Freddy Coignoul

Comparative pathogenesis of acute and latent infections of calves with bovine herpesvirus types 1 and 5

Summary. This study was conducted to compare the pathogenesis of acute and latent infections with closely related bovine herpesvirus types 1 (BHV-1) and 5 (BHV-5) in their natural host. Two groups of eight calves were inoculated intranasally with BHV-1 or BHV-5. Although BHV-1 and BHV-5 similarly replicate in the nasal mucosa after inoculation, both viruses differ markedly in their ability to cause disease, BHV-5 being responsible of some fatal encephalitis while BHV-1 inducing rhinotracheitis. Virus isolation and immunohistochemistry demonstrated that BHV-5 replicates extensively in neurons of the central nervous system (CNS) and in respiratory cells of lungs, tracheal and nasal mucosae. Invasion of the CNS likely occurs through the trigeminal and olfactory pathways. Both groups developed cross-neutralising antibodies during this experiment suggesting partial clinical cross-protection afforded by the two infections. Three months after primary infection, experimental reactivation showed that BHV-5 was able to establish latency in the trigeminal ganglia but also the CNS of surviving calves. Moreover, laboratory findings suggested that BHV-5 could also persist in the tracheal and nasal mucosae. These results indicate that, after primary infection, BHV-1 and BHV-5 displayed similar biological features and consequently need to be considered together for the control of BHV-1 infection.

Canine hemorrhagic enteritis: Detection of viral particles by electron microscopy

SummaryAt necropsy, several dogs which died showing symptoms of hemorrhagic diarrhea, had significant lesions of the mucosa that were found especially in the duodenum and upper part of the small bowel.Study of ultrathin sections from the diseased mucosa revealed particles resembling parvoviruses in altered nuclei of cells of the intestinal crypts.Electron microscopic examination of intestinal contents by negative staining has shown the presence of many viral particles which have a diameter of 24 nm and whose profile is consistent with an icosahedral shape. These virions float at a density of 1.43 g/cm3 in cesium chloride and agglutinate rhesus monkey and swine red blood cells at 4° C.A possible etiological role in discussed.This virus is compared with the minute virus of canines and the Feline Panleukopenia virus.

Experimental infection of bulls with a genital isolate of bovine herpesvirus-4 and reactivation of latent virus with dexamethasone

Five 13- to 18-month old Belgian Blue bulls were used in this experiment. Four bulls (Nos. 2, 3, 4 and 5) were inoculated intratesticularly with 10(5) plaque-forming units of bovine herpesvirus-4 (BHV-4) in each testicle (Day 0). The challenge BHV-4 strain was previously isolated from testicle cells of a bull exhibiting orchitis and azoospermia. The fifth bull (No. 1) was used as a control and received the same volume of uninfected cell culture supernatant. For 5 days, beginning on Day 51 post-infection, two bulls (Nos. 4 and 5) and the control bull (No. 1) received 0.1 mg kg-1 of dexamethasone. Unilateral castrations were then performed at regular intervals for viral examination. Treatment with dexamethasone reactivated latent BHV-4, but no clinical signs were observed in treated bulls until the end of the experiment (Day 93). Only Bull 3 showed conjunctivitis and temporary azoospermia. The virus was recovered from various samples showing that: (i) BHV-4 can be present in a latent state in the testicles and mononuclear blood cells; (ii) dexamethasone reactivates the virus; (iii) the virus is excreted by nasal and ocular routes. Each infected bull seroconverted and a booster antibody response appeared after dexamethasone treatment as shown by immunofluorescence. Neutralizing antibodies were detected in each bull by complement-dependent neutralization test with titres higher than those obtained by a classical neutralization test. No booster response of neutralizing antibodies was observed after dexamethasone treatment. The antigenically relevant envelope BHV-4 proteins were identified by Western blotting using sera samples from the animals. DNA restriction endonuclease profiles of viruses reisolated after primary infection and reactivation showed only small differences.

Bluetongue in Eurasian lynx.

To the Editor: Bluetongue is an infectious disease of ruminants; it is caused by bluetongue virus (BTV), has 24 known serotypes, and is transmitted by several species of Culicoides biting midges. The disease mainly affects sheep and occurs when susceptible animals are introduced to areas where BTV circulates or when BTV is introduced to naive ruminant populations. The natural host range is strictly limited to ruminants, although seroconversion without disease has been reported in carnivores (1). We report BTV infection, disease, and death in 2 Eurasian lynx (Lynx lynx) and the isolation of BTV serotype 8 (BTV-8) from this carnivorous species. The 2 Eurasian lynx, held in the same cage in a zoo in Belgium, became lethargic in September 2007; animal 1 died after 2 days, and animal 2 died in February 2008. Both had been fed ruminant fetuses and stillborns from surrounding farms in an area where many bluetongue cases had been confirmed (2). Necropsy findings for animal 1 were anemia, subcutaneous hematomas, petechial hemorrhages, and lung congestion with edema. Necropsy findings for animal 2 were emaciation, anemia, enlarged and gelatinous lymph nodes, petechial hemorrhages, and pneumonia. For each animal, microscopic examination showed edematous vascular walls; enlarged endothelial cells; and evidence of acute to subacute vasculitis in muscle, myocardium, peritoneum, and lung. Tissue samples (spleen, lung, intestine) were analyzed by using 2 real-time reverse transcriptase–quantitative PCR techniques targeting BTV segment 5 and host β-actin mRNA as a control. BTV RNA was found in all samples from animal 1; cycle threshold values (3) ranged from 28.6 to 36.2. Tissues from animal 2 were negative for BTV RNA. Although the internal control was originally designed to detect β-actin mRNA of bovine or ovine species, clear positive signals were noted in all lynx samples, which indicated that this was a reliable control procedure. Infectious virus was subsequently isolated from the lung sample of animal 1 after inoculation of embryonated chicken eggs and amplification in baby hamster kidney–21 cell cultures (4). The specificity of the cytopathic effect, observed 48 hours after passage on baby hamster kidney–21 cells, was confirmed by real-time reverse transcriptase–quantitative PCR. Virus neutralization using specific reference serum (5) proved that the isolated virus was BTV-8. Anti-BTV antibodies were detected in lung tissue fluid from animal 2 (ID Screen Bluetongue Competition assay, ID VET, Monpellier, France) (6). We describe a natural, wild-type infection of a carnivorous species. Although deaths have been documented in dogs accidentally infected with a BTV-contaminated vaccine (7), the 2 lynx in this report were neither vaccinated nor medically treated by injection. BTV-8 was first introduced to northern Europe in 2006 and has subsequently spread rapidly to many countries on that continent. During 2007, a total of 6,870 bluetongue cases were reported in Belgium (2); animal 1 died in September 2007, which corresponded to the peak of bluetongue outbreaks in that region. No deaths were reported during that period among other animals, including ruminants, held in the same zoo as the 2 lynx reported here. The time lapse between initial clinical signs and death could explain the failure to detect BTV-8 RNA in animal 2. Although speculative, the suspicion of bluetongue in this animal is based on the presence of anti–BTV-8 antibodies, vasculitis, and pneumonia, which have been found in dogs accidentally infected with BTV (7). This report raises questions about the current knowledge of the epidemiology of bluetongue. Bluetongue in lynx indicates that the list of known susceptible species must be widened, at least for serotype 8. Although infection of a susceptible host by an insect vector is the only proven natural transmission mechanism for wild-type BTV, transplacental transmission of BTV-8, resulting in the birth of seropositive (8) or virus-positive calves (9), has recently been described in cattle. Although infection by an insect vector cannot be excluded, transmission by the oral route must be strongly suspected because the lynx described in this report had been fed ruminant fetuses and stillborn animals from surrounding farms. This possibility is supported by a previous suspicion that seroconversion to BTV in carnivores was a result of oral infection (1). The possibility of oral transmission is also supported by evidence of lateral transmission of BTV infection to cattle having occurred, in the absence of insect vectors, as a result of direct contact with newborn viremic calves born to infected dams that had been imported to Northern Ireland from a bluetongue-infected region of continental Europe (S. Kennedy, unpub. data). The role of wildlife, especially carnivores, in the epidemiology of bluetongue deserves further study to elucidate their role as either dead-end hosts or new sources of infection for livestock and to help determine the risks for wildlife populations. Our findings clearly indicate that a novel transmission pathway enables the virus to cross species. Consequently, transmission to other species, including domestic animals, can no longer be excluded. Moreover, oral transmission is likely to have considerable implications for disease control, including vaccination, because BTV-8 is a fast-emerging virus with major financial consequences.

Evidence for transplacental transmission of the current wild-type strain of bluetongue virus serotype 8 in cattle.

DURING the winter 2007/08, an outbreak of unprecedented development lesions of the central nervous system was detected in newborn or stillborn calves and lambs among the routine submissions to the Faculty of Veterinary Medicine, Liege, Belgium, for postmortem examination. The congenital

POSTMORTEM INVESTIGATIONS ON WINTER STRANDED SPERM WHALES FROM THE COASTS OF BELGIUM AND THE NETHERLANDS

During winter 1994–95, four and three sperm whales (Physeter niacrocephalus) were stranded along the Belgian and the Dutch coasts, respectively. Necropsies and tissue samplings were collected 24 hrs post mortem. Lesions on several whales included round and linear skin scars, ventral skin abrasions, acute skin ulcers, acute ulcerative stomatitides, acute to chronic external otitides, and passive visceral congestion. In addition, these sperm whales appeared to be debilitated with severe weight deficit, had blubber thickness reduction, the absence of abdominal fat, and the intestinal tracts were almost empty. Three categories of lesions and their possible relation with the stranding were evaluated. Cutaneous scars observed on the seven whales appeared to have no relation with the stranding. The poor body condition and acute integument ulcerative lesions were present before the stranding. Ventral skin abrasions and visceral passive congestion were caused by the strandings. Absence of food in the alimentary tracts, evidence of weight loss and blubber thickness reduction were compatible with an extended presence of the sperm whales in the North Sea, where adequate food is not available. This might lead to progressive weakness, predisposing the animals to secondary pathogens such as viral diseases. Finally, the coastal configuration of the southern North Sea makes it a trap for sperm whales which have entered the area during their wanderings.

Bronchopulmonary and Disseminated Granulomatous Disease Associated with Aspergillus Fumigatus and Candida Species Infection in a Golden Retriever

A seven-year-old, female golden retriever was referred for a paroxysmal, chronic cough and dyspnea, dysphagia, facial pruritus, anterior uveitis, and deteriorating general condition. A severe, mixed interstitial and alveolar pattern, with poorly defined amorphous lesions, was seen on thoracic radiographs. Multiple, whitish nodules disseminated on the hyperemic respiratory mucosa were noted on bronchoscopy. Escherichia coli and Aspergillus fumigatus were cultured from the bronchoalveolar lavage. Granulomatous lesions in numerous organs were identified during necropsy, and Aspergillus fumigatus and Candida spp. were cultured from lung and kidney tissues. Microscopic granulomatous lesions were compatible with mycotic infection; however fungal organisms were not observed.

Morbillivirus in common seals stranded on the coasts of Belgium and northern France during summer 1998

Sixteen common seals (Phoca vitulina) were stranded on the Belgian and northern French coasts during the summer of 1998. Eleven (10 pups and one adult) were sampled for histopathological, immunohistochemical, serological, bacteriological, parasitological and virological investigations. The main gross findings were severe emaciation, acute haemorrhagic enteritis, acute pneumonia, interstitial pulmonary emphysema and oedema, and chronic ulcerative stomatitis. Microscopical lung findings were acute to subacute pneumonia with interstitial oedema and emphysema. Severe lymphocytic depletion was observed in lymph nodes. Severe acute to subacute meningoencephalitis was observed in one animal. Specific staining with two monoclonal antibodies directed against canine distemper virus (cDv) and phocine distemper virus was observed in a few lymphocytes in the spleen and lymph nodes of three seals. Anti-cDv neutralising antibodies were detected in sera from six animals. Seven of the seals were positive by reverse transcriptase- PCR for the morbillivirus phosphoprotein gene. The lesions observed were consistent with those in animals infected by a morbillivirus, and demonstrated that distemper has recently recurred in North Sea seals.

Brucella ceti Infection in Harbor Porpoise (Phocoena phocoena)

We describe Brucella sp. infection and associated lesions in a harbor porpoise (Phocoena phocoena) found on the coast of Belgium. The infection was diagnosed by immunohistochemistry, transmission electron microscopy, and bacteriology, and the organism was identified as B. ceti. The infection’s location in the porpoise raises questions of abortion and zoonotic risks.

Lesions of morbillivirus infection in a fin whale (Balaenoptera physalus) stranded along the Belgian coast

with allopurinol, so that the xanthine urolithiasis was probably due to a defect in the enzyme itself. The fact that the concentrations of xanthine in blood and urine were about 10 times higher than the corresponding concentrations of hypoxanthine indicates that there was no absolute lack of xanthine oxidase. Most of the hypoxanthine was presumably transformed to xanthine by the xanthine oxidase, the same enzyme which catalyses the transformation of xanthine to uric acid. It is likely, as postulated by Ayvazian and Skupp (1965) for human beings, that there was a partial defect in the enzyme molecule. Because there are different binding sites on the molecule, the xanthine oxidase might have transformed hypoxanthine normally but xanthine only to a limited extent (Bergmann and Dickstein 1956). This possibility is supported by the fact that in human beings with hereditary xanthinuria the plasma concentration of xanthine increases much more than the concentration of hypoxanthine (Strauven and others 1989), as was the case in this dachshund. Kucera and others (1997) have suggested that a congential xanthinuria may occur in dachshunds.