Serum periostin levels in early in pregnancy are significantly altered in women with miscarriage.

BACKGROUND: Miscarriage is a common complication in pregnancy and there is still a lack of biomarkers usable in asymptomatic patients before the event occurs. Periostin (PER), whose levels rise particularly during injury or inflammation, has been shown to play an important local role in implantation and early embryonic development. As PER has been described as a biomarker in various medical conditions we intended to evaluate if changes in PER serum levels may help to identify women at risk for spontaneous abortion in the first trimester. METHODS: Women between 18 and 42 years without confounding comorbidities who conceived by IVF/ICSI and ovarian hyperstimulation were analysed in the study after informed consent. Maternal serum samples from 41 patients were assessed at the time of pregnancy testing (PT) and the following first ultrasound checkup (US). Patients were subsequently divided in two groups: (1) patients with subsequent miscarriage in the first trimester (n = 18) and (2) patients with ongoing pregnancy (n = 23), allowing for statistical analysis and investigating the change of PER levels per individual. PER levels were measured using enzyme-linked immunosorbent assay. Statistical analysis was performed using the Fisher exact and Student's t test. p ≤ 0.05 was considered to be significant. RESULTS: There was no significant difference concerning possible confounders between the two groups. We did not find any significant difference in PER levels at the time point of PT or US. By investigating the interindividual changes of PER between the two time points however, we observed that patients with a following miscarriage showed increasing levels of PER at the time point of PT compared to US in contrast to patients with an ongoing pregnancy who demonstrated a decrease in PER levels. These alterations were significant in the absolute as well as in the relative comparison. CONCLUSION: The relative expression of PER between PT and US is significantly altered in asymptomatic women with subsequent miscarriage compared to women with ongoing pregnancy. Therefore systemic PER levels might represent a potential promising biomarker for the assessment of pregnancy outcome. TRIAL REGISTRATION: Not applicable.

Effect of dietary methionine content on growth performance, carcass traits, and feather growth of Pekin duck from 15 to 35 days of age.

A study was conducted to establish the response of Pekin ducks to dietary Met from 15 to 35 d age. Experimental diets were formulated to contain 0.35, 0.45, 0.55, 0.65, and 0.75% Met (0.30, 0.39, 0.45, 0.56, and 0.68% on an analyzed basis, respectively) and 0.3% cysteine (0.25, 0.27, 0.26, 0.26, and 0.28% on an analyzed basis, respectively). Each diet was fed to 10 pens of 55 ducks/pen. Carcass yields and feather growth were determined at 28 and 35 d. Results showed that feeding 0.30% Met (0.55% Met+Cys) significantly impaired ADG, feed-to-gain (F:G) ratio, breast meat yield, and feather growth in comparison to the other dietary treatments (P < 0.05). BW, ADG, F: G, carcass and breast meat weight and yield, breast skin and subcutaneous fat weight and yield, the fourth primary wing feather length, and feather coverage showed significant quadratic broken-line or quadratic polynomial response to increasing dietary Met (P < 0.05). From 15 to 28 d age, the optimal Met requirement for the BW, breast meat yield, and the fourth primary wing feather length were 0.510, 0.445, and 0.404%, respectively, based on quadratic broken-line model, and correspondingly were 0.606, 0.576, and 0.559% by quadratic regression. For ducks from 15 to 35 d age, the optimal Met requirement for BW, breast meat yield, and feather coverage were 0.468, 0.408, and 0.484%, respectively, by quadratic broken-line model, and 0.605, 0.564, and 0.612%, by quadratic regression, respectively.

Effect of dietary energy and protein content on growth and carcass traits of Pekin ducks.

A study was conducted to determine the influence of dietary energy and protein concentrations on growth performance and carcass traits of Pekin ducks from 15 to 35 d of age. In experiment 1, 14-d-old ducks were randomly assigned to 3 dietary metabolizable energy (11.8, 12.8, and 13.8 MJ/kg) and 3 crude protein concentrations (15, 17, and 19%) in a 3×3 factorial arrangement (6 replicate pens; 66 ducks/pen). Carcass characteristics were evaluated on d 28, 32, and 35. In Experiment 2, 15-d-old ducks (6 replicate cages; 6 ducks/cage) were randomly allotted to the 9 diets that were remixed with 0.5% chromic oxide. Excreta were collected from d 17 to 19, and ileal digesta was collected on d 19 to determine AMEn and amino acid digestibility. In Experiment 1, there were interactions (P<0.05) between dietary metabolizable energy and crude protein (CP) on body weight (BW) gain and feed intake, wherein BW gain increased more to increasing dietary CP as dietary metabolizable energy increased. However, feed intake was only influenced by dietary crude protein at 11.8 MJ ME/kg and not 12.8 or 13.8 MJ/kg. As dietary CP increased from 15 to 19%, breast meat yield increased by 10.8% on d 35 (P<0.01). Conversely, increasing metabolizable energy from 11.8 to 13.8 MJ/kg increased dressing percentage, breast skin, and subcutaneous fat, but decreased breast meat yield (% but not weight) on d 35 (P<0.01). In Experiment 2, the determined AMEn for diets formulated to contain 11.8, 12.8, or 13.8 MJ ME/kg were 11.66, 12.68, and 13.75 MJ/kg, respectively; determined standardized ileal digestible Lys was 0.95, 1.00, and 1.21% for diets formulated to contain 15, 17, or 19% crude protein, respectively. The best body weight gain and feed conversion ratio was obtained when ducks were fed a high dietary AMEn (13.75 MJ/kg) and high CP (19%, 1.21% SID Lys). These results provide a framework for subsequent modeling of amino acid and energy inputs and the corresponding outputs of growth performance and carcass components.

Dietary threonine requirement of Pekin ducks from 15 to 35 days of age based on performance, yield, serum natural antibodies, and intestinal mucin secretion.

A study was conducted to establish the dietary Thr requirement of Pekin ducks from 15 to 35 d of age. Experimental diets were formulated to contain 0.55, 0.60, 0.65, 0.75, and 0.85% Thr (0.57, 0.60, 0.64, 0.72, and 0.80% on an analyzed basis) and were studied in 2 experiments. In experiment 1, each diet was fed to 10 pens of 52 drakes per pen. Samples were collected at d 35 for determinations of carcass yields, serum immune parameters, and intestinal characteristics. Experiment 2 was a digestibility study, wherein 0.5% chromic oxide was mixed into the experimental diets and fed from 15 to 19 d. Ileal digesta were collected at d 19 to analyze mucin secretions and apparent ileal Thr digestibility. The results showed that feeding 0.72% versus 0.64% Thr improved 15 to 35 d BW gain by 55 g (P < 0.05), reduced feed-to-gain by 0.04 (P < 0.05), as well as increased carcass and breast meat yields by 22 and 24 g, respectively. Also, 0.72% Thr had the highest crude mucin secretion on a DM intake (DMI) basis (P < 0.05), although Thr had no effect on villus height, crypt depth, goblet cells, and MUC2 gene expression in the jejunum and ileum. In addition, serum natural IgY linearly increased (P < 0.0001) with dietary Thr increase. Using nonlinear regressions, Thr requirement was estimated to range from a low of 0.70% to maximize dry crude mucin secretion on a DMI basis to a high of 0.80% to maximize carcass weight and serum IgY production by the linear or quadratic regression. Equivalently, Thr requirement varied between a low of 0.62% to minimize mortality and a high of 0.73% to maximize dry crude mucin secretion expressed as DMI using the quadratic broken-line model. Correspondingly, the apparent ileal digestible Thr requirements were estimated to be 0.52 to 0.66% (0.70 to 0.80% dietary Thr) by quadratic and 0.47 to 0.56% (0.62 to 0.73% dietary Thr) by quadratic broken-line model.

Angiogenesis and expression of vascular endothelial growth factor, tumour necrosis factor-α and hypoxia inducible factor-1α in canine renal cell carcinoma.

The aim of the present study was to determine the distribution and characteristics of microvessels in various histological types of canine renal cell carcinoma (RCC). The study compared microvessel density (MVD) and distribution of blood vessels according to histological type and evaluated the presence of angiogenesis-related proteins. Nine archival samples of canine RCC were studied. MVD was calculated as the mean number of blood vessels per mm(2). The diameter of blood vessels was calculated by determining either the length of the long axis of blood vessels (diameter(max)) or the mean distance from the centre of each blood vessel to the tunica adventia (diameter(mean)). A significant difference in MVD was evident between RCCs and normal kidneys (46.6 ± 28.0 versus 8.4 ± 2.2 microvessels/mm(2)). Diameter(max) in canine RCCs (34.1 ± 14.7 µm) was also significantly different from normal canine kidney (23.2 ± 3.4 µm). Vascular endothelial growth factor (VEGF) was expressed by tumour cells and vascular endothelial cells and tumour necrosis factor (TNF)-α expression was observed in vascular endothelial cells in both neoplastic and normal kidney. Although VEGF is involved in angiogenesis and correlates with tumour stage of development, no correlation was found between VEGF expression and MVD. Tumour-associated macrophages expressing TNF-α and hypoxia inducible factor 1α were identified in peritumoural tissue and may play an important role in angiogenesis.

The bovine herpesvirus type 1 envelope protein Us9 acidic domain is crucial for anterograde axonal transport.

In this study, we examined the functional role of bovine herpesvirus type 1 (BHV-1) Us9 acidic domain residues 83-90 in the anterograde axonal transport of the virus in calves (natural host), rabbits, and in cultured neurons. A mutant virus strain lacking Us9 residues 83-90 (BHV-1 Us9 Δ83-90) and the rescued virus (BHV-1 Us9 R83-90) replicated efficiently in the nasal and ocular epithelium during primary infection and established latency in the trigeminal ganglia (TG). However, upon reactivation from latency, only the BHV-1 Us9 R83-90 virus was detected in nasal and ocular swabs of animals. In compartmentalized, rabbit primary dorsal root ganglia (DRG) neuron cultures, the Us9-deleted BHV-1, BHV-1 Us9 Δ83-90 and BHV-1 Us9 R83-90 viruses were transported efficiently in the retrograde direction. However, only the BHV-1 Us9 R83-90 virus was transported in an anterograde direction. These studies suggested that the Us9 acidic domain residues located between 83 and 90 were required for axonal anterograde transport.

CDX-2 and HER-3 expression in canine gastric and colorectal adenocarcinomas.

CDX-2 is used as a specific cell marker for human intestinal adenocarcinoma. In human studies, HER-3 overexpression predicts poor survival for patients with various cancers including gastric cancer. Gastrointestinal adenocarcinoma is less common in dogs than in man and the expression of immunological markers by the canine tumours has not yet been extensively studied. CDX-2 and HER-3 expression was determined in 18 canine gastrointestinal adenocarcinomas: 13 were of colorectal origin and five were of gastric origin. CDX-2 expression was predominantly observed in the nuclei of normal colonic epithelium and in neoplastic epithelium and neoplastic gastric epithelial cells that which had metastasized to the gastric lymph node. CDX-2 was expressed in 11 of 13 (84.6%) colorectal adenocarcinomas and in all five (100%) gastric adenocarcinomas. HER-3 was consistently expressed in the cytoplasm of neoplastic epithelial cells. HER-3 expression was detected in 12 of 13 (92.3%) colorectal and in all five (100%) gastric adenocarcinomas. CDX-2 and HER-3 may be useful markers for canine gastrointestinal adenocarcinoma.

Immunohistochemical application of an antibody specific for human CD1a to the diagnosis of canine mast cell tumour.

Canine mast cell tumours (MCTs) may be graded microscopically for prognostic purposes. Grade I (well-differentiated) and grade II (intermediate differentiation) tumours have an abundance of metachromatic granules within the cytoplasm; however, grade III (poorly differentiated) MCTs may be difficult to diagnose as they frequently have fewer discernable granules. Herein we report that a cross-reactive anti-human CD1a monoclonal antibody (clone O10) may be used in immunohistochemistry to identify canine MCTs of all grades. The antibody was applied to tissue sections from 48 canine MCTs of different histological grades. Serial sections from each tumour were stained with toluidine blue and safranin O to compare diagnostic sensitivity. All MCTs were labelled positively by the CD1a antibody, but histochemical staining was often equivocal and identification of mast cells was extremely difficult in some cases. This antibody did not label neoplastic cells in cases of canine histiocytoma, plasmacytoma or amelanotic melanoma; therefore, the reagent may be a valuable marker for the diagnosis of canine MCTs, especially those tumours of histological grade III.

A bovine herpesvirus type 1 mutant virus specifying a carboxyl-terminal truncation of glycoprotein E is defective in anterograde neuronal transport in rabbits and calves.

Bovine herpesvirus type 1 (BHV-1) is an important component of the bovine respiratory disease complex (BRDC) in cattle. The ability of BHV-1 to transport anterogradely from neuronal cell bodies in trigeminal ganglia (TG) to nerve ending in the noses and corneas of infected cattle following reactivation from latency plays a significant role in the pathogenesis of BRDC and maintenance of BHV-1 in the cattle population. We have constructed a BHV-1 bacterial artificial chromosome (BAC) clone by inserting an excisable BAC plasmid sequence in the long intergenic region between the glycoprotein B (gB) and UL26 genes. A BAC-excised, reconstituted BHV-1 containing only the 34-bp loxP sequence within the gB-UL26 intergenic region was highly infectious in calves, retained wild-type virulence properties, and reactivated from latency following treatment with dexamethasone. Using a two-step Red-mediated mutagenesis system in Escherichia coli, we constructed a gE cytoplasmic tail-truncated BHV-1 and a gE-rescued BHV-1. Following primary infection, the gE cytoplasmic tail-truncated virus was efficiently transported retrogradely from the nerve endings in the nose and eye to cell bodies in the TG of calves and rabbits. However, following dexamethasone-induced reactivation from latency, the gE mutant virus was not isolated from nasal and ocular sheddings. Reverse transcriptase PCR assays detected VP5 transcription in the TG of rabbits infected with gE-rescued and gE cytoplasmic tail-truncated viruses during primary infection and after dexamethasone treatment but not during latency. Therefore, the BHV-1gE cytoplasmic tail-truncated virus reactivated in the TG; however, it had defective anterograde transport from TG to nose and eye in calves and rabbits.

Pulmonary lymphomatoid granulomatosis in a dog: evidence of immunophenotypic diversity and relationship to human pulmonary lymphomatoid granulomatosis and pulmonary Hodgkin's disease.

We describe a 10-month-old, intact female American Cocker Spaniel with pulmonary lymphomatoid granulomatosis (PLG). On clinical examination, this dog presented with nonproductive dry cough, serous nasal discharge, dyspnea, and lack of appetite. Radiography showed a consolidated lesion in the left cranial lung lobe. Histopathologic examination showed a mixed population of atypical lymphoid cells that had infiltrated into the pulmonary blood vessels angiocentrically. The lymphocytes were CD3 positive, consistent with a pan-T-cell phenotype. The lymphoid cells in the lesion were also positive for CD20cy and CD79a, indicative of the presence of B cells. We also observed large Reed-Sternberg-like cells that were positive for CD15 and CD30, similar to observations in human pulmonary Hodgkin's disease (PHD). In conclusion, canine PLG in this Cocker Spaniel was associated with B and T cells, which is first identified in a case of canine PLG. It was histopathologically similar to human lymphomatoid granulomatosis and immunophenotypically similar to human PHD.

Envelope protein Us9 is required for the anterograde transport of bovine herpesvirus type 1 from trigeminal ganglia to nose and eye upon reactivation.

In this study, the authors examined the role of bovine herpesvirus type 1 (BHV-1) Us9 in the anterograde transport of the virus from trigeminal ganglia (TG) to nose and eye upon reactivation from latency. During primary infection, both BHV-1 Us9-deleted and BHV-1 Us9-rescued viruses replicated efficiently in the nasal and ocular epithelium. However, upon reactivation from latency, only the BHV-1 Us9-rescued virus could be isolated in the nasal and ocular shedding. By real-time polymerase chain reaction, comparable DNA copy numbers were detected in the TGs during latency and reactivation for both the viruses. Therefore, Us9 is essential for reactivation of the virus in the TG and anterograde axonal transport from TG to nose and eye.

Protection against porcine reproductive and respiratory syndrome virus (PRRSV) infection through passive transfer of PRRSV-neutralizing antibodies is dose dependent.

Previous work in our laboratory demonstrated that passive transfer of porcine reproductive and respiratory syndrome virus (PRRSV)-neutralizing antibodies (NA) protected pregnant sows against reproductive failure and conferred sterilizing immunity in sows and offspring. We report here on the dose requirement for protection by passive transfer with NA in young weaned pigs. The presence of a 1:8 titer of PRRSV-NA in serum consistently protected pigs against viremia. Nevertheless, their lungs, tonsils, buffy coat cells, and peripheral lymph nodes contained replicating PRRSV similar to the infected control group. Likewise, these animals excreted infectious virus to sentinels similar to the infectivity control animals. In an attempt to reach complete protective immunity equivalent to that previously observed in sows, the pigs were transferred with a higher titer of PRRSV-NA (1:32), and even then apparent sterilizing immunity was attained in only 50% of the animals. In conclusion, the presence of anti-PRRSV-NA in serum with a titer of 1:8 is enough to block viremia but not peripheral tissue seeding and transmission to contact animals. While a relatively low level of NA in blood is capable of conferring sterilizing immunity against PRRSV in sows, the amount of NA necessary to obtain full protection of a young weaned pig would be significantly higher, suggesting that differences exist in the PRRSV pathogenesis between both age groups. In addition, the titer of NA could be a helpful parameter of protection in the assessment of PRRSV vaccines.

Biological responses to porcine respiratory and reproductive syndrome virus in pigs of two genetic populations.

One hundred pigs from the NE Index Line (NEI) and 100 Hampshire-Duroc cross pigs (HD) were inoculated intranasally with porcine respiratory and reproductive syndrome virus (PRRSV 97-7895 strain) at 26 d of age to determine whether genetic variation in response to PRRSV exists. An uninfected littermate to each infected pig served as a control. Pigs were from 163 dams and 83 sires. Body weight and rectal temperature were recorded, and blood samples were drawn from each pig on d 0 before inoculation and on d 4, 7, and 14 after inoculation. Pigs were sacrificed on d 14. Lung and bronchial lymph nodes were collected, placed in optimal cutting temperature compound, and frozen at -80 degrees C. The presence of PRRSV in serum and in lung tissue and bronchial lymph nodes was determined by isolation in cell culture. The presence of antibodies in serum collected on d 14 was determined by a commercial ELISA test. Lung tissue was examined microscopically and scored for incidence and severity of lesions (score of 1 to 3; 1 = no or few lesions, and 3 = severe interstitial pneumonia). Data were analyzed with a mixed model that included random sire and dam effects. The interaction of line x treatment was significant (P < 0.001) for weight change and rectal temperature. Un-infected HD pigs gained 0.67 kg more from d 0 to 14 and averaged 0.32 degrees C higher rectal temperature than uninfected NEI pigs (P < 0.001), whereas infected NEI pigs gained 0.34 kg more and had -0.54 degrees C lower temperature than infected HD pigs (P < 0.001). Viremic titer (cell culture infectious dose 50%/mL) was greater (P < 0.05) in HD than NEI at d 4 (10(4.52) vs. 10(4.22)), 7 (10(4.47) vs. 10(3.99)), and 14 (10(3.49) vs. 10(3.23)). Viral titer loads in lung (P = 0.11) and bronchial lymph nodes tended (P = 0.07) to be greater in HD than NEI pigs. Antibody signal-to-positive (S/P) ELISA ratios in infected pigs ranged from 0.18 to 3.38, and 88% had levels > or = 0.40, which is the positive threshold for this ELISA. The S/P range in uninfected pigs was 0 to 1.11, and 99% had levels < or = 0.40. Mean S/P ratio for infected pigs was 0.23 units higher in HD than in NEI (P < 0.001). The HD pigs had a greater incidence of interstitial pneumonia and 0.65 higher mean lesion scores than NEI pigs (P < 0.001). In summary, responses of pigs of the two lines to infection with PRRSV differed, indicating that underlying genetic variation existed.

Vesicular stomatitis virus infection and neuropathogenesis in the murine model are associated with apoptosis.

This study examines apoptosis and viral neuropathogenesis in a murine model infected with vesicular stomatitis virus (VSV). VSV induces apoptotic cell death in cultured cell lines, raising the possibility that apoptosis of infected neurons and other target cells may contribute to disease and mortality. To determine whether or not VSV induces apoptosis in neural tissues, mice were inoculated intranasally with VSV. At 24, 48, 72, 96, and 120 hours postinfection, brain tissues were assayed for the presence of viral RNA by in situ hybridization and viral antigen by immunohistochemistry. Apoptosis was identified by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling and electron microscopy. Viral replication and lesions were observed predominantly in central nervous system neurons. Apoptotic cell death was restricted to the same regions of the brain in which infected cells and tissue injury were identified. Results suggest that VSV-induced apoptosis is a mechanism causing cell death, tissue injury, and mortality in VSV-infected mice.

Passive transfer of virus-specific antibodies confers protection against reproductive failure induced by a virulent strain of porcine reproductive and respiratory syndrome virus and establishes sterilizing immunity.

Immune mechanisms mediating protective immunity against porcine reproductive and respiratory syndrome virus (PRRSV) are not well understood. The PRRSV-specific humoral immune response has been dismissed as being ineffective and perhaps deleterious for the host. The function of PRRSV antibodies in protective immunity against infection with a highly abortifacient strain of this virus was examined by passive transfer experiments in pregnant swine. All of a group of pregnant gilts (n = 6) that received PRRSV immunoglobulin (Ig) from PRRSV-convalescent, hyperimmune animals were fully protected from reproductive failure as judged by 95% viability of offspring at weaning (15 days of age). On the other hand, the totality of animals in a matched control group (n = 6) receiving anti-pseudorabies virus (PRV) Ig exhibited marked reproductive failure with 4% survival at weaning. Besides protecting the pregnant females from clinical reproductive disease, the passive transfer of PRRSV Ig prevented the challenge virus from infecting the dams and precluded its vertical transmission, as evidenced by the complete absence of infectious PRRSV from the tissues of the dams and lack of infection in their offspring. In summary, these results indicate that PRRSV-Igs are capable of conferring protective immunity against PRRSV and furthermore that these Igs can provide sterilizing immunity in vivo.

Analysis of bovine trigeminal ganglia following infection with bovine herpesvirus 1.

Following primary infection of the eye, oral cavity, and/or nasal cavity, bovine herpesvirus 1 (BHV-1) establishes latency in trigeminal ganglionic (TG) neurons. Virus reactivation and spread to other susceptible animals occur after natural or corticosteroid-induced stress. Infection of calves with BHV-1 leads to infiltration of lymphocytes in TG and expression of IFN-gamma (interferon-gamma), even in latently infected calves. During latency, virus antigen and nucleic acid positive non-neural cells were occasionally detected in TG suggesting there is a low level of spontaneous reactivation. Since we could not detect virus in ocular or nasal swabs, these rare cells do not support high levels of productive infection and virus release or they do not support virus production at all. Dexamethasone (DEX) was used to initiate reactivation in latently infected calves. Foci of mononuclear or satellite cells undergoing apoptosis were detected 6h after DEX treatment, as judged by the appearance of TUNEL+ cells (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling). BHV-1 antigen expression was initially detected in lymphocytes and other non-neural cells in latently infected calves following DEX treatment. At 24h after DEX treatment, viral antigen expression and nucleic acid were readily detected in neurons. Our data suggest that persistent lymphocyte infiltration and cytokine expression occur during latency because a low number of cells in TG express BHV-1 proteins. Induction of apoptosis and changes in cytokine expression following DEX treatment correlates with reactivation from latency. We hypothesize that inflammatory infiltration of lymphoid cells in TG plays a role in regulating latency.

A mutation in the latency-related gene of bovine herpesvirus 1 leads to impaired ocular shedding in acutely infected calves.

Bovine herpesvirus 1 (BHV-1) is an important pathogen of cattle, and infection is usually initiated in the ocular or nasal cavity. Like other alphaherpesviruses, BHV-1 establishes latency in sensory neurons but has the potential of reactivating from latency and spreading. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA, which is alternatively spliced in trigeminal ganglia during acute infection (L. R. Devireddy and C. Jones, J. Virol. 72:7294-7301, 1998). LR gene products inhibit cell cycle progression (Y. Jiang, A. Hossain, M. T. Winkler, T. Holt, A. Doster, and C. Jones, J. Virol. 72:8133-8142, 1998) and chemically induced apoptosis (J. Ciacci-Zannela, M. Stone, G. Henderson, and C. Jones. J. Virol. 73:9734-9740, 1999). Although these studies suggest that LR gene products play an important role in the latency/pathogenesis of BHV-1, construction of a mutant is necessary to test this hypothesis. Because the bICP0 gene overlaps and is antisense to the LR gene, it was necessary to mutate the LR gene without altering bICP0 expression. This was accomplished by inserting three stop codons near the beginning of the LR RNA, thus interfering with expression of proteins expressed by the LR RNA. The LR mutant virus grew with wild-type (WT) efficiency in bovine kidney (MDBK) cells and expressed bICP0 at least as efficiently as WT BHV-1 or the LR rescued virus. When calves were infected with the LR mutant, we observed a dramatic decrease (3 to 4 log units) in ocular shedding during acute infection relative to WT or the LR rescued virus. In contrast, shedding of the LR mutant from the nasal cavity was not significantly different from that of the WT or the LR rescued virus. Calves infected with the LR mutant exhibited mild clinical symptoms, but they seroconverted. Neutralizing antibody titers were lower in calves infected with the LR mutant, confirming reduced growth. In summary, this study suggests that an LR protein promotes ocular shedding during acute infection of calves.

Evidence for the localization of porcine reproductive and respiratory syndrome virus (PRRSV) antigen and RNA in ovarian follicles in gilts.

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) infection in ovary was studied in sexually mature, cycling, nonsynchronized gilts infected with the PRRSV 16244B, a virulent field strain. Previous studies have shown that PRRSV can be isolated from ovaries and is transplacentally passed from gilts to the fetuses. The cause of infertility following PRRSV infection is not known. In this study, we identified the tropism of PRRSV in ovarian tissue from experimentally infected gilts in samples collected between 7 and 21 days postinfection (DPI). Tissues were collected and examined by virus isolation, in situ hybridization (ISH), immunohistochemistry (IHC), and double labeling to identify PRRSV-infected cell types. PRRSV was isolated in ovarian follicles at 7 days DPI. The IHC and ISH indicated that PRRSV-positive cells in ovaries were predominantly macrophages, which were numerous in atretic follicles. No evidence of infection and/or perpetuation of PRRSV in ova was observed, indicating that the female gonad is an unlikely site of persistence. No alteration of the normal ovarian architecture that would support a possible role of PRRSV infection in porcine female infertility was observed.

Analysis of latency in cattle after inoculation with a temperature sensitive mutant of bovine herpesvirus 1 (RLB106).

Calves were inoculated with the bovine herpes virus 1 (BHV-1) vaccine strain (RLB 106), which is a temperature sensitive mutant. The route of inoculation was intranasal instillation or intramuscular (i.m.) injection (flank or neck). As a control, five calves were given placebo by i.m. injection of the neck. Regardless of the infection route, clinical symptoms did not occur. However, BHV-1 neutralizing antibodies were detected after inoculation demonstrating that sero-conversion occurred. At 60 days post-inoculation, dexamethasone was given by i.m. injection to attempt reactivation of RLB 106. Only those calves inoculated by the intranasal route shed virus leading to an increase in BHV-1 specific antibodies. As expected, viral DNA and the latency related-RNA were detected in trigeminal ganglia (TG) of calves inoculated by the intranasal route. In contrast, viral nucleic acid was not detected in TG of calves inoculated by the i.m. route or in calves inoculated with placebo. In cervical ganglia or sacral dorsal root ganglia, viral nucleic acid was not consistently detected. This study provides evidence that efficient latency and reactivation does not occur following i.m. inoculation. Since serum-neutralizing antibodies were detected in all inoculated calves, i.m. inoculation led to sero-conversion.