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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., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory "anti-CRISPR" (Acr) proteins, including six inhibitors (AcrIIA1-AcrIIA6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its more potent homolog, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially, and conditionally., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Bacterial CRISPR-Cas systems protect their host from bacteriophages and other mobile genetic elements. Mobile elements, in turn, encode various anti-CRISPR (Acr) proteins to inhibit the immune function of CRISPR-Cas. To date, Acr proteins have been discovered for type I (subtypes I-D, I-E, and I-F) and type II (II-A and II-C) but not other CRISPR systems. Here, we report the discovery of 12 acr genes, including inhibitors of type V-A and I-C CRISPR systems. AcrVA1 inhibits a broad spectrum of Cas12a (Cpf1) orthologs-including MbCas12a, Mb3Cas12a, AsCas12a, and LbCas12a-when assayed in human cells. The acr genes reported here provide useful biotechnological tools and mark the discovery of acr loci in many bacteria and phages., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The methionine salvage pathway (MSP) is critical for regeneration of S-adenosyl-l-methionine (SAM), a widely used cofactor involved in many essential metabolic reactions. The MSP has been completely elucidated in aerobic organisms, and found to rely on molecular oxygen. Since anaerobic organisms do not use O2, an alternative pathway(s) must be operating. We sought to evaluate whether the functions of two annotated MSP enzymes from Methanocaldococcus jannaschii, a methylthioinosine phosphorylase (MTIP) and a methylthioribose 1-phosphate isomerase (MTRI), are consistent with functioning in a modified anaerobic MSP (AnMSP). We show here that recombinant MTIP is active with six different purine nucleosides, consistent with its function as a general purine nucleoside phosphorylase for both AnMSP and purine salvage. Recombinant MTRI is active with both 5-methylthioribose 1-phosphate and 5-deoxyribose 1-phosphate as substrates, which are generated from phosphororolysis of 5'-methylthioinosine and 5'-deoxyinosine by MTIP, respectively. Together, these data suggest that MTIP and MTRI may function in a novel pathway for recycling the 5'-deoxyadenosine moiety of SAM in M. jannaschii. These enzymes may also enable biosynthesis of 6-deoxy-5-ketofructose 1-phosphate (DKFP), an essential intermediate in aromatic amino acid biosynthesis. Finally, we utilized a homocysteine auxotrophic strain of Methanosarcina acetivorans Δma1821-22Δoahs (HcyAux) to identify potential AnMSP intermediates in vivo. Growth recovery experiments of the M. acetivorans HcyAux were performed with known and proposed intermediates for the AnMSP. Only one metabolite, 2-keto-(4-methylthio)butyric acid, rescued growth of M. acetivorans HcyAux in the absence of homocysteine. This observation may indicate that AnMSP pathways substantially differ among methanogens from phylogenetically divergent genera.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

The mechanisms of sulfur uptake and trafficking in methanogens inhabiting sulfidic environments are highly distinctive. In aerobes, sulfur transfers between proteins occur via persulfide relay, but direct evidence for persulfides in methanogens has been lacking. Here, we use mass spectrometry to analyze tryptic peptides of the Methanosarcina acetivorans SepCysS and MA1821 proteins purified anaerobically from methanogen cells. These enzymes insert sulfide into phosphoseryl(Sep)-tRNA Cys and aspartate semialdehyde, respectively, to form Cys-tRNA Cys and homocysteine. A high frequency of persulfidation at conserved cysteines of each protein was identified, while the substantial presence of persulfides in peptides from other cellular proteins suggests that this modification plays a general physiological role in the organism. Purified native SepCysS containing persulfide at conserved Cys260 generates Cys-tRNA Cys in anaerobic single-turnover reactions without exogenously added sulfur, directly linking active-site persulfide formation in vivo with catalytic activity.

Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Conserved genes essential to sulfur assimilation and trafficking in aerobic organisms are missing in many methanogens, most of which inhabit highly sulfidic, anaerobic environmental niches. This suggests that methanogens possess distinct pathways for the synthesis of key metabolites and intermediates, including cysteine, homocysteine, and protein persulfide groups. Prior work identified a novel tRNA-dependent two-step pathway for cysteine biosynthesis and a new metabolic transformation by which sulfur is inserted into aspartate semialdehyde to produce homocysteine. Homocysteine biosynthesis requires two of the three proteins previously identified in our laboratory by a comprehensive bioinformatics approach. Here, we show that the third protein identified in silico, the ApbE-like protein COG2122, facilitates sulfide assimilation in Methanosarcina acetivorans Knockout strains lacking the gene encoding COG2122 are severely impaired for growth when sulfide is provided as the sole sulfur source. However, rapid growth is recovered upon supplementation with cysteine, homocysteine, or cystathionine, suggesting that COG2122 is required for efficient biosynthesis of both cysteine and homocysteine. Deletion of the gene encoding COG2122 does not influence the extent of sulfur modifications in tRNA or the prevalence of iron-sulfur clusters, indicating that the function of COG2122 could be limited to sulfide assimilation for cysteine and homocysteine biosynthesis alone., Importance: We have found that the conserved M. acetivorans ma1715 gene, which encodes an ApbE-like protein, is required for optimal growth with sulfide as the sole sulfur source and supports both cysteine and homocysteine biosynthesis in vivo Together with related functional-genomics studies in methanogens, these findings make a key contribution to elucidating the novel pathways of sulfide assimilation and sulfur trafficking in anaerobic microorganisms that existed before the advent of oxygenic photosynthesis. The data suggest that the MA1715 protein is particularly important to sustaining robust physiological function when ambient sulfide concentrations are low. Phylogenetic analysis shows that MA1715 and other recently discovered methanogen sulfur-trafficking proteins share an evolutionary history with enzymes in the methanogenesis pathway. The newly characterized genes thus likely formed an essential part of the core metabolic machinery of the ancestral euryarchaeote., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The Methanocaldcoccus jannaschii tyrosyl-tRNA synthetase (TyrRS):tRNA(Tyr) cognate pair has been used to incorporate a large number of noncanonical amino acids (ncAAs) into recombinant proteins in Escherichia coli. However, the structural elements of the suppressor tRNA(Tyr) used in these experiments have not been examined for optimal performance. Here, we evaluate the steady-state kinetic parameters of wild-type M. jannaschii TyrRS and an evolved 3-nitrotyrosyl-tRNA synthetase (nitroTyrRS) toward several engineered tRNA(Tyr) suppressors, and we correlate aminoacylation properties with the efficiency and fidelity of superfolder green fluorescent protein (sfGFP) synthesis in vivo. Optimal ncAA-sfGFP synthesis correlates with improved aminoacylation kinetics for a tRNA(Tyr) amber suppressor with two substitutions in the anticodon loop (G34C/G37A), while four additional mutations in the D and variable loops, present in the tRNA(Tyr) used in all directed evolution experiments to date, are deleterious to function both in vivo and in vitro. These findings extend to three of four other evolved TyrRS enzymes that incorporate distinct ncAAs. Suppressor tRNAs elicit decreases in amino acid Km values for both TyrRS and nitroTyrRS, suggesting that direct anticodon recognition by TyrRS need not be an impediment to superior performance of this orthogonal system and offering insight into novel approaches for directed evolution. The G34C/G37A tRNA(Tyr) may enhance future incorporation of many ncAAs by engineered TyrRS enzymes.

The biosynthetic route for homocysteine, intermediate in methionine biosynthesis, is unknown in some methanogenic archaea because homologues of the canonical required genes cannot be identified. Here we demonstrate that Methanocaldococcus jannaschii can biosynthesize homocysteine from aspartate semialdehyde and hydrogen sulfide. Additionally, we confirm the genes involved in this new pathway in Methanosarcina acetivorans. A possible series of reactions in which a thioaldehyde is formed and then reduced to a thiol are proposed. This represents a novel route for the biosynthesis of homocysteine and exemplifies unique aspects of sulfur chemistry occurring in prebiotic environments and in early life forms.

The metabolic network for sulfide assimilation and trafficking in methanogens is largely unknown. To discover novel proteins required for these processes, we used bioinformatics to identify genes co-occurring with the protein biosynthesis enzyme SepCysS, which converts phosphoseryl-tRNA(Cys) to cysteinyl-tRNA(Cys) in nearly all methanogens. Exhaustive analysis revealed three conserved protein families, each containing molecular signatures predicting function in sulfur metabolism. One of these families, classified within clusters of orthologous groups (COG) 1900, possesses two conserved cysteine residues and is often found in genomic contexts together with known sulfur metabolic genes. A second protein family is predicted to bind two 4Fe-4S clusters. All three genes were also identified in more than 50 strictly anaerobic bacterial genera from nine distinct phyla. Gene-deletion and growth experiments in Methanosarcina acetivorans, using sulfide as the sole sulfur source, demonstrate that two of the proteins (MA1821 and MA1822) are essential to homocysteine biosynthesis in a background lacking an additional gene for sulfur insertion into homocysteine. Mutational analysis confirms the importance of several structural elements, including a conserved cysteine residue and the predicted 4Fe-4S cluster-binding domain., (© 2014 John Wiley & Sons Ltd.)

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., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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.

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.