Detection of turkey coronaviral enteritis (bluecomb) in field epiornithics, using the direct and indirect fluorescent antibody tests.

In Minnesota, efforts have been made over the past 10 years to eliminate turkey coronaviral enteritis (TCE, bluecomb) by controlled depopulation and decontamination with a rest period before restocking. In 1973, clinical observations indicated that bluecomb was restricted to one limited area in Minnesota. Five epiornithics occurred during late 1973 and 1974, involving 5 different farms in this limited geographic area. During 1975, 3 epiornithics of TCE were investigated, involving 185,000 turkeys in 17 flocks, of which approximately 17,000 died. Naturally infected turkeys representing 7 operations between 1973 and 1976 were examined by both the direct fluorescent antibody test and indirect fluorescent antibody test (IFAT). The direct fluorescent antibody test detected coronaviral antigen in intestinal tissues during the acute phase of the disease, and the IFAT was highly useful in detecting TCE serum antibodies of turkey flocks that had recovered and were potential carriers. Therefore, an IFAT surveillance program was instituted for replacement flocks on farms where clinical epiornithics of TCE had occurred in 1974 through 1976. Operation 5 involved TCE epiornithics over a 2-year period and illustrate the importance of complete depopulation with an intensive decontamination program.     

Coronaviral enteritis of young calves: virologic and pathologic findings in naturally occurring infections.

Coronavirus was detected in 113 (16.4%) of 689 calf enteric disease accessions received at the South Dakota Animal Disease Research and Diagnostic Laboratory from Mar 1, 1975 to Oct 31, 1976. During this period, modified live coronaviral vaccine was not in common use in this area. The virus was detected by negative stain electron microscopy of feces and intestinal contents and by fluorescent antibody examination of intestine and colon sections. Calves with coronaviral enteritis ranged in age from 1 day to 3 months, with most cases occurring between 1 and 7 days. At necropsy, a green, mucoid fecal cast was commonly seen in the colon. Histopathologic examination revealed necrotizing enterocolitis.     

Clinical and pathologic effects of oral administration of transmissible gastroenteritis vaccine to gnotobiotic pigs.

Pigs from 3 litters kept under gnotobiotic conditions were inoculated orally with virulent transmissible gastroenteritis (TGE) virus, a TGE vaccine, or Hank's balanced salt solution at 2 days of age and then euthanatized at intervals ranging from 1 to 7 days after inoculation. Pigs exposed to the vaccine had clinical evidence of diarrhea and weakness. Lesions resembling those of TGE were revealed grossly, microscopically, and by scanning electron microscopy. Viral antigen was seen in intestinal epithelial cells by the direct fluorescent antibody technique. The disease induced by the vaccine virus had a longer incubation period and lesions were less severe than that induced by the virulent virus.     

水貂肠炎细小病毒灭活疫苗抗体消长规律的研究

应用血凝抑制试验对水貂肠炎细小病毒灭活疫苗免疫水貂进行抗体水平动态监测,结果表明,疫苗接种14 d抗体达到保护,21~30 d抗体水平达到高峰;免疫180 d抗体仍在保护值以上。攻毒试验证实,免疫180 d后90%免疫水貂获得保护,确定水貂细小病毒性肠炎灭活疫苗免疫保护期可持续6个月。     

A comparison of gel diffusion, fluorescent antibody and virus isolation methods in experimental and natural cases of infectious bursal disease.

In studies with chicks inoculated with the Sk-1 strain of infectious bursal agent the bursa of Fabricius was found to be the tissue of choice for virus isolation as well as for use in the fluorescent antibody test and the agar gel diffusion test. In separate experiments positive results were obtained until postinoculation days 3 or 4 by the agar gel diffusion test, 5 or 6 by the fluorescent antibody test and 14 by the virus isolation method, respectively. Bursas from chickens involved in seven natural outbreaks of infectious bursal disease were then examined by these three methods. Virus was isolated from six outbreaks and infectious bursal agent antigen was demonstrated in three by the agar gel diffusion test method and seven (three by direct examination and four after one passage in chicks) by the fluorescent antibody test method. Passage in chicks was required when nonspecific fluorescence complicated the interpretation of fluorescent antibody test results.     

Immune response of ponies to experimental infection with Ehrlichia equi.

Four ponies experimentally infected with Ehrlichia equi developed substantial cell-mediated immune responses, as measured by the leukocyte migration-inhibition test. Serum anti-E equi antibodies up to 1:1,280 were demonstrated by the indirect fluorescent antibody test. Cell-mediated immune responses returned to a base-line value by day 200 after primary inoculation, but serum antibody titers persisted for at least 300 days after inoculation. Two additional susceptible ponies, which were inoculated with convalescent blood or organ homogenates from ponies recovered from acute equine ehrlichiosis, treated with tetracycline, and subsequently challenge exposed with E equi-infective blood, did not develop clinical disease. This study suggested that ponies are resistant to reinfection with E equi following clinical ehrlichiosis.     

Detection of mink enteritis virus in mink feces, using enzyme-linked immunosorbent assay, hemagglutination, and electron microscopy

Twenty-five mink were inoculated with mink enteritis virus (MEV). Fecal specimens were collected daily and were simultaneously evaluated for MEV antigen by use of a direct enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA), and electron microscopy. Results of the evaluations indicated that MEV was shed in the feces on postinoculation days 5 and 6. The virus was not detectable by ELISA or HA after postinoculation day 6, although viruses were found in reduced numbers by use of electron microscopy. The ELISA was specific for MEV, and the sensitivity of the ELISA for MEV was comparable with that of HA.     

Isolation and immunisation studies of a canine parco-like virus from dogs with haemorrhagic enteritis

A newly recognised canine parvo like virus was isolated from faeces of dogs with haemorrhagic enteritis. Cell cultures from several species were susceptible to it. Virus infected cells could be demonstrated by staining with fluorescent antibody reagents (prepared against canine virus or feline panleucopenia virus) or by haemagglutination with pig or rhesus monkey red blood cells. Inhibition of haemagglutination by specific antiserum prepared in specific-pathogen-free beagles provided a convenient method for viral identification. Experimental inoculation of specific-pathogen-free beagles resulted in elevated body temperatures and caused lymphopenia lasting one to three days. Feline panleucopenia virus vaccines protected dogs against challenge with virulent canine parvo-like virus.     

Epizootic hemorrhagic disease virus in Montana: isolation and serologic survey

Epizootic hemorrhagic disease virus (EHDV) was isolated in Vero cell culture from the spleen and whole blood of a white-tailed deer (Odocoileus virginianus). A 10% spleen suspension caused acute hemorrhagic disease (HD) when inoculated into an experimental white-tailed deer and resulted in the recovery of EHDV from the blood of the experimental animal at 5 days after inoculation. The virus was identified as EHDV serotype 2 through indirect fluorescent antibody tests, electron microscopy, and reciprocal cross-neutralization tests. Approximately 73% (36/49) of the mule deer, 5% (2/42) of the white-tailed deer, and 79% (249/314) of the cattle samples tested from areas where HD had been reported were EHDV seropositive. Although none of the white-tailed deer was bluetongue virus seropositive, 29% of the mule deer and 3% of the cattle tested from "active" HD areas possessed bluetongue virus precipitating antibody.     

Kinetics of antibody response to Ehrlichia canis assayed by the indirect fluorescent antibody method.

The kinetics of antibody production response to experimentally induced infection of dogs with Ehrlichia canis was determined by ion-exchange and molecularsieve chromatography and by indirect fluorescent antibody (IFA) test. The first IFA antibody at 7 days after inoculation resided in immunoglobulin M (IgM) and immunoglobulin A (IgA) classes. At approximately 21 days after inoculation, the antibody was in IgM, IgA, and immunoglobulin G (IgG) classes. Thereafter, antibody concentrations continued to increase in the IgG class; those in the other 2 immunoglobulin classes had a variable pattern. In 2 dogs which died 60 and 114 days after inoculation, a decrease of antibody concentration in the 3 immunoglobulin classes was evident at the time of death. In the carrier dog, however, which was killed 147 days after inoculation, antibody concentrations sustained increasing titers in the 3 immunoglobulin classes.     

Direct diagnosis of herpesvirus in equine foetal organs by means of electron microscopy

A technique for direct electron microscopy of EHV-1 infected foetal organs is described. Of 124 cases shown to be positive by fluorescent antibody procedurees, 67 (54%) were positive by direct electron microscopy.     

Intestinal infection of neonatal dogs with canine coronavirus 1-71: studies by virologic, histologic, histochemical, and immunofluorescent techniques.

Enteritis induced in neonatal dogs by oral inoculation of coronavirus 1-71 was studied by virologic, histologic, histochemical, and immunofluorescent antibody methods. The enteritis, accompanied by diarrhea, developed in 4 to 7 days and was characterized by atrophy and fusion of intestinal villi and deepening of crypts, increase in cellularity of the lamina propria, flattening of epithelial cells, and discharge of goblet cells. Intra-epithelial enzyme activities generally were depressed. Specific immunofluorescent antibody reactions were present in villous epithelium of the duodenum starting on postinoculation day 2 and of the ileum on day 4. Epiehtlium of the colon showed no reaction. Virus was present in the feces for 6 to 9 days and was recovered from the small and large intestines from day 2 through day 10. The infection was not fatal and was self limiting; the diarrhea stopped between 1 and 2 weeks. The intestinal mucosa also started recovering by the end of week 1, proceeding caudally from the duodenum.     

Negative contrast electron microscopic diagnosis of viruses of neonatal calf diarrhea.

Ninety-one cases of neonatal calf diarrhea were examined for viruses with negative contrast electron microscopy. Viruses were demonstrated in 41% of the cases. Reo-like viruses and corona-like viruses, and mixed virus populations were observed in 12%, 20% and 9%, respectively. Twenty-six of the cases were examined by negative contrast electron microscopy, and by virus isolation or by fluorescent antibody technique. There was an 81% agreement in obtained results. The disagreements resulted from the demonstration of a viral agent by negative contrast electron microscopy while the other techniques did not indicate a virus. The results suggest that negative contrast electron microscopy is a more sensitive diagnostic tool for demonstration of viruses associated with neonatal calf diarrhea than are viral isolation or the fluorescent antibody technique.     

Pathogenesis of Rabies in the Rat

Three strains of rabies virus, one fixed and two street, were compared for invasiveness in neurectomized rats following foot pad inoculation. Approximately one-fifth of test rats studied revealed that the virus bypassed the severed sciatic and saphenous nerves and reached the brain.

All rats dying within 20 days were positive for rabies upon fluorescent antibody and mouse mortality tests.

Viremia was demonstrated in 20 per cent of both neurectomized or non-neurectomized rats one hour after inoculation of rabies virus, both fixed and street strains, into the foot pad.

     

Encephalitozoonosis in the blue fox. Comparison between the india-ink immuno-reaction and the indirect fluorescent antibody test in detecting Encephalitozoon cuniculi antibodies

Sera from 32 foxes sampled at intervals varying from 20 to 70 days after oral inoculation with E. cuniculi spores were tested by the india-ink immunoreaction (IIR) and the indirect fluorescent antibody test (IFAT). Using the IFAT, antibodies were detected at low levels in sera sampled on days 20 and 29 post inoculation, whereas the IIR failed to reveal antibodies in the same sera. In sera sampled from day 35 until day 70 post inoculation, antibodies were detected by both tests, the IIR-titres reaching the magnitude of the IFAT-titres after about 50 days posit inoculation.In 14 sera sampled from foxes of at least 46 days of age and with signs of encephalitozoonosis, the tests gave almost identical results.Comparing IIR- and IFAT-determined antibody titres using E. cuniculi antigens of blue fox and rabbit origin in the test, the antigens seemed to be closely related, supporting the suggestion that the isolates are strains of the same microsporidian species.     

Serological reactions in sheep and cattle experimentally infected with three Australian isolates of bluetongue virus

Serums from 103 sheep and 24 cattle experimentally infected with one of 3 serotypes of bluetongue virus isolated in Australia were tested for antibody to bluetongue virus in the serum neutralisation test and the agar gel diffusion precipitin test. Antibody to bluetongue virus was first detected by these tests 8 to 10 days after intravenous infection in 4 sheep that were bled daily for serum analysis. The agar gel diffusion test failed to detect antibody in 28% (29/103) of sheep which had seroconverted in the serum neutralisation test. A further 7% (7/103) of sheep serums were negative in both tests 14 to 22 d after infection. Both tests detected antibody to bluetongue virus in all cattle serums by 10 days after detection of viraemia. In comparison with the intravenous route of infection, extended prepatent periods for the commencement of viraemia resulting from intradermal, subcutaneous and intrauterine routes of infection in the cattle caused corresponding delays in the detection of antibody. For example, one cow that was infected by intrauterine inoculation did not become viraemic until 22 d after inoculation and antibody was not detected until 32 d after inoculation.     

Antibody response and duration of latent infection in sheep following experimental infection with Babesia ovis

Babesia ovis isolated in Extremadura (Spain) was the subject of a serological study in experimentally inoculated sheep. The first antibody titres, determined by the indirect fluorescent antibody (IFA) test, were observed 7-8 days post infection (p.i.) in all animals except the splenectomized group, in which the only animal that survived showed antibody response 10 days p.i. A faster response following challenge was observed in sheep which were seropositive before inoculation, which suggests the existence of an antigen memory. The highest titres were reached 16-25 days p.i., and subsequently began to fall, reaching minima at the end of the experimental period (330 days p.i.). The chronic carrier state in experimental B. ovis infection had a duration of at least 2 years. Passive transmission of antibodies from experimentally infected mothers to newborn lambs was also detected. Antibody levels were observed for a period not longer than 2 months after birth.     

Lesions of transmissible gastroenteritis virus infection in experimentally inoculated pigs suckling immunized sows

Intestinal lesions of transmissible gastroenteritis (TGE) virus infection in conventionally reared pigs suckling either nonvaccinated, vaccinated, or previously infected sows were studied by scanning electron microscopy, light microscopy, and immunofluorescent microscopy for TGE viral antigen. Pigs were inoculated with virulent TGE virus when they were 5 or 21 days old and were euthanatized shortly after the onset of diarrhea or 96 hours after inoculation if no diarrhea developed. Pigs inoculated when they were either 5 or 21 days old and suckling nonvaccinated sows developed severe lesions, including swelling and necrosis of enterocytes and severe villus atrophy. Pigs inoculated when they were 5 days old and suckling sows vaccinated with attenuated vaccines developed less-severe villus atrophy, and those suckling sows immunized by exposure to nonattenuated TGE virus developed moderate or no villus atrophy. Pigs inoculated when they were 21 days old and suckling sows vaccinated with attenuated vaccines had severe villus atrophy, whereas those suckling sows immunized by exposure to nonattenuated virus had more-moderate villus lesions. Villus atrophy was inhibited to various degrees in pigs suckling immunized sows, depending in part on the antibody titer in the colostrum and milk.     

Prolonged excretion and failure of cross-protection between distinct serotypes of bovine rotavirus

Four newborn calves were experimentally infected with two distinct serotypes of bovine rotavirus (BRV-1 and BRV-2). Initially, three colostrum-deprived calves were inoculated orally with either BRV-1 or BRV-2; all developed severe diarrhea and produced serotype-specific neutralizing antibodies. Fecal virus was first demonstrated by immunofluorescence the day after inoculation. The virus titers reached a maximum of 10(5.2)-10(6.6) fluorescent focus forming units g-1 of feces 2-5 days after inoculation and then decreased. Fecal virus was detected in low titers beyond 28 days after inoculation despite the development of serum neutralizing antibodies. One calf, which had acquired specific active immunity against BRV-1 following oral infection, was further infected orally with BRV-2 4 weeks later. The calf again manifested diarrhea, excreted BRV-2 and showed an increase in serum neutralizing antibody against BRV-2. These results indicated that calves infected with either BRV-1 or BRV-2 do not have cross-protection to infection with heterologous BRV, and that recurrence of the disease can occur. The possible mechanisms of the persistence of BRV in calves and its role in the epidemiology of this infection are discussed.     

Effect of age on the immune response of cattle experimentally infected with Babesia bigemina

Two different age groups of Holstein Friesian cattle were experimentally infected with Babesiabigemina. Calves of group A (6 months old) did not show noticeable symptoms of babesiosis and had relatively low (0.6%) numbers of parasites in their red blood cells (RBCs). Group B calves (1 year old) had typical signs of the disease; parasites were found in 6.6% of their RBCs. Blood from both groups inoculated into splenectomized calves at 3, 6, 12 and 18 months following initial inoculation demonstrated the presence of B. bigemina, while after 22 months no parasites could be demonstrated.The indirect fluorescent antibody (IFA) test detected babesial antibodies at 4–5 days post inoculation (PI) and reached a maximum titre of 1 : 640 at 2 weeks PI. Following challenge at 2–3 months after initial inoculation, the antibody titre rose sharply to 1 : 2560, then decreased gradually but was still detectable 22 months PI. No correlation was found between antibody titre and the presence of the parasite hin the peripheral blood.