Effectiveness of Intramammary Antibiotics, Internal Teat Sealants, or Both at Dry-Off in Dairy Cows: Clinical Mastitis and Culling Outcomes.

Intramammary antibiotic (AB) and internal teat sealants (TS) infusion at dry-off have been used to prevent intramammary infections (IMI) in dairy cows during the dry period and reduce the risk of mastitis during the dry period and subsequent lactation. A randomized clinal trial was completed on eight California dairy herds to estimate the effects of different dry cow therapies (AB, TS, AB + TS or None) on clinical mastitis and culling. A total of 1273 cows were randomized to one of the four treatment groups over summer and winter seasons. For each enrolled cow, microbiological testing was done on quarter milk samples collected from the first detection of clinical mastitis within the first 150 days in milk (DIM) in the subsequent lactation. Statistical analysis was done using generalized linear mixed models. There were no significant differences in the odds of clinical mastitis or culling between cows treated with AB, TS, or AB + TS compared to the controls. Dry cow therapy with AB and/or TS had no statistically significant effect on clinical mastitis and cow culling during the first 150 DIM.

Bayesian estimation of sensitivity and specificity of a rapid mastitis test kit, bacterial culture, and PCR for detection of Staphylococcus aureus, Streptococcus species, and coliforms in bovine milk samples.

Our objectives were to evaluate the diagnostic accuracy of a rapid and novel immunochromatography-based mastitis kit that includes 3 independent tests to detect coliforms (Escherichia coli or Klebsiella pneumoniae), Streptococcus spp., and Staphylococcus aureus. The kit was developed to facilitate diagnostic-based mastitis treatment. Validation of the kit was based on 154 aseptically collected mastitis samples from 2 clinical herds (clinical population) and 120 milk samples from 3 nonclinical herds (nonclinical population) without clinical cases at the time of enrollment. One herd sampled at different times was common to both populations. A 3-test in 2-population Bayesian latent class model with uniform priors for all test parameters except specificity of culture, which was modeled informatively, was used to estimate sensitivity (Se) and specificity (Sp) of the test kit, culture, and PCR at the cow level. The mastitis test kit's 96.9% Sp for Streptococcus spp. had a low false positive percentage (3.1%), which, together with the kit's rapid turnaround time for results, makes it a suitable initial screening test that producers can use to identify clinical cows to treat based on Streptococcus spp. mastitis in kit-positive results. Due to the 60.4% kit Se, producers should follow up on Streptococcus spp. kit-negative cows using a confirmatory test such as PCR (Sp of 98.4%) or culture (Sp of 99.6%). In contrast, aerobic culture had Se of 76.5% and Sp of 99.6% for Streptococcus spp. Similarly, the Sp of the kit (98.2%) and culture (99.8%) for Staph. aureus were particularly high, and even though the kit's Se (61.0%) was lower than culture (88.4%; posterior probability of difference 98%), the kit could be beneficial before use of a confirmatory test for kit-negative samples due to its ease and rapid turnaround time. Mostly, quantitative real-time (q)PCR outperformed the kit's Se (37.7%) and Sp (92.9%) for coliforms, as well as the kit's Se (60.4%) for Streptococcus spp. However, qPCR may require more technical skills and turnaround time for final results. Use of the on-farm mastitis test kit evaluated in the present study could enhance sustainable antimicrobial drug use by rapidly identifying Streptococcus mastitis for targeted treatment. Furthermore, the kit may be used in a Staph. aureus outbreak where cows can be rapidly screened to identify cases for segregation or culling during an outbreak and kit-negative cows further confirmed by milk culture or qPCR. However, the cost-effectiveness of such an approach has not been investigated.

Development of a multiplex qPCR assay for the simultaneous detection of Mycoplasma bovis, Mycoplasma species, and Acholeplasma laidlawii in milk.

Contagious bovine mastitis caused by Mycoplasma bovis and other Mycoplasma species including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma canadense is an economical obstacle affecting many dairy herds throughout California and elsewhere. Routine bacteriological culture-based assays for the pathogens are slow and subject to false-positive results due to the presence of the related, non-pathogenic species Acholeplasma laidlawii. To address the need for rapid and accurate detection methods, a new TaqMan multiplex, quantitative real-time PCR (qPCR) assay was developed that targets the 16S rRNA gene of Mycoplasma, rpoB gene of M. bovis, and the 16S to 23S rRNA intergenic transcribed spacer (ITS) region of A. laidlawii. qPCR amplification efficiency and range of detection were similar for individual assays in multiplex as when performed separately. The multiplex assay was able to distinguish between M. bovis and A. laidlawii as well as detect Mycoplasma spp. collectively, including Mycoplasma californicum, Mycoplasma bovigenitalium, Mycoplasma canadense, Mycoplasma arginini and Mycoplasma alkalescens. In milk, the lower limit of detection of M. bovis, M. californicum, and A. laidlawii with the multiplex assay was between 120 to 250 colony forming units (CFU) per mL. The assay was also able to simultaneously detect both M. bovis and A. laidlawii in milk when present in moderate (103 to 104 CFU/mL) to high (106 to 107 CFU/mL) quantities. Compared to laboratory culture-based methods, the multiplex qPCR diagnostic specificity (Sp) was 100% (95% CI [86.8-100]; n = 26) and diagnostic sensitivity (Se) was 92.3% (95% CI [74.9-99.1]; n = 26) for Mycoplasma species in milk samples collected from California dairy farms. Similarly, the Sp was 100% (95% CI [90.5-100]; n = 37) and Se was 93.3% (95% CI [68.1-99.8]; n = 15) for M. bovis. Our assay can detect and distinguish among M. bovis, other prevalent Mycoplasma spp., and non-pathogenic Acholeplasma laidlawii for effective identification and control of mycoplasma mastitis, ultimately supporting dairy cattle health and high-quality dairy products in California.

Development and comparison of loop-mediated isothermal amplification and quantitative polymerase chain reaction assays for the detection of Mycoplasma bovis in milk.

Bovine mastitis is an economic burden for dairies worldwide. Mycoplasma species, and especially Mycoplasma bovis, are among the most important causative agents, and rapid, precise, and low-cost methods for Mycoplasma detection are urgently needed. For this purpose, loop-mediated isothermal amplification (LAMP) and quantitative PCR (qPCR) assays were developed and compared. The LAMP assay was designed and primer concentrations optimized to M. bovis oppD, encoding oligopeptide permease D. For qPCR, a Taqman assay (Applied Biosystems, Carlsbad, CA) targeting M. bovis gltX, encoding glutamate transfer RNA ligase, was optimized for primer concentration, annealing temperature, and DNA polymerase. Both assays were similarly sensitive, with a detection limit of approximately 104 to 105M. bovis cells/mL. Both assays were also successful in confirming M. bovis identity in laboratory culture suspensions and in bovine milk. The LAMP and qPCR assays combined with the MoBio DNA extraction kit (MoBio Laboratories Inc., Carlsbad, CA) resulted in the correct detection of 13 out of 13 M. bovis isolates and 14 out of 16 M. bovis-positive milk samples collected from commercial dairies in California. When combined with the PrepMan Ultra reagent (Applied Biosystems), the qPCR assay resulted in confirming 21 out of 21 M. bovis-positive milk samples. Comparison of the assays to milk containing either Mycoplasma arginini, Mycoplasma bovigenitalium, Mycoplasma californicum, M. alkalescens, or Acholeplasma laidlawii or milk lacking any detectable Mycoplasma species or relatives resulted in 3 out of 17 (LAMP with MoBio), 1 out of 17 (qPCR with MoBio), and 2 out of 36 (qPCR with PrepMan Ultra) false positives. Overall, the qPCR assay was more robust than LAMP and could be used on DNA recovered from milk prepared with the PrepMan Ultra reagent, a method that does not include a DNA purification step. The use of this qPCR method enables M. bovis detection in bovine milk in 40 to 55 min, and therefore provides new opportunities to accelerate and simplify M. bovis detection in unpasteurized milk to reduce the incidence of M. bovis mastitis outbreaks.

Cost-effectiveness of diagnostic strategies using quantitative real-time PCR and bacterial culture to identify contagious mastitis cases in large dairy herds.

Diagnostic strategies to detect contagious mastitis caused by Mycoplasma bovis, Staphylococcus aureus, and Streptococcus agalactiae in dairy herds during an outbreak have been minimally studied with regard to cost and diagnostic sensitivity. The objective of this cross-sectional study was to compare the cost-effectiveness of diagnostic strategies for identification of infected cows in two California dairy herds during contagious mastitis outbreaks. M. bovis was investigated in a subset of a herd (n=1210 cows) with an estimated prevalence of 2.8% (95% CI=1.9, 3.7), whereas Staph. aureus and Strep. agalactiae were studied in a second herd (n=351 cows) with an estimated prevalence of 3.4% (95% CI=1.5, 5.3) and 16.8% (95% CI=12.9, 20.7), respectively. Diagnostic strategies involved a combination of testing stages that utilized bacterial culture, quantitative real-time PCR (qPCR), or both. Strategies were applied to individual or pooled samples of 5, 10, 50 or 100 samples. Culture was considered the gold standard for sensitivity estimation of each strategy. The reference strategy was the strategy with the lowest cost per culture-positive cow which for both M. bovis and Strep. agalactiae consisted of 2 stages, culture of samples in pools of 5 followed by culture of individual samples in positive pools with a sensitivity of 73.5% (95% CI: 55.6, 87.1) and 96.6% (95% CI: 27.7, 84.8), respectively. The reference strategy for Staph. aureus consisted of 3 stages, culture of individual samples in pools of 100 (stage 1), culture constituents of those positive from stage 1 in pools of 5 (stage 2), culture constituents of those positive from stage 2 individually (stage 3) which resulted in a sensitivity of 58.3% (95% CI: 88.3, 99.6). The most cost-effective alternative to the reference strategy was whole herd milk culture for all 3 pathogens. QPCR testing was a component of the second most cost-effective alternative for M. bovis and the third most cost-effective alternatives for the 3 pathogens. A stochastic model was used to assess the effect of prevalence or herd size on the cost-effectiveness of diagnostic strategies. In the current study, increasing the prevalence of mastitis did not alter the ranking of strategies by cost-effectiveness. However, larger herds could benefit from testing larger pools such as 50 or 100 samples to improve cost-effectiveness. Several diagnostic strategy options exist to identify contagious mastitis in herds, decisions should be based on cost and sensitivity of the strategies available.