An enzyme-linked immunosorbent assay for the detection of canine antibodies to canine adenoviruses.

An enzyme-linked immunosorbent assay was used to detect canine immunoglobulin G antibodies specific for infectious canine hepatitis virus and the serologically related canine adenovirus Type 2. The sequential development of homologous and heterologous antibodies was measured by the enzyme-linked immunosorbent assay and serum neutralization tests in two groups of dogs which were experimentally infected with either infectious canine hepatitis virus or canine adenovirus Type 2. Both tests were comparable in their abilities to detect the development of homologous and heterologous antibodies. Homologous antibodies were detected earlier and to a higher titer in both tests. There was a 98% agreement between the serum neutralization test and the enzyme-linked immunosorbent assay when sera from 224 random-source dogs were examined for infectious canine hepatitis virus antibodies. The enzyme-linked immunosorbent assay was found to be a highly efficient and rapid test to determine the immune status of dogs to infectious canine hepatitis virus and canine adenovirus Type 2.     

犬传染性肝炎病毒在体外细胞质内的发生

通过对犬传染性肝炎病毒（ＩＣＨＶ）在犬肾传代细胞内形态发生及其抗原定位的电镜和免疫胶体金电镜研究,发现ＩＣＨＶ除了在宿主细胞核内发生外,还有一条细胞质内的发生途径。在细胞质内病毒核壳体的装配是以均质致密包涵体和副晶格包涵体为“基地”,这与人们熟知的细胞核内形态发生方式相似。免疫胶体金标记显示,细胞质包涵体中含有大量的ＩＣＨＶ抗原成分,显核壳体在细胞质内装配病毒的结构蛋白来源。此外,在感染的细胞质内还观察到与核内相同的病毒核心样结构。     

Continuous-Flow Ultracentrifugation of Canine Distemper Virus and Infectious Canine Hepatitis Virus

The use of a Spinco L-4 zonal centrifuge with the B-XVI continuous-flow rotor for the purification of canine distemper and infectious canine hepatitis viruses is described. Up to 68 liters of virus was processed at one time. Infectious canine hepatitis virus was found to band at 39% sucrose and canine distemper virus banded between 32 and 48% sucrose. The virus was concentrated 10-fold, and the purity of the virus, as measured by protein concentration, was increased by 99%.     

Viricidal effects of Lactobacillus and yeast fermentation

The survival of selected viruses in Lactobacillus- and yeast-fermented edible waste material was studied to determine the feasibility of using this material as a livestock feed ingredient. Five viruses, including Newcastle disease virus, infectious canine hepatitis virus, a porcine picornavirus, frog virus 3, and bovine virus diarrhea, were inoculated into a mixture of ground food waste (collected from a school lunch program) containing Lactobacillus acidophilus. Mixtures were incubated at 20, 30, and 40 degrees C for 216 h. In a second trial, four viruses, including Newcastle disease virus, infectious canine hepatitis virus, frog virus 3, and a porcine picornavirus, were inoculated into similar edible waste material containing Saccharomyces cerevisiae. Mixtures were incubated at 20 and 30 degrees C for 216 h. Samples were obtained daily for quantitative (trial 1) and qualitative (trial 2) virus isolation. Temperature, pH, and redox potential were monitored. Controlled pH and temperature studies were also done and compared with the inactivation rates in the fermentation processes. In trial 1 (Lactobacillus fermentation), infectious canine hepatitis virus survived the entire test period in the fermentation process but was inactivated below pH 4.5 in the controlled studies. Newcastle disease virus was inactivated by day 8 in the fermentation process and appeared to be primarily heat sensitive and secondarily pH sensitive in the controlled studies. The porcine picornavirus survived the fermentation process for 8 days at 20 degrees C but was inactivated more rapidly at 30 and 40 degrees C. The controlled studies verified these findings. Frog virus 3 was inactivated by day 3 in the fermentation process and appeared to be sensitive to low pH in the controlled studies. Bovine virus diarrhea was rapidly inactivated in the fermentation process (less than 2 h) and was pH and temperature sensitive. In trial 2 (yeast fermentation), infectious hepatitis virus survived the entire test period in the fermentation process. Newcastle disease virus was inactivated by day 7 at 20 degrees C and day 6 at 30 degrees C. The porcine picornavirus was inactivated by day 7 at 30 degrees C but survived the entire test period at 20 degrees C. Frog virus 3 was inactivated by day 3 at 20 degrees C and day 2 at 30 degrees C.     

A Sarcocystis sp.-like protozoan and concurrent canine distemper virus infection associated with encephalitis in a raccoon (Procyon lotor).

A raccoon (Procyon lotor) with signs of weakness was captured in upstate New York (USA). Despite attempted care in a rehabilitation facility, the animal died and was examined because of suspected infectious neurologic disease. The cerebrum had a marked, locally extensive, neutrophilic, necrotizing encephalitis with numerous associated intralesional protozoal organisms, and a moderate to marked multifocal perivascular nonsuppurative meningoencephalitis. Based on morphology and immunohistochemical staining, the organism was a Sarcocystis sp.-like protozoan. Rabies antigen and canine distemper virus (CDV) inclusions were not detected. However, the animal was positive for canine distemper virus based on peroxidase anti-peroxidase staining.     

Macromolecular Content of Inclusions Produced by a Canine Adenovirus

Early inclusions induced by a canine adenovirus in a canine cell line, appearing before the formation of infectious virus particles, were purified by differential centrifugation in sucrose followed by CsCl density gradient centrifugation. Chemical analysis of these inclusions revealed that they contained deoxyribonucleic acid (DNA), ribonucleic acid, and protein. On the basis of density gradient centrifugation, the DNA extracted from the inclusions was found to be viral DNA. Electron microscope autoradiography showed that these inclusions were the sites of DNA synthesis. In addition, association of DNA polymerase activity with the inclusions was detected by incorporation of radioactivity from (3)H-thymidine triphosphate into a DNA product. The in vitro product of the enzyme had a density equal to that of viral DNA rather than host DNA. The level of DNA polymerase activity in exponentially growing infected and uninfected whole cells was similar, but in cells in stationary phase the enzyme activity of infected cells was twice that in noninfected cells. Furthermore, nuclei isolated from infected cells showed a fourfold increase in DNA polymerase activity over the noninfected cells.     

Viricidal Effects of Lactobacillus and Yeast Fermentation

The survival of selected viruses in Lactobacillus- and yeast-fermented edible waste material was studied to determine the feasibility of using this material as a livestock feed ingredient. Five viruses, including Newcastle disease virus, infectious canine hepatitis virus, a porcine picornavirus, frog virus 3, and bovine virus diarrhea, were inoculated into a mixture of ground food waste (collected from a school lunch program) containing Lactobacillus acidophilus. Mixtures were incubated at 20, 30, and 40°C for 216 h. In a second trial, four viruses, including Newcastle disease virus, infectious canine hepatitis virus, frog virus 3, and a porcine picornavirus, were inoculated into similar edible waste material containing Saccharomyces cerevisiae. Mixtures were incubated at 20 and 30°C for 216 h. Samples were obtained daily for quantitative (trial 1) and qualitative (trial 2) virus isolation. Temperature, pH, and redox potential were monitored. Controlled pH and temperature studies were also done and compared with the inactivation rates in the fermentation processes. In trial 1 (Lactobacillus fermentation), infectious canine hepatitis virus survived the entire test period in the fermentation process but was inactivated below pH 4.5 in the controlled studies. Newcastle disease virus was inactivated by day 8 in the fermentation process and appeared to be primarily heat sensitive and secondarily pH sensitive in the controlled studies. The porcine picornavirus survived the fermentation process for 8 days at 20°C but was inactivated more rapidly at 30 and 40°C. The controlled studies verified these findings. Frog virus 3 was inactivated by day 3 in the fermentation process and appeared to be sensitive to low pH in the controlled studies. Bovine virus diarrhea was rapidly inactivated in the fermentation process (less than 2 h) and was pH and temperature sensitive. In trial 2 (yeast fermentation), infectious hepatitis virus survived the entire test period in the fermentation process. Newcastle disease virus was inactivated by day 7 at 20°C and day 6 at 30°C. The porcine picornavirus was inactivated by day 7 at 30°C but survived the entire test period at 20°C. Frog virus 3 was inactivated by day 3 at 20°C and day 2 at 30°C.     

Biophysical Comparison of Two Canine Adenoviruses

The two canine adenoviruses, infectious canine hepatitis (ICH) virus and infectious canine laryngotracheitis (ICL) virus (also designated as Toronto A26/61 virus), were studied with respect to their morphology and the biological properties of their soluble components. The two viruses were found to be composed of soluble components similar to, and carrying biological activities corresponding to, those of the human adenoviruses after fractionation by rate zonal centrifugation and anion-exchange chromatography. The following soluble components were identified: hexons (carrying a common group-specific complement-fixing antigen), penton oligomers (complete soluble hemagglutinin), and penton and fiber monomers (incomplete soluble hemagglutinins). The latter were indicated by hemagglutination enhancement with selected antisera directed against human adenovirus soluble components. Elution characteristics of corresponding components of the two viruses in anion-exchange chromatography experiments were distinctly different. Electron microscopic examination of purified virions and soluble components revealed the fiber component of ICL virus to be 35 to 37 nm in length, and that of ICH virus to be 25 to 27 nm long.     

Diseases, parasites and survival of coyotes in south-central Georgia.

Serologic testing, radio-telemetry and post-mortem diagnostic evaluations were used to investigate survival and causes of mortality among 17 coyotes (Canis latrans) in south-central Georgia (USA). Prevalence of canine heartworm (Dirofilaria immitis) microfilariae was lower (P = 0.057) among fall-captured (22%) than among winter-captured (75%) coyotes. Prevalence of heartworm was higher among adults than juveniles in the fall, but no significant difference was detected between animals captured in winter. Antibodies were found against canine parvovirus (65%), canine parainfluenza virus (59%), infectious canine hepatitis virus (41%), and Toxoplasma gondii (18%). Antibodies were not found to Brucella canis, canine coronavirus, five serovars of Leptospira interrogans, or canine distemper virus. Seroprevalence of canine parvovirus was lower (P = 0.009) among fall-captured animals (33%) than winter-captured animals (100%). The Kaplan-Meier estimate of annual survival was 0.500 for all animals. Juvenile survival did not differ (P = 0.79) from adult survival, but male survival (S = 0.217) was lower (P = 0.11) than female survival (S = 0.804). Two of nine (22%) mortalities were human-caused, one was due to concurrent canine parvovirus and canine distemper virus infections, one animal died of trauma, two were considered natural mortalities of unknown cause, and no cause of death could be determined for the remaining three animals. Natural mortality may be significant for coyotes in south-central Georgia, although there was no apparent link between exposure to pathogens and the animals' subsequent fate in our small sample.     

Isolation of a novel adenovirus from California sea lions Zalophus californianus

Viral hepatitis associated with adenoviral infection has been reported in California sea lions Zalophus californianus admitted to rehabilitation centers along the California coast since the 1970s. Canine adenovirus 1 (CAdV-1) causes viral hepatitis in dogs and infects a number of wildlife species. Attempts to isolate the virus from previous sea lion hepatitis cases were unsuccessful, but as the hepatitis had morphologic features resembling canine infectious hepatitis, and since the virus has a wide host range, it was thought that perhaps the etiologic agent was CAdV-1. Here, we identify a novel adenovirus in 2 stranded California sea lions and associate the infection with viral hepatitis and endothelial cell infection. Phylogenetic analysis confirmed the classification of the sea lion adenovirus in the Mastadenovirus genus with the most similarity to tree shrew adenovirus 1 (TSAdV-1, 77%). However, as the sea lion adenovirus appeared to be equally distant from the other Mastadenovirus species based on phylogenetic analysis, results indicate that it represents an independent lineage and species. Although sequences from this novel virus, otarine adenovirus 1 (OtAdV-1), show some similarity to CAdV-1 and 2, it is clearly distinct and likely the cause of the viral hepatitis in the stranded California sea lions.     

Serologic survey for infectious canine hepatitis virus in grizzly bears (Ursus arctos) from Alaska, 1973 to 1987

Serum antibody prevalence of infectious canine hepatitis virus was 12% (90 of 725) for grizzly bears (Ursus arctos) from Alaska (USA) during the period 1973 to 1987. Prevalence was highest on Kodiak Island at 29% (37 of 127). Prevalence of exposure at individual collection areas did not change significantly over time. There were no significant sex-specific differences in prevalence. Prevalence was directly related to age, but it was 0% for bears less than 2-yr-old. Young bears which are exposed to the virus may develop clinical disease and die as a result of the infection. This disease may be a factor affecting grizzly bear population dynamics.     

Serologic survey for selected disease agents in wolves (Canis lupus) from Alaska and the Yukon Territory, 1984-2000

Wolves (Canis lupus) were captured in several geographic areas of Alaska (USA) and the Yukon Territory (Canada) during 1984-2000. Blood was collected from 1,122 animals. Sera were tested for antibodies against infectious canine hepatitis virus (ICH), canine distemper virus (CDV), canine parvovirus (CPV), Francisella tularensis, and serovars of Leptospira interrogans. Antibody prevalence for ICH was >84% for all areas. Area-specific prevalences of antibodies ranged from 12% to 70% for CPV, from 0% to 41% for CDV, and from 4% to 21% for F. tularensis. There was no evidence of CDV exposure at the two southernmost locations in Alaska. Prevalence of antibodies for ICH increased slightly during the 16-yr course of the survey. There was essentially no evidence of exposure to L. interrogans. Prevalences of antibodies for both CPV and CDV were age-specific, with higher values in the adult cohort compared with the pup cohort. There were no sex-specific differences in prevalence of antibodies for any of the five disease agents.     

Serologic survey for canine distemper and infectious canine hepatitis in wolves in Alaska

Sera from 57 wolves (Canis lupus) in three areas of Alaska were evaluated for evidence of previous exposure to infectious canine hepatitis virus (ICHV) and canine distemper virus (CDV). Fifty-four sera (94.7%) were positive for ICHV exposure and four (7%) were positive for CDV exposure. All four CDV-reacting wolves also had titres to ICHV. The relatively common occurrence of ICHV exposure may be due to the greater resistance of ICHV to chemical and physical agents and its transmissibility via the urine of infected animals. The ICHV titres observed could indicate enzootic pathogenic ICHV, or exposure to the mildly pathogenic vaccine strain of CAV-1 through contact with the urine of domestic dogs. If CAV-1 is the original source of exposure, the titres could represent an ICHV-protected wolf population.     

Prevalence of selected pathogenic microbial agents in the red fox (Vulpes fulva) and gray fox (Urocyon cinereoargenteus) of southwestern Wisconsin

Free-ranging red foxes (Vulpes fulva) and gray foxes (Urocyon cinereoargenteus) were trapped in southwestern Wisconsin. Fox sera were tested to determine the prevalence of antibody for five different Leptospira interrogans serovars, canine distemper virus (CDV), infectious canine hepatitis virus (ICHV), and Franciscella tularensis infections. Grippotyphosa was the most prevalent leptospiral serovar antibody observed. Twenty-five of 53 (47%) red foxes and 11 of 36 (31%) gray foxes had specific antibodies to grippotyphosa. Juvenile foxes had geometric mean antibody titers to grippotyphosa significantly higher (P less than 0.05) than those of the adults of both species. CDV antibody was detected in sera of red foxes only. Six of 57 (11%) red foxes had CDV antibody. ICHV antibody was detected in 2 of 57 (3%) red foxes and 3 of 32 (9%) gray foxes. Antibody to F. tularensis was not detected in any fox sera.     

Virological, Immunochemical, and Cytochemical Studies of Four HeLa Cell Lines Infected with Two Strains of Influenza Virus

The production of infectious virus, hemagglutinin, and viral (V) antigens and the changes in ribonucleoprotein (RNP) and lipoprotein metabolism have been studied in four sublines of HeLa cells infected with the PR8 and a PR8 recombinant strain of influenza virus. Much greater amounts of infectious virus and much less hemagglutinin were produced by the PR8 recombinant than by PR8 virus in all four cell lines. Different amounts of infectious virus per infected cell were produced by the recombinant in the four cell lines, whereas very little infectious virus was produced by the PR8 strain in any of the HeLa cells. In all cell lines infected with both strains of virus, "soluble" (S) antigen appeared early in the nucleolus. In cells infected with PR8 recombinant, S antigen subsequently filled the nucleus and later appeared in the cytoplasm. In most cells infected with PR8 virus, nuclear S antigen did not fuse to fill the nucleus, and S antigen was not detected in the cytoplasm. V antigen was observed in the cytoplasm of cells when diffuse nuclear S antigen had formed. The earliest and most frequent change in the RNP of the infected cells was a decrease in stainable RNP spherules (nucleolini) in the nucleolus. This was followed, in a smaller proportion of cells, by the appearance of nuclear and cytoplasmic inclusions containing RNP. There was a characteristic difference in the morphology of the cytoplasmic inclusions produced by the two strains of virus, but the same types of inclusions were observed in all four HeLa lines. A significant increase in lipoprotein was observed only in association with the cytoplasmic inclusions produced by PR8 recombinant virus. There was a striking difference in the proportion of cells with cytochemical changes in RNP in the four cell lines. A significant cytopathic effect (CPE) was observed only in three virus-cell systems in which a high proportion of cells exhibited changes in nucleolinar RNP. It is suggested that disappearance of RNP in the nucleolini may be an indication of shutdown of host ribonucleic acid synthesis and that this in turn results in a CPE. Virus infection resulted in a C-mitotic block that was followed by karyorrhexis. Infection of the cell did not always result in the production of infectious virus, in changes in the RNP of the nucleolini, in the development of nuclear or cytoplasmic RNP inclusions, or in CPE. The results suggest that production of infectious virus, shutdown of cellular RNP synthesis with accompanying CPE, and the formation of inclusions appear to be independent events.     

Hepatitis B virus DNA and e antigen in serum from blood donors in the United Kingdom positive for hepatitis B surface antigen.

Serum samples from 214 blood donors in the United Kingdom who were carriers of hepatitis B surface antigen (HBsAg) were examined for hepatitis B virus deoxyribonucleic acid (DNA) by DNA:DNA hybridisation and for hepatitis B e antigen (HBeAg) and its antibody. One fifth of the donors carried infectious virus in their circulation. The presence of hepatitis B virus DNA correlated well with that of HBeAg, although hepatitis B virus DNA was found in five serum samples that were negative for HBeAg. It is concluded that analysis of serum samples for hepatitis B virus DNA by hybridisation should be the method of choice for determining whether carriers of HBsAg are infectious.     

Macrotiter assay for coronavirus-neutralizing activity in cats using a canine continuous cell line (A-72)

A heterologous neutralization assay for feline infectious peritonitis virus serology was developed using a single continuous cell line of canine origin, A-72, which is susceptible to cytopathic infection with both transmissible gastroenteritis virus of pigs and canine coronavirus. Of several coronavirus isolates tested, the 1-71 isolate of canine coronavirus demonstrated the most effective neutralization by serum and body fluids of cats with histopathologically confirmed feline infectious peritonitis.     

Serological survey for selected diseases in the endangered San Joaquin kit fox (Vulpes macrotis mutica)

Blood from endangered San Joaquin kit foxes (Vulpes macrotis mutica) inhabiting the Elk Hills Naval Petroleum Reserve, Kern County, and the Elkhorn Plain, San Luis Obispo County, California, was collected in 1981, 1982 and 1984 and sera were tested for antibodies against 10 selected pathogens. Proportions of kit fox sera containing antibodies against pathogens were: canine parvovirus, 100% in 1981-1982 and 67% in 1984; infectious canine hepatitis virus, 6% in 1981-1982 and 21% in 1984; canine distemper virus, none in 1981-1982 and 14% in 1984; Francisella tularensis, 8% in 1981-1982 and 31% in 1984; Brucella abortus, 8% in 1981-1982 and 3% in 1984; Brucella canis, 14% in 1981-1982 and none in 1984; Toxoplasma gondii, 6% in 1981-1982; Coccidioides immitis, 3% in 1981-1982; and Yersinia pestis and Leptospira interrogans serotypes canicola, grippotyphosa, hardjo, icterohaemorrhagiae, and pomona, none in 1981-1982. Although antibodies against selected pathogens were present, no clinical indications of disease were observed in these fox populations.     

新生幼犬的被动免疫研究

新生幼犬通过初乳获得的被动免疫,对它的生存非常重要,本研究用犬的多价免疫血清代替初乳作为幼犬保护性免疫球蛋白,对生后2 d内不能获得初乳的幼犬,以及虽然获得初乳但初乳中特异性免疫球蛋白含量较低的幼犬进行了被动免疫试验。对5窝35只幼犬在生后,随机分为吃初乳的对照组、口服血清组、皮下注射血清组、以及口服和皮下注射血清又吃初乳5个组,在幼犬出生时、及出生后48 h和第5天采血,测定犬细小病毒、犬传染性肝炎、和犬副流感的血凝抑制（HI）抗体效价。结果表明：对没有吃到初乳的仔犬,通过早期口服或皮下注射途径给予成年犬的血清,均能代替初乳供给幼犬免疫球蛋白。对吃到初乳的仔犬,能进一步提高幼犬体内特异性抗体水平,但以皮下注射途径最佳。