Development of well validated and standardised serological assays for the detection of Leptospira, Trichinella and A. cantonensis infection in livestock (Objective 1)

ELISA tests are under development for the detection of infection with Leptospira, Trichinella and Angiostrongylus cantonensis in livestock in the Pacific islands. Sources of antigen and reference serum have been identified and each assay will be optimised and validated in the next twelve months.

In addition, PCR-based tests for the detection of Leptospira and A. cantonensis infection have been developed that will be used to test samples collected on surveys conducted in Pacific island countries in 2003.

Transfer of skills to partner institutions necessary to perform Trichinella detection techniques and post mortem examination of rodents to collect parasites and serum (Objective 1)

A workshop to train technicians from NAQIA and SPC in the skills necessary to detect Leptospira, Angiostrongylus and Trichinella infection was postponed from November 2002 until mid-2003 because a survey on the Bula Plain earlier in the year failed to provide a new isolate of T. papuae was that was to be used for experimental studies at the NVL. The workshop will be conducted once a sample of T. papuae is made available by the International Trichinella reference Centre in Rome.

Information on the prevalence and distribution of Trichinella, Leptospira and A. cantonensis in the Pacific Islands (Objective 2)

Two villages in Fiji have been comprehensively studied to date. Serum was collected from every animal in each village and anti-Leptospira antibodies detected using the microscopic agglutination technique (MAT) at the WHO reference centre for leptospirosis in Brisbane. Results show that prevalence of Leptospira infection was higher in cattle in Naduri compared to Burebusaga. In Naduri, the prevalence of infection in cattle and dogs was higher than in pigs. Although not significant the same trend appears to exist in Burebusaga. At this stage it is not possible to interpret these data fully. This will be done once information on infecting serovars and data from testing the human population are available.

Collection and identification of new isolates of Trichinella from pigs and rodents in an area in PNG where human infection is suspected (Objective 4)

One survey was conducted in collaboration with the Northern Australia Quarantine Strategy (NAQS) to recover a new isolate of T. papuae from pigs on the Bula Plain, Western Province. One pig sampled was found to be infected but only with approximately 1 larvae per gram of muscle. This larval burden was too low to enable passage to a rat for further experimental studies. Future experimental work at the NVL will be done using a sample of T. papuae supplied by the International Trichinella Reference Centre in Rome, which holds the original isolate recovered in 1997.

Information on the impact of A. cantonensis infection in ruminants (Objective 4)

The life cycle of A. cantonensis has been established at Murdoch University including maintenance of infected snails (intermediate host) and infection of rats (definitive hosts). Two experiments have been conducted to determine the pathogenesis of A. cantonensis infection in sheep. Results to date are not conclusive. No evidence of eosinophilic meningitis has been observed in experimentally infected sheep This could be due to a number of factors including; the isolate used is not infective for sheep, the dose given is not sufficiently high to elicit an immune response, the method of preparation of larvae reduced their infectivity, sheep are refractory to A. cantonensis infection. We do not have sufficient data to fully interpret our data as yet. Experiments are planned for 2003 to attempt to address some of these issues.

Research conducted by at Murdoch University showed that a large number of snail and slug species commonly found in Perth are able to act as intermediate hosts for A. cantonensis. Snail species included small aquatic snails that are likely to be accidentally ingested by ruminants grazing near sources of standing water.

Year 2 (01/01/2003-31/12/2003)
An ELISA test was successfully developed for Leptospira pomona. This ELISA has now been standardised for use in cattle and pigs. The results from testing serum collected cattle on Numundo Farm in PNG show that the test has good agreement with the standard serological test and that it had high specificity. Further work is required to test sufficient samples from PNG to determine the diagnostic sensitivity and specificity compared to the standard test. The test is now in a format suitable for transfer to partner laboratories.
A two-day workshop on diagnostic techniques for Trichinella infection was held at the National Veterinary Laboratory (NVL) in PNG.
Results of a retrospective serological survey of 479 people living in the Balimo area showed that the prevalence of infection with Trichinella of 29.5%. Antibody prevalence did not differ according to sex but there was a significant difference between age groups, and also appeared to differ significantly according to geographic region and age. These results (and those from other surveys) show that T. papuae is probably widely distributed in wild pigs (the most likely source of infection for humans) in the south coastal area of PNG at least as far east as Gulf Province.
At the NVL, examination of muscle digests from 31 pigs and 15 rats from the Goroka area found no evidence of T. papuae.
Following the discovery of Trichinella zimbabwensis in crocodiles in Zimbabwe AQIS has required PNG to confirm freedom from Trichinella infection in its crocodile meat exported to Australia from Mainland Holdings Crocodile Farm in Lae. A total of 112 samples of crocodile muscle have been tested at the NVL from captive bred and wild-caught animals. The majority of wild-caught crocodiles originate from the Kikori area (n=52) in Gulf Province. Sixteen of the 52 (30.8%) samples of meat taken from crocodiles from Kikori contained T. papuae larvae.
Surveys to determine the prevalence and identity of infecting Leptospira serovars began in 2003. Results of a cross-sectional survey of breeding cattle on Numundo Beef (West New Britain Palm Oil) show that there is a high prevalence of Leptospira infection in cattle older than two years of age and that the predominant infecting serovars were hardjo, pomona, swajizack and medanensis. These data confirm that active Leptospira infection exists in cattle herds in PNG and may be a factor in the infertility observed in cattle in PNG.

Development of the capacity for effective surveillance of Trichinella, Leptospira and A. cantonensis infection in the South Pacific region through the development and transfer of diagnostic technologies

Training was provided to a technician from the NVL in the use of an antibody detection ELISA to detect infection with Leptospira spp and Trichinella spp during a workshop at Murdoch University.

Peter Wai'in from the NVL is completing a PhD on techniques to detect infection with Leptospira (PCR, culture and ELISA) and the epidemiological principles required to design, implement and analyse data from surveys to determine the incidence of infection and its association with infertility. Dr Nime Kapo (NAQIA) has also received hands-on training in the sampling strategies for disease surveys in PNG.

To determine the prevalence of Trichinella, Leptospira and A. cantonensis infection in livestock in Fiji and Kiribati and risk factors associated with their transmission

Results of surveys conducted in PNG show that there is a high prevalence of infection with Leptospira spp in cattle in PNG and it is a significant cause of infertility in affected herds. These results show that the prevalence of infection is higher in large commercial herds and that there is evidence that attempts to control infection using vaccination have failed. However, the reasons for this apparent failure may relate to management issues and not the failure of vaccination per se. Further work is required to determine the optimal vaccination strategy.

A comparison of data from testing humans and animals in two villages in Fiji show that in the village that experienced human cases of leptospirosis the pattern of serovars affecting the human population most closely resembles the profile found in domestic dogs. This opens up the possibility of interventions by vaccination of village dogs or by modifying the contact between the species.

Results from testing a total of 108, 48 and 73 serum samples collected from pigs from Fiji, Tonga and Kiribati respectively showed that the prevalence of antibodies to Trichinella were low. Only three pigs and one mongoose from Fiji were shown to have high levels of antibodies, which suggest that infection, if present is sporadic and may be present in Mongoose. Further work is required to determine the presence or absence of Trichinella in Mongoose because they are abundant and relatively easy to trap.

A field visit to Kikori in April 2004 to investigate the potential source of Trichinella isolated from farmed crocodiles in Lae revealed that crocodiles held in the villages are fed them on wild pig meat and fish prior to transport to the crocodile farm in Lae. It is likely that pigs are the source of Trichinella because larvae were recovered from the meat of a wild pig sold at Kikori market.

To determine which species of Trichinella are present in Fiji and Kiribati

No further progress was made towards fulfilling this objective due to staff shortages in the SPC

To investigate the pathogenesis of T. papuae in pigs and Angiostrongylus cantonensis in ruminant

Experimental studies in PNG showed that the larvae of T. papuae remain infective in pig meat for up to 26 days at 5C and for 9 days in meat maintained outdoors at ambient temperature (32C). This is important because it shows that surveys to recover Trichinella from animals in remote locations are possible if tissue samples can be refrigerated.

There was no evidence of clinical or subclinical disease induced by experimental infection of two merino cross-bred ewes with at least 1,000 L3 larvae of A. cantonensis. This suggests that A. cantonensis is not a significant pathogen of sheep.

The majority of project activities are due to begin in 2006. There has been limited progress towards Objective 1 mainly because of delays in the finalisation of project documents for this extension. Preliminary discussions with cattle producers in the Markham Valley have been conducted and their cooperation achieved. The first survey is planned for May 2006.

Research activity to perform a cross-sectional survey of domestic pigs in PNG to prove freedom from Trichinella infection will commence in 2006. In 2005 a total of 327 crocodiles were screened for the presence of infection with Trichinella using the digest method. A total of 44 crocodiles were shown to be infected with Trichinella larvae, of which 27 came from Kikori, 12 from other locations and 5 were born on the farm. Larvae from the crocodiles from Ambunti (Sepik) were analysed at the International Trichinella Reference Centre, Rome and found to be genetically identical to larvae isolated crocodiles from Kikori. These data suggest that Kikori is the only source of infected crocodiles at Mainland Holdings. In addition, meat from 3 wild pigs from Ambunti was also negative for Trichinella infection.

Progress towards project milestones has been hampered by technical issues in PNG related to the mergence of avian influenza as a high priority disease, which reduced the time project staff had to devote to project activities.

Longitudinal and cross-sectional surveys to determine the prevalence of Leptospira infection in animals in smallholder villages and to determine the dynamics of transmission on commercial cattle properties have progressed well. This work has so far demonstrated that there is a very low prevalence of Leptospira infection in animals in smallholder villages associated with commercial properties. This means that the animals do not pose a risk to humans and that leptospirosis is unlikely to be a major constraint to livestock production. Samples collected from commercial properties are in the process of being tested.