for a maximum of 96 h (four days) until they were transported in a cool box with ice packs to the Clinical Microbiology Laboratory, Department of Clinical Studies, Faculty of Veterinary Medicine, University of Nairobi, for bacterial culture.

Samples were classified as having significant growth if the growth was considered of ‘probable significance’ or ‘highly significant’ based on National Mastitis Council Guidelines for significance (National Mastitis Council 1987). Samples that had two colony types were considered mixed growth, and samples with three or more colony types were considered contaminated.

All Gram-negative rods were divided into lactose and non-lactose fermenters, based on their growth characteristics on MacConkey agar; lactose fermenters had pink-red colonies, whilst non-lactose fermenters were colourless and/or translucent colonies. The lactose fermenters were subjected to the citrate fermentation test to determine whether they were E. coli or Klebsiella. Those that tested negative with the citrate test were classified as E. coli, and those that tested positive was classified as Klebsiella.

The small to medium-sized colonies that were haemolytic or non-haemolytic on 5% sheep blood agar, and yielding Gram-positive cocci, were subjected to catalase and coagulase tests. All colonies that tested negative for catalase were identified as Streptococci. Those that tested positive for catalase were further tested with rabbit plasma for coagulase activity, and those found to be positive (ability to produce coagulase enzyme that clots rabbit plasma) were confirmed to be S. aureus. Those that tested negative were confirmed to be coagulase-negative Staphylococci. The catalase-negative Streptococci were further tested with bacitracin and those testing negative were classified as S. agalactiae.

The bacterial isolates were tested for antibiotic sensitivity through a panel of antimicrobial drugs on the disks available at the laboratory, using a panel of 8 locally available chemotherapeutics: ampicillin, gentamycin, ceforclor, cotrimazole, kanamycin, tetracycline, norfloxacin and streptomycin. Commercially available drug-impregnated paper disks, either singly or combined, as in Multodisk^® (Oxoid), Mastring-S^® (Mast Laboratories) and Octodics^® (HI Media), were applied onto the surface of 5% sheep blood agar and MacConkey agar that were inoculated uniformly with the pathogen and then incubated overnight at 37 °C. The effectiveness of a drug was determined by measuring the diameter of the zone of inhibition around the disc – the larger the diameter, the more effective the drug was considered to be. The following standard criteria were used to summarise the various sensitivity classes for each of the antibiotics used:

• A zone diameter of 0 mm – 8 mm scored 0 or ‘R’ for resistant.
• A zone diameter of 9 mm – 15 mm scored + or slightly sensitive.
• A zone diameter of 16 mm – 22 mm scored ++ or sensitive.
• A zone diameter of 23 mm and above scored +++ or very sensitive.

Two-by-two tables of districts versus infection status were constructed using the CMT and laboratory culture results, as described by Dohoo, Martin and Stryhn (2009) for the calculation of the probability of infection (prevalence) in each district. A Chi-square was computed to establish whether there was an association between the district and infection status (Dohoo, Martin & Stryhn 2009). If there was a significant association, a relative risk was calculated (a strength of association) to compare the risk between the two districts (Dohoo et al. 2009).

Descriptive statistics were computed for the laboratory culture results to establish the frequencies and percentages of quarters and cows infected, based on the clinical signs (clinical mastitis), CMT score (subclinical mastitis), and organisms isolated (culture-based mastitis), for the first and second visits. Descriptive statistics were also computed for antibiotic sensitivity, based on the sensitivity of different organisms isolated, as well as the overall sensitivity for all organisms isolated. These statistics were performed for the two districts combined and later for each district separately.

Table 1 shows the CMT results, by level of CMT score, during the first and second farm visits. Based on the CMT, the overall quarter prevalence of subclinical mastitis was 30.5% and 34.3% during the first and second visits, respectively (scores 1, 2 and 3 combined). A CMT score of 1 was the most common score, making up two thirds of the CMT-positive quarters. The differences between visits were not statistically significant (p > 0.05).

Table 2 shows the distribution of cow infections, by number of CMT-positive quarters, during the first and second visits. Of the 130 and 139 cows with CMT-positive quarters, over one third were test-positive on only one quarter during the first and the second visits, respectively, whilst just over 20% tested positive on all 4 quarters during both the first and the second visits. Some of these cows with 4 positive quarters may not have had mastitis. Again, the differences between visits were not statistically significant (p > 0.05).

Table 3 shows the CMT results and the combined CMT and laboratory culture results of the first and second farm visits. The estimated prevalence of subclinical mastitis based on CMT results increased from first to second sampling in Nakuru District first from 52% to 69%, whilst this prevalence stayed the same in Mukurwe-ini District at 61%. Overall, there was no significant difference in subclinical mastitis prevalence between the two districts based on the CMT results of the first sampling (χ² = 2.56, p = 0.11) or the second sampling (χ² = 1.30, p > 0.05).

The estimated prevalence of subclinical mastitis based on culture results increased from first to second sampling in Nakuru District from 47% to 60%, whilst this prevalence only increased from 53% to 57% in Mukurwe-ini District. Overall, there was no significant difference between the two districts based on the culture results for the first sampling (χ² = 0.832, p = 0.505) or the second sampling (χ² = 0.178, p > 0.050).

Table 4 provides a summary of the cow-level culture results for the two combined districts during the first, second, and both visits combined. S. aureus was the most prevalent organism at 68.5% and 77.7% during the first and second visits, respectively, followed by S. agalactiae and other Streptococci. Of the CMT-positive samples collected from the first and second visits, 11.5% and 9.4%, respectively, did not yield any organism after laboratory bacterial culture.

Table 5 shows the cow-level sensitivity of the S. aureus isolates to antimicrobials during the first and second visits in Mukurwe-ini and Nakuru Districts combined. During the first visit, S. aureus was most sensitive to gentamycin at 88%, followed by norfloxacin at 83%. The highest resistance was seen for cotrimazole and ampicillin at 76% and 57%, respectively. During the second visit, the antimicrobial sensitivity patterns were similar to those from the first visit.

Table 6 shows the cow-level sensitivity of the S. agalactiae isolates to antimicrobials during the first visit in Mukurwe-ini and Nakuru Districts combined. Streptococcus agalactiae was most sensitive to norfloxacin at 90%, followed by gentamycin at 70% and ampicillin at 50%. The highest resistance was seen for cotrimazole, kanamycin and ceforclor at 80%, 40% and 40%, respectively. During the second visit, the antibiotic sensitivity patterns were similar.

The prevalence of clinical mastitis at the two points in time of sampling was quite low (< 1%). These findings were in agreement with Harmon (1994) and Gitau et al. (2003), who concluded that the subclinical form of bovine mastitis is 15–40 times more prevalent than the clinical form of bovine mastitis.

The frequency and distribution of subclinical mastitis amongst the sampled dairy farms indicated that it was very common and widespread, both at the quarter level (Table 1 and Table 2) and at the cow level (Table 3). The percentage of positive quarters (Table 1) was lower than the Egyptian findings of Attia, El-Rashidy and Metias (2003), who reported 75% and 30% of quarters with the CMT score of ++ and +++, respectively. This difference could be because of the subjective nature of the CMT interpretations by those carrying out the CMT test in the field or from real differences in the epidemiology of mastitis between the two studies.

The overall quarter prevalence of subclinical mastitis in this study was lower than the 37.0% reported by Nessru, Teshome and Getachew (1997) but higher than that reported by Biffa, Debela and Beyene (2005) and Haftu et al. (2012), who reported a prevalence of 17.9% and 28.2%, respectively. The cow-level prevalence of subclinical mastitis estimated in the present study was lower than that reported by Biru (1989) and Bishi (1998) in dairy cows on farms around Addis Ababa, Ethiopia, who reported a mastitis prevalence of 67.4% and 69.8%, respectively. On the other hand, our findings were higher than the reports of Nessru et al. (1997) and Haftu et al. (2012), who reported a prevalence of 33.0% and 34.0% in Ethiopia, respectively. The variability in the prevalence of bovine mastitis amongst the reports could be attributed to differences in the sampling schemes, management of the farms in the study areas, breeds considered, or the technical methods used by the investigators.

The percentage of cows positive on the laboratory bacterial cultures in this study (Table 3) was lower than the cow-level prevalence reported in Egypt by Morcos et al. (1991) at 66.8% and in Algeria by Ghazi and Niar (2006) at 81.4%. The non-significant differences in the prevalence of subclinical mastitis based on laboratory culture results between Mukurwe-ini and Nakuru Districts in Kenya may be explained by the similarity in the smallholder dairy management in these regions.

In the present study, the most common bacterium isolated (Table 4) was S. aureus (73.2%). This finding was higher than the 36.0% of isolates found by Haftu et al. (2012) in Ethiopia, but similar to studies by Omore et al. (1996) and Gitau et al. (2011) in Kenya. Shekimweri, Kurwijila and Mgongo (1998) and Gitau et al. (2003) in Tanzania and Zanzibar, respectively, reported higher prevalence of S. aureus mastitis in the smallholder dairy sector. This high prevalence may be partly attributed to the fact that S. aureus is one of the most prevalent bacteria of subclinical mastitis in dairy cows and in this investigation, it may have been spread by milkers’ hands (no farmers used gloves) and wash cloths, which are considered the main tools in the distribution of microorganisms from teat to teat and from cow to cow, in addition to lack of hygiene, as reported by El-Balkemy et al. (1997). Small herd sizes would limit the potential of exposure to S. aureus amongst cows on the same farm. However, most of the dairy farms were in close proximity to other dairy farms, especially in the Mukurwe-ini District, and neighbours frequently provide milking assistance to each other in these close-knit dairying communities, with limited attention to biosecurity, thereby increasing the number of other cows to which a cow is exposed. Poor treatment success and limited use of dry cow therapy would also contribute to chronic infections that would act as a reservoir (Radostits 2001). Minimal use of post-milking teat dip by the farmers (22%) was also likely contributing to the spread of this contagious pathogen in the present study.

The other moderately prevalent isolated organisms in the present study were S. agalactiae, coagulase negative Staphylococcus spp. and other Streptococcus spp. (Table 4). This agrees with the findings of Dego and Tareke (2003) and El-Attar, Salama and Abd El Samie (2002), who reported that Staphylococci and Streptococci caused up to 90% of bovine mastitis in Egypt and Ethiopia.

The pathogens isolated were generally more sensitive to gentamycin and norfloxacin, with high resistance to ampicillin and cotrimoxazole, but the sensitivity was moderate too low for the other antibacterial drugs under study (Table 5 and Table 6). This is in agreement with Kumar and Sharma (2002) and Haftu et al. (2012) in India and Ethiopia, respectively, as well as a study carried out by De Oliveira et al. (2000) that showed a high resistance to drugs routinely used for S. aureus mastitis therapy in 11 different western countries. The moderate to low sensitivity to some antimicrobials could be explained by the fact that there has been availability of tetracyclines and beta-lactams for many years in the areas under study, whilst gentamycin and norfloxacin were only introduced recently.

In the present study, 10.4% of the CMT-positive milk samples did not yield any organisms after laboratory bacterial culture (Table 4), in agreement with the findings reported by Sori, Zerihun and Abdicho (2005) of 9.8%. The reasons why the CMT-positive milk samples did not yield bacterial growth were probably failure to isolate organisms by the culture techniques employed (selective media for mycoplasma, Haemophilus spp. and fungi were not employed), as suggested by Ismail and Hatem (1998); short-lived recent infections that have been cleared by treatment or by the cow; infections that are characterised by intermittent shedding of bacteria, such as S. agalactiae, S. aureus, and Mycoplasma; and other reasons for having an elevated CMT, unrelated to mastitogenic microbes.

The study had some limitations as the CMT only provides an indication of increased somatic cell counts in milk, for which mastitis is the main cause. However, the concentration of white blood cells in colostrum is elevated, and the concentration of glandular cells in milk can be increased with low milk production volumes (due to a relatively constant rate of glandular cell shedding). Milk production levels are affected by many factors other than mastitis, including parity, days-in-milk, nutrition, stress or concomitant disease (Radostits 2001). All cows in the study were clinically healthy upon examination. Also, in our statistical analyses of risk factors associated with CMT status (unpublished data), breed, milk production volume, days-in-milk and parity were not significantly associated with CMT status. Furthermore, nearly 90% of CMT-positive cows had positive culture results. If the CMT scores were due to a systemic cause, the CMT scores on all quarters would likely be the same. There were only 10 cows that had CMT scores equal on all 4 quarters. Of these 10 cows, only two had no growth on the culture. Therefore, the authors are reasonably confident that the results provided by the CMT represented udder infections, although there is a possibility of a small number of false positives.

California Mastitis Test-negative cows were not cultured in this study owing to financial constraints. This may have contributed to the low prevalence of bacteriological cultures if some of the CMT-negative cows were false negatives. However, false negatives could have occurred only if the undetected bacterial infections produced a limited inflammatory response at the time of the sampling.

Whilst this epidemiological study provides useful information for dairy farmers and animal health personnel treating dairy cows for mastitis, the results should not be over-interpreted. The cows and farms were probably reasonably representative due to the large number of farms that were randomly selected (Nakuru District) or quasi-randomly selected (Mukurwe-ini District). However, the samples were taken during the cool dry season, during which environmental mastitis is less likely to occur, and therefore the results probably underestimate environmental mastitis prevalence.

The high prevalence of S. aureus infections found in this population of dairy cows indicated that additional preventive measures need to be taken to limit the spread of this host-adapted pathogen. It is recommended that cows with normal-looking milk that is rejected, based on an alcohol test, should be tested with a CMT, and if CMT-positive, should be cultured to determine the pathogen, if possible. Cows with known infections should be milked last when there is more than one lactating cow on a farm, and a teat with a known infection should be milked last, after the other teats are milked. Latex gloves should be worn when milking the cows, and post-milking teat dip should be routinely used if possible. Infected quarters with positive cultures for S. aureus should be treated as soon as possible, either with extended lactation therapy or dry cow therapy, using an antibiotic that is likely to be effective (Barkema, Schukken & Zadoks 2006).

From the total number of samples collected in the two districts during the first and the second visits combined, S. aureus was the most prevalent organism isolated, followed by S. agalactiae and other Streptococci, whilst 10% of samples had no growth. The isolated S. aureus and S. agalactiae were most sensitive to gentamycin and norfloxacin, and least sensitive to cotrimoxazole and ampicillin.

These findings provide information about dry season mastitis for improved treatment efficacy and pro-active approaches towards mastitis prevention in Kenya and possibly elsewhere in sub-Saharan Africa. Sampling in both the dry and rainy seasons would provide a more complete understanding of udder health throughout the year.

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