S. Mapes

Detection of Lawsonia intracellularis by Real-time PCR in the Feces of Free-living Animals from Equine Farms with Documented Occurrence of Equine Proliferative Enteropathy

The objective of this study was to determine whether Lawsonia intracellularis was present in the feces of free-living animals collected on two equine premises with documented occurrence of equine proliferative enteropathy (EPE). Fresh feces from black-tailed jackrabbits (Lepus californicus, n=100), striped skunks (Mephitis mephitis, n=22), feral cats (Felis catus, n=14), Brewers Blackbirds (Euphagus cyanocephalus, n=10), Virginian opossums (Didelphis virginiana, n=9), raccoons (Procyon lotor, n=4), California ground squirrels (Spermophilus beecheyi, n=3), and coyotes (Canis latrans, n=2) were collected from August 2006 to January 2007 either from the ground while walking the premises or after trapping the animals using live traps. Nucleic acid purified from feces was directly processed for polymerase chain reaction (PCR) analysis using a real-time PCR assay targeting the aspartate ammonia lyase gene of L. intracellularis. Purified DNA samples were also precipitated, preamplified for L. intracellularis, and analyzed using the same real-time PCR assay, to increase the detection limit to one L. intracellularis organism per extracted sample. Feces from jackrabbits, striped skunks, Virginian opossums, and coyotes tested PCR positive for L. intracellularis, whereas all feces from feral cats, Brewers Blackbirds, raccoons, and ground squirrels tested PCR negative for L. intracellularis. PCR testing on DNA extracted directly from feces was positive for L. intracellularis in six of 164 fecal samples. When DNA purification from feces was followed by a precipitation and preamplification step, five additional fecal samples tested PCR positive for L. intracellularis (11/164). The largest number of PCR positive L. intracellularis fecal samples was observed in striped skunks, followed by Virginian opossums, jackrabbits, and coyotes. This is the first description of L. intracellularis in these four species. Because the fecal samples were collected at equine farms with confirmed cases of EPE, striped skunks, Virginian opossums, jackrabbits, and coyotes may act as potential sources of infection to susceptible weanlings.

Surveillance programme for important equine infectious respiratory pathogens in the USA

The prevalence and epidemiology of important viral (equine influenza virus [EIV], equine herpesvirus type 1 [EHV-1] and EHV-4) and bacterial (Streptococcus equi subspecies equi) respiratory pathogens shed by horses presented to equine veterinarians with upper respiratory tract signs and/or acute febrile neurological disease were studied. Veterinarians from throughout the USA were enrolled in a surveillance programme and were asked to collect blood and nasal secretions from equine cases with acute infectious upper respiratory tract disease and/or acute onset of neurological disease. A questionnaire was used to collect information pertaining to each case and its clinical signs. Samples were tested by real-time PCR for the presence of EHV-1, EHV-4, EIV and S equi subspecies equi. A total of 761 horses, mules and donkeys were enrolled in the surveillance programme over a 24-month study period. In total, 201 (26.4 per cent) index cases tested PCR-positive for one or more of the four pathogens. The highest detection rate was for EHV-4 (82 cases), followed by EIV (60 cases), S equi subspecies equi (49 cases) and EHV-1 (23 cases). There were 15 horses with double infections and one horse with a triple infection. The detection rate by PCR for the different pathogens varied with season and with the age, breed, sex and use of the animal.

Use of quantitative real-time PCR for the detection of Salmonella spp. in fecal samples from horses at a veterinary teaching hospital.

A quantitative real-time (RT)-PCR assay was developed to detect Salmonella spp. in the feces of 911 equine species admitted to a veterinary hospital. Fresh feces and feces enriched for 24h in selenite broth were assessed by conventional culture and by RT-PCR targeting the Salmonella invA gene. The detection limit for the RT-PCR assay was 3 and 10 organisms, respectively, when spiked samples were purified from selenite broth and feces. The analytical specificity was 100% based on the detection of a panel of 40 salmonella serotypes from five serogroups and the lack of cross-reactivity with non-related micro-organisms. Although Salmonella spp. were not cultured from fresh feces, the organism was cultured from 6/911 (0.6%) of broth-enriched samples. The bacterial load in enriched samples varied from 3 to 861,037 salmonella invA gene copies/μL DNA. The RT-PCR assay had an overall relative accuracy of 98%, a relative sensitivity of 100% and a relative specificity of 98%, when compared to conventional culture. The judicious use of such a RT-PCR method has the potential to reduce the risk of nosocomial infections such as salmonellosis through the provision of highly accurate and rapid pathogen detection.

Oral infection of weanling foals with an equine isolate of Lawsonia intracellularis, agent of equine proliferative enteropathy

BACKGROUND Equine proliferative enteropathy (EPE) is an emerging disease of weanling foals. OBJECTIVES Describe clinical, hematologic, biochemical, serologic, molecular, and ultrasonographic findings in foals experimentally infected with Lawsonia intracellularis. ANIMALS Eight foals. METHODS Recently weaned foals were assigned to either the challenge (n = 3), the sentinel (n = 3), or the control (n = 2) group. Foals were experimentally challenged via intragastric inoculation of 3 x 10(10)L. intracellularis organisms grown in culture. Each experimentally infected foal was housed with a sentinel foal in order to assess feco-oral transmission. All foals were monitored daily for the development of clinical abnormalities and were weighed once weekly for the duration of the study (90 days). Abdominal ultrasound examination was performed weekly. Feces were collected every other day for 60 days, then weekly for an additional 30 days for the quantitative molecular detection of L. intracellularis. Blood was collected weekly for hematologic, biochemical, and serologic analysis. RESULTS Only challenged foals developed transient clinical signs of EPE consisting of anorexia, lethargy, fever, loose feces, and peripheral edema. Two challenged foals developed transient hypoalbuminemia. Fecal shedding of L. intracellularis was first detected in the challenged foals between days 12 and 18 postinoculation and lasted for 7-21 days. Seroconversion was documented in all challenged foals and in 1 sentinel foal. The remaining sentinel and control foals remained unaffected. CONCLUSIONS AND CLINICAL IMPORTANCE Clinical EPE of variable severity was induced in all foals infected with L. intracellularis. Furthermore, L. intracellularis can be transmitted via the feco-oral route to susceptible herdmates.

Biochemical Assessment of Limits to Estrogen Synthesis in Porcine Follicles

Abstract Limits to estrogen production by early and late preovulatory porcine follicles were assessed by comparing enzymatic capacities for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa, support of enzyme activities by the redox partner proteins NADPH-cytochrome P450 oxidoreductase (reductase) and cytochrome b5, and tissue-specific expression and regulation of these proteins. Parameters included follicular fluid (FF) estradiol and progesterone levels, theca and granulosa aromatase and reductase activities, and theca 17,20-lyase activity. Expression of proteins responsible for these activities, aromatase (P450arom) and 17α-hydroxylase/17,20-lyase (P450c17) cytochromes P450, reductase, and for the first time in ovarian tissues cytochrome b5, were examined by Western immunoblot and immunocytochemistry. Theca and granulosa aromatase activities were as much as 100-fold lower than theca 17,20-lyase activity, but aromatase was correlated with only the log of FF estradiol. Granulosa reductase activity was twice that of the theca, and cytochrome b5 expression was clearly identified in both the theca and granulosa layers, as was P450arom, but was not highly correlated with either 17,20-lyase or aromatase activities. Reductase expression did not change with stage of follicular development, but cytochrome b5, P450c17, and P450arom were markedly lower in post-LH tissues. These data indicate that aromatase and not 17,20-lyase must limit porcine follicular estradiol synthesis, but this limitation is not reflected acutely in FF steroid concentrations. Neither reductase nor cytochrome b5 appear to regulate P450 activities, but the expression of cytochrome b5 in granulosa and theca suggests possible alternative roles for this protein in follicular development or function.

Emerging outbreaks associated with equine coronavirus in adult horses.

Abstract The purpose of this study was to describe clinical, hematological and fecal PCR results from 161 horses involved in outbreaks associated with ECoV. The outbreaks happened at four separate boarding facilities between November 2011 and April 2012 in the States of CA, TX, WI and MA. Following the molecular detection of ECoV in the feces from the initial index cases, the remaining herdmates were closely observed for the development of clinical signs. Fecal samples were collected from sick and healthy horses for the PCR detection of ECoV. All four outbreaks involved primarily adult horses. Fifty-nine horses developed clinical signs with 12–16 sick horses per outbreak. The main clinical signs reported were anorexia, lethargy and fever. Four horses from 3 different outbreaks were euthanized or died due to rapid progression of clinical signs. The cause of death could not be determined with necropsy evaluation in 2 horses, while septicemia secondary to gastrointestinal translocation was suspected in 2 horses. Blood work was available from 10 horses with clinical disease and common hematological abnormalities were leucopenia due to neutropenia and/or lymphopenia. Feces were available for ECoV testing by real-time PCR from 44 and 96 sick and healthy horses, respectively. 38/44 (86%) horses with abnormal clinical signs tested PCR positive for ECoV, while 89/96 (93%) healthy horses tested PCR negative for ECoV. The overall agreement between clinical status and PCR detection of ECoV was 91%. The study results suggest that ECoV is associated with self-limiting clinical and hematological abnormalities in adult horses.

Epidemiological survey on farms with documented occurrence of equine proliferative enteropathy due to Lawsonia intracellularis.

PROLIFERATIVE enteropathy caused by the obligate intra-cellular organism Lawsonia intracellularis has been described in a number of domestic and wild animal species ([Lawson and Gebhart 2000][1]). In horses, the disease is known as equine proliferative enteropathy (epe) and has been reported from

Temporal detection of Lawsonia intracellularis using serology and real-time PCR in Thoroughbred horses residing on a farm endemic for equine proliferative enteropathy

The goals of this study were to evaluate titers of antibodies against Lawsonia intracellularis in 68 resident broodmares from a farm known to be endemic for equine proliferative enteropathy (EPE) and to evaluate maternal antibodies, occurrence of seroconversion and fecal shedding in their foals. Serum samples collected from mares at delivery and from foals pre- and post-colostrum ingestion and monthly thereafter were tested for the presence of L. intracellularis antibodies by immunoperoxidase monolayer assay (IPMA). Further, feces collected from mares at delivery and foals post-partum and monthly thereafter were assayed for L. intracellularis using real-time PCR. Thirty-seven mares (54.4%) had detectable antibody titers (> or =60) against L. intracellularis by IPMA at the time of foaling. Passive transfer of colostral antibodies against L. intracellularis was documented in 37 foals (54.4%) and the colostral antibodies remained detectable in the serum of foals for 1-3 months. Overall, 22 foals (33.3%) showed evidence of natural exposure to L. intracellularis throughout the study period, however, none of the study foals developed signs compatible with EPE. The serological results showed that mares residing on a farm known to be endemic for EPE are routinely exposed to L. intracellularis and that antibodies against L. intracellularis are passively transferred to 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.

Structural and functional differences among purified recombinant mammalian aromatases: glycosylation, N-terminal sequence and kinetic analysis of human, bovine and the porcine placental and gonadal isozymes

Recombinant porcine gonadal and placental, and the human and bovine, isozymes of aromatase cytochrome P450 (P450arom) were over-expressed in insect cells, purified and quantified by difference spectroscopy. Human and bovine P450arom exhibited greater apparent molecular size than either porcine isozyme prompting an examination of N-linked glycosylation and amino-terminal peptide sequence. Comparisons of substrate affinities and turnover were also made. In contrast to human and bovine P450arom which are N-linked glycoproteins, neither isozyme of porcine P450arom is glycosylated, explaining in part their lower molecular size. Differences found in N-terminal peptide sequences were unlikely to influence apparent molecular size or enzyme function. Human and bovine P450arom had similar affinities and turnovers for androstenedione (approximately 200 nM, 3/min) and testosterone (approximately 350 nM, 2/min). The porcine isozymes had 10-fold higher affinities but correspondingly lower turnovers, particularly the gonadal P450arom. Overall, the catalytic efficiency (Vmax/Km) was similar for all but porcine gonadal P450arom which was much lower. These data emphasize the structural and functional variability of even the most conserved of proteins among diverse species wherein such differences have previously remained unexpected.