Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk

Coagulase-negative staphylococci (CNS; n=417) were isolated from bovine milk and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Nineteen different species were identified, and Staphylococcus xylosus, Staphylococcus chromogenes, Staphylococcus haemolyticus, and Staphylococcus sciuri were the most prevalent species. Resistance to oxacillin (47.0% of the isolates), fusidic acid (33.8%), tiamulin (31.9%), penicillin (23.3%), tetracycline (15.8%), streptomycin (9.6%), erythromycin (7.0%), sulfonamides (5%), trimethoprim (4.3%), clindamycin (3.4%), kanamycin (2.4%), and gentamicin (2.4%) was detected. Resistance to oxacillin was attributed to the mecA gene in 9.7% of the oxacillin-resistant isolates. The remaining oxacillin-resistant CNS did not contain the mecC gene or mecA1 promoter mutations. The mecA gene was detected in Staphylococcus fleurettii, Staphylococcus epidermidis, Staph. haemolyticus, and Staph. xylosus. Resistance to tetracycline was attributed to the presence of tet(K) and tet(L), penicillin resistance to blaZ, streptomycin resistance to str and ant(6)-Ia, and erythromycin resistance to erm(C), erm(B), and msr. Resistance to tiamulin and fusidic acid could not be attributed to an acquired resistance gene. In total, 15.1% of the CNS isolates were multidrug resistant (i.e., resistant to 2 or more antimicrobials). The remaining CNS isolates were susceptible to antimicrobials commonly used in mastitis treatment. Methicillin-resistant CNS isolates were diverse, as determined by mecA gene sequence analysis, staphylococcal cassette chromosome mec typing, and pulsed-field gel electrophoresis. Arginine catabolic mobile element types 1 and 3 were detected in both methicillin-resistant and methicillin-susceptible Staph. epidermidis and were associated with sequence types ST59 and ST111. Because this study revealed the presence of multidrug-resistant CNS in a heterogeneous CNS population, we recommend antibiogram analysis of CNS in persistent infections before treatment with antimicrobials.

Antimicrobial susceptibility of gram-positive udder pathogens from bovine mastitis milk in Switzerland

We evaluated the susceptibility of the gram-positive mastitis pathogens S. aureus, Str. uberis, Str. dysgalactiae, E. faecalis and L. garviae to antibiotics that are of epidemiological interest or are critically important for mastitis therapy and human medicine. Penicillin resistance was found to be most frequent in S. aureus, and nearly 5 % of the Str. uberis strains displayed a decreased susceptibility to this antibiotic. Resistance to aminoglycosides and macrolides was also detected in the strains tested. The detection of methicillin-resistant S. aureus (MRSA) and of a ciprofloxacin-resistant Str. dysgalactiae isolate corroborated the emergence of mastitis pathogens resistant to critically important antibiotics and underscores the importance of susceptibility testing prior to antibiotic therapy. The monitoring of antibiotic susceptibility patterns and antibiogram analyses are strongly recommended for targeted antimicrobial treatment and to avoid the unnecessary use of the latest generation of antibiotics.

Evaluation of PCR electrospray-ionization mass spectrometry for rapid molecular diagnosis of bovine mastitis

Bovine mastitis, an inflammatory disease of the mammary gland, is one of the most costly diseases affecting the dairy industry. The treatment and prevention of this disease is linked heavily to the use of antibiotics in agriculture and early detection of the primary pathogen is essential to control the disease. Milk samples (n=67) from cows suffering from mastitis were analyzed for the presence of pathogens using PCR electrospray-ionization mass spectrometry (PCR/ESI-MS) and were compared with standard culture diagnostic methods. Concurrent identification of the primary mastitis pathogens was obtained for 64% of the tested milk samples, whereas divergent results were obtained for 27% of the samples. The PCR/ESI-MS failed to identify some of the primary pathogens in 18% of the samples, but identified other pathogens as well as microorganisms in samples that were negative by culture. The PCR/ESI-MS identified bacteria to the species level as well as yeasts and molds in samples that contained a mixed bacterial culture (9%). The sensitivity of the PCR/ESI-MS for the most common pathogens ranged from 57.1 to 100% and the specificity ranged from 69.8 to 100% using culture as gold standard. The PCR/ESI-MS also revealed the presence of the methicillin-resistant gene mecA in 16.2% of the milk samples, which correlated with the simultaneous detection of staphylococci including Staphylococcus aureus. We demonstrated that PCR/ESI-MS, a more rapid diagnostic platform compared with bacterial culture, has the significant potential to serve as an important screening method in the diagnosis of bovine clinical mastitis and has the capacity to be used in infection control programs for both subclinical and clinical disease.

Short communication: differential immunoglobulin transfer during mastitis challenge by pathogen-specific components

Mastitis induced by Escherichia coli is often characterized by severe clinical signs, indicating a more powerful combat of the immune system against the pathogen compared with Staphylococcus aureus infections, which are often represented by chronic and subclinical diseases. The aim of this study was to test the major pathogenic component lipopolysaccharide (LPS) from E. coli and lipoteichoic acid (LTA) from Staph. aureus for their effects on blood-milk barrier integrity and the related transfer of immunoglobulins and lactate from blood into milk. A similar somatic cell count (SCC) increase was achieved by intramammary challenge of 1 quarter of 5 cows with 20 µg of LTA, and 8 cows with 0.2 µg of LPS (maximum log SCC/mL: 7). Milk IgG(1) concentrations increased in LPS- but not in LTA-challenged quarters. Milk IgG(2) concentrations were increased in treated quarters at 3h after LPS, and 6h after LTA challenge. Higher maximum levels of IgG(2) were reached in milk of LPS-treated quarters (173 ± 58 μg/mL) than of LTA-challenged quarters (62 ± 13 μg/mL). Immunoglobulin G(1) and IgG(2) levels did not change in control quarters. l-Lactate concentrations in milk increased 4h after LPS and 5h after LTA challenge and reached higher maximum levels in LPS- (221 ± 48 mg/L) than in LTA-treated quarters (77 ± 18 mg/L). In conclusion, a mammary inflammation on a quantitatively similar level based on SCC increase achieves a more efficient transfer of blood components such as IgG(2) via the blood-milk barrier if induced by LPS from E. coli than by LTA from Staph. aureus. This pathogen-specific difference may play an important role in the cure rate of the respective intramammary infection, which is usually lower in Staph. aureus- than in E. coli-induced mastitis.

Questionnaire-based study to assess the association between management practices and mastitis within tie-stall and free-stall dairy housing systems in Switzerland.

BACKGROUND Prophylactic measures are key components of dairy herd mastitis control programs, but some are only relevant in specific housing systems. To assess the association between management practices and mastitis incidence, data collected in 2011 by a survey among 979 randomly selected Swiss dairy farms, and information from the regular test day recordings from 680 of these farms was analyzed. RESULTS The median incidence of farmer-reported clinical mastitis (ICM) was 11.6 (mean 14.7) cases per 100 cows per year. The median annual proportion of milk samples with a composite somatic cell count (PSCC) above 200,000 cells/ml was 16.1 (mean 17.3) %. A multivariable negative binomial regression model was fitted for each of the mastitis indicators for farms with tie-stall and free-stall housing systems separately to study the effect of other (than housing system) management practices on the ICM and PSCC events (above 200,000 cells/ml). The results differed substantially by housing system and outcome. In tie-stall systems, clinical mastitis incidence was mainly affected by region (mountainous production zone; incidence rate ratio (IRR) = 0.73), the dairy herd replacement system (1.27) and farmers age (0.81). The proportion of high SCC was mainly associated with dry cow udder controls (IRR = 0.67), clean bedding material at calving (IRR = 1.72), using total merit values to select bulls (IRR = 1.57) and body condition scoring (IRR = 0.74). In free-stall systems, the IRR for clinical mastitis was mainly associated with stall climate/temperature (IRR = 1.65), comfort mats as resting surface (IRR = 0.75) and when no feed analysis was carried out (IRR = 1.18). The proportion of high SSC was only associated with hand and arm cleaning after calving (IRR = 0.81) and beef producing value to select bulls (IRR = 0.66). CONCLUSIONS There were substantial differences in identified risk factors in the four models. Some of the factors were in agreement with the reported literature while others were not. This highlights the multifactorial nature of the disease and the differences in the risks for both mastitis manifestations. Attempting to understand these multifactorial associations for mastitis within larger management groups continues to play an important role in mastitis control programs.

[Mastitis management in Swiss dairy farms with udder health problems]

The objective of this study was to describe the udder health management in Swiss dairy herds with udder health problems. One hundred dairy herds with a yield-corrected somatic cell count of 200'000 to 300'000 cells/ml during 2010 were selected. Data concerning farm structure, housing system, milking technique, milking procedures, dry-cow and mastitis management were collected during farm visits between September and December 2011. In addition, quarter milk samples were collected for bacteriological culturing from cows with a composite somatic cell count ≥ 150'000 cells/ml. The highest quarter level prevalence was 12.3 % for C. bovis. Eighty-two percent of the pipeline milking machines in tie-stalls and 88 % of the milking parlours fulfilled the criteria for the vacuum drop, and only 74 % of the pipeline milking machines met the criteria of the 10-l-water test. Eighty-five percent of the farms changed their milk liners too late. The correct order of teat preparation before cluster attachment was carried out by 37 % of the farmers only. With these results, Swiss dairy farmers and herd health veterinarians can be directed to common mistakes in mastitis management. The data will be used for future information campaigns to improve udder health in Swiss dairy farms.

Improving bovine udder health: a national mastitis control program in the Netherlands

Because of increasing bulk milk somatic cell counts and continuous clinical mastitis problems in a substantial number of herds, a national mastitis control program was started in 2005 to improve udder health in the Netherlands. The program started with founding the Dutch Udder Health Centre (UGCN), which had the task to coordinate the program. The program consisted of 2 parts: a research part and a knowledge-transfer part, which were integrated as much as possible. The knowledge-transfer part comprised 2 communication strategies: a central and a peripheral approach. The central approach was based on educating farmers using comprehensive science-based and rational argumentation about mastitis prevention and included on-farm study group meetings. Comprehensive education materials were developed for farmers that were internally motivated to improve udder health. In the peripheral approach it was tried to motivate farmers to implement certain management measures using nontechnical arguments. Mass media campaigns were used that focused on one single aspect of mastitis prevention. These communication strategies, as well as an integrated approach between various stakeholders and different scientific disciplines were used to reach as many farmers as possible. It should be noted that, because this intervention took place at a national level, no control group was available, as it would be impossible to isolate farmers from all forms of communication for 5 years. Based on several studies executed during and after the program, however, the results suggest that udder health seemed to have improved on a national level during the course of the program from 2005 to 2010. Within a cohort of dairy herds monitored during the program, the prevalence of subclinical mastitis did not change significantly (23.0 in 2004 vs. 22.2 in 2009). The incidence rate of clinical mastitis, however, decreased significantly, from 33.5 to 28.1 quarter cases per 100 cow years at risk. The most important elements of the farmers' mindset toward mastitis control also changed favorably. The simulated costs of mastitis per farm were reduced compared with a situation in which the mastitis would not have changed, with € 400 per year. When this amount is extrapolated to all Dutch farms, the sector as a whole reduced the total costs of mastitis by € 8 million per year. It is difficult to assign the improved udder health completely to the efforts of the program due to the lack of a control group. Nevertheless, investing € 8 million by the Dutch dairy industry in a 5-yr national mastitis control program likely improved udder health and seemed to pay for itself financially.

Herd-specific strains of Mycoplasma bovis in outbreaks of mycoplasmal mastitis and pneumonia

Mycoplasma bovis causes severe economic losses in livestock production, particularly on the Northern American continent and more recently also in continental Europe. The aim of the current study was to evaluate whether the recently emerging outbreaks were due to a particular clone or strain of M. bovis or whether these outbreaks are due to multiple infectious strains of M. bovis. The study is based on the analysis M. bovis isolated from cattle of herds with outbreaks of mycoplasmal mastitis or pneumonia from geographically non related parts of Switzerland. M. bovis isolates were typed by insertion sequence (IS) element analysis based upon ISMbov1 and ISMbov2 southern-blot hybridization. We observed a strong divergence of M. bovis strains among affected herds which mostly were herd specific. This argues against the assumption that a recent infiltration of a particular clone of M. bovis is the cause of the perilous emerging outbreaks. The study suggests that transmission occurs from animal to animal most probably via milk.

Bovine mastitis: the diagnostic properties of a PCR-based assay to monitor the Staphylococcus aureus genotype B status of a herd, using bulk tank milk

Staphylococcus aureus genotype B (GTB) is a contagious mastitis pathogen in cattle, occurring in up to 87% of individuals. Because treatment is generally insufficient, culling is often required, leading to large economic loss in the Swiss dairy industry. As the detection of this pathogen in bulk tank milk (BTM) would greatly facilitate its control, a novel real-time quantitative PCR-based assay for BTM has previously been developed and is now being evaluated for its diagnostic properties at the herd level. Herds were initially classified as to their Staph. aureus GTB status by a reference method. Using BTM and herd pools of single-quarter and 4-quarter milk, the herds were then grouped by the novel assay, and the resulting classifications were compared. A total of 54 dairy herds were evaluated. Using the reference method, 21 herds were found to be GTB positive, whereas 33 were found to be negative. Considering the novel assay using both herd pools, all herds were grouped correctly, resulting in maximal diagnostic sensitivities (100%) and specificities (100%). For BTM samples, diagnostic sensitivities and specificities were 90 and 100%, respectively. Two herds were false negative in BTM, because cows with clinical signs of mastitis were not milked into the tank. Besides its excellent diagnostic properties, the assay is characterized by its low detection level, high efficiency, and its suitability for automation. Using the novel knowledge and assay, eradication of Staph. aureus GTB from a dairy herd may be considered as a realistic goal.

Mastitis in dairy heifers: Prevalence and risk factors

Poor udder health represents a serious problem in dairy production and has been investigated intensively, but heifers generally have not been the main focus of mastitis control. The aim of this study was to evaluate the prevalence, risk factors and consequences of heifer mastitis in Switzerland. The study included 166,518 heifers of different breeds (Swiss Red Pied, Swiss Brown Cattle and Holstein). Monthly somatic cell counts (SCCs) provided by the main dairy breeding organisations in Switzerland were monitored for 3 years; the prevalence of subclinical mastitis (SCM) was determined on the basis of SCCs ≥100,000 cells/mL at the first test date. The probability of having SCM at the first test date during lactation was modelled using logistic regression. Analysed factors included data for the genetic background, morphological traits, geographical region, season of parturition and milk composition. The overall prevalence of SCM in heifers during the period from 2006 to 2010 was 20.6%. Higher frequencies of SCM were present in heifers of the Holstein breed (odds ratio, OR, 1.62), heifers with high fat:protein ratios (OR 1.97) and heifers with low milk urea concentrations combined with high milk protein concentrations (OR 3.97). Traits associated with a low risk of SCM were high set udders, high overall breeding values and low milk breeding values. Heifers with SCM on the first test day had a higher risk of either developing chronic mastitis or leaving the herd prematurely.

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

BackgroundInfection 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.ResultsThermisation 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.Quarter milk samples revealed that two heifers had a S. aureus intramammary infection, but caused by a genotype different from genotype B.During the suckling period, the UMG had a significantly higher incidence rate of 1.09 diarrhoea cases per 100 calf days at risk compared to 0.26 cases per 100 calf days in the HMG (p¿<¿0.05).ConclusionsUnder 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.

Associations between the decrease in bovine clinical mastitis and changes in dairy farmers' attitude, knowledge, and behavior in the Netherlands.

The aim of this study was to associate changes in dairy farmers' self-reported attitude, knowledge, and behavior with the decrease in incidence rate of clinical mastitis (IRCM). Farmer-diagnosed clinical mastitis cases were obtained from two surveys conducted before (July 2004-June 2005) and at the end (2009) of a mastitis control program in the Netherlands. Information on farmers' attitude, knowledge, and behavior was also obtained by sending the farmers the same questionnaire during both surveys. Multivariable linear regression models identified that the herd level 2004 IRCM explained 28% of the variation in the decrease of IRCM. Changes in farmers' attitude and knowledge, and changes in farmers' behavior additionally explained 24% and 5%, respectively. These results suggest that the way management measures are executed may be at least as important as the fact that they are executed. No control group was available for this study because the intervention was applied at the national level. We therefore do not claim any causal relationships.

Hyperketonemia during lipopolysaccharide-induced mastitis affects systemic and local intramammary metabolism in dairy cows

Hyperketonemia interferes with the metabolic regulation in dairy cows. It is assumed that metabolic and endocrine changes during hyperketonemia also affect metabolic adaptations during inflammatory processes. We therefore studied systemic and local intramammary effects of elevated plasma β-hydroxybutyrate (BHBA) before and during the response to an intramammary lipopolysaccharide (LPS) challenge. Thirteen dairy cows received intravenously either a Na-DL-β-OH-butyrate infusion (n = 5) to achieve a constant plasma BHBA concentration (1.7 ± 0.1 mmol/L), with adjustments of the infusion rates made based on immediate measurements of plasma BHBA every 15 min, or an infusion with a 0.9% NaCl solution (control; n = 8) for 56 h. Infusions started at 0900 h on d 1 and continued until 1700 h 2 d later. Two udder quarters were challenged with 200 μg of Escherichia coli LPS and 2 udder quarters were treated with 0.9% saline solution as control quarters at 48 h after the start of infusion. Blood samples were taken at 1 wk and 2h before the start of infusions as reference samples and hourly during the infusion. Mammary gland biopsies were taken 1 wk before, and 48 and 56 h (8h after LPS challenge) after the start of infusions. The mRNA abundance of key factors related to BHBA and fatty acid metabolism, and glucose transporters was determined in mammary tissue biopsies. Blood samples were analyzed for plasma glucose, BHBA, nonesterified fatty acid, urea, insulin, glucagon, and cortisol concentrations. Differences were not different for effects of BHBA infusion on the mRNA abundance of any of the measured target genes in the mammary gland before LPS challenge. Intramammary LPS challenge increased plasma glucose, cortisol, glucagon, and insulin concentrations in both groups but increases in plasma glucose and glucagon concentration were less pronounced in the Na-DL-β-OH-butyrate infusion group than in controls. In response to LPS challenge, plasma BHBA concentration decreased in controls and decreased also slightly in the BHBA-infused animals because the BHBA concentration could not be fully maintained despite a rapid increase in BHBA infusion rate. The change in mRNA abundance of citrate synthase in LPS quarters was significant between the 2 treatment groups. The results indicate that elevated circulating BHBA concentration inhibits gluconeogenesis before and during immune response to LPS challenge, likely because BHBA can replace glucose as an energy source.

[Evaluation of risk factors for Mastitis-Metritis-Agalactia in pig farms in Switzerland].

Mastitis-Metritis-Agalactia (MMA), also known as postpartum dysgalactia syndrome (PPDS) is the most important disease complex in sows after birth. The present study compared 30 MMA problem herds (over 12% of farrowing sows affected) with 30 control farms (less than 10% of farrowing sows affected) to identify risk factors and treatment incidence. Important risk factors identified were in gilts the integration into the herd after the first farrowing, in gestating sows firm fecal consistency as well as in lactating sows soiled troughs, a low flow rate (<2 liters per minute) in drinking nipples and a high prevalence of lameness. The treatment incidence was also significantly different between the two groups. The MMA prevalence could be reduced through optimization of husbandry, feeding and management, which could essentially diminish the use of antibiotics.

Fecal coliform contamination in child day care environments in Houston, Texas

Outbreaks of diarrhea are common among children in day care centers (DCC). Enteropathogens associated with these outbreaks are spread by the fecal-oral route through contaminated hands or environmental objects. This prospective study was undertaken to determine the prevalence of fecal coliform (FC) contamination in the DCC environment. Ten rooms in 6 DCC housing 121 children $<$2 years of age were studied for 13 weeks. Inanimate objects (1275), toy balls (724), and hands (954) were cultured 1-3 times per week. FC contamination was common during each week of study and was significantly (p $<$ 0.05) greater for objects, toy balls, and hands of children in toddler compared to infant rooms. In 5 rooms in which clothes were worn over diapers, there was a significantly lower prevalence of FC of toy balls (p $<$ 0.005), inanimate objects (p $<$ 0.05), and hands of children (p $<$ 0.001) and caregivers (p $<$ 0.05) when compared to rooms in which overclothes were not worn. Occurrence of diarrhea was significantly associated with increased contamination of caregivers' and children's hands. Using plasmid analysis of trimethoprim (TMP)-resistant Escherichia coli, stool and environmental isolates from individual DCC rooms had the same plasmid patterns, which were unique to each center. In summary, FC of environmental isolates and hands of children and caregivers in DCC is common; toy balls can serve as sentinels of contamination; FC can be significantly decreased by use of clothes worn over diapers; and plasmid analysis of E. coli strains showed the same patterns from stool and environmental isolates. ^