Comparison of Minimum Inhibitory Concentrations of Selected Antimicrobials for Non-Aureus Staphylococci, Enterococci, Lactococci, and Streptococci Isolated from Milk Samples of Cows with Clinical Mastitis.

The objective of this study was to compare the minimum inhibitory concentrations of antimicrobials included in a commercial broth microdilution panel among Gram-positive pathogens that caused non-severe clinical mastitis on three Michigan dairy farms. Duplicate quarter milk samples were collected from eligible quarters of cows enrolled in a randomized clinical trial, cultured in a university laboratory, and identified using MALDI-TOF. Etiologies were grouped by genus as Enterococcus species (n = 11), Lactococcus species (n = 44), non-aureus Staphylococcus species (n = 39), or Streptococcus species (n = 25). Minimum inhibitory concentrations (MICs) were determined using the mastitis panel of a commercially available broth microdilution test. In vitro susceptibility was determined using approved guidelines and included breakpoints for mastitis pathogens, or when not available, breakpoints from other species. Most isolates were inhibited at or below breakpoints that demonstrated in vitro susceptibility. The proportions of susceptible isolates varied among pathogens for pirlimycin, penicillin, and tetracycline. The greatest proportion of resistance was observed for pirlimycin, tetracycline, and sulfadimethoxine. Survival analysis was performed to evaluate differences in MICs among pathogen groups. MIC values varied among pathogens for ceftiofur, cephalothin, erythromycin, penicillin, pirlimycin, and tetracycline. However, nearly all isolates were susceptible to ceftiofur and cephalothin, indicating that pathogen differences in MIC are not likely clinically relevant, as these are the two most commonly administered mastitis treatments in the United States. While differences in vitro susceptibility were observed for some antimicrobials, susceptibility was high to cephalosporin-based IMM treatments that are most commonly used and did not vary among pathogens.

Options for the control of bovine leukemia virus in dairy cattle.

The subclinical impact of bovine leukemia virus (BLV) on the sustainability of the US dairy industry is only now being fully recognized. Findings of recent longitudinal studies conducted in Michigan dairy herds were consistent with the results of previous studies in showing that within-herd prevalence of BLV-infected cattle was negatively associated with milk production and cow longevity. Risk factors relating to routes of hematogenous transmission such as the use of shared hypodermic needles, shared reproductive examination sleeves, and natural breeding were associated with BLV within-herd prevalence. Few US dairy producers know the prevalence of BLV-infected cattle in their herds or are aware of the insidious economic impact of BLV or the options for BLV control. As an increasing number of countries eradicate BLV from their cattle populations, restrictions on the movement of US cattle and cattle products will likely increase. Veterinarians should be aware of recent developments for screening serum and milk samples for antibodies against BLV and the results of research regarding the economic impact of BLV so they can advise their dairy clients of available alternatives for monitoring and controlling BLV infection.

Impact of bovine leukemia virus infection on neutrophil and lymphocyte concentrations in dairy cattle.

OBJECTIVE: To determine the effect of bovine leukemia virus (BLV) infection on absolute neutrophil and lymphocyte concentrations in healthy lactating Holstein dairy cattle. DESIGN: Observational cross-sectional survey. ANIMALS: 311 healthy lactating Holstein dairy cattle from herds in Michigan (n = 2), Wisconsin (1), Iowa (1), and Pennsylvania (1). PROCEDURES: Whole and anticoagulated (EDTA) blood samples were collected. Serum samples were tested for antibody against BLV by use of an ELISA. Absolute neutrophil and lymphocyte concentrations were measured in EDTA blood samples with an automated hematology analyzer and manual differential cell counts. RESULTS: 208 cows tested positive and 103 cows tested negative for anti-BLV antibodies. Neutrophil concentration was not significantly different between BLV-positive versus BLV-negative cattle. The distribution of lymphocyte concentration was positively skewed for the entire cow population (n = 311) and the BLV-positive subset (208). In contrast, lymphocyte concentration distribution was approximately normal for BLV-negative cows (n = 103). Consequently, the presence or absence of BLV infection strongly influenced the calculated neutrophil-to-lymphocyte concentration ratio. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that absolute lymphocyte concentration is significantly affected by BLV infection in dairy cattle. Accordingly, hematologic reference intervals should be derived from healthy animals that are not infected with BLV and patient BLV status must be considered for meaningful interpretation of lymphocyte concentration. We recommend that the calculated neutrophil-to-lymphocyte ratio be abandoned because it does not provide more information than direct comparison of patient absolute leukocyte concentration with updated reference intervals from healthy BLV-negative cattle.

Herd-level determinants of bovine leukaemia virus prevalence in dairy farms.

The prevalence of bovine leukaemia virus (BLV) was determined in 113 Michigan dairy herds by ELISA testing for anti-BLV antibodies in milk. Additionally, an interview regarding management practices with cooperating herd managers identified farm-level variables thought to be associated with prevalence of BLV. Twenty-three risk factors (P ≤ 0·1) were identified on one-way ANOVA or simple linear regression. Multivariate analysis identified several management practices whose predictive value for increased prevalence of BLV may relate to transmission among herd mates, e.g. reuse of hypodermic needles, lack of fly control, gouge dehorning and increased use of injections in dry cows. Additionally, exclusive breeding of heifers with artificial insemination was associated with decreased BLV prevalence, as compared with at least some use of natural service by a bull. Although intervention studies are needed before causal relationships can be concluded, and unaccounted variables related to transmission exist among dairy herds, these findings suggest management practices that may help dairy producers reduce the transmission of BLV within their herds.

Using a herd profile to determine age-specific prevalence of bovine leukemia virus in michigan dairy herds.

Enzootic bovine leukosis is a contagious disease of cattle caused by the retrovirus, bovine leukemia virus (BLV) and is the most common cause of malignant neoplasm in cattle. In order to facilitate surveillance of this disease in dairy herds, we developed a method to combine ELISA of milk collected during routine production testing with a prescribed sampling of cows that is independent of the proportion of cows within each lactation. In 113 Michigan dairy herds, milk samples from ten cows in each of the 1st, 2nd, 3rd, and ≥4th lactations were analyzed for anti-Bovine Leukemia Virus (BLV) antibodies by milk ELISA. For each herd, a BLV herd profile (BHP) was calculated as the simple average of the percent of BLV-positive cows within each of the four lactation groups. The mean BHP for all herds was 32.8%, with means of 18.5, 28.8, 39.2, and 44.8% of 1st, 2nd, 3rd, and ≥4th lactation animals infected, respectively. In eight herds, we determined the correlation between the BHP, and true herd prevalence by testing the entire lactating herd (r = 0.988, P < 0.0001). The BHP allows discrimination of lactation-specific BLV prevalence within a dairy herd, to help identify risk factors and management plans that may be important in transmission of BLV.

Effect of infection with bovine leukosis virus on lymphocyte proliferation and apoptosis in dairy cattle.

OBJECTIVE: To determine effects of infection with bovine leukosis virus (BLV) on lymphocyte proliferation and apoptosis in dairy cattle. ANIMALS: 27 adult Holstein cows. PROCEDURES: Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood from lactating Holstein cows seronegative for BLV (n = 9 cows), seropositive for BLV and aleukemic (aleukemic; 9), and seropositive for BLV and persistently lymphocytotic (PL; 9). Isolated PBMCs were assayed for mitogen-induced proliferation and were analyzed by means of flow cytometry. The PBMCs from a subset of each group were assayed for apoptosis, caspase-9 activity, and expression of selected genes related to apoptosis. RESULTS: PL cows had significantly higher total lymphocyte counts and significantly lower proportions of T-lymphocyte populations than did BLV-negative and aleukemic cows. Both groups of BLV-infected cows had significantly higher proportions of B cells and major histocompatibility complex II-expressing cells than did BLV-negative cows. Proliferation with concanavalin A was significantly lower for PL cows, compared with proliferation for BLV-negative cows. Pokeweed mitogen-induced proliferation was significantly higher for aleukemic and PL cows than for BLV-negative cows. Gene expression of apoptosis-inhibitory proteins BCL2 and BCL2L1 was significantly higher for aleukemic cows and expression of BCL2 was significantly higher for PL cows than for BLV-negative cows. CONCLUSIONS AND CLINICAL RELEVANCE: Cattle infected with BLV had marked changes in PBMC populations accompanied by alterations in proliferation and apoptosis mechanisms. Because the relative distribution and function of lymphocyte populations are critical for immune competence, additional studies are needed to investigate the ability of BLV-infected cattle to respond to infectious challenge.

Bovine Leukemia Virus Infection in Dairy Cattle: Effect on Serological Response to Immunization against J5 Escherichia coli Bacterin.

Thirteen bovine leukemia virus- (BLV-) negative and 22 BLV-positive Holstein cows were immunized with J5 Escherichia coli bacterin at dry off, three weeks before calving, during the second week after calving, and three weeks after the third immunization. Serum was collected before the initial immunization, immediately before the third and fourth immunizations, and 21 days after the fourth immunization. Anti-J5 E. coli IgM, IgG1, and IgG2 titers were determined by ELISA. Anti-J5 E. coli IgM titers did not differ significantly (P = .98) between groups. Increases in anti-J5 E. coli IgG1 titers were higher in the BLV-negative cows (P = .057). Geometric mean anti-J5 E. coli IgG2 titers increased fourfold in the BLV-negative cows, which was significantly higher (P = .007) than the twofold increase in the BLV-positive cows. Cattle infected with BLV may have impaired serologic responses following immunization with J5 bacterin, and response may differ according to antibody isotype.

Effect of changes in number of doses and anatomic location for administration of an Escherichia coli bacterin on serum IgG1 and IgG2 concentrations in dairy cows.

OBJECTIVE: To determine effects of injection site on antibody response to J5 Escherichia coli bacterin. ANIMALS: 28 adult Holstein cows. PROCEDURES: Cows were randomly assigned as control cattle (n = 4 cows), not administered J5 E coli bacterin; 3X (8), administered 3 doses of bacterin SC in the left side of the neck; 5XN (8), administered 5 doses of bacterin SC in the left side of the neck; or 5XSR (8), administered 5 doses of bacterin SC sequentially in the left side of the neck, right side of the neck, right side of the thorax, left side of the thorax, and left side of the neck. Blood samples were collected from the cows to determine anti-J5 E coli IgG1 and IgG2 concentrations. RESULTS: Vaccinated cows had higher mean serum anti-J5 E coli IgG1 concentrations than did control cows. The 5XN and 5XSR cows had higher mean serum anti-J5 E coli IgG1 concentrations than did 3X cows. Additionally, 5XSR cows had higher mean serum anti-J5 E coli IgG1 concentrations than did 5XN cows. Vaccinated cows had higher mean serum anti-J5 E coli IgG2 concentrations than did control cows. The 5XN and 5XSR cows had higher mean serum anti-J5 E coli IgG2 concentrations than did 3X cows. The 5XSR cows had higher mean serum anti-J5 E coli IgG2 concentrations than did all other groups at 84 days after the fifth vaccination. CONCLUSIONS AND CLINICAL RELEVANCE: Sequential doses of core-antigen bacterins administered at different anatomic locations may improve antibody response in dairy cattle.

Changes in multidrug resistance of enteric bacteria following an intervention to reduce antimicrobial resistance in dairy calves.

An intervention study was conducted to determine whether discontinuing the feeding of milk replacer medicated with oxytetracycline and neomycin to preweaned calves reduced antimicrobial resistance in Salmonella, Campylobacter, and Escherichia coli bacteria. Results demonstrated that the intervention did reduce multidrug resistance in these bacteria but that other factors also influenced multidrug resistance.

Changes in tetracycline susceptibility of enteric bacteria following switching to nonmedicated milk replacer for dairy calves.

A randomized intervention study was conducted to determine if discontinuing use of calf milk replacer medicated with oxytetracycline results in increased tetracycline susceptibility in Salmonella and Campylobacter spp. and Escherichia coli in dairy calves over a 12-month period. Dairy herds with enteric bacteria with known low tetracycline susceptibility were enrolled for the study. Fecal samples from preweaned calves and environmental samples were collected from eight dairy herds in Michigan and New York State. Samples were collected monthly for 3 months prior to and 12 months after four of the eight herds discontinued medicated milk replacer feeding. Salmonella and Campylobacter spp. and E. coli were isolated, and antimicrobial susceptibility testing was conducted using automated broth microdilution. A total of 804 intervention and 1,026 control calf fecal samples and 122 intervention and 136 control environmental samples were collected for testing. No differences in owner-reported morbidity and mortality between treatment groups were seen. The intervention was significantly associated with increasing tetracycline susceptibility in E. coli and Salmonella. Tetracycline susceptibility increased in intervention herds for the first 3 months after switching to nonmedicated milk replacer but declined in subsequent months. Discontinuing the practice of feeding medicated milk replacers to calves increased tetracycline susceptibility in E. coli and Salmonella on dairy farms, without increasing cattle disease, but declines in effectiveness after 3 months suggest that other factors contribute to decreasing susceptibility on the farm.

Epidemiologic attributes of invasive non-typhoidal Salmonella infections in Michigan, 1995--2001.

OBJECTIVES: To determine: (1) the incidence of invasive salmonellosis, (2) the distribution of specific Salmonella serotypes associated with invasive disease, and (3) the role of demographic characteristics in invasive salmonellosis in Michigan. DESIGN: We conducted a cross-sectional study using laboratory-confirmed cases of salmonellosis reported to the Michigan Department of Community Health between 1995 and 2001. METHODS: Average annual incidences (AAIs) for invasive salmonellosis were computed, and Poisson regression analysis was used to model the association between demographic attributes and invasive salmonellosis. RESULTS: Of 6797 cases of salmonellosis, 347 (5.1%) were characterized as invasive having an AAI of 0.5/100000. A covariate-adjusted Poisson model showed children aged <1 year to be at higher risk for invasive salmonellosis compared to adults aged 40-49 years (rate ratio (RR) 8.98, 95% confidence interval (CI) 5.69-14.17). No significant differences were found between males and females (RR 1.02, 95% CI 0.83-1.26), African-Americans and Caucasians (RR 0.95, 95% CI 0.64-1.42), and urban and rural residents (RR 0.98, 95% CI 0.76-1.26). Among the most common serotypes associated with invasive salmonellosis (Heidelberg, Typhimurium, and Enteritidis), Salmonella serotype Heidelberg had the highest blood invasiveness ratio (15.79/100). CONCLUSIONS: Children aged <1 year, especially of minority groups, are at higher risk for invasive Salmonella infections. Invasive salmonellosis may be included in the differential diagnosis of acute bacterial infections in young children with positive stool cultures for Salmonella.

Effect of hyperimmunization with an Escherichia coli J5 bacterin in adult lactating dairy cows.

OBJECTIVE: To determine the effect of hyperimmunization with an Escherichia coli J5 bacterin on serum IgG2 concentration, incidence of clinical mastitis, and rate of survival to the end of the lactation period (ie, day 305) in adult lactating dairy cattle. DESIGN: Randomized controlled trial. ANIMALS: 1,012 Holstein cows in their second lactation and greater. PROCEDURES: All cows were given 3 doses of the J5 bacterin; cows in the hyperimmunization group were given an additional 3 doses during the first 3 months of lactation. Blood was collected from a small sample of cows to determine anti-J5 IgG2 concentrations. RESULTS: Cows in the hyperimmunization group had higher mean serum anti-J5 IgG2 concentrations than did control cows 28 days after administration of the fourth, fifth, and sixth doses of the J5 bacterin. However, mean serum anti-J5 concentrations during the subsequent lactation were not significantly different between groups. The proportions of cows that developed clinical mastitis were not significantly different between groups. However, control cows were more likely to have severe clinical mastitis than were cows in the hyperimmunization group. The percentage of control cows that remained in the herd to day 305 was significantly lower than the percentage of cows in the hyperimmunization group that did. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that hyperimmunization of mature lactating dairy cattle was associated with increased serum anti-J5 IgG2 concentrations and decreased incidence of severe clinical mastitis, but did not alter survival rate of cows that developed severe clinical mastitis.

Prevalence and pattern of antimicrobial susceptibility in Escherichia coli isolated from pigs reared under antimicrobial-free and conventional production methods.

OBJECTIVE: To determine and compare levels and patterns of antimicrobial resistance among Escherichia coli isolated from pigs on farms that did not use antimicrobial agents versus pigs produced under conventional methods. DESIGN: Cross-sectional study. Sample Population-35 antimicrobial-free and 60 conventional swine farms. PROCEDURES: Farms were visited once, and fecal samples were collected from 15 finisher pigs if available. One E coli isolate from each sample was tested for susceptibility pattern to 14 antimicrobial agents by use of microbroth dilution. RESULTS: E coli isolates were recovered from 1,381 (97.1%) of 1,422 fecal samples. Herd size was significantly larger for conventional swine farms. Resistance to ceftriaxone, ciprofloxacin, or nalidixic acid was not observed on any of the 95 farms. Three isolates from 2 conventional farms were resistant to ceftiofur. Conventional farms had significantly higher levels of resistance to ampicillin, sulfamethoxazole, tetracycline, and chloramphenicol, compared with antimicrobial-free farms. Fourteen percent of E coli isolates were susceptible or had intermediate resistance to all the tested antimicrobial agents. The 3 most frequent patterns of multiple resistance were streptomycin-tetracycline, sulfamethoxazole-tetracycline, and kanamycin-streptomycin-sulfamethoxazole-tetracycline. CONCLUSIONS AND CLINICAL RELEVANCE: Cessation of antimicrobial use did not appear to result in an immediate reduction in antimicrobial resistance in swine farms. Prospective studies of long-term antimicrobial usage and cessation are needed to estimate the extent to which food animal production may be contributing to antimicrobial drug resistance and might provide a direct measure of the rates of reversibility of antimicrobial drug resistance that might be achieved by curtailing antimicrobial usage.

A registry-based study on the association between human salmonellosis and routinely collected parameters in Michigan, 1995-2001.

PURPOSE: Salmonella serotypes are among the most common bacterial causes of foodborne gastroenteritis in the United States, associated with approximately 1.4 million human illnesses annually. Studies on trends of the serotypes and host-related factors are necessary for the development of effective prevention plans for foodborne diseases caused by these pathogens. MATERIALS AND METHODS: To determine the epidemiologic trends of human infections with the most common Salmonella serotypes in Michigan, we analyzed cases of culture-confirmed salmonellosis at the Michigan Department of Community Health (MDCH) from 1995 to 2001. RESULTS: A total of 6797 cases were reported, with an average annual incidence per 100,000 population (AAI) of 9.9. Among cases for which information on Salmonella serotype were available (6292 cases), the most common serotypes were S. Typhimurium (1596 cases, 26%), followed by S. Enteritidis (1309, 22%), S. Heidelberg (466, 8%) and S. Newport (222, 4%). From 1998 to 2001, the incidence of S. Typhimurium and S. Enteritidis decreased significantly by 39% (95% confidence interval [CI], 49% to 26% decrease) and 32% (95% CI, 44% to 18% decrease) respectively. Whereas the incidence of S. Newport increased by 101% (95% CI, 25% to 225% increase) and S. Heidelberg remained stable. Infection with these serotypes frequently occurred in the summer months. As a group, infants had the highest AAI for all Salmonella serotypes (75.0), S. Typhimurium (21.9), S. Enteritidis (14.0), S. Heidelberg (5.4), and S. Newport (1.7). Among patients whose race was known, blacks had a significantly higher AAI compared to whites for S. Typhimurium (2.5 vs. 1.3; RR = 2.3, 95% CI, 1.6-3.3), S. Enteritidis (1.4 vs. 1.1; relative rate (RR) = 1.4; 95% CI, 1.1-1.6), S. Heidelberg (0.8 vs. 0.3; RR = 3.6; 95% CI, 2.8-4.6), and S. Newport (0.3 vs. 0.1; RR = 2.8; 95% CI, 1.9-4.2). Among patients whose ethnicity was known, Hispanics had a significantly higher AAI for S. Enteritidis compared to non-Hispanics (1.0 vs. 0.5; RR = 1.9; 95% CI, 1.2-3.0), but not different significantly for S. Typhimurium, S. Heidelberg, and S. Newport. CONCLUSION: This study revealed the emergence of S. Newport and the high incidence of the most common Salmonella serotypes among infants, people of African descent, and Hispanics. This information can be used by the state and local health departments of Michigan to enhance salmonellosis prevention efforts by rationalizing the allocation of appropriate public health resources and personnel.

Immunity and mastitis. Some new ideas for an old disease.

The biggest challenge for host immune defense against mastitis-causing bacteria in dairy cows is to quickly recruit large enough numbers of opsonizing molecules and mature neutrophils into milk such that intramammary pathogens are cleared before they multiply significantly and the inflammatory response gets out of control. Currently, this challenge is best facilitated when established mastitis control procedures are practiced on the farm, including proper hygiene, milking procedures, and regular administration of approved mastitis vaccines. However, mastitis is still a significant problem. New animal functional genomics research is beginning to allow scientists to solve the puzzle of mastitis susceptibility. Results of this type of research offer the hope of giant leaps toward a clear identification of molecular genetic variation and potential gene targets for therapies and immune manipulations that could significantly reduce the risk of clinical mastitis in traditionally susceptible cows.

Mastitis therapy and pharmacology.

Mastitis remains the most frequent cause of antibacterial use on dairy farms and contributes to a substantial portion of total drug and veterinary costs incurred by the dairy industry. Ultimately, the best outcome of mastitis therapy is a positive effect on the amount of marketed milk harvested and long-term survival of the cow. This article describes the strategies of therapy for bovine mastitis, with an emphasis on antibacterial and anti-inflammatory therapy.

Enzyme-Linked Immunosorbent Assay and Microbiologic Culture for Diagnosis of Staphylococcus aureus Intramammary Infection in Cows.

Recent reports have indicated that the relative sensitivity and specificity of the ELISA test for detection of intramammary infection of cows with Staphylococcus aureus is not as high as originally reported. It has been suggested that antibodies measured by enzyme-linked immunosorbent assay (ELISA) more closely reflect previous infection status rather than current infection status, and that the delay in antibody formation following infection and the persistence of antibodies after elimination of infection may be responsible for some of the discrepancy observed between ELISA and bacterial culture results conducted on the same milk sample. This study (n = 209 cows) was undertaken to determine if an ELISA for S. aureus intramammary infection more closely reflects previous infection status than it does current infection status, and to ascertain whether correction of this time-delay factor substantially improves calculated values of ELISA relative sensitivity and specificity. Receiver-operator curves were constructed to compare different time-related definitions of microbiologic culture results used for comparison with ELISA results. A greater degree of curvature in receiver-operator curves indicated that ELISA results did more closely reflect culture results performed on milk samples taken 1 and 3 weeks previously. Insignificant improvement in sensitivity and specificity occurred when the database was limited to cows (n = 140) with milk production greater than 13.6 kg/day. However, values of sensitivity were all less than or equal to 90%, and values of specificity were all less than 54%.

Incidence of False-Positive Results for Assays Used To Detect Antibiotics in Milk.

The incidence of false-positive results from milk assays for antimicrobial agents was determined for composite milk samples collected from 407 lactating dairy cows with a history of no antibiotic treatment for a minimum of 30 days. Milk samples were also cultured for bacteria and analyzed for somatic cell count. Mean herd prevalence of intramammary infections (±SEM) caused by Streptococcus agalactiae and Staphylococcus aureus was 3.3 ± 2.8 and 20.2 ± 9.5% of lactating cows, respectively. All 407 milk samples were assayed for antibiotics with three commercial tests; a fourth test was used to assay 391 samples. Samples were assayed twice with each test, and if the results from these repetitions did not agree, a third assay was performed and the result obtained in two of the three repetitions was reported. Because samples were only collected from cows with no antimicrobial treatment for a minimum of 30 days, positive assays were considered to be false-positive results. Three test kits did not yield any false-positive results, one test kit had 5 false-positive results of 407 samples collected (specificity, 98.8%). Although there was a trend for false-positive samples to have a higher somatic cell count than negative samples, the low incidence of false-positive results did not allow a meaningful comparison. We conclude that the incidence of false-positive results is very low when testing milk from cows that have a history of no clinical mastitis or antimicrobial treatment.