SURVEY FOR EQUINE HERPESVIRUSES IN POLAR BEARS ( URSUS MARITIMUS) AND EXOTIC EQUIDS HOUSED IN US AZA INSTITUTIONS.

Infection by equine herpesvirus (EHV) strains (EHV-1, EHV-9) in ursid species, including polar bears ( Ursus maritimus), has been associated with neurological disease and death. A serosurvey of captive exotic equid and polar bear populations in US Association of Zoos and Aquaria institutions was performed to determine the prevalence of EHV strains using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) tests. Equid species surveyed included zebra ( Equus spp.), Przewalski's wild horse ( Equus ferus przewalskii), Persian onager ( Equus hemionus), and Somali wild ass ( Equus africanus somaliensis). A questionnaire regarding husbandry and medical variables was distributed to institutions housing polar bears. No polar bears tested positive for EHVs on qPCR of blood or nasal swabs. No exotic equids tested positive for EHVs on qPCR of blood, but two exotic equids ( n = 2/22; 9%) tested positive for EHVs on qPCR of nasal swabs. On ELISA, polar bears infrequently were positive for EHV-1 ( n = 5/38; 13%). Exotic equids were positive for EHV-4 on ELISA more frequently ( n = 30/43; 70%) than for EHV-1 ( n = 8/43; 19%). Nine institutions submitted samples from both exotic equids and polar bears, two of which had both exotic equids and polar bears positive for EHVs by ELISA. Each of these institutions reported that the polar bear and exotic equid exhibits were within 80 m of each other and that risk factors for fomite transmission between exhibits based on husbandry practices were present. One institution that did not house exotic equids had a polar bear test positive for EHV-1 on ELISA, with no history of exposure to exotic equids. Further testing of captive polar bears and exotic equids is recommended, as is modification of husbandry practices to limit exposure of polar bears to exotic equids.

Development of an equine coronavirus-specific enzyme-linked immunosorbent assay to determine serologic responses in naturally infected horses.

Equine coronavirus (EqCoV) infection has been documented in most reports through quantitative qPCR analysis of feces and viral genome sequencing. Although qPCR is used to detect antigen during the acute disease phase, there is no equine-specific antibody test available to study EqCoV seroprevalence in various horse populations. We developed an enzyme-linked immunosorbent assay (ELISA) targeting antibodies to the spike (S) protein of EqCoV and validated its use, using acute and convalescent sera from 83 adult horses involved in 6 outbreaks. The EqCoV S protein-based ELISA was able to reliably detect antibodies to EqCoV in naturally infected horses. The greatest seroconversion rate was observed in horses with clinical signs compatible with EqCoV infection and EqCoV qPCR detection in feces. The EqCoV S protein-based ELISA could be used effectively for seroepidemiologic studies in order to better characterize the overall infection rate of EqCoV in various horse populations.

Pyrosequencing as a fast and reliable tool to determine clade affiliation for equine Influenza A virus.

The objective of our study was to determine the clade affiliation of 116 contemporary equine Influenza A virus (EIV) isolates using pyrosequencing. The EIV isolates originated from horses with clinical signs of equine influenza and laboratory confirmation of EIV by real-time quantitative PCR (qPCR) in nasal secretions. Clade affiliation was performed on the basis of a single nucleotide polymorphism at 2 positions of the hemagglutinin 1 gene. Pyrosequencing was able to clearly classify EIV Florida sublineage prototype A/equine/Ohio/1/2003 and prototype A/equine/Richmond/1/2007 as clade 1 and 2, respectively. Out of the 116 EIV qPCR-positive samples, 113 (97.4%) were classified as belonging to clade 1 Florida sublineage, whereas 3 (2.6%) were classified as clade 2. All clade 1 EIV strains were detected in domestic horses, whereas the 3 clade 2 EIV strains originated from horses recently imported to the United States. Although clade 1 EIV strains are endemic in the United States, international transportation of horses represents a real risk in introducing clade 2 EIV strains into North America.

Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy.

Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.

Evidence of host adaptation in Lawsonia intracellularis infections.

BACKGROUND: Lawsonia intracellularis is the causative agent of proliferative enteropathy, an endemic disease in pigs and an emerging concern in horses. Enterocyte hyperplasia is a common lesion in every case but there are differences regarding clinical and pathological presentations among affected species. We hypothesize that host susceptibility to L. intracellularis infection depends on the species of origin of the bacterial isolate. The objective of this study was to evaluate the susceptibilities of pigs and horses to L. intracellularis infection using either a porcine or an equine isolate. MATERIALS AND METHODS: Twelve foals and eighteen pigs were equally divided into three groups and infected with either a porcine or an equine isolate (109L. Intracellularis/challenged animal), and a saline solution (negative control group). The animals were monitored regarding clinical signs, average of daily weight gain, fecal shedding of the bacteria by PCR and humoral serological response. RESULTS: Foals infected with the equine isolate developed moderate to severe clinical signs and maintained a lower average of weight gain compared to control foals. Fecal quantitative PCR in equine isolate-infected foals revealed higher amounts of bacterial DNA associated with longer duration of shedding compared with porcine isolate-infected foals. All four foals infected with the equine isolate demonstrated higher IgG titers in the serum compared with porcine isolate-infected foals. In the pig trial, diarrhea and seroconversion were only observed in animals infected with the porcine isolate. Pathological changes typical of proliferative enteropathy were observed in the necropsied foal infected with equine isolate and in the two necropsied pigs infected with the porcine isolate. CONCLUSIONS: Evident clinical signs, longer periods of bacterial shedding and stronger serologic immune responses were observed in animals infected with species-specific isolates. These results show that host susceptibility is driven by the origin of the isolated L. intracellularis strain.

Efficacy of an avirulent live vaccine against Lawsonia intracellularis in the prevention of proliferative enteropathy in experimentally infected weanling foals.

OBJECTIVE: To determine the efficacy of an avirulent Lawsonia intracellularis vaccine in preventing proliferative enteropathy in weanling foals. ANIMALS: 12 healthy weanling foals. PROCEDURES: Foals were randomly assigned to a vaccinated, nonvaccinated, or control group. Vaccinated foals received an avirulent porcine L intracellularis frozen-thawed vaccine intrarectally 60 and 30 days prior to experimental challenge. On day 1, vaccinated and nonvaccinated foals were challenged via nasogastric intubation with a virulent heterologous isolate of L intracellularis. Control foals were not challenged. Clinical observation and ultrasonographic evaluation of the small intestine were performed, and body weight, serum concentration of total solids, fecal excretion of L intracellularis, and seroconversion were measured for each foal until day 56. Diseased foals were treated with antimicrobials and supportive care. RESULTS: None of the 4 vaccinated foals developed clinical disease following challenge with virulent L intracellularis. Three of 4 nonvaccinated foals developed moderate to severe clinical signs compatible with proliferative enteropathy, hypoproteinemia, and thickened small intestinal loops. Vaccinated foals had significantly less fecal shedding of L intracellularis than nonvaccinated foals. Serologic responses between vaccinated and nonvaccinated foals after challenge were similar. Control foals remained clinically unaffected with no evidence of fecal shedding and seroconversion. CONCLUSIONS AND CLINICAL RELEVANCE: Intrarectal administration of a commercial avirulent porcine vaccine against L intracellularis resulted in complete protection against proliferative enteropathy in the foals in this study and may also reduce environmental contamination with the organism on endemic farms.

Serial use of serologic assays and fecal PCR assays to aid in identification of subclinical Lawsonia intracellularis infection for targeted treatment of Thoroughbred foals and weanlings.

OBJECTIVE: To assess the serial use of serum immunoperoxidase monolayer assays (IPMAs) and fecal PCR assays, combined with other diagnostic methods, to identify subclinical Lawsonia intracellularis infections for targeted treatment of Thoroughbred foals and weanlings at farms in which the pathogen was endemic or nonendemic. DESIGN: Evaluation study. ANIMALS: 100 foals and weanlings (53 and 47 at farms in which L intracellularis was endemic and nonendemic, respectively). PROCEDURES: Serum was collected every 4 weeks and tested via IPMA, for antibodies against L intracellularis. Fecal samples were collected every 2 weeks and tested by use of an L intracellularis-specific PCR assay. When results for IPMAs or PCR assays were positive or clinical signs compatible with equine proliferative enteropathy (EPE) were detected, clinicopathologic testing was performed to determine treatment. RESULTS: No foals had positive results for the L intracellularis-specific IPMA until after weaning; 32 of 53 (60.4%) weanlings at the farm in which L intracellularis was endemic and 8 of 47 (170%) at the farm in which L intracellularis was nonendemic had positive IPMA results, whereas the number of weanlings that tested positive via fecal PCR assays at those farms was 6 and 0, respectively. Nineteen of 32 weanlings with positive IPMA results at the farm in which L intracellularis was endemic were treated for EPE; 5 of these had clinical signs of EPE. No weanlings at the nonendemic farm had clinical signs of or were treated for EPE. CONCLUSIONS AND CLINICAL RELEVANCE: IPMA appeared to be a useful means of identifying weanlings exposed to L intracellularis.

The immune response of foals to natural infection with equid herpesvirus-2 and its association with febrile illness.

Equid herpesvirus-2 (EHV-2) infection is ubiquitous in horses. Although EHV-2 infection has been associated with several disease syndromes, its true pathogenic significance in horses remains uncertain. Epstein-Barr virus (EBV), another gammaherpesvirus, has been shown to cause febrile illness in humans related to its immunopathologic effects. Thus, the purpose of this study was to describe the ontogeny of the immune response of a cohort of 9 foals to natural infection with EHV-2 by evaluating serial complete blood counts, lymphocyte morphology, cytokine gene expression in peripheral blood mononuclear cells (PBMC), viral load in nasal swabs and blood, and antigen-specific cellular immune responses of PBMC, in conjunction with clinical evaluation of the foals. The occurrence of fever in foals was not related to lymphocytosis or specific changes in lymphocyte morphology, cytokine gene expression, or viral load, but tended to be associated (P

Lawsonia intracellularis: humoral immune response and fecal shedding in weanling foals following intra-rectal administration of frozen-thawed or lyophilized avirulent live vaccine.

The humoral immune response and fecal shedding of Lawsonia intracellularis was investigated in 20 weanling foals following intra-rectal administration of frozen-thawed or lyophilized avirulent live L. intracellularis vaccine. Foals received either 30 mL frozen-thawed or lyophilized vaccine intra-rectally, given twice, 4 weeks apart. Serum samples from each foal were collected every 4 weeks for 16 weeks following the first vaccination and tested for anti-L. intracellularis specific IgG by immunoperoxidase monolayer assay. Rectal swabs were collected every other day following the first vaccination for 4 weeks for detection of L. intracellularis by real-time PCR. Both vaccine formulations administered intra-rectally in weanling foals were safe and led to similar onset and duration of fecal shedding and measurable serum IgG response against L. intracellularis.

Evaluation of the humoral immune response and fecal shedding in weanling foals following oral and intra-rectal administration of an avirulent live vaccine of Lawsonia intracellularis.

Equine proliferative enteropathy (EPE) caused by Lawsonia intracellularis has recently been recognized as an emerging disease in foals. Whilst the clinical entity, diagnostic evaluation and treatment of affected foals have been well established and described, preventive measures for EPE have remained largely unaddressed. The objectives of this study were to investigate the humoral immune response and onset and duration of fecal shedding in foals after oral and intra-rectal administration of a modified-live vaccine of L. intracellularis. Foals were vaccinated twice, 3 weeks apart, via oral drenching after pre-medication with a proton-pump inhibitor (omeprazole; group 1), intra-rectally (group 2) or orally without any pre-medication (group 3). The health status of the foals was monitored daily, with feces and serum collected at regular intervals for Polymerase Chain Reaction (PCR) and serology. All foals remained healthy and no adverse vaccine reactions were observed. Fecal shedding lasted from 1 to 12 days and was mainly detected in foals receiving the intra-rectal vaccine 11-15 days following the first vaccine administration. Serological responses were measured in the majority of the vaccinated foals. All foals vaccinated intra-rectally seroconverted after the first vaccine, compared to 50% and 0% of foals in groups 1 and 3, respectively. Pre-medication with omeprazole prior to oral vaccination in group 1 foals led to an earlier and stronger detectable humoral response compared to non pre-medicated foals.

Morphological adrenarche in rhesus macaques: development of the zona reticularis is concurrent with fetal zone regression in the early neonatal period.

Human adrenarche is associated with the establishment of a functional zona reticularis (ZR) and increasing secretion of dehydroepiandrosterone (DHEA) in sulfated form (DS). Like most non-human primates, rhesus macaques are not believed to undergo adrenarche, though they clearly establish a functional ZR after birth. However, the origins of the rhesus ZR are not well defined. Therefore, we investigated the zonal development, steroidogenic enzyme expression and morphology of rhesus adrenals from 1 day to 14 months of age. Immunohistochemistry was conducted to determine expression profiles of the steroidogenic enzymes 17alpha-hydroxylase/17,20-lyase cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1), cytochrome P450, family 21, subfamily A, polypeptide 2 (CYP21A2), hydroxy-Delta-5-steroid dehydrogenase, 3beta- and steroid Delta-isomerase 2 (HSD3B2), the redox partner NADPH-cytochrome P450 oxidoreductase (CPR), as well as the accessory protein cytochrome b5 (b5), a marker of the primate ZR. The rhesus ZR is mature by 3 months of age based on differentiation of the innermost zone that lacks HSD3B2, but exhibits increased b5 expression during this period. Further, the ZR develops in neonates from a previously described dense band of cells which we show expresses b5, CYP17A1, CPR, and CYP21A2 throughout maturation. The fetal zone (FZ) is distinguished from the ZR by its lack of CYP21A2, and ZR development proceeded as the FZ regressed with two important implications: neither FZ regression nor ZR maturation can be monitored independently by circulating adrenal androgens, and these events must be induced by different factors in rhesus, and likely humans. Collectively these data demonstrate that ZR development begins before birth in the rhesus, proceeding concomitantly with FZ regression post-natally, suggesting that rhesus experiences morphological adrenarche during the first three months of life.

Isolation of a gammaherpesvirus similar to asinine herpesvirus-2 (AHV-2) from a mule and a survey of mules and donkeys for AHV-2 infection by real-time PCR.

Equids are commonly infected by herpesviruses, but isolation of herpesviruses from mules has apparently not been previously reported. Furthermore, the genomic relationships among the various equid herpesviruses are poorly characterized. We describe the isolation and preliminary characterization of a mule gammaherpesvirus tentatively identified as asinine herpesvirus-2 (AHV-2; also designated equid herpesvirus-7 (EHV-7)) from the nasal secretions (NS) of a healthy mule in northern California. The virus was initially identified by transmission electron microscopic examination of lysates of cell culture inoculated with NS collected from the mule. A 913 nucleotide sequence of the DNA polymerase gene was amplified using degenerate primers, and comparison of this sequence with those of various other herpesviruses showed that the mule herpesvirus was most closely related to EHV-2 (AHV-2 sequences were not available for comparison). The sequence of a shorter portion (166 nucleotides) of the mule herpesvirus DNA polymerase gene was identical to that of the published sequence of an asinine gammaherpesvirus, previously designated as AHV-4-3 (AY054992). AHV-2 was detected by real-time polymerase chain reaction assay in the NS of approximately 8% of a cohort of 114 healthy mules and 13 donkeys.