Latent class analysis of the diagnostic characteristics of PCR and conventional bacteriological culture in diagnosing intramammary infections caused by Staphylococcus aureus in dairy cows at dry off

Background

Staphylococcus aureus is one of the most common causes of intramammary infections in dairy cows at dry off. Reliable identification is important for disease management on herd level and for antimicrobial treatment of infected animals. Our objective was to evaluate the test characteristics of PathoProof ™ Mastitis PCR Assay and bacteriological culture (BC) in diagnosing bovine intramammary infections caused by S. aureus at dry off at different PCR cycle threshold (Ct)-value cut-offs.

Methods

Sterile quarter samples and non-sterile composite samples from 140 animals in seven herds were collected in connection with the dairy herd improvement (DHI) milk recording. All quarter samples were analyzed using BC whereas all composite samples were analyzed with PathoProof ™ Mastitis PCR Assay. Latent class analysis was used to estimate test properties for PCR and BC in the absence of a perfect reference test. The population was divided into two geographically divided subpopulations and the Hui-Walter 2-test 2-populations model applied to estimate Se, Sp for the two tests, and prevalence for the two subpopulations.

Results

The Se for PCR increased with increasing Ct-value cut-off, accompanied by a small decrease in Sp. For BC the Se decreased and Sp increased with increasing Ct-value cut-off. Most optimal test estimates for the real-time PCR assay were at a Ct-value cut-off of 37; 0.93 [95% posterior probability interval (PPI) 0.60-0.99] for Se and 0.95 [95% PPI 0.95-0.99] for Sp. At the same Ct-value cut-off, Se and Sp for BC were 0.83 [95% PPI 0.66-0.99] and 0.97 [95% PPI 0.91-0.99] respectively. Depending on the chosen PCR Ct-value cut-off, the prevalence in the subpopulations varied; the prevalence increased with increasing PCR Ct-value cut-offs.

Conclusion

Neither BC nor real-time PCR is a perfect test in detecting IMI in dairy cows at dry off. The changes in sensitivity and prevalence at different Ct-value cut-offs for both PCR and BC may indicate a change in the underlying disease definition. At low PCR Ct-value cut-offs the underlying disease definition may be a truly/heavily infected cow, whereas at higher PCR Ct-value cut-offs the disease definition may be a S. aureus positive cow.     

Genetic basis of penicillin resistance of S. aureus isolated in bovine mastitis

Background

The blaZ gene encoding penicillin resistance can be located either chromosomally or on plasmids. The aim of this study was to investigate the genetic relationships and to determine the location of the blaZ gene in S. aureus isolated in bovine mastitis in Finland and Sweden.

Methods

Seventy-eight β-lactamase positive S. aureus isolates from bovine mastitis (34 from Finland and 44 from Sweden) were included in the study. The localization of blaZ gene was determined by Southern blotting. The blaZ genes of the isolates were sequenced and the sequences were translated to beta-lactamase proteins and further grouped as different protein signatures. The isolates and, as control, 33 Swedish and 36 Finnish beta-lactamase negative isolates were typed with pulsed-field gel electrophoresis (PFGE).

Results

In 26 out of 34 Finnish isolates (76.5%) and in 25 out of 44 Swedish isolates (56.8%) the blaZ gene was localized on a plasmid. Six different protein signatures were found. One signature was found only in four Swedish isolates, but all other signatures were found both in Finnish and Swedish isolates. The PFGE results revealed a diversity of S. aureus clones. The protein signatures were not clearly associated with certain pulsotypes.

Conclusions

The plasmid location of the blaZ gene was not statistically significantly more common in Finland than in Sweden, and hence does not explain the higher proportion of penicillin-resistant isolates of S. aureus causing bovine mastitis in Finland compared to Sweden.     

Equine mammary gland disease with a focus on botryomycosis: A review and case study

Mammary gland problems occur incidentally in horses and one of the rarer conditions is botryomycosis (bacterial pseudomycosis, bacterial granuloma, staphylococcal pseudomycetoma). This article includes a short review of equine mammary gland problems inappropriate lactation, mastitis and neoplasia and botryomycosis, and additionally 2 clinical cases of botryomycosis of the udder resulting from Staphyloccocus aureus infection will be discussed. Both cases involved nonpregnant, nonlactating mares referred for chronic mammary inflammation with draining abcessation. In both mares, botryomycosis caused by S. aureus was confirmed by histopathology and a bacterial culture. Both mares recovered fully after surgical hemimastectomy under general anaesthesia.     

甘草查尔酮E对金黄色葡萄球菌的抗菌活性

应用肉汤微量稀释法测定甘草查尔酮E对金黄色葡萄球菌（简称金葡菌）的抗菌活性,并通过溶血试验、Western blot分析和荧光定量PCR试验考察亚抑菌浓度的甘草查尔酮E对金葡菌α-溶血素表达的影响。结果显示,甘草查尔酮E对7株金葡菌的最低抑菌质量浓度为2～8mg/L;亚抑菌浓度的甘草查尔酮E能显著降低金葡菌α-溶血素的表达,且呈现剂量依赖性。     

Prevalence of methicillin-resistant Staphylococci on a farm: staff can harbour MRS when animals do not

The aim of this work was to establish the prevalence of methicillin-resistant Staphylococci (MRS) in the animals and staff of a teaching and research farm. Samples of dairy cattle (36), beef cattle (26), sheep (19), horses (21), pigs (23), goats (23) and humans (13) were collected and screened for the presence of MRS. The detection of mecA gene was performed by PCR to determine the resistance of the samples to methicillin. Antimicrobial-resistance testing to penicillin, meropenem, ceftriaxone, cephalothin, oxacillin, levofloxacin, enrofloxacin, chloramphenicol, ciprofloxacin, gentamicin, clindamycin, erytromycin, linezolid, sulfamethoxazole/trimethoprim, tetracycline, doxycycline and vancomycin was performed on the mecA+ isolates. From the 161 samples, four methicillin-resistant coagulase-negative Staphylococci (MRCoNS) were isolated from human beings (31%), whereas none was isolated from animals (0%). No methicillin-resistant Staphylococcus aureus (MRSA) were isolated. All of the MRCoNS isolates from this work presented different antimicrobial resistance patterns. MRCoNS may be present in humans associated with animals while not present in the animals. Selective pressure outside of the farm and a lack of MRCoNS transmission between humans and animals may be responsible for this lack of correlation.     

广东省宠物源伪中间型葡萄球菌的耐药性调查研究

为调查广东省宠物源伪中间型葡萄球菌(Staphylococcus pseudintermedius)的携带和耐药情况,从小动物临床采集皮肤、耳部和鼻腔等部位的样品,分离鉴定伪中间型葡萄球菌,并采用琼脂稀释法和纸片扩散法对伪中间型葡萄球菌进行抗菌药物敏感性检测,采用PCR方法检测mecA基因。结果表明,从898个样品中共分离到144株伪中间型葡萄球菌,检出率为16.0%,皮肤、耳部和鼻腔的伪中间型葡萄球菌检出率分别为20%(64/319)、17.8%(64/359)和7.5%(3/40)。抗菌药物敏感性检测和mecA基因检测结果显示有89株(61.8%)为耐甲氧西林伪中间型葡萄球菌(MRSP)。伪中间型葡萄球菌对氨苄西林、红霉素、阿奇霉素、克林霉素和四环素的耐药率均在80%以上,对利福平和阿米卡星的耐药率低于8%,对万古霉素、利奈唑胺、喹奴普汀/达福普汀和替考拉宁均敏感。大部分(>69%)MRSP对红霉素、克林霉素、泰乐菌素、阿奇霉素、复方新诺明、四环素、环丙沙星和恩诺沙星等抗菌药物表现耐药。本研究结果表明宠物源伪中间型葡萄球菌的耐药情况严重且MRSP的检出率高,应高度重视宠物源细菌耐药问题。     

黑麦草-耳葡萄球菌对水体中Cu的联合修复效应研究

采用水培实验方法模拟不同程度的Cu污染状况,研究了黑麦草与耳葡萄球菌联合修复作用对Cu的去除效果。结果表明,联合修复作用对Cu的去除效果明显优于单一的黑麦草修复,Cu初始浓度在5-30mg.L-1时,联合修复对Cu的去除率均达85%以上。耳葡萄球菌对Cu表现出较强的耐受性和吸附性,Cu浓度为100mg.L-1时其干重积累量达到40.6mg.g-1。耳葡萄球菌的存在降低了黑麦草根系及茎叶中Cu的积累量,联合修复组的黑麦草的生物量和叶绿素含量明显高于单一黑麦草修复组。该菌在一定程度上减轻了黑麦草所受Cu的毒害性,提高了水中Cu的联合修复效果。     

10种中草药对金黄色葡萄球菌的体外抗菌试验

采用试管法和平板法,分别测定了10种中草药对临床分离的一株鸡关节炎源性金黄色葡萄球菌的最小抑菌浓度(MIC)和最低杀菌浓度(MBC),从而为临床预防和治疗鸡金黄色葡萄球菌性关节炎提供理论依据。结果发现,10种单味中草药对临床分离的球菌均有一定的抗菌活性,尤其是黄连、大青叶、艾叶、连翘4 种中草药的体外抗菌效果更明显。     

Culture results from milk samples submitted to veterinary diagnostic laboratories from August 2003 to December 2006 in New Zealand

Abstract

AIMS: To determine the pattern of isolation of major mastitis-causing organisms isolated from milk samples submitted to five veterinary diagnostic laboratories in New Zealand.

METHODS: The culture results of 25,288 milk samples that were collected from dairy cows throughout New Zealand from August 2003 to December 2006 and submitted to a group of veterinary diagnostic laboratories were assembled, reviewed and summarised. Logistic regression was used to analyse the effect of year, region (i.e. North vs South Island), and season on the probability of isolating the two most common organisms.

RESULTS: The most commonly isolated mastitis causing organisms from all samples were: Streptococcus uberis (23.6%), Staphylococcus aureus (23.5%), coagulase-negative staphylococci (CNS; 7.2%), Strep. dysgalactiae (6.2%), Bacillus spp. (4.0%), and coliforms (3.7%). The percentage of samples with isolates of Strep. uberis or Staph. aureus was affected by island, year and season (p<0.001). For most of the year, except in late winter and early spring when Strep. uberis was much more common, the percentage of isolates of Strep. uberis and Staph. aureus were not apparently different despite the former being an environmental pathogen and the other a contagious one.

CONCLUSION: The pattern of isolation of major mastitis-causing organisms, as determined from culture of milk samples submitted to diagnostic laboratories in New Zealand, has changed significantly over the last 40 years, with a substantial increase in the percentage of isolates that are Strep. uberis and a decrease in isolates of Strep. agalactiae. There is a clear seasonal pattern to the isolation of both Strep. uberis and Staph. aureus, particularly the former.

CLINICAL RELEVANCE: Knowledge of the aetiological agents causing bovine mastitis on a farm is of value in determining the choice of treatment. This dataset shows that, although there is seasonal pattern to the isolation of mastitis-causing organisms in New Zealand, both Strep. uberis and Staph. aureus are isolated throughout the year, so bacteriology is of value in determining aetiology even in late winter/early spring.