Richard J. Whittington

Comparative studies of piscine and amphibian iridoviruses

Summary. A total of 30 iridoviruses collected from Australia, South-East Asia, North America, South America and Europe were characterised. With the exception of the South-East Asian iridoviruses all viruses were found to belong to the genus Ranavirus. All viruses, except those originating from South-East Asia, cross-reacted with antisera against epizootic haematopoietic necrosis virus (EHNV). Viruses or virus-infected cells were examined using electron microscopy, SDS PAGE, restriction endonuclease (RE) digestion, DNA hybridisation, and DNA sequencing. Data from RE digestion of genomic DNA, and from the sequencing of PCR products indicated that the viruses generally grouped according to their geographic and taxonomic (i.e. amphibian or fish) origin. The one exception to this was the viruses from the United Kingdom that grouped with the North American ranaviruses. The differences between specified genomic regions were small. To assess the validity of the differences in sequence homology, similar studies were performed with different isolates from two viruses (EHNV and Guatopo virus (GV), collected from different animals at different locations and time). The sequence data showed complete homology for the isolates for any one virus over the 200 and 586 bp regions examined. Collectively, the data showed that the coding region for the major coat protein (MCP) is stable for any one species (e.g. EHNV).

Survival and dormancy of Mycobacterium avium subsp. paratuberculosis in the environment.

ABSTRACT The survival of Mycobacterium avium subsp. paratuberculosis was studied by culture of fecal material sampled at intervals for up to 117 weeks from soil and grass in pasture plots and boxes. Survival for up to 55 weeks was observed in a dry fully shaded environment, with much shorter survival times in unshaded locations. Moisture and application of lime to soil did not affect survival. UV radiation was an unlikely factor, but infrared wavelengths leading to diurnal temperature flux may be the significant detrimental component that is correlated with lack of shade. The organism survived for up to 24 weeks on grass that germinated through infected fecal material applied to the soil surface in completely shaded boxes and for up to 9 weeks on grass in 70% shade. The observed patterns of recovery in three of four experiments and changes in viable counts were indicative of dormancy, a hitherto unreported property of this taxon. A dps-like genetic element and relA, which are involved in dormancy responses in other mycobacteria, are present in the M. avium subsp. paratuberculosis genome sequence, providing indirect evidence for the existence of physiological mechanisms enabling dormancy. However, survival of M. avium subsp. paratuberculosis in the environment is finite, consistent with its taxonomic description as an obligate parasite of animals.

Evidence for age susceptibility of cattle to Johne's disease.

Calf rearing programs for the control of bovine Johnes disease (BJD) in dairy farms have been widely adopted globally and are based on evidence that the most significant risk factor for developing the disease is exposure of young calves to infectious doses of the causative organism Mycobacterium avium subsp. paratuberculosis (Mptb). Hygienic calf rearing practices aim to break the transmission cycle of Mptb by removing neonatal calves from their dams within 12h of birth and segregating replacement heifers from the herd until they are 12 months of age. But compliance with these interventions is difficult for many producers and delaying the removal of calves from their dams and earlier return of heifers to the herd are common practices. However, would changing these practices increase the risk of animals contracting BJD? Evidence for age susceptibility of calves and young adults to Mptb is reviewed. The experimental studies selected for inclusion in an analysis of the evidence were those designed specifically to address the issue and were confined to examination of 140 cattle in experiments conducted by eight groups of workers between the years 1938 and 2006. Approximately 75% of calves <6 months of age, 50% of those aged between 6 and 12 months, and just less than 20% of cattle >12 months old developed lesions indicative of BJD infection when exposed to any of the tested routes of Mptb infection. No direct evidence was found to support the commonly held view that calf removal from the dam for a maximum period of 12h is preferable to 24h. However the studies did show that if exposure to infection occurs at birth, then the risk of infection progressing to BJD is high, particularly in a highly contaminated environment or if the dam is infected.

Iridovirus infections in finfish – critical review with emphasis on ranaviruses

Viruses in three genera of the family Iridoviridae (iridoviruses) affect finfish. Ranaviruses and megalocytiviruses are recently emerged pathogens. Both cause severe systemic disease, occur globally and affect a diversity of hosts. In contrast, lymphocystiviruses cause superficial lesions and rarely cause economic loss. The ranavirus epizootic haematopoietic necrosis virus (EHNV) from Australia was the first iridovirus to cause epizootic mortality in finfish. Like other ranaviruses, it lacks host specificity. A distinct but closely related virus, European catfish virus, occurs in finfish in Europe, while very similar ranaviruses occur in amphibians in Europe, Asia, Australia, North America and South America. These viruses can be distinguished from one another by conserved differences in the sequence of the major capsid protein gene, which informs policies of the World Organisation for Animal Health to minimize transboundary spread of these agents. However, limited epidemiological information and variations in disease expression create difficulties for design of sampling strategies for surveillance. There is still uncertainty surrounding the taxonomy of some putative ranaviruses such as Singapore grouper iridovirus and Santee-Cooper ranavirus, both of which cause serious disease in fish, and confusion continues with diseases caused by megalocytiviruses. In this review, aspects of the agents and diseases caused by ranaviruses are contrasted with those due to megalocytiviruses to promote accurate diagnosis and characterization of the agents responsible. Ranavirus epizootics in amphibians are also discussed because of possible links with finfish and common anthropogenic mechanisms of spread. The source of the global epizootic of disease caused by systemic iridoviruses in finfish and amphibians is uncertain, but three possibilities are discussed: trade in food fish, trade in ornamental fish, reptiles and amphibians and emergence from unknown reservoir hosts associated with environmental change.

Survival of Mycobacterium avium subsp. paratuberculosis in Dam Water and Sediment

ABSTRACT In a previous longitudinal study, Mycobacterium avium subsp. paratuberculosis survived for 55 weeks in fecal material in the shade, but for much shorter periods in exposed locations. In this experiment, the survival of the organism was studied in 250 liters of dam water and sediment in large water troughs that were placed in either a semiexposed location or in a shaded location and compared to survival in fecal material and soil in the shaded location. Survival in water and/or sediment in the shade was for up to 48 weeks compared to 36 weeks in the semiexposed location. Survival in sediment was 12 to 26 weeks longer than survival in the water column. Survival in soil and fecal material in the terrestrial environment in the shaded location was only 12 weeks. Although disturbance to sediment could not be ruled out as a factor, there was evidence of dormancy in both the water column and the sediment, since the organism could not be recovered for several months before again becoming detectable. The results suggest that water may be a significant reservoir of M. avium subsp. paratuberculosis infection. Further research on the biology of the organism in aquatic environments is warranted. Animal health authorities will need to provide appropriate advice to farmers to minimize exposure of livestock to potentially infected water sources. Survival of the organism in water destined for human consumption will need to be addressed if the organism is found to be involved in the etiology of Crohns disease.

The type IV fimbrial subunit gene (fimA) of Dichelobacter nodosus is essential for virulence, protease secretion, and natural competence.

Dichelobacter nodosus is the essential causative agent of footrot in sheep. The major D. nodosus-encoded virulence factors that have been implicated in the disease are type IV fimbriae and extracellular proteases. To examine the role of the fimbriae in virulence, allelic exchange was used to insertionally inactivate the fimA gene, which encodes the fimbrial subunit protein, from the virulent type G D. nodosus strain VCS1703A. Detailed analysis of two independently derived fimA mutants revealed that they no longer produced the fimbrial subunit protein or intact fimbriae and did not exhibit twitching motility. In addition, these mutants were no longer capable of undergoing natural transformation and did not secrete wild-type levels of extracellular proteases. These effects were not due to polar effects on the downstream fimB gene because insertionally inactivated fimB mutants were not defective in any of these phenotypic tests. Virulence testing of the mutants in a sheep pen trial conducted under controlled environmental conditions showed that the fimA mutants were avirulent, providing evidence that the fimA gene is an essential D. nodosus virulence gene. These studies represent the first time that molecular genetics has been used to determine the role of virulence genes in this slow growing anaerobic bacterium.

Does a Th1 over Th2 dominancy really exist in the early stages of Mycobacterium avium subspecies paratuberculosis infections

The immune response of ruminants to Johnes disease has been long associated with a cell mediated immune (CMI) response in the early stages of infection with a switch to an antibody response later as the disease manifests. This study examines the immune response in sheep to Mycobacterium avium subspecies paratuberculosis (Map) infections, specifically the antigen-specific interferon gamma (IFN-γ) and antibody responses as surrogates of T helper-1 (Th1) and Th2 immunity. The difference in IFN-γ production between paucibacillary and multibacillary diseased animals was also examined. The results show that sheep are more likely to have a combined antibody and IFN-γ response (seen in 50% of the animals) rather than a switch from an IFN-γ to antibody response (39%). Multibacillary diseased animals were found to have a decrease in functional ability to produce IFN-γ from cells stimulated with MAP-specific antigens and non-specific mitogens. This indicates that the immune responses to Map infections are more complex than thought, where both antibody and cellular immunity may play key roles in the early stages of disease manifestation or resistance. The loss of the cellular response in multibacillary animals may be an indication that the entire immune response is dysfunctional, with the cell mediated responses becoming affected first.

The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated from tissues and faeces

The effect of decontamination protocols on the numbers of sheep strain Mycobacterium avium subsp. paratuberculosis isolated in BACTEC cultures from clinical samples was assessed by spiking tissues and faeces at various points during the decontamination procedure. Routine protocols in the laboratory were shown to decrease the number of organisms isolated per sample by about 2.7 log(10) and 3.1 log(10) for faeces and tissues, respectively. These findings are important for the interpretation of negative culture results and may be useful in epidemiological studies. Addition of a centrifugation step to the tissue protocol increased the recovery by about 1 log, but resulted in increased contamination of BACTEC cultures. These studies may also facilitate future improvements to decontamination procedures.

The Subtilisin-Like Protease AprV2 Is Required for Virulence and Uses a Novel Disulphide-Tethered Exosite to Bind Substrates

Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals.

Genomic Comparison of Mycobacterium avium subsp. paratuberculosis Sheep and Cattle Strains by Microarray Hybridization

Microarray-based comparisons of three Mycobacterium avium subsp. paratuberculosis isolates, including one sheep strain and two cattle strains, identified three large genomic deletions in the sheep strain, totaling 29,208 bp and involving 24 open reading frames. These deletions may help explain some of the differences in pathogenicity and host specificity observed between the cattle and sheep strains of Mycobacterium avium subsp. paratuberculosis.