Contact transmission of vesicular stomatitis virus New Jersey in pigs

OBJECTIVE: To determine how viral shedding and development or lack of clinical disease relate to contact transmission of vesicular stomatitis virus New Jersey (VSV-NJ) in pigs and determine whether pigs infected by contact could infect other pigs by contact. ANIMALS: 63 pigs. PROCEDURE: Serologically naive pigs were housed in direct contact with pigs that were experimentally inoculated with VSV-NJ via ID inoculation of the apex of the snout, application to a scarified area of the oral mucosa, application to intact oral mucosa, or ID inoculation of the ear. In a second experiment, pigs infected with VSV-NJ by contact were moved and housed with additional naive pigs. Pigs were monitored and sampled daily for clinical disease and virus isolation and were serologically tested before and after infection or contact. RESULTS: Contact transmission developed only when vesicular lesions were evident. Transmission developed rapidly; contact pigs shed virus as early as 1 day after contact. In pens in which contact transmission was detected, 2 of 3 or 3 of 3 contact pigs were infected. CONCLUSIONS AND CLINICAL RELEVANCE: Transmission was lesion-dependent; however, vesicular lesions often were subtle with few or no clinical signs of infection. Contact transmission was efficient, with resulting infections ranging from subclinical (detected only by seroconversion) to clinical (development of vesicular lesions). Long-term maintenance of VSV-NJ via contact transmission alone appears unlikely. Pigs represent an efficient large-animal system for further study of VSV-NJ pathogenesis and transmission.     

Experimental vesicular stomatitis virus infection of swine: Extent of infection and immunological response

Swine, a natural host species for infection by vesicular stomatitis virus (VSV), were infected with VSV-New Jersey (VSV-NJ) serotype virus obtained from a recent field isolate. Tissues collected from the infected pigs were examined for the presence of infective virus, for viral antigens, and/or for viral nucleic acid. Infective virus could be recovered from tissues near the site of infection for as long as 6 days after the primary infection with VSV. However, no infective virus was recovered following hypothermia induced 11 weeks after infection, or following a secondary challenge with virus 22 weeks after initial infection. Immunofluorescence tests for viral antigens and nucleic acid hybridization assays failed to detect viral antigens or nucleic acids in tissues from which no infective virus could be recovered. Titers of serum-neutralizing antibody peaked 3–5 weeks after infection and then fell slightly until the secondary infection which caused a rapid anamnestic response. Peripheral blood mononuclear cells (PBM) tested 3, 5, 8 or 18 weeks after primary infection all produced readily detectable antigen-specific proliferative responses when cultured with VSV. Thus, although direct tests failed to demonstrate persistence of virus after infection, the humoral and cellular immune response remained elevated for months. Infective VSV was not required to stimulate the proliferative response since UV-inactivated VSV was immunogenic in these in vitro tests. Following primary infection, antigen-specific proliferative responses could be stimulated by several strains of VSV-NJ, but not by VSV-Indiana (VSV-Ind) serotype virus. Secondary infection had relatively little effect on the proliferative response to VSV-NJ strains, but it did cause the PBM to gain responsiveness to VSV-Ind.     

An enzyme-linked immunosorbent assay for the detection of vesicular stomatitis virus antibodies

A liquid-phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection of vesicular stomatitis virus (VSV) types New Jersey (VSV-NJ) and Indiana subtype Indiana I (VSV-IND₁) antibodies in the sera of naturally and experimentally infected cattle, horses and swine. Four different VSV preparations were compared for use as antigen in the ELISA: virus used in neutralization tests, complement-fixation antigen, immunodiffusion ager gel antigen and viral glycoprotein. Comparative antibody titration results of virus neutralization (VN) and ELISA showed no statistically significant difference between serum titers obtained with the four antigens to VSV-NJ (P=0.21) and VSV-IND₁ (P=0.14). The viral glycoprotein antigen was incorporated in the ELISA system because it is non-infectious and induces neutralizing antibodies. The reliability and variability of the ELISA was determined by testing 516 bovine, equine and swine sera which originated from areas free of vesicular stomatitis, and by testing 186 sera from areas where outbreaks occur. ELISA and VN results were correlated (P < 0.001 for both serotypes), and no statiscafically difference was found between replications of the tests. The ELISA allows the testing of a larger number of sera in a shorter time than is possible with the VN test and it can be used in diagnostic laboratories in VSV-free areas for the support of epidemiological surveillance programs.     

Brain and spinal cord lesions in pigs inoculated with swine vesicular disease (UKG strain) virus and coxsackievirus B5.

Pigs inoculated intravenously with swine vesicular disease virus (UKG strain), those inoculated with coxsackievirus B5, and other pigs exposed by pen contact to the same viruses developed diffuse encephalomyelitis. Perivascular cuffing, with lymphocytes and formation of neuroglia cell foci, were most prominent in telencephalon, diencephalon, and mesencephalon. Encephalitis was of mild to severe intensity. Severity of lesions was more extensive and severe in the pigs exposed to swine vesicular disease virus. Pen contact exposure to either of the 2 viruses caused a more severe central nervous system reaction than did intravenous inoculation. The type and the distribution of lesions produced by the 2 viruses indicate that they may be related.     

Pathogenesis of experimental vesicular stomatitis virus (New Jersey serotype) infection in the deer mouse (Peromyscus maniculatus)

The pathogenesis of vesicular stomatitis virus (VSV) infection has not been investigated previously in native New World rodents that may have a role in the epidemiology of the disease. In the present study, 45 juvenile and 80 adult deer mice (Peromyscus maniculatus) were inoculated intranasally with VSV New Jersey serotype (VSV-NJ) and examined sequentially over a 7-day period. Virus was detected by means of immunohistochemistry and in situ hybridization in all tissues containing histologic lesions. Viral antigen and mRNA were observed initially in olfactory epithelium neurons, followed by olfactory bulbs and more caudal olfactory pathways in the brain. Virus also was detected throughout the ventricular system in the brain and central canal of the spinal cord. These results support both viral retrograde transneuronal transport and viral spread within the ventricular system. Other tissues containing viral antigen included airway epithelium and macrophages in the lungs, cardiac myocytes, and macrophages in cervical lymph nodes. In a second experiment, 15 adult, 20 juvenile, and 16 nestling deer mice were inoculated intradermally with VSV-NJ. Adults were refractory to infection by this route; however, nestlings and juveniles developed disseminated central nervous system infections. Viral antigen also was detected in cardiac myocytes and lymph node macrophages in these animals. Viremia was detected by virus isolation in 35/72 (49%) intranasally inoculated juvenile and adult mice and in 17/36 (47%) intradermally inoculated nestlings and juveniles from day 1 to day 3 postinoculation. The documentation of viremia in these animals suggests that they may have a role in the epidemiology of vector-borne vesicular stomatitis.     

Genetic analysis of vesicular stomatitis virus-New Jersey from the 1995 outbreak in the western United States

OBJECTIVE: To compare molecular associations between the vesicular stomatitis virus (VSV)-New Jersey isolates of the 1995 outbreak with those from previous outbreaks between 1982 and 1985 in the western United States. SAMPLE POPULATION: 23 virus isolates considered representative of the 1995 outbreak of vesicular stomatitis. PROCEDURE: Viral gene coding for surface-envelope protein G was evaluated by use of nucleotide sequencing and phylogenetic analysis. RESULTS: Changes in up to 0.77% of the nucleotide bases and 1.35% of the amino acids were detected among the 1995 viral isolates, whereas changes in up to 3.2 and 2.9% of the nucleotides and amino acids, respectively, were found, compared with the 1982 to 1985 viruses. Insertions or deletions were not found in the entire gene, which spanned 1,554 nucleotide bases. CONCLUSIONS AND CLINICAL RELEVANCE: Phylogenetic analysis indicated that the 1995 VSV-New Jersey belongs to a lineage distinct from that of the 1982 to 1985 viruses that caused previous outbreaks in the western United States. Furthermore, it also is distinct from strains from Central America and from the Georgian Hazelhurst strain.     

Effect of vaccination with a modified-live porcine reproductive and respiratory syndrome virus vaccine on dynamics of homologous viral infection in pigs

OBJECTIVE: To determine effects of vaccination protocols with modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine on persistence and transmission of virus in pigs infected with a homologous isolate and determine clinical and virologic responses following heterologous viral challenge. ANIMALS: Four hundred forty 6- to 8-week-old PRRSV-na?ve pigs. PROCEDURES: Pigs were allocated into 5 groups. Groups A to D were inoculated with wild-type PRRSV VR2332. Group A (positive control pigs) received PRRSV only. Groups B, C, and D received modified-live PRRSV vaccine (1, 2, or 3 doses). Group E served as a negative control group. To evaluate viral transmission, sentinel pigs were introduced into each group at intervals from 37 to 67, 67 to 97, and 97 to 127 days postinoculation (DPI). To evaluate persistence, pigs were euthanized at 37, 67, 97, or 127 DPI. To assess clinical and virologic response after challenge, selected pigs from each group were inoculated at 98 DPI with a heterologous isolate (PRRSV MN-184). RESULTS: Mass vaccination significantly reduced the number of persistently infected pigs at 127 DPI. Vaccination did not eliminate wild-type PRRSV; administration of 2 or 3 doses of modified-live virus vaccine reduced viral shedding after 97 DPI. Previous exposure to wild-type and vaccine virus reduced clinical signs and enhanced growth following heterologous challenge but did not prevent infection. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggest that therapeutic vaccination may help to reduce economic losses of PRRSV caused by infection; further studies to define the role of modified-live virus vaccines in control-eradication programs are needed.     

Comparison of the serum neutralization test and a competitive enzyme-linked immunosorbent assay for the detection of antibodies to vesicular stomatitis virus New Jersey and vesicular stomatitis virus Indiana.

A competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of antibodies against vesicular stomatitis virus New Jersey (VSV-NJ) and vesicular stomatitis virus Indiana (VSV-IN) was compared with the serum neutralization test (SNT) using 1,106 serum samples obtained from dairy cattle on sentinel study farms in the Poás region of Costa Rica. Kappa coefficients between the C-ELISA and the SNT were 0.8871 (95% confidence interval [CI]: 0.8587-0.9155) and 0.6912 (95% CI: 0.6246-0.7577) for the VSV-NJ and VSV-IN tests, respectively. These results indicate good to excellent agreement between the 2 tests under these conditions.     

Pathogenesis of swine vesicular disease in pigs.

Pigs exposed to swine vesicular disease virus developed vesicular lesions by postinoculation day 2. Lesions first appeared on the coronary band and then on the dewclaw, tongue, snout, lips, and bulbs of the heels. The onset of viremia coincided with febrile response and the appearance of vesicles. Virus was isolated from the nasal discharge, esophageal-pharyngeal fluid, and feces as early as postinoculation day 1. Greater amounts of virus were isolated from samples collected during the first week of infection, and lesser amounts from samples collected during the second week. The appearance and the distribution of specific fluorescence in various tissues indicated that during the development of swine vesicular disease virus infection, the epithelial tissues were initially involved, followed by a generalized infection of lymph tissues, and subsequently, a primary viremia. Seroconversion was detectable as early as postinoculation day 4. A mild nonsuppurative meningoencephalomyelitis throughout the CNS was observed in both inoculated and contact-exposed pigs. The olfactory bulbs were most severely and were frequently affected, particularly in contact pigs. The most severe brain lesions were found in pigs 3 to 4 days after the onset of viremia; contact pigs showed more severe brain lesions than inoculated pigs. Microscopic changes were also found in the coronary band, snout, tongue, and heart.     

An enzyme-linked immunosorbent assay for the detection of bovine antibodies to vesicular stomatitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine antibody to vesicular stomatitis virus (VSV). Serum samples from cows experimentally infected with the New Jersey serotype of VSV (VSV-NJ) were assayed by the ELISA and serum-neutralization (SN) assay. The ELISA was as sensitive as the SN assay in detecting bovine antibody to VSV. The correlation between SN titers and ELISA values at absorbance at 405 nm was statistically significant. The ELISA was not specific for VSV-NJ, however, and could detect serum samples positive to the Indiana serotype of VSV that had SN titers of greater than or equal to 480. Nonspecific reactions were due to cross-reactive group-specific viral proteins that are shared by both serotypes. The cross-reactivity allows the use of a single rapid test in identifying both serotypes of VSV from the other exotic vesicular diseases, especially foot-and-mouth disease. The ELISA titers of serum samples positive for VSV-NJ were comparable with the corresponding SN titers of each sample. The sensitivity, rapidity, and ease of the ELISA system and the use of a single test in identifying both serotypes of VSV from the other exotic vesicular diseases make this ELISA suitable as a rapid diagnostic assay for VS.     

The pathogenesis of vesicular exanthema of swine virus and San Miguel sea lion virus in swine

Vesicular exanthema of swine virus type A48 or San Miguel sea lion virus type 2, when inoculated intradermally into swine, resulted in fluid-filled vesicles at the sites of inoculation in the snout, coronary band, and tongue. Pigs that developed vesicles also had fevers. Secondary vesicle formation varied, depending on virus serotype. Viremia was found in one pig infected with San Miguel sea lion virus five days after infection. Virus was recovered from nasal-oral passages for up to five days after infection in both groups of pigs and from the gastrointestinal and urinary tracts of pigs infected with San Miguel sea lion virus. Neutralizing antibodies began to increase three days after inoculation and reached peak titers in seven to ten days. In the absence of secondary bacterial infection, healing was well advanced by ten days after inoculation. Lesions usually were limited to nonhaired portions of the integument and tongue. Individual epithelial cells became infected when a break in the skin allowed virus access to susceptible epithelial cells from either exogenous or endogenous sources. Individual infected cells ruptured and adjacent cells were infected, resulting in the formation of multiple microvesicles. Centrifugal coalescence of microvesicles led to formation of grossly visible macrovesicles. Lesions rarely developed from viral contamination of intact hair follicles. A mild virus-induced encephalitis was seen in pigs infected with vesicular exanthema of swine virus, and virus was recovered from brain tissue of pigs infected with San Miguel sea lion virus.     

Evaluation of transmission of swine influenza type A subtype H1N2 virus in seropositive pigs

OBJECTIVE: To examine clinical signs, virus infection and shedding, and transmission of swine influenza virus (SIV) subtype H1N2 among seropositive pigs. ANIMALS: Eighteen 3-week-old pigs with maternal antibodies against SIV subtypes H1N1, H3N2, and H1N2. PROCEDURE: Ten pigs (principal) were inoculated intranasally with subtype H1N2 and 2 groups of contact pigs (n = 4) each were mixed with principal pigs on day 7 (group 1) or 28 (group 2). Two principal pigs each were necropsied on days 4, 14, 21, 28, and 42 days after inoculation. Four pigs in each contact group were necropsied 35 and 14 days after contact. Virus excretion was evaluated after inoculation or contact. Lung lesions and the presence of SIV in various tissues were examined. RESULTS: Mild coughing and increased rectal temperature were observed in principal pigs but not in contact pigs. Nasal virus shedding was detected in all principal pigs from day 2 for 3 to 5 days, in group 1 pigs from day 2 for 4 to 9 days after contact, and in group 2 pigs from day 4 for 2 to 6 days after contact. Trachea, lung, and lymph node specimens from infected pigs contained virus. Antibody titers against all 3 subtypes in all pigs gradually decreased. CONCLUSIONS AND CLINICAL RELEVANCE: Protection from viral infection and shedding was not observed in pigs with maternal antibodies, but clinical disease did not develop. Vaccination programs and good management practices should be considered for control of SIV subtype H1N2 infection on swine farms.     

Antigen-specific proliferative responses to vesicular stomatitis virus following immunization of cattle with inactive virus

Peripheral blood mononuclear cells (PBM) from four normal cows with no known exposure to vesicular stomatitis virus (VSV) were cultured with a New Jersey (NJ) serotype (Ogden) VSV that had been UV-irradiated and inactivated. PBM from these animals produced no detectable proliferative response when incubated with varying concentrations of VSV-NJ (Ogden) ranging from 10 ng to 10 μg protein/ml. Two of these cows were immunized with an experimental VSV-NJ vaccine and their PBM were tested at various intervals after immunization. PBM tested 14 days after the initial immunization produced readily detectable antigen-specific proliferative responses when cultured with UV-irradiated strains of VSV-NJ. Following a second immunization, lower concentrations of antigen were sufficient to stimulate the proliferative response and the magnitude of the proliferative response was increased. The responsiveness persisted for at least 6 months after these two immunizations. The specificity of the proliferative response was examined by comparing the responses stimulated by one VSV-Ind and four VSV-NJ serotype strains. The PBM from the immunized cows produced proliferative responses that were essentially specific for the VSV-NJ serotype antigens. In dose titrations, the VSV-NJ antigens were 300–1000-fold more effective than was the VSV-Ind antigen. Thus, persistent antigen-specific proliferative responsiveness that is serotype specific can be stimulated by immunizing cattle with an inactivated VSV vaccine.     

Development of an immunoelectroosmophoresis test for the detection and typing of antibodies to vesicular stomatitis viruses.

This study was conducted to develop a rapid test to detect type specific antibodies to various strains of vesicular stomatitis virus. Glycoprotein was extracted from purified vesicular stomatitis virus-Indiana 3 and used as antigen in an immunoelectroosmophoresis test (counter immunoelectrophoresis) to test reactivity with homologous hyperimmune serum and antisera to vesicular stomatitis virus-New Jersey, Indiana 1 and Indiana 2. A reaction was only detected with the homologous antiserum. A simpler preparation of antigen, a detergent extract of infected cells was also evaluated and shown to have less specificity in reactions with hyperimmune sera. This infected cell extract was, however, useful in detecting homologous antibodies in convalescent sera (natural vesicular stomatitis virus-New Jersey infection) and no reactions were detected with infected cell extracts of vesicular stomatitis virus-Indiana 1, 2 and 3.     

Time-dependent infection probability of classical swine fever via excretions and secretions

Several routes contribute to the spread of classical swine fever (CSF) during outbreaks of this disease. However, for many infected herds in recent epidemics, no route of virus introduction could be indentified. To obtain more insight into the relative importance of secretions and excretions in transmission of CSF virus, a model was developed. This model quantified the daily transmission probabilities from one infectious pig to one susceptible pig, using quantitative data on: (a) virus excretion by infected pigs, (b) survival of virus in the environment and (c) virus dose needed to infect susceptible pigs. Furthermore, the model predicted the relative contribution of secretions and excretions to this daily probability of infection of a susceptible pig. Three virus strains that differed in virulence were evaluated with the model: the highly virulent strain Brescia, the moderately virulent strain Paderborn and the low virulent strain Zoelen. Results suggest that it is highly probable that susceptible pigs in contact with Brescia or Paderborn infected pigs will be infected. For a pig in contact with a Zoelen infected pig, infection is less likely. When contact with blood is excluded, the predicted overall probability of infection was only 0.08 over the entire infectious period. The three strains differed in the relative contribution of secretions and excretions to transmission, although blood had a high probability of causing infection of a susceptible pig when in contact with a pig infected with any strain. This supports the statement that during outbreaks, control measures should ideally be based on the characteristics of the specific virus strain involved, which implies the development of strain-specific measures.     

Microarray-based detection of viruses causing vesicular or vesicular-like lesions in livestock animals

Definitive diagnosis of vesicular or vesicular-like lesions in livestock animals presents challenges both for veterinary clinicians and diagnostic laboratories. It is often impossible to diagnose the causative disease agent on a clinical basis alone and difficult to collect ample vesicular epithelium samples. Due to restrictions of time and sample size, once laboratory tests have ruled out foot-and-mouth disease, vesicular stomatitis and swine vesicular disease a definitive diagnosis may remain elusive. With the ability to test a small quantity of sample for a large number of pathogens simultaneously, DNA microarrays represent a potential solution to this problem. This study describes the application of a long oligonucleotide microarray assay to the identification of viruses known to cause vesicular or vesicular-like lesions in livestock animals. Eighteen virus isolates from cell culture were successfully identified to genus level, including representatives of each foot-and-mouth disease virus serotype, two species of vesicular stomatitis virus (VSV), swine vesicular disease virus, vesicular exanthema of swine virus (VESV), bovine herpesvirus 1, orf virus, pseudocowpox virus, bluetongue virus serotype 1 and bovine viral diarrhoea virus 1. VSV and VESV were also identified in vesicular epithelium samples, with varying levels of sensitivity. The results indicate that with further development this microarray assay could be a valuable tool for the diagnosis of vesicular and vesicular-like diseases.     

Multiplication of attenuated and virulent porcine parvoviruses in colostrum-deprived, neonatal pigs

Colostrum-deprived, neonatal, 2 days old pigs were inoculated with the attenuated HT-/SK or the virulent 90HS strain of porcine parvovirus (PPV) by the oral or subcutaneous route and sacrificed 2, 4 or 6 days after inoculation. Then, comparison was made on viral multiplication in pigs between the two strains. Pigs inoculated with the HT-/SK strain showed no detectable viremia or HI antibody responses against PPV within 6 days after inoculation. Only in pigs inoculated by the subcutaneous route, a small amount of virus was recovered from the spleen, liver, or mesenteric lymph nodes. These viruses were distinguished from the parental virulent 90HS strain, as examined for rct maker in vitro. When pigs were inoculated with the virulent 90HS strain, viremia appeared in all of them 1 day after inoculation and continued for up to the sacrificed day. Moreover, a considerable amount of virus was also detected from all tissues, including brain, lung, liver, spleen, pancreas, small intestine, and lymph node tissues, in all pigs tested. HI antibodies were first detected 6 days after inoculation.     

Studies on the pathogenesis of chicken infectious anaemia virus infection in six-week-old SPF chickens

The pathogenesis of chicken infectious anaemia virus (CAV) infection was studied in 6-week-old and one-day-old SPF chickens inoculated intramuscularly with graded doses of Cux-1 strain (10(6)-10(2) TCID50/chicken). Viraemia, virus shedding, development of virus neutralizing (VN) antibodies and CAV distribution in the thymus were studied by virus isolation, polymerase chain reaction (PCR), immunocytochemistry (IP) and in situ hybridization until postinfection day (PID) 28. In 6-week-old chickens infected with high doses of CAV, viraemia and VN antibodies could be detected 4 PID and onward without virus shedding or contact transmission to sentinel birds. However, virus shedding and contact transmission were demonstrated in one-day-old infected chickens. In the 6-week-old groups infected with lower doses, VN antibodies developed by PID 14, transient viraemia and virus shedding were detected. The thymus cortex of all 1-day-old inoculated chickens stained with VP3-specific mAb. Cells with positive in situ hybridization signal were fewer and scattered throughout the thymus tissue of the one-day-old inoculated chickens as compared to IP-positive cells. These results suggest that early immune response induced by high doses of CAV in 6-week-old chickens curtails viral replication and prevents virus shedding.