Metritis and the uterine disease microbiome are associated with long-term changes in the endometrium of dairy cows†.

Cows with metritis (uterine disease) during the first 1 to 2 weeks postpartum have lower pregnancy rates when inseminated later postpartum (typically >10 weeks). We hypothesized that metritis and the disease-associated uterine microbiome have a long-term effect on endometrial gene expression. Changes in gene expression may inform a mechanism through which disease lowers pregnancy rates. A total of 20 cows were enrolled at 1 to 2 weeks postpartum to either metritis (clinical disease; n = 10) or healthy (control; n = 10) groups and randomly assigned to be slaughtered at approximately 80 and 165 dpp (mid-lactation). The microbiome of the reproductive tract was sampled to confirm the presence of pathogens that are typical of metritis. In addition to the original clinical diagnosis, study cows were retrospectively assigned to uterine-disease and control groups based on the composition of their microbiome. There was no effect of early postpartum uterine disease on the uterine microbiome at mid-lactation (time of slaughter). Nonetheless, early postpartum metritis and the disease microbiome were associated with a large number of differentially-expressed genes at mid-lactation primarily in the caruncular compared with the inter-caruncular endometrium. Gene enrichment analysis identified oxidative phosphorylation as the primary pathway increased in caruncular endometrium of diseased cows whereas growth factor signaling pathways were reduced. The current study demonstrated that metritis and a uterine disease microbiome leave a sustained imprint on gene expression in the caruncular endometrium that may explain lower fertility in cows with postpartum uterine disease.

One Health Approach for Reporting Veterinary Carbapenem-Resistant Enterobacterales and Other Bacteria of Public Health Concern.

A carbapenem-resistant Enterobacterales outbreak at a veterinary teaching hospital in the United States increased urgency for improved communication among diagnostic laboratories, public health authorities, veterinarians, and pet owners. Kansas State University, University of Missouri, Kansas Department of Health and Environment, and Veterinary Laboratory Investigation and Response Network created a surveillance, storage, and reporting protocol for veterinary antimicrobial-resistant bacteria; determined frequency of those bacteria in companion animals during 2018-2021; and created educational flyers for veterinarians and pet owners. We recommend a One Health strategy to create efficient surveillance programs to identify and report antimicrobial-resistant bacteria and educate veterinarians and pet owners about transmission risks.

Non-carbapenemase producing carbapenem-resistant Klebsiella pneumoniae isolated from the urinary tract of a dog.

Objective: Carbapenems are broad-spectrum ß-lactams with excellent activity against multidrug-resistant (MDR) Enterobacterales. Unfortunately, resistance to carbapenems within this bacterial family, known as carbapenem-resistant Enterobacterales (CRE), occurs and challenges the ability to treat difficult MDR infections. Although the impact of carbapenem-resistance has been greatest in human medicine, reports in the veterinary literature are increasing especially as national veterinary antimicrobial resistance surveillance programs are now in place. In this brief communication, we report the isolation of a non-carbapenemase-producing, carbapenem-resistant Klebsiella pneumoniae from the urine of a dog, discuss the likely mechanism of resistance, and wider implications. Animal: Canine. Procedure: Whole genome sequencing and phenotypic antimicrobial susceptibility testing was performed on a K. pneumoniae isolated from the urine of a dog. Results: Antimicrobial susceptibility testing identified phenotypic resistance to imipenem and meropenem. Phenotypic detection of carbapenemase production was negative. Whole genome sequencing identified efflux pump genes associated with carbapenem resistance and point mutations in membrane porin genes. No carbapenemase gene was identified. Conclusion: Phenotypic antimicrobial susceptibility testing identified the K. pneumoniae as a non-carbapenemase producing carbapenem-resistant organism with the proposed genotypic mechanism including alteration of efflux pumps and membrane porin activity and/or expression. Clinical significance: Currently, there is limited use of carbapenem antimicrobial drugs in veterinary medicine, and practitioners may be unfamiliar or unaware of this type of resistance, its significance on routine antimicrobial susceptibility test reports, and implications for antimicrobial therapy and public health. Carbapenem-resistant Enterobacterales are infrequently isolated from companion animals; however, due to increasing adoption of advanced medical and surgical interventions, they may become more prevalent.

Objectif: Les carbapénèmes sont des ß-lactamines à large spectre avec une excellente activité contre les Enterobacterales multirésistantes (MDR). Malheureusement, la résistance aux carbapénèmes au sein de cette famille bactérienne, connue sous le nom d'Enterobacterales résistantes aux carbapénèmes (CRE), se produit et remet en question la capacité de traiter les infections MDR difficiles. Bien que l'impact de la résistance aux carbapénèmes ait été plus important en médecine humaine, les rapports dans la littérature vétérinaire se multiplient, d'autant plus que des programmes nationaux de surveillance de la résistance aux antimicrobiens vétérinaires sont désormais en place. Dans cette brève communication, nous rapportons l'isolement d'une Klebsiella pneumoniae non-productrice de carbapénémase et résistante aux carbapénèmes à partir de l'urine d'un chien, discutons du mécanisme probable de résistance et des implications plus larges. Animal: Canin. Procédure: Le séquençage du génome entier et les tests de sensibilité phénotypique aux antimicrobiens ont été effectués sur un isolat de K. pneumoniae provenant de l'urine d'un chien. Résultats: Les tests de sensibilité aux antimicrobiens ont identifié une résistance phénotypique à l'imipénème et au méropénème. La détection phénotypique de production de carbapénèmase était négative. Le séquençage du génome entier a identifié des gènes de pompe à efflux associés à la résistance aux carbapénèmes et à des mutations ponctuelles dans les gènes des porines membranaires. Aucun gène de carbapénémase n'a été identifié. Conclusion: Les tests de sensibilité phénotypique aux antimicrobiens ont identifié cet isolat de K. pneumoniae comme un organisme résistant aux carbapénèmes ne produisant pas de carbapénémase avec le mécanisme génotypique proposé, y compris l'altération des pompes à efflux et l'activité et/ou l'expression de porines membranaires. Signification clinique: Actuellement, l'utilisation des médicaments antimicrobiens à base de carbapénème en médecine vétérinaire est limitée, et les praticiens peuvent ne pas être familiers ou ne pas être au fait de ce type de résistance, de son importance dans les rapports de routine sur les tests de sensibilité aux antimicrobiens et de ses implications pour la thérapie antimicrobienne et la santé publique. Les Enterobacterales résistantes aux carbapénèmes sont rarement isolées des animaux de compagnie; cependant, en raison de l'adoption croissante d'interventions médicales et chirurgicales avancées, elles peuvent devenir plus répandues.(Traduit par Dr Serge Messier).

Influence of intraoperative closed glove exchange on glove contamination during clean soft tissue surgeries.

OBJECTIVES: To determine the influence of intraoperative glove exchange on glove contamination during clean soft tissue surgery. STUDY DESIGN: Prospective clinical study. SAMPLE POPULATION: Two hundred pairs of gloves and gowns from 50 clean soft tissue surgeries. METHODS: Gloves and gown cuffs were cultured from the primary surgeon and first assistant using a standardized protocol. Cultures were taken from outer surface of both gown cuffs prior to surgery and after gloves were removed at the end of surgery; gloves were cultured prior to surgery, at end of surgery and after a new pair was donned after closed glove exchange. Cultures were evaluated for colony-forming units after 72 h of inoculation. RESULTS: Bacterial contamination was documented in 41 of the 50 surgeries (82%). The most common species cultured was Streptocococcus spp. There was no difference (p = .719) in the bacterial contamination rate of gown cuffs prior to surgery (10%; 20/200) compared to after surgery (9.5%; 19/200). The bacterial contamination rate for gloves was 10.5% (21/200) prior to surgery, 19.5% (39/196) after surgery, and 11% (22/200) after regloving. Gloves cultured following surgery were significantly more contaminated than gloves cultured preoperatively (p = .010) or gloves cultured following regloving (p = .018). CONCLUSION: Glove exchange did not increase bacterial contamination of gloves during the clean soft tissue surgeries tested here. CLINICAL SIGNIFICANCE: The outside of the gown cuff does not seem to represent a major source of contamination during clean procedures. This study does not provide evidence to support a change in current practices for intraoperative closed glove exchange.

Stability of 1% voriconazole solution in a constant-rate infusion pump for topical ocular delivery to horses.

OBJECTIVE: To establish the effect of storage in a constant-rate infusion (CRI) pump on the sterility and stability of voriconazole 1% solution. PROCEDURE: Nine vials of voriconazole (Vfend(®) I.V.) 1% solution were prepared. Approximately half of each solution was used to prime a commercially available CRI pump with attached subpalpebral lavage system (CRI/SPL unit) with the remaining solution stored in the commercial glass vial. Three CRI/SPL units and their three corresponding vials were stored at one of three temperatures: 23 °C, 33 °C, and 40 °C. The CRI pumps ran for 7 days, and the vials were stored for 30 days. Fungal and aerobic bacterial cultures were performed on the first and last day of the storage period for each vessel. Samples were obtained at regular intervals for determination of voriconazole concentration using high-performance liquid chromatography. RESULTS: No bacterial or fungal contamination was identified in any solution at any time point. All solutions stored in the commercial glass vial remained stable throughout the study period. Multiple CRI/SPL units became blocked with crystallized voriconazole. There was a significant increase in voriconazole concentration after passage through the CRI/SPL units. CONCLUSIONS: Voriconazole 1% solution is not compatible for use in a CRI/SPL unit at temperatures between 23 and 40 °C. Voriconazole 1% solution is stable in the commercial glass vial when stored at controlled temperatures as high as 40 °C for up to 30 days.

Comparison of genotypic and phenotypic antimicrobial resistance profiles of Salmonella enterica isolates from poultry diagnostic specimens.

The spread of antimicrobial-resistant bacteria is a significant concern, as it can lead to increased morbidity and mortality in both humans and animals. Whole-genome sequencing (WGS) is a powerful tool that can be used to conduct a comprehensive analysis of the genetic basis of antimicrobial resistance (AMR). We compared the phenotypic and genotypic AMR profiles of 97 Salmonella isolates derived from chicken and turkey diagnostic samples. We focused AMR analysis on 5 antimicrobial classes: aminoglycoside, beta-lactam, phenicol, tetracycline, and trimethoprim. The overall sensitivity and specificity of WGS in predicting phenotypic antimicrobial resistance in the Salmonella isolates were 93.4% and 99.8%, respectively. There were 16 disagreement instances, including 15 that were phenotypically resistant but genotypically susceptible; the other instance involved phenotypic susceptibility but genotypic resistance. Of the isolates examined, 67 of 97 (69%) carried at least 1 resistance gene, with 1 isolate carrying as many as 12 resistance genes. Of the 31 AMR genes analyzed, 16 were identified as aminoglycoside-resistance genes, followed by 4 beta-lactam-resistance, 3 tetracycline-resistance, 2 sulfonamide-resistance, and 1 each of fosfomycin-, quinolone-, phenicol-, trimethoprim-, bleomycin-, and colistin-resistance genes. Most of the resistance genes found were located on plasmids.

The microbiome of the pregnant uterus in Holstein dairy heifers and cows assessed by bacterial culture and 16S ribosomal RNA gene sequencing.

Introduction: The possibility that there is a resident and stable commensal microbiome within the pregnant uterus has been supported and refuted by a series of recent studies. One element of most of the initial studies was that they were based primarily on 16S rRNA gene sequencing from bacteria. To account for this limitation, the current study performed both bacterial culture and 16S rRNA gene sequencing in a side-by-side manner (e.g., same tissues isolated from the same animal). Methods: The uteruses of 10 mid-pregnant (156 ± 5 d of gestation) Holstein heifers and cows were collected following slaughter. The external surface of the reproductive tract (positive control for contamination during tissue collection) as well as tissues within the pregnant uterus (placentome, inter-cotyledonary placenta, inter-caruncular endometrium, amnionic fluid, allantoic fluid, fetal abomasum content, and fetal meconium) were sampled for bacterial culture and 16S rRNA gene sequencing. Results: There were 87 unique bacterial species cultured from the external surface of the pregnant reproductive tract (contamination control) and 12 bacterial species cultured from pregnancy tissues. Six out of 10 cattle (60%) exhibited bacterial growth in at least one location within the pregnant uterus. For the metataxonomic results (16S rRNA gene sequencing), a low targeted microbial biomass was identified. Analyses of the detected amplicon sequence variants (ASV) revealed that there were: (1) genera that were prevalent on both the external surface and within the pregnant uterus; (2) genera that were prevalent on the external surface but either not detected or had very low prevalence within the pregnant uterus; and (3) genera that were either not detected or had low prevalence on the external surface but found with relatively high prevalence within the pregnant uterus. Conclusion: There are a small number of viable bacteria in the pregnant uterus. The 16S rRNA gene sequencing detected a microbial community within the pregnant uterus but with a low biomass. These results are consistent with recent studies of the pregnant bovine uterus and leave open the question of whether there is adequate microbial mass to significantly affect the biology of the normal healthy bovine pregnancy.

Causes of mortality in farmed white-tailed deer in the midwestern United States, 2004-2023.

Farmed cervids are of growing economic importance in the midwestern United States. Although diseases of wild and captive cervids have been examined in more northerly climates, little information exists on the health challenges of deer in the Midwest. We characterized and summarized the causes of mortality in farmed white-tailed deer (Odocoileus virginianus) submitted to the University of Missouri Veterinary Medical Diagnostic Laboratory (Columbia, MO, USA) over a 19-y period (2004-2023). Of 388 cases examined, 253 (65%) were carcasses submitted for autopsy, and 135 (35%) cases were tissue samples harvested by field veterinarians. Infectious disease was the most common cause of mortality (n = 335; 86.3%). Of infectious causes, primary pneumonia was most common (n = 140; 41.7%), followed by septicemia (n = 68; 20.1%), and primary enteritis (n = 64; 19.1%). Viruses were detected in 18% of pneumonia cases. The most common non-infectious diagnoses were emaciation and trauma (both 4 each; 1%). Thirteen animals (3.4%) died of unknown causes. Forty-nine percent of cases were juvenile deer <1-y-old. Most cases were received in the summer (212; 54.6%). Infectious diseases, particularly bacteria and viruses, pose a significant health challenge to farmed deer in the midwestern United States.

Establishment of the uterine microbiome following artificial insemination in virgin heifers.

Introduction: The concept of a sterile uterus was challenged by recent studies that have described the microbiome of the virgin and pregnant uterus for species including humans and cattle. We designed two studies that tested whether the microbiome is introduced into the uterus when the virgin heifer is first inseminated and whether the origin of the microbiome is the vagina/cervix. Methods: The uterine microbiome was measured immediately before and after an artificial insemination (AI; Study 1; n = 7 AI and n = 6 control) and 14 d after insemination (Study 2; n = 12 AI and n = 12 control) in AI and non-AI (control) Holstein heifers. A third study (Study 3; n = 5 Holstein heifers) that included additional negative controls was subsequently conducted to support the presence of a unique microbiome within the uterus despite the low microbial biomass and regardless of insemination. Traditional bacteriological culture was performed in addition to 16S rRNA gene sequencing on the same samples to determine whether there were viable organisms in addition to those detected based on DNA sequencing (16S rRNA gene sequence). Results and discussion: Inseminating a heifer did not lead to a large change in the microbiome when assessed by traditional methods of bacterial culture or metataxonomic (16S rRNA gene) sequencing (results of Studies 1 and 2). Very few bacteria were cultured from the body or horn of the uterus regardless of whether an AI was or was not (negative control) performed. The cultured bacterial genera (e.g., Bacillus, Corynebacterium, Cutibacterium, Micrococcus, Staphylococcus, and Streptococcus) were typical of those found in the soil, environment, skin, mucous membranes, and urogenital tract of animals. Metataxonomic sequencing of 16S rRNA gene generated a large number of amplicon sequence variants (ASV), but these larger datasets that were based on DNA sequencing did not consistently demonstrate an effect of AI on the abundance of ASVs across all uterine locations compared with the external surface of the tract (e.g., perimetrium; positive control samples for environment contamination during slaughter and collection). Major genera identified by 16S rRNA gene sequencing overlapped with those identified with bacterial culture and included Cutibacterium, Staphylococcus, and Streptococcus.

Systemic antibiotic treatment of cows with metritis early postpartum does not change the progression of uterine disease or the uterine microbiome at 1 month postpartum.

Background: Postpartum uterine disease (metritis) is common in dairy cows. The disease develops within 1 week after calving and is associated with microbial dysbiosis, fever, and fetid uterine discharge. Cows with metritis have a greater likelihood of developing endometritis and infertility later postpartum. Antibiotic treatment is used to relieve symptoms of metritis but the capacity of antibiotic treatment to improve fertility later postpartum is inconsistent across published studies. We hypothesized that an antibiotic has only a short-term effect on the uterine microbiome and does not change the progression of disease from metritis to endometritis. To test this hypothesis, we studied the effects of systemic antibiotic given to cows diagnosed with metritis and healthy cows early postpartum on the development of endometritis and the uterine microbiome at 1 month postpartum. Results: Cows diagnosed with metritis were compared to healthy ones in a 2 × 2 factorial design, where they were either treated with an antibiotic (ceftiofur hydrochloride) at 7 to 10 days postpartum or left untreated. Cows were slaughtered at one month postpartum and the uterus was assessed for endometritis (presence of purulent material in the uterine lumen and inflammation in the endometrium) and uterine samples were collected for bacteriology and metagenomics (16S rRNA gene sequencing). As expected, the uterine microbiome at disease diagnosis had dysbiosis of typical metritis pathogens (e.g., Fusobacterium, Bacteroides, and Porphyromonas) in diseased compared with healthy cows. At one month postpartum, there was a tendency for more endometritis in metritis cows compared with healthy but antibiotic treatment had no effect on endometritis prevalence regardless of the original disease diagnosis. Likewise, when bacteria were cultured or sequenced, there were a greater number of species (culture) or amplicon sequence variants (ASV; sequencing) in the uterine lumen of cows with metritis. However, antibiotic treatment had no effect on the prevalence of cultured species or the composition of the detected ASV. The uterine microbiome at 1 month postpartum was associated with the clinical observation of the uterus (endometritis or healthy). Conclusions: Early postpartum antibiotic treatment only provides temporary resolution of uterine dysbiosis that is not sustained long-term. Failure to resolve the dysbiosis is associated with a greater prevalence of endometritis in cows with metritis, and the occurrence of endometritis significantly impacts fertility later postpartum.

Bacillary hemoglobinuria in beef cattle infected with Fascioloides magna in Missouri.

Bacillary hemoglobinuria (BH) is an infectious disease, mostly affecting cattle, caused by Clostridium haemolyticum (C. novyi type D), with acute hepatic necrosis and intravascular hemolysis. Cattle are typically predisposed to BH by liver injury caused by Fasciola hepatica, although cases have been reported in cattle without evidence of this parasite. Here we describe a cluster of 14 BH cases from 7 counties in north-central to central Missouri submitted to a veterinary diagnostic laboratory between December 2020 and April 2023. Postmortem examination in all cases revealed hemoglobinuria and acute hepatic necrosis with large numbers of gram-positive bacilli with terminal-to-subterminal spores. Flukes, fluke ova, and/or fluke pigment consistent with Fascioloides magna were identified in 12 of 14 cases. Sequences of the nuclear ribosomal internal transcribed spacer 1 (ITS1) from one fluke had 100% identity to F. magna. C. novyi was detected by fluorescent antibody testing of liver impression smears (11 of 12 cases) and by immunohistochemistry of liver sections (7 of 7 cases). PCR on formalin-fixed, paraffin-embedded tissues amplified the C. haemolyticum beta toxin gene in each of the 7 cases tested. To our knowledge, a confirmed cluster of BH associated with F. magna has not been reported previously in cattle.

Comparison of four endotracheal tube cleaning protocols in anesthetized dogs.

OBJECTIVE: To compare the efficacy of 4 cleaning protocols applied to endotracheal tubes (ETTs) collected from anesthetized dogs. SAMPLE: 100 ETTs (25 per protocol). PROCEDURES: A 10-question survey designed to determine ETT reuse and cleaning practices was distributed via email to a sample of veterinary anesthesiologists. Informed by survey results, 4 ETT cleaning protocols were selected for use in a prospective clinical study. Dogs were intubated with sterile polyvinyl chloride ETTs. At extubation, each ETT was cultured for bacterial growth, randomly assigned to 1 of 4 protocols [water scrub (P1), detergent scrub (P2), detergent scrub and chlorhexidine gluconate (CHG) soak (P3), or detergent scrub and bleach soak (P4)], and cultured again after drying. Bacterial genera were identified using mass spectrometry and 16s rRNA sequencing. Proportions of ETTs exhibiting no post-cleaning growth were compared between protocols using the Fisher exact test with Bonferroni correction. RESULTS: Half of survey respondents that reused ETTs did not sterilize them before reuse, cleaning methods varied widely, and no reported methods were evidence-based. After use, the number of ETTs exhibiting no post-cleaning bacterial growth were 15/25 (60%), 14/25 (56%), 20/25 (80%), and 17/25 (68%) for protocols P1, P2, P3, and P4, respectively. Pairwise comparisons did not reveal any statistically significant differences between protocols. CLINICAL RELEVANCE: In small animal patients, some veterinary anesthesiologists reuse ETTs without sterilization and cleaning protocols vary widely. No differences between the studied protocols were identified. Further research is necessary to identify a safe, efficacious ETT cleaning protocol for use in small animal practice.

Bacterial culture and susceptibility of samples taken from septic foot lesions of adult beef cattle.

BACKGROUND: Lameness is an economically important and common disease of cattle, and foot disease is the most common cause of lameness in cattle. Limited data is available regarding lameness in cow-calf operations. OBJECTIVES: Describe the bacteria most commonly isolated from septic lesions of the feet of adult beef cattle and the antimicrobial susceptibility patterns of the isolated bacteria. ANIMALS: Fifty-four adult cattle from cow-calf operations and diagnosed with a sole abscess or distal interphalangeal joint sepsis were enrolled. METHODS: Prospective observational study. Abscess fluid from a convenience sample of clinical cases was cultured. Isolated bacteria were identified using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry or 16s rRNA gene sequencing. Antimicrobial susceptibility profiling was performed on isolates when a bacterial species was identified from ≥5 samples. RESULTS: Fifty of the 54 samples were polymicrobial. Trueperella pyogenes (22/54), Streptococcus uberis (16/54), and Bacteroides pyogenes (14/54) were the most commonly isolated bacteria. Eighty-one of 96 tested isolates were resistant to at least 1 antimicrobial; multidrug resistance was identified in 37/96 isolates. Oxytetracycline (50/96), tylosin (40/96), and florfenicol (37/96) resistance was commonly identified. Resistance to ceftiofur (5/96) was rare. CONCLUSIONS AND CLINICAL IMPORTANCE: Septic processes of the foot in these adult beef cattle frequently were polymicrobial. Most of the isolated bacteria were resistant to at least 1 antimicrobial with over one-third being multidrug resistant. Although simple sole abscesses do not require antimicrobial treatment, deep septic processes of the foot often are treated with antimicrobials. Culture and susceptibility of deep septic lesions may guide judicious antimicrobial usage.

Mycobacterium haemophilum infection in canine cerebrospinal fluid.

A 4-year-old female spayed Australian cattle dog was presented to the Emergency Service at the University of Missouri Veterinary Health Center Small Animal Hospital for generalized pain and lethargy. At presentation, the dog showed severe cervical spinal pain and thoracic limb deficits consistent with a multifocal neuroanatomic localization. Magnetic resonance imaging of the cervical spine revealed T2 and T1 postcontrast intense signal extending from the level of the medulla through C5 most marked in the caudal brainstem and cranial cervical spinal cord. The suspected diagnosis was severe meningoencephalomyelitis and secondary edema. Analysis of cerebrospinal fluid (CSF) collected from the cerebellomedullary cistern revealed a marked mixed pleocytosis with intralesional structures morphologically consistent with Mycobacterium sp. Standard DNA PCR assay performed on the CSF yielded the presence of Mycobacterium haemophilum. To the authors' knowledge, this is the first reported case of CNS mycobacteriosis diagnosed on CSF analysis in a dog.

Metagenomic identification of a novel oomycete cultured from a pyogranulomatous mass on the tail of a domestic cat.

We report here a transiently culturable oomycete pathogen isolated from a pyogranulomatous tail mass in a cat. The organism was morphologically and genetically distinct from Lagenidium and Pythium species. Following next-generation sequencing (NGS) and assembly of contigs, initial phylogenetic analysis using fragments of the cox1 mitochondrial gene identified this specimen as Paralagenidium sp. after nucleotide alignments with sequences obtained from the Barcode of Life Data System (BOLD). However, further analysis of a concatenation of 13 different mitochondrial genes showed that this organism is unique and different from all known oomycetes. A negative PCR result using primers targeting known oomycete pathogens may not be enough to rule out oomycosis in a suspected case. Additionally, the use of a single gene to classify oomycetes may produce misleading results. The advent of metagenomic sequencing and NGS provides a unique opportunity to further explore the diversity of oomycetes as plant and animal pathogens beyond the current capabilities of global barcoding projects that are based on partial genomic sequences.

Bacterial Causes of Intestinal Disease in Dairy Calves: Acceptable Control Measures.

Although diarrhea in dairy calves is common, it is not always due to bacteria. Escherichia coli, Salmonella, and Clostridium perfringens are the most commonly implicated bacteria, but an etiologic diagnosis should be sought before specific treatment is instituted. Nonspecific treatment such as fluid, electrolyte, and nutritional support should be accomplished while diagnostics are pending. Antimicrobials should not be a first-line therapy for calf diarrhea. Control measures are discussed.

Systemic coccidioidomycosis in a llama cria native to Missouri.

A 3-mo-old male llama was examined because of a 4-wk history of lethargy and ill thrift. Clinical examination revealed subcutaneous masses in the left prescapular and right inguinal regions, mild ataxia, a slight head tilt to the right, and right ear droop. The cria died before clinical workup was complete. At autopsy, there was generalized lymphadenomegaly, a hepatic nodule, a midbrain mass causing rostral compression of the cerebellum, and internal hydrocephalus. Microscopic findings included pyogranulomatous lymphadenitis, meningoencephalitis, hepatitis, and bronchopneumonia. Intralesional fungal spherules, most consistent with Coccidioides spp., were identified in the lymph nodes, lung, and brain. Fungal culture, single-nucleotide variation genotyping real-time PCR, and DNA sequencing confirmed Coccidioides posadasii. The dam of the cria was native to Arizona and had been moved to Missouri ~2.5 y previously. Agar gel immunodiffusion assay of the herd revealed that only the dam was positive for Coccidioides spp.; 6 herdmates were negative. Computed tomography of the dam revealed multiple nodules within the lungs and liver, which were presumed to be an active coccidioidomycosis infection. This case of systemic coccidioidomycosis in a llama native to Missouri was presumably acquired by vertical transmission from the dam.

Systems Biology Analysis of Temporal In vivo Brucella melitensis and Bovine Transcriptomes Predicts host:Pathogen Protein-Protein Interactions.

To date, fewer than 200 gene-products have been identified as Brucella virulence factors, and most were characterized individually without considering how they are temporally and coordinately expressed or secreted during the infection process. Here, we describe and analyze the in vivo temporal transcriptional profile of Brucella melitensis during the initial 4 h interaction with cattle. Pathway analysis revealed an activation of the "Two component system" providing evidence that the in vivo Brucella sense and actively regulate their metabolism through the transition to an intracellular lifestyle. Contrarily, other Brucella pathways involved in virulence such as "ABC transporters" and "T4SS system" were repressed suggesting a silencing strategy to avoid stimulation of the host innate immune response very early in the infection process. Also, three flagellum-encoded loci (BMEII0150-0168, BMEII1080-1089, and BMEII1105-1114), the "flagellar assembly" pathway and the cell components "bacterial-type flagellum hook" and "bacterial-type flagellum" were repressed in the tissue-associated B. melitensis, while RopE1 sigma factor, a flagellar repressor, was activated throughout the experiment. These results support the idea that Brucella employ a stealthy strategy at the onset of the infection of susceptible hosts. Further, through systems-level in silico host:pathogen protein-protein interactions simulation and correlation of pathogen gene expression with the host gene perturbations, we identified unanticipated interactions such as VirB11::MAPK8IP1; BtaE::NFKBIA, and 22 kDa OMP precursor::BAD and MAP2K3. These findings are suggestive of new virulence factors and mechanisms responsible for Brucella evasion of the host's protective immune response and the capability to maintain a dormant state. The predicted protein-protein interactions and the points of disruption provide novel insights that will stimulate advanced hypothesis-driven approaches toward revealing a clearer understanding of new virulence factors and mechanisms influencing the pathogenesis of brucellosis.

Systems Analysis of Early Host Gene Expression Provides Clues for Transient Mycobacterium avium ssp avium vs. Persistent Mycobacterium avium ssp paratuberculosis Intestinal Infections.

It has long been a quest in ruminants to understand how two very similar mycobacterial species, Mycobacterium avium ssp. paratuberculosis (MAP) and Mycobacterium avium ssp. avium (MAA) lead to either a chronic persistent infection or a rapid-transient infection, respectively. Here, we hypothesized that when the host immune response is activated by MAP or MAA, the outcome of the infection depends on the early activation of signaling molecules and host temporal gene expression. To test our hypothesis, ligated jejuno-ileal loops including Peyer's patches in neonatal calves were inoculated with PBS, MAP, or MAA. A temporal analysis of the host transcriptome profile was conducted at several times post-infection (0.5, 1, 2, 4, 8 and 12 hours). When comparing the transcriptional responses of calves infected with the MAA versus MAP, discordant patterns of mucosal expression were clearly evident, and the numbers of unique transcripts altered were moderately less for MAA-infected tissue than were mucosal tissues infected with the MAP. To interpret these complex data, changes in the gene expression were further analyzed by dynamic Bayesian analysis. Bayesian network modeling identified mechanistic genes, gene-to-gene relationships, pathways and Gene Ontologies (GO) biological processes that are involved in specific cell activation during infection. MAP and MAA had significant different pathway perturbation at 0.5 and 12 hours post inoculation. Inverse processes were observed between MAP and MAA response for epithelial cell proliferation, negative regulation of chemotaxis, cell-cell adhesion mediated by integrin and regulation of cytokine-mediated signaling. MAP inoculated tissue had significantly lower expression of phagocytosis receptors such as mannose receptor and complement receptors. This study reveals that perturbation of genes and cellular pathways during MAP infection resulted in host evasion by mucosal membrane barrier weakening to access entry in the ileum, inhibition of Ca signaling associated with decreased phagosome-lysosome fusion as well as phagocytosis inhibition, bias toward Th2 cell immune response accompanied by cell recruitment, cell proliferation and cell differentiation; leading to persistent infection. Contrarily, MAA infection was related to cellular responses associated with activation of molecular pathways that release chemicals and cytokines involved with containment of infection and a strong bias toward Th1 immune response, resulting in a transient infection.

Development of real-time diagnostic assays specific for Mycoplasma mycoides subspecies mycoides Small Colony.

Rapid and specific detection of Mycoplasma mycoides subsp. mycoides Small Colony (M. mycoides SC) is important for the effective control of contagious bovine pleuropneumonia. Although the United States has been free of this disease for over 100 years, it is necessary to develop modern diagnostic assays that are sensitive and specific for biological agents that would affect the US agricultural industry following accidental or intentional introduction into the US agricultural population. With this aim in mind, we have identified M. mycoides SC-specific genetic loci and developed TaqMan-based PCR assays for the detection of M. mycoides SC. The TaqMan assay allows for real-time detection of specific, amplified PCR products using portable equipment, enabling testing to be performed in the field. These assays are specific for M. mycoides SC, failing to amplify DNA from other organisms belonging to the M. mycoides cluster or two phylogenetically unrelated bovine mycoplasma species. Standard curves were drawn based on the linear relationships measured between the threshold fluorescence (C(T)) values and a measured quantity of genomic DNA. M. mycoides SC was successfully detected in bronchoalveolar lavage samples obtained from experimentally infected cattle. These TaqMan-based real-time PCR assays will allow for the rapid and specific detection of M. mycoides SC.