Craig N. Carter

Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky

REASON FOR PERFORMING STUDY An emerging problem of equine herpesvirus-1 (EHV-1) infection in horses in the USA is a high-mortality myeloencephalopathy that commonly occurs where large numbers of horses are stabled. EHV-1 isolates recovered from recent neurological outbreaks represent a mutant virus strain that possesses enhanced neuropathogenicity. A central question of EHV-1 myeloencephalopathy is the latency carriage rate for these mutants of EHV-1 in USA horse populations. OBJECTIVE To estimate the prevalence of neuropathogenic strains of EHV-1 as latent infections in the Thoroughbred broodmare population of central Kentucky. METHODS Submandibular lymph nodes (SMLN) were collected during post mortem examination of 132 Thoroughbred broodmares. Total DNA purified from SMLN tissue was tested for the presence of latent EHV-1 DNA by an ultrasensitive magnetic bead-based, sequence-capture, nested PCR method. Differentiation of active from latent infections by EHV-1 was achieved by detection of transcripts of EHV-1 glycoprotein B by reverse transcription PCR. RESULTS Latent EHV-1 DNA was detected in the SMLN tissues of 71 (54%) of the 132 mares submitted for necropsy. Thirteen (18%) of the 71 latently infected horses harboured the neuropathogenic biovar of EHV-1. Of the 13 horses latently infected with an ORF30 mutant strain of EHV-1, 11 also carried a latent, wild-type strain of the virus in their SMLN tissues. CONCLUSIONS Neuropathogenic strains of EHV-1 have established a significant presence in the Thoroughbred broodmare population of central Kentucky as latently infected carrier horses. The data also indicate that a highly sensitive DNA detection method is required to identify many instances of EHV-1 latency. POTENTIAL RELEVANCE The presence of a relatively large biological reservoir of latent, neuropathogenic EHV-1 has the potential for posing emerging equine health and economic threats to the future prosperity of the USA horse industry.

OAS1 Polymorphisms Are Associated with Susceptibility to West Nile Encephalitis in Horses

West Nile virus, first identified within the United States in 1999, has since spread across the continental states and infected birds, humans and domestic animals, resulting in numerous deaths. Previous studies in mice identified the Oas1b gene, a member of the OAS/RNASEL innate immune system, as a determining factor for resistance to West Nile virus (WNV) infection. A recent case-control association study described mutations of human OAS1 associated with clinical susceptibility to WNV infection. Similar studies in horses, a particularly susceptible species, have been lacking, in part, because of the difficulty in collecting populations sufficiently homogenous in their infection and disease states. The equine OAS gene cluster most closely resembles the human cluster, with single copies of OAS1, OAS3 and OAS2 in the same orientation. With naturally occurring susceptible and resistant sub-populations to lethal West Nile encephalitis, we undertook a case-control association study to investigate whether, similar to humans (OAS1) and mice (Oas1b), equine OAS1 plays a role in resistance to severe WNV infection. We identified naturally occurring single nucleotide mutations in equine (Equus caballus) OAS1 and RNASEL genes and, using Fishers Exact test, we provide evidence that mutations in equine OAS1 contribute to host susceptibility. Virtually all of the associated OAS1 polymorphisms were located within the interferon-inducible promoter, suggesting that differences in OAS1 gene expression may determine the hosts ability to resist clinical manifestations associated with WNV infection.

Association of soil concentrations of Rhodococcus equi and incidence of pneumonia attributable to Rhodococcus equi in foals on farms in central Kentucky

OBJECTIVE To determine whether soil concentrations of total or virulent Rhodococcus equi differed among breeding farms with and without foals with pneumonia caused by R equi. SAMPLE POPULATION 37 farms in central Kentucky. Procedures-During January, March, and July 2006, the total concentration of R equi and concentration of virulent R equi were determined by use of quantitative bacteriologic culture and a colony immunoblot technique, respectively, in soil specimens obtained from farms. Differences in concentrations and proportion of virulent isolates within and among time points were compared among farms. RESULTS Soil concentrations of total or virulent R equi did not vary among farms at any time point. Virulent R equi were identified in soil samples from all farms. Greater density of mares and foals was significantly associated with farms having foals with pneumonia attributable to R equi. Among farms with affected foals, there was a significant association of increased incidence of pneumonia attributable to R equi with an increase in the proportion of virulent bacteria between samples collected in March and July. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that virulent R equi were commonly recovered from soil of horse breeding farms in central Kentucky, regardless of the status of foals with pneumonia attributable to R equi on each farm. The incidence of foals with pneumonia attributable to R equi can be expected to be higher at farms with a greater density of mares and foals.

Beta-hemolytic Streptococcus spp. from horses a retrospective study (2000–2010)

The goal of this retrospective study was to have a comprehensive picture of the β-hemolytic streptococci of horses including tissue/organ distributions and susceptibility patterns against specific antimicrobials between January 1, 2000 and December 31, 2010. A total of 2,497 β-hemolytic streptococci were isolated from 2,391 cases, of which Streptococcus equi subsp. zooepidemicus was the most frequent isolate (72.0%). Other species isolated were Streptococcus dysgalactia subsp. equisimilis (21.3%), Streptococcus equi subsp. equi (5.8%), and unidentified β-hemolytic streptococci (0.9%). As expected, S. equi was mostly isolated from lymph node abscesses and the respiratory tract in foals and adult horses. Streptococcus equi subsp. zooepidemicus and S. equisimilis were mostly isolated from placenta, fetal tissues, and genital tract of horses; S. zooepidemicus and S. equisimilis were also recovered in significant numbers from a number of other organs including lung, liver, brain, kidney, and joints, indicating a much broader tissue tropism than S. equi. In addition, more than 1 Streptococcus spp. was recovered in 106 cases, indicating the co-existence of these bacteria in some horses. This data also suggested that S. equisimilis is a major bacterial agent of horses, contrary to present knowledge. Based on Kirby-Bauer antimicrobial susceptibility data, streptococci were found to be generally susceptible to cephalothin, erythromycin, nitrofurantoin, penicillin, and ticarcillin and clavulanate. Resistance to antimicrobials has not developed over the years, except for gentamicin and tetracycline against S. equisimilis.

Frequency of Corynebacterium pseudotuberculosis infection in horses across the United States during a 10-year period

OBJECTIVE To quantify the number of horses with Corynebacterium pseudotuberculosis infection identified in the United States from January 2003 through December 2012. DESIGN Cross-sectional study. SAMPLE State veterinary diagnostic laboratory records of 2,237 C pseudotuberculosis culture-positive samples from horses. PROCEDURES 44 state veterinary diagnostic laboratories throughout the United States were invited by mail to participate in the study. Data requested included the number of C pseudotuberculosis culture-positive samples from horses identified per year, geographic location from which the C pseudotuberculosis culture-positive sample was submitted, month and year of sample submission, breed and age of horses, and category of clinical manifestation (ie, internal infection, external infection, or ulcerative lymphangitis). RESULTS Of the 44 invited laboratories, 15 agreed to participate and provided data on affected horses from 23 states. The proportion of C pseudotuberculosis culture-positive samples submitted during 2011 through 2012 (1,213/2,237 [54%]) was significantly greater than that for the period from 2003 through 2010 (1,024/2,237 [46%]). Corynebacterium pseudotuberculosis was recovered from horses in states where the disease has not been previously recognized as endemic. Affected horses were identified year-round. The greatest proportion of C pseudotuberculosis culture-positive samples was identified during November, December, and January (789/2,237 [35%]). No significant association between the clinical form of disease and age or breed of horse was observed. CONCLUSIONS AND CLINICAL RELEVANCE The occurrence of C pseudotuberculosis infection in horses increased during the 10-year period, and affected horses were identified throughout the United States. Further studies to determine changes in annual incidence and to identify potential changing climatic conditions or vector populations associated with disease transmission are warranted to help control the occurrence and spread of C pseudotuberculosis infection in horses.

A diagnostic evaluation of real-time PCR, fluorescent antibody and microscopic agglutination tests in cases of equine leptospiral abortion

REASONS FOR PERFORMING STUDY A comprehensive evaluation of the real-time PCR assay for leptospirosis in comparison with other diagnostic assays on a large-scale basis is fundamental in validating the assay and determining the causes of equine abortions. OBJECTIVES To compare and evaluate the diagnostic value of real-time PCR assay for leptospirosis with traditional methods in equine leptospiral abortions. STUDY DESIGN Cross-sectional observational study. METHODS A Leptospira spp. fluorescent antibody test (FAT), microscopic agglutination test (MAT) and real-time PCR (targeting the LipL32 gene) were compared and evaluated in equine fetal necropsy specimens (placenta, kidney, liver and heart blood) and maternal serum (when available) in 339 equine fetuses. RESULTS From a total of 339 equine fetuses necropsied, 21 cases (6.19%) were diagnosed as leptospiral abortion. The majority of leptospiral abortions occurred in January (8 cases) and February (5 cases). Real-time PCR detected 21 of 21 cases, whereas MAT and FAT detected 19 and 18 (including 2 suspicious cases) cases, respectively. Comparing tissues, placenta yielded somewhat similar cycle of threshold values by real-time PCR compared with kidney, whereas kidney was the best specimen for the diagnosis of leptospirosis by the FAT test. In all MAT positive cases, the predominant titre in fetal heart blood was to serovar Pomona (ranging 1:100 to 1:204,800) with little or no cross-reaction to serovar Grippotyphosa. CONCLUSIONS The results indicate that real-time PCR is an effective method for the diagnosis of leptospiral abortion in horses. However, MAT should continue to be used in clinical cases for serovar determination.

An investigation of a recent outbreak of nocardioform placentitis caused abortions in horses

Nocardioform placentitis associated with gram positive branching actinomycetes caused a record number of abortions in mares diagnosed by the University of Kentucky Veterinary Diagnostic Laboratory (UKVDL) affecting the 2011 foal crop (2011 foal crop: the cohort of foals conceived during the 2010 breeding season). The goal of the present study is to make a comprehensive analysis of this outbreak in terms of frequencies of the bacteria causing nocardioform placentitis mediated abortions and to investigate the ages of fetuses, abortion months and breeding times. In the present study, characteristic slow-growing, pungent/soil odor gram positive branching actinomycetes were recovered in high numbers in placental specimens in 76 abortion cases diagnosed as nocardioform placentitis by pathologists. To determine the type of actinomycetes responsible for the abortions, PCR assays were performed on the gram positive branching bacilli. The most prominent actinomycetes species were Amycolatopsis spp. (37 cases, 48.7%) and Crossiella equi (C. equi) (22 cases, 28.9%). Six cases (7.9%) contained both Amycolatopsis spp., and C. equi. 10 isolates were unidentified by PCR assays and shown to have high DNA sequence homology to Streptomyces species, Microbacterium species, Nocardia species and Allokutzneria species, as evidenced by 16 rRNA gene sequencing and phylogenetic analysis. Nocardioform placentitis related abortions occurred mostly between December 2010 and April 2011 happening exclusively in the last trimester. Breeding time of aborted pregnancies ranged from March 2010 to July 2010, suggesting that if transmission of the actinomycetes agents occurred during breeding, it was not related to a specific season.

Effects of location for collection of air samples on a farm and time of day of sample collection on airborne concentrations of virulent Rhodococcus equi at two horse breeding farms

OBJECTIVE To determine whether airborne concentrations of virulent Rhodococcus equi at 2 horse breeding farms varied on the basis of location, time of day, and month. SAMPLE POPULATION 2 farms in central Kentucky with recurrent R equi-induced pneumonia in foals. PROCEDURES From February through July 2008, air samples were collected hourly for a 24-hour period each month from stalls and paddocks used to house mares and their foals. Concentrations of airborne virulent R equi were determined via a modified colony immunoblot technique. Differences were compared by use of zero-inflated negative binomial methods to determine effects of location, time, and month. RESULTS Whether mares and foals were housed predominantly in stalls or paddocks significantly affected results for location of sample collection (stall vs paddock) by increasing airborne concentrations of virulent R equi at the site where horses were predominantly housed. Airborne concentrations of virulent R equi were significantly higher from 6:00 pm through 11:59 pm than for the period from midnight through 5:59 am. Airborne concentrations of virulent R equi did not differ significantly between farms or among months. CONCLUSIONS AND CLINICAL RELEVANCE Airborne concentrations of virulent R equi were significantly increased when horses were predominantly housed at the site for collection of air samples (ie, higher in stalls when horses were predominantly housed in stalls and higher in paddocks when horses were predominantly housed in paddocks). Concentrations of virulent R equi among air samples collected between the hours of 6:00 am and midnight appeared similar.

Application of an automated surveillancedata–analysis system in a laboratory-based early-warning system for detection of an abortion outbreak in mares

OBJECTIVE To develop an early-warning automated surveillance-data-analysis system for early outbreak detection and reporting and to assess its performance on an abortion outbreak in mares in Kentucky. SAMPLE POPULATION 426 data sets of abortions in mares in Kentucky during December 2000 to July 2001. PROCEDURES A custom software system was developed to automatically extract and analyze data from a Laboratory Information Management System database. The software system was tested on data on abortions in mares in Kentucky reported between December 1, 2000, and July 31, 2001. The prospective space-time permutations scan statistic, proposed by Kulldorff, was used to detect and identify abortion outbreak signals. RESULTS Results indicated that use of the system would have detected the abortion outbreak approximately 1 week earlier than traditional surveillance systems. However, the geographic scale of analysis was critical for highest sensitivity in outbreak detection. Use of the lower geographic scale of analysis (ie, postal [zip code]) enhanced earlier detection of significant clusters, compared with use of the higher geographic scale (ie, county). CONCLUSIONS AND CLINICAL RELEVANCE The automated surveillance-data-analysis system would be useful in early detection of endemic, emerging, and foreign animal disease outbreaks and might help in detection of a bioterrorist attack. Manual analyses of such a large number of data sets (ie, 426) with a computationally intensive algorithm would be impractical toward the goal of achieving near real-time surveillance. Use of this early-warning system would facilitate early interventions that should result in more positive health outcomes.

Antibiotic susceptibility patterns of Crossiella equi and Amycolatopsis species causing nocardioform placentitis in horses

Nocardioform actinomycetes are significant causes of placentitis and abortions in horses. In the current study, antimicrobial susceptibility patterns of 38 Amycolatopsis spp. and 22 Crossiella equi isolates, the most common nocardioform actinomycetes causing placentitis in horses, were evaluated. Antimicrobial susceptibilities of these isolates were tested by broth microdilution method in a commercial system, which was designed for Nocardia spp., fast-growing Mycobacterium spp., and other aerobic actinomycetes. The minimum inhibitory concentration required to inhibit the growth of 90% of organisms (MIC90) of the following antibiotics tested for Amycolatopsis spp. were: 4 µg/ml for linezolid, trimethophrim–sulfametaxazole (TMP-SMX), and ciprofloxacin; 8 µg/ml for ceftriaxone, doxycycline, and minocycline; 16 µg/ml for amoxicillin–clavulanic acid, clarithromycin, and imipenem; >16 µg/ml for tobramycin; 32 µg/ml for amikacin and cefepime; and 128 µg/ml for cefoxitin. The MIC90 levels for C. equi were 0.25 µg/ml for doxycycline; ≤1 µg/ml for minocycline; 2 µg/ml for linezolid and TMP-SMX; 4 µg/ml for ciprofloxacin; 8 µg/ml for amoxicillin–clavulanic acid, ceftriaxone, and imipenem; 16 µg/ml for clarithromycin; >16 µg/ml for tobramycin; 32 µg/ml for cefepime; >64 µg/ml for amikacin; and 128 µg/ml for cefoxitin.