Gap analysis of Mycoplasma bovis disease, diagnosis and control: An aid to identify future development requirements.

There is a worldwide problem of disease caused by Mycoplasma (M.) bovis in cattle; it has a significant detrimental economic and animal welfare impact on cattle rearing. Infection can manifest as a plethora of clinical signs including mastitis, pneumonia, arthritis, keratoconjunctivitis, otitis media and genital disorders that may result in infertility and abortion. Current diagnosis and control information are reviewed and analysed to identify gaps in knowledge of the causative organism in respect of the disease pathology, diagnosis and control methods. The main considerations are as follows: no vaccines are commercially available; antimicrobial resistance is increasing; diagnostic and antimicrobial sensitivity testing needs to be improved; and a pen-side test would facilitate more rapid diagnosis and implementation of treatment with antimicrobials. More data on host susceptibility, stress factors, immune response and infectious dose levels are required. The impact of asymptomatic carriers, M. bovis survival in the environment and the role of wildlife in transmitting the disease also needs investigation. To facilitate development of vaccines, further analysis of more M. bovis genomes, its pathogenic mechanisms, including variable surface proteins, is required, along with reproducible disease models.

Effect of atmospheric carbon dioxide concentration on the cultivation of bovine Mycoplasma species.

Recommendations for bovine mycoplasma culture CO2 concentrations are varied and were not empirically derived. The objective of this study was to determine whether the growth measures of bovine mycoplasma isolates differed when incubated in CO2 concentrations of 10 or 5% or in candle jars (2.7 ± 0.2% CO2). Growth of Mycoplasma bovis (n = 22), Mycoplasma californicum (n = 18), and other Mycoplasma spp. (n = 10) laboratory isolates was evaluated. Isolate suspensions were standardized to approximately 108 cfu/mL and serially diluted in pasteurized whole milk to achieve test suspensions of 102 and 106 cfu/mL. One hundred microliters of each test dilution was spread in duplicate onto the surface of a modified Hayflick's agar plate. Colony growth was enumerated on d 3, 5, and 7 of incubation. A mixed linear model included the fixed effects of CO2 treatment (2.7, 5, or 10%), species, day (3, 5, or 7), and their interactions, with total colony counts as the dependent variable. Carbon dioxide concentration did not significantly affect overall mycoplasma growth differences, but differences between species and day were present. Colony counts (log10 cfu/mL) of M. bovis were 2.6- and 1.6-fold greater than M. californicum and other Mycoplasma spp., respectively. Growth at 7 d of incubation was greater than d 3 and 5 for all species. These findings were confirmed using field isolates (n = 98) from a commercial veterinary diagnostic laboratory. Binary growth responses (yes/no) of the field isolates were not different between CO2 treatments but did differ between species and day of incubation. On average, 57% of all field isolates were detected by 3 d of incubation compared with 93% on d 7. These results suggest that the range of suitable CO2 culture conditions and incubation times for the common mastitis-causing Mycoplasma spp. may be broader than currently recommended.

Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis.

Staphylococcus hyicus and Staphylococcus agnetis are two coagulase-variable staphylococcal species that can be isolated from bovine milk and are difficult to differentiate. The objectives of this study were to characterize isolates of bovine milk origin from a collection that had previously been characterized as coagulase-positive S. hyicus based on phenotypic species identification methods and to develop a PCR-based method for differentiating S. hyicus, S. agnetis, and Staphylococcus aureus Isolates (n = 62) were selected from a previous study in which milk samples were collected from cows on 15 dairy herds. Isolates were coagulase tested and identified to the species level using housekeeping gene sequencing. A multiplex PCR to differentiate S. hyicus, S. agnetis, and S. aureus was developed. Pulsed-field gel electrophoresis was conducted to strain type the isolates. Based on gene sequencing, 44/62 of the isolates were determined to be either S. agnetis (n = 43) or S. hyicus (n = 1). Overall, 88% (37/42) of coagulase-positive S. agnetis isolates were found to be coagulase positive at 4 h. The herd-level prevalence of coagulase-positive S. agnetis ranged from 0 to 2.17%. Strain typing identified 23 different strains. Six strains were identified more than once and from multiple cows within the herd. Three strains were isolated from cows at more than one time point, with 41 to 264 days between samplings. These data suggest that S. agnetis is likely more prevalent on dairy farms than S. hyicus Also, some S. agnetis isolates in this study appeared to be contagious and associated with persistent infections.

Identification of factors influencing teat dip efficacy trial results by meta-analysis.

Two meta-analyses were conducted using data from peer-reviewed natural exposure (NE) and experimental challenge (EC) teat dip efficacy trials to identify factors influencing the new intramammary infection (IMI) rate. A NE data set containing 16 studies and an EC data set containing 21 studies were created. New IMI rate was calculated based on the percentage of new quarter infections per month (PNQI/mo) for each observation, in both data sets, and used as the dependent variable for model derivation. A linear, mixed-effects model with a random study effect, weighted by number of quarters eligible for infection, was derived for each data set. The final NE model included the effects of experimental design (split herd or split udder), mastitis pathogen group (Staphylococcus aureus, Streptococcus agalactiae, environmental streptococci, gram-negative species, Corynebacterium spp., or coagulase-negative staphylococci), postmilking treatment (iodine, chlorhexidine, linear dodecyl benzene sulfonic acid, chlorine compounds, phenol compounds, or undipped negative controls), and the interaction between mastitis pathogen group and postmilking treatment. Overall, Corynebacterium spp. had the highest new IMI rate (0.0139±0.0018 PNQI/mo), and environmental streptococci and gram-negative species had the lowest (0.0023±0.0022 PNQI/mo). Additionally, trials utilizing a split herd experimental design had a 2-fold higher new IMI rate than trials using a split udder design. The final EC model included the effects of mastitis pathogen (Staph. aureus and Strep. agalactiae), postmilking treatment (iodine, chlorine compounds, "other" active ingredients, or undipped negative controls), geographic region of study (Eastern, Southern, and Pacific Northwest), and the 2-way interactions of region and pathogen group and postmilking treatment and pathogen group. Overall, Staph. aureus and Strep. agalactiae had similar new IMI rates. Quarters dipped postmilking in either iodine (0.0127±0.0099 PNQI/mo), chlorine compounds (0.0258±0.0095 PNQI/mo), or "other" active ingredient teat dips (0.0263±0.0106 PNQI/mo) had lower new IMI rates than undipped quarters (0.0859±0.0087 PNQI/mo). These results indicate that experimental design influences the new IMI rate of teat dip efficacy trials and that using an effective postmilking teat dip has a greater effect on controlling the new Staph. aureus and Strep. agalactiae IMI rate than the teat dip's active ingredient.

Short communication: Removal of hair from the mammary gland: Recovery of bacteria from teat skin and milk.

It has been assumed that the presence of udder hair can interfere with safe milking practices and reduce the wholesomeness of milk relative to bacterial content. This study determined the effect of removal by singeing udder hair on the microflora of teat skin (total bacteria, coliform, and esculin-positive and esculin-negative streptococci) and milk (total bacteria, coliform, psychrotrophic, and thermoduric counts) as opposed to not singeing udder hair, using different pre-and postmilking disinfection (predip, postdip, or both) combinations. The 4 different pre-and postmilking disinfection combinations were predip and postdip, postdip only, predip only, and no predip and no postdip. Differences in bacterial numbers recovered from teat skin and milk in singed and not singed glands were not significantly affected by treatment. Findings of this trial do not support the concept that udder hair removal results in improved milk quality as measured by bacterial content.

Reduction of teat skin mastitis pathogen loads: differences between strains, dips, and contact times.

The purpose of these experiments was to (1) assess differences in mastitis pathogen strain sensitivities to teat disinfectants (teat dips), and (2) determine the optimum time for premilking teat dips to remain in contact with teat skin to reduce pathogen loads on teat skin. Two experiments were conducted using the excised teat model. In experiment 1, the differences in mastitis pathogen strain sensitivities to 4 commercially available dips (dip A: 1% H2O2; dip B: 1% chlorine dioxide; dip C: 1% iodophor; and dip D: 0.5% iodophor) were evaluated. Four strains of 11 common mastitis pathogens (Staphylococcus aureus, Streptococcus agalactiae, Mycoplasma bovis, Streptococcus dysgalactiae, Streptococcus uberis, Escherichia coli, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus xylosus, and Staphylococcus haemolyticus) were tested. In experiment 2, the percentage log reduction of mastitis pathogens (Escherichia coli, Streptococcus uberis, Streptococcus dysgalactiae, Klebsiella species, Staphylococcus chromogenes, Staphylococcus haemolyticus, Staphylococcus xylosus, and Staphylococcus epidermidis) on teat skin with 3 commercially available teat dips: dip A; dip D; and dip E: 0.25% iodophor, using dip contact times of 15, 30, and 45 s, was evaluated. Experiment 1 results indicated significant differences in strain sensitivities to dips within pathogen species: Staphylococcus aureus, Staphylococcus chromogenes, and Streptococcus uberis. Species differences were also found where Mycoplasma bovis (97.9% log reduction) was the most sensitive to tested teat dips and Staphylococcus haemolyticus (71.4% log reduction) the most resistant. Experiment 2 results indicated that contact times of 30 and 45 s were equally effective in reducing recovered bacteria for dips D and E and were also significantly more effective than a 15-s contact time. No differences were seen in recovered bacteria between tested contact times after treatment with dip A. It can be concluded that different mastitis pathogen species and strains within species may possess different sensitivities to teat dips, which may have implications in selection of teat dips on dairies. Furthermore, a 30-s premilking dip contact time for iodophors and 15 s for H2O2 dips may be optimal in reducing pathogen load in the shortest amount of time. A reduction in premilking teat dip contact time may improve milking parlor efficiency.

Evaluation of a clay-based acidic bedding conditioner for dairy cattle bedding.

This study investigated the effects of a clay-based acidic bedding conditioner on sawdust bedding pH, dry matter (DM), environmental pathogen counts, and environmental bacterial counts on teat ends of lactating dairy cows. Sixteen lactating Holstein cows were paired based on parity, days in milk, milk yield, and milk somatic cell count, and were negative for the presence of an intramammary pathogen. Within each pair, cows were randomly assigned to 1 of 2 treatments with 3-wk periods in a crossover design. Treatment groups consisted of 9 freestalls per group bedded with either untreated sawdust or sawdust with a clay-based acidic bedding conditioner, added at 3- to 4-d intervals over each 21-d period. Bedding and teat ends were aseptically sampled on d 0, 1, 2, 7, 14, and 21 for determination of environmental bacterial counts. At the same time points, bedding was sampled for DM and pH determination. The bacteria identified in the bedding material were total gram-negative bacteria, Streptococcus spp., and coliform bacteria. The bacteria identified on the teat ends were Streptococcus spp., coliform bacteria, and Klebsiella spp. Teat end score, milk somatic cell count, and intramammary pathogen presence were measured weekly. Bedding and teat cleanliness, environmental high and low temperatures, and dew point data were collected daily. The bedding conditioner reduced the pH, but not the DM, of the sawdust bedding compared with untreated sawdust. Overall environmental bacterial counts in bedding were lower for treated sawdust. Total bacterial counts in bedding and on teat ends increased with time over both periods. Compared with untreated sawdust, the treated bedding had lower counts of total gram-negative bacteria and streptococci, but not coliform counts. Teat end bacterial counts were lower for cows bedded on treated sawdust for streptococci, coliforms, and Klebsiella spp. compared with cows bedded on untreated sawdust. The clay-based acidic bedding conditioner reduced environmental pathogens in sawdust bedding and teat ends without affecting teat end integrity.

Intramammary infections and teat canal colonization with coagulase-negative staphylococci after postmilking teat disinfection: species-specific responses.

Coagulase-negative staphylococci (CNS) are the most common pathogens associated with intramammary infections (IMI) in dairy cows. We hypothesized that postmilking teat disinfection would reduce microbial colonization of the teat canal and thus reduce the prevalence of IMI caused by certain CNS species. The efficacy of iodine postmilking teat dip was tested against CNS colonization of the teat canal, and incidence of IMI was measured. Using an udder-half model, 43 Holstein cows at the Washington State University Dairy were enrolled in the trial; postmilking teat dip was applied to one udder-half, treatment (TX), and the remaining half was an undipped control (CX). Teat canal swabbing and mammary quarter milk samples were taken in duplicate once a week for 16 wk for microbial culture. Isolates from agar cultures were presumptively identified as CNS and then speciated using PCR-RFLP and agarose gel electrophoresis. Colonization of the teat canal and IMI by CNS were assessed. Thirty CNS IMI were diagnosed and the number of new IMI in CX quarters (21) was significantly greater than that in TX mammary quarters (9). The majority of CNS IMI were caused by Staphylococcus chromogenes (30%) and Staphylococcus xylosus (40%), and the latter were appreciably reduced by teat dip. Except for S. xylosus, an association was observed between teat canal colonization and IMI by all CNS species in this study, in which the majority of IMI were preceded by teat canal colonization. The total number of CNS IMI was greater for CX group cows compared with TX group cows. However, the effect of disinfection on IMI did not appear to be the same for all CNS species.

Invited review: Mastitis in dairy heifers: nature of the disease, potential impact, prevention, and control.

Heifer mastitis is a disease that potentially threatens production and udder health in the first and subsequent lactations. In general, coagulase-negative staphylococci (CNS) are the predominant cause of intramammary infection and subclinical mastitis in heifers around parturition, whereas Staphylococcus aureus and environmental pathogens cause a minority of the cases. Clinical heifer mastitis is typically caused by the major pathogens. The variation in proportions of causative pathogens between studies, herds, and countries is considerable. The magnitude of the effect of heifer mastitis on an individual animal is influenced by the form of mastitis (clinical versus subclinical), the virulence of the causative pathogen(s) (major versus minor pathogens), the time of onset of infection relative to calving, cure or persistence of the infection when milk production has started, and the host's immunity. Intramammary infection in early lactation caused by CNS does not generally have a negative effect on subsequent productivity. At the herd level, the impact will depend on the prevalence and incidence of the disease, the nature of the problem (clinical, subclinical, nonfunctional quarters), the causative pathogens involved (major versus minor pathogens), the ability of the animals to cope with the disease, and the response of the dairy manager to control the disease through management changes. Specific recommendations to prevent and control mastitis in late gestation in periparturient heifers are not part of the current National Mastitis Council mastitis and prevention program. Control and prevention is currently based on avoidance of inter-sucking among young stock, fly control, optimal nutrition, and implementation of hygiene control and comfort measures, especially around calving. More risk factors for subclinical and clinical heifer mastitis have been identified (e.g., season, location of herd, stage of pregnancy) although they do not lend themselves to the development of specific intervention strategies designed to prevent the disease. Pathogen-specific risk factors and associated control measures need to be identified due to the pathogen-related variation in epidemiology and effect on future performance. Prepartum intramammary treatment with antibiotics has been proposed as a simple and effective way of controlling heifer mastitis but positive long-lasting effects on somatic cell count and milk yield do not always occur, ruling out universal recommendation of this practice. Moreover, use of antibiotics in this manner is off-label and results in an increased risk of antibiotic residues in milk. Prepartum treatment can be implemented only as a short-term measure to assist in the control of a significant heifer mastitis problem under supervision of the herd veterinarian. When CNS are the major cause of intramammary infection in heifers, productivity is not affected, making prepartum treatment redundant and even unwanted. In conclusion, heifer mastitis can affect the profitability of dairy farming because of a potential long-term negative effect on udder health and milk production and an associated culling risk, specifically when major pathogens are involved. Prevention and control is not easy but is possible through changes in young stock and heifer management. However, the pathogenesis and epidemiology of the disease remain largely unknown and more pathogen-specific risk factors should be identified to optimize current prevention programs.

A PCR assay and PCR-restriction fragment length polymorphism combination identifying the 3 primary Mycoplasma species causing mastitis.

The focus of the current research was to develop real-time PCR assays with improved sensitivity and the capacity to simultaneously speciate the 3 most common mycoplasma mastitis agents: Mycoplasma bovis, Mycoplasma californicum, and Mycoplasma bovigenitalium. Real-time PCR was chosen because it provides rapid results. Partial 16S rRNA gene sequencing was used as the gold standard for evaluating candidate real-time PCR assays. To ascertain the real-time PCR assay specificity, reference strains of Mycoplasma species, Acholeplasma axanthum, and common gram-positive and gram-negative mastitis pathogens were tested. No cross-reactions were observed. Mycoplasma spp. isolated from bovine milk samples (n=228) and other organ sites (n=40) were tested by the real-time PCR assays and the partial 16S rRNA gene sequencing assay. Overall accuracy of this novel real-time PCR was 98.51%; 4 of 228 isolates identified as M. bovis by the partial 16S rRNA gene sequencing assay were identified as both M. bovis and M. californicum by real-time PCR. Subsequent amplicon sequencing suggested the presence of both M. bovis and M. californicum in these 4 samples. Using a cycle threshold of 37, the detection limits for real-time PCR were 10 copies of DNA template for both M. bovis and M. bovigenitalium, and 1 copy for M. californicum. This real-time PCR assay is a diagnostic technique that may be used as a screening tool or as a confirmation test for mycoplasma mastitis.

Incidence and transmission of Mycoplasma bovis mastitis in Holstein dairy cows in a hospital pen: A case study.

The objective was to determine the incidence and transmission of mycoplasma mastitis in the hospital pen in a dairy herd of 650 lactating cows after a hospital pen was established following an outbreak of this disease. Mycoplasma mastitis status was monitored for 3 months through repeated collection of milk samples from cows with clinical mastitis (CM) and from bulk tank milk. During the outbreak 13 cows were diagnosed with Mycoplasma bovis CM, 1 cow with Mycoplasma sp. mastitis and 8 cows showed signs of arthritis, 3 of which were confirmed as having M. bovis arthritis. M. bovis isolates from cows with CM, arthritis and bulk tank milk had indistinguishable chromosomal digest pattern fingerprints. Incidence rates of M. bovis CM cases in the milking and hospital pens were 0.01 and 1.7 cases per 100 cow-days at risk. Approximately 70% of cows with M. bovis CM became infected within 12 days of entering the hospital pen. Transmission of M. bovis in the hospital pen occurred as 3 episodes. Each episode corresponded to the introduction of a cow with M. bovis CM from a milking pen. Evidence indicates that cows with M. bovis CM from milking pens were the source of transmission of the disease in the hospital pen and thus their presence in the hospital pen appeared to be a risk factor for transmission of M. bovis mastitis in this single case study herd.

Factors associated with concentrations of select cytokine and acute phase proteins in dairy cows with naturally occurring clinical mastitis.

The objective of the current observational study was to determine the potential associations between cow factors, clinical mastitis (CM) etiology, and concentrations of select acute phase proteins and cytokines in milk from affected quarters of cows with CM. Cows with CM (n=197) were grouped based on systemic disease severity, milk culture result, parity, days in milk (DIM), previous CM occurrence, and season of the year when CM occurred. Concentrations of lipopolysaccharide-binding protein (LBP), haptoglobin (Hp), BSA, IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-8, IL-10, IL-12, transforming growth factor (TGF)-alpha, and TGF-beta and activity of lactate dehydrogenase (LDH) were evaluated. Differences in the least squares means log(10) transformed concentrations of these proteins were compared using multiple linear regression mixed models. The milk concentrations of LBP, Hp, IL-1beta, IL-10, and IL-12, and activity of LDH in milk were higher in cows with moderate to severe versus mild systemic disease. The concentrations of Hp, BSA, IL-1beta, and IL-10 in milk were higher in cows with a gram-negative versus gram-positive milk culture result. Season of the year when CM occurred was associated with the concentration of all proteins evaluated except for IL-1beta and IL-12. Concentrations were higher in the winter versus summer except for Hp and TGF-beta, for which the opposite was true. Concentrations of LBP, IL-10, and IL-12, and LDH activity in milk were associated with DIM group. Except for LBP, these proteins were lower in cows with CM during the first 60 DIM versus those in mid or later lactation. Interferon-gamma, TNF-alpha, and IL-8 were undetectable in 67, 31, and 20% of samples, respectively. Detection of IFN-gamma and IL-8 was associated with season, and detection of TNF-alpha and IL-8 was associated with systemic disease severity. The current study provides the most comprehensive report of milk concentrations of innate immune response proteins in cows with naturally occurring CM and identifies factors that potentially influence those concentrations. Further investigation into the seasonal variation of cytokine production and its potential effect on the outcome of CM is warranted. Furthermore, the results of this study provide useful data for planning future studies examining the role of the innate immune response in CM.

Effects of storage methods on the recovery of Mycoplasma species from milk samples.

Mycoplasma species are fastidious microorganisms causing mastitis in dairy cows. Storage by freezing milk samples affects their viability. The purpose of this study was to compare the effect of alternative storage methods on their recoverability. In Experiment I, mycoplasma counts from fresh milk samples were compared to those same samples stored for 1, 3, and 5 days at refrigerated (5 degrees C) temperatures. Experiment II was done to compare the mycoplasma counts of fresh milk samples with those stored frozen (-20 degrees C) with addition of 0%, 10%, 30% and 50% glycerol (v/v). Two strains of each of 5 species: M. bovis, M. californicum, M. bovigenitalium, M. canadense and M. alkalescens, were selected and inoculated into bulk tank milk free of this pathogen. Compared to those in fresh milk samples, counts were approximately reduced by: 0.3 log(10)CFU/ml in 5 day refrigerated milk (P<0.05) and by 1.0 log(10)CFU/ml in milk frozen without glycerol (P<0.05). Addition of glycerol (10% and 30%, v/v) to milk samples increased the number of recovered Mycoplasma by up to 0.4 log(10)CFU/ml in frozen milk samples (P<0.05). No significant interactions were detected between either Mycoplasma species or starting concentration and survival as effected by storage method. Refrigerating milk samples for 5 days and freezing milk samples lowers the number of recovered Mycoplasma species. The addition of glycerol to achieve 10% and 30% v/v solutions improves the recovery of Mycoplasma species from frozen milk samples. To maximize detection of this pathogen, fresh milk samples should be cultured without storage.

Association between an outbreak strain causing mycoplasma bovis mastitis and its asymptomatic carriage in the herd: a case study from Idaho, USA.

The objective of this study was to determine the association between mycoplasma mastitis and colonization of mycoplasma organisms at body sites of asymptomatic carriers. The investigation was done in a dairy herd with a first outbreak of mycoplasma mastitis. Milk and swab solution specimens from accessible mucosal surfaces of body sites from cows and replacements were sampled at quarterly intervals (Herd Samplings 1-4). Samples were cultured and Mycoplasma spp. were isolated, speciated and fingerprinted. During Herd Sampling 1 two cows with mycoplasma bovis mastitis were identified and all swabbing solutions of body site samples from 18 of 84 cows and 36 of 77 replacements were positive to Mycoplasma bovis and fingerprinted as the same strain. A case of clinical M. bovis mastitis developed during Herd Sampling 3. During Herd Samplings 2-4, 4 lactating cows and 12 replacements were positive to M. bovis at various body sites with 4 different strains. Three isolates of Mycoplasma californicum were found from swabbing solutions of three cows during Herd Samplings 3 and 4. Only one strain of M. bovis caused mastitis although four strains were isolated from body sites of animals. Isolation of M. bovis from a body site never preceded mastitis. No lactating cow developed mastitis during Herd Sampling 4 although some animals were colonized with the organism. It appears that during the initial outbreak of M. bovis mastitis colonization of body sites by the outbreak strain may be common. However, the prevalence of colonization subsides and colonization does not appear to precede mastitis.

Multilocus variable-number tandem-repeat analysis and plasmid profiling to study the occurrence of blaCMY-2 within a pulsed-field gel electrophoresis-defined clade of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium circulating in food animal populations and carrying resistance to antimicrobial agents represents a human health risk. Recently, a new clade of S. Typhimurium, WA-TYP035/187, was reported in cattle and humans in the Pacific Northwest, United States of America. The objective of this study was to describe a possible mechanism of acquisition of expanded-spectrum cephalosporin resistance in this clade. Ceftazidime resistance increased steadily among WA-TYP035/187 isolates, from 0% (0/2) in 1999 to 77.8% (28/36) in 2006 (chi2 for linear trend, P value of <0.001). Among 112 bovine-source and 18 human-source isolates, 49 (43.8%) and 12 (66.7%) were resistant to ceftazidime, respectively. Multiple-locus variable-number tandem-repeat analysis (MLVA) and plasmid profiling suggested that resistance was acquired by multiple independent genetic events within the WA-TYP035/187 clade. Given the lack of an obvious reservoir in species other than cattle and a parallel rise in ceftiofur resistance in the bovine-specific serovar Salmonella enterica serovar Dublin in the same time frame and region, selection pressure due to the use of the expanded-spectrum cephalosporin drug ceftiofur in cattle is a likely factor driving the increasing cephalosporin resistance of WA-TYP035/187.

Introduction of new multidrug-resistant Salmonella enterica strains into commercial dairy herds.

A longitudinal observational study of 59 dairy herds was conducted in Washington State to estimate the rate of introduction of new multidrug-resistant (MDR) Salmonella enterica strains onto commercial dairy herds. Samples were collected on these herds over 7 visits separated by intervals of 2 to 4 mo over a period of 15 to 21 mo. Samples were cultured for Salmonella spp. and serogroup, serovar, and antimicrobial susceptibility patterns were identified for MDR Salmonella isolates. Fingerprinting generated by pulsed-field gel electrophoresis (PFGE) using XbaI restriction enzyme digestion generated genotyping profiles for all MDR isolates identified in the study. The rate of new MDR Salmonella strain introduction was 0.9 per herd-year (95% confidence interval: 0.6-1.4). The rates for the most commonly introduced MDR Salmonella serovars were 0.4/herd-year for Typhimurium, 1.2/herd-year for Newport, and 0.1/herd-year for Dublin. Thirty-three of 59 herds (56%) had at least one new MDR Salmonella introduction during the study period. The number of new MDR Salmonella strains acquired by dairy herds ranged from zero to 8. Thirteen of the 59 herds had a history of clinical salmonellosis. Among these 13 herds, 6 herds acquired new MDR Salmonella strains, although these strains were different than historical clinical strains. These data indicate that acquisition of new MDR Salmonella strains by dairy herds was a common event in participating herds, although the number of strains introduced varied greatly among herds.

The role of animal movement, including off-farm rearing of heifers, in the interherd transmission of multidrug-resistant Salmonella.

Fifty-nine commercial dairy farms were sampled 7 times over 15 to 21 mo to determine the role of animal movement, including off-farm rearing of heifers, in the interherd transmission of multidrug-resistant (MDR) Salmonella spp. Farm management data were collected by on-site inspections and questionnaires on herd management practices before and after the study. Forty-four percent (26/59) of herds did not acquire any new MDR Salmonella strains. The number of newly introduced MDR Salmonella strains acquired by the remaining 56% (33/59) of herds ranged from 1 to 8. Logistic regression models indicated that off-farm heifer raising, including contract heifer raising where heifers commingle with cattle from other farms [commingled heifers, odds ratio (OR) = 8.9, 95% confidence interval (CI): 2.4, 32.80], and herd size per 100-animal increment (herd size, OR = 1.04, 95% CI, 1.01, 1.05) were significantly associated with the introduction of new MDR Salmonella strains. The negative binomial regression similarly revealed that commingled heifers [relative risk (RR) = 2.3, 95% CI: 1.1, 4.7], herd size per 100 animals (RR = 1.02, 95% CI, 1.01, 1.03), and a history of clinical salmonellosis diagnosed before the study (RR = 2.5, 95% CI, 1.3, 5.0) were significantly associated with the number of new MDR Salmonella strains that were introduced. Factors not associated with the introduction of new MDR Salmonella strains were housing of heifers and cows in the same close-up pen, a common hospital-maternity pen, and the number of purchased cattle. This study highlights the role of animal movement in the interherd transmission of MDR Salmonella spp.

Short communication: The effect of centrifugation and resuspension on the recovery of Mycoplasma species from milk.

Low sensitivity of a single bulk tank milk culture is a major limitation for detection of mycoplasma organisms. We hypothesized that sedimentation of Mycoplasma spp. in a milk sample by centrifugation followed by resuspension in a small volume of fluid before agar plating would increase the ability to detect Mycoplasma spp. compared with direct conventional culture. The experiment was conducted to determine recovery of Mycoplasma spp. from milk as affected by 1) treatment (centrifugation vs. conventional method); 2) 2 species (Mycoplasma bovis and Mycoplasma californicum and 4 strains for each species); and 3) 4 different concentrations of Mycoplasma spp. (1,000, 100, 10, and 1 cfu/mL). A 5-mL portion of mycoplasma suspension from each strain was inoculated into 45 mL of fresh bulk tank milk to achieve concentrations of 1,000, 100, 10, and 1 cfu/mL. Treatment samples were vigorously mixed and centrifuged at 5,000 x g for 30 min. Control samples were vigorously mixed. All samples were plated on modified Hayflick agar. Plates were incubated at 37 degrees C and 5% CO(2) for 5 d. Mean (+/-SE) log(10) mycoplasma counts (cfu/mL) in the treatment groups (1.91 +/- 0.15) were higher than those in the control groups (1.70 +/- 0.16). Recovery of at least 1 mycoplasma colony on agar culture was 100% in both treatment and control groups at high, medium, and low concentrations. At the lowest concentration, recovery of at least 1 mycoplasma colony on agar culture in treatment and control groups was 75% (n = 12/16) and 18.75% (n = 3/16), respectively. Centrifugation of milk followed by suspension in a smaller volume of saline before conventional culture increased the ability to detect mycoplasma microorganisms in the milk sample compared with controls. Recovery by centrifugation appeared best at the lowest concentration where detection of a positive sample was 4 times more likely than when conventional methods were used.

Factors associated with the risk of antibiotic residues and intramammary pathogen presence in milk from heifers administered prepartum intramammary antibiotic therapy.

Heifers (n=136) from 5 herds were treated with a commercially available beta-lactam intramammary (IMM) antibiotic preparation containing cephapirin sodium at 10-21 d prior to anticipated parturition to evaluate the risk of antibiotic residues occurring in milk postpartum and to determine factors associated with antibiotic residues and IMM pathogen presence in milk postpartum. Mammary secretions collected from quarters before antibiotic administration and during weeks 1, 2 and 3 postpartum were analyzed for mastitis pathogens. Composite milk was collected at milkings 3, 6 and 10 postpartum and analyzed for beta-lactam residues using a microbial inhibition antibiotic residue screening test. Antibiotic residues were confirmed with beta-lactamase treatment and re-tested for residues. Residues were detected in 28.0, 8.82 and 3.68% of milk samples obtained at the third, sixth, and tenth milking postpartum, respectively. Increases in interval between prepartum antibiotic therapy and parturition and an increase in the postpartum interval to sampling were associated with a decrease in risk of antibiotic residues. The presence of antibiotic residues in milk at the third milking was associated with a reduced risk for IMM pathogen prevalence in the first 21 d postpartum. Lower somatic cell counts, an increase in mean milk yield over 200 days in milk and a reduction in IMM pathogen prevalence were associated with the presence of an antibiotic in milk postpartum. Screening milk for antibiotic residues in milk postpartum following prepartum antibiotic therapy in heifers is recommended to reduce the risk for antibiotic residue contamination of milk.

Prevalence, incidence and risk factors of heifer mastitis.

Traditionally heifers, as calves and as primiparae, have been thought of as a group as free of mastitis. Without appreciable lacteal secretion, there is reduced nutrient fluid available to support growth of intramammary pathogens. Contagious mastitis is primarily transmitted at milking time and the milking process affects the patency of the teat orifice which can increase the risk of development of environmental mastitis. Logically therefore prepartum heifers should be free of intramammary infections. During the last 20 years there have been numerous investigations describing the nature of mastitis in heifers and thus the dogma that heifers are free of this disease has been challenged. The purpose of this manuscript is to review that literature describing heifer intramammary infections that cause both subclinical and clinical disease. Mammary quarter infection prevalence ranges between 28.9-74.6% prepartum, and 12.3-45.5% at parturition. Generally, the pathogens that cause mastitis in heifers are the same as those that cause infections in the older cows. In all but one study reviewed, coagulase-negative staphylococci (CNS) are the most prevalent cause of subclinical intramammary infections in heifers. Coagulase-positive staphylococci (CPS) in some studies are the second most prevalent pathogens, while in other studies the environmental mastitis pathogens are more prevalent. The risk factors for subclinical mastitis appear to be season, herd location, and trimester of pregnancy; all suggesting that management can have an impact in control of this disease prepartum. With respect to clinical mastitis, the most prevalent mastitis pathogen has been reported to be CNS in one study and CPS, or environmental mastitis pathogens, in other studies. The heifer is most at risk for clinical mastitis during the periparturient period. Risk factors found are related to diet, mammary gland factors such as edema and leaking of milk, and factors associated with the change in management and introduction of the heifer to the milking herd.