Detection of bovine herpesvirus 1 in the semen of experimentally infected bulls by dot-blot hybridisation, polymerase chain reaction and virus isolation.

Two 18-month-old bovine herpesvirus 1 (BHV1)-seronegative bulls were inoculated experimentally with BHV1 via their prepuces. Semen collected at intervals was examined by optimised virus isolation, dot-blot hybridisation and the polymerase chain reaction (PCR) for detection of BHV1, and the infection was monitored serologically by using a virus neutralisation test. Antibodies were first detected 10 days after inoculation and were still present 40 days after inoculation. Semen collected from four to 40 days after inoculation was positive by PCR with Southern blot hybridisation whereas only the semen collected on day 4 was positive by dot-blot hybridisation, virus isolation and PCR with ethidium bromide staining. These results indicate that the bulls started to shed the virus in semen before they developed any detectable antibody. PCR with Southern blot hybridisation was the most sensitive of the three methods and detected virus for the longest period.

Comparison of dot blot hybridization, polymerase chain reaction, and virus isolation for detection of bovine herpesvirus-1 (BHV-1) in artificially infected bovine semen.

Bovine semen samples spiked with bovine herpesvirus 1 (BHV-1) were used to compare dot blot hybridization, polymerase chain reaction (PCR), and virus isolation for detection of BHV-1 in bovine semen. The PCR amplification used primers targeting the BHV-1 thymidine kinase gene and a nucleic acid releasing cocktail (GeneReleaser); the PCR product was used as the DNA probe in dot blot hybridization; virus isolation was done in primary bovine fetal testis (BFT) cell cultures. Semen diluted 1:20 in tissue culture medium had the least cytotoxicity and inhibition of viral cytopathic effects in BFT cells, allowing detection of 1 TCID50/100 microL of BHV-1 suspension by virus isolation. The presence of foreign DNA such as bovine sperm DNA or salmon sperm DNA increased the sensitivity of dot blot hybridization in detecting BHV-1, allowing detection of 20,000 TCID50/100 microL of neat semen. The inhibition of PCR amplification of BHV-1 DNA in bovine semen was eliminated by diluting the samples 1:20 in tissue culture medium. The best PCR amplification was obtained when semen was diluted 1:20 and when a reaction buffer of pH 9.0, with 1.0 mM MgCl2 was used. Under these conditions, the PCR followed by ethidium bromide staining of agarose gels could detect 1 TCID20/100 microL of sample, whereas PCR followed by Southern blot hybridization could detect 0.01 TCID50/100 microL of sample.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of conditions for the detection of bovine herpesvirus-1 by polymerase chain reaction using primers in the thymidine kinase region.

Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaserTM + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by Gene-ReleaserTM, a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaserTM showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaserTM has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization.

Amplification of strains of bovine herpesvirus 1 by use of polymerase chain reaction with primers in the thymidine kinase region.

A primer pair was designed from the published nucleotide sequence of the coding region of the bovine herpesvirus 1 (BHV-1) thymidine kinase (tk) gene for use in detection of the virus by use of polymerase chain reaction (PCR) amplification of 12 BHV-1 strains (3 ATCC and 9 local isolates). A tk deletion mutant BHV-1, and 2 BHV-4 strains from ATCC were used as negative controls. One strain each of feline herpes-virus, equine herpesvirus, and bovine adenovirus, and 2 noninoculated bovine cultured cells--bovine fetal testis and Madin-Darby bovine kidney--also were examined to verify specificity of the primers. A PCR product, 183 bp long, was detected by ethidium bromide staining after agarose gel electrophoresis, when purified DNA from cell cultures infected with BHV-1 strain LA was used as template. Specificity of the PCR product was confirmed by restriction digestion with Sac II enzyme and Southern blot hybridization. Amplification was detected by ethidium bromide staining of agarose gels and/or Southern blot hybridization with the radiolabeled PCR product of the LA strain in similarly prepared DNA templates of 5 other BHV-1 strains, 2 obtained from ATCC and 3 of the 9 local isolates. In a modified PCR protocol, using virus suspensions treated with a nucleic acid-releasing cocktail, substantial amplification was obtained for the 3 BHV-1 strains from ATCC and for all 9 local bovine herpesvirus field isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Proventricular impaction associated with nonsuppurative encephalomyelitis and ganglioneuritis in two Canada geese.

Two wild Canada geese (Branta canadensis) in an extremely emaciated state and with severe proventricular food impaction also had a nonsuppurative encephalomyelitis and ganglioneuritis. The condition in these two birds was morphologically similar to psittacine proventricular dilatation, a recently identified disease of psittacine birds.

Pathogenesis of rotavirus infection in various age groups of chickens and turkeys: pathology.

Various age groups of turkeys, White Leghorn chickens, and broiler chickens were inoculated with turkey rotavirus strain Tu-2 or with chicken rotavirus Ch-2, and the development of rotavirus-induced lesions were evaluated macroscopically and microscopically (light microscopy and scanning electron microscopy). Morphometric evaluations were conducted to determine morphologic changes in the villi of infected turkeys. Macroscopic lesions that were found in turkeys, but not in chickens, consisted of pallor of the intestinal tract and distension of the cecum with frothy or nonfrothy fluid contents. Histologic lesions in turkeys consisted of basal vacuolation of enterocytes, separation of enterocytes from the lamina propria (with subsequent desquamation), villus atrophy accompanied by widening of the lamina propria, scalloping of the villus surface, fusion of the villi, and leukocytic infiltration of the lamina propria. Scanning electron microscopy indicated roughened villus surfaces, distortion of the normal morphologic features of the villi, and loss of microvilli in cells located on the tips of the villi. Most of the lesions disappeared by 8 days after inoculation. Results of the morphometric evaluations indicated that the crypt length had increased and the villus-to-crypt ratio had significantly decreased, compared with that of noninoculated control turkeys. Broilers greater than or equal to 21 days old and White Leghorn chickens greater than or equal to 35 days old had minimal leukocytic infiltration of the lamina propria and minimal loss of microvilli in cells located on the tips of the villi. The loss of microvilli was more extensive in chickens greater than or equal to 119 days old than in younger birds. Generally, turkeys 1 to 112 days old developed more severe lesions than did chickens, and lesions were more pronounced in turkeys at 112 days of age.

Pathogenesis of rotavirus infection in various age groups of chickens and turkeys: clinical signs and virology.

Age-related susceptibility patterns of turkeys, broilers, and specific pathogen-free (SPF) White Leghorn chickens to experimentally induced infection with turkey or chicken rotavirus isolates were compared. The following determinants were evaluated: clinical signs, onset and duration of virus production, viral titers, involvement of intestinal villi in the replication of the virus, and the development of antibodies against the virus. Older turkeys and chickens were more susceptible than were their younger counterparts, turkeys were more susceptible than were broiler and White Leghorn chickens (regardless of age), and broiler chickens were slightly more susceptible than were age-matched White Leghorn chickens. Turkeys developed diarrhea, accompanied by high viral titers within 1 day after inoculation with virus. Viral antigen was found in the epithelial cells of the intestinal villi throughout the intestinal tract and some cells of the cecal tonsils. Antibodies could be detected as early as 4 to 5 days after inoculation. These findings were more pronounced in turkeys inoculated at 112 days of age than in birds inoculated at a younger age. Age-related susceptibility patterns were similar in White Leghorn and broiler chickens. Infection was subclinical in birds less than 56 days old, whereas older birds developed soft feces. Egg production in the White Leghorn chickens decreased after being inoculated with virus at 350 days of age.

Experimental infection of specific-pathogen-free chickens with avian rotaviruses.

One-to-70-day-old specific-pathogen-free chickens were infected with rotaviruses isolated from chickens, turkeys, and pheasants. Chicks infected at 1 or 7 days of age remained largely free of infection: virus could be isolated from a few chicks of these ages, but virus antigen could not be detected in the epithelial cells of the intestinal villi. Treating the virus with trypsin before inoculation did not markedly enhance virus infectivity. The same batches of virus successfully infected chickens at 56 and 70 days of age: virus could be isolated between 1 and 5 days postinfection, and virus antigen was detected in the epithelial cells of the intestinal villi. Rotaviruses isolated from pheasants were infectious for 49-day-old chickens. All infections in the older chickens remained subclinical.

Pathogenesis of rotavirus infection in turkey poults.

The pathogenesis of rotavirus infection was examined after experimental infection of conventional and specific-pathogen-free (SPF) turkey poults. In six experiments birds were exposed to turkey rotavirus isolates Tu-1 or TU-2 or the chicken isolate Ch-1 at 7, 10 or 42 days of age. Poults were examined between 1 and 24 days post-infection (dpi) for diarrhoea, gross and histopathologic lesions, virus excretion in the intestinal tract, viral antigen in intestinal epithelial cells, and the development of serum antibodies. Between 2 and 5 dpi watery droppings were observed in conjunction with remarkable paleness of the intestinal tract which was grossly observable. Maximum viral replication occurred between 2 and 5 dpi, during which period viral antigen could be demonstrated in the epithelial cells of the duodenum, jejunum, ileum and colon. Sporadically, virus antigen-positive cells were seen in the cecum. As early as 4 to 5 dpi rotavirus antibodies could be detected by indirect immunofluorescence assays. Remarkable leukocyte infiltration of the lamina propria, vacuolation of the epithelial cells and scalloping of the villous surface at the tips were observed in the intestine of infected birds. Infection with rotavirus caused a significant impairment at 2 and 4 dpi of absorption of D-xylose from the intestinal tract.

Isolation and characterization of avian rotaviruses.

Rotaviruses were isolated from intestinal contents obtained from flocks of turkey poults and pheasant poults with diarrhea and from different age groups of chickens showing various signs of intestinal disorders. The incorporation of 5 micrograms trypsin/ml in the inoculum and medium was essential for virus isolation in chicken kidney cells. All isolates were identified as rotaviruses by fluorescent-antibody technique using a National Institutes of Health reference rotavirus antiserum against human rotavirus strain "D," Type 2. Negative-staining and transmission electron microscopy showed the presence of rotavirus particles. Furthermore, polyacrylamide gel electrophoresis pattern of viral RNA segments of our isolates confirmed that they are rotaviruses. Seven-day-old turkey poults could be infected with turkey and chicken rotavirus isolates; in contrast, chicks of the same age were refractory.

Clinical and pathologic features of experimental Schistosoma japonicum infection in pigs.

Nine piglets, 2-5 months old and weighing 12-15 kg were infected percutaneously with 5000 to 6000 cercariae of Schistosoma japonicum. Three similar piglets which were not infected served as controls. Infected animals were necropsied at 5, 10, 30, 40, 59, 90, 120, 150 and 180 days post-infection (PI) and the controls at 5, 60 and 180 days PI. The prepatent period varied from 27 to 33 days after infection. Clinical signs observed, coincident with egg production, were loss of appetite, lethargy, pallor of mucous membranes, diarrhea, progressive emaciation and dehydration. One piglet was moribund when killed for necropsy at 40 days and another piglet died 59 days PI. Pathological changes induced by S. japonicum included erythematous papules on the site of inoculation, petechial hemorrhages in the lungs, catarrhal to hemorrhagic enteritis, bluish-gray discoloration of the liver, and egg granulomas in the liver, lungs, spleen, intestines, pancreas and mesenteric lymph nodes. Endophlebitis with intimal hyperplasia was occasionally observed in veins harboring adult schistosomes.