C. Gradil

Porcine reproductive and respiratory syndrome virus: seminal transmission

PORCINE reproductive and respiratory syndrome is a highly contagious viral disease present in pig populations in North America and Europe (Collins and others 1992). The signs of the disease are reproductive failure, including abortion, stillbirth and neonatal death, and respiratory disease in piglets (Pol and others 1991). Genome organisation, strategy of gene expression and the sequence of deduced proteins show that porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the arterivirus

Cloning and expression of ADAM-related metalloproteases in equine laminitis.

Equine laminitis is a debilitating disease affecting the digital laminae that suspend the distal phalanx within the hoof. While the clinical progression of the disease has been well documented, the molecular events associated with its pathogenesis remain largely unknown. Using real time quantitative PCR (RT-qPCR), we have investigated the expression of genes coding for proteins containing a Disintegrin and Metalloprotease domain (ADAM), as well as genes encoding the natural inhibitors of these enzymes (tissue inhibitor of metalloprotease; TIMP) in horses with naturally-acquired (acute, chronic and aggravated chronic clinical cases) or experimentally-induced (black walnut extract (BWE) and starch gruel models) laminitis. Changes in expression of these enzymes and regulators may underlie the pathologic remodeling of lamellar tissue in laminitis. Genes encoding ADAMs involved in inflammation (ADAM-10 and ADAM-17), as well as those implicated in arthritis (ADAMTS-1, ADAMTS-4 and ADAMTS-5) were cloned, and the sequences used to generate specific oligonucleotide primers for the RT-qPCR experiments. Our results show that genes encoding ADAM-10 and ADAM-17 were not induced in most laminitic animals, whereas ADAMTS-4 gene expression was strongly upregulated in nearly all horses with experimentally-induced and naturally-acquired laminitis. The expression of matrix metalloproteases (MMP)-9 and ADAMTS-5 was also increased in many of the laminitic horses. In addition, TIMP-2 gene expression was decreased in most laminitic horses, whereas expression of genes encoding other TIMPs, namely TIMP-1 and TIMP-3, was randomly increased or decreased in the various models. We conclude that increased expression of lamellar ADAMTS-4 is a common feature of laminitis consistent with a central role of the gene product in the pathophysiology of the disease.

In vitro exposure of preimplantation porcine embryos to porcine parvovirus

Early porcine embryos at the four- to eight-cell stage can be infected with either the virulent (NADL-8) or avirulent KBSH strain of porcine parvovirus (PPV) by microinjection or by incubation of embryos with virus. Treatment of embryos by microinjection of virus or incubation in media with virus did not significantly inhibit in vitro development of the embryos when compared with untreated controls. RNA-DNA hybridization was used to identify the presence of virus associated with embryos. It was found that PPV-DNA was present in viable embryos after microinjection of embryos with KBSH and NADL-8 strains of PPV and after incubation of embryos with KBSH strain. The data indicated the presence of replicative virus associated with viable porcine embryos.

THE USE OF PENTOXIFYLLINE TO IMPROVE MOTILITY OF CRYOPRESERVED EQUINE SPERMATOZOA

Pentoxifylline was evaluated as a method to increase motility of cryopreserved equine spermatozoa. In a preliminary experiment, pentoxifylline (3.5 mM or 7.0 mM) was added to extended semen that was chilled to 4 degrees C. Motility was evaluated at 8-h intervals for 48 h. The addition of 3.5 or 7.0 mM pentoxifylline appeared to increase the motility of chilled spermatozoa compared to controls. Based on these results, similar concentrations of pentoxifylline were added to semen either before or after cryopreservation. The addition of pentoxifylline (3.5 or 7.0 mM) to semen before cryopreservation significantly (P < 0.001) decreased total and progressive motility compared to controls. However, the addition of pentoxifylline (3.5 or 7.0 mM) to cryopreserved semen immediately after thawing significantly (P < 0.01) increased total and progressive motility compared to controls. These results indicate that pentoxifylline enhanced the postthaw motility of cryopreserved equine semen when added after thawing. Further research is required to evaluate the effect of pentoxifylline on the fertility of cryopreserved equine semen.

Detection of bovine immunodeficiency virus DNA in the blood and semen of experimentally infected bulls

Five 18- to 24-month-old bulls were inoculated with either a cell suspension containing bovine immunodeficiency virus (BIV-FL112; 3 bulls) or a BIV-free cell suspension (2 bulls). Blood and semen specimens were collected once a week for 14 weeks, and seroconversion was confirmed by indirect immunofluorescent antibody (IFA) testing. The presence of BIV in blood and semen was determined by virus isolation and/or polymerase chain reaction (PCR) assays. Antibodies to BIV were detected in the 3 experimentally infected bulls as early as day post inoculation (DPI) 17, and levels peaked at DPI 37-58. BIV was isolated from the peripheral blood mononuclear cells (MNCs) of the infected bulls at DPI 9 (2 bulls) and DPI 23 (1 bull), and could be isolated from one animal up to DPI 65. PCR analysis of MNC DNA, using BIV pol gene primers, detected virus in all three of the experimentally infected bulls from DPI 9 until the termination of the experiment at DPI 98. Efforts to isolate a significant number of non-spermatozoal cells (NSC) by gradient separation from the semen of the experimentally infected bulls were unsuccessful. Two methods for the extraction of total NSC DNA from up to 2 ml of non-extended semen were employed; however, no BIV pol fragment was amplified from these DNA preparations. Additionally, 30 bulls from artificial insemination (AI) centers were evaluated for BIV infection by PCR. No amplification products were obtained from MNC DNA from the AI submissions using primer sets for both the BIV pol and env genes.

Manual corneal thickness measurements of healthy equine eyes using a portable spectral-domain optical coherence tomography device

REASONS FOR PERFORMING STUDY Corneal thickness measurements of the equine globe using spectral-domain optical coherence tomography (SD-OCT) have not been reported. OBJECTIVES To determine corneal thickness measurements and the intra- and interoperator reliability of a portable SD-OCT device in equine eyes. STUDY DESIGN Prospective observational study. METHODS Horses free of ocular disease were used for this study. Gentle manual restraint, in combination with detomidine hydrochloride and a head stand, were employed to ensure proper animal positioning. Corneal pachymetry measurements were obtained from both eyes of each animal 3 times by 2 operators in succession. A 6 mm corneal pachymetry protocol was performed using a portable SD-OCT device. All measurements were obtained manually by one operator (C.G.P.) using the integrated calliper function. Measurements included epithelial thickness, stromal thickness, Descemets membrane thickness and total corneal thickness. All recorded measurements were analysed to determine both intra- and interoperator reliability. RESULTS Thirty horses with a mean age of 10.6 ± 6.4 years were examined. Mean epithelial, stromal, Descemets membrane and total corneal thickness values obtained were, respectively, 174.7 ± 13.6, 599.2 ± 45.4, 38.4 ± 15.3 and 812.0 ± 44.1 μm for operator A and 175.9 ± 12.9, 599.2 ± 44.9, 38.4 ± 15.0 and 812.9 ± 42.9 μm for operator B. A positive correlation was found between Descemets membrane thickness and age, whereby Descemets membrane thickness increased by 2 μm/year (P<0.0001). The coefficients of variation for both operators were <4% for all measurements. Intraclass correlations ranged from 0.92 to 0.98. CONCLUSIONS Manual corneal thickness measurement using a portable SD-OCT device provides epithelial, stromal, Descemets membrane and total corneal thickness measurements with clinically acceptable intra- and interoperator reliability in healthy equine eyes.

Evaluation of Nucleic Acid Amplification Methods for the Detection of Hog Cholera Virus

A blind panel was tested in a diagnostic evaluation of a reverse transcription (RT) polymerase chain reaction (PCR) method for detecting hog cholera virus (HCV) from pig tissues. The capability of the RT-PCR test to discriminate between HCV and related pestiviruses, bovine viral diarrhea virus (BVDV), and those viruses causing similar diseases in swine, including African swine fever virus (ASFV) and pseudorabies virus (PRV), was also considered. Nucleic acid extraction involved either kit-based or conventional phenol: chloroform: isoamyl alcohol methods. A single-round PCR assay, using primers that hybridize to the conserved p 120 nonstructural gene region, was 82.5% sensitive (n = 17) and 100% specific (n = 18) in the detection of the presence of HCV RNA. However, the sensitivity was increased to 100% following a second PCR test. In all, 4 HCV, 7 BVDV, 2 ASFV, and 1 PRV isolates were studied. Novel nucleic acid sequences were generated for 9 HCV strains. Analysis of a portion of the p120 region using these methods was suitable for HCV isolate characterization.

Detection of verotoxigenic Escherichia coli in bull semen using the polymerase chain reaction

Oligonucleotide primers used in a polymerase chain reaction (PCR) protocol detected the verotoxin 2 (VT2) gene in E. coli present in experimentally contaminated bull semen. The VT2 (Shiga-like toxin II [SLT-II]) primers targeted a 346-bp fragment of the gene coding for the A subunit of the toxin. PCR products, corresponding to the VT2 gene sequence, were amplified from template E. coli nucleic acid extracted from 18-h broth culture and from E. coli in contaminated semen in the undiluted state, diluted in egg yolk-Tris and diluted in milk. The sensitivity of the assay to detect E. coli was determined to be 1 pg of nucleic acid, and as few as 10-20 E. coli organisms/ml could be detected in raw and diluted semen. Preliminary confirmation of the PCR product was accomplished by slot blot hybridization to a radiolabeled specific oligoprobe. Sequencing of the PCR products identifying VT2 gene sequence revealed 99.7% homology with published gene sequences for VT2. This study demonstrates the feasibility of applying PCR technology for the detection of E. coli in bovine semen. This technique may find wide application for the detection of other pathogens that may be present in semen.

A radial immunodiffusion enzyme assay for detection of antibody to equine rhinopneumonitis virus (EHV-1) in horse serum

A radial immunodiffusion enzyme assay (RIDEA) was developed for detection and quantitation of antibodies to equine herpes virus-1 (EHV-1) in horse sera. The detection and quantitation of EHV-1 antibody levels were based on the diameter of the radial diffusion zone of specific antibody in each serum sample reacting with EHV-1 antigen. The circular zone was made visible using peroxidase-conjugated rabbit anti-horse immunoglobulin G and a substrate containing hydrogen peroxide. The results of the RIDEA were compared with those of virus neutralization (VN) and enzyme-linked immunosorbent assay (ELISA) and found to be highly correlated. The relative sensitivity and specificity (percentage of agreement with VN test) were found to be 98.2 and 92.5%, respectively. Because the test procedure is relatively easy to perform, the RIDEA could be used as a field test to detect antibodies to EHV-1 in horses.

Trisomy-X with estrous cycle anomalies in two female dogs.

Two female dogs were presented with a history of abnormal estrous cycles and infertility, despite multiple breedings. Medical therapy to correct the cycle anomalies did not result in pregnancy. Cytogenetic analysis of blood lymphocyte cultures in each dog revealed three copies of the X chromosome in each cell, constituting a 79,XXX karyotype (trisomy-X). Both dogs were eventually ovariohysterectomised and histological evaluation revealed hypoplastic ovaries and an absence of normal follicular structures. However, partial or immature follicles were noted, which may have been sufficient to cause both females to initiate cycling. The history and clinical characteristics found in these dogs were compared to those described in three other dogs reported with trisomy-X, as well as those reported in other species. These findings highlighted the importance of cytogenetic studies in fertility evaluation and achieving a definitive diagnosis for infertility in the bitch.