Detection of immunoperoxidase labelled mycoplasmas in cell culture by light microscopy and electronmicroscopy.

A McCoy cell line persistently infected with Mycoplasma orale was examined by light microscopy and electronmicroscopy after specific labelling with a direct immunoperoxidase conjugate. Mycoplasmas were readily detected in monolayer cultures in bright-field conditions and these were related to labelled organisms observed by electronmicroscopy in thin sections of similar cells. The specificity of the conjugate for M. orale was demonstrated by blocking with unconjugated antiserum, and by its inability to detect M. bovirhinis. Non-specific background labelling was consistently absent.

A comparison of three rapid diagnostic methods for the detection of rotavirus infection in calves.

Three techniques for the detection of rotavirus in faecal samples from calves with neonatal gastroenteritis were compared. A preliminary study indicated that reverse passive haemagglutination (RPHA) was at least as sensitive as the enzyme-linked immunosorbent assay (ELISA). These two immunoassays were compared with the detection of viral RNA by polyacrylamide gel electrophoresis (PAGE) on 209 field samples. Of the 77 samples in which at least one test gave a positive result, 69 were positive by both RPHA and PAGE, but only 49 were also positive by ELISA, indicating a lower sensitivity for the latter test. The overall agreement between RPHA and PAGE was 96%. The reasons for the discrepancies between the tests are discussed.

Haemorrhagic disease of lagomorphs: evidence for a calicivirus.

Studies on the aetiological agents of rabbit haemorrhagic disease (RHD) and European brown hare syndrome show that the viruses responsible for these infections can be placed in the family Caliciviridae. Established members of this group are vesicular exanthema virus (prototype), San Miguel sea lion virus and feline calcivirus. The human hepatitis E virus and the Norwalk agent may soon be included. The RHD virus genome consists of a positive stranded RNA molecule composed of 7437 nucleotides. A major subgenomic RNA of 2.2 kb, colinear with the 3' end of the genomic RNA, can also be recovered from infected liver tissue, and both RNAs are enclosed within viral capsids formed by a single major protein of approximately 60 kDa. Electron microscopic examination of organ suspensions from diseased animals shows two types of particle; 35-40 nm complete virions have the regularly arranged cup-shaped depressions typical of calcivirus morphology, and 23-25 nm smooth particles resulting from degradation of the outer surface structures of the complete virions.

Atypical rotavirus and villous epithelial cell syncytia in piglets.

Histopathological examination of small and large intestine from piglets with enteritis has shown the presence of epithelial multinucleate syncytia. Syncytia were associated with a specific type of atypical rotavirus infection, determined by electron microscopy and polyacrylamide gel electrophoresis analysis of viral RNA. The observations are consistent with similar previously described natural or experimental infections in other animals.

Detection of rotavirus in experimentally infected piglets.

Scouring and vomiting was induced in piglets by experimental infection with a field strain of rotavirus. Virus or viral antigen was detected in the small intestine by the fluorescent antibody technique and virus could also be demonstrated in infected tissue culture cells by immuno-fluorescence. Progeny particles in the epithelial layer of the small intestine were identified by electron microscopy. Three morphological types could be distinguished, often associated with electron-dense inclusion, and infected cells, though small in number, were present throughout the length of the small intestine.

Morphological defects of the acrosome in boar spermatozoa.

Morphological examination of semen from 17 boars of five breeds showed the presence of acrosome defects in 11 boars from four breeds. Two distinct types were seen; 'knobbed' sperm (type 1), of which two forms were found to be present by electron microscopy, and an uneven swellling of part of the acrosome (type 2) whose contents consisted of cytoplasmic and membrane-like material. The incidence of 'knobbed' sperm ranged from 0.2 to 6.3 per cent. Type 2 abnormalities were seen in only two boars, at 0.66 and 1.33 per cent.

Virus-like particles associated with porcine epidemic diarrhoea.

Porcine epidemic diarrhoea type II was reproduced in experimental pigs of various ages by oral dosing with minced intestine from a naturally occurring case of the disease. Virus-like particles which probably represent an unidentified coronavirus were seen by electron microscopy in the faeces and intestinal epithelium of infected animals.

Experimental infectious bovine rhinotracheitis: comparison of four antigen detection methods.

The duration of detectable viral antigen in nasal secretions was studied by four methods in calves experimentally infected with infectious bovine rhinotracheitis. Virus isolation in cell culture was the most sensitive and was successful for up to 11 days after inoculation. Three direct rapid antigen detection systems (immunofluorescence, immunoperoxidase and enzyme-linked immunosorbent assay [ELISA]) were all successful during the pyrexic phase, but less useful in the later stages of the disease. Virus isolation and ELISA remained positive in strongly positive samples stored for a week at room temperature.

Development of monoclonal antibody ELISA for simultaneous detection of bovine coronavirus, rotavirus serogroup A, and Escherichia coli K99 antigen in feces of calves.

A rapid ELISA was developed for simultaneous detection of bovine coronavirus (BCV), rotavirus (RV) serogroup A, and Escherichia coli K99 antigen in feces of calves. A mixture of 3 monoclonal antibodies specific for BCV, RV, or K99 was used successfully to capture the antigens; the same antibodies labeled with peroxidase were used to detect BCV, RV, or K99. The triple ELISA was compared with standard reference diagnostic methods by examining feces from experimentally and naturally infected and healthy calves. All the components of the test were highly specific (greater than 90%) and sensitive (BCV, 77%; K99, 93%; RV, 100%) when used in a format requiring short incubation steps at 20 C and visual recording of results.

Rabbit haemorrhagic disease: the new scourge of Oryctolagus cuniculus.

A new, widespread and important disease of rabbits, rabbit haemorrhagic disease (RHD), is concisely reviewed and discussed. RHD is an acute, infectious condition of adult rabbits and morbidity and mortality, after a relatively short incubation period, can be very high. The disease appears typically as a necrotizing hepatitis with associated haemorrhaging, and death occurs as a result of generalized organ dysfunction. RHD is caused by a calicivirus, antigenically related to a similar virus found in brown hares but distinct from other known caliciviruses, and is spread to susceptible rabbits by a number of routes and vectors. The disease is easily identified and can be effectively controlled in commercial and domestic rabbit populations by slaughter and vaccination regimes. The occurrence of pre-existing cross-reacting antibody in a proportion of rabbits unchallenged by the disease implies the presence of non-pathogenic strains of the virus. This antibody protects against disease on subsequent exposure to RHD. Uniquely, pre-existing antibody does not occur in rabbits in Australia where, after accidental release, the virus is currently spreading rapidly.

Possible origin of rabbit haemorrhagic disease in the United Kingdom.

Rabbit haemorrhagic disease was reported for the first time in the United Kingdom in 1992, and there followed four further small localised outbreaks, all in the south of England. Investigation has revealed no definitive mechanism for the introduction of the disease but it is considered that it probably originated from the near continent. The possibility of the transmission of the virus across the Channel by aerosol, birds and south coast ferry traffic is discussed, particularly with respect to the 1993 outbreaks of the disease in Kent and East Sussex. There may be implications for infection of wild rabbits, although preliminary investigation has shown no direct evidence of the disease in them.

Some observations on the semen of bulls persistently infected with bovine virus diarrhoea virus.

As a result of screening procedures employed for animals entering the AI service, two bulls were identified as being persistently infected with bovine virus diarrhoea virus by isolation of the virus from blood. Semen was collected on two occasions from these bulls; its quality as measured by density and motility was poor. Gross abnormalities of the sperm head, termed 'collapsed' heads, were seen in 28 to 45 per cent of sperm from one bull and in 1 per cent of sperm from the other. The collapsed heads were small and the whole head or its anterior part had the appearance of a dried pea. Electron microscopy showed the defect to consist of convoluted nuclear material with membrane-bound vacuoles and invaginations containing membranous debris and lamellar structures. In the 'high incidence' bull there was a corresponding increase in enlarged sperm heads. The 'low incidence' bull had sperm with heads of similar mean size to sperm from control bulls but with an increased variance. The semen was diluted in a lactose diluent, frozen and stored in liquid nitrogen. The distribution of viral antigen was determined and virus was isolated from several fractions of the semen, both before and after processing and cryopreservation. In one animal raw semen failed to yield virus but virus was recovered after processing, suggesting that raw semen may not be suitable for the efficient detection of the virus.

Isolation of a syncytium-forming virus from a goat with polyarthritis.

Virus particles, morphologically indistinguishable from maedi-visna virus or caprine arthritis-encephalitis virus, were detected in tissue explant cultures prepared post mortem from a goat with joint, lung and kidney lesions similar to those associated with the caprine arthritis-encephalitis complex. Serum from this goat and others in its herd of origin gave positive reactions in an agar gel immunodiffusion test for maedi-visna virus antibodies.

Evaluation of ELISA and electron microscopy for the detection of coronavirus and rotavirus in bovine faeces.

Enzyme-linked immunosorbent assay (ELISA) was compared with electron microscopy in the examination of faeces from experimental calves and showed 100 per cent agreement in the detection of 19 bovine coronavirus and 15 bovine rotavirus electron microscope positive samples. In a limited field survey of calf diarrhoea 75 selected faeces were examined independently by ELISA and electron microscopy and the agreement between the two tests was 95 per cent for coronavirus and 84 per cent for rotavirus. A further comparison was made with 74 samples submitted for routine diagnosis and this yielded agreements of 82 per cent (coronavirus) and 89 per cent (rotavirus). Factors contributing to discrepant results were examined and the relative advantages and disadvantages of the two tests for routine detection of these enteric viruses are discussed.

Rabbit haemorrhagic disease in the United Kingdom.

In April 1992, an investigation of sudden, increased mortality among adult rabbits on two premises in the south of England revealed clinical and pathological findings consistent with rabbit haemorrhagic disease. Virus particles morphologically resembling caliciviruses were detected in the livers of affected rabbits, and further studies confirmed the first recorded cases of rabbit haemorrhagic disease in the United Kingdom.

European brown hare syndrome in England.

The livers from 50 brown hares (Lepus europaeus) were examined by electron microscopy for calicivirus-like particles typical of the virus of European brown hare syndrome (EBHS). The virus was visible in 23 of the livers which included four taken from hares which died in 1982 and 1985 and which had been stored at -20 degrees C. The clinical histories, post mortem and other findings associated with these 23 hares were similar to those described in cases of European brown hare syndrome in other European countries. The findings were compared with those associated with viral haemorrhagic disease (VHD) of rabbits which, although it did not occur in the United Kingdom until 1992, is also caused by a calicivirus believed to be related to EBHS virus. The finding of the virus in hares which died from the disease in 1982 is the earliest recorded isolation of the organism and predates the description of VHD virus and VHD in China in 1984. A retrospective review of post mortem reports revealed cases of disease between 1976 and 1977 which may have been due to EBHS and it is considered that the disease has occurred sporadically in England for several years. The results from suspected and confirmed cases of EBHS in England between 1976 and 1990 are reported and the epidemiology and emergence of the disease is considered.

An investigation into the susceptibility of cattle to bovine leukosis virus following inoculation by various routes.

The effect of different routes of inoculation on the incubation period, as indicated by the detection of antibody and by the detection of bovine leukosis virus (BLV) in lymphocytes, were compared. None of the 12-month-old steers exposed to BLV by the oral route developed BLV infection. Intratracheal, subcutaneous and intradermal inoculations were found to be particularly effective in establishing BLV infection, which was detected three to four weeks after inoculation. In the majority of animals, serum antibody and virus were detected at the same time. One out of four in-oestrus heifers inoculated via the uterus with mixtures of BLV infected lymphocytes and semen became infected. It appears that there is an inhibitory factor in fresh semen that prevents BLV infection from becoming established. Viral antigen was detected earlier in BLV infected lymphocytes using the cocultivation method than by electron microscopy to demonstrate BLV particles in mitogen stimulated lymphocytes.

Development of a diagnostic approach to the identification of rabbit haemorrhagic disease.

Haemagglutination and ELISA tests, and negative contrast electron microscopy, have been used to identify rabbit haemorrhagic disease virus in naturally occurring cases of the disease and in experimentally infected rabbits in the United Kingdom. Haemagglutination tests alone are not satisfactory for the diagnosis because non-haemagglutinating isolates of the virus, otherwise indistinguishable from others, have been found in some outbreaks. Haemagglutination inhibition tests have shown that a proportion of both commercial laboratory and wild rabbits in the UK are seropositive to the virus although they have not been associated with clinical disease. This observation, made previously in other parts of Europe, may indicate the longstanding circulation of a related but non-pathogenic strain of virus. Naturally occurring antibody appears to afford a high degree of protection against experimental challenge with virulent virus.

The demonstration of maedi/visna virus in sheep in Great Britain.

A virus indistinguishable from that causing maedi/visna has been isolated from a sheep flock in Great Britain. The virus was identified in cell cultures using immunofluorescent and electron microscopical techniques. The complement fixation test and the agar gel immunodiffusion test were used to assess the proportion of serological reactors within the flock. There has been no evidence of clinical disease in the flock into which foreign imports were introduced in the years prior to 1976.

Investigation of the possible role of the tuberculin intradermal test in the spread of enzootic bovine leukosis.

The single intradermal comparative test was used with both avian and bovine tuberculin. Three cattle infected with bovine leukosis virus (BLV) were used as a source of infection. BLV-positive and susceptible animals were tuberculin tested alternately. Fifteen susceptible calves and 15 susceptible sheep were tested. A further three valves and three sheep were used as controls; the needles of the tuberculin syringes were deliberately contaminated with blood from the BLV-infected cattle, before being used in the test. Whereas all three calves and the three sheep inoculated intradermally with contaminated needles developed BLV infections, all of the other 30 animals have remained serologically negative to BLV for 10 months. Transmission of BLV with needles contaminated with BLV-infected blood was prevented by wiping the needles with absorbent cotton wool.