Drosophila melanogaster mounts a unique immune response to the rhabdovirus Sigma virus

Rhabdoviruses are important pathogens of humans, livestock, and plants that are often vectored by insects. Rhabdovirus particles have a characteristic bullet shape with a lipid envelope and surface-exposed transmembrane glycoproteins. Sigma virus (SIGMAV) is a member of the Rhabdoviridae and is a naturally occurring disease agent of Drosophila melanogaster. The infection is maintained in Drosophila populations through vertical transmission via germ cells. We report here the nature of the Drosophila innate immune response to SIGMAV infection as revealed by quantitative reverse transcription-PCR analysis of differentially expressed genes identified by microarray analysis. We have also compared and contrasted the immune response of the host with respect to two nonenveloped viruses, Drosophila C virus (DCV) and Drosophila X virus (DXV). We determined that SIGMAV infection upregulates expression of the peptidoglycan receptor protein genes PGRP-SB1 and PGRP-SD and the antimicrobial peptide (AMP) genes Diptericin-A, Attacin-A, Attacin-B, Cecropin-A1, and Drosocin. SIGMAV infection did not induce PGRP-SA and the AMP genes Drosomycin-B, Metchnikowin, and Defensin that are upregulated in DCV and/or DXV infections. Expression levels of the Toll and Imd signaling cascade genes are not significantly altered by SIGMAV infection. These results highlight shared and unique aspects of the Drosophila immune response to the three viruses and may shed light on the nature of the interaction with the host and the evolution of these associations.

An evaluation of the apxIVA based PCR-REA method for differentiation of Actinobacillus pleuropneumoniae

A restriction analysis of PCR (PCR-REA) amplified apxIVA gene has been suggested as an alternative method for serotyping of Actinobacillus pleuropneumoniae by Jaglic et al. [Jaglic, Z., Svastova, P., Rychlik, I., Nedbalcova, K., Kucerova, Z., Pavlik, I., Bartos, M., 2004. Differentiation of Actinobacillus pleuropneumoniae by PCR-REA based on sequence variability of the apxIVA gene and by ribotyping. Vet. Microbiol. 103, 63-69]. The current study investigated whether this alternative method could distinguish between the reference strains of serovars 13-15 and the value of the method when applied to 47 field isolates representing serovars 1-3, 5, 7-9, 12 and 15 as well as non-typable isolates. The reference strains of serovars 13 and 14 had the same sized product after the apxIVA PCR, while the product for serovar 15 was of different size compared to all the other serovar reference strains. The CfoI digest profiles of the reference serovars 13 and 14 strains were different from each other and from all other serovars. The HpaII digest profiles of these two serovars were very similar to each other, but both were distinctively different from the other serovar profiles. The CfoI digest profile of serovar 15 strain was very similar to the serovars 3 and 12 strains except for two faint extra bands for serovar 15. The HpaII digest profiles of serovars 12 and 15 reference strains were identical. The PCR-REA method correctly recognized the serovar of 21 of 43 field isolates. It was concluded that the method was a useful additional tool to support, but could not replace, conventional serotyping.

Comparison of sampling sites and detection methods for Haemophilus parasuis

Objective To improve the isolation rate and identification procedures for Haemophilus parasuis from pig tissues. Design Thirteen sampling sites and up to three methods were used to confirm the presence of H. parasuis in pigs after experimental challenge. Procedure Colostrum-deprived, naturally farrowed pigs were challenged intratracheally with H parasuis serovar 12 or 4. Samples taken during necropsy were either inoculated onto culture plates, processed directly for PCR or enriched prior to being processed for PCR. The recovery of H parasuis from different sampling sites and using different sampling methods was compared for each serovar. Results H parasuis was recovered from several sample sites for all serovar 12 challenged pigs, while the trachea was the only positive site for all pigs following serovar 4 challenge. The method of solid medium culture of swabs, and confirmation of the identity of cultured bacteria by PCR, resulted in 38% and 14% more positive results on a site basis for serovars 12 and 4, retrospectively, than direct PCR on the swabs. This difference was significant in the serovar 12 challenge. Conclusion Conventional culture proved to be more effective in detecting H parasuis than direct PCR or PCR on enrichment broths. For subacute (serovar 4) infections, the most successful sites for culture or direct PCR were pleural fluid, peritoneal fibrin and fluid, lung and pericardial fluid. For acute (serovar 12) infections, the best sites were lung, heart blood, affected joints and brain. The methodologies and key sampling sites identified in this study will enable improved isolation of H parasuis and aid the diagnosis of Glässer's disease.

The effects of salivary gland extracts from Boophilus microplus ticks on mitogen-stimulated bovine lymphocytes

The effect of salivary gland extract (SGE) from the tick Boophilus microplus was examined in mitogen-stimulated lymphocytes in vitro. SGE was added to lymphocytes of seven cattle together with the mitogens concanavalin A (ConA), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM). Semi-purified B cells from another seven cattle were stimulated with the mitogen lipopolysaccharide (LPS). PHA and ConA stimulated proliferation of lymphocytes to the same extent, but the inhibition due to SGE of Boophilus microplus on the proliferative response stimulated by PHA (39.0% ± 9.3%) was less than the inhibition of proliferative response stimulated by ConA (75.4% ± 6.9%). In contrast, SGE of B. microplus stimulated the proliferation of B cells in the presence of LPS in a dose-dependent manner. Enhanced stimulation of B cells by SGE at >4 μg in culture was greater than twice that observed when B cells were stimulated by LPS alone. SGE does not have a direct suppressive effect on bovine B cell proliferation; however, in vivo the effectiveness of B cell responses might be influenced by other immune factors, such as cytokine profiles.

Salmonella and on-farm risk factors in healthy slaughter-age cattle and sheep in eastern Australia

To examine healthy slaughter-age cattle and sheep on-farm for the excretion of Salmonella serovars in faeces and to identify possible risk factors using a questionnaire. The study involved 215 herds and flocks in the four eastern states of Australia, 56 with prior history of salmonellosis. Production systems examined included pasture beef cattle, feedlot beef cattle, dairy cattle, prime lambs and mutton sheep and animals were all at slaughter age. From each herd or flock, 25 animals were sampled and the samples pooled for Salmonella culture. All Salmonella isolated were serotyped and any Salmonella Typhimurium isolates were phage typed. Questionnaires on each production system, prepared in Epi Info 6.04, were designed to identify risk factors associated with Salmonella spp excretion, with separate questionnaires designed for each production system. Salmonellae were identified in all production systems and were more commonly isolated from dairies and beef feedlots than other systems. Statistical analysis revealed that dairy cattle were significantly more likely to shed Salmonella in faeces than pasture beef cattle, mutton sheep and prime lambs (P < 0.05). A wide diversity of Salmonella serovars, all of which have been isolated from humans in Australia, was identified in both cattle and sheep. Analysis of the questionnaires showed access to new arrivals was a significant risk factor for Salmonella excretion on dairy properties. For beef feedlots, the presence of large numbers of flies in the feedlot pens or around stored manure were significant risk factors for Salmonella excretion. Dairy cattle pose the highest risk of all the slaughter-age animals tested. Some of the identified risk factors can be overcome by improved management practices, especially in relation to hygiene.

Identification and immunogenicity of an immunodominant mimotope of Avibacterium paragallinarum from a phage display peptide library

Avibacterium paragallinarum is the causative agent of infectious coryza. The protective antigens of this important pathogen have not yet been clearly identified. In this paper, we applied phage display technique to screen the immunodominant mimotopes of a serovar A strain of A. paragallinarum by using a random 12-peptide library, and evaluated the immunogenicity in chickens of the selected mimotope. Polyclonal antibody directed against A. paragallinarum strain 0083 (serovar A) was used as the target antibody and phage clones binding to this target were screened from the 12-mer random peptide library. More than 50% of the phage clones selected in the third round carried the consensus peptide motif sequence A-DP(M)L. The phage clones containing the peptide motif reacted with the target antibody and this interaction could be blocked, in a dose-dependent manner, by A. paragallinarum. One of the peptide sequences, YGLLAVDPLFKP, was selected and the corresponding oligonucleotide sequence was synthesized and then inserted into the expression vector pFliTrx. The recombinant plasmid was transferred into an expression host Escherichia coli GI826 by electroporation, resulting in a recombinant E. coli expressing the peptide on the bacterial surface. Intramuscular injection of the epitope-expressing recombinant bacteria into chickens induced a specific serological response to serovar A. A. paragallinarum. The chickens given the recombinant E. coli showed significant protection against challenge with A. paragallinarum 0083. These results indicated a potential for the use of the mimotope in the development of molecular vaccines for infectious coryza.

Genetic characterisation of bovine herpesvirus 1 in New Zealand

AIM: To genotype bovine herpesvirus type 1 (BHV-1) isolates from cattle in New Zealand. METHODS: Twenty-eight BHV-1 isolates were collected from clinical samples from cattle over 28 years. They were characterised and compared using restriction endonuclease analysis (REA), and polymerase chain reaction (PCR) and DNA sequencing. RESULTS: Twenty-four isolates were classified as bovine herpesvirus subtype 1.2b (BHV-1.2b) by REA. The remaining four isolates were distinct from the others in REA profiles of one of the major enzymes (HindIII) by which the classification was made. However, these four isolates were closely related to others when the REA profiles of other restriction enzymes were studied, and therefore were regarded as divergent strains of BHV-1.2b. All BHV-1 isolates were detectable by PCR, and sequence analysis of selected PCR products did not indicate any significant differences between isolates. CONCLUSION: BHV-1.2b appears to be the predominant strain of BHV-1 in cattle in New Zealand. There was no evidence that more virulent strains of BHV-1, e.g. subtype 1.1 and BHV type 5, are, or have been, present in New Zealand. Genetic variations exist among these BHV-1.2b isolates.

Review of bats and SARS

Bats have been identified as a natural reservoir for an increasing number of emerging zoonotic viruses, including henipaviruses and variants of rabies viruses. Recently, we and another group independently identified several horse-shoe bat species (genus Rhinolophus) as the reservoir host for a large number of viruses that have a close genetic relationship with the coronavirus associated with severe acute respiratory syndrome (SARS). Our current research focused on the identification of the reservoir species for the progenitor virus of the SARS coronaviruses responsible for outbreaks during 2002-2003 and 2003-2004. In addition to SARS-like coronaviruses, many other novel bat coronaviruses, which belong to groups 1 and 2 of the 3 existing coronavirus groups, have been detected by PCR. The discovery of bat SARS-like coronaviruses and the great genetic diversity of coronaviruses in bats have shed new light on the origin and transmission of SARS coronaviruses.

Global trade in ornamental fish from an Australian perspective: The case for revised import risk analysis and management strategies

Over 1 billion ornamental fish comprising more than 4000 freshwater and 1400 marine species are traded internationally each year, with 8-10 million imported into Australia alone. Compared to other commodities, the pathogens and disease translocation risks associated with this pattern of trade have been poorly documented. The aim of this study was to conduct an appraisal of the effectiveness of risk analysis and quarantine controls as they are applied according to the Sanitary and Phytosanitary (SPS) agreement in Australia. Ornamental fish originate from about 100 countries and hazards are mostly unknown; since 2000 there have been 16-fold fewer scientific publications on ornamental fish disease compared to farmed fish disease, and 470 fewer compared to disease in terrestrial species (cattle). The import quarantine policies of a range of countries were reviewed and classified as stringent or non-stringent based on the levels of pre-border and border controls. Australia has a stringent policy which includes pre-border health certification and a mandatory quarantine period at border of 1-3 weeks in registered quarantine premises supervised by government quarantine staff. Despite these measures there have been many disease incursions as well as establishment of significant exotic viral, bacterial, fungal, protozoal and metazoan pathogens from ornamental fish in farmed native Australian fish and free-living introduced species. Recent examples include Megalocytivirus and Aeromonas salmonicida atypical strain. In 2006, there were 22 species of alien ornamental fish with established breeding populations in waterways in Australia and freshwater plants and molluscs have also been introduced, proving a direct transmission pathway for establishment of pathogens in native fish species. Australia's stringent quarantine policies for imported ornamental fish are based on import risk analysis under the SPS agreement but have not provided an acceptable level of protection (ALOP) consistent with government objectives to prevent introduction of pests and diseases, promote development of future aquaculture industries or maintain biodiversity. It is concluded that the risk analysis process described by the Office International des Epizooties under the SPS agreement cannot be used in a meaningful way for current patterns of ornamental fish trade. Transboundary disease incursions will continue and exotic pathogens will become established in new regions as a result of the ornamental fish trade, and this will be an international phenomenon. Ornamental fish represent a special case in live animal trade where OIE guidelines for risk analysis need to be revised. Alternatively, for countries such as Australia with implied very high ALOP, the number of species traded and the number of sources permitted need to be dramatically reduced to facilitate hazard identification, risk assessment and import quarantine controls. Lead papers of the eleventh symposium of the International Society for Veterinary Epidemiology and Economics (ISVEE), Cairns, Australia

Pimelea trichostachya poisoning (St George disease) in horses

A dense population of Pimelea trichostachya plants (Family Thymelaeaceae) in pasture poisoned a horse herd in southern inland Queensland in October-November 2005. Plant density was 2 to 45 g wet weight/m2 (mean 16 g/m2) from 5 to 69 plants/m2 (mean 38 plants/m2) representing 3 to 20% (mean 9%) of the volume of pasture on offer. Ten of 35 mares, fillies and geldings were affected. Clinical signs were loss of body weight, profound lethargy, serous nasal discharge, severe watery diarrhoea and subcutaneous oedema of the intermandibular space, chest and ventral midline. Pathological findings were anaemia, leucocytopenia, hypoproteinaemia, dilatation of the right ventricle of the heart, dilated hepatic portal veins and periportal hepatic sinusoids (peliosis hepatis), alimentary mucosal hyperaemia and oedema of mesenteric lymph nodes. Cattle grazing the same pasture were affected by Pimelea poisoning simultaneously. Removal of the horses to Pimelea-free pasture initiated recovery. The one other incident of this syndrome, previously only recognised in cattle in Australia, occurred in horses, in South Australia in 2002, with access to a dense Pimelea simplex population.

Rapid and sensitive detection of Avibacterium paragallinarum in the presence of other bacteria using a 5' Taq nuclease assay: A new tool for diagnosing infectious coryza

A 5' Taq nuclease assay specific for Avibacterium paragallinarum was designed and optimized for use in diagnosing infectious coryza. The region chosen for assay design was one of known specificity for Av. paragallinarum. The assay detected Av. paragallinarum reference strains representing the three Page and the eight Kume serovars, and field isolates from diverse geographical locations. No cross-reactions were observed with other Avibacterium species, with other bacteria taxonomically related to Av. paragallinarum nor with bacteria and viruses likely to be present in swabs collected from suspected infectious coryza cases. The detection limit for the assay was 6 to 60 colony-forming units per reaction. Twenty-two out of 53 swabs collected from sick birds reacted in the 5' Taq nuclease assay, whereas Av. paragallinarum was not isolated from any of the swabs. All of the 22 swabs yielded other bacteria in culture. The presence of Av. paragallinarum in the swabs was also demonstrated by sequencing, thereby confirming the ability of the assay to detect Av. paragallinarum in the presence of other bacteria. The ability to quantify bacterial load in the swabs using the 5' Taq nuclease assay was demonstrated.

Leptospira weilii serovar Topaz, a new member of the Tarassovi serogroup isolated from a bovine source in Queensland, Australia

This paper reports on a Leptospira isolate of bovine origin and its identification as belonging to a previously unknown serovar, for which the name Topaz is proposed. The isolate (94-79970/3) was cultured from bovine urine from a north Queensland dairy farm in Australia. Strain 94-79970/3 grew at 30 °C in Ellinghausen McCullough Johnson Harris (EMJH) medium but failed to grow at 13 °C in EMJH medium or in the presence of 8-azaguanine. Serologically, strain 94-79970/3 produced titres against the Leptospira borgpetersenii serovar Tarassovi, the reference strain for the Tarassovi serogroup; however, no significant titres to any other serovars within the serogroup were obtained. Using 16S rRNA and DNA gyrase subunit B gene analysis, strain 94-79970/3 was identified as a member of the species Leptospira weilii. We propose that the serovar be named Topaz, after the location where the original isolate was obtained. The reference strain for this serovar is 94-79970/ 3 (=KIT 94-79970/35LT722).

Characterisation of chicken Campylobacter jejuni isolates using resolution optimised single nucleotide polymorphisms and binary gene markers

The principal objective of this study was to determine if Campylobacter jejuni genotyping methods based upon resolution optimised sets of single nucleotide polymorphisms (SNPs) and binary genetic markers were capable of identifying epidemiologically linked clusters of chicken-derived isolates. Eighty-eight C. jejuni isolates of known flaA RFLP type were included in the study. They encompassed three groups of ten isolates that were obtained at the same time and place and possessed the same flaA type. These were regarded as being epidemiologically linked. Twenty-six unlinked C. jejuni flaA type I isolates were included to test the ability of SNP and binary typing to resolve isolates that were not resolved by flaA RFLP. The remaining isolates were of different flaA types. All isolates were typed by real-time PCR interrogation of the resolution optimised sets of SNPs and binary markers. According to each typing method, the three epidemiologically linked clusters were three different clones that were well resolved from the other isolates. The 26 unlinked C. jejuni flaA type I isolates were resolved into 14 SNP-binary types, indicating that flaA typing can be unreliable for revealing epidemiological linkage. Comparison of the data with data from a fully typed set of isolates associated with human infection revealed that abundant lineages in the chicken isolates that were also found in the human isolates belonged to clonal complex (CC) -21 and CC-353, with the usually rare C-353 member ST-524 being especially abundant in the chicken collection. The chicken isolates selected to be diverse according to flaA were also diverse according to SNP and binary typing. It was observed that CC-48 was absent in the chicken isolates, despite being very common in Australian human infection isolates, indicating that this may be a major cause of human disease that is not chicken associated.

Infection with Menangle virus in flying foxes (Pteropus spp.) in Australia

Objective: To examine flying foxes (Pteropus spp.) for evidence of infection with Menangle virus. Design: Clustered non-random sampling for serology, virus isolation and electron microscopy (EM). Procedure: Serum samples were collected from 306 Pteropus spp. in northern and eastern Australia and tested for antibodies against Menangle virus (MenV) using a virus neutralisation test (VNT). Virus isolation was attempted from tissues and faeces collected from 215 Pteropus spp. in New South Wales. Faecal samples from 68 individual Pteropus spp. and four pools of faeces were examined by transmission EM following routine negative staining and immunogold labelling. Results: Neutralising antibodies (VNT titres ≥ 8) against MenV were detected in 46% of black flying foxes (P. alecto), 41% of grey-headed flying foxes (P. poliocephalus), 25% of spectacled flying foxes (P. conspicillatus) and 1% of little red flying foxes (P. scapulatus) in Australia. Positive sera included samples collected from P. poliocephalus in a colony adjacent to a piggery that had experienced reproductive disease caused by MenV. Virus-like particles were observed by EM in faeces from Pteropus spp. and reactivity was detected in pooled faeces and urine by immunogold EM using sera from sows that had been exposed to MenV. Attempts to isolate the virus from the faeces and tissues from Pteropus spp. were unsuccessful. Conclusion: Serological evidence of infection with MenV was detected in Pteropus spp. in Australia. Although virus-like particles were detected in faeces, no viruses were isolated from faeces, urine or tissues of Pteropus spp.

Gene expression in the skin of Bos taurus and Bos indicus cattle infested with the cattle tick, Rhipicephalus (Boophilus) microplus

The cattle tick Rhipicephalus microplus (formerly Boophilus microplus) is responsible for severe production losses to the cattle industry worldwide. It has long been known that different breeds of cattle can resist tick infestation to varying degrees; however, the mechanisms by which resistant cattle prevent heavy infestation are largely unknown. The aim of this study was to determine whether gene expression varied significantly between skin sampling sites (neck, chest and tail region), and whether changes in gene expression could be detected in samples taken at tick attachment sites (tick attached to skin sample) compared with samples taken from non-attachment sites (no tick attachment). We present here the results of an experiment examining the expression of a panel of forty-four genes in skin sections taken from Bos indicus (Brahman) cattle of known high resistance, and Bos taurus (Holstein-Friesian) cattle of known low resistance to the cattle tick. The forty-four genes chosen for this study included genes known to be involved in several immune processes, some structural genes, and some genes previously suggested to be of importance in tick resistance by other researchers. The expression of fifteen gene transcripts increased significantly in Holstein-Friesian skin samples at tick attachment sites. The higher expression of many genes involved in innate inflammatory processes in the Holstein-Friesian animals at tick attachment sites suggests this breed is exhibiting a non-directed pathological response to infestation. Of the forty-four genes analysed, no transcripts were detected in higher abundance at tick attachment sites in the Brahman cattle compared with similar samples from the Holstein-Friesian group, nor difference between attachment site and non-attachment site samples within the Brahman group. The results presented here suggest that the means by which these two cattle breeds respond to tick infestation differ and warrant further investigation.