Classification of Bisgaard taxon 6 and taxon 10 as Exercitatus varius gen. nov., sp. nov.

Forty-one isolates of Bisgaard taxon 6 obtained from guinea pigs, pandas, pigs and muskrat and isolates of taxon 10 from horses and horse bites in humans were subjected phenotypic characterization. Production of acid from (-)-d-mannitol, (-)-d-sorbitol and (+)-d-glycogen separated taxon 10 (positive) from taxon 6 (negative), while from two to 11 phenotypic characteristics separated taxa 6 and 10 from the 32 genera of Pasteurellaceae reported so far. Forty-four strains were genetically characterized. Sequencing of 16S rRNA genes documented a monophyletic relationship at the species level and the highest 16S rRNA gene sequence similarity of 95.6 % to other species was found between strain CCUG 15568T and the type strain of Mannheimia glucosida (CCUG 38457T). Digital DNA-DNA hybridization (dDDH) values predicted from whole genomic sequences between CCUG 15568T and other characterized strains of taxa 6 and 10 were 69.3-99.9 %. The average nucleotide identity values were higher than 95 % for all strains. The highest dDDH value of 29 % outside the taxa 6 and 10 group was obtained with the genome of the type strain of [Actinobacillus] succinogenes, indicating a separate taxonomic status at species level to taxa 6 and 10. The phylogenetic comparison of concatenated conserved protein sequences showed the unique position of the taxa investigated in the current study which qualified for the status of a new genus since the highest identity was found with Basfia with 79 %, well below the upper threshold between genera of 85 %. Based upon the low genetic similarity to other genera of the family Pasteurellaceae and a unique phenotype, we suggest that Bisgaard taxa 6 and 10 should be classified as Exercitatus varius gen. nov., sp. nov. The G+C of the type strain of Exercitatus varius, 8.5T (=CCUG 15568T=DSM 115565T), is 46.2 mol%, calculated from the whole genome.

Bacterial and fungal microflora on the external genitalia of male donkeys (Equus asinus).

This study was undertaken to investigate the bacterial and fungal microflora on the external genitalia of a population of healthy male donkeys in the state of Michigan, USA. The aim was to identify and determine the frequency of occurrence of these microorganisms using seven different isolation media and standard microbiological procedures. The sites (urethral fossa [fossa glandis], dorsal diverticulum of the urethral sinus, distal urethra, and penile surface) in the distal reproductive tract were cultured and each isolated microorganism identified. Ten different genera of gram-positive bacteria, eight different genera of gram-negative bacteria, and two genera of fungi were isolated from the external genitalia of the 43 donkeys in this study. All 43 donkeys yielded gram-positive bacteria (2-8 species) from all four sites sampled. Arcanobacterium spp., Corynebacterium spp., and Bacillus spp. were the most frequently isolated gram-positive bacteria. Gram-negative bacteria were cultured from 16 (37.2%) of the 43 donkeys, with Acinetobacterlwoffii (16.3%), Oligella urethralis (11.6%), and Taylorellaasinigenitalis (9.3%), the most frequently isolated. Fungi were cultured from only 5 (11.6%) of the 43 donkeys, with Rhizopus spp. isolated from 3 (7.0%) and Cladosporium spp. from 2 (4.7%) individuals. The testes and epididymides collected from 40 donkeys at time of castration were culture negative. Few differences were found in the bacterial flora between prepubertal and mature intact and castrated donkeys. Of notable interest was the scarcity of known equine pathogens across the population tested and isolation of T. asinigenitalis from normal donkeys, especially prepubertal individuals and previously castrated males.

Bartonella bovis isolated from a cow with endocarditis.

A 7-year-old pregnant Angus cow was found dead in the field. At necropsy, the aortic valve was expanded by moderate fibrous connective tissue and acidophilic coagulum containing multifocal marked bacteria, mineral, neutrophils, and red blood cells. Numerous tiny grayish, opaque bacterial colonies were detected on blood agar plates at 7 days after inoculation with a swab of the heart valve of the cow. The bacterium was a Gram-negative, very small coccobacillus that was catalase, oxidase, and urease negative, and did not change litmus milk, triple sugar iron agar, and sulfide-indole-motility medium. The bacterium was negative for esculin hydrolysis, phenylalanine deaminase, nitrate reduction, and gelatin hydrolysis. The isolate did not produce acid from glycerol, inulin, lactose, maltose, mannose, raffinose, salicin, sorbitol, sucrose, trehalose, glycogen, ribose, or starch. Polymerase chain reaction tests for the gltA, ssrA, ftsZ, ribC, rpoB, and 16S ribosomal RNA genes of Bartonella species were positive for the isolate. Amplicons were sequenced, and the gltA, ribC, ssrA, and 16S ribosomal RNA gene sequences were found to have 100% homology to the type strain of Bartonella bovis, whereas the fts and rpoB sequences showed 99.9% and 99.6% homology, respectively, to the type strain of Bartonella bovis. Diagnosticians should be aware of slow-growing microorganisms, and culture media should be incubated beyond the standard period to enhance the recovery of Bartonella species.

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 (MIC(90)) 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 MIC(90) 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.

Prevalence and persistence of Taylorella asinigenitalis in male donkeys.

This study was undertaken to investigate the prevalence of Taylorella asinigenitalis in a subset of the donkey population of Michigan and in other equids on farms on which the organism was identified. Other aims were to further characterize the carrier state in terms of persistence and preferred sites of colonization of T. asinigenitalis in the male donkey as well as determine the genotype of any isolates of the organism. Initial testing of 43 donkeys and 1 mule turned up 4 (9.3%) donkeys culture positive for T. asinigenitalis. The 4 culture-positive donkeys resided on 2 farms accommodating a collective total of 89 equids, of which 23 (25.8%) were confirmed positive for T. asinigenitalis. The positive equid population on the 2 farms comprised 14 (67%) of 21 gelded donkeys, 8 (36.4%) of 22 intact male donkeys, and 1 (25%) of 4 gelded horses. T. asinigenitalis was not isolated from 27 female donkeys, 11 female horses, 2 female mules, 1 male horse, or 1 male mule resident on these premises. Isolations of the bacterium were obtained from a number of male donkeys whenever they were sampled over a span of 33 months; preferential sites of isolation were the urethral fossa (fossa glandis), dorsal diverticulum of the urethral sinus, and terminal urethra. Isolates of T. asinigenitalis from the 23 culture-positive equids comprised 2 genotypes, one identical to the type strain isolated in California in 1997, and the other identical to 2 strains isolated from donkey jacks in Kentucky in 1998.

Infection of an equine placenta with a novel mycobacterial species leading to abortion.

A 25-year-old pregnant American Quarter Horse mare presented with a 1-week history of progressively worsening vaginal discharge. Transrectal ultrasound revealed increased thickness of the combined uterus and placenta with evidence of chorioallantoic edema but no placental separation. A thickened amnion was visible on transabdominal ultrasound. Abortion occurred 2 days after presentation despite medical treatment. At necropsy, the chorioallantois had variable but diffuse thickening with focally extensive browning of the chorionic surface in the right horn and adjacent body. There were fluid-filled sacculations on the allantoic surface of the umbilical cord, allantoamnion, and chorioallantois associated with diffuse perivascular fluid microscopically. A nonbranching acid-fast bacterium identified as belonging to the genus Mycobacterium Runyon group IV was isolated from the chorioallantois and uterine fluid. Ziehl-Neelsen stain confirmed the presence of intracellular acid-fast bacilli in trophoblasts of the gravid horn and the cervical star area. The current case is unique in that the mycobacteria did not initiate a significant granulomatous inflammatory response in the chorion unless villar necrosis occurred. Sequence analysis of the 16S ribosomal RNA gene and the rpoß gene, encoding the ß subunit of RNA polymerase, indicated that the strain of mycobacteria isolated in this case belonged to a novel species of rapidly growing mycobacteria and not to an established species. Mycobacteria are an uncommon and sporadic cause of placentitis and abortion, but should be suspected in cases of chronic placentitis that are not restricted to the cervical star area.

New real-time PCR assay using allelic discrimination for detection and differentiation of equine herpesvirus-1 strains with A2254 and G2254 polymorphisms.

A single-nucleotide polymorphism (A(2254) or G(2254)) in open reading frame 30 (ORF30) has been linked to the neuropathogenic phenotype of equine herpesvirus-1 (EHV-1). Identification of this polymorphism led to the development of a real-time PCR (rPCR) assay using allelic discrimination (E(2)) to distinguish between potentially neuropathogenic and nonneuropathogenic EHV-1 strains (G. P. Allen, J. Vet. Diagn. Invest. 19:69-72, 2007). Although this rPCR assay can detect and genotype EHV-1 strains, subsequent studies demonstrated that it lacks the sensitivity for the routine detection of viral nucleic acid in clinical specimens. Therefore, a new allelic discrimination EHV-1 rPCR assay (E(1)) was developed by redesigning primers and probes specific to ORF30. The E(1) and E(2) rPCR assays were evaluated using 76 archived EHV isolates and 433 clinical specimens from cases of suspected EHV-1 infection. Nucleotide sequence analysis of ORF30 was used to confirm the presence of EHV-1 and characterize the genotype (A(2254) or G(2254)) in all archived isolates plus 168 of the clinical samples. The E(1) assay was 10 times more sensitive than E(2), with a lower detection limit of 10 infectious virus particles. Furthermore, all A(2254) and G(2254) genotypes along with samples from three cases of dual infection (A(2254)+G(2254)) were correctly identified by E(1), whereas E(2) produced 20 false dual positive results with only one actual mixed A(2254)+G(2254) genotype confirmed. Based on these findings, E(1) offers greater sensitivity and accuracy for the detection and A/G(2254) genotyping of EHV-1, making this improved rPCR assay a valuable diagnostic tool for investigating outbreaks of EHV-1 infection.

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.

Evaluation of two magnetic-bead-based viral nucleic acid purification kits and three real-time reverse transcription-PCR reagent systems in two TaqMan assays for equine arteritis virus detection in semen.

This study showed that under specifically defined conditions with respect to nucleic acid extraction method and testing reagents, a previously described real-time reverse transcription-PCR (rRT-PCR) assay (T1 assay) provides sensitivity equal to or higher than that of virus isolation for the detection of equine arteritis virus in semen.

Diagnostic application of H3N8-specific equine influenza real-time reverse transcription polymerase chain reaction assays for the detection of Canine influenza virus in clinical specimens.

The objective of the current study was to determine the capability of 3 recently described one-step TaqMan real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of H3N8 Equine influenza virus (EIV NP, EIV M, and EIV HA3 assays, respectively) to detect Canine influenza virus (CIV). The assays were initially evaluated with nucleic acid extracted from tissue culture fluid (TCF) containing the A/canine/FL/43/04 strain of Influenza A virus associated with the 2004 canine influenza outbreak in Florida. The EIV NP, EIV M, and EIV HA3 assays could detect CIV nucleic acid at threshold cycle (Ct) values of 16.31, 23.71, and 15.28, respectively. Three assays using TCF or allantoic fluid (AF) samples containing CIV (n  =  13) and archived canine nasal swab samples (n  =  20) originally submitted for laboratory diagnosis of CIV were further evaluated. All TCF and AF samples, together with 10 nasal swab samples that previously tested positive for virus by attempted isolation in embryonated hens' eggs or Madin-Darby canine kidney cells, were positive in all 3 real-time RT-PCR assays. None of the 3 assays detected the H1N1 Swine influenza virus strain in current circulation. These findings demonstrate that previously described real-time RT-PCR assays targeting NP, M, and H3 HA gene segments of H3N8 EIV are also valuable for the diagnosis of CIV infection in dogs. The assays could expedite the detection and identification of CIV.

Development and evaluation of one-step TaqMan real-time reverse transcription-PCR assays targeting nucleoprotein, matrix, and hemagglutinin genes of equine influenza virus.

The objective of this study was to develop and evaluate new TaqMan real-time reverse transcription-PCR (rRT-PCR) assays by the use of the minor groove binding probe to detect a wide range of equine influenza virus (EIV) strains comprising both subtypes of the virus (H3N8 and H7N7). A total of eight rRT-PCR assays were developed, targeting the nucleoprotein (NP), matrix (M), and hemagglutinin (HA) genes of the two EIV subtypes. None of the eight assays cross-reacted with any of the other known equine respiratory viruses. Three rRT-PCR assays (EqFlu NP, M, and HA3) which can detect strains of the H3N8 subtype were evaluated using nasal swabs received for routine diagnosis and swabs collected from experimentally inoculated horses. All three rRT-PCR assays have greater specificity and sensitivity than virus isolation by egg inoculation (93%, 89%, and 87% sensitivity for EqFlu NP, EqFlu M, and EqFlu HA3 assays, respectively). These assays had analytical sensitivities of >or=10 EIV RNA molecules. Comparison of the sensitivities of rRT-PCR assays targeting the NP and M genes of both subtypes with egg inoculation and the Directigen Flu A test clearly shows that molecular assays provide the highest sensitivity. The EqFlu HA7 assay targeting the H7 HA gene is highly specific for the H7N7 subtype of EIV. It should enable highly reliable surveillance for the H7N7 subtype, which is thought to be extinct or possibly still circulating at a very low level in nature. The assays that we developed provide a fast and reliable means of EIV diagnosis and subtype identification of EIV subtypes.

Classification of Actinobacillus spp isolates from horses involved in mare reproductive loss syndrome.

OBJECTIVE: To identify Actinobacillus spp isolates recovered from fetuses and pericardial fluid from horses affected with mare reproductive loss syndrome (MRLS) and determine whether these bacterial species are the same as those isolated from clinically normal horses. SAMPLE POPULATION: Isolates of actinobacilli recovered from 18 horses with pericarditis and 109 fetuses aborted by mares affected by MRLS. Procedures-Actinobacillus spp isolates were identified to the level of species or subspecies by use of conventional phenotypic tests and biochemical and enzyme test kits. The 16S rRNA gene from selected isolates was amplified, purified, and sequenced. Sequence data were compared with sequence data for actinobacilli in GenBank. RESULTS: Of the 109 isolates obtained from fetuses, 14 were Actinobacillus equuli subsp equuli, 65 were A equuli subsp haemolyticus, 28 were Bisgaard taxon 10-like bacterium, and 2 were Actinobacillus genomospecies 1. Of the 18 isolates from horses with pericarditis, 4 were A equuli subsp equuli, 13 were A equuli subsp haemolyticus, and 1 was Bisgaard taxon 10-like bacterium. Comparisons with published data and GenBank data revealed that the isolates recovered from horses with MRLS were the same as those isolated from the oral cavity or alimentary tract of healthy horses. CONCLUSIONS AND CLINICAL RELEVANCE: Actinobacillus spp isolates recovered from fetuses and pericardial fluid samples of horses affected by MRLS in 2001 to 2003 were identical to Actinobacillus spp found in the oral cavity and alimentary tracts of healthy horses.

Nocardioform placentitis with isolation of Amycolatopsis spp in a Florida-bred mare.

CASE DESCRIPTION: A 4-year-old Thoroughbred mare was evaluated because of placental abnormalities and a retained placental remnant. CLINICAL FINDINGS: Microbial culture of the placenta yielded pure growth of Amycolatopsis spp. Histologic examination of the placenta revealed a focally expanding chorionitis with intralesional gram-positive filamentous bacilli and multifocal allantoic adenomatous hyperplasia on the apposing allantoic surface. TREATMENT AND OUTCOME: Treatment with lavage and oxytocin resulted in expulsion of the placental remnant within hours of parturition. The mare did not become pregnant again despite multiple breedings. The foal appeared healthy but died of complications during an elective surgical procedure at 7 weeks of age. CONCLUSIONS AND CLINICAL RELEVANCE: To the author's knowledge, all previously confirmed cases of nocardioform placentitis have been in mares bred in the central Kentucky region. Indications that the pathogen in the mare reported here is a different species than that isolated in Kentucky suggest that this is an emerging disease. Mares with nocardioform placentitis usually do not have the same clinical signs as mares with placentitis resulting from an ascending pathogen.