Transcriptome profile and cytogenetic analysis of immortalized neuronally restricted progenitor cells derived from the porcine olfactory bulb.

Recently, we established and phenotypically characterized an immortalized porcine olfactory bulb neuroblast cell line, OBGF400 (1). To facilitate the future application of these cells in studies of neurological dysfunctions and neuronal pathogen interactions, a comprehensive knowledge of their genomic variability and overall gene expression capacity was pursued. Accordingly, the OBGF400 cells were subjected to karyotyping and more extensive transcriptome analyses. Cytogenetic characterization of these cells revealed a genetic mosaicism of neuronal hyperdiploidy. A direct comparison of the OBGF400 cell transcriptome pattern, generated by utilizing the Affymetrix GeneChip(R) Porcine Genome Array, to that of a non-neural, porcine epithelial cell line facilitated the identification of 831 probe sets preferentially hybridized by the neuroblast transcripts. Subsequent functional annotation of these OBGF400 RNAs using the Database for Annotation, Visualization and Integrated Discovery 2008 enabled their allocation to the corresponding gene ontology biological process term, thereby assisting the recognition of key elements involved in the regulation of neuronal signal transduction and neurogenesis.

Vaccination against the avian infectious bronchitis virus affects sperm concentration, sperm quality and blood testosterone concentrations in cockerels.

1. It was previously found that cockerels vaccinated with live attenuated avian infectious bronchitis virus (AIBV) have decreased serum testosterone concentrations, epididymal stones and reduced fertility. The objectives of this study were twofold: to determine if reduced fertility following vaccination with live attenuated virus was the result of reduced sperm concentration or reduced sperm quality and to determine if vaccination with a killed strain of virus caused a similar reduction in sperm function in vivo. 2. Specific-pathogen-free Single Comb White Leghorn cockerels were divided into three treatment groups: no vaccination (NONVAC), vaccination with killed AIBV virus (KVAC) or vaccination with live attenuated AIBV virus (LVAC). Semen was collected daily from 17 to 27 weeks of age, and semen quality was assessed frequently by analysing sperm concentration, viability, motility, and ability to reach and interact with the ovum in vivo. Blood plasma was assayed for testosterone concentration. 3. Differences in sperm analysis among treatment groups were limited. Sperm viability was increased in NONVAC during week 20 which then decreased in week 22 when compared to vaccinated cockerels. Acrosome damage was increased in vaccinated cockerels in week 22, and decreased in weeks 25 and 27 when compared to controls, which correlate to the period of epididymal stone development. Plasma testosterone concentrations and sperm concentrations among treatment groups were different only at 16 and 19 weeks of age, respectively. There were no differences across treatment groups in sperm mobility through Accudenz or in numbers of sperm holes in perivitelline membranes of eggs following insemination with semen from 27-week-old cockerels. No differences were observed in viability or acrosome integrity between cockerels with and without epididymal stones within treatment groups. 4. In conclusion, pre-pubertal vaccination against AIBV and subsequent epididymal stone formation had a limited effect on sperm concentration, sperm quality and plasma testosterone concentrations. Vaccination with killed AIBV vaccine did not diminish effects on sperm function in vivo.

Comparative utility of restriction fragment length polymorphism analysis and gene sequencing to the molecular epidemiological investigation of a viral outbreak.

Restriction fragment length polymorphism (RFLP) analysis and partial-genome DNA sequencing are commonly used to infer genetic relationships among pathogens. This study compares the application of both techniques to the analysis of 16 pseudorabies virus isolates collected during a 1989 outbreak. Genetic distances derived from RFLP and DNA sequence data were not significantly correlated with geographic distances between farms from which isolates were collected. RFLP-based genetic distance was, however, strongly correlated with temporal distance between isolates (days separating time of isolation). Sequence-based genetic distance was significantly correlated with temporal distance only when synonymous changes (nucleotide changes not leading to amino acid changes) were considered separately. Conversely, non-synonymous changes were correlated with the host species of origin of the viral isolate. These results indicate that selectively-neutral genetic changes most accurately reflect historical relationships, but that non-neutral changes most accurately reflect the biological environment of the viral isolate (e.g. host immune system).

Comparison of serologic testing and slaughter evaluation for assessing the effects of subclinical infection on growth in pigs.

OBJECTIVE: To compare serologic testing with slaughter evaluation in assessing effects of subclinical infection on average daily weight gain (ADG) in pigs. DESIGN: Cohort study. ANIMALS: 18 cohorts (30 to 35 pigs/cohort) of pigs on/farms. PROCEDURE: Blood samples were collected, and pigs were weighed at 8, 16, and 24 weeks of age. Sera were tested for antibodies to porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), transmissible gastroenteritis virus (TGEV), pseudorabies virus, Mycoplasma hyopneumoniae, and Actinobacillus pleuropneumoniae. At slaughter, skin, nasal turbinates, lungs, and liver were examined. Associations between ADG and results of serologic testing and slaughter evaluation were examined by use of multiple linear regression. RESULTS: Pathogens that had a significant effect on any given farm during any given year and the magnitude of that effect varied. However, at 16 and 24 weeks of age, a higher antibody titer was consistently associated with a lower ADG. Mean differences in ADG between seropositive and seronegative pigs were 18 g/d (0.04 lb/d) for SIV, 40 g/d (0.09 lb/d) for PRRSV, 38 g/d (0.08 lb/d) for M hyopneumoniae, and 116 g/d (0.26 lb/d) for TGEV. Of the evaluations performed at slaughter, only detection of lung lesions was consistently associated with a decrease in ADG. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that subclinical infection with any of a variety of pathogens commonly found in swine herds was associated with a decrease in ADG. Serologic testing was more effective than slaughter evaluation in assessing the impact of subclinical infection on ADG in these pigs.

Identification of the pseudorabies virus promoter required for latency-associated transcript gene expression in the natural host.

Expression of the latency-associated transcript (LAT) gene is a hallmark of alphaherpesvirus latency, and yet its control and function remain an enigma. Resolution of this problem will require verification and subsequent elimination or disabling of elements regulating LAT gene transcription so that the influence of the resultant RNA can be evaluated. Toward this end, we generated a novel pseudorabies virus (PrV) recombinant in which a 282-bp region containing the LAP1 (first latency-active promoter) consensus sequence was replaced by a reporter cassette. Despite this substitution, replication of the recombinant was comparable to that of the parental and rescuant viruses both in cultured mammalian cells and in the natural host, swine. Furthermore, production of the LAT gene-associated 2.0- and 8.0-kb RNAs during an in vitro lytic infection of cultured neuronal cells was unaffected. However, the otherwise constitutively produced and processed 8.4-kb LAT was not detected in porcine trigeminal ganglia latently infected with this novel recombinant, although the viral genome was shown to be present. Therefore, LAP1 is apparently the basal promoter for PrV LAT gene expression during viral latency but is not required for such activity during an in vitro lytic infection of neuronal cells. More importantly, the ability of PrV to persist in a latent state in the absence of LAT suggests that other factors are responsible for this event in the natural host.

Associations between genetics, farm characteristics and clinical disease in field outbreaks of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is a disease of domestic swine characterized by exceptionally high clinical variability. This study addresses the question of whether clinical variability in PRRS results from (a) genetic variation among viral isolates and/or (b) variation in management practices among farms on which isolates are found. Genetic data (open reading frame 5 gene sequences) and data on farm characteristics and associated clinical disease signs were collected for 62 PRRS virus (PRRSV) field isolates, representing 52 farms. Clinical disease signs were interrelated--confirming that a true reproductive syndrome exists (involving abortions, infertility in sows, deaths of sows and preweaning mortality). Pairs of farms experiencing deaths in their sow populations also tended to share viral isolates which were more similar to one another than expected by chance alone. This implies that sow death (one of the more-severe manifestations of PRRS) is under genetic influence. Large herd size was a significant risk factor for the death of sows and for respiratory disease in nursery pigs. All-in-all-out management practices in the nursery were protective against reproductive signs in the sow herd. All-in-all-out management practices in the finishing stages of production were protective against respiratory disease in nursery pigs--but were paradoxically associated with an increased risk of infertility in sows. These results suggest that farm-management practices can also influence which PRRS clinical signs are manifested during an outbreak. In general, signs associated with PRRS appear to result from a combination of genetic factors and herd-management characteristics. The relative contributions of these two influences differ depending on the specific clinical sign in question.

Genetic, geographical and temporal variation of porcine reproductive and respiratory syndrome virus in Illinois.

Porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 gene sequences were generated by RT-PCR from 55 field isolates collected in Illinois and eastern Iowa. Spatial and temporal patterns of genetic variation in the virus were examined on a local geographical scale in order to test the hypothesis that the genetic similarity of PRRSV isolates (measured as their percentage pairwise ORF5 nucleotide similarity) was positively correlated with their geographical proximity. Levels of genetic variability in the Illinois/eastern Iowa PRRSV sample were similar to levels of variability seen across broader geographical regions within North America. The genetic similarity of isolates did not correlate with their geographical distance. These results imply that the movement of PRRSV onto farms does not generally occur via distance-limited processes such as wind or wildlife vectors, but more typically occurs via the long-distance transport of animals or semen. Genetic distances between PRRSV isolates collected from the same farms at different times increased as the time separating the collection events increased. This result implies rapid movement of new genetic types of PRRSV into and out of farms. PRRSV ORF5 displayed a pattern of third-codon-position diversity bias that was not evident in a geographically comparable sample of pseudorabies virus (a swine alphaherpesvirus) gC gene sequences. This result provides evidence that PRRSV ORF5 is experiencing stabilizing selection against structural novelty. Despite high genetic variability at all geographical levels, PRRSV ORF5 nevertheless contained potentially antigenic regions that were invariant at the amino acid level. These regions should make effective vaccine targets if they prove to be immunogenic.

Expression of the pseudorabies virus latency-associated transcript gene during productive infection of cultured cells.

Like other alphaherpesviruses, pseudorabies virus (PrV) exhibits restricted gene expression during latency. These latency-associated transcripts (LATs) are derived from the region located within 0.69 to 0.77 map units of the viral genome. However, the presence of such viral RNAs during a productive infection has not been described. Although several transcripts originating between 0.706 to 0.737 map units have been detected in PrV-infected cultured cells, their relationship to the LATs has not been examined. Therefore, to determine if any correlation exists between PrV LAT gene expression in the natural and laboratory systems, transcription from the LAT gene region during lytic infection of cultured neuronal and nonneuronal cells was evaluated. A Northern blot assay using single-stranded RNA probes complementary to the spliced in vivo 8. 4-kb largest latency transcript (LLT) detected 1.0-, 2.0-, and 8. 0-kb poly(A) RNAs in all PrV-infected cells lines. The 1.0- and 8. 0-kb transcripts partially overlapped the first and second exons of the LLT, respectively. In contrast, portions of both LLT exons comprised the 2.0-kb RNA sequence, which lacked the same intron as the LLT. Generation of this transcript began about 243 bp downstream of the LLT initiation site and terminated near the junction of BamHI fragments 8' and 8. Its synthesis was inhibited by cycloheximide but not by cytosine beta-D-arabinofuranoside, which suggests that the 2. 0-kb RNA is not an immediate-early gene product. Thus, although the PrV LAT gene is transcriptionally active during a productive infection of cultured cells, the resulting RNAs are distinctive from the LLT.

Application of a quantitative algorithm to restriction endonuclease analysis of Aujeszky's disease (pseudorabies) virus from a geographically localized outbreak.

A retrospective epidemiologic study was conducted to evaluate the application of an objective quantitative algorithm for estimating genetic similarity from restriction endonuclease analysis data. The analysis was performed to assist the determination of chronologic trends in an Aujeszky's disease viral epidemic in a geographic region. DNA from each viral isolate obtained during the epidemic was digested with 4 restriction endonucleases and molar ratio labeled to generate separate fragment patterns that were simultaneously compared using the algorithm. The resultant estimates of genetic similarity were then used in conjunction with time of virus isolation and specific geographic location of the outbreaks to identify the probable sources of infection and the patterns of spread among swine herds. This type of quantitative analysis enabled a more precise and objective approach than previously has been applied to the interpretation of restriction endonuclease data, thereby demonstrating the benefit of this methodology for the investigation of infectious disease outbreaks.

Quantitative assessment of genomic similarity from restriction fragment patterns.

A quantitative algorithm for comparing restriction fragment patterns (RFPs) was developed and used to estimate the genomic similarity of 18 isolates of pseudorabies virus of known origin. Variants of this algorithm using either untransformed or square-root-transformed differences between fragment sizes were investigated with regard to their ability to classify RFPs. Multidimensional scaling was used to represent spatially the genomic relatedness among samples, with 3 dimensions producing the most meaningful results. The square-root transformation provided more interpretable dimensions. The first dimension differentiated samples geographically, separating North American from European isolates. The second and third dimensions differentiated isolates with specific gene deletions (gE and gG, respectively) from those not having these deletions. Clusters of isolates were identified that were related either by collection from the same geographic area during a specific time period, or by laboratory intervention to create vaccines. These methods offer increased precision in the determination of genetic relatedness based on RFPs, and thus offer increased diagnostic accuracy for the determination of sources of infection.

Characterization of paramyxoviruses isolated from three snakes.

Multiple epizootics of pneumonia in captive snakes have been attributed to viruses which have been tentatively placed in the family Paramyxoviridae. Viruses isolated from an ill Neotropical rattlesnake (Crotalus durissus terrificus), from an Aruba Island rattlesnake (Crotalus unicolor), and from a bush viper (Atheris sp.) were propagated in Vero cells and characterized. Viral particles produced in Vero cells were pleomorphic, enveloped, and contained helical nucleocapsids. The viruses were sensitive to ether and to acidic and basic pH. Moreover, they had neuraminidase activity and were able to agglutinate erythrocytes from chicken and a variety of species of mammals. Hemagglutination was inhibited with rabbit antiserum raised against each virus. The buoyant densities of the three isolates ranged from 1.13/cm3 to 1.18/cm3, values consistent with that for an enveloped virus. The nucleic acid in the virion was determined to be RNA by [3H]uridine incorporation. Viral proteins characteristic of paramyxoviruses were immunoprecipitated from cells infected with each of the three isolates using rabbit anti-Neotropical virus serum. The morphologic appearance, physico- and biochemical properties, and cytopathologic effects of these snake viruses were consistent with those of certain members of the family Paramyxoviridae.

Presence of wild type Aujeszky's disease virus in swine identified as subclinical low-prevalent serological test reactors within qualified virus-negative herds.

Subclinical low-prevalent Aujeszky's disease (AD) serological test reactors are defined as those few swine within a qualified AD virus (ADV)-negative herd that have antibodies to wild type virus. However, clinical signs of the associated disease are not observed in these putatively infected swine or elsewhere in the herd. Twelve such animals, including 7 previously vaccinated with a genetically modified ADV, were identified in Illinois (USA) during a 2.5 year period. The humoral immune responses of the 5 nonvaccinated swine were assessed by an enhanced virus neutralization test and a radioimmunoassay. Anti-ADV antibodies were determined to be present in the serum from 4 of these swine. Attempts to isolate ADV by in vitro and in vivo inoculations of cell cultures and weanling mice, respectively, of tonsillar and trigeminal nerve ganglionic tissue preparations from each animal (vaccinated and nonvaccinated) were unsuccessful. Tonsillar and trigeminal nerve ganglionic tissues of each animal were screened for the presence of wild type and/or vaccine viral genomes by a soluble polymerase chain reaction (PCR) coupled with Southern hybridization. Unique PCR primers were used to distinguish between wild type and vaccine viral DNAs. An additional PCR procedure, which amplifies a portion of the essential and highly conserved viral gp50 gene, also was employed in an effort to detect viral genomes. Wild type viral DNA was found in the tissues from at least 5 of the vaccinated and 3 of the nonvaccinated swine. These results indicate that such animals should be considered as being infected with ADV. Further, these findings emphasize the need to develop highly specific and sensitive antemortem testing methods for accurate assessment of ADV infection in herds containing such subclinical, yet serologically positive, swine.

Chlamydiosis in mariculture-reared green sea turtles (Chelonia mydas).

From August 1990 to June 1991, a moderate die-off of 4- to 5-year-old green sea turtles (Chelonia mydas) occurred at Cayman Turtle Farm, Grand Cayman, British West Indies. Clinical signs included lethargy, anorexia, and inability to dive. Many of the ill turtles floated on the surface of their tanks. There was no apparent sex predilection. Complete necropsies, including histopathologic examination of tissues, were performed on eight turtles. Necropsies revealed multiple irregular discrete to patchy 1-10 mm pale gray foci throughout the hearts of four turtles. By light microscopic examination, the most severe and consistent lesions were necrotizing myocarditis, histiocytic to fibrinous splenitis, and hepatic lipidosis and necrosis. A mixed leukocytic infiltrate of acidophils, macrophages, and lymphocytes was present in affected areas of the heart. Other lesions included lymphocytic/plasmacytic interstitial nephritis, subacute interstitial pneumonia, subacute mesenteric vasculitis, chronic/active enteritis of the small intestine, and occasional granulomas associated with spirorchid trematode ova. Chlamydiae could be demonstrated in macrophages in sections of paraffin-embedded heart, liver, and spleen and in myocardial fibers and hepatocytes using a modified Macchiavello's stain. Chlamydial antigen was detected by light microscopic examination in the cytoplasm of myocardial fibers and in occasional hepatocytes using a commercially available genus-specific antichlamydial monoclonal antibody and the avidin biotin peroxidase complex staining method. Electron microscopic examination of the heart of the most severely affected turtle revealed developmental stages of chlamydial organisms. A suspension of heart from this turtle was inoculated into the yolk sacs of chicken embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of the sensitivity and specificity of two diagnostic tests for antibodies to pseudorabies virus glycoprotein X.

The diagnostic performance of 2 enzyme-linked immunosorbent assays (gX-T, gX-H) for antibodies to pseudorabies virus (PRV) glycoprotein X (gX) were evaluated using 311 serum samples from a nonvaccinated quarantined herd. When the standardized virus neutralization (VN) test, which uses the Shope strain (VN Shope), was used as the comparative diagnostic standard, the gX-T test had a 7% false-negative rate and a 52% false-positive rate, and the gX-H test had a 19% false-negative rate and a 19% false-positive rate. When the VN test with a Bartha recombinant strain (VN Bartha gIIIKa) was used as the diagnostic standard, the gX-T test had a 9% false-negative rate and a 26% false-positive rate, and the gX-H test had a 24% false-negative rate and a 11% false-positive rate. Thus, the gX-T test was more sensitive and the gX-H test was more specific. Additional diagnostic tests on 79 serum samples from a noninfected herd did not produce false positives for the gX-H test, but there was an 8% false-positive rate for the gX-T test. Previous studies from our laboratory have demonstrated that VN Bartha gIIIKa has higher sensitivity than VN Shope, without losing specificity, and thus is a better comparative diagnostic standard. When adding a suspect range to the gX-T test, using the same criteria as the suspect range for the gX-H test, the false-positive rate of the gX-T test was reduced to 5% when evaluated versus VN Bartha gIIIKa in the infected herd and to 1% for the PRV-negative herd.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential polymerase chain reaction for detection of wild-type and a vaccine strain of Aujeszky's disease (pseudorabies) virus.

A polymerase chain reaction (PCR) strategy for differentiating between a vaccine mutant strain and wild-type (WT) strains of Aujesky's disease (pseudorabies) virus (ADV) was evaluated. With this approach, a single virus or a concurrent WT and vaccine virus infection could be distinguished by targeting the genomic alteration within the vaccine strain. PCR primers were designed for a recombinant vaccine virus that has almost all of the WT gX gene replaced by the lacZ gene. One primer, corresponding to a conserved sequence upstream of the altered region, was selected for common use. The differentiating primers were chosen from the unique WT gX and vaccine lacZ gene sequences. The sensitivity of the differential PCR was analyzed using extracted viral DNA and in vitro infected cell lysates. Approximately 10 and between 10 to 100 molecules of WT and vaccine viral DNAs, respectively, could be detected, regardless of the presence or absence of uninfected cell lysates. Detection of viral DNA from in vitro infected cell cultures approximated this level of sensitivity. The specificity of the amplifications was verified by restriction endonuclease analysis and Southern hybridization. Although the vaccine primer pair target was amplified to a lesser degree as compared to the WT primer pair product, utility of the differential PCR was demonstrated using trigeminal nerve ganglia from swine infected with vaccine virus and WT virus. Both viral targets were detected only by their specific primer pair, in either the single or dual infection.

Apparent retrovirus-induced immunosuppression in a yearling llama.

Immunosuppression suspected to be associated with retrovirus infection was diagnosed in an 18-month-old female llama. The llama had a 6-month history of weight loss, intermittent lameness, and infections that were nonresponsive to treatment. Serial CBC indicated persistent nonregenerative anemia and leukopenia characterized by absolute neutropenia and lymphopenia. Functional hypoplasia of myeloid and erythroid cell lines was detected in serial bone marrow biopsy specimens. Notable pathologic findings included inadequate hematopoiesis, generalized lymphoid hypoplasia and plasma cell depletion, and pulmonary alveolar histiocytosis. Pneumocystis carinii cysts and viral particles of the size and morphologic features consistent with the retrovirus family were observed in lung sections examined by transmission electron microscopy. Antemortem macrophage and postmortem lymph node cultures were positive for reverse transcriptase activity.

Sensitivity of the standardized pseudorabies virus neutralization test varies with the test strain used.

The effect of altering the strain of the test virus used in the standardized pseudorabies virus neutralization (VN) test on the sensitivity of the assay was evaluated. Comparative VN tests were performed using 4 different strains: the avirulent Bartha parental, the avirulent recombinant Bartha gIIIKa, the moderately virulent Shope (currently used for the VN test at the National Veterinary Services Laboratory, Ames, IA), and the highly virulent P2208 (Funkhauser). A radioimmunoassay and a Western immunoblotting technique were employed to verify the presence of anti-pseudorabies virus (PrV) antibodies in sera. Statistical analysis indicated that replacement of the Shope strain by the Bartha gIIIKa or the P2208 strain resulted in VN titers that were 4.23- and 2.00-fold higher, respectively. Despite these differences, specificity with regard to PrV diagnosis was unaltered. This apparent enhancement of the sensitivity of the PrV VN test would be beneficial for the serologic identification of PrV-infected animals during an eradication effort.

Quantitative assessment of the germicidal efficacy of ultrasonic energy.

Propagated (free-field) ultrasonic energy at a frequency of 26 kHz was used to expose aqueous suspensions of bacteria (Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa), fungus (Trichophyton mentagrophytes), and viruses (feline herpesvirus type 1 and feline calicivirus) to evaluate the germicidal efficacy of ultrasound. There was a significant effect of time for all four bacteria, with percent killed increasing with increased duration of exposure, and a significant effect of intensity for all bacteria except E. coli, with percent killed increasing with increased intensity level. There was a significant reduction in fungal growth compared with that in the controls, with decreased growth with increased ultrasound intensity. There was a significant reduction for feline herpesvirus with intensity, but there was no apparent effect of ultrasound on feline calicivirus. These results suggest that ultrasound in the low-kilohertz frequency range is capable to some degree of inactivating certain disease agents that may reside in water. The physical mechanism of inactivation appears to be transient cavitation.

Persistent cutaneous ulcers associated with feline herpesvirus type 1 infection in a cheetah.

Persistent cutaneous ulcers developed in a female cheetah cub after an episode of rhinotracheitis. When they were 3 weeks old, the cub and a male littermate developed mucopurulent oculonasal discharge consistent with feline herpesvirus type 1 infection (feline viral rhinotracheitis). The male cub was weaned and its lesions resolved. The female cub remained with the dam until the cub was 3 months old, at which time plaque-like lesions developed on the eye margins and muzzle. These plaques regressed over the next month and were replaced with cutaneous ulcers ranging from 1 to 10 mm in diameter. Feline herpesvirus type 1 was isolated from biopsy specimens collected from the ulcers. Cutaneous ulcers are uncommon manifestations of feline herpesvirus infections and have not been reported in other exotic fields. A proposed susceptibility to viral infections related to low genetic diversity has been proposed in cheetahs, and may be involved in the pathogenesis of persistent herpetic ulcers.