Anthony E. Castro

Circovirus-Like Infection in a Pigeon:

4,7,10-13 These viruses are similar in that they are small (15-17 nm), icosahedral, and nonenveloped and contain single-stranded circular DNA. Each virus, however, has distinct DNA sequences and antigenicity. Psittacine beak and feather disease virus has the largest host range and has been reported in over 35 species of Old World and New World psittaciforms but has never been reported in other avian families. 4 This is the first case report of circovirus-like infection in a pigeon (Columbiformes). A young female racing pigeon was submitted for necropsy to the Davis branch of the California Veterinary Diagnostic Laboratory System in the fall 1990. The owner had 50 birds (adult and young), and mortality in the squabs was 100%. Three to 5 birds had died per week. Squabs exhibited anorexia and lethargy and died 3-4 days after clinical signs appeared. On gross examination, the pigeon was emaciated, with pectoral muscle atrophy. The mucosal wall of the intestinal tract was slightly red, and luminal contents were foamy and yellow-green. The spleen was enlarged. Air sacs were edematous, and there were caseous cores within the primary bronchi of both lungs. The right lung was consolidated and red. A defined bursa of Fabricius was not evident on gross examination of the site dorsal to the cloaca. The lung and liver were cultured aerobically on blood agar. A Pasteurella sp. was isolated from the lung. Bacteria were not isolated from the liver. Air sacs cultured on selective media for Mycoplasma sp. exhibited no growth. Chlamydia was not identified in Gimenez-stained impression smears of the liver, air sac, and spleen nor in liver, air sac, and spleen impression smears stained with fluorescence-labeled monoclonal antibody specific for Chlamydia a and examined with ultraviolet light. However, Chlamydia psittaci was isolated in a McCoy cell culture system from an organ pool of liver, air sac, and spleen. Viruses were not isolated from pooled tissue homogenates of liver, spleen, lung, brain, and intestine

Evaluation of the infectivity, length of infection, and immune response of a low-pathogenicity H7N2 avian influenza virus in specific-pathogen-free chickens

Abstract The H7N2 subtype of avian influenza virus (AIV) field isolate (H7N2/chicken/PA/3779-2/97), which caused the 1997–98 AIV outbreak in Pennsylvania, was evaluated for its infectivity, length of infection, and immune response in specific-pathogen-free (SPF) chickens. The composite findings of three clinical trials with various concentrations of virus indicated that this H7N2 subtype contained minimal pathogenicity for chickens. The concentration of the virus in the inoculum proved critical in the establishment of a productive infection in a chicken. Seven-day-old SPF chickens were not infected when inoculated with 100.7–2.0 mean embryo lethal dose (ELD50) of the H7N2 virus per bird. At this dose level, the immune response to this virus was not detected by the hemagglutination-inhibition (HI) test. Nonetheless, chickens at ages of 5 and 23 wk old tested were successfully infected when exposed to 104.7–5.7 ELD50 of H7N2 infectious doses per bird by various routes of administration and also by direct contact. Infected birds started shedding virus as early as 2 days postinoculation, and the period of virus shedding occurred mostly within 1 or 2 wk postinoculation (WPI). This H7N2 subtype of AIV induced a measurable immune response in all birds within 2 wk after virus exposure. Antibody titers were associated with AIV infectious doses and age of exposure of birds. Challenge of these infected birds with the same H7N2 virus at 5 and 10 WPI indicated the infective virus was recoverable from cloacal swabs at 3 days postchallenge and disappeared thereafter. In these challenged birds, the antibody levels as measured by the HI test spiked within 1–2 wk.

Detection of Bluetongue Virus in Clinical Samples by Polymerase Chain Reaction

A previously described bluetongue virus (BTV) serogroup polymerase chain reaction (PCR) assay was applied to clinical samples. The sensitivity of the BTV serogroup PCR was increased by the use of non-radioactive chemiluminescent hybridization. Unfractionated whole blood samples from rams experimentally inoculated with cell culture-adapted BTV- 11 UC-8 were analyzed by virus isolation (VI) on Vero cells and PCR. VI and PCR were in agreement, with the exception of 3 blood samples that were VI negative and PCR positive. In semen spiked with BTV- 11 UC-8, PCR detected as little as 1.6 × 102 plaque-forming units of BTV/ml of semen. BTV in the spleen of a sheep submitted for necropsy for suspect BTV infection was detected by both PCR and VI in embryonated chicken eggs. BTV PCR with nonradioactive chemiluminescent hybridization resulted in a level of sensitivity comparable to that of VI and likly more sensitive than VI on Vero cells for blood. This BTV PCR has great promise for rapid, sensitive, and specific detection of active BTV infection in a variety of clinical samples.

Comparison of polyacrylamide gel electrophoresis, an enzyme-linked-immunosorbent assay, and an agglutination test for the direct identification of bovine rotavirus from feces and coelectrophoresis of viral RNA's.

The dsRNA concentrated polyacrylamide gel electrophoresis (CPAGE) detected rotavirus directly from 19% of 77 stool specimens from diarrheic calves. A commercial enzyme-linked immunosorbent assay (ELISA) detected 25%, latex agglutination test, 23%, and polyacrylamide gel electrophoresis (PAGE), 19%. Establishing CPAGE as the “standard,” the commercial ELISA and the latex agglutination test both had higher sensitivity (84%) than PAGE (79%). However, PAGE produced the highest specificity (100%), followed by agglutination (88%) and ELISA (84%). The commercial ELISA had a slightly higher sensitivity than agglutination, PAGE, and CPAGE, but the ELISA specificity was generally lower. The latex agglutination test had a lower sensitivity than ELISA, but specificity was higher. Agglutination had similar negative predictive values (94%), compared with agglutination and PAGE, but had the lowest positive predictive value (a measure of accuracy) (70%). Agreement with CPAGE was highest for PAGE (94.8%), followed by agglutination (87%) and ELISA (84.4%). The calculated percentages of total disagreement with all other tests indicated that ELISA differed from the other rotavirus detection assays in 10.4% of the cases, agglutination in 7.8%, PAGE in 2.6%, and CPAGE in 1.3%. The 2 PAGE assays allowed the detection of atypical rotaviruses from feces based on the characteristic “super-short” migration pattern of the 11 genomic segments of rotaviruses and of other members of the Reoviridae.

PCR Detection of Group A Bovine Rotaviruses in Feces

A polymerase chain reaction (PCR) protocol has been developed for identification of bovine group A rotavirus infection in feces. Primers (20mers) complementary to 3′ ends of double-stranded RNA genome segment 6 of bovine rotavirus NCDV strain were synthesized and used in PCR. Bovine rotavirus RNA from infected cell culture was employed to optimize the PCR protocol. Rotavirus-negative fecal samples were spiked with known quantities of bovine rotavirus, and the sensitivity of the PCR assay was determined. Fecal samples were extracted with phenol and treated to eliminate unidentified PCR inhibitor(s) in feces, and PCR was performed. PCR products were either visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the assay was 6 × 104 viral particles/ml of feces with ethidium bromide-stained agarose gel visualization or 6 × 102 viral particles/ml of feces with chemiluminescent hybridization. The PCR assay was applied to 18 fecal specimens from clinical cases. All 16 clinical samples that were positive for rotavirus by enzyme-linked immunosorbent assay (ELISA) or by ELISA and electron microscopy (EM) were positive by PCR. The 2 samples that were rotavirus negative by ELISA or by ELISA and EM were also negative on PCR analysis.

Association of Reoviridae particles in an enteric syndrome of poults observed in turkey flocks during 1988

An enteric syndrome of turkey poults, characterized by enteritis, crop mycosis, intestinal changes (pale, thin-walled ballooning with watery contents), and rickets, occurred during 1988 in 74 turkey flocks from different farms belonging to 9 California turkey growers. The flocks ranged in size from 9,000 to 120,000 birds. Pools of intestine sections from 618 birds, representing 78 field cases, were examined. Histopathological examination of the intestines showed a mild to severe atrophy with a reduced depth of crypts, which was more prominent in the distal part of the small intestine. Viral isolation attempts with primary cell cultures of chicken embryo kidney cells were negative. Examination by electron microscopy of negatively stained intestinal specimens revealed the presence of Reoviridae particles of 58.8 to 80 nm in diameter. Enzyme-linked immunosorbent assay results on the intestinal pools for mammalian and group A avian rotaviruses were negative. A statistically significant relationship was found for the presence of Reoviridae particles in the intestines of 10-21-day-old birds. Of the 7 most common pathological conditions analyzed, 2, rickets and intestinal changes (thin-walled ballooning intestine with watery contents), showed a statistically significant association with the presence of Reoviridae particles.

RESTRICTION ENDONUCLEASE ANALYSIS OF HERPESVIRUSES ISOLATED FROM TWO PENINSULAR BIGHORN SHEEP (OVIS CANADENSIS CREMNOBATES)

In 1989, herpesviruses were isolated from nasal swabs taken from two peninsular bighorn sheep (Ovis canadensis cremnobates) in the Anza-Borrego Desert State Park, San Diego County, California (USA). Using restriction endonuclease analysis (REA) with Pst1 enzyme, each isolate was found to be similar to the Cooper strain of infectious bovine rhinotracheitis virus (IBRV). The REA patterns of the two herpesviruses from bighorn sheep were typical of either field strains or vaccine strains of IBRV commonly associated with cattle in the USA.

Comparison of electron microscopy and polyacrylamide gel electrophoresis in the diagnosis of avian reovirus and rotavirus infections.

Electron microscopy (EM) and genome electropherotyping by polyacrylamide gel electrophoresis (PAGE) for the detection of avian rotaviruses and reoviruses in intestinal specimens and cell cultures were compared. Fifty-eight field samples of intestine with intestinal contents, referred to as direct specimens, from turkey and chicken flocks located in different regions of California and submitted during 1989 for virus isolation were randomly selected as test samples. Also, 38 field intestinal specimens with suspected viral infection that had been passaged three times in primary chicken embryo kidney (CEK) cell cultures were used in their third passage. The percentage of agreement and the Kappa statistic of positive and negative results between these two tests were calculated. In the comparison, EM was considered the standard test. By statistical analysis, an agreement of 87% was observed in cell-culture samples analyzed by the two virus-detection methods, as contrasted with an agreement of 72% for direct specimens. The analysis of the number of segments and band migration profiles of reference and field virus strains indicated that only reoviruses replicated in CEK cell cultures and mainly rotaviruses were detected by both tests in direct specimens. The Kappa statistic analysis indicated substantial agreement (0.69) between the two tests for CEK samples, with moderate agreement (0.45) for the direct specimens examined.

Herpes viral hepatitis in a toucan.

Herpesvirus infection was diagnosed in a toucan. The herpesvirus was isolated from the liver and identified by electron microscopy in the liver and in cell culture. A negative immunofluorescent reaction was obtained when virus-infected cell cultures were reacted with a conjugate to the herpesvirus of Pachecos disease. The main pathologic finding in the toucan consisted of a severe necrotizing hepatitis with intranuclear inclusions in the liver and spleen. A presumptive diagnosis of chlamydiosis was also made, based on a positive direct fluorescent monoclonal antibody reaction to chlamydial antigens in impression smears of liver and spleen. Chlamydial isolation attempts were unsuccessful. The toucan had been in contact with two macaws that had died 5 days before the toucan died and were diagnosed by histology as having herpesvirus hepatitis.

Susceptibility of a fetal tongue cell line derived from bighorn sheep to five serotypes of bluetongue virus and its potential for the isolation of viruses

A cell line (BHFTE) was derived from a tongue explant of a bighorn sheep fetus (Ovis canadensis nelsoni). The cells have been maintained through 23 serial passages, and the modal number of chromosomes was calculated to be 55. Monolayer cultures were shown to be susceptible to various viruses, including bluetongue virus (BTV). Of 5 BTV serotypes (2, 10, 11, 13, and 17) tested, each produced a cytopathic effect (CPE) on initial passage at 33 C. A field isolate (serotype 10) of BTV from a black-tailed deer (Odocoileus hemionus columbianus) in its second passage in Vero-M cells also produced CPE when inoculated into BHFTE cells. Antigens of BTV were demonstrated by direct immunofluorescence in the cytoplasm of BHFTE cells inoculated with homogenates of chicken embryos injected with clinical specimens from a domestic sheep and an Arabian oryx (Oryx gazella leucoryx). A suspension of BTV-infected gnats (Culicoides spp.) produced CPE and BTV-specific fluorescence on the first passage in cells inoculated with a suspension of blood from sheep experimentally infected with BTV. Additionally, selected bovine viruses induced CPE in the cells. The cell line, which is free of mycoplasma and bovine viral diarrhea virus contamination, may be useful in diagnostic medicine and research involving the ruminant species.