Diagnosis and surgical management of a retrobulbar abscess causing unilateral exophthalmos in a Boer goat.

A 10-year-old Boer goat wether presented for unilateral exophthalmos of 2- to 3-week duration. Ocular ultrasonography and computed tomography (CT) were utilized in the diagnosis of the patient's orbital disease and surgical planning. Exenteration was performed under the same general anesthetic event as CT. Cytology, culture, and histopathology were performed after exenteration. Cytology was consistent with a mixed bacterial infection. Culture confirmed the presence of Streptococcus ovis. Histopathology on the enucleated globe and mass revealed no evidence of tumor and confirmed intraocular extension of retrobulbar inflammation. Histopathologic diagnosis was consistent with severe chronic orbital pyogranuloma and fibrinosuppurative endophthalmitis confined to the subretinal space. The abscess recurred in the orbital space 2 weeks postoperatively; the orbit was explored. Repeat culture was consistent with S. ovis, Staphylococcus schleigeri subspecies coagulans, and Fusobacterium necrophorum. Complete resolution was obtained after drainage and lavage of the orbit. Abscess is cited as a cause of exophthalmos in small ruminants, but no individual case reports exist. Advanced imaging allowed presumptive diagnosis and surgical planning. Histopathology confirmed intraocular extension of retrobulbar disease.

Whole-Genome Comparisons of Staphylococcus agnetis Isolates from Cattle and Chickens.

Staphylococcus agnetis has been previously associated with subclinical or clinically mild cases of mastitis in dairy cattle and is one of several staphylococcal species that have been isolated from the bones and blood of lame broilers. We reported that S. agnetis could be obtained frequently from bacterial chondronecrosis with osteomyelitis (BCO) lesions of lame broilers (A. Al-Rubaye et al., PLoS One 10:e0143336, 2015 [https://doi.org/10.1371/journal.pone.0143336]). A particular isolate, S. agnetis 908, can induce lameness in over 50% of exposed chickens, exceeding normal BCO incidences in broiler operations. We reported the assembly and annotation of the genome of isolate 908. To better understand the relationship between dairy cattle and broiler isolates, we assembled 11 additional genomes for S. agnetis isolates, an additional chicken BCO strain, and ten isolates from cattle milk, mammary gland secretions, or udder skin from the collection at the University of Missouri. To trace phylogenetic relationships, we constructed phylogenetic trees based on multilocus sequence typing and genome-to-genome distance comparisons. Chicken isolate 908 clustered with two of the cattle isolates, along with three isolates from chickens in Denmark and an isolate of S. agnetis we isolated from a BCO lesion on a commercial broiler farm in Arkansas. We used a number of BLAST tools to compare the chicken isolates to those from cattle and identified 98 coding sequences distinguishing isolate 908 from the cattle isolates. None of the identified genes explain the differences in host or tissue tropism. These analyses are critical to understanding how staphylococci colonize and infect different hosts and potentially how they can transition to alternative niches (bone versus dermis).IMPORTANCEStaphylococcus agnetis has been recently recognized as associated with disease in dairy cattle and meat-type chickens. The infections appear to be limited in cattle and systemic in broilers. This report details the molecular relationships between cattle and chicken isolates in order to understand how this recently recognized species infects different hosts with different disease manifestations. The data show that the chicken and cattle isolates are very closely related, but the chicken isolates all cluster together, suggesting a single jump from cattle to chickens.

Test Agreement among Biochemical Methods, Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry, and 16S rRNA Sequencing for Identification of Microorganisms Isolated from Bovine Milk.

The objective of this prospective study was a blinded comparison of three methods for the identification of bacteria isolated on Columbia blood agar from milk samples of dairy cows. Basic biochemical testing, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and 16S rRNA partial genome sequence analysis were compared for bacterial identification to the genus or species level. Milk samples submitted from a commercial dairy farm from recently calved cows or clinical mastitis cases were cultured, and 181 isolates were identified by biochemical testing, MALDI-TOF MS, and 16S rRNA sequence analysis (179 isolates; 2 isolates could not be recovered from storage). For Staphylococcus aureus and Escherichia coli, agreement was determined at the species level. For other microbes, agreement was determined at the genus level or at the group level for streptococcus-like organisms. The positive agreement among all 3 diagnostic methods was 94%, with 95% to 98% between each pair of methods. The overall (including negative agreement) agreement among all 3 methods ranged from 97% to 100%. MALDI-TOF MS is becoming more commonplace for the genus- and/or species-level identification of bacteria isolated from milk samples and, in some laboratories, has replaced conventional biochemical methods. The results of the present study suggest that when identifying pathogens at the genus or group level, conventional culture followed up with either secondary biochemical testing or MALDI-TOF MS is of practical value. For purposes of milk quality and udder health monitoring or research, any of the 3 methods is a valuable tool for genus-level identification of bacteria isolated from dairy cow milk.

Staphylococcal intramammary infection dynamics and the relationship with milk quality parameters in dairy goats over the dry period.

The objectives of this study were (1) to report the rates of new intramammary infection (IMI) and spontaneous IMI cure over the dry period in 3 dairy goat herds; (2) to evaluate the factors predicting infection dynamics over the dry period; and (3) to define milk quality parameter thresholds that predict infection dynamics over the dry period. Two consecutive udder-half milk samples were collected 10 to 14 d apart before dry-off from 288 goats in 3 herds, and 2 consecutive udder-half samples were collected 7 to 14 d apart in the following lactation, with the first sample being collected ≤10 d in milk, from 200 of the same goats. In 2 of the herds, udder-half milk samples were also collected at the same time points (n = 312 halves; 157 goats) for measurement of milk quality parameters. Standard aerobic culture of milk samples was performed for the detection of mastitis pathogens. To rule out the presence of Mycoplasma spp. IMI, milk samples were also cultured on modified Hayflick medium. Non-Mycoplasma isolates were speciated using MALDI-TOF mass spectrometry. Staphylococcal isolates, when not identified by MALDI-TOF, were speciated using partial gene sequence analysis of rpoB or tuf. When >1 sample from an udder half yielded the same species, available isolates from the first and last positive samples for that species were strain-typed using pulsed-field gel electrophoresis. Incidence of new IMI and cure rate were computed. Generalized linear mixed regression models were built to evaluate the associations between new IMI and pre-dry somatic cell score (SCS), between IMI persistence and half-level SCS, and between IMI persistence and pre-dry IMI species. Thresholds for pre-dry SCS and lactose concentration were computed to predict IMI persistence. Overall, 12.6% (48/380) of halves had a persistent IMI. Cumulative incidence of new IMI over the dry period was 13.2%, and cure rate was 52.0%. Pre-dry SCS was not associated with odds of new IMI or IMI persistence. Pre-dry IMI species was not associated with odds of persistence. Lactose concentration was not associated with odds of persistence. Regardless of culture data, the optimal pre-dry SCS threshold to detect IMI that would persist into the next lactation was 8.7, with sensitivity and specificity of 50 and 73.8%, respectively. Further studies on the effect of control measures on species-specific incidence and cure rates during the dry period are warranted.

Persistence of coagulase negative staphylococcal intramammary infections in dairy goats.

The objectives of the research described here were to describe the persistence of intramammary infections (IMI) caused by coagulase negative staphylococci (CNS) in goats using strain-typing, and to evaluate the relationship between species-specific CNS IMI and somatic cell score (SCS) at the udder-half level. Udder-half milk samples were collected from all 909 lactating goats (1817 halves; 1 blind half) in a single herd. Milk samples were cultured on Columbia blood agar, and 220 goats with at least one half yielding a single colony type CNS were enrolled for two additional half-level samplings at approximately 1-month intervals. Isolates were identified to the species level by matrix-assisted laser desorption-ionisation time-of-flight mass spectrometry or PCR amplification and partial sequencing of tuf or rpoB. An IMI was defined as persistent when ≥1 follow-up sample yielded the same species and strain as on Day 0 based on pulsed-field gel electrophoresis. A generalised mixed linear model was used to evaluate the odds of persistence as a function of CNS species. A mixed linear model was used to evaluate the relationship between IMI status on a given day and SCS. Among 192 IMI, 69.8% were persistent based on species and strain-type. Staphylococcus simulans IMI had higher odds of persistence than Staphylococcus arlettae IMI. In primiparous goats, Staphylococcus epidermidis IMI was associated with higher SCS than S. arlettae, Staphylococcus xylosus and 'other CNS' IMI. The differences detected in the present study between CNS species, with regard to persistence of IMI and association with SCS, highlight the need to study CNS at the species and strain level to understand the pathogenicity and epidemiology of CNS in goats.

Use of MALDI-TOF to characterize staphylococcal intramammary infections in dairy goats.

The most common pathogens causing intramammary infections (IMI) in dairy goats are staphylococci. Gene sequencing has been the reference method for identification of staphylococcal species, but MALDI-TOF mass spectrometry could represent a rapid and cost-effective alternative method. The objectives were to evaluate the typeability and accuracy of partial gene sequencing and MALDI-TOF for identifying staphylococci isolated from caprine milk samples, and to evaluate the relationship between staphylococcal species IMI, milk somatic cell score (SCS), and milk yield (MY). A composite (goat-level) milk sample was collected from all 940 lactating goats in a single herd. Dairy Herd Information Association test-day data for parity, days in milk, SCS, and MY were retrieved from Dairy Herd Information Association records. Milk samples were cultured on Columbia blood agar, and isolates from samples that yielded a single colony type of a presumptively identified Staphylococcus spp. were identified by PCR amplification and partial sequencing of rpoB, tuf, or 16S-rRNA, and MALDI-TOF. Mixed linear models were used to evaluate the relationship between staphylococcal IMI, SCS, and MY. The goat-level prevalence of staphylococcal IMI based on isolation of a single colony type was 24.4% (213/874). Seventeen goats had a contaminated sample. Among the remaining goats (n = 857), the most common species causing single colony-type IMI were Staphylococcus simulans (7.9%), Staphylococcus xylosus (3.5%), Staphylococcus caprae (3.6%), Staphylococcus chromogenes (2.9%), and Staphylococcus epidermidis (2.2%). The typeability of staphylococcal isolates with partial housekeeping gene sequence analysis (rpoB, complemented by tuf and 16S as needed) was 97.7%. The typeability and accuracy of MALDI-TOF were 84 and 100%, respectively. Overall, only Staphylococcus chromogenes IMI was associated with a higher SCS than goats with no growth. After adjusting for parity and stage of lactation, staphylococcal IMI status was not significantly associated with MY. For the staphylococci isolated from goats in this herd, MALDI-TOF proved an accurate method of speciation with a relatively high typeability. An association between staphylococcal IMI, SCS, and MY was not defined using goat-level data with the exception of S. chromogenes IMI, which was associated with a higher SCS than goats with no growth.

Comparison of Virulence Gene Identification, Ribosomal Spacer PCR, and Pulsed-Field Gel Electrophoresis for Typing of Staphylococcus aureus Strains Isolated from Cases of Subclinical Bovine Mastitis in the United States.

Staphylococcus aureus is one of the most important pathogens causing contagious mastitis in dairy cattle worldwide. The objectives of this study were to determine if recently described S. aureus genotype B was present among previously characterized isolates from cases of bovine intramammary infection in the United States and to compare pulsed-field gel electrophoresis (PFGE) to the combination of ribosomal spacer PCR (RS-PCR) and virulence gene identification for typing of S. aureus strains. The hypothesis was that isolates that were previously characterized as contagious would be identified as genotype B and that the results of the two strain-typing methods would be comparable. Isolates were selected from a collection of S. aureus isolates from eight dairy farms. Mammary quarter milk somatic cell count (SCC) and N-acetyl-ß-d-gluconaminidase (NAGase) activity data were known and used to evaluate strain pathogenicity. RS-PCR was performed with conventional gel electrophoresis, and PCR was used for toxin gene identification. RS-PCR patterns were associated with a specific virulence gene pattern, as previously reported. Five RS-PCR banding patterns were identified. None of the isolates were characterized as genotype B. No association between RS-PCR types and milk SCC was found; however, NAGase activity was significantly higher in milk from mammary glands infected with RS-PCR banding type 1 (RSP type 1) than in milk from those infected with RSP type 2. The discriminatory power values were 1.0 and 0.46 for PFGE and RS-PCR, respectively. These data suggest that genotype B may have a limited geographic distribution and that PFGE is more discriminatory than RS-PCR performed with conventional gel electrophoresis for typing of S. aureus isolates of bovine origin.

Current and Emerging Diagnostic Approaches to Bacterial Diseases of Ruminants.

The diagnostic approaches and methods to detect bacterial pathogens in ruminants are discussed, with a focus on cattle. Conventional diagnostic methods using culture, isolation, and characterization are being replaced or supplemented with new methods. These include molecular diagnostics such as real-time polymerase chain reaction and whole-genome sequencing. In addition, methods such as matrix-assisted laser desorption ionization-time-of-flight mass spectrometry enable rapid identification and enhanced pathogen characterization. These emerging diagnostic tools can greatly enhance the ability to detect and characterize pathogens, but performance and interpretation vary greatly across sample and pathogen types, disease syndromes, assay performance, and other factors.

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.

Cryptosporidiosis.

Cryptosporidiosis is a common cause of diarrhea among preweaned dairy calves. In the United States, the most common species of Cryptosporidium found in dairy calves is Cryptosporidium parvum, an important zoonotic species. Cryptosporidiosis is spread by fecal-oral transmission. Calves begin shedding the oocysts as early as 2 days of age, with peak shedding occurring at 14 days of age. Diarrhea generally starts 3 to 4 days after ingestion of the oocysts. Risk factors for the disease include large dairy farms, summer months, feeding of milk replacer, and early feeding of starter grain. Concrete flooring and appropriate cleaning of feeding utensils decreases the risk of disease.

Gross necropsy, histopathology, and ancillary test results from neonatal beef calves submitted to a veterinary diagnostic laboratory.

Objective: The objective of this study was to evaluate the prevalence of abnormal findings in gross necropsy, histopathology, and ancillary test results from neonatal beef calves submitted to a veterinary diagnostic laboratory. Samples: This retrospective clinical case study was conducted by reviewing necropsy reports submitted between 2015 to 2020. Case inclusion criteria were animals had to be a bovine, 2 to 21 days of age, and a nondairy breed. Procedures: Gross necropsy, histopathology, and laboratory test results were recorded. Identified lesions and abnormal test results were categorized based on body systems and infectious agent type. Age and system affected were analyzed using a 1-way ANOVA and Bonferonni pairwise comparisons. Results: Overall, 1,060 reports were reviewed and 95 met the inclusion criteria. Median age of enrolled calves was 9 days (range, 2 to 21). A total of 252 lesions were identified with a median of 3 lesions/calf (range, 0 to 7) and 2 different body systems involved/calf (range, 0 to 5). The most common disorders were classified as digestive (42.1% [106/252]), respiratory (12.7% [32/252]), and multisystemic (11.1% [28/252]). With respect to age and system affected, calves with neurologic lesions were significantly younger (mean age, 5.1 days) than calves with digestive lesions (mean age 9.6 days). Clinical Relevance: These data suggest a high prevalence of infectious diseases, mainly digestive, respiratory and multisystemic in origin. These findings could help guide producers and veterinarians when assessing factors contributing to neonatal beef calf loss.

Assessment of cerebrospinal fluid analysis and short-term survival outcomes in South American camelids: A retrospective study of 54 cases (2005-2021).

BACKGROUND: Cerebrospinal fluid (CSF) is commonly analyzed in South American camelids with suspected neurologic disease because of ease of collection and characteristic findings associated with certain diseases. OBJECTIVES: To assess CSF findings associated with short-term survival or non-survival in South American camelids in which neurologic disease was a differential diagnosis based on history and physical examination. ANIMALS: Twenty-one llamas and 33 alpacas that underwent CSF analysis at the University of Missouri Veterinary Health Center. METHODS: Retrospective study. Medical records of camelids that underwent CSF analysis between January 2005 and September 2021 were studied. Short-term survival was defined as survival to discharge from the Veterinary Health Center. A Fisher's exact test was used to compare species, CSF results, and survival. RESULTS: Odds of survival were 3.9 times higher in camelids with a total nucleated cell count (TNCC) <3 cells/µL (P = .04). No significant association was found between survival and total protein concentration (TPC; P = .15) or percentage of eosinophils (P = 1.0). No significant correlation was found between species and increased TNCC (P = .63), TPC (P = .55), or percentage of eosinophils (P = .30). Among camelids diagnosed with Paralephostrongylus tenuis infestation, odds of survival were 4.95 times higher in alpacas (P = .05). CONCLUSIONS: Cerebrospinal fluid TNCC ≥3 cells/µL is associated with decreased odds of short-term survival in South American camelids.

Diagnosing Intramammary Infection: Meta-Analysis and Mapping Review on Frequency and Udder Health Relevance of Microorganism Species Isolated from Bovine Milk Samples.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry provides accurate species-level identification of many, microorganisms retrieved from bovine milk samples. However, not all those microorganisms are pathogenic. Our study aimed to: (1) determine the species-specific prevalence of microorganisms identified in bovine milk of apparently healthy lactating quarters vs. quarters with clinical mastitis (CM); and (2) map current information and knowledge gaps on udder health relevance of microorganisms retrieved from bovine milk samples. A mixed study design (meta-analysis and mapping review) was chosen. We gathered several large Canadian, US and Brazilian data sets of MALDI-TOF results for organisms cultured from quarter milk samples. For meta-analysis, two datasets (apparently healthy quarters vs. CM samples) were organized. A series of meta-analyses was conducted to determine microorganisms' prevalence. Then, each species reported was searched through PubMed to investigate whether inflammation (increased somatic cell count (SCC) or signs of CM) was associated with microorganism's recovery from milk. A total of 294 different species of microorganisms recovered from milk samples were identified. Among 50,429 quarter-milk samples from apparently healthy quarters, the 5 most frequent species were Staphylococcus chromogenes (6.7%, 95% CI 4.5-9.2%), Aerococcus viridans (1.6%, 95% CI 0.4-3.5%), Staphylococcus aureus (1.5%, 95% CI 0.5-2.8%), Staphylococcus haemolyticus (0.9%, 95% CI 0.4-1.5%), and Staphylococcus epidermidis (0.7%, 95% CI 0.2-1.6%). Among the 43,924 quarter-milk CM samples, the 5 most frequent species were Escherichia coli (11%, 95% CI 8.1-14.3%), Streptococcus uberis (8.5%, 95% CI 5.3-12.2%), Streptococcus dysgalactiae (7.8%, 95% CI 4.9-11.5%), Staphylococcus aureus (7.8%, 95% CI 4.4-11.9%), and Klebsiella pneumoniae (5.6%, 95% CI 3.4-8.2%). When conducting the PubMed literature search, there were 206 species identified by MALDI-TOF for which we were not able to find any information regarding their association with CM or SCC. Some of them, however, were frequently isolated in our multi-country dataset from the milk of quarters with CM (e.g., Citrobacter koseri, Enterococcus saccharolyticus, Streptococcus gallolyticus). Our study provides guidance to veterinarians for interpretation of milk bacteriology results obtained using MALDI-TOF and identifies knowledge gaps for future research.

Medical management of an osseous sequestrum in an alpaca cria.

CASE DESCRIPTION: A 2-month-old 12.0-kg (26.4-lb) sexually intact male alpaca was evaluated for a 1-week history of progressive forelimb lameness with no known history of trauma. CLINICAL FINDINGS: The cria had toe-touching lameness in the right forelimb with a firm swelling at the distal dorsomedial metacarpal region. Signs of pain were elicited on palpation of the swollen region. There was no associated wound or draining tract. Radiographic examination revealed soft tissue swelling and osteomyelitis of the distal portion of the third metacarpal bone with a possible medial cortical sequestrum. TREATMENT AND OUTCOME: The cria was hospitalized and treated with meloxicam (1 mg/kg [0.45 mg/lb], PO, q 72 h) and ceftiofur sodium administered SC (2.2 mg/kg [1 mg/lb], q 12 h for 8 days) and by means of regional limb perfusion (1.25 mg/kg [0.57 mg/lb], IV, q 48 h for 8 days). Lameness and swelling improved, and the cria was discharged from the hospital with meloxicam (1 mg/kg, PO, q 72 h for 2 weeks) and ceftiofur crystalline free acid (1.5 mg/kg [0.68 mg/kg], SC, q 5 d for 2 weeks). At a recheck examination 17 days later, there was radiographic evidence of a well-defined 3.4 × 0.3-cm osseous sequestrum in the distal aspect of the affected third metacarpal bone. The owner declined further treatment and elected to monitor the cria at home. One year later, radiographic examination revealed nearly complete resolution of the sequestrum. CLINICAL RELEVANCE: Results for this patient suggested that osseous sequestra in some camelids may resolve following medical treatment without surgical intervention.

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.

Influence of PCR cycle number on 16S rRNA gene amplicon sequencing of low biomass samples.

The objective of this study was to evaluate the effects of increased PCR cycle number on sequencing results from samples with low microbial biomass, including bovine milk, and murine pelage and blood. We hypothesized that subjecting DNA from such samples to higher PCR cycle numbers would increase 16S rRNA sequencing coverage. DNA was extracted from matched samples of each type and multiple PCR cycle numbers were evaluated to generate a total of 96 libraries from 24 milk samples, 46 libraries from 23 pelage samples, and 170 libraries from 85 blood samples. 16S rRNA sequencing was performed on the Illumina MiSeq platform, and the coverage per sample, detected richness, and beta-diversity were evaluated. Across all sample types, higher PCR cycle numbers were associated with increased coverage. Surprisingly however, while higher PCR cycle numbers resulted in greater number of useable datapoints, no differences were detected in metrics of richness or beta-diversity. While reagent controls amplified for 40 cycles yielded similarly increased coverage, control and experimental samples were clearly differentiated based on beta-diversity. The results from this study support the use of higher PCR cycle numbers to evaluate samples with low microbial biomass.

Longitudinal microbiological evaluation of subclinical non-aureus staphylococcal intramammary infections in a lentivirus-infected dairy goat herd.

The objectives of this study were to 1) correlate pre-partum teat skin colonization with non-aureus staphylococcal (NAS) intramammary infection (IMI) in early lactation, and 2) evaluate infection dynamics of subclinical NAS IMI in goats during lactation in a small ruminant lentivirus-infected herd. Pre-partum teat skin swabs (41 goats, 82 halves) and post-partum half-level milk samples (106 goats, 203 halves) were collected at various intervals starting at ≤10 days in milk (DIM) until ≥120 DIM. Teat skin colonization and IMI were defined by culture and strain-typing. The association between the pre-kidding udder-half teat skin sample status and early lactation IMI status for a given species was investigated using McNemar's exact test or logistic regression. Time to IMI elimination and time to new IMI were evaluated by discrete-time survival analysis. Halves with S. caprae isolated from teat skin prior to kidding had increased odds of S. caprae IMI ≤ 10 DIM. Time to IMI elimination varied as a function of NAS species. Intramammary infections detected >10 DIM had a higher hazard of elimination (hazard ratio [HR] 5.6, 95% CI 2.8-11.2) than IMI detected ≤10 DIM. The presence of an IMI in the contralateral half was associated with a higher hazard of new IMI (HR 2.1, 95% CI 1.3-3.4) in an uninfected half. Further studies on interventional strategies targeting early IMI and IMI caused by persistent species are warranted.

Methods for Diagnosing Mastitis.

A diagnosis of mastitis is based on clinical observations or direct/indirect measures of the inflammatory response to infection, whereas a diagnosis of an intramammary infection is based on identification of the infectious agent. Somatic cell count/somatic cell score are common diagnostic tests for the detection of subclinical mastitis. Culture and polymerase chain reaction can be useful in the diagnosis of an intramammary infection; however, both have their advantages and disadvantages. Diagnosing the bacterial agent causing the intramammary infection can help to determine treatment and prevention strategies on the farm, which in turn can help to reduce incidence and prevalence.

The use of pulsed-field gel electrophoresis for genotyping of Clostridium difficile.

Genotyping approaches are important for tracking infectious agents and can be used for various purposes. Pulsed-Field Gel Electrophoresis (PFGE) is among the highly discriminatory genotyping approaches commonly used for characterizing Clostridium difficile. Other genotyping methods used for C. difficile include Ribotyping, Restriction Endonuclease Assay (REA), Multilocus Variable Number Tandem Repeats (VNTR) Assay, and others. PFGE has a high discriminatory power, high reproducibility, and typeability. We utilized PFGE for typing C. difficile isolates of porcine and human origin. We used a macrorestriction fragment analysis of an intact genomic DNA using SmaI, a rare cutting restriction endonuclease. Using a Contour-Clamped Homogeneous Electric Field (CHEF) system with running conditions of 120° angle; initial switch time of 5 s; final switch time of 40 s with a run time of 18 h in a low-melting temperature agarose (Seakem Gold); and 0.5× TBE circulated in the CHEF system at 6 V/cm [CDC (2014) Pulsenet. http://www.cdc.gov/pulsenet/index.html . Accessed 22 Aug 2014] supported by 14 °C cooling module, we were able to separate very large DNA fragments (up to 2 Mb).

Sequence Analysis of Staphylococcus hyicus ATCC 11249T, an Etiological Agent of Exudative Epidermitis in Swine, Reveals a Type VII Secretion System Locus and a Novel 116-Kilobase Genomic Island Harboring Toxin-Encoding Genes.

Staphylococcus hyicus is the primary etiological agent of exudative epidermitis in swine. Analysis of the complete genome sequence of the type strain revealed a locus encoding a type VII secretion system and a large chromosomal island harboring the genes encoding exfoliative toxin ExhA and an EDIN toxin homolog.