Carriage of Brachyspira hyodysenteriae on common insect vectors.

The interactions of likely insect and murine vectors of the causative agent of swine dysentery, Brachyspira hyodysenteriae, were investigated. Insects were collected and analysed from 3 pig farms positive for B hyodysenteriae. Within these farms, several Musca domestica and Orphyra adult fly, Blatta sp. cockroach digestive tracts and hover fly (Eristalis sp) pupal form contents were positive in a standard PCR assay for B hyodysenteriae, whereas all other insect samples on these and case control farms were negative. In challenge exposure studies, B hyodysenteriae DNA was detected in the digestive tract of cockroaches and M domestica flies from day 1 post-inoculation with cultured B hyodysenteriae, for up to 5 days or 10 days respectively, while control non-inoculated insects remained negative. Isolates consistent with B hyodysenteriae were only cultured from frass samples of these inoculated cockroach and flies on days 1-3 post-inoculation. Isolates consistent with B hyodysenteriae were detected by analysis of agar plates exposed to live B hyodysenteriae-inoculated adult flies wandering and feeding on these plates for 20 min per day. In generational challenge inoculation studies, B hyodysenteriae was detected in the adult emergent flies, and internal components of fly pupae on days 1-7 of the pupation period, after being inoculated with B hyodysenteriae as larvae. Five-week-old conventional mice (C3H) that consumed 2 meals of B hyodysenteriae-infected flies remained negative for B hyodysenteriae throughout the next 10 days. The results indicated that pathogenic Brachyspira sp have a limited ability to internally colonise likely insect vectors and do not readily transmit infection to mice. However, the insect vectors analysed were demonstrably capable of mechanical carriage and likely on-farm involvement in consequence.

Modern pig farming in the People's Republic of China: growth and veterinary challenges.

Cyclical oversupply and non-profitability situations have led to pig industry consolidations in the People's Republic of China, with many smaller farmers leaving the industry. In 2007, pork supply worsened due to outbreaks of 'high fever blue-ear disease', a complex disease issue that includes highly virulent porcine reproductive and respiratory syndrome, porcine circovirus and classical swine fever. Best estimates suggest that 50 million pigs were affected. More recent natural disasters (earthquakes/freezing winters) have also limited pig production in some areas. Overall expansion of the Chinese breeding herd is now continuing at a good pace and is likely to be sufficient to supply the predicted 7% annual increase in demand for pork. High prices of feed ingredients (cereals and soybean) continue to create cost-of-production issues. Authorities have instigated many helpful measures over the past decade, including insurance for farm breeder stock, direct subsidies for farm expansions and breeding programmes, free supplies of some vaccines, and taxation exemptions. Specific challenges remaining include: the high levels of spread, persistence and on-farm impact of key virus infections on single-site farm systems; the variable titre and potency of some local vaccines; the low level of technical capacity in laboratories and the lack of training and expertise among farm staff; and the lack of a distinctive representative voice for pig farmers.

House fly vector for porcine circovirus 2b on commercial pig farms.

We investigated the on-farm potential of common farm invertebrates to transmit porcine circovirus genotype 2 (PCV2) and other non-enveloped viruses. In 2007 (pre-PCV2 vaccination) and 2008 (post-PCV2 vaccination), invertebrate communities were trap-collected (8 trap-dates per year), counted and sorted into genus and species groups on 5 farm study sites within England. Total DNA was extracted from feces of representational cross-sections of pigs on each farm in each year and also from intact samples of Diptera flies (ca. 20 flies per trap) and dissected viscera of any cockroaches (ca. 5 per trap). Each DNA sample was tested for the presence of PCV2 DNA by separate PCRs for ORF1 and ORF2. Positive samples were sub-typed via DNA sequencing of PCR products. The pig-associated Diptera fly community was dominated by Musca domestica (house fly) in both years on all 5 farms; numerous Blatta orientalis cockroaches were only noted on 1 farm throughout. Specific PCV2b DNA elements were routinely detected (25-60% of samples) in weaner/nursery pig feces in 2007, but not in other age groups. Musca collected on 4 of the 5 farms in 2007 was also positive for PCV2b DNA elements. Comparison of ORF2 sequences indicated that ORF2 sequences indicating PCV2b genotype were identical in pigs and flies. Minor changes were noted in ORF1 sequences from different samples. Flies collected in the weaner/nursery area were most likely to be positive (22-50% of fly-trap samples). DNA extracted from all cockroaches (2007 and 2008) and all flies and pig feces in 2008 were also negative throughout. We suggest that Musca flies have the most likely on-farm potential to carry and transmit PCV2b due to their life cycle incorporating stages in close association with pigs and their habitat. Vaccination appeared to reduce environmental load of PCV2b.

Lipopolysaccharide-based enzyme-linked immunosorbent assay for experimental use in detection of antibodies to Lawsonia intracellularis in pigs.

An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis.

Prevalence of exposure and infection of Lawsonia intracellularis among slaughter-age pigs.

The extent of clinical or subclinical infection associated with Lawsonia intracellularis within Dutch pig herds was uncertain. A case-control study of slaughter age pigs was used to study natural infection within Dutch herds and to compare diagnostic methods. From six case herds where clinical disease had been identified recently, and six disease-free herds, 40 pigs of slaughter-age were examined postmortem. The diagnostic methods used were: serology, gross examination, Haematoxylin and Eosin stain (HE), Warthin-Starry silver stain, Lawsonia-specific indirect immunoperoxidase of the ileum, and PCR of ileum mucosa and colon contents. There were 59% seropositive pigs in case herds and 26% seropositive pigs in control herds. Using immunohistochemistry, 57% of case herds and 46% of control herds were bacteria positive in the ileum mucosa. It was concluded that a majority of Dutch herds contain L. intracellularis infected finisher pigs. In some herds this is associated with clinical outbreaks of acute haemorrhagic enteropathy but in other herds no clinical disease is apparent. Many seropositive pigs in herds without clinical disease had evidence of Lawsonia antigen in sites other than the apical cytoplasm of proliferating epithelial cells, particularly the supranuclear region. It was uncertain whether to classify these pigs as having "recovered" from an infection or whether they have a sub-clinical or chronic form of the disease. We concluded that PCR examination of faeces and serology probably provide more specific results than gross examinations at slaughter, and that a monoclonal antibody-based examination of ileum mucosa should be the accepted screening method for this infection.

Patterns of exposure to Lawsonia intracellularis infection on European pig farms.

A serological investigation was made of the patterns of exposure of pigs to Lawsonia intracellularis, the causative agent of proliferative enteropathy (ileitis), on farms in France and Spain. Blood samples from groups of adult female pigs in breeding programmes and from postweaning pigs were monitored, the latter every month for five months, by a L. intracellularis-specific immunofluorescence seroassay. Four of 33 farms monitored in France (12 per cent) and three of 29 farms monitored in Spain (10.3 per cent) remained free of clinical signs and seronegative throughout the study. The postweaning pigs on all of the remaining French farms and on 20 of the 26 remaining Spanish farms had a pattern of infection characterised by seroconversion in the grower period, generally between eight and 16 weeks of age. The seroprevalence in these groups ranged from 8 to 20 per cent. On all of these farms at least 15 per cent of the breeding females tested were seropositive, and the farms were under similar management systems, with a continuous flow of pigs or between buildings on one site, so-called 'one site, farrow-to-finish'. On the six remaining Spanish farms, under two management groups, a multiple-site system was used, with the piglets being separated from the adults at weaning and moved to a separate location. On three of these farms, the pattern of infection was characterised by seroconversion later in the finisher period, at between 16 and 20 weeks of age, and none of the breeding females was seropositive. On the three other multiple-site farms the pattern of infection resembled that on the one-site farms. On all of the farms, the seroconversion of groups of pigs was frequently associated with clinical or subclinical signs of ileitis.

Risk-based evaluation of postmortem inspection procedures for pigs in Australia.

The results of traditional (incision) and risk-based (visual) postmortem inspection procedures were compared on groups of approximately 30,000 pigs. The performance characteristics used as a basis for comparison included the non-detection rates of grossly detectable abnormalities, the microbiological contamination rates of carcases and boned product, the association of reactive lymph nodes with carcase condemnation and the achievement of 'finished product standards' for 'wholesomeness'. It was estimated that 6 per cent of all cases of abscessation and 28 per cent of all cases of arthritis were undetected by the traditional method, and the comparable figures for the risk-based procedure were 19 per cent and 39 per cent. However, when the rates of contamination of undetected abnormalities with foodborne hazards and other carcase contamination parameters were taken into account, it was concluded that both inspection systems were likely to result in a very similar level of consumer protection. Any increase in potential exposure to foodborne hazards in the abnormalities undetected by risk-based inspection would be insignificant in comparison with the potential exposure to foodborne hazards resulting from contaminated 'normal' lymph nodes and carcase surfaces. There were no statistically significant differences between the two procedures in the contamination rates of pre-chill carcases or boned retail products with Salmonella and Yersinia species.

Colonic infection by Bilophila wadsworthia in pigs.

Bilophila wadsworthia is a common inhabitant of the human colon and has been associated with appendicitis and other local sites of inflammation in humans. Challenge-exposure or prevalence studies in laboratory and other animals have not been reported. B. wadsworthia is closely related phylogenetically to Desulfovibrio sp. and Lawsonia intracellularis, which are considered colon pathogens. We developed a PCR specific for B. wadsworthia DNA. Samples of bacterial DNA extracted from the feces of pigs on six farms in Australia and four farms in Venezuela were examined. Specific DNA of B. wadsworthia was detected in the feces of 58 of 161 Australian and 2 of 45 Venezuelan pigs, results comprising 100% of the neonatal pigs, 15% of the weaned grower pigs, and 27% of the adult sows tested. Single-stranded conformational polymorphism analysis of PCR product DNA derived from pigs or from known human strains showed an identical pattern. Histologic examination of the intestines of weaned B. wadsworthia-positive pigs found no or minor specific lesions in the small and large intestines, respectively. B. wadsworthia is apparently a common infection in neonatal pigs, but its prevalence decreases after weaning. The possible role of B. wadsworthia as an infection in animals and in human colons requires further study.

Identification of canine coronavirus strains from feces by S gene nested PCR and molecular characterization of a new Australian isolate.

A nested PCR (nPCR) assay for the detection of canine coronavirus (CCV) in fecal samples is described. The target sequence for the assay was a 514-bp fragment within the spike (S) glycoprotein gene. The sensitivity of the assay is extremely high, detecting as little as 25 50% tissue culture infective doses per g of unprocessed feces. A clinical trial using dogs challenged orally with CCV SA4 and CCV NVSL was used to compare viral isolation and the nPCR assay as detection techniques over a 2-week period of infection. Virus isolation detected CCV shedding from day 4 to 9 postchallenge, while the nPCR assay detected CCV shedding from day 4 to 13 postchallenge. Cloning and sequencing of the nPCR assay product enabled investigation of the evolutionary relationships between strains within the S gene. The simple and rapid procedure described here makes this assay an ideal alternative technique to electron microscopy and viral isolation in cell culture for detection of CCV shedding in feces. The described assay also provides a method of identifying new strains of CCV without the complicated and time-consuming practice of raising antibodies to individual strains. This is illustrated by the identification, for the first time, of an Australian isolate of CCV (UWSMN-1).

Actinobacillus suis infection in pigs in Australia.

This is the first report of the isolation of Actinobacillus suis in association with significant disease among preweaned pigs in Australia. Sudden deaths occurred in preweaned piglets at one facility and enlarged joints, particularly the stifles and tarsi, occurred at another. Isolates with the biochemical phenotype and apx genotype profile of A suis were cultured from affected piglets. Both facilities were of high health status and one had undergone a recent depopulation, disinfection and sow repopulation process. Reviewing initial reports of A suis disease overseas, it is apparent that outbreaks occurred sporadically in various locations, with disease occurring among the herds involved for some months only, before development of immunity. A suis disease is now considered an emerging disease on high health status farms.

Therapeutic efficacy of water-soluble lincomycin-spectinomycin powder against porcine proliferation enteropathy in a European field study.

Controlled clinical trials to a standardised protocol were conducted into the effect of a water-soluble antibiotic on proliferative enteropathy and its causative agent (Lawsonia intracellularis) on commercial pig farms at six sites in four European countries. Clinical signs of the disease and L intracellularis-specific polymerase chain reaction (PCR)-positive pigs were detected in pens of six- to 12-week-old pigs (weighing 5 to 55 kg) immediately before each trial. Matched pens of randomised pigs were either left unmedicated (32 to 59 pigs per trial), or medicated orally with 10 mg/kg of a water-soluble combination of lincomycin and spectinomycin powder (21 and 42 mg, respectively, of antibiotic activity per litre) for either seven days (33 to 61 pigs per trial), or 14 days (33 to 61 pigs per trial), delivered via the drinking water. Investigators did not know which pens received which treatment In most of the affected pigs in each trial, diarrhoea due to L intracellularis resolved within three to seven days after the medication began, whereas most unmedicated pigs remained diarrhoeic for at least 10 days. On average the medicated pigs gained more weight than the unmedicated pigs over the 21-day trial period (P=0.01). In two trials, the absence of L intracellularis after the treatment ended was confirmed by the PCR.

Monitored control programme for proliferative enteropathy on British pig farms.

The effect of control programmes on proliferative enteropathy and its causative agent (Lawsonia intracellularis) was investigated on four farrow-to-finish pig farms in Britain. Faeces samples from groups of boars and gilts in breeding programmes, and from preweaning and postweaning pigs were monitored prospectively every month for six months by a L intracellularis-specific polymerase chain reaction (PCR). On one farm with 150 sows, an outbreak of acute proliferative enteropathy in boars and gilts was controlled clinically by the use of tiamulin and chlortetracycline. The percentage of detectable PCR-positive pigs decreased from between 50 to 70 per cent to zero in the treated pigs and their progeny less than 14 weeks old, but clinical signs of the disease and PCR-positive pigs were detected in some 14-week-old pigs derived from the treated groups. On another farm with 160 sows, an outbreak of chronic proliferative enteropathy in six-week-old pigs (23 to 26 per cent PCR-positive) was controlled by the use of oral tylosin phosphate. Faeces samples from the medicated pigs on this farm remained PCR-negative during the study period, whereas samples from unmedicated control pigs showed that the infection persisted in some pigs for at least six weeks. The two other monitored farms remained PCR-negative and clinically negative for the disease during the study period. These farms treated the pigs regularly with oral chlortetracycline.

Tissue damage in cattle infected with Theileria annulata accompanied by metastasis of cytokine-producing, schizont-infected mononuclear phagocytes.

The distribution of schizont-infected cells in six calves undergoing acute, lethal sporozoite-induced infections with Theileria annulata was examined, the calves being killed in the early, middle or late stages of disease. A combination of histological and immunocytochemical techniques showed that schizont-infected cells became disseminated rapidly through the lymphoid tissues from the prescapular lymph node draining the site of inoculation to distant lymph nodes (e.g., precrural, mesenteric and mediastinal) and to the spleen and thymus. The parasitized cells also spread rapidly into non-lymphoid organs, being found in the liver, kidney, lung, abomasum, adrenal glands and pituitary gland by day 7, in the brain by day 12 and in the heart by day 14 after infection. As infection progressed, the schizonts differentiated into merozoites. By the late stages of disease, the cells containing merozoites greatly out-numbered schizont-infected cells. The parasitized mononuclear cells were labelled by antibodies to bovine interferon-alpha1 and tumour necrosis factor-alpha and, during the later stages of the disease, contained erythrocytes parasitized by piroplasms. The results suggested that the parasitized mononuclear cells themselves played a role in the development of clinical disease and in tissue damage. These findings provide new evidence that tropical theileriosis can no longer be viewed as a lymphoproliferative disease resulting from the uncontrolled multiplication and metastasis of lymphoid cells infected with T. annulata schizonts, but is caused by a parasite that lives in, and is disseminated by, cytokine-secreting, proliferating mononuclear phagocytes.