Polymerase chain reaction and other laboratory techniques in the diagnosis of long incubation rabies in Australia

SUMMARY Blood and post-mortem tissues from a 10-years-old girl were submitted to the Australian Animal Health Laboratory. Clinical signs and histopathological lesions had suggested a diagnosis of rabies, but, an unusually long incubation period of at least 5 years did not encourage such a diagnosis. Serological examinations by the rapid fluorescent focus inhibition test revealed a dramatic increase in rabies virus-neutralising antibody during the 10-day period of hospitalisation. The results of a fluorescent antibody test on brain smears, and an immunoperoxidase test on formalin-fixed sections of brain were also consistent with a diagnosis of rabies. Attempts to isolate virus were unsuccessful. Polymerase chain reactions (PCRs) were conducted on a 10% suspension of a post-mortem sample from the patient's brain, using primers based on the published sequence of the Pasteur virus strain of rabies virus. 413 and 513 bp fragments from the nucleoprotein gene and a 403 bp fragment from the glycoprotein gene were amplified. Subsequent sequencing of these fragments, and comparison with equivalent regions of known rabies viruses, confirmed that the fragments originated from a virus belonging to the rabies virus serotype. This case demonstrated the advantage of using a range of laboratory techniques to obtain a definitive diagnosis. In particular, a PCR-based test may allow a diagnosis, even in the face of conditions that preclude virus isolation such as apparently occurred in this case. Finally, this case demonstrated that an unusually long incubation period should not discourage a tentative clinical diagnosis of rabies.     

Transmission studies of Hendra virus (equine morbilli-virus) in fruit bats, horses and cats

Objective To determine the infectivity and transmissibility of Hendra virus (HeV). Design A disease transmission study using fruit bats, horses and cats. Procedure Eight grey-headed fruit bats (Pteropus poliocephalus) were inoculated and housed in contact with three uninfected bats and two uninfected horses. In a second exper iment, four horses were inoculated by subcutaneous injection and intranasal inoculation and housed in contact with three uninfected horses and six uninfected cats. In a third experi ment, 12 cats were inoculated and housed in contact with three uninfected horses. Two surviving horses were inoculated at the conclusion of the third experiment: the first orally and the second by nasal swabbing. All animals were necropsied and examined by gross and microscopic pathological methods, immunoperoxidase to detect viral antigen in formalin-fixed tissues, virus isolation was attempted on tissues and SNT and ELISA methods were used to detect HeV-specific antibody. Results Clinical disease was not observed in the fruit bats, although six of eight inoculated bats developed antibody against HeV, and two of six developed vascular lesions which contained viral antigen. The in-contact bats and horses did not seroconvert. Three of four horses that were inoculated devel oped acute disease, but in-contact horses and cats were not infected. In the third experiment, one of three in-contact horses contracted disease. At the time of necropsy, high titres of HeV were detected in the kidneys of six acutely infected horses, in the urine of four horses and the mouth of two, but not in the nasal cavities or tracheas. Conclusions Grey-headed fruit bats seroconvert and develop subclinical disease when inoculated with HeV. Horses can be infected by oronasal routes and can excrete HeV in urine and saliva. It is possible to transmit HeV from cats to horses. Transmission from P poliocephalus t o horses could not be proven and neither could transmission from horses to horses or horses to cats. Under the experimental conditions of the study the virus is not highly contagious.     

Epidemic of blindness in kangaroos - evidence of a viral aetiology

OBJECTIVE: To determine the cause of an epidemic of blindness in kangaroos. DESIGN AND PROCEDURES: Laboratory examinations were made of eyes and brains of a large number of kangaroos using serological, virological, histopathological, electron microscopical, immunohistochemical methods, and PCR with cDNA sequencing. In addition, potential insect viral vectors identified during the disease outbreak were examined for specific viral genomic sequences. SAMPLE POPULATION: For histopathological analysis, 55 apparently blind and 18 apparently normal wild kangaroos and wallabies were obtained from New South Wales, Victoria, South Australia, and Western Australia. A total of 437 wild kangaroos and wallabies (including 23 animals with apparent blindness) were examined serologically. RESULTS: Orbiviruses of the Wallal and Warrego serogroups were isolated from kangaroos affected with blindness in a major epidemic in south-eastern Australia in 1994 and 1995 and extending to Western Australia in 1995/96. Histopathological examinations showed severe degeneration and inflammation in the eyes, and mild inflammation in the brains. In affected retinas, Wallal virus antigen was detected by immunohistochemical analysis and orbiviruses were seen in electron microscopy. There was serological variation in the newly isolated Wallal virus from archival Wallal virus that had been isolated in northern Australia. There were also variations of up to 20% in genotype sequence from the reference archival virus. Polymerase chain reactions showed that Wallal virus was present during the epidemic in three species of midges, Culicoides austropalpalis, C dycei and C marksi. Wallal virus nucleic acid was also detected by PCR in a paraffin-embedded retina taken from a blind kangaroo in 1975. CONCLUSION: Wallal virus and perhaps also Warrego virus are the cause of the outbreak of blindness in kangaroos. Other viruses may also be involved, but the evidence in this paper indicates a variant of Wallal virus, an orbivirus transmitted by midges, has the strongest aetiological association, and immunohistochemical analysis implicates it as the most damaging factor in the affected eyes.     

The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A

Initiation and maintenance of mitosis require the activation of protein kinase cyclin B-Cdc2 and the inhibition of protein phosphatase 2A (PP2A), which, respectively, phosphorylate and dephosphorylate mitotic substrates. The protein kinase Greatwall (Gwl) is required to maintain mitosis through PP2A inhibition. We describe how Gwl activation results in PP2A inhibition. We identified cyclic adenosine monophosphate-regulated phosphoprotein 19 (Arpp19) and α-Endosulfine as two substrates of Gwl that, when phosphorylated by this kinase, associate with and inhibit PP2A, thus promoting mitotic entry. Conversely, in the absence of Gwl activity, Arpp19 and α-Endosulfine are dephosphorylated and lose their capacity to bind and inhibit PP2A. Although both proteins can inhibit PP2A, endogenous Arpp19, but not α-Endosulfine, is responsible for PP2A inhibition at mitotic entry in Xenopus egg extracts.     

Protocol: Optimised methodology for isolation of nuclei from leaves of species in the <Emphasis Type="Italic">Solanaceae</Emphasis> and <Emphasis Type="Italic">Rosaceae</Emphasis> families

In this study, a protocol is described for rapid preparation of an enriched, reasonably pure fraction of nuclear proteins from the leaves of tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and apple (Malus domestica). The protocol gives reproducible results and can be carried out quickly in 2 hours. Tissue extracts clarified with filtration were treated with non-ionic detergent (Triton X-100) to lyse membranes of contaminating organelles. Nuclei were collected from a 60% Percoll layer of density gradient following low-speed centrifugation. Western blot analysis using antibodies to marker proteins of organelles indicated that the nuclear protein fractions were highly enriched and free or nearly free of proteins from the endoplasmic reticulum and chloroplasts.     

Involvement of Guggenheimella bovis in digital dermatitis lesions of dairy cows

Digital dermatitis (DD) of cattle leads to lameness and a decrease of milk production and is responsible for major economic losses worldwide. Although a bacterial aetiology is generally accepted, it still is unclear which microorganisms cause and/or maintain the disease. Recently, a previously undiscovered bacterial species, Guggenheimella bovis, has been isolated from the front of two DD lesions in Swiss cattle and suggested as a potential pathogen. The aims of the present study were to determine the prevalence of G. bovis in 58 German cows suffering from DD via dot blot hybridization, and to analyse the spatial distribution of G. bovis within the affected tissue by fluorescence in situ hybridization (FISH). A species-specific probe, GUBO1, was designed and evaluated. In none of the 58 samples Guggenheimella could be detected, while cultured G. bovis was reliably identified by GUBO1. Further FISH experiments were carried out on two additional biopsies of Swiss cattle tested positive for G. bovis by quantitative PCR and permitted visualization of the newly discovered bacteria in situ. In these biopsies G. bovis proved to be tissue invasive forming characteristic spherical microcolonies not only within the bacterial biofilm but also in seemingly unaffected parts of the tissue not yet reached by the advancing bacterial front. Although the presence of G. bovis does not constitute an essential premise for DD, it seems likely that the bacterial species involved in DD vary, and that in some cases G. bovis is crucial for the development of DD lesions.     

Epidemiological investigation of the buller steer syndrome (riding behaviour) in a western Canadian feedlot

Objective To describe the buller steer syndrome in a Western Canadian feedlot. Design A retrospective epidemiological study. Animals 78,445 male cattle that entered a 24,000-head feedlot in western Canada from 1991 to 1993. Procedure All cattle were given a hormonal growth promotant containing 20 mg oestradiol benzoate and 200 mg progesterone within 24 h of arrival at the feedlot. A ‘buller’ was a steer that was observed at daily pen checking to be ridden persistently by pen mates or had evidence of having been persistently ridden by pen mates. At the completion of the feeding period, animal health records for bullers were collected and analysed. Results The prevalence of bullers in the total population was 2139/78,445 (2.7%, range per pen 0 to 11.2%). The prevalence of bullers increased with increasing weight and age. The relapse risk after first treatment (three days in the feedlot hospital plus treatment for concurrent disease) was 30% on average (27 to 35%). Individual records from 9734 yearling steers that entered the feedlot in 1991 and 1992 showed that bullers were significantly (P < 0.05) heavier at processing than non-bullers. Bullers occurred as a point source epidemic with the cause occurring soon after cattle arrived at the feedlot and were mingled into pen groups. This gave a ‘days on feed’ distribution. The peak incidence of bullers occurred much sooner after arrival and dropped off much quicker in older cattle. The daily incidence of bullers was temporal, but was not related to season of the year, weather conditions or any other feedlot management practice. It was related to the seasonal arrival of cattle at the feedlot, their age at entry to the feedlot and the post arrival occurrence of bullers. Reimplantation with hormonal growth promotants and castration of intact bulls did not produce an epidemic of bullers. Conclusion The findings of this study support the theory that bullers are the result of agonistic interactions, which occur concurrent with the establishment and maintenance of a social hierarchy within pens of feedlot cattle.