Experimental infection and horizontal transmission of Modoc virus in deer mice (Peromyscus maniculatus)

Deer mice (Peromyscus maniculatus) were inoculated with a sublethal dose of a field strain of Modoc virus to determine patterns of viral persistence, shedding, and transmission. Blood, serum, urine, fecal, and oral swab samples were collected at selected intervals until 63 days postinoculation (PI) after which lung, liver, spleen, kidney, and salivary glands were explanted. Viral assays were conducted by intracranial inoculations of suckling mice and antibody titers were determined by the micro-complement-fixation test. Viremias lasted for up to 4 days PI. Antibody titers were present by day 8 PI, peaked at day 13-20 PI, and persisted until day 63 PI. There was no evidence of viral shedding in urine, fecal, or oral swab samples. Virus was detected in explanted lungs only. In a separate experiment, deer mice were inoculated with virus and lungs were removed from five mice per wk for 10 wk. Indirect fluorescent antibody (IFA) techniques were used to determine the location of virus in lung tissue and to examine fixed tissue for lesions. IFA showed virus in lung parenchymal cells beginning 42 days PI and persisting at least 70 days PI. No histopathologic changes were seen. Horizontal transmission of the virus was studied by placing uninoculated mice with inoculated mice for 42 days and determining if the test animals developed antibodies or had virus in their lungs. Fifty-percent of the uninoculated mice developed antibody. One of these animals had virus in its lungs. Therefore, Modoc virus may be transmitted by direct contact.     

Viremia and nasal and rectal shedding of rotavirus in gnotobiotic pigs inoculated with Wa human rotavirus

Respiratory symptoms with rotavirus shedding in nasopharyngeal secretions have been reported in children with and without gastrointestinal symptoms (Zheng et al., 1991, J. Med. Virol. 34:29-37). To investigate if attenuated and virulent human rotavirus (HRV) strains cause upper respiratory tract infections or viremia in gnotobiotic pigs, we inoculated them with attenuated or virulent HRV intranasally, intravenously, or orally or via feeding tube (gavage) and assayed virus shedding. After oral or intranasal inoculation with attenuated HRV, the pigs remained asymptomatic, but 79 to 95% shed virus nasally and 5 to 17% shed virus rectally. After inoculation by gavage, no pigs shed virus nasally or rectally, but all pigs seroconverted with antibodies to HRV. No viremia was detected through postinoculation day 10. Controls inoculated intranasally with nonreplicating rotavirus-like particles or mock inoculated did not shed virus. In contrast, 100% of pigs inoculated with virulent HRV (oral, intranasal, or gavage) developed diarrhea, shed virus nasally and rectally, and had viremia. The infectivity of sera from the viremic virulent HRV-inoculated pigs was confirmed by inoculating gnotobiotic pigs orally with pooled HRV-positive serum. Serum-inoculated pigs developed diarrhea and fecal and nasal virus shedding and seroconverted with serum and intestinal HRV antibodies. Pigs inoculated intravenously with serum or intestinal contents from the viremic virulent HRV-inoculated pigs developed diarrhea, virus shedding, and viremia, similar to the orally inoculated pigs. This study provides new evidence that virulent HRV causes transient viremia and upper respiratory tract infection in addition to gastrointestinal infection in gnotobiotic pigs, confirming previous reports of rotavirus antigenemia (Blutt et al., Lancet 362:1445-1449, 2003). Our data also suggest that intestinal infection might be initiated from the basolateral side of the epithelial cells via viremia. Additionally, virus shedding patterns indicate a different pathogenesis for attenuated versus virulent HRV.     

Serological Diagnosis of Classical Swine Fever

Antibody levels in post-infection sera from a pig inoculated with a low virulent strain of classical swine fever virus (Hannover 62) and in sera from two pigs inoculated with another low virulent strain (Spielbach 66) and from an in-contact pig were assayed by complement fixation and immunofluorescence using classical swine fever virus (ALD strain) and bovine virus diarrhoea virus (UG 59 strain) as antigens. The complement fixation test used was modified by addition of a preparation of porcine Glq to the complement and by mercaptoethanol treatment of the immune serum before use. The mercaptoethanol treatment of the immune serum resulted in complete elimination of a haemolytic prozone often seen with porcine immune sera. In the sera from the inoculated animals complement-fixing antibodies appeared earlier than neutralizing antibodies. A few weeks after inoculation there was a correlation between the presence of complement-fixing and neutralizing antibodies. During the entire observation period of 13 weeks it was not possible to demonstrate complement-fixing or neutralizing antibodies in serum from a pig exposed to infection by contact with the two pigs inoculated with the Spièlbach 66 strain of classical swine fever virus.     

Susceptibility of mink to certain viral animal diseases foreign to the United States.

Mink (Mustela vison) were inoculated with viruses: African horse sickness (AHS), African swine fever (ASF), bovine herpes virus II (BHV2), foot-and-mouth disease (FMD), goat pox (GP), hog cholera (HC), peste des petits ruminants (PPR), rinderpest (RP), swine vesicular disease (SVD), vesicular exanthema of swine (VES) and vesicular stomatitis (VS). Their susceptibility was measured by development of clinical signs, virus isolation and detection of precipitin and/or virus neutralizing antibodies. SVD virus produced a lesion in one mink. Virus was isolated from mink inoculated with SVD, FMD and BHV2. Neutralizing and/or precipitin antibodies were detected in mink inoculated with ASF, FMD, GP, RP, SVD and VS viruses. Mink were not susceptible to AHS, HC, PPR and VES viruses.     

Passive immunization against transmissible gastroenteritis virus in piglets by ingestion of milk of sows inoculated with attenuated virus.

Pregnant sows were inoculated with the attenuated strain, TO--163, of swine transmissible gastroenteritis virus. Suckling piglets born from them received challenge inoculation with the virulent virus at 3 days after birth, and examined for ability to prevent infection and the immunoglobulin (Ig) classes of antibody in milk. A pregnant sow was inoculated intramuscularly with a dose of 10(8.0) TCID50 and intranasally with a dose of 10(9.3) TCID50 of attenuated virus. Piglets born from it suffered from diarrhea after challenge inoculation, but none of them died eventually. Their dam was also affected with diarrhea for 4 to 7 days after challenge inoculation of them. Another pregnant sow was inoculated twice with 10(9.3) TCID50 of attenuated virus, first by the intramuscular and secondly by the intranasal route. Of nine piglets born from it, one excreted soft feces after challenge inoculation, but all survived to grow normally. Their dam manifested no clinical symptoms at all after challenge inoculation of them. The higher the titer of virus inoculated into pregnant sows, the higher the neutralizing antibody titer in serum and milk of the sows after farrowing. The puerperal sow which had received two doses of 10(9.3) TCID50 each of attenuated virus by the intramuscular and intranasal route, respectively, presented the highest neutralizing antibody titer of all the inoculated sows. This titer was 2,048 in serum and 14,183 in colostrum immediately after farrowing. In that sow IgG was the main class of immunoglobulins in neutralizing antibody in milk. Even the IgA antibody titer of that sow was higher than that of any other sow which had been administered with virus of low titer. It was 392 and 19 3 and 9 days, respectively, after farrowing.     

The Effect of Eimeria nieschulzi Infection on Leukocyte Levels in the Rat*

SYNOPSIS. Rats inoculated with 10,000 sporulated oocysts of Eimeria nieschulzi had significantly higher total leukocyte counts on postinoculation days (PI) 1, 5, 6 and 7 when compared to control rats. Relative and absolute neutrophil counts increased concomitantly with a decrease in the relative lymphocyte levels in E. nieschulzi-infected rats on PI day 7. Absolute and relative neutrophil counts in infected rats on PI days 7 and 8 were closely correlated with the host's total oocyst discharge. The E. nieschulzi infection had no significant effect on the relative or absolute levels of monocytes or eosinophils. The described changes in leukocyte levels were not paralleled by a significant change in the erythrocyte count.     

Pathogenicity and distribution of avian nephritis virus (G-4260 strain) in inoculated laying hens

Specific-pathogen-free laying hens were inoculated intravenously with the G-4260 strain of avian nephritis virus (ANV). The distribution of the virus in organs, histological changes in main organs, the condition of laying, and egg transmission of the virus were examined in them. Over an experimental period of 27 days, no clinical sings were observed. In a chronological study on the distribution of the virus in organs, the virus was recovered from liver, kidney, jejunum, and rectum for 6 days postinoculation (PI). The virus titer in organ emulsion was the highest in the jejunum of all the main organs. The virus was recovered from the kidney for 8 days PI, although it was not so high in this organ. It was not recovered from the ovary or oviduct. Fluorescent antigens were not observed at all in any material. In a pathological examination, some local inflammatory changes were observed only in the kidney. There were no significant changes in the ovary, oviduct, or any other organ. Antibody appeared 10 days PI and was detectable even 27 days PI, although it was not so high in titer. There was no significant difference in the rate of egg-production between the infected and the sham inoculated groups. No virus was isolated from 111 fertile eggs laid by infected hens over a period from 2 to 27 days PI.     

Effect of diamfenetide on experimental infections of Fasciola hepatica in lambs: anthelmintic and clinical investigations.

The activity of diamfenetide (N,N'-[oxybis(2,1-ethan diyloxy-4,1-phenylene)] bis acetamide) was studied in lambs experimentally inoculated with Fasciola hepatica. The drug was given orally at a dose level of 100 mg/kg either 1,3,5,7, or 9 weeks postinoculation. It was 100% effective 1, 3, and 5 weeks postinoculation, 73% effective 7 weeks postinoculation, and 57% effective 9 weeks postinoculation. Serum gamma-glutamyl transpeptidase activity remained normal in all lambs for 5 weeks after infection; it then began to increase in infected, untreated lambs at 6 weeks, and had increased 5- to 6-fold 9 weeks postinoculation in infected lambs. This enzyme activity was the most sensitive hematologic parameter used in this test to detect hepatobiliary damage by the parasite. The drug was well tolerated at the dose level used.     

Experimental infection of white-tailed deer with rinderpest virus.

White-tailed deer (Odocoileus virginianus) succumbed to experimental infection with virulent rinderpest (RP) virus that was also lethal to cattle and goats. The deer developed clinical signs typical of RP and died 5 and 6 days post-inoculation. Infection was confirmed by recovery of virus from blood before death, from lymph node tissue after necropsy, and demonstration of specific complement fixing antigen in those tissues. Electron micrographs of infected Vero cell cultures revealed extracellular virions and intracytoplasmic and intranuclear inclusions made of randomly distributed fibrillar strands.     

Animal model of mucosally transmitted human immunodeficiency virus type 1 disease: intravaginal and oral deposition of simian/human immunodeficiency virus in macaques results in systemic infection, elimination of CD4+ T cells, and AIDS.

Chimeric simian/human immunodeficiency virus (SHIV) consists of the env, vpu, tat, and rev genes of human immunodeficiency virus type 1 (HIV-1) on a background of simian immunodeficiency virus (SIV). We derived a SHIV that caused CD4+ cell loss and AIDS in pig-tailed macaques (S. V. Joag, Z. Li, L. Foresman, E. B. Stephens, L. J. Zhao, I. Adany, D. M. Pinson, H. M. McClure, and O. Narayan, J. Virol. 70:3189-3197, 1996) and used a cell-free stock of this virus (SHIV(KU-1)) to inoculate macaques by the intravaginal route. Macaques developed high virus burdens and severe loss of CD4+ cells within 1 month, even when inoculated with only a single animal infectious dose of the virus by the intravaginal route. The infection was characterized by a burst of virus replication that peaked during the first week following intravenous inoculation and a week later in the intravaginally inoculated animals. Intravaginally inoculated animals died within 6 months, with CD4+ counts of <30/microl in peripheral blood, anemia, weight loss, and opportunistic infections (malaria, toxoplasmosis, cryptosporidiosis, and Pneumocystis carinii pneumonia). To evaluate the kinetics of virus spread, we inoculated macaques intravaginally and euthanized them after 2, 4, 7, and 15 days postinoculation. In situ hybridization and immunocytochemistry revealed cells expressing viral RNA and protein in the vagina, uterus, and pelvic and mesenteric lymph nodes in the macaque euthanized on day 2. By day 4, virus-infected cells had disseminated to the spleen and thymus, and by day 15, global elimination of CD4+ T cells was in full progress. Kinetics of viral replication and CD4+ loss were similar in an animal inoculated with pathogenic SHIV orally. This provides a sexual-transmission model of human AIDS that can be used to study the pathogenesis of mucosal infection and to evaluate the efficacy of vaccines and drugs directed against HIV-1.     

Pathogenicity for chicks of line cells from lymphoma of Marek's disease.

Pathogenicity for chicks of the MSB-1 line, a cell line derived from the tumorous tissue of a chick with Marek's disease (MD) and established by Akiyama & Kato, was studied. Five groups, including a control one, of 20 chicks each were inoculated with 1 X 10(3), 1 X 10(4), 1 X 10(5), 1 X 10(6) and no cells of a 180-day culture of the cell line at one day of age. They were housed all together in an isolation unit. An attempt was first made successfully to isolate MD virus (MDV) directly in culture of kidney cells 3 weeks after inoculation. Horizontal infection was first detected 4 weeks after inoculation. From 3 weeks after inoculation on, the disease with almost the same clinical and pathological pictures as the infection with a virulent strain of MDV showed a high incidence. Morbidity was closely related to the number of MSB-1 line cells inoculated. Parenchymal destruction was conspicuous in the central lymphoid organs of four chicks given the largest number of MSB-1 line cells and sacrificed in extremis about 4 weeks after inoculation. Establishment of MD in chicks inoculated with MSB-1 line cells carrying MDV genome seemed to be initiated under the circumstances where the line cells which had come into contact with susceptible cells in the peritoneal cavity released virulent MDV per se. Then host chicks might be infected with MDV and suffer from MD at a high rate. There was no great difference in oncogenic potential between MSB-1 line cells cultivated in vitro for 180 days and virulent MDV serially passaged through one-day-old chicks.     

Suppression and regeneration of rat bone marrow under the influence of methotrexate. An EM study.

Adult male rats were given daily injections of methotrexate for 12 days, during which time specimens of femoral bone marrow were taken for correlative light- and electron-microscopic study. After 12 days methotrexate was discontinued and specimens were taken the marrow recovered from the effect of methotrexate. Normal rat bone marrow was used as a basis of comparison. As methotrexate was administered, cell division ceased and the immature erythroblasts and myelocytes proceeded in the maturational process and a rapid hypocellularity of the marrow developed. Eosinophilic leukocytes, lymphocytes, large fat cells, erythrocytes, and phagocytic reticuloendothelial cells were the only formed elements remaining after 12 days of methotrexate treatment. Two days after methotrexate was discontinued, myeloblasts, myelocytes and proerythroblasts were plentiful, and on the third day the marrow exhibited an erythroid hyperplasia. By the eighth day all cell types were present though the marrow still was hypocellular. 14 days post methotrexate the marrow returned to normal.     

Serologic and mucosal immune response to rotavirus infection in the rabbit model.

We examined the humoral immune response to rotavirus infection in specific pathogen-free rabbits inoculated and challenged orally with rabbit Ala rotavirus (7.5 x 10(5) to 1 x 10(7) PFU). The humoral immune response in both serologic and mucosal samples was monitored by using total antibody enzyme-linked immunosorbent assays (ELISAs), isotype-specific ELISAs, and plaque reduction neutralization assays. Following a primary infection, all rabbits shed virus and serologic and mucosal antibody responses were initially detected by 1 week postinoculation. Intestinal immunoglobulin M was detected by 3 days postinoculation, and secretory immunoglobulin A was detected by 6 days postinoculation. Following challenge, rabbits were protected (no detectable virus shedding) from infection. An anamnestic immune response was observed only with mucosal neutralizing antibodies, and all serologic and mucosal immune responses persisted at high levels until at least 175 days postchallenge (204 days postinoculation). Detection of neutralization responses was influenced by the virus strain used in the neutralization assay; all inoculated rabbits developed detectable serum and intestinal neutralizing antibodies against the infecting (Ala) virus strain. Neutralization activity in both serum and mucosal samples was generally, but not exclusively, homotypic (VP7 serotype 3) after both primary and challenge inoculations with Ala virus. Heterotypic serum neutralization activity was observed with serotype 8 (9 of 12 rabbits) and 9 (12 of 12 rabbits) viruses and may be based on reactivity with the outer capsid protein VP4 or on a shared epitope in the C region of VP7. Comparisons of heterologous (serotype 3) and heterotypic neutralizing responses in mucosal and serologic samples revealed that 43% (21 of 49) of the responses were discordant. In 19 of 49 (39%) of these cases, a heterotypic serologic response was seen in the absence of a heterotypic mucosal response, but in 2 of 49 (4%) instances, a heterotypic mucosal response was seen in the absence of a concomitant serologic response. These results provide insight into factors which may affect detection of heterotypic responses.     

Immune and antibody responses to an isolated capsid protein of foot-and-mouth disease virus.

The purified capsid proteins VP1, VP2, and VP3 of foot-and-mouth disease virus type A12 strain 119 emulsified with incomplete Freund's adjuvant were studied in swine and guinea pigs. Swine inoculated on days 0, 28, and 60 with 100-mug doses of VP3 were protected by day 82 against exposure to infected swine. Serums from animals inoculated with VP3 contained viral precipitating and neutralizing antibodies, but such serums recognized fewer viral antigenic determinants than did antiviral serums. Capsid proteins VP1 and VP2 did not produce detectable antiviral antibody in guinea pigs, and antiviral antibody responses in swine to a mixture of VP1, VP2, and VP3 were lower than the responses to VP3 alone. However, when swine were inoculated with VP1, VP2, and VP3 separately at different body sites, no interference with the response to VP3 was observed. Vaccine containing VP3 isolated from acetylethylenimine-treated virus appeared less protective for swine than vaccine containing VP3 from nontreated virus. Trypsinized virus, which contains the cleaved peptides VP3a and VP3b rather than intact VP3, produced approximately the same levels of antiviral antibody responses in guinea pigs as did virus. Conversely, an isolated mixture of VP3a and VP3b did not produce detectable antiviral antibody responses in guinea pigs. The VP3a-VP3b mixture did, however, sensitize guinea pigs to elicit such responses following reinoculation with a marginally effective dose of trypsinized virus.     

In vitro splenic T cell responses of diverse mouse genotypes after oronasal exposure to mouse hepatitis virus, strain JHM.

Mortality rates among BALB/cByJ, A/JCr, C3H/HeSnJ, and C57BL/6NCr mice inoculated oronasally with mouse hepatitis virus (MHV) strain JHM, ranged from 25 to 67%. Spleen cells harvested from the first three genotypes at 5 days postinoculation proliferated poorly in response to concanavalin A stimulation and produced significantly less interleukin (IL) 2 than cells from uninfected control mice. The function of spleen cells harvested at 14 days postinoculation varied and was host genotype-dependent. Despite clinical signs among some infected C57BL/6NCr mice, spleen cell function was relatively unaffected. C57BL/10ScNCr, B10.A, and SJL/JCr mice remained clinically normal after MHV inoculation. Proliferation and IL2 production by cells from inoculated C57BL/10ScNCr and B10.A mice were similar to responses of their respective controls. In contrast, cells from inoculated SJL/JCr mice were hyper-responsive and produced peak levels of IL2 earlier than control cells. Among the seven genotypes tested, only BALB/cByJ and C3H/HeSnJ spleen cells produced detectable IL4 after primary stimulation with concanavalin A or after priming and restimulation. Primary IL4 production by cells from these two genotypes was significantly reduced if donors were inoculated with MHV 5 days prior to spleen harvest. IL4 production by cells from acutely infected BALB/cByJ mice was considerably enhanced by priming and restimulation.     

Effect of a bioengineered pseudorabies (Aujeszky's disease) vaccine on the semen quality of boars

Eight boars 11 months of age that were seronegative to pseudorabies virus were trained for hand collection of semen. A genetically engineered pseudorabies virus vaccine was given to 7 of 8 boars, while the eighth was left unvaccinated to serve as a control. Semen samples were collected at 7 day intervals from 42 days prior to vaccination through 49 days after vaccination. Rectal temperatures and general health of the boars were clinically normal throughout the trial, and no clear differences were observed in the quality of semen collected from the boars before or after vaccination. The semen samples were tested for vaccine virus and none was detected.     

Duration of infection of jirds (Meriones unguiculatus) with Trichostrongylus colubriformis

Jirds (Meriones unguiculatus) were infected with 700 infective larvae (L3) of Trichostrongylus colubriformis. The fecal egg counts (epg) were followed regularly until 340 days postinoculation, and worm numbers were determined on necropsy at 315 and 350 days postinoculation. The epg increased to a peak of 2,600 on day 24 postinoculation. Thereafter, the epg showed continuous decrease from day 31 until day 318 postinoculation. The rates of inoculum recovery were 0.29-1.43% on day 315 and 0.142-0.428% on day 350 postinoculation. On day 315 postinoculation, 12 male worms were recovered from 2 of 3 animals necropsied. On day 350 postinoculation, all 6 animals were infected, and 6 female and 4 male worms were recovered. The systemic IgM, IgG, and intestinal mucous IgA responses were significantly higher in infected groups compared with negative control groups necropsied on 315 and 350 days postinoculation (P < 0.001). These results confirm that the T. colubriformis-jird system is a useful model for studies on the various host-parasite relationships of chronic trichostrongylosis.     

Studies on pathogenesis of buffalo pox virus in rabbits: quantitative assay of virus in different organs

The buffalo pox virus was found to multiply in the skin, the primary site of inoculation with an eclipse phase of 12 hr. The virus was then detected in the skin after 15 hr followed by its appearance in regional lymph nodes 36 hr postinoculation. Primary viremia was detected 48 hr postinoculation, followed by detection of virus in the lungs, liver, and spleen. The virus multiplied in the lungs on day 4 and in the liver and spleen on day 5 postinoculation and its release led to secondary viremia. In a follow-up from day 7 to 14 postinoculation, the virus was detected in the kidneys, stomach, intestines, and gonads.