Molecular epidemiology of Streptococcus uberis intramammary infections: Persistent and transient patterns of infection in a dairy herd.

A longitudinal observational study was carried out to explore transmission dynamics and duration of infection of Streptococcus uberis. Quarter milk samples were collected aseptically for bacterial culture from all lactating cows once a month over a 10-mo period. Molecular typing of S. uberis mastitis was performed using pulsed-field gel electrophoresis (PFGE). Molecular typing was used to determine episodes of S. uberis intramammary infection (IMI). Comparisons of spontaneous cure among PFGE types were performed using Fisher's exact chi-squared tests. Differences of duration among PFGE types and between periods of lactation were tested with Kaplan-Meier survival curves and Cox's proportional hazard model. Among a total of 851 quarter samples, 145 milk samples were detected with S. uberis presence. Based on results of PFGE, 66 episodes of S. uberis IMI were determined. From the 8 main PFGE types (A-H), PFGE type D, E, F1, F2, G, and H had only one episode indicating no evidence for transmission, subsequently defined as environmental S. uberis strains. In contrast, PFGE types A1, A2, B, C1, and C2 had at least 2 infection episodes caused by the same strain in different quarters or cows, indicating that these strains would be able to transmit to other quarters or cows. These strains were defined as contagious strains. The majority of IMI were attributable to PFGE type A1 (55%), B (17%), and A2 (11%). Spontaneous cures were observed in 35 IMI episodes. Of these 35 IMI cures, 91.4% were in IMI with duration of infection of 1 mo, n = 25, and 2 mo, n = 6. The remaining 8.6% was in IMI with duration of infection >2 mo, n = 4. Based on results from Cox's proportional hazard model, environmental S. uberis episodes were likely to have spontaneous cure with shorter duration compared with contagious S. uberis with PFGE type B (hazard ratio = 8.4). Quarters infected with S. uberis strain PFGE type A in early lactation were more likely to persist compared with those infected in late lactation (hazard ratio = 7.57). In conclusion, the majority of S. uberis IMI in this herd were transient and showed spontaneous cure. In addition to environmental S. uberis IMI, at least 3 types of contagious IMI S. uberis can be defined as (1) short duration of IMI and likely to have spontaneous cure, (2) long duration and unlikely to have spontaneous cure, and (3) wide range of duration of IMI either transient or persistent where spontaneous cure may occur depending on host defense capacity.

Dynamics of somatic cell count patterns as a proxy for transmission of mastitis pathogens.

Management of udder health is particularly focused on preventing new infections. Data from the DeLaval Online Cell Counter (DeLaval, Tumba, Sweden) may be used in forecasting to improve decision support for improved udder health management. It provides online cell counts (OCC) as a proxy for somatic cell counts from every milking at the cow level. However, these values are typically too insensitive and nonspecific to indicate subclinical intramammary infection (IMI). Our aim was to describe and evaluate use of dynamic transmission models to forecast subclinical IMI episodes using milk cultures or changes in OCC patterns over time. The latter was expressed by an elevated mastitis risk variable. Data were obtained from the dairy herd of the Norwegian University of Life Sciences (Oslo, Norway). In total, 173 cows were sampled monthly for bacteriological milk culture during a 17-mo study period and 5,330 quarter milk samples were cultured. Mastitis pathogens identified were assigned to 1 of 2 groups, Pat 1 or Pat 2. Pathogens from which a high cell count would be expected during a subclinical IMI episode were assigned to the Pat 1 group. Pathogens not in the Pat 1 group were assigned to the Pat 2 group. Staphylococcus epidermidis, Staphylococcus aureus, and Streptococcus dysgalactiae were the most common Pat 1 pathogens. Corynebacterium bovis, Staphylococcus chromogenes, and Staphylococcus haemolyticus were the most common Pat 2 pathogens. The OCC were successfully recorded from 82,182 of 96,542 milkings. The current study included 324 subclinical IMI episodes. None of the mastitis pathogens demonstrated a basic reproduction number (R0) >1. Patterns of OCC change related to an episode of Pat 1 subclinical IMI at specificity levels of 80, 90, and 95% at sensitivity levels of 69, 59, and 48% respectively, demonstrated an R0 >1. An existing infection was significant for transmission for several Pat 2 pathogens, but only for Staphylococcus aureus and Staphylococcus epidermidis among Pat 1 pathogens. Dynamic transmission models showed that patterns of OCC change related to an episode of Pat 1 subclinical IMI were significantly related to the same pattern occurring in susceptible cows at specificity levels of 80, 90, and 99% at sensitivity levels of 69, 48, and 8%, respectively. We conclude that changes in herd prevalence of subclinical IMI can be predicted using dynamic transmission models based on patterns of OCC change. Choice of specificity level depends on management goals and tolerance for false-positive alerts.

Transmission dynamics of Staphylococcus aureus within two Danish dairy cattle herds.

Staphylococcus aureus is a major pathogen causing intramammary infections (IMI) in dairy cattle herds worldwide. Simulation models can be used to investigate the epidemiologic and economic outcomes of different control strategies against IMI. The transmission rate parameter is one of the most influential parameters on the outcomes of these simulation models. Very few studies have estimated the transmission rate parameter and investigated the transmission dynamics of Staph. aureus IMI in dairy cattle herds. The objective of our study was therefore to analyze the transmission dynamics of Staph. aureus in 2 Danish dairy herds participating in a longitudinal study. The 2 herds had 180 and 360 milking cows, and animals were tested at quarter level once per month over a period of 1 yr. We estimated the quarter-level prevalence to be 34% for herd 1 and 2.57% for herd 2. The daily quarter-level transmission rate was estimated to be 0.0132 and 0.0077 cases/quarter-day for herds 1 and 2, respectively, and the median duration of infection was estimated to be 91 and 64 d for herds 1 and 2, respectively. We also estimated the reproductive ratio at 1.21 for herd 1 and 0.52 for herd 2. The results can provide valuable information for simulation models to aid decision-making in terms of the prevention and control of Staph. aureus IMI in dairy cattle herds.

Characteristics of Escherichia coli Isolated from Bovine Mastitis Exposed to Subminimum Inhibitory Concentrations of Cefalotin or Ceftazidime.

Escherichia coli is a major udder pathogen causing clinical mastitis in dairy cattle and its heat stable endotoxin in powdered infant formula milk is a potential risk factor in neonatal infections. Cephalosporins are frequently used for treatment of mastitis caused by mastitis; however, use of these antimicrobials may induce antimicrobial resistance in E. coli. The objective of this study was to explore the in vitro effect of subminimum inhibitory concentrations (sub-MIC) of cefalotin (CF) and ceftazidime (CAZ) on the morphology, antimicrobial resistance, and endotoxin releasing characteristics of 3 E. coli isolates recovered from bovine clinical mastitis. The parent E. coli isolates, which were susceptible to CF and CAZ, were exposed to CF or CAZ separately at sub-MIC levels to produce 9 generations of induced isolates. Colonies of the CAZ-induced isolates from all 3 parent E. coli were smaller on blood agar and the bacteria became filamentous, whereas the CF-induced isolates did not demonstrate prominent morphological changes. After induction by CF or CAZ, many induced isolates showed resistance to cefoxitin, CAZ, CF, kanamycin, ampicillin, and amoxicillin/clavulanic acid while their parent isolates were susceptible to these antimicrobials. Notably, 5 CAZ-induced isolates from the same parent isolate were found to produce extended-spectrum beta-lactamase (ESBL) though none of the tested ESBL related genes could be detected. All CAZ-induced isolates released more endotoxin with a higher release rate, whereas endotoxin release of CF-induced E. coli isolates was not different from parent isolates. The exposure of cephalosporins at sub-MIC levels induced resistant Escherichia coli. We inferred that cephalosporins, especially CAZ, should be used prudently for treatment of clinical E. coli mastitis.

Discrimination of contagious and environmental strains of Streptococcus uberis in dairy herds by means of mass spectrometry and machine-learning.

Streptococcus uberis is one of the most common pathogens of clinical mastitis in the dairy industry. Knowledge of pathogen transmission route is essential for the selection of the most suitable intervention. Here we show that spectral profiles acquired from clinical isolates using matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) can be used to implement diagnostic classifiers based on machine learning for the successful discrimination of environmental and contagious S. uberis strains. Classifiers dedicated to individual farms achieved up to 97.81% accuracy at cross-validation when using a genetic algorithm, with Cohen's kappa coefficient of 0.94. This indicates the potential of the proposed methodology to successfully support screening at the herd level. A global classifier developed on merged data from 19 farms achieved 95.88% accuracy at cross-validation (kappa 0.93) and 70.67% accuracy at external validation (kappa 0.34), using data from another 10 farms left as holdout. This indicates that more work is needed to develop a screening solution successful at the population level. Significant MALDI-TOF spectral peaks were extracted from the trained classifiers. The peaks were found to correspond to bacteriocin and ribosomal proteins, suggesting that immunity, growth and competition over nutrients may be correlated to the different transmission routes.

Methicillin-resistant Staphylococcus aureus CC22-MRSA-IV as an agent of dairy cow intramammary infections.

Methicillin-resistant S. aureus (MRSA) lineages have become major responsible of healthcare- and community-associated infections in human population. Bovine MRSA are sporadically detected in the dairy herd, but its presence enhances the risk of zoonosis. Some lineages are able to lose the specific host tropism, being easily transmitted from animals to humans and vice-versa. The present study aims at clarifying the epidemiology of MRSA intramammary infections in a closed dairy herd, which was running a mastitis control program since years. Quarter milk samples were collected from all lactating cows once a week for 9 weeks and bacteriologically tested. At the end of the follow-up period, also a self-taken nasal swab of the milker was analysed. Three cows (12.5%) were MRSA positive, a fourth showed a transient infection and MRSA was isolated also from the milker's nose. Somatic cell counts of infected quarters fluctuated from 1000 to 1,800,000 cells/mL. The isolates were genotyped using DNA microarrays and identified as the epidemic UK-EMRSA-15 grouping in CC22. All strains carried the genes for ß-lactam and macrolide resistance. The milker isolate differed from cow isolates mainly for the absence of the untruncated ß-haemolysin and the presence of the immune evasion cluster. The milker had been volunteering in a nursing home since months, thus playing the role of MRSA vector into the herd. Our results showed the adaptive capacity of such MRSA to the bovine host. Therefore, we suggest that CC22-MRSA should be regarded as a potential cause of reverse zoonosis in dairy cattle herds.

Identification and antimicrobial suceptibility profile of bacteria causing bovine mastitis from dairy farms in Pelotas, Rio Grande do Sul / Identificação e perfil de suscetibilidade antimicrobiana de bactérias causadoras de mastite bovina em propriedades leiteiras de Pelotas, Rio Grande do Sul

Abstract Mastitis is an inflammatory process of the udder tissue caused mainly by the bacteria Staphylococcus aureus. The indiscriminate use of antibiotics fosters conditions that favor the selection of resistant microorganisms, suppressing at the same time susceptible forms, causing a serious problem in dairy cattle. Given the importance in performing an antibiogram to select the most adequate antimicrobial therapy, the aim of this study was to identify bacteria isolated from cow's milk with mastitis, in dairy farms situated in the city of Pelotas, Rio Grande do Sul, and to determinate the susceptibility profile of these isolates against the antibiotics used to treat this illness. A total of 30 isolates of Staphylococcus spp., were selected from milk samples from the udder quarters with subclinical mastitis whose species were identified through the Vitek system. The susceptibility profile was performed by the disk diffusion assay, against: ampicillin, amoxicillin, bacitracin, cephalexin, ceftiofur, enrofloxacin, gentamicin, neomycin, norfloxacin, penicillin G, tetracycline and trimethoprim. In the antibiogram, 100.0% of the isolates were resistant to trimethoprim and 96.7% to tetracycline and neomycin, three strains of Staphylococcus spp., (10.0%) presented resistance to the 12 antibiotics tested and 24 (80.0%) to at least eight. These results showed the difficulty in treating mastitis, due to the pathogens' resistance.

Resumo A mastite se constitui no processo inflamatório da glândula mamária causada principalmente por bactérias Staphylococcus aureus. O uso indiscriminado dos antibióticos promove condições que favorecem a seleção de micro-organismos resistentes e, ao mesmo tempo, suprime formas suscetíveis, causando um grave problema para a bovinocultura leiteira. Tendo em vista a importância da realização do antibiograma para a seleção da terapia antimicrobiana mais adequada, o objetivo deste estudo foi identificar bactérias isoladas de leite de vaca com mastite, oriundas de propriedades leiteiras localizadas na cidade de Pelotas, RS, bem como determinar o perfil de suscetibilidade desses isolados frente a antibióticos usados para o tratamento desta doença. Foram selecionados 30 isolados de Staphylococcus spp. de amostras de leite provenientes de quartos mamários com mastite subclínica, cujas espécies foram identificadas através do sistema Vitek. O perfil de suscetibilidade foi realizado pela técnica de difusão em disco, frente a: ampicilina, amoxicilina, bacitracina, cefalexina, ceftiofur, enrofloxacina, gentamicina, neomicina, norfloxacina, penicilina G, tetraciclina e trimetoprima. No antibiograma, 100,0% dos isolados foram resistentes a trimetoprima e 96,7% a tetraciclina e a neomicina, três cepas (10,0%) foram resistentes aos 12 antibióticos testados e 24 (80,0%) a pelo menos oito. Esses resultados demonstram a dificuldade encontrada no tratamento da mastite devido à resistência dos agentes patológicos.

Distinct phenotypic traits of Staphylococcus aureus are associated with persistent, contagious bovine intramammary infections.

Staphylococcus aureus causing persistent, recurrent bovine intramammary infections are still a major challenge to dairy farming. Generally, one or a few clonal lineages are predominant in dairy herds, indicating animal-to-animal transfers and the existence of distinct pathotypic traits. The aim of this study was to determine if long term persistence and spreading of S. aureus are associated with specific phenotypic traits, including cellular invasion, cytotoxicity and biofilm formation. Mastitis isolates were collected over a 3-years period from a single dairy herd, resulting in two persistent subtypes, the high within-herd prevalent subtype ST9 (CC9)-methicillin-susceptible S. aureus (MSSA), designated HP/ST9, and the low within-herd prevalent subtype ST504 (CC705)-MSSA, designated LP/ST504. Characterization of the two different coexisting persistent subtypes showed that the following phenotypic traits are particularly associated with high within-herd prevalence: lack of capsular polysaccharide expression, high cellular invasiveness, low cytotoxicity and high biofilm/ poly-N-acetylglucosamine (PNAG) production, which may concomitantly contribute to the spreading of HP/ST9 within the herd. By contrast to HP/ST9, LP/ST504 is characterized by the formation of colony dendrites, which may help the bacteria to access deeper tissues as niches for persistence in single animals. Thus, within a single herd, two different types of persistence can be found in parallel, allowing longtime persistence of S. aureus in dairy cattle. Furthermore, this study indicates that ST9 (CC9)-MSSA strains, which are currently thought to have their primary reservoir in swine and humans, can also successfully spread to new hosts and persist in dairy herds for years.

A strain-, cow-, and herd-specific bio-economic simulation model of intramammary infections in dairy cattle herds.

Intramammary infections (IMI) in dairy cattle lead to economic losses for farmers, both through reduced milk production and disease control measures. We present the first strain-, cow- and herd-specific bio-economic simulation model of intramammary infections in a dairy cattle herd. The model can be used to investigate the cost-effectiveness of different prevention and control strategies against IMI. The objective of this study was to describe a transmission framework, which simulates spread of IMI causing pathogens through different transmission modes. These include the traditional contagious and environmental spread and a new opportunistic transmission mode. In addition, the within-herd transmission dynamics of IMI causing pathogens were studied. Sensitivity analysis was conducted to investigate the influence of input parameters on model predictions. The results show that the model is able to represent various within-herd levels of IMI prevalence, depending on the simulated pathogens and their parameter settings. The parameters can be adjusted to include different combinations of IMI causing pathogens at different prevalence levels, representing herd-specific situations. The model is most sensitive to varying the transmission rate parameters and the strain-specific recovery rates from IMI. It can be used for investigating both short term operational and long term strategic decisions for the prevention and control of IMI in dairy cattle herds.

Semen as a source of Mycoplasma bovis mastitis in dairy herds.

Mycoplasma bovis infections are responsible for substantial economic losses in the cattle industry, have significant welfare effects and increase antibiotic use. The pathogen is often introduced into naive herds through healthy carrier animals. In countries with a low prevalence of M. bovis, transmission from less common sources can be better explored as the pathogen has limited circulation compared to high prevalence populations. In this study, we describe how M. bovis was introduced into two closed and adequately biosecure dairy herds through the use of contaminated semen during artificial insemination (AI), leading to mastitis outbreak in both herds. Epidemiological analysis did not reveal an infection source other than semen. In both farms the primary clinical cases were M. bovis mastitis in cows inseminated with the semen of the same bull four weeks before the onset of the disease. One semen straw derived from the semen tank on the farm and other semen lots of this bull were positive for M. bovis. In contrast, semen samples were negative from other bulls that had been used for insemination in previous or later oestrus to those cows with M. bovis mastitis. Furthermore, cgMLST of M. bovis isolates supported the epidemiological results. To our knowledge this is the first study describing the introduction of M. bovis infection into a naive dairy herd via processed semen. The antibiotics used in semen extenders should be re-evaluated in order to provide farms with M. bovis-free semen or tested M. bovis-free semen should be available.

Genomic investigation of Staphylococcus aureus isolates from bulk tank milk and dairy cows with clinical mastitis.

Staphylococcus aureus is one of the most common pathogens that cause mastitis in dairy cows. Various subtypes, virulence genes and mobile genetic elements have been associated with isolates from bulk tank milk and clinical mastitis. So far, no Danish cattle associated S. aureus isolates have been whole-genome sequenced and further analyzed. Thus, the main objective was to investigate the population structure and genomic content of isolates from bulk tank milk and clinical mastitis, using whole-genome sequencing. This may reveal the origin of strains that cause clinical mastitis. S. aureus isolates from bulk tank milk (n = 94) and clinical mastitis (n = 63) were collected from 91 and 24 different farms, respectively and whole-genome sequenced. The genomic content was analyzed and a phylogenetic tree based on single nucleotide polymorphisms was constructed. In general, the isolates from both bulk tank milk and clinical mastitis were of similar genetic background. This suggests that dairy cows are natural carriers of the S. aureus subtypes that cause clinical mastitis if the right conditions are present and that a broad range of subtypes cause mastitis. A phylogenetic cluster that mostly consisted of ST151 isolates carried three mobile genetic elements that were primarily found in this group. The prevalence of resistance genes was generally low. However, the first ST398 methicillin resistant S. aureus isolate from a Danish dairy cow with clinical mastitis was detected.

Identification and antimicrobial suceptibility profile of bacteria causing bovine mastitis from dairy farms in Pelotas, Rio Grande do Sul.

Mastitis is an inflammatory process of the udder tissue caused mainly by the bacteria Staphylococcus aureus. The indiscriminate use of antibiotics fosters conditions that favor the selection of resistant microorganisms, suppressing at the same time susceptible forms, causing a serious problem in dairy cattle. Given the importance in performing an antibiogram to select the most adequate antimicrobial therapy, the aim of this study was to identify bacteria isolated from cow's milk with mastitis, in dairy farms situated in the city of Pelotas, Rio Grande do Sul, and to determinate the susceptibility profile of these isolates against the antibiotics used to treat this illness. A total of 30 isolates of Staphylococcus spp., were selected from milk samples from the udder quarters with subclinical mastitis whose species were identified through the Vitek system. The susceptibility profile was performed by the disk diffusion assay, against: ampicillin, amoxicillin, bacitracin, cephalexin, ceftiofur, enrofloxacin, gentamicin, neomycin, norfloxacin, penicillin G, tetracycline and trimethoprim. In the antibiogram, 100.0% of the isolates were resistant to trimethoprim and 96.7% to tetracycline and neomycin, three strains of Staphylococcus spp., (10.0%) presented resistance to the 12 antibiotics tested and 24 (80.0%) to at least eight. These results showed the difficulty in treating mastitis, due to the pathogens' resistance.

Transmission dynamics of intramammary infections caused by Corynebacterium species.

The development of reliable models for transmission of intramammary infections (IMI) is the subject of extensive research. Such models are useful to enhance the identification and understanding of factors that affect pathogen-specific IMI dynamics. Longitudinal transmission models are valuable for predicting infection outbreak risks, quantifying the effectiveness of response tactics, and performing response planning. In this work, we focused on modeling Corynebacterium spp. by using a compartmental model. Previous investigations have considered modeling the transmission dynamics of several bacterial pathogens, but not Corynebacterium spp. We established a Corynebacterium spp. Susceptible-Infectious-Susceptible (SIS) model. We simulated the model numerically by using parameters that we estimated by a generalized linear model approach, using month of study as the time variable. The data, from which the parameters of the model were estimated, were obtained in a field trial conducted in 2 US dairy herds. Altogether, 786 cows were sampled at least once during the 13-mo study period. The total number of quarter milk cultures and cases of IMI caused by Corynebacterium spp. were 11,744 and 556, respectively, in farm A; the corresponding figures for farm B were 11,804 and 179. Our modeling study included only transmission from persistent IMI caused by Corynebacterium spp. within the lactation pens. The rate of new infections was significantly related to preexisting IMI in both farms, underscoring the importance of preexisting Corynebacterium spp. IMI for the transmission of Corynebacterium spp. within lactation pens. The estimated basic reproduction numbers (R0) in the 2 farms were 1.18 and 0.98, respectively. The nonsignificant disparity in R0 was associated with significant differences in cure rates between farms.

Molecular Characterization of Multidrug-Resistant Escherichia coli Isolates from Bovine Clinical Mastitis and Pigs in the Vojvodina Province, Serbia.

The aim of the study was to characterize multidrug-resistant (MDR) Escherichia coli isolates collected in Serbia from bovine clinical mastitis cases and diseased pigs, mainly with molecular methods. A total of 48 E. coli isolates was collected during the years 2013-2014, of which 22 were MDR and were included in further analysis. Phylogenetic typing showed that 17 isolates belonged to group A, while two isolates were classified in group B1 and a single one in group D. All isolates showed unique macrorestriction patterns. Phenotypic susceptibility testing revealed resistances of the isolates against up to 13 antimicrobial agents, including resistance to fluoroquinolones. A wide variety of resistance genes was detected by PCR amplification and sequencing of amplicons. Sequence analysis of the quinolone resistance determining regions of topoisomerase genes revealed mutations in gyrA, parC, and/or parE. Plasmid-mediated quinolone resistance genes were detected in two porcine (aac-6'-Ib-cr and qnrS, respectively) isolates and a single bovine (aac-6'-Ib-cr) isolate. Resistance genes were found to be located on conjugative plasmids in 16 cases, many of which conferred a multidrug resistance phenotype. In conclusion, the plentitude of resistance genes located on conjugative plasmids and integrons in E. coli from cows and pigs in Vojvodina, Serbia, pose a high risk for horizontal gene transfer in bacteria from livestock husbandry.

Virulence Genes of S. aureus from Dairy Cow Mastitis and Contagiousness Risk.

Staphylococcus aureus (S. aureus) is a major agent of dairy cow intramammary infections: the different prevalences of mastitis reported might be related to a combination of S. aureus virulence factors beyond host factors. The present study considered 169 isolates from different Italian dairy herds that were classified into four groups based on the prevalence of S. aureus infection at the first testing: low prevalence (LP), medium-low (MLP), medium-high (MHP) and high (HP). We aimed to correlate the presence of virulence genes with the prevalence of intramammary infections in order to develop new strategies for the control of S. aureus mastitis. Microarray data were statistically evaluated using binary logistic regression and correspondence analysis to screen the risk factors and the relationship between prevalence group and gene. The analysis showed: (1) 24 genes at significant risk of being detected in all the herds with infection prevalence >5%, including genes belonging to microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), immune evasion and serine proteases; and (2) a significant correlation coefficient between the genes interacting with the host immune response and HP isolates against LP ones. These results support the hypothesis that virulence factors, in addition to cow management, could be related to strain contagiousness, offering new insights into vaccine development.

DNA carryover in milk samples from routine milk recording used for PCR-based diagnosis of bovine Staphylococcus aureus mastitis.

Real-time PCR techniques are increasingly used to detect udder pathogens from milk samples collected non-aseptically at routine milk recording. The objectives of this study were (1) to estimate the statistical associations between cycle threshold (Ct) values for Staphylococcus aureus in non-aseptically collected composite samples taken at routine milk recording from cows milked consecutively with the same milking unit and milk meter; and (2) to formulate practical and plausible guidelines for understanding the diagnostic implications of PCR testing for Staph. aureus intramammary infection at routine milk recording. The study included 4 herds with conventional milking parlors and repeatedly low Ct-values for Staph. aureus (representing a high DNA load) in bulk tank milk. Composite milk samples were collected from all cows at all milking units during routine milk recording using the Tru-Test electronic milk meter (Tru-Test Group, Auckland, New Zealand) and analyzed using the PathoProof PCR (Thermo Fisher Scientific, Vantaa, Finland) assay. Milking clock times were retrieved at each milk meter to establish the milking order of the cows at each unit. A multinomial logistic regression was applied to estimate the association between Ct-values from cows milked consecutively with the same milking unit and milk meter. The following groups were selected based on Ct-values: (1) 0-31.3, (2) 31.4-33.9, (3) 34.0-37, (4) 37.1-39.9, and (5) 40 (negative result). The association between groups from cows milked consecutively with the same milking unit and milk meter was statistically significant. Approximately 60% of cows were in Ct group 5 if the antecedent cow was also in Ct group 5, but only 20% of cows were in Ct group 5 if the antecedent cow was in Ct group 1. The probability of cows being in Ct group 1 was not markedly influenced by the group of the antecedent cow. Statistical relationships in the intermediate range gave a plausible indication of a dose-response relationship. Carryover of bacterial DNA via the milking unit and milk meter is very likely to affect PCR results for Staph. aureus. Therefore, information about milking order must be considered in mastitis control efforts. We suggest a practical interpretation of PCR results: cows with a Ct-value <32 can be labeled "very likely to be infected with Staph. aureus," but cows with Ct-values of >37 and 32-37 can be labeled "very likely to be negative for Staph. aureus" and "uncertain Staph. aureus status," respectively.

Prevalence and antimicrobial resistance profile of Staphylococcus in dairy farms, abattoir and humans in Addis Ababa, Ethiopia.

BACKGROUND: Staphylococcus species cause mastitis and wound infection in livestock and food poisoning in humans through ingestion of contaminated foods, including meat and dairy products. They are evolving pathogens in that they readily acquire drug resistance, and multiple drug-resistant (MDR) isolates are increasing in human and veterinary healthcare. Therefore, this study was conducted to evaluate the prevalence of Staphylococci and their drug resistance in dairy farms and abattoir settings of Addis Ababa. METHODS: In this cross-sectional study, 193 samples of milk, meat, equipment and humans working in the dairy farms and abattoir were collected (dairy farms = 72 and abattoir sources = 121). Staphylococcus isolation and identification at the species level was done according to ISO-6888-3 using biochemical characteristics. An antimicrobial susceptibility test was conducted for 43 of the isolates using 15 antimicrobial agents commonly used for humans and livestock by the Kirby Bauer disk diffusion method following CLSI guidelines. RESULTS: Staphylococcus organism were isolated from 92 (47.7%) of the total 193 samples, 50% in the dairy farms and 46.3% in the abattoir. The isolated species were S. aureus (n = 31; 16.1%), S. intermedius (n = 21; 10.9%), S. hyicus (n = 16; 8.3%), and coagulase negative Staphylococcus (CNS) (n = 24; 12.4%). Gentamycin was effective drug as all isolates (n = 43; 100%) were susceptible to it and followed by kanamycin (n = 39; 90.7%). However, the majority of the isolates showed resistance to penicillin-G (95.3%), nalidixic acid (88.4%), cloxacillin (79.1%), vancomycin (65.1%) and cefoxitin (55.8%). Of the 15 S. aureus tested for drug susceptibility, 73.3% of them were phenotypically resistant to vancomycin (VRSA) and all of the 15 isolates showed multi-drug resistance (MDR) to >3 drugs. Also, all of the tested CNS (100%), S. hyicus (100%) and the majority of S. intermedius isolates (88.9%) developed MDR. CONCLUSION: Alarmingly, the Staphylococcus isolates circulating in the dairy farms and abattoir in the study area harbor MDR. High level of Staphylococcus species isolation from personnel and equipment besides food (meat and milk) samples in dairy farms and abattoir settings reveals that the hygiene practice in the dairy farm and abattoir is substandard. Prudent drug use and improved hygienic practice is recommended in the dairy farms and abattoir to safeguard the public from the risk of acquiring infections and MDR pathogenic Staphylococcus.

Prevalence of contagious and environmental mastitis-causing bacteria in bulk tank milk and its relationships with milking practices of dairy cattle herds in São Miguel Island (Azores).

This study aimed to assess the degree of contamination of bulk tank milk (BTM) by Staphylococcus spp. and coliform bacteria and to identify major milking practices that help perpetuate them in dairy cattle herds in São Miguel Island. In July 2014, BTM was sampled and a survey concerning local milking practices was conducted on 100 herds. Semi quantitative multiplex polymerase chain reaction detected coagulase-negative staphylococci, Escherichia coli, Staphylococcus aureus, and other coliform bacteria (Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia marcescens) in 100, 75, 59, and 35 % of BTM, respectively. According to multivariable univariate models, on herds not using hot water for cleaning the milking machine and teat liners, there was at least 3.4 more odds (P < 0.01) to have S. aureus or coliform bacteria contamination in BTM. The likelihood of finding S. aureus in BTM was higher (P < 0.001) on herds without high hygiene during milking, when milking mastitic cows at the end, on abrupt cessation of milking at dry-off, and official milk control implementation. The glove use also favored (odds ratio (OR) 5.8; P < 0.01) the detection of coliform bacteria in BTM. Poor milking practices identified in this study should be avoided in order to decrease S. aureus and coliform bacteria contamination of BTM. Other factors associated with milk quality in São Miguel Island also should be further investigated.

Molecular Epidemiology of Streptococcus uberis Clinical Mastitis in Dairy Herds: Strain Heterogeneity and Transmission.

Multilocus sequence typing was successfully completed on 494 isolates of Streptococcus uberis from clinical mastitis cases in a study of 52 commercial dairy herds over a 12-month period. In total, 195 sequence types (STs) were identified. S. uberis mastitis cases that occurred in different cows within the same herd and were attributed to a common ST were classified as potential transmission events (PTEs). Clinical cases attributed to 35 of the 195 STs identified in this study were classified PTE. PTEs were identified in 63% of the herds. PTE-associated cases, which include the first recorded occurrence of that ST in that herd (index case) and all persistent infections with that PTE ST, represented 40% of all the clinical mastitis cases and occurred in 63% of the herds. PTE-associated cases accounted for >50% of all S. uberis clinical mastitis cases in 33% of the herds. Nine STs (ST-5, -6, -20, -22, -24, -35, -233, -361, and -512), eight of which were grouped within a clonal complex (sharing at least four alleles), were statistically overrepresented (OVR STs). The findings indicate that 38% of all clinical mastitis cases and 63% of the PTEs attributed to S. uberis in dairy herds may be caused by the nine most prevalent strains. The findings suggest that a small subset of STs is disproportionally important in the epidemiology of S. uberis mastitis in the United Kingdom, with cow-to-cow transmission of S. uberis potentially occurring in the majority of herds in the United Kingdom, and may be the most important route of infection in many herds.

Feeding mastitis milk to organic dairy calves: effect on health and performance during suckling and on udder health at first calving.

BACKGROUND: Infection pathways of S. aureus udder infections in heifers are still not well understood. One hypothesis is that calves become infected with S. aureus via feeding mastitis milk. Especially on small-scale farms, pasteurisers are not economic. The purpose of this randomised comparative study was to investigate the influence of feeding milk containing S. aureus genotype B (SAGTB) on the health and development of calves and udder health of the respective heifers. Additionally, a method reducing the bacterial load to obtain safer feeding milk was tested. Thirty-four calves were fed mastitis milk from cows with subclinical SAGTB mastitis. One group was fed untreated milk (UMG). For the other group, milk was thermised at 61°C for one minute (heat treated milk group = HMG). After weaning, calves were followed up until first calving. A milk sample of these heifers was taken at first milking to compare udder health of both groups. RESULTS: Thermisation of milk led to an effective reduction of S. aureus in the feeding milk. 78% of the analysed pools were free of S. aureus, a reduction of at least one log was obtained in the other pools. CONCLUSIONS: Under the conditions of this study, no effects of feeding milk containing SAGTB on udder health after first calving were observed. But a power analysis indicated that the sample size in the current setup is insufficient to allow for assessment on mastitis risk after SAGTB exposition, as a minimal number of 4 calves infected (vs. 0 in the HMG) would have shown significant effects. High bacterial load, however, was associated with an increased incidence rate of diarrhoea. Thus, thermisation as a minimal preventive measure before feeding mastitis milk to calves might be beneficial for maintaining calf health.