Development and evaluation of a quantitative polymerase chain reaction for aquatic Streptococcus agalactiae based on the groEL gene.

AIMS: The aim of this study was to develop a TaqMan quantitative polymerase chain reaction (qPCR), based on the Streptococcus agalactiae groEL gene, to specifically quantify levels of bacteria within samples derived from aquatic sources, particularly aquaculture. Enumeration of bacteria by qPCR was compared with culture-based methods. METHODS AND RESULTS: The qPCR was sensitive to 33 isolates of S. agalactiae, representing 11 clonal complexes from aquatic, bovine and human hosts. The specificity of the assay was 92·5% at a threshold Cq value of 35. No cross-reaction with Streptococcus iniae was noted and of the 22 comparator species screened to test assay specificity, Streptococcus porcinus had a Cq value of 33·7 S, while Streptococcus gallolyticus subsp. macedonicus and Streptococcus ictaluri had one replicate value above the Cq threshold of 35 (34·5 and 34·4 respectively), while only S. agalactiae were detected with a Cq value of 30. The limit of detection of the assay was 1·7 copies per µl at Cq 35. Discrepancies between molecular and culture-based methods of enumeration were noted. CONCLUSIONS: The qPCR was able to detect a diverse range of S. agalactiae isolates from different clonal complexes (CCs) and could distinguish between S. agalactiae and closely related species, notably S. iniae. The results suggest that a Cq 30 would be a very meaningful cut-off, allowing the detection of infected fish while ruling out all false positives. SIGNIFICANCE AND IMPACT OF THE STUDY: This rapid and sensitive qPCR assay is useful to quantify DNA copy number in the laboratory and could prove useful for detecting low levels of S. agalactiae in aquaculture systems, including Oreochromis niloticus culture.

Evaluation of PCR primers targeting the groEL gene for the specific detection of Streptococcus agalactiae in the context of aquaculture.

AIMS: The aim of this study was to design a set of primers for specific detection and identification of Streptococcus agalactiae in polymerase chain reaction (PCR) that can detect a diverse range of S. agalactiae isolates from different hosts and that it is capable of discriminating between S. agalactiae and other species that are closely related or potentially present in aquaculture environments, notably Streptococcus iniae. METHODS AND RESULTS: Primers, based on the groEL2 gene of S. agalactiae, were shown to be epidemiologically sensitive to 97 isolates of S. agalactiae, representing 11 clonal complexes derived from piscine, terrestrial and aquatic mammalian host species. The primers were tested with 10 S. iniae isolates and 22 other comparator species with no cross-reaction observed after optimization of reaction conditions. They have a high analytical sensitivity, detecting as few as 10 copies of S. agalactiae genomic DNA per reaction and are capable of detecting the target in DNA extracted from the brains of infected fish. CONCLUSIONS: The primers proved suitable for the sensitive and specific detection of S. agalactiae from dairy-, human- and fish-related origins by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the importance of S. agalactiae as a pathogen, many PCR primers have been published for this bacterium, designed largely for its detection in dairy and human samples, but many cross-reacting with S. iniae. The ability to differentiate between S. agalactiae and S. iniae in aquaculture derived samples is important as both infect fish, causing similar disease symptoms and are phenotypically similar, yet control strategies and zoonotic risk are species specific.

Genomic comparison of virulent and non-virulent Streptococcus agalactiae in fish.

Streptococcus agalactiae infections in fish are predominantly caused by beta-haemolytic strains of clonal complex (CC) 7, notably its namesake sequence type (ST) 7, or by non-haemolytic strains of CC552, including the globally distributed ST260. In contrast, CC23, including its namesake ST23, has been associated with a wide homeothermic and poikilothermic host range, but never with fish. The aim of this study was to determine whether ST23 is virulent in fish and to identify genomic markers of fish adaptation of S. agalactiae. Intraperitoneal challenge of Nile tilapia, Oreochromis niloticus (Linnaeus), showed that ST260 is lethal at doses down to 10(2) cfu per fish, whereas ST23 does not cause disease at 10(7) cfu per fish. Comparison of the genome sequence of ST260 and ST23 with those of strains derived from fish, cattle and humans revealed the presence of genomic elements that are unique to subpopulations of S. agalactiae that have the ability to infect fish (CC7 and CC552). These loci occurred in clusters exhibiting typical signatures of mobile genetic elements. PCR-based screening of a collection of isolates from multiple host species confirmed the association of selected genes with fish-derived strains. Several fish-associated genes encode proteins that potentially provide fitness in the aquatic environment.

Molecular epidemiology and strain-specific characteristics of Streptococcus agalactiae at the herd and cow level.

Host-adaptation of Streptococcus agalactiae subpopulations has been described whereby strains that are commonly associated with asymptomatic carriage or disease in people differ phenotypically and genotypically from those causing mastitis in dairy cattle. Based on multilocus sequence typing (MLST), the most common strains in dairy herds in Denmark belong to sequence types (ST) that are also frequently found in people. The aim of this study was to describe epidemiological and diagnostic characteristics of such strains in relation to bovine mastitis. Among 1,199 cattle from 6 herds, cow-level prevalence of S. agalactiae was estimated to be 27.4% based on PCR and 7.8% based on bacteriological culture. Quarter-level prevalence was estimated at 2.8% based on bacteriological culture. Per herd, between 2 and 26 isolates were characterized by pulsed-field gel electrophoresis (PFGE) and MLST. Within each herd, a single PFGE type and ST predominated, consistent with a contagious mode of transmission or point source infection within herds. Evidence of within-herd evolution of S. agalactiae was detected with both typing methods, although ST belonged to a single clonal complex (CC) per herd. Detection of CC23 (3 herds) was associated with significantly lower approximate count (colony-forming units) at the quarter level and significantly lower cycle threshold value at the cow level than detection of CC1 (2 herds) or CC19 (1 herd), indicating a lower bacterial load in CC23 infections. Median values for the number of infected quarters and somatic cell count (SCC) were numerically but not significantly lower for cows infected with CC23 than for cows with CC1 or CC19. For all CC, an SCC threshold of 200,000 cells/mL was an unreliable indicator of infection status, and prescreening of animals based on SCC as part of S. agalactiae detection and eradication campaigns should be discouraged.

Detection of livestock-associated meticillin-resistant Staphylococcus aureus CC398 in retail pork, United Kingdom, February 2015.

Livestock-associated meticillin-resistant Staphylococcus aureus belonging to clonal complex 398 (LA-MRSA CC398) is an important cause of zoonotic infections in many countries. Here, we describe the isolation of LA-MRSA CC398 from retail meat samples of United Kingdom (UK) farm origin. Our findings indicate that this lineage is probably established in UK pig farms and demonstrate a potential pathway for the transmission of LA-MRSA CC398 from livestock to humans in the UK.

Prevalence and properties of mecC methicillin-resistant Staphylococcus aureus (MRSA) in bovine bulk tank milk in Great Britain.

OBJECTIVES: mecC methicillin-resistant Staphylococcus aureus (MRSA) represent a newly recognized form of MRSA, distinguished by the possession of a divergent mecA homologue, mecC. The first isolate to be identified came from bovine milk, but there are few data on the prevalence of mecC MRSA among dairy cattle. The aim of this study was to conduct a prevalence study of mecC MRSA among dairy farms in Great Britain. METHODS: Test farms were randomly selected by random order generation and bulk tank samples were tested for the presence of mecC MRSA by broth enrichment and plating onto chromogenic agar. All MRSA isolated were screened by PCR for mecA and mecC, and mecC MRSA were further characterized by multilocus sequence typing, spa typing and antimicrobial susceptibility testing. RESULTS: mecC MRSA were detected on 10 of 465 dairy farms sampled in England and Wales (prevalence 2.15%, 95% CI 1.17%-3.91%), but not from 625 farms sampled in Scotland (95% CI of prevalence 0%-0.61%). Seven isolates belonged to sequence type (ST) 425, while the other three belonged to clonal complex 130. Resistance to non-ß-lactam antibiotics was uncommon. All 10 isolates produced a negative result by slide agglutination for penicillin-binding protein 2a. mecA MRSA ST398 was detected on one farm in England. CONCLUSIONS: mecC MRSA is widely distributed among dairy farms in Great Britain, but this distribution is not uniform across the whole country. These results provide an important baseline dataset to monitor the epidemiology of this emerging form of MRSA.

Prevalence and characterization of human mecC methicillin-resistant Staphylococcus aureus isolates in England.

OBJECTIVES: There are limited data available on the epidemiology and prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the human population that encode the recently described mecA homologue, mecC. To address this knowledge gap we undertook a prospective prevalence study in England to determine the prevalence of mecC among MRSA isolates. PATIENTS AND METHODS: Three hundred and thirty-five sequential MRSA isolates from individual patients were collected from each of six clinical microbiology laboratories in England during 2011-12. These were tested by PCR or genome sequencing to differentiate those encoding mecA and mecC. mecC-positive isolates were further characterized by multilocus sequence typing, spa typing, antimicrobial susceptibility profile and detection of PBP2a using commercially available kits. RESULTS: Nine out of the 2010 MRSA isolates tested were mecC positive, indicating a prevalence among MRSA in England of 0.45% (95% CI 0.24%-0.85%). The remainder were mecA positive. Eight out of these nine mecC MRSA isolates belonged to clonal complex 130, the other being sequence type 425. Resistance to non-ß-lactam antibiotics was rare among these mecC MRSA isolates and all were phenotypically identified as MRSA using oxacillin and cefoxitin according to BSAC disc diffusion methodology. However, all nine mecC isolates gave a negative result using three different commercial PBP2a detection assays. CONCLUSIONS: mecC MRSA are currently rare among MRSA isolated from humans in England and this study provides an important baseline prevalence rate to monitor future changes, which may be important given the increasing prevalence of mecC MRSA reported in Denmark.

Short communication: comparison of virulence factors in Klebsiella pneumoniae strains associated with multiple or single cases of mastitis.

Klebsiella pneumoniae mastitis in dairy cattle is generally due to an opportunistic infection from the environment, resulting in large heterogeneity among mastitis-causing strains within a herd. However, in mastitis outbreaks in 4 herds, several strains of K. pneumoniae were identified as the cause of infection in multiple cows, suggesting increased ability to either cause disease or evade host defenses. In this study, differences in capsule formation and immune evasion were compared in 5 pairs of K. pneumoniae strains, where one strain in each pair was associated with multiple cases of mastitis and the other with a single case of mastitis. Production of capsular polysaccharide, ability to evade killing by polymorphonuclear neutrophilic leukocytes (PMNL), and the relationship between the 2 were evaluated for each strain grown in broth or milk. Growth of isolates in skim milk increased capsule size and ability to evade killing by PMNL, depending on strain type. Specifically, strains associated with multiple cases of mastitis had increased capsule size in skim milk. Strains associated with single cases of mastitis were better able to evade killing by PMNL when grown in skim milk. Our results, although preliminary, suggest that the 2 groups of strains may constitute different subpopulations of K. pneumoniae. However, our findings do not indicate that capsule or evasions of killing by PMNL explain increased mastitis outbreaks with Klebsiella. Further work will explain the enhanced ability of some strains to cause mastitis in dairy cows.

Milk as diagnostic fluid for udder health management.

BACKGROUND: Mastitis is the major disease affecting milk production of dairy cattle, and milk is an obvious substrate for the detection of both the inflammation and its causative infectious agents at quarter, cow, or herd levels. In this review, we examine the use of milk to detect inflammation based on somatic cell count (SCC) and other biomarkers, and for the detection of mastitis pathogens through culture-based and culture-free methods. FINDINGS: The use of SCC at a cow or bulk milk level to guide udder health management in lactation is well-established, and SCC is increasingly used to guide selective dry cow treatment. Other markers of inflammation include electrical conductivity, which is used commercially, and markers of disease severity such as acute phase proteins but are not pathogen-specific. Some pathogen-specific markers based on humoral immune responses are available, but their value in udder health management is largely untested. Commercial pathogen detection is based on culture or polymerase chain reaction, with other tests, for example, loop-mediated isothermal amplification or 16S microbiome analysis still at the research or development stage. Matrix-assisted laser desorption ionisation time of flight (MALDI-ToF) is increasingly used for the identification of cultured organisms whilst application directly to milk needs further development. Details of test sensitivity, specificity, and use of the various technologies may differ between quarter, cow, and bulk milk applications. CONCLUSIONS: There is a growing array of diagnostic assays that can be used to detect markers of inflammation or infection in milk. The value of some of these methods in on-farm udder health improvement programs is yet to be demonstrated whilst methods with proven value may be underutilised.

Comparative molecular analysis of ovine and bovine Streptococcus uberis isolates.

Streptococcus uberis causes clinical and subclinical mastitis in cattle and sheep, but it is unknown whether the composition of Strep. uberis populations differs between host species. To address this, we characterized a collection of bovine and ovine Strep. uberis isolates with shared geographical and temporal origins by means of an expanded multilocus sequence typing scheme. Among 14 ovine and 35 bovine isolates, 35 allelic profiles were detected. Each allelic profile was associated with a single host species and all but one were new to the multilocus sequence typing database. The median number of new alleles per isolate was higher for ovine isolates than for bovine isolates. None of the ovine isolates belonged to the global clonal complexes 5 or 143, which are commonly associated with bovine mastitis and which have a wide geographical distribution. Ovine isolates also differed from bovine isolates in carriage of plasminogen activator genes, with significantly higher prevalence of pauB in ovine isolates. Isolates that were negative for yqiL, one of the targets of multilocus sequence typing, were found among ovine and bovine isolates and were not associated with a specific sequence type or global clonal complex. One bovine isolate carried a gapC allele that was probably acquired through lateral gene transfer, most likely from Streptococcus salivarius. We conclude that ovine isolates are distinct from bovine isolates of Strep. uberis, and that recombination between isolates from different host species or bacterial species could contribute to changes in virulence gene profiles with relevance for vaccine development.

Strain-specific pathogenicity of putative host-adapted and nonadapted strains of Streptococcus uberis in dairy cattle.

Streptococcus uberis is an important cause of intramammary infection in dairy cattle. Strains of Strep. uberis appear to differ in their ability to cause disease based on previous epidemiological studies. We explored the pathogenicity of 2 strains of Strep. uberis, where one strain represented a putatively host-adapted type based on its ability to cause persistent infection and to spread from cow to cow in a lactating herd. This type was part of a clonal complex that is commonly associated with bovine mastitis. The other strain, which was isolated from a transient infection in a single animal in the same herd and did not belong to any known clonal complex, was selected as putatively nonadapted type. Cows (6 per strain) were experimentally challenged in a single hind quarter and the adjacent hind quarter was used as mock challenged control quarter. Both strains showed an equal ability to grow in the milk of challenge animals in vitro. All cows that were challenged with the putatively host-adapted strain developed clinical signs of mastitis, including fever and milk yield depression as well as elevated somatic cell count due to influx of polymorphonuclear leucocytes and lymphocytes. The cytokine response followed a specific order, with an increase in IL-1ß, IL-6, and IL-8 levels at the time of first SCC elevation, followed by an increase in IL-10, IL-12p40, and tumor necrosis factor-α levels approximately 6h later. In 4 of 6 animals, IL-17A was detected in milk between 57 and 168 h postchallenge. The increase in IL-17A levels coincided with inversion of the prechallenge CD4(+)-to-CD8(+) T lymphocyte ratio, which was observed from 96 h postchallenge. This was followed by normalization of the CD4(+)-to-CD8(+) ratio due to continued increase of the CD8(+) concentration up to 312 h postchallenge. Spontaneous resolution of infection was observed in 5 animals and coincided with a measurable IL-17A response in 4 animals, suggesting that IL-17 may be involved in the resolution of intramammary infection. With the exception of minor elevation of IL-8 levels, no clinical, cytological, or immunological response was detected in quarters challenged with the nonadapted strain. The observed strain-specific pathogenicity was consistent across animals, implying that it is determined by pathogen factors rather than host factors.

Low prevalence of antimicrobial resistant organisms (methicillin resistant Staphylococcus aureus, extended beta-lactamase producing Enterobacteriaceae, and vancomycin resistant enterococci) in bulk tank milk in New South Wales, Australia.

OBJECTIVE: Estimate the presence of methicillin resistant Staphylococcus aureus (MRSA), extended beta-lactamase (ESBL) producing Enterobacteriaceae, and vancomycin resistant enterococci (VRE) in bulk tank milk in dairy herds in New South Wales (NSW), Australia. METHODS: Bulk tank milk samples (n = 80) were collected from dairy farms (n = 40, i.e. 2 per farm) in NSW during 2021. Bacteria were cultured using selective chromogenic indicator media with isolate identity confirmed using biochemical testing, Gram stain, and MALDI-TOF mass spectroscopy. Antimicrobial resistance (AMR) was confirmed using antibiotic disk diffusion. RESULTS: No samples tested positive to the targeted AMR organisms. CONCLUSION: The prevalence of MRSA, ESBL-producing Enterobacteriaceae, and VRE is low in NSW dairy herds.

A framework for evaluation of on-farm mastitis diagnostics in Australia.

Numerous culture-based diagnostics are available on the Australian and international markets for on-farm detection of bacterial pathogens in milk. Use of such diagnostics may provide an opportunity to improve the prudent use of antimicrobials in udder health management. Farms are low-resource settings in terms of diagnostic microbiology capacity. The World Health Organisation has identified criteria for the evaluation of diagnostic tests in low resource settings based on Accuracy, Sensitivity, Specificity, User-friendliness, being Rapid or Robust, Equipment-free and being Deliverable (ASSURED). Here, we review how those criteria can be interpreted in the context of microbiological diagnosis of mastitis pathogens, and how on-farm diagnostics that are currently available in Australia perform relative to ASSURED criteria. This evaluation identifies multiple trade-offs, both with regard to scientific criteria and with regards to convenience criteria. More importantly, the purpose of testing may differ between farms, and test performance should be evaluated relative to its intended use. The ability of on-farm mastitis diagnostics to inform mastitis treatment decision-making in a timely and cost-effective manner depends not just on test characteristics but also on farm-specific pathogen prevalence, and on the farm enterprise's priorities and the farm manager's potential courses of action. With most assay evaluations to date conducted in professional laboratories, there is a surprising dearth of information on how well any of the diagnostic tests perform on-farm and, indeed, of the on-farm decision-making processes that they aim to inform.

The newly described mecA homologue, mecALGA251, is present in methicillin-resistant Staphylococcus aureus isolates from a diverse range of host species.

OBJECTIVES: A previously unidentified mecA homologue, mecA(LGA251), has recently been described in methicillin-resistant Staphylococcus aureus (MRSA) from humans and dairy cattle. The origin and epidemiology of this novel homologue are unclear. The objective of this study was to provide basic descriptive information of MRSA isolates harbouring mecA(LGA251) from a range of host animal species. METHODS: A number of S. aureus isolates from historical animal isolate collections were chosen for investigation based on their similarity to known mecA(LGA251) MRSA isolates. The presence of mecA(LGA251) was determined using a multiplex PCR and antimicrobial susceptibility testing performed by disc diffusion. RESULTS: MRSA harbouring mecA(LGA251) were found in isolates from a domestic dog, brown rats, a rabbit, a common seal, sheep and a chaffinch. All of the isolates were phenotypically MRSA, although this depended on which test was used; some isolates would be considered susceptible with certain assays. All isolates were susceptible to linezolid, rifampicin, kanamycin, norfloxacin, erythromycin, clindamycin, fusidic acid, tetracycline, trimethoprim/sulfamethoxazole and mupirocin. Five multilocus sequence types were represented (2273, 130, 425, 1764 and 1245) and six spa types (t208, t6293, t742, t6594, t7914 and t843). CONCLUSIONS: The discovery of MRSA isolates possessing mecA(LGA251) from a diverse range of host species, including different taxonomic classes, has important implications for the diagnosis of MRSA in these species and our understanding of the epidemiology of this novel mecA homologue.

First detection of livestock-associated meticillin-resistant Staphylococcus aureus CC398 in bulk tank milk in the United Kingdom, January to July 2012.

Livestock-associated meticillin-resistant Staphylococcus aureus belonging to clonal complex 398 (LA-MRSA CC398) is an important cause of zoonotic infections in several countries, but there is only a single published report of this lineage from the United Kingdom (UK). Here, we describe the isolation of LA-MRSA CC398 from bulk tank milk from five geographically dispersed farms in the UK. Our findings suggest that LA-MRSA CC398 is established in livestock in the UK. Awareness of the potential occupational risks and surveillance in other food-producing animal species should be promoted.

Some coagulase-negative Staphylococcus species affect udder health more than others.

A longitudinal study in 3 dairy herds was conducted to profile the distribution of coagulase-negative Staphylococcus (CNS) species causing bovine intramammary infection (IMI) using molecular identification and to gain more insight in the pathogenic potential of CNS as a group and of the most prevalent species causing IMI. Monthly milk samples from 25 cows in each herd as well as samples from clinical mastitis were collected over a 13-mo period. Coagulase-negative staphylococci were identified to the species level using transfer-RNA intergenic spacer PCR. The distribution of CNS causing IMI was highly herd-dependent, but overall, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus cohnii, and Staphylococcus simulans were the most prevalent. No CNS species were found to cause clinical mastitis. The effect of the most prevalent species on the quarter milk somatic cell count (SCC) was analyzed using a linear mixed model, showing that Staph. chromogenes, Staph. simulans, and Staph. xylosus induced an increase in the SCC that is comparable with that of Staphylococcus aureus. Almost all CNS species were able to cause persistent IMI, with Staph. chromogenes causing the most persistent infections. In conclusion, accurate species identification cannot be ignored when studying the effect of CNS on udder health, as the effect on SCC differs between species and species distribution is herd-specific. Staphylococcus chromogenes, Staph. simulans, and Staph. xylosus seem to be the more important species and deserve special attention in further studies. Reasons for herd dependency and possible cow- and quarter-level risk factors should be examined in detail for the different species, eventually leading to cost-benefit analyses for management changes and, if needed, treatment recommendations.

Sources of Klebsiella and Raoultella species on dairy farms: be careful where you walk.

Klebsiella spp. are a common cause of mastitis, milk loss, and culling on dairy farms. Control of Klebsiella mastitis is largely based on prevention of exposure of the udder to the pathogen. To identify critical control points for mastitis prevention, potential Klebsiella sources and transmission cycles in the farm environment were investigated, including oro-fecal transmission, transmission via the indoor environment, and transmission via the outdoor environment. A total of 305 samples was collected from 3 dairy farms in upstate New York in the summer of 2007, and included soil, feed crops, feed, water, rumen content, feces, bedding, and manure from alleyways and holding pens. Klebsiella spp. were detected in 100% of rumen samples, 89% of water samples, and approximately 64% of soil, feces, bedding, alleyway, and holding pen samples. Detection of Klebsiella spp. in feed crops and feed was less common. Genotypic identification of species using rpoB sequence data showed that Klebsiella pneumoniae was the most common species in rumen content, feces, and alleyways, whereas Klebsiella oxytoca, Klebsiella variicola, and Raoultella planticola were the most frequent species among isolates from soil and feed crops. Random amplified polymorphic DNA-based strain typing showed heterogeneity of Klebsiella spp. in rumen content and feces, with a median of 4 strains per 5 isolates. Observational and bacteriological data support the existence of an oro-fecal transmission cycle, which is primarily maintained through direct contact with fecal contamination or through ingestion of contaminated drinking water. Fecal shedding of Klebsiella spp. contributes to pathogen loads in the environment, including bedding, alleyways, and holding pens. Hygiene of alleyways and holding pens is an important component of Klebsiella control on dairy farms.

Randomized clinical trial to evaluate the efficacy of a 5-day ceftiofur hydrochloride intramammary treatment on nonsevere gram-negative clinical mastitis.

The objective of this study was to evaluate the efficacy of intramammary treatment with ceftiofur hydrochloride of nonsevere, clinical coliform mastitis. One hundred four cases on 5 farms met the enrollment criteria for the study. Escherichia coli was the most common coliform species identified in milk samples from cows with mild to moderate clinical mastitis, followed by Klebsiella spp. and Enterobacter spp. At enrollment, a milk sample from the affected quarter was taken and used for on-farm culture or submitted to the laboratory. For cows in the treatment group, treatment was initiated with ceftiofur hydrochloride via intramammary infusion at 24-h intervals for 5 d according to label standards. Cows in the control group did not receive treatment. Culture results were available on the day after enrollment and only cows with coliform mastitis continued in the treatment and untreated control groups. Bacteriological cure was defined based on 2 posttreatment milk samples. Molecular typing was used for final definition of bacteriological cure. Treatment of nonsevere clinical gram-negative mastitis with ceftiofur hydrochloride resulted in a significant increase in bacteriological cure compared with nontreated controls in animals infected with E. coli or Klebsiella spp. Treated animals clinically improved significantly more compared with control cows. No significant differences were observed between treated and control animals in milk production or linear score before or after clinical mastitis. Treated animals left the study less frequently compared with control animals.

Host adaptation of bovine Staphylococcus aureus seems associated with bacteriological cure after lactational antimicrobial treatment.

Staphylococcus aureus causes a wide range of diseases in multiple species. Some sequence types (ST) are observed in a variety of hosts, whereas other strains are mainly associated with bovine mastitis, suggesting host adaptation. We propose that host adaptation of Staph. aureus may influence bacteriological cure of bovine subclinical mastitis after antimicrobial treatment. To test this hypothesis, multilocus sequence typing was performed on Staph. aureus isolates from 60 treated and 79 untreated control quarters that were obtained from well-defined cohorts of dairy cows from a recently conducted randomized field trial on early treatment of subclinical mastitis. Bovine-associated ST were distinguished from non-bovine-associated ST based on the literature and public databases. The association between host adaptation and bacteriological cure was investigated using population-averaged logistic regression models. Thirteen ST were identified, with approximately 80% of isolates belonging to bovine-associated ST. The odds for cure were around 2.5 times as high for non-bovine-associated ST as for bovine ST in treated quarters, whereas no difference in spontaneous cure was observed in untreated control quarters. In addition, host adaptation was related to known predictors of cure, such as penicillin susceptibility and somatic cell count. All isolates belonging to non-bovine-associated ST were resistant to penicillin, whereas the majority of isolates belonging to bovine-associated ST were penicillin susceptible. Penicillin-resistant bovine-associated strains were associated with high somatic cell counts compared with other strains. The correlation between penicillin resistance, cell counts, and host adaptation may affect the association between host adaptation and cure. For diagnostic purposes, a simple and fast alternative to multilocus sequence typing of Staph. aureus to determine host adaptation may be valuable.

Performance of API Staph ID 32 and Staph-Zym for identification of coagulase-negative staphylococci isolated from bovine milk samples.

In this study, the accuracy of two phenotypic tests, API Staph ID 32 and Staph-Zym, was determined for identification of coagulase-negative staphylococci (CNS) from bovine milk samples in comparison with identification based on DNA-sequencing. A total of 172 CNS isolated from bovine milk were classified into 17 species. The most frequently isolated species based on rpoB sequencing were Staphylococcus chromogenes and Staphylococcus epidermidis, followed by Staphylococcus xylosus, Staphylococcus warneri and Staphylococcus equorum (37, 13, 9, 8 and 6% of isolates, respectively). The API Staph ID 32 correctly identified 41% of the CNS isolates. Best agreement with rpoB sequence based species identification was found for S. epidermidis, Staphylococcus hyicus and S. xylosus (100, 89 and 87%, respectively). The positive predictive value was 89, 100 and 52%, respectively. Poor sensitivity was observed for 3 of the 5 most frequently found species, S. chromogenes (37%), Staphylococcus warneri (15%) and S. equorum (0%) albeit with specificity of 100%. The Staph-Zym needed additional tests for 66% of the isolates and identified 31% of the CNS isolates correctly. Good sensitivity was found for S. epidermidis, S. simulans and S. xyloxus (100, 78 and 73%, respectively). The positive predictive value was 89, 78 and 98%, respectively. Poor sensitivity was observed for S. chromogenes, S. warneri and S. equorum (0, 54 and 0%, respectively) but with a specificity of 100, 99 and 100%, respectively. Both phenotypic tests misidentified a large proportion of CNS isolates and were thus unsuitable for identification of CNS species from bovine milk samples.