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.
Contagious bovine mastitis caused by
and other
species including
,
, and
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
. 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
gene of
, and the 16S to 23S rRNA intergenic transcribed spacer (ITS) region of
. 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
and
as well as detect
spp. collectively, including
and
. In milk, the lower limit of detection of
, and
with the multiplex assay was between 120 to 250 colony forming units (CFU) per mL. The assay was also able to simultaneously detect both
and
in milk when present in moderate (10
to 10
CFU/mL) to high (10
to 10
CFU/mL) quantities. Compared to laboratory culture-based methods, the multiplex qPCR diagnostic specificity (Sp) was 100% (95% CI 86.8-100;
= 26) and diagnostic sensitivity (Se) was 92.3% (95% CI 74.9-99.1;
= 26) for
species in milk samples collected from California dairy farms. Similarly, the Sp was 100% (95% CI 90.5-100;
= 37) and Se was 93.3% (95% CI 68.1-99.8;
= 15) for
. Our assay can detect and distinguish among
, other prevalent
spp., and non-pathogenic
for effective identification and control of mycoplasma mastitis, ultimately supporting dairy cattle health and high-quality dairy products in California.
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.
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.
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.
The objective of this prospective study was to determine the prevalence of potential environmental bacterial mastitis pathogens in bulk tank milk, and to determine their antibiotic susceptibility ...patterns. Bulk tank milk samples from over 400 California dairies routinely submitting samples to the Milk Quality Laboratory were initially screened. Once potential environmental bacterial mastitis pathogens were found in bulk milk from a dairy, the dairy was repeatedly sampled on a monthly basis. Over a nine-month period, 93 dairies were identified with these bacteria, and 381 isolates were collected. Most common isolates were Streptococcus uberis (42.3%), Streptococcus dysgalactiae (16.3%) and Enterococcus faecium (10.5%). Bacterial isolates (335) from 73 dairies which had at least three isolates were subjected to Minimum Inhibitory Concentration (MIC) testing using 10 antibiotics. A wide range of susceptibility to antibiotics was found. Streptococcus dysgalactiae tended to have lower MIC values to the test antibiotics than the other isolates. The non-streptococcal bacteria tended to have the highest MIC values.
To determine the prevalence of Mycoplasma spp in herds that were members of a milk cooperative.
Epidemiologic study.
267 dairy herds that were members of a milk cooperative.
Bulk-tank milk samples ...were collected monthly during a 6-year period from all dairies in the cooperative. Samples were submitted to the cooperative's laboratory for bacterial culture for Mycoplasma spp, using direct plating. Milk samples positive for Mycoplasma organisms were speciated.
Prevalence of positive samples varied from 1.8 to 5.8% for all species of Mycoplasma and from 1.2 to 3.1% for Mycoplasma spp known to be mastitis pathogens. One mycoplasmal species was isolated initially on 99 of 198 (50.0%) dairies, but 68 of 198 (34.3%) dairies had 2 species isolated. Mycoplasma bovis, M californicum, and M bovigenitalium were consistently isolated, but M bovis (243/499; 48.6%) was the most commonly isolated species. Acholeplasma laidlawii was more prevalent in 1989 and 1995 than other years. Mycoplasma bovigenitalium and M californicum had a seasonal distribution. Less than 50 colonies per plate were isolated for most (317/500; 63.4%) bulk-tank samples. Of the milk samples with > 100 colonies/plate, Mycoplasma bovis was isolated most frequently (73/243; 30.0%).
Distribution of Mycoplasma spp varied by year, number of colonies isolated per sample, season, and herd. Therefore, it may be necessary to routinely sample bulk-tank milk, and all isolates should be speciated. Culture results from milk cooperatives should be used with other monitoring information to determine the Mycoplasma status of herds.
In a phase 3 trial involving more than 15,000 participants, two doses of NVX-CoV2373, a recombinant SARS-CoV-2 nanoparticle vaccine, administered 21 days apart had a vaccine efficacy of 89.7%. ...Reactogenicity was generally mild and transient, and adverse events were infrequent and of low grade.
The transmembrane 6 superfamily member 2 (TM6SF2) loss‐of‐function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically ...associated with lower levels of hepatically derived triglyceride‐rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites for triglyceride‐rich lipoprotein biogenesis and export. In light of this, we hypothesized that TM6SF2 may exhibit analogous effects on both liver and intestine lipid homeostasis. To test this, we genotyped rs58542926 in 983 bariatric surgery patients from the Geisinger Medical Center for Nutrition and Weight Management, Geisinger Health System, in Pennsylvania and from 3,556 study participants enrolled in the Amish Complex Disease Research Program. Although these two cohorts have different metabolic profiles, carriers in both cohorts had improved fasting lipid profiles. Importantly, following a high‐fat challenge, carriers in the Amish Complex Disease Research Program cohort exhibited significantly lower postprandial serum triglycerides, suggestive of a role for TM6SF2 in the small intestine. To gain further insight into this putative role, effects of TM6SF2 deficiency were studied in a zebrafish model and in cultured human Caco‐2 enterocytes. In both systems TM6SF2 deficiency resulted in defects in small intestine metabolism in response to dietary lipids, including significantly increased lipid accumulation, decreased lipid clearance, and increased endoplasmic reticulum stress. Conclusions: These data strongly support a role of TM6SF2 in the regulation of postprandial lipemia, potentially through a similar function for TM6SF2 in the lipidation and/or export of both hepatically and intestinally derived triglyceride‐rich lipoproteins. (Hepatology 2017;65:1526‐1542).
Increasingly, the effort is being fought on the ground to get out loyal voters, not only on the airwaves. Printing and mailing literature, recruiting and overseeing volunteers, setting up phone banks ...and, most importantly, combing voter databases in search of likely supporters, are ever more sophisticated and expensive undertakings. Though grass-roots efforts traditionally are more a tried-and-true Democratic Party tactic, the Republicans are getting into the act in a big way. The state Republican Party figures it already has spent upward of $1 million on voter mobilization this year and plans to spend a lot more before it is all over. The GOP's Chester County organization near Philadelphia alone recently augmented its $450,000 election budget by $50,000 to pay for signs, phone banks and other turnout efforts. Catholics, about a third of the state's electorate, are a major focus of the GOP's efforts. The Republican National Committee's newly formed Catholic Task Force is expected to spend as much as $1 million in Pennsylvania. The archdiocese itself is closely, if not explicitly, aligned with the GOP. Cardinal Anthony Bevilacqua made two appearances at the Republican National Convention in Philadelphia this summer, giving the benediction and holding a mass. The archdiocese's political operation of six full-time employees plans to distribute nearly 400,000 voter guides this weekend and to visit about 100 parishes before Election Day, says Guy Ciarrocchi, the lawyer-lobbyist who manages the archdiocese's efforts. John Dougherty, an official in both the Philadelphia Democratic Party and the local International Brotherhood of Electrical Workers, says he expects two-thirds of building-trade union members to help out that day, giving rides and distributing free lunches to voters. Arlene Holt, assistant to AFL-CIO President John Sweeney, has been in Philadelphia since mid-September coordinating union efforts and hopes to hit 200,000 Philadelphia area union members with at least one phone call and three or four pieces of mail each.
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