A note on effectiveness of dry cow therapy in New Zealand dairy herds

Two dry-cow therapy products were evaluated in seven factory-supply dairy herds in the Waikato area. A product containing neomycin sulphate and the benzathine salt of penicillin (Neopen D.C. White; Smith-Biolab) was used in five herds, and one containing benzathine cloxacillin (Orbenin, Beecham) was used in two herds. Non-treated control cows were included in each herd. Both products were effective in eliminating intramammary infections with Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae. Efficacy of dry-cow therapy against S. aureus was 83.8% and 85.2% respectively. Spontaneous cure rate among controls was 30.8% for S. aureus during the dry period. Spontaneous cure rate for Str. uberis was 50%, while dry-cow therapy eliminated 100% and 77.8%, respectively, for the two products. Dry-cow therapy with either product eliminated more than 90% of Str. agalactiae infections while spontaneous cure rate was only 28.6%. These results further support the effectiveness of dry-cow therapy in reducing the level of subclinical mastitis in dairy herds by shortening the duration of intramammary infections.     

Udder pathogens and their resistance to antimicrobial agents in dairy cows in Estonia

Background

The goal of this study was to estimate the distribution of udder pathogens and their antibiotic resistance in Estonia during the years 2007-2009.

Methods

The bacteriological findings reported in this study originate from quarter milk samples collected from cows on Estonian dairy farms that had clinical or subclinical mastitis. The samples were submitted by local veterinarians to the Estonian Veterinary and Food Laboratory during 2007-2009. Milk samples were examined by conventional bacteriology. In vitro antimicrobial susceptibility testing was performed with the disc diffusion test. Logistic regression with a random herd effect to control for clustering was used for statistical analysis.

Results

During the study period, 3058 clinical mastitis samples from 190 farms and 5146 subclinical mastitis samples from 274 farms were investigated. Positive results were found in 57% of the samples (4680 out of 8204), and the proportion did not differ according to year (p > 0.05). The proportion of bacteriologically negative samples was 22.3% and that of mixed growth was 20.6%. Streptococcus uberis (Str. uberis) was the bacterium isolated most frequently (18.4%) from cases of clinical mastitis, followed by Escherichia coli (E. coli) (15.9%) and Streptococcus agalactiae (Str. agalactiae) (11.9%). The bacteria that caused subclinical mastitis were mainly Staphylococcus aureus (S. aureus) (20%) and coagulase-negative staphylococci (CNS) (15.4%). The probability of isolating S. aureus from milk samples was significantly higher on farms that had fewer than 30 cows, when compared with farms that had more than 100 cows (p < 0.005). A significantly higher risk of Str. agalactiae infection was found on farms with more than 600 cows (p = 0.034) compared with smaller farms. The proportion of S. aureus and CNS isolates that were resistant to penicillin was 61.4% and 38.5%, respectively. Among the E. coli isolates, ampicillin, streptomycin and tetracycline resistance were observed in 24.3%, 15.6% and 13.5%, respectively.

Conclusions

This study showed that the main pathogens associated with clinical mastitis were Str. uberis and E. coli. Subclinical mastitis was caused mainly by S. aureus and CNS. The number of S. aureus and Str. agalactiae isolates depended on herd size. Antimicrobial resistance was highly prevalent, especially penicillin resistance in S. aureus and CNS.     

The association between quarter somatic-cell counts and clinical mastitis in three British dairy herds

The association between quarter somatic-cell counts (QSCCs) of milk and the risk of clinical mastitis (CM) was investigated in a 1-year study on three dairy herds in Somerset, UK. The three herds had 95-130 milking cows and an annual mean bulk milk somatic-cell count (BMSCC) of <150 x 10(3)cells/ml. The farms were visited every 4-6 weeks at morning milking when quarter-milk samples were collected. The farmers recorded all cases of CM and were trained to collect sterile milk samples from affected quarters, before treatment for bacteriology.The three herds had CM incidence rates of 25.4, 55.2, and 67.6 quarter-cases per 100 cow-years. Escherichia coli and Streptococcus uberis were cultured from approximately 50% of cases. QSCC was categorised and the risk of CM occurring in the month after the QSCC was examined using multilevel models to account for the correlated nature of the dependent data. Three models were developed: one for all cases of CM, one for those caused by coliforms and one for those caused by S. uberis. When all cases of CM were considered, quarters with somatic-cell count (SCC) 21-100 x 10(3)cells/ml had reduced odds (OR=0.60, P=0.06) and quarters with SCC >200 x 10(3)cells/ml has over three time the odds (OR=3.7, P<0.01) of CM compared with QSCC 1-20 x 10(3)cells/ml. When only coliform CM were investigated, quarters with SCC 6-200 x 10(3)cells/ml had reduced odds of coliform CM (OR=0.47, P=0.04) compared with QSCC 1-5 x 10(3)cells/ml, and SCC >200 x 10(3)cells/ml were not significantly different from the baseline. Finally, when S. uberis CM were investigated, quarters with SCC >200 x 10(3)cells/ml had more than three times the odds of S. uberis CM compared with QSCC 1-20 x 10(3)cells/ml (OR=3.73, P<0.01). QSCC <21 x 10(3) and >200 x 10(3)cells/ml are associated with increased odds of CM in the following 4-6 weeks; this association may be pathogen specific.     

Prevalence and associations between bacterial isolates from dry mammary glands of dairy cows

To assess the prevalence and patterns of bacterial isolates, cultures were made from the dry mammary glands of dairy cows in six commercial dairy herds in the UK. Milk samples were taken from all four quarters of 480 cows at drying off and at weekly intervals from 14 days before to seven days after calving. A major mastitis pathogen was isolated from at least one quarter of 220 (45.8 per cent) of the cows and from more than one quarter of 90 (18.8 per cent) of them. During the late dry to calving period, of the 957 quarters with three culture results, a major mastitis pathogen was cultured from 236 (24.7 per cent) quarters of 186 (38.8 [corrected] per cent) cows. The most commonly isolated major pathogen was Escherichia coli, followed by Streptococcus uberis and coagulase-positive staphylococci. There were significant differences between the patterns of isolates from different farms and in different calving months, suggesting that the rate of infection was partially dependent on external conditions. The isolation of E. coli, S. uberis or coagulase-positive staphylococci from a cow during the late dry/periparturient period was associated with an increased risk of that cow being culled in the next lactation. Bayesian general linear mixed models were used to assess the associations between the different bacterial species. The probability of isolating either E. coli or S. uberis was significantly greater when the other organism was cultured in a milk sample; this was also true of coagulase-positive staphylococci and S. uberis. When Corynebacterium species were isolated from a milk sample, the probability of isolating coagulase-positive staphylococci or S. uberis decreased significantly, and when coagulase-negative staphylococci were isolated the probability of isolating coagulase-positive staphylococci was reduced.     

Bactericidal activity of macrophages against Streptococcus uberis is different in mammary gland secretions of lactating and drying off cows

The aim of this study was to compare the ability of milk macrophages and macrophages from the mammary gland secretions during the mid-dry period for their interaction with the mastitis-causing Streptococcus uberis. We also aimed to determine if S. uberis induced the release of the cytokine tumour necrosis alpha (TNF-alpha) and the bactericidal moiety nitric oxide (NO) from milk macrophages of lactating cows and macrophages from the mammary gland secretions at the mid-dry period. Macrophages were isolated from the mammary gland secretions of cows during the mid-lactation or mid-dry period, and compared with blood monocytes for their interaction with the important mastitis-causing pathogen S. uberis. When infected in vitro with S. uberis, milk macrophages from lactating cows with S. uberis released modest amounts of the cytokine tumour necrosis factor alpha (TNF-alpha) (139 pg/ml) and the bactericidal moiety nitric oxide (NO) (3-4 microM of nitrite). Blood monocytes from lactating cows released significantly higher amounts of TNF-alpha (345 +/- 143 pg/ml) and NO (7 +/- 2 microM of nitrite) after interaction with S. uberis, compared to milk macrophages (P < 0.01 for both TNF-alpha and NO). Stimulation of blood monocytes with the cytokine interferon-gamma (IFN-gamma) enhanced significantly the release of NO and TNF-alpha, but IFN-gamma did not significantly enhance the production of NO and TNF-alpha by milk macrophages from lactating cows. Milk macrophages from all lactating cows failed to kill S. uberis efficiently, and this lack of killing was unaffected by prior treatment with gamma interferon (IFN-gamma) (P > 0.05). Rather, S. uberis multiplied significantly inside infected milk macrophages from lactating cows, with a two-fold increase in bacterial numbers at 2 h post-infection. Milk macrophages from lactating cows were able however, to kill a significant proportion (50-60%, P < 0.01) of phagocytosed Staphylococcus aureus. Blood monocytes from all cows were found to exert significant bactericidal activity against S. uberis. There were no significant differences in the bactericidal activity of milk macrophages obtained from lactating cows with low somatic cell counts (SCC; < 10(5) ml(-1)) compared with those with a mildly elevated SCC (> 10(5) ml(-1)) (P > 0.05). In contrast, mammary gland secretion macrophages isolated from the same cows in the mid-dry period killed a significant proportion of phagocytosed S. uberis (50-65% of ingested S. uberis killed, P < 0.01) although cytokine production in response to in vitro bacterial infection was low. We conclude that the bactericidal activity of mammary gland secretion macrophages against a virulent strain of S. uberis is low during the lactation period. In addition, our data indicate that S. uberis is not a strong inducer of NO and TNF-alpha in macrophages from the milk or mammary gland secretions of cows during the drying off period. Finally, IFN-gamma does not activate milk macrophages or macrophages from cows during the lactating period or mammary gland secretions during the drying off period.     

A retrospective study of the aetiology and temporal distribution of bovine clinical mastitis in smallholder dairy herds in the Dar es Salaam region of Tanzania

A 31-year record-based retrospective study was carried out to determine the aetiology and temporal distribution of bovine clinical mastitis in smallholder dairy herds in the Dar es Salaam region of Tanzania over the period November 1971-December 2002. Laboratory information on 1964 quarter samples from 1365 cows in 281 smallholder dairy herds were retrieved, compiled and studied. Eighty-eight percent of the quarter samples were culture-positive and the predominant mastitis pathogens isolated were Staphylococcus aureus (25.7%), Streptococcus agalactiae (15.4%), Klebsiella pneumoniae (14.3%) and Escherichia coli (14.1%). Other isolates included Pseudomonas aeruginosa (7.5%), Streptococcus dysgalactiae (5.2%) and Streptococcus uberis (4.2%). Contagious mastitis pathogens were isolated from 45.6% of the culture-positive samples, whereas environmental and miscellaneous pathogens were isolated from 48.2% and 5.7%, respectively. Thirty percent of the miscellaneous mastitis pathogens were Candida species. The results demonstrate a steady increase in clinical Candida albicans mastitis. The prevalence of Candida albicans has increased from 1% in 1971 to 17.0% in November 2002. Conversely, despite some fluctuations, the prevalence of Staphylococcus aureus, Streptococcus agalactiae, E. coli and K. pneumoniae remain above 10%. The possible risk factors for these observations are discussed.     

Etiology and antimicrobial susceptibility of udder pathogens from cases of subclinical mastitis in dairy cows in Sweden

Background

A nationwide survey on the microbial etiology of cases of subclinical mastitis in dairy cows was carried out on dairy farms in Sweden. The aim was to investigate the microbial panorama and the occurrence of antimicrobial resistance. Moreover, differences between newly infected cows and chronically infected cows were investigated.

Methods

In total, 583 quarter milk samples were collected from 583 dairy cows at 226 dairy farms from February 2008 to February 2009. The quarter milk samples were bacteriological investigated and scored using the California Mastitis Test. Staphylococci were tested for betalactamase production and presence of resistance was evaluated in all specific udder pathogens. Differences between newly infected cows and chronically infected cows were statistically investigated using logistic regression analysis.

Results

The most common isolates of 590 bacteriological diagnoses were Staphylococcus (S) aureus (19%) and coagulase-negative staphylococci (CNS; 16%) followed by Streptococcus (Str) dysgalactiae (9%), Str. uberis (8%), Escherichia (E.) coli (2.9%), and Streptococcus spp. (1.9%). Samples with no growth or contamination constituted 22% and 18% of the diagnoses, respectively. The distribution of the most commonly isolated bacteria considering only bacteriological positive samples were: S. aureus - 31%, CNS - 27%, Str. dysgalactiae - 15%, Str. uberis - 14%, E. coli - 4.8%, and Streptococcus spp. - 3.1%. There was an increased risk of finding S. aureus, Str. uberis or Str. dysgalactiae in milk samples from chronically infected cows compared to findings in milk samples from newly infected cows. Four percent of the S. aureus isolates and 35% of the CNS isolates were resistant to penicillin G. Overall, resistance to other antimicrobials than penicillin G was uncommon.

Conclusions

Staphylococcus aureus and CNS were the most frequently isolated pathogens and resistance to antimicrobials was rare.     

Within herd comparison of teat dipping and dry cow therapy with only selective dry cow therapy in six herds

A within herd comparison of teat dipping and dry cow therapy (full treatment) with only selective dry cow therapy (partial treatment) was carried out in six commercial dairy herds for a two year period. In four herds, the incidence of clinical mastitis was 2 to 12 per cent higher in the partial treatment group. In another herd, in which the pattern of clinical mastitis isolates was unusual in that minor pathogens were isolated from 30 per cent of mastitis cases, the incidence was 43 per cent higher in the partial treatment group. In the remaining herd the incidence was 10 percent higher in the full treatment group. Streptococcus uberis mastitis was more common in the partial treatment groups of five herds; coliform mastitis was more common in the full treatment groups of two herds and similar in both groups in the other herds. High rates of coliform mastitis were associated with poor herd environmental conditions but this was not true for Strep uberis mastitis. Rates of staphylococcus aureus and Strep dysgalactiae mastitis were low in all herds. The level of major pathogen infection in cows completing the trial in all herds increased in the partial treatment group from 5 per cent of quarters at the start to 12 per cent at the finish of the trial. In the full treatment group, however, there was only a small increase in this level. In contrast, levels of Corynebacterium bovis infection increased by 17 per cent in both treatment groups. Continued use of teat dipping and dry cow therapy was associated with a higher rate of coliform mastitis in two of the three herds where there were poor standards of hygiene and husbandry.     

Udder shape and teat-end lesions as potential risk factors for high somatic cell counts and intra-mammary infections in dairy cows

The association of common bacterial pathogens in milk samples during calving with udder shape or the presence of 'teat-end' lesions was investigated in 240 dairy cows from two herds. Sixty-three of 120 cows (53%) in one herd (herd A) and 54/120 animals (45%) in a second herd (herd B) had normal-shaped udders. The remaining animals had udder shapes defined as follows: large pendulous (18% herd A, 26% herd B); large between hindquarter (10% herd A, 17% herd B); overall small (8% herd A, 5% herd B); or small but pendulous (11% herd A, 7% herd B). At calving teat-end lesions were present in 63% and 76% of the quarters of herd A and B animals, respectively. There was no herd effect on udder shape or teat-end lesions. Analysis of variance revealed that udder shape and teat-end lesions did not have a significant association with quarter somatic cell count. However there was some association between mammary infection and udder shape and teat-end lesions. Compared to other udder shapes, cows with large between hindquarter shape had significantly less Staphylococcus aureus and Streptococcus uberis infection (P<0.001). There was a similar albeit less significant negative association with Escherichia coli infection (P<0.01). Infection with Streptococcus agalactiae and Streptococcus dysgalactiae was more frequent in cows with large pendulous and overall small udder conformations. The results also suggest an association between intra-mammary infection at calving and the presence of hyperkeratotic teat-end lesions, given that S. aureus, coagulase-negative staphylococci, S. uberis, S. agalactiae and E. coli were cultured from significantly more quarters with such lesions than from quarters without lesions or with other types of lesion (P<0.001).     

Antimicrobial resistance in streptococcal species isolated from bovine mammary glands

Streptococcal species isolated from dairy cows with clinical mastitis were obtained from mastitis research workers in Florida, Louisiana, New York, Vermont, Washington, and West Virginia. Seventy-one streptococcal isolates were tested, including 39 strains of Streptococcus agalactiae, 21 strains of S dysgalactiae, and 11 strains of S uberis. The minimal inhibitory concentration of erythromycin, lincomycin, oxytetracycline, penicillin, spectinomycin, streptomycin, and tetracycline was determined for each isolate. Differences were not detected among strains with respect to geographic origin. None of the strains was resistant to penicillin. Lincomycin was the next most effective antimicrobial, with only 2 resistant strains of each streptococcal species. There were no differences among the streptococcal species with respect to resistance to either penicillin or lincomycin. Streptococcus uberis was more likely to be resistant to erythromycin than were S agalactiae and S dysgalactiae (P less than 0.02). Streptococcus agalactiae and S uberis had similar distributions for resistance to oxytetracycline, tetracycline, spectinomycin, and streptomycin. Strains of S dysgalactiae were more likely to have intermediate resistance to oxytetracycline and streptomycin than were strains of S agalactiae and S uberis, which were highly resistant to oxytetracycline and streptomycin (P less than 0.001). Differences were not detected among the streptococcal species with respect to resistance to spectinomycin. Resistance to multiple antimicrobials was observed in all streptococcal species tested. Although S dysgalactiae appeared to have a greater percentage of strains (73%) that were resistant to multiple antimicrobials than did S agalactiae (31%) or S uberis (45%), differences were not statistically significant.     

Efficacy of extended pirlimycin therapy for treatment of experimentally induced Streptococcus uberis intramammary infections in lactating dairy cattle

Streptococcus uberis is an important cause of mastitis in dairy cows throughout the world, particularly during the dry period, around the time of calving, and during early lactation. Strategies for controlling S. uberis mastitis have not received adequate research attention and are therefore poorly defined and inadequate. Objectives of the present study were to evaluate the efficacy of extended therapy regimens with pirlimycin for treatment of experimentally induced S. uberis intramammary infections in lactating dairy cows during early lactation and to evaluate the usefulness of the S. uberis experimental infection model for evaluating antimicrobial efficacy in dairy cows. The efficacy of extended pirlimycin intramammary therapy regimens was investigated in 103 mammary glands of 68 dairy cows that became infected following experimental challenge with S. uberis during early lactation. Cows infected with S. uberis in one or both experimentally challenged mammary glands were randomly allocated to three groups, representing three different treatment regimens with pirlimycin, including 2-day (n = 21 cows, 31 mammary quarters), 5-day (n = 21 cows, 32 quarters), and 8-day (n = 26 cows, 40 quarters). For all groups, pirlimycin was administered at a rate of 50 mg of pirlimycin hydrochloride via intramammary infusion. A cure was defined as an experimentally infected mammary gland that was treated with pirlimycin and was bacteriologically negative for the presence of S. uberis at 7, 14, 21, and 28 days after treatment. Experimental S. uberis intramammary infections were eliminated in 58.1% of the infected quarters treated with the pirlimycin 2-day regimen, 68.8% for the 5-day regimen, and 80.0% for the 8-day regimen. Significant differences (P <.05) in efficacy were observed between the 2-day and 8-day treatment regimens. The number of somatic cells in milk decreased significantly following therapy in quarters for which treatment was successful in eliminating S. uberis. However, there was no evidence to suggest that extended therapy with pirlimycin resulted in a greater reduction in somatic cell counts in milk than the 2-day treatment. The S. uberis experimental infection model was a rapid and effective means of evaluating antimicrobial efficacy during early lactation at a time when mammary glands are highly susceptible to S. uberis intramammary infection.     

Epidemiology of Streptococcus uberis intramammary infections in a dairy herd.

From 1987 to 1991, almost 36,000 quarter samples of mammary secretion representing 1790 lactations of 510 dairy cows from a research herd were collected for bacteriological examination. The percentage of cows infected with Streptococcus uberis ranged from 12 to 16% of cows/year. S. uberis was isolated from 14.2% of lactations over the 5-year period. The prevalence of S. uberis intramammary infection (IMI) was significantly higher in cows with > or = 4 lactations than in cows with 3 or fewer lactations. Regardless of lactation number, the prevalence of S. uberis was highest before parturition, during early lactation and near drying off. The prevalence of S. uberis infected quarters ranged from 1.3 to 2.3% of quarters/year; the prevalence rate for the 5-year period was 2% of quarters. The quarter prevalence of S. uberis was lowest in cows with < or = 3 lactations, increased significantly with lactation number and was highest in cows with > or = 6 lactations. The percentage of quarters infected with S. uberis varied significantly by year. The majority (95%) of S. uberis IMI were subclinical. The ratio of subclinical IMI to clinical IMI was lowest during early lactation, and increased with days in milk, and with lactation age except for cows in their 5th and 6th lactations. Results of this epidemiological investigation suggest that opportunities exist where suitable control measures could be applied to reduce the impact of S. uberis infections in the dairy herd.     

Genomic typing of Streptococcus uberis isolates from cases of mastitis, in New Zealand dairy cows, using pulsed-field gel electrophoresis

Three hundred and forty-two Streptococcus uberis isolates were cultured from milk samples from subclinical and clinical cases of dairy cattle mastitis. The samples were collected from 15 different New Zealand farming regions, including eight specific farms, during field research trials and veterinary diagnostic investigations. Pulsed-field gel electrophoresis was used to determine and compare the degree of genetic dissimilarity between the restriction endonuclease fragment pattern of the 342 New Zealand and a single United States S. uberis isolate. The 343 isolates exhibited 330 different restriction endonuclease fragment patterns. The United States isolate had a pattern unlike any of the New Zealand isolates. Most of the isolates were genetically different strains (pattern deferred by at least 33%), but identical patterns were noted within the same or different quarters of an individual cow, different cows within the same farm, and from different cows from the same or different districts, farming regions or islands. Seven of the eight selected farms had at most only one pair of isolates with banding patterns, which differed by less than 33%. A high degree of dissimilarity was noted in individual herds in which all the samples were collected on the same day or over a 2-year period. The high degree of dissimilar isolates is an indication that S. uberis infections in New Zealand dairy cattle are largely due to the opportunistic nature of the organism in the cows' environment. Prevention and treatment of S. uberis mastitis will therefore need to be directed at a multitude of different strains present throughout the country as well as in individual herds.     

Staphylococcus aureus associated with mammary glands of cows: genotyping to distinguish different strains among herds

The hypothesis that strains of Staphylococcus aureus are more likely to be unique to a herd than common to several herds was tested. Herds (n=28) from nine geographic areas of Korea, with elevated milk somatic cell counts (>500000 cells/ml) were enrolled in this study. Mammary quarter milk samples were aseptically collected from all lactating cows (n=616) with at least three functional quarters. Milk was cultured and S. aureus isolates were typed using pulse field gel electrophoresis of DNA SmaI digests. A total of 181 cows were identified as having S. aureus intramammary infections. A total of 52 different types of S. aureus were identified and 34 (65.4%) were associated with a single herd. A total of 18 types of S. aureus were found in multiple herds; 14 types were found in two herds, and four types were found in three herds. Herds with 1, 2, 3, and more than 3 types, were: four (14.3%); eight (28.6%); nine (32.1%); and seven (25.0%). The data indicate that the majority of strains were found in one herd only, and more than 90% were found in two or less herds, suggesting that strains of S. aureus are more likely to be restricted to a single herd, than found in multiple herds.     

Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences

Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with HRMA can be used as a powerful, fast and low-cost tool for the differentiation of clinically important bacterial mastitis pathogens.     

Phenotypic characteristics of Staphylococcus aureus isolated from bovine mastitis in Israeli dairy herds

A study of the characterization of the phenotypic patterns of Staphylococcus aureus strains isolated from bovine subclinical mastitis in Israeli dairy herds and their correlation with the severity of the disease was undertaken. A total of 400 chronically S. aureus-infected Israeli-Holstein cows, from 15 dairy herds were included in this study. Based on the results of the biochemical reactions, of the anti-biogram and phage typing, one major type of S. aureus was determined in each herd, its prevalence being between 54 and 100% of the total isolates from that same herd. The majority of the isolates were found to be non-haemolytic (62.7%). The most common phage type was 3/A,3/C,55,71, which was predominant in five herds. In two herds none of the isolates (24) were typable by this set of phages. All isolates were susceptible to methicillin, erythromycin, cephalotin, norfloxacin, trimethoprin-sulphamethoxazole and novobiocin. Most isolates were resistant to penicillin (96.6%) and 52% to oxytetracyclin. Differences in protein patterns between 50 and 36 kDa were found by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis. No correlation between any combination of the phenotypic characteristics was found when correlation was done with milk yield and somatic cell count, corresponding to the 6 months before sampling. Otherwise, a positive correlation was found between type of haemolysis and the N-acetyl-beta-glucosaminidase (NAGase) values. In milk from quarters infected with the-non-haemolytic strains, the level of NAGase was significantly lower (P < 0.05) than that from quarters infected with the haemolytic strains (69.7 and 105.9, respectively). However, the level of NAGase activity in the milk of the quarters infected with the non-haemolytic strains was significantly higher (P < 0.05) when compared to the milk of quarters infected with coagulase-negative staphylococci (43.5).     

THE NEW SOUTH WALES MASTITIS CONTROL PROGRAM

Bacteriological examinations were made on quarter samples from cows in 35 herds over a 3 year period to monitor the response in a mastitis control program. Initially, Staphylococcus aureus predominated in 32 of the herds and the mean herd prevalence was 26%. The control measures halved this rate but there was considerable variation in response between herds. The decline occurred rapidly and there was a significant reduction (P less than 0.01) by 3 months. Streptococcus agalactiae predominated in 3 herds and the overall infection rate was 4.9%. Control measures eliminated the infection completely from most herds but reinfection occurred in 2 herds. The greatest decline occurred in the first 6 months and was significant (P less than 0.05). The measures had little effect upon Str. uberis and Str. dysgalactiae which remained fairly consistently at low levels. Initially, strains of Staph. aureus resistant to penicillin were dominant in most herds. In a minority of herds strains resistant to streptomycin predominated and in these herds there was a concurrent resistance to penicillin. These patterns did not change greatly over the control period. Resistance by Str. agalactiae to streptomycin occurred in most herds at the start of the program.     

Elucidation of the DNA sequence of Streptococcus uberis adhesion molecule gene (sua) and detection of sua in strains of Streptococcus uberis isolated from geographically diverse locations

Streptococcus uberis is an important environmental pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows throughout the world. S. uberis adhesion molecule (SUAM) was identified recently by our laboratory and we hypothesize that SUAM is a potential virulence factor involved in the pathogenesis of S. uberis mastitis. The first objective of the present study was to clone and sequence the SUAM gene (sua) from S. uberis UT888. The second objective was to determine the prevalence of sua in strains of S. uberis isolated from geographically diverse locations. The 20 amino acid N-terminal sequence of purified SUAM was utilized to identify a single open reading frame (ORF) in the S. uberis O140J (ATCC BAA-854) genome database. Three sets of primers were identified from this sequence for amplification of sub-fragments and the complete gene encoding SUAM. Restriction fragment analysis of the largest polymerase chain reaction (PCR) product confirmed the desired fragment had been amplified. This 2970bp PCR fragment was cloned into plasmid pCR-XL-TOPO and sequenced. The S. uberis UT888 sua sequence (NCBI Accession no. DQ232760) was 99% similar to the S. uberis O140J database sequence. The three pairs of PCR primers were used in a subsequent experiment to identify sua in 12 strains of S. uberis isolated in milk from dairy cows with mastitis in Tennessee (n=6), Colorado (n=1), Washington (n=1), New Zealand (n=1) and from the American Type Culture Collection (n=3). Primer pairs yielded the expected 2970, 2639 and 2362bp PCR fragments in all strains evaluated. In conclusion, we cloned and sequenced sua, which codes for the first described S. uberis adhesin, SUAM. sua was detected in all strains of S. uberis evaluated suggesting that it is conserved.     

Influence of the genotype of Staphylococcus aureus, determined by pulsed-field gel electrophoresis, on dry-period elimination of subclinical mastitis in Canadian dairy herds

By combining information from 2 databases, we investigated the possibility of an association between the genotype of Staphylococcus aureus causing bovine intramammary infection and dry-period cure of subclinical infection. The 1st database contained bacteriologic and cow data from a field study evaluating the efficacy in such infections of a new intramammary dry-cow therapy (DCT) containing tilmicosin phosphate, in comparison with a commercially available DCT containing benzathine cloxacillin. Isolates of S. aureus from that study were frozen and later independently analyzed by pulsed-field gel electrophoresis (PFGE) and macrorestriction DNA fingerprinting. The molecular information, summarized and published elsewhere, constituted the 2nd database. Data from 121 subclinically infected quarters of 92 cows from 40 herds were studied by univariate and multivariable regression analysis. Infection by an isolate of PFGE lineage group D was more likely than infection by an isolate of group A or F to be cured (P < 0.05). Cows infected by lineage group D had a higher linear somatic cell count score (LS) from the last Dairy Herd Improvement test before the dry period than did cows infected by the other lineage groups (P = 0.04). Although the probability of cure was significantly lower for cows with an LS at or above the mean of 5.7 for the study population (P = 0.05), when such a cow was infected with lineage group D, cure was significantly more likely (P < 0.001) than when it was infected by another lineage group. Significantly more (P = 0.02) of the infections treated with tilmicosin (74%) than of those treated with benzathine cloxacillin (53%) were cured, and significantly more (P = 0.05) of the infections by group D (81%) than of those by group A (57%) or group F (54%) were cured. However, there was no difference in cure rate for any PFGE genotype when tilmicosin phosphate was administered; when benzathine cloxacillin was administered, 87% of lineage group D isolates were eliminated, as compared with 46% of group A and 33% of group F isolates (P < 0.05). This research demonstrates that certain genotypes of S. aureus may naturally elicit a greater inflammatory response, yet be more susceptible to elimination by antibiotics in the dry period, than other genotypes.     

A SURVEY OF CLINICAL MASTITIS IN SOUTH-EAST QUEENSLAND DAIRY HERDS

SUMMARY A survey of clinical mastitis in 26 South-East Queensland dairy herds supplying more than 300,000 litres of milk annually, revealed a quarter incidence of 2.6% with a cow incidence of 9% over a 3-month period. Secretion samples from cows yielded Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, coliforms, Pseudomonas spp and other bacteria in 38.5%, 12.5%, 6.6%, 3.4%, 2.6%. 0.6% and 2.0% of cases respectively. Samples from 38% of the cases were culturally negative while at least 38% of the clinical quarters had shown a previous clinical episode of mastitis. There was a significant association (P < 0.025) between herd and the likelihood of a negative culture result on samples from clinical cases. Twenty-one percent of cases occurred in the first month of lactation and 10.3% in the first week. There was a significant association (P < 0.005) between the age of cow and the stage of lactation in which clinical cases occurred in that cows in the 2 to 5 year age group were more likely to suffer attacks in the earlier part of lactation. There was a highly significant relationship (P < 0.005) between cow age group and the proportion of affected quarters and it was calculated that the change with each year of age was a 55% increase over the previous year. Forty-five percent of the isolates of S. aureus were resistant to penicillin.