Persistent infection of turkeys with an avirulent strain of turkey hemorrhagic enteritis virus.

The Virginia avirulent strain (VAS) of turkey hemorrhagic enteritis virus (THEV), which is commonly used in live vaccines for commercial turkeys, was studied to determine characteristics of infection. It has been observed that turkeys infected with the VAS maintain protective antibody levels in excess of 20 wk postvaccination. It is theorized that this immune response is modulated by either a persistent or latent infection. A series of studies have been undertaken to determine changes in virus location and serology over time. A trial was also conducted to evaluate the effect of corticosteroid administration on viral recrudescence, and an attempt was made to isolate live virus from tissues of birds 10 wk postinfection (pi). Antibody titers were determined by enzyme-linked immunosorbent assay, and PCR was used to detect viral DNA. Histopathology was performed on formalin-fixed paraffinized tissues. Viral DNA was detected in various tissues through 15 wk pi in the presence of high antibody titers. Viral DNA was detected at 3-5 days pi in the spleens of susceptible turkeys inoculated with tissues collected from infected birds at 10 wk pi. It is unknown whether the viral DNA is associated with live virus or rather is the result of persistent maintenance of the viral genome within lymphoid/macrophage target cells. Future studies will test for viral RNA in order to confirm the presence of replicating THEV. Regardless of the actual status of the THEV DNA detected at 10-15 wk pi, it is clear that THEV does not cause a simple acute infection. The characteristics of THEV infection are identical to the nonlytic persistent infections seen in human adenoviruses, and therefore THEV may serve as a model for the study of virus-cell interactions mediating persistence.

Development and validation of a negative-strand-specific reverse transcription-PCR assay for detection of a chicken strain of hepatitis E virus: identification of nonliver replication sites.

As a positive-strand RNA virus, hepatitis E virus (HEV) produces an intermediate negative-strand RNA when it replicates. Thus, the detection of negative-strand viral RNA is indicative of HEV replication. The objective of this study was to develop a negative-strand-specific reverse transcription-PCR (RT-PCR) assay for the identification of extrahepatic sites of HEV replication. Briefly, a 494-bp fragment within the orf1 gene of a chicken strain of HEV (designated avian HEV) was amplified and cloned into a pSK plasmid. A synthetic negative-strand viral RNA was generated from the plasmid by in vitro transcription and was used to standardize the assay. A nested set of primers was designed to amplify a 232-bp fragment of the negative-strand viral RNA. The assay was found to detect up to 10 pg and 10(-5) pg of negative-strand HEV RNA in first- and second-round PCRs, respectively. The standardized negative-strand-specific RT-PCR assay was subsequently used to test 13 conveniently obtained tissue specimens collected sequentially on different days postinoculation from chickens experimentally infected with avian HEV. In addition to the liver, the negative-strand-specific RT-PCR assay identified replicative viral RNA in gastrointestinal tissues, including the colorectal, cecal, jejunal, ileal, duodenal, and cecal tonsil tissues. The detection of replicative viral RNA in these tissues indicates that after oral ingestion of the virus, HEV replicates in the gastrointestinal tract before it reaches the liver. This is the first report on the identification of extrahepatic sites of HEV replication in animals after experimental infection via the natural route. The assay should be of value for studying HEV replication and pathogenesis.

Utility of diagnostic tests used in diagnosis of infection in dogs experimentally inoculated with a North American isolate of Leishmania infantum infantum.

Eight female beagles were infected with 1 x 10(7) (low dose, LD) or 2 x 10(8) (high dose, HD) promastigotes of a North American isolate of Leishmania infantum infantum (LIVT-1 strain) isolated from naturally infected Virginia Foxhounds. Two female beagles served as negative controls and 2 male beagles chronically infected (> 3 years) with Leishmania infantum chagasi were positive controls. Bone marrow (BM) and lymph node (LN) aspirates were collected every 6-8 weeks for cytologic evaluation, parasite culture, and polymerase chain reaction (PCR). Serum samples were collected monthly for determination of serologic responses by indirect fluorescent antibody test (IFAT) and diagnostic rK39 antigen. Cultures of BM and LN aspirates and cytology evaluation were consistently positive in positive control dogs during the course of study. Negative control dogs were negative on BM and LN cultures and on cytologic evaluation of aspirates. Amastigotes were present on cytological examination of BM aspirates in 2 experimentally infected dogs. Cultures of LN aspirates were positive on 22 samples, whereas BM cultures were positive on 12 samples for all dogs. IFA titers ranged from 0 to 1 :400 in experimentally infected dogs during the course of the study. Recombinant K39 immunoassay tests were consistently positive in positive control dogs and in the HD dogs by approximately 8 weeks after infection. BM PCR products were identified more consistently in the HD dogs compared with the LD dogs. Kappa statistics indicated PCR correlated better with cultures and cytology than did IFAT or the rK39 immunoassay results in the experimentally infected dogs.

Antigenic diversity of respiratory syncytial viruses and its implication for immunoprophylaxis in ruminants.

Bovine respiratory syncytial virus (BRSV) is a very important pathogen of cattle and perhaps other ruminants. It is a major contributor to the incidence of respiratory tract disease in nursing beef and feedlot and dairy calves. The genome of respiratory syncytial viruses encodes 10 proteins translated from 10 unique mRNAs. The major glycoprotein (G), fusion protein (F), 1A protein and the 22K protein are components of the viral envelope. The nucleocapsid contains the nucleocapsid protein (N), the phosphoprotein (P), and the large protein (L). The matrix protein (M) forms a structural layer between the envelope and the nucleocapsid. Antibodies to all the structural proteins develop in convalescent calves. However, evidence suggests that immunity develops primarily as a result of the antigenic stimulus by the major glycoprotein G and the fusion glycoprotein F. It is known also that activated cytotoxic T cells interact with N and F protein antigens and helper T cells interact with N, F, and 1A protein antigens. With the exception of the major glycoprotein, the respective proteins of various respiratory syncytial viruses share major antigenic domains. Based on antigenic differences of the major glycoprotein, at least 3 subgroups of RSV are recognized; human A, human B, and bovine RSV. Indirect evidence suggests that a second subgroup of BRSV exists. However, we have identified only one BRSV subgroup based on our work with RNase mismatch cleavage analysis of the G protein gene from a limited number of strains. Furthermore, our data indicated that a caprine RSV isolate is closely related to the bovine strains, but an ovine isolate is not. The latter may constitute yet another subgroup of RSV. These data affect decisions on optimization of immunoprophylaxis since evidence suggests that protection against a homologous RSV subgroup virus is superior to that against a heterologous strain in immune subjects.

Analysis of ruminant respiratory syncytial virus isolates by RNAse protection of the G glycoprotein transcripts.

Two different respiratory syncytial virus (RSV) radiolabeled probes were used to characterize the genetic heterogeneity of 25 ruminant RSV isolates by the ribonuclease protection assay. A 32P-radiolabeled antisense RNA probe was transcribed from cloned ovine and bovine RSV G glycoprotein genes and then hybridized with total RNA isolated from infected cells with various ruminant RSV isolates. The results of this study, along with previously published nucleotide sequence data of the ovine RSV G glycoprotein gene, suggest the presence of at least 2 ruminant RSV subgroups. One subgroup is represented by RSV isolated from respiratory disease outbreaks from calves and goats, and the other is represented by RSV isolated from sheep.

Complex vertebral malformation in a holstein calf: report of a case in the USA.

Complex vertebral malformation (CVM), a familial syndrome of Holstein calves, has been reported in aborted fetuses and in prematurely born, stillborn, and neonatal calves. Affected calves have anomalies in the vertebral column, including hemivertebrae, fused and misshapen vertebrae and ribs, scoliosis, and vertebral synostosis. Concurrent low body weight, symmetrical arthrogryposis, and cardiac anomalies have been documented in affected calves. The syndrome was identified and characterized in Holstein cattle in Denmark; however, a global distribution of this genetic disorder is likely based on identification of common ancestral sires widely used for artificial insemination. This is the first documented case of CVM in a Holstein calf in the USA.

Confirmation that the dog is a definitive host for Neospora caninum.

Two mixed-breed littermate dogs were fed mouse brains containing tissue cysts of the NC-beef isolate of Neospora caninum. Both dogs excreted N. caninum oocysts in their feces. Dog 1 which was given methylprednisolone acetate (MPA) prior to ingesting tissue cysts, excreted oocysts on days 5 to 10 inclusive and on day 17 after ingesting tissue cysts. Dog 1 had a serum antibody titer of 1:200 in the indirect fluorescent antibody test (IFAT) 35 days after it was fed tissue cysts. Dog 2, which was not treated with MPA, excreted oocysts on Day 6 and Day 9 after ingesting tissue cysts. Antibodies to N. caninum were not found in a 1:25 dilution of serum on any examination period for Dog 2 during the study. Neospora caninum was not found in the tissues of either dog by histological or immunohistochemical means following necropsy 42 days after being fed tissue cysts. The identity of the oocysts excreted in the feces of the dogs was confirmed by mouse inoculation studies.

Neospora hughesi: experimental infections in mice, gerbils, and dogs.

Neospora hughesi is a recently described cause of equine protozoal myeloencephalitis (EPM). A rodent model for pathogenicity would facilitate development of therapies to be used in horses. In the present study, we examined the susceptibility of BALB/c gamma-interferon gene knockout (gamma-INFKO), BALB/c, CD-1, and C57BL/6 strains of mice and gerbils to infection with tachyzoites of the Nh-A1 strain of N. hughesi isolated from a horse from AL, USA. Only the gamma-IFNKO mice developed severe clinical disease following infection with N. hughesi and died 19-25 days after infection and exhibited severe cardiac lesions. In contrast, experimental infection of gamma-INFKO mice with tachyzoites of the NC-1 or NC-Liverpool strains of Neospora caninum resulted in deaths 8-10 days after infection. The most severe lesions were in the livers, spleens, and lungs of these mice. Gerbils inoculated with N. hughesi did not develop clinical disease, had few microscopic lesions, but did seroconvert. Two dogs fed the brains of mice, shown to contain N. hughesi tissue stages by cell culture and gamma-IFNKO mouse bioassay, did not shed N. caninum-like oocysts over a 23 days observation period. The marked difference in pathogenicity between the two species of Neospora in gamma-IFNKO mice, and lack of oocyst excretion by dogs fed N. hughesi infected mice provide additional evidence that the species distinction between N. caninum and N. hughesi is valid.

Malleus handle probe: a middle ear diagnostic procedure.

Manual probing of the malleus handle (MH) of 477 patients suggests a protective middle ear reflex with a variable tonic contraction of the tensor tympani muscle (TTM). The degree and type of MH retraction suggests three stages of progressive middle ear inflammation. Prediction accuracy of 250 myringotomy findings was 60% with impedance audiometry and 92% with MH probing. Mechanical pressure gauge readings (grams) of 202 ears correlated with finger pressure estimations. The TTM tendons were sectioned in ten children with persisting otitis media with effusion (OME) and seven obtained benefit. The three diagnostic methods, pneumatic otoscopy, impedance audiometry and MH probing, assess similar and separate perimeters and are complementary. Malleus handle probing alone detects attic loculation of OME, 54 of 954 ears (5.6%). it extends the diagnostic range to the TTM activities.

Bony auditory tube and otitis media with effusion.

The author has for 25 years manually probed the malleus handle for indications of middle ear pathology. Dissection of the bony auditory tube (BAT) and isthmus comprised 120 fresh autopsy and 26 anatomically prepared temporal bones. The semicanal lamina was not completely ossified in all infants and children and 52% of adults. The tensor tympani muscle was edematous and prolapsed into the superior portion of the BAT in eight ears with otitis media with effusion. Infants and children have the tendon of the tensor tympani muscle inserting into the neck of the malleus at an angle of 60 degrees or greater, possibly explaining the relatively large muscle bulk. It is suggested from these autopsy findings that the BAT and isthmus lumen is a functional 2-way channel for air superiorly and mucus streaming inferiorly. Manual palpation of the cartilaginous auditory tube in infants did not suggest malacia.

Molecular characterization of a Cryptosporidium isolate from a black bear.

To further validate the observation of the existence of host-adapted strains of Cryptosporidium parvum, we genetically characterized an isolate of Cryptosporidium parasite from a black bear. Sequence analysis of the ribosomal RNA small subunit and the 70-kDa heat shock protein (HSP70) showed that this parasite represents a new genotype of C. parvum and is related to the C. parvum dog genotype. This finding is helpful for clarifying Cryptosporidium taxonomy.

The gastroduodenal effects of buffered aspirin, carprofen, and etodolac in healthy dogs.

Twenty-four healthy mixed-breed dogs were divided into 4 groups. Group 1 received a placebo p.o. q12h, group 2 received an average of 16.5 (15.1-17.8) mg/kg buffered aspirin p.o. q12h, group 3 received an average of 2.2 (2.0-2.4) mg/kg carprofen p.o. q12h, and group 4 received an average of 12.8 (11.7-13.8) mg/kg etodolac p.o. q24h (with a placebo in the PM). All treatments continued for 28 consecutive days. Gastroduodenal endoscopy was performed on days -9, 0, 5, 14, and 28. Multiple gastric biopsies were obtained endoscopically on day -9 to determine each dog's Helicobacter infection status. Four regions in the stomach and 1 region in the proximal duodenum were evaluated endoscopically, and each was assigned a score from 1 to 11. Scores for each region then were summed to give a total score for each endoscopic evaluation. Erosions and submucosal hemorrhages were seen in all dogs receiving aspirin. Only minor gastric lesions were observed in the carprofen, etodolac, and control groups. No adverse clinical signs were noted in any dog given any treatment. Median total score on days 0, 5, 14, and 28, respectively, were as follows: group 1: 5.0, 5.0, 5.0, 5.0; group 2: 5.0, 27.0, 26.0, 27.5; group 3: 5.0, 5.0, 6.0, 5.0, group 4: 5.0, 7.0, 5.0, 5.0. There was no significant difference among dogs receiving carprofen, etodolac, or placebo. The administration of carprofen, etodolac, or placebo to healthy dogs resulted in significantly less gastroduodenal lesion development than in dogs receiving buffered aspirin.

Fatal lungworm infection in an opossum.

A 3-mo-old male, feral opossum (Didelphis virginiana) was presented to the University of Tennessee College of Veterinary Medicine (USA) for evaluation of prolonged dyspnea. The animal died shortly after examination and a necropsy was performed. The cause of death was attributed to a verminous pneumonia, associated with numerous intrabronchial and intrabronchiolar nematodes, subsequently identified as Didelphostrongylus hayesi.

Cryptosporidiosis in a black bear in Virginia.

Cryptosporidiosis has not been previously reported in black bears in North America, either free-roaming or captive. However, oocysts have been documented in two captive Malayan sun bears (Helarctos malayanus) located in zoological parks in Taiwan. Developmental stages of Cryptosporidium parvum were observed in tissue sections from the small intestine of a black bear cub found dead in Virginia (USA).

Acute sarcocystosis in a captive white-tailed deer in Virginia.

A 19-mo-old female captive white-tailed deer in a public wild animal park in Richmond (Virginia, USA) was necropsied and evaluated histologically following spontaneous death after a 1 wk period of lethargy in a captive herd of 22 deer. An acute necrotizing pneumonia was associated with intraendothelial protozoal schizonts that were identified immunohistochemically as Sarcocystis spp. This is the first confirmed report of acute sarcocystosis in a wild ruminant.

Pulmonary hypertension in a group of dairy calves.

An episode of pulmonary arteritis and sclerosis in twenty 5- to 6-month-old dairy calves was investigated. Sixteen of the calves died acutely, without marked premonitory signs of disease. Four calves evaluated clinically had lethargy, pallor, weakness, tachycardia, tachypnea, and jugular venous distention. Cardiac catheterization performed in 3 of the calves revealed pulmonary hypertension; 1 of these calves survived. Necropsy findings in 19 calves included pale lungs and excess free fluid in the pleural and abdominal cavities. In addition, 13 of 19 calves had a dilated and thin-walled right ventricle; 4 of the calves had right-sided cardiac hypertrophy, and 2 had dilatation of the pulmonary artery. Microscopically, pulmonary arteritis and sclerosis of the small to medium-sized arteries were evident in all calves submitted for necropsy. A lung biopsy specimen from a surviving calf had similar lesions. Centrilobular hepatic necrosis was found in 17 of 19 calves. Investigation of the disease episode, including feed analysis for toxins and serologic and microbiological studies of clinically affected calves and clinically normal in-contact penmates, failed to reveal any associated risk factor. The pulmonary arterial changes in the calves were similar to lung lesions in rats fed monocrotaline.

Otolaryngeal allergy in Wellington. 1971-1975.

Two thousand and twenty-nine patients received allergic therapy 1971-1975. Nine hundred and eighty-nine were followed up six to 24 months later. All received Rinkel's inhalant skin titration method. The first group 1971-1972 of 309 patients were investigated with Lee's food provocation method and useful relief of symptoms occurred in 27.6 per cent of patients. The second group 1973-1975 of 680 patients were investigated with Bryan's cytotoxic food test, 60 percent reported very good relief and 26 percent useful relief of symptoms. Successfully treated patients were able to curtail or abandon drug therapy and the frequency of surgery was decreased. The authors believe allergic symptoms are of common occurrence in patients frequently seeking medical attention.

Inferolateral ST-segment elevation associated with a gastric variceal bleed and the use of a Minnesota tube.

The use of Minnesota and modified Sengstaken-Blakemore tubes for balloon tamponade in acute variceal haemorrhage has declined with the availability of modern endoscopic techniques. However, in massive uncontrolled haemorrhage their use may still be required. They are very effective in controlling acute bleeding, but are associated with a range of potentially serious complications. This case demonstrates an unusual complication of the use of a Minnesota tube in a gentleman with a large gastric variceal bleed. The patient developed inferolateral ST-segment elevation on a 12-lead ECG which resolved rapidly following aspiration of 2000 mL of blood from the gastric port of the Minnesota tube. It was thought that the distension of the stomach, along with the traction applied to the Minnesota tube, resulted in external compression of the diaphragmatic surface of the heart and the observed ECG changes.