Distribution of Lactococcus spp. in New York State dairy farms and the association of somatic cell count resolution and bacteriological cure in clinical mastitis samples.

We investigated the distribution of pathogenic non-agalactiae gram-positive, catalase-negative cocci (GPCN) in a convenience sample of New York State dairy farms. Our primary objective with the clinical mastitis (CM) GPCN samples was to evaluate somatic cell count (SCC) resolution and bacteriological cure of Streptococcus dysgalactiae or Streptococcus uberis versus Lactococcus lactis or Lactococcus garvieae in cows that received an approved intramammary treatment. In phase I, we assessed the distribution of the GPCN and SCC resolution. In phase II, we evaluated the SCC resolution and bacteriological cure in CM samples from the 4 farms with the highest prevalence of L. lactis or L. garvieae in phase I. In phase I, 8,868 CM and subclinical mastitis (SCM) milk samples were received from 143 farms. The GPCN samples identified by culture were confirmed with MALDI-TOF. From the 473 MALDI-TOF-confirmed GPCN samples, 155 were S. dysgalactiae (33%); 150, S. uberis (32%); 112, L. lactis (24%); 16, L. garvieae (3%); and 40, other GPCN (8%). From these, 277 were CM samples and 127 were eligible for the evaluation of SCC resolution, which was defined as SCC ≤200,000 cells/mL in a composite sample 15 to 60 d post-diagnosis. The odds of SCC resolution in CM samples was evaluated with multivariable logistic regression, and the odds were 6.1 [95% confidence interval (CI):2.7-13.9] times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. In phase II, a total of 1,662 CM and SCM samples were evaluated with microbiological methods as in phase I, of which 211 samples were confirmed by MALDI-TOF: 39% were S. dysgalactiae (n = 61) and S. uberis (n = 21); 55%, L. lactis (n = 114) and L. garvieae (n = 2); and 6%, other GPCN (n = 13). In total, 168 CM samples were eligible for analysis and 118 were included in the final SCC resolution model. Similar statistical methods as in phase I were performed, and the odds of SCC resolution were 2.4 (95% CI: 1.1-5.5) times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. Bacteriological cure was defined as having a different or negative culture on a quarter sample taken 14 to 28 d after initial diagnosis. The odds of bacteriological cure (n = 121) were 8.0 (95% CI: 2.5-25.6) times higher for S. dysgalactiae or S. uberis compared with L. lactis or L. garvieae. Differences in SCC resolution and bacteriological cure between these groups may dictate a different management approach.

Randomized noninferiority trial comparing 2 commercial intramammary antibiotics for the treatment of nonsevere clinical mastitis in dairy cows.

The purpose was to evaluate 2 intramammary treatments for mild-to-moderate cases of clinical mastitis in a noninferiority comparison. Noninferiority trials are intended to show whether a given treatment, hetacillin potassium, has at least comparable efficacy as the reference treatment, ceftiofur hydrochloride. Treatments can be deemed inferior to the reference treatment by an amount less than the margin of noninferiority, or inconclusive if the confidence interval crosses the margin of noninferiority. Cows with clinical mastitis from 6 farms were considered for enrollment. Using a randomized design, cows with mild or moderate mastitis in 1 quarter were assigned to on-label treatment with either ceftiofur or hetacillin. A total of 596 cows met the criteria needed for continued enrollment. Treatment distribution resulted in 309 cows in the ceftiofur group and 287 cows in the hetacillin group. Mixed regression analysis was performed for the following outcomes: bacteriological cure, pathogen cure, clinical cure, postevent milk production and linear score, and survival to d 30 and 60. Cox proportional hazards analysis was used to describe treatment effect on survival and mastitis risks. Bacteriological cure, defined as absence of causative organism in samples retrieved at d 14 and 21 postmastitis, was similar between groups. No significant statistical differences were found in cure risk, and noninferiority of hetacillin relative to ceftiofur for bacteriological cure was conclusive (hetacillin=67%, ceftiofur=72%). Absence of a pathogen on both follow-up samples designated a cow as a pathogen cure. Pathogen cure was similar between treatment groups and noninferiority of hetacillin relative to ceftiofur was shown (hetacillin=35%, ceftiofur=32%). Clinical cure (hetacillin=68%, ceftiofur=64%), postevent milk production (hetacillin=37.0kg, ceftiofur=38.2kg), and linear scores (hetacillin=3.4, ceftiofur=3.1) were also not statistically different between treatment groups. Noninferiority of hetacillin relative to ceftiofur was shown for survival to d 30 and survival to d 60, whereas hetacillin was more likely to have a clinical cure than ceftiofur by d 4. No differences were seen between groups when Cox proportional hazards were performed, neither for exit from the herd in the 60 d following the event nor in the risk for a subsequent mastitis event. These findings can be used to develop farm-specific protocols for clinical mastitis treatment.

Noninferiority trial on the efficacy of premilking teat disinfectant against naturally occurring new intramammary infections using a novel 2-step diagnostic process.

The objective of the study was to evaluate premilking teat disinfectant efficacy with the use of a novel diagnostic protocol against a positive control. The evaluation of efficacy was based on establishing noninferiority of a new premilking teat disinfectant compared with an existing premilking teat disinfectant. Approximately 200 cows were randomly allocated to 1 of 2 groups. Study personnel were blinded as to cow allocation and identification of the premilking disinfectants. Quarters were disinfected using a dip cup before milking with either the control or experimental product. The disinfectant was left on the teat for approximately 30s before being wiped off in preparation for milking. Quarter-level bacteriological infection status was established at the beginning of the study and quarter milk samples were collected biweekly thereafter. Bacteriological analyses were performed only when somatic cell counts in milk samples crossed a parity-specific threshold. Poisson regression models were used to analyze data. The difference in the rate of new intramammary infections was small and the upper bound of the 95% confidence interval of the rate difference was smaller than a predefined noninferiority limit. Based on the observed difference in new intramammary infections rate between the experimental and control product, it was concluded that the experimental disinfectant was not inferior compared with the positive control. The protocol used in this study proposes a valid and economically attractive methodology to evaluate the efficacy of teat disinfectants relative to a positive control.

Noninferiority trial comparing a first-generation cephalosporin with a third-generation cephalosporin in the treatment of nonsevere clinical mastitis in dairy cows.

The objective of this study was to evaluate the noninferiority of 2 intramammary treatments for nonsevere clinical mastitis. The 2 treatments were a first-generation cephalosporin (cephapirin sodium, 2 treatments 12h apart) and a third-generation cephalosporin (ceftiofur hydrochloride, treatments once a day for 5d). A total of 296 cases on 7 farms met the enrollment criteria for the study. Streptococcus dysgalactiae was the most common bacterial species identified in milk samples from cows with mild to moderate clinical mastitis, followed by Escherichia coli, other esculin-positive cocci, Streptococcus uberis, and Klebsiella spp. Treatment was randomly allocated as either cephapirin sodium or ceftiofur hydrochloride via intramammary infusion according to label standards. Bacteriological cure was defined based on 2 posttreatment milk samples taken at 10 and 17d after enrollment. Noninferiority of cephapirin relative to ceftiofur was shown for bacteriological cure of gram-positive cases and for clinical cure of all cases. Ceftiofur showed a significantly higher bacteriological cure in gram-negative cases. Treatments showed no significant difference in bacteriological cure of all cases and in time to exit from the study, where the absence of a difference does not imply noninferiority. Based on the findings from this study, farm-specific treatment protocols that differ for gram-positive and gram-negative cased may be developed.

Defining standardized protocols for determining the efficacy of a postmilking teat disinfectant following experimental exposure of teats to mastitis pathogens.

The objective of this paper was to define standardized protocols for determining the efficacy of a postmilking teat disinfectant following experimental exposure of teats to both Staphylococcus aureus and Streptococcus agalactiae. The standardized protocols describe the selection of cows and herds and define the critical points in performing experimental exposure, performing bacterial culture, evaluating the culture results, and finally performing statistical analyses and reporting of the results. The protocols define both negative control and positive control trials. For negative control trials, the protocol states that an efficacy of reducing new intramammary infections (IMI) of at least 40% is required for a teat disinfectant to be considered effective. For positive control trials, noninferiority to a control disinfectant with a published efficacy of reducing new IMI of at least 70% is required. Sample sizes for both negative and positive control trials are calculated. Positive control trials are expected to require a large trial size. Statistical analysis methods are defined and, in the proposed methods, the rate of IMI may be analyzed using generalized linear mixed models. The efficacy of the test product can be evaluated while controlling for important covariates and confounders in the trial. Finally, standards for reporting are defined and reporting considerations are discussed. The use of the defined protocol is shown through presentation of the results of a recent trial of a test product against a negative control.

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

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

The effect of recurrent episodes of clinical mastitis caused by gram-positive and gram-negative bacteria and other organisms on mortality and culling in Holstein dairy cows.

The objective of this study was to estimate the effects of recurrent episodes of different types of clinical mastitis (CM) caused by gram-positive (Streptococcus spp., Staphylococcus aureus, Staphylococcus spp.) and gram-negative (Escherichia coli, Klebsiella, Citrobacter, Enterobacter, Pseudomonas) bacteria, and other organisms (Arcanobacterium pyogenes, Mycoplasma, Corynebacterium bovis, yeast, miscellaneous) on the probability of mortality and culling in Holstein dairy cows. Data from 30,233 lactations in cows of 7 dairy farms in New York State were analyzed. Cows were followed for the first 10 mo in lactation, or until death or culling occurred, or until the end of our study period. Generalized linear mixed models with a Poisson error distribution were used to study the effects of recurrent cases of the different types of CM and several other factors (herd, parity, month of lactation, current year and season, profitability, net replacement cost, other diseases) on cows' probability of death (model 1) or being culled (model 2). Primiparous and multiparous cows were modeled separately because they had different risks of mortality and culling and potentially different CM effects on mortality and culling. Approximately 30% of multiparous cows had at least one case of CM in lactation compared with 16.6% of primiparous cows. Multipara also had higher lactational incidence risks of second (10.7%) and third (4.4%) cases than primipara (3.7% and 1.1%, respectively). For primipara, CM increased the probability of death, with each successive case occurring in a month being increasingly lethal. In multipara, gram-negative CM increased the probability of death, especially when the gram-negative case was the first or second CM case in lactation. Primiparous cows with CM were more likely to be culled after CM than if they did not have CM, particularly after a second or third case. In multipara, any type of CM increased the probability of being culled. Gram-negative CM cases were associated with the numerically highest risk of culling.

Effects of clinical mastitis caused by gram-positive and gram-negative bacteria and other organisms on the probability of conception in New York State Holstein dairy cows.

The objective of this study was to estimate the effects of different types of clinical mastitis (CM) on the probability of conception in New York State Holstein cows. Data were available on 55,372 artificial inseminations (AI) in 23,695 lactations from 14,148 cows in 7 herds. We used generalized linear mixed models to model whether or not a cow conceived after a particular AI. Independent variables included AI number (first, second, third, fourth), parity, season when AI occurred, farm, type of CM (due to gram-positive bacteria, gram-negative bacteria, or other organisms) in the 6 wk before and after an AI, and occurrence of other diseases. Older cows were less likely to conceive. Inseminations occurring in the summer were least likely to be successful. Retained placenta decreased the probability of conception. Conception was also less likely with each successive AI. The probability of conception associated with the first AI was 0.29. The probability of conception decreased to 0.26, 0.25, and 0.24 for the second, third, and fourth AI, respectively. Clinical mastitis occurring any time between 14 d before until 35 d after an AI was associated with a lower probability of conception; the greatest effect was an 80% reduction associated with gram-negative CM occurring in the week after AI. In general, CM due to gram-negative bacteria had a more detrimental effect on probability of conception than did CM caused by gram-positive bacteria or other organisms. Furthermore, CM had more effect on probability of conception immediately around the time of AI. Additional information about CM (i.e., its timing with respect to AI, and whether the causative agent is gram-positive or gram-negative bacteria, or other organisms) is valuable to dairy personnel in determining why some cows are unable to conceive in a timely manner. These findings are also beneficial for the management of mastitic cows (especially those with gram-negative CM) when mastitis occurs close to AI.

Effects of repeated gram-positive and gram-negative clinical mastitis episodes on milk yield loss in Holstein dairy cows.

The objective of this study was to estimate the effects of recurrent episodes of gram-positive and gram-negative cases of clinical mastitis (CM) on milk production in Holstein dairy cows. We were interested in the severity of repeated cases in general, but also in the severity of the host response as judged by milk production loss when a previous case was caused by a similar or different microorganism. The results were based on data from 7,721 primiparous lactations and 13,566 multiparous lactations in 7 large dairy herds in New York State. The distribution of organisms in the CM cases showed 28.5% gram-positive cases, 31.8% gram-negative cases, 15.0% others, and 24.8% with no organism identified. Mixed models, with a random herd effect and an autoregressive covariance structure to account for repeated measurements, were used to quantify the effect of repeated CM and several other control variables (parity, week of lactation, other diseases) on milk yield. Our data indicated that repeated CM cases showed a very similar milk loss compared with the first case. No reduction of severity was present with increasing count of the CM case. Gram-negative cases had more severe milk loss compared with gram-positive and other cases irrespective of the count of the case in lactation. Milk loss in multipara (primipara) due to gram-negative CM was approximately 304 kg (228 kg) in the 50 d following CM. This loss was approximately 128 kg (133 kg) for gram-positive cases and 92 kg (112 kg) for other cases. The severity of a second case of gram-negative CM was not reduced by previous cases of gram-negative CM in multipara and only slightly less severe in a similar scenario in primipara cows. Similarly, a previous gram-positive case did not reduce severity of a second or third gram-positive case. Hence, our data do not support that immunological memory of previous exposure to an organism in the same generic class provides protection for a next case of CM with an organism in the same class.

Fecal shedding of Klebsiella pneumoniae by dairy cows.

Klebsiella pneumoniae is a common cause of clinical mastitis in dairy cattle. Wood products are considered to be the main source of Klebsiella on dairy farms. Environmental hygiene and use of inorganic bedding materials such as sand are recommended to control Klebsiella mastitis. However, Klebsiella mastitis still occurs on well-managed dairy farms that use sand as bedding material. In a 5-mo study in a New York State dairy herd performed during the summer of 2005, all of 9 samples of unused sand bedding tested negative for Klebsiella, whereas 14 of 18 samples of used sand bedding contained Klebsiella at a median level of 10(4.6) cfu/g. We hypothesized that fecal shedding of Klebsiella by dairy cows contributes to the presence of Klebsiella in the environment. Using a cheap and simple method based on ampicillin-containing MacConkey agar for screening, and biochemical tests for confirmation of species identity, 595 fecal samples from healthy dairy cattle were screened for presence of Klebsiella. In a longitudinal study of 100 cows followed over 5 mo, more than 80% of fecal samples tested positive for K. pneumoniae. The average prevalence of K. pneumoniae-positive fecal samples was also above 80% in a cross-sectional study of 100 cows from 10 herds across New York and Massachusetts. Fecal shedding of K. pneumoniae by a large proportion of dairy cows may explain why Klebsiella mastitis occurs in herds that use inorganic bedding material or other bedding material that is free from Klebsiella upon introduction into the barn.