The project aimed to develop accurate diagnostic tests for this parasitic disease, to establish more effective ways of controlling it, and to transfer this technology to neighbouring countries to improve surveillance and reduce the risk of disease entry to Australia.
The disease of animal trypanosomiasis (or surra) is caused by the protozoan parasite Trypanosoma evansi, transmitted by biting tabanid flies (March flies). The result of infection is an acute or chronic wasting, depending on host susceptibility and the virulence of the parasite strain. Many animals can be afflicted, including livestock and horses. The disease is found in much of the world, including all of the Philippines and Indonesia. Evidence from antibodies in blood samples shows that T. evansi may be present in parts of Irian Jaya. However, it is believed that Australia and Papua New Guinea may no longer be free of this disease.
Recent research has shown that pigs and deer could be efficient reservoir hosts for the parasite. Pigs and two species of wallaby common to Papua New Guinea and northern Australia, are believed to be highly susceptible to infection and suffer acute disease with a high mortality rate. Therefore if the disease is found in PNG or enters Australia, it would have a devastating effect on livestock and probably on many native marsupials.
Before any control programs could be designed, more knowledge was needed about the impact of T. evansi on livestock and the determinants of clinical disease. This would require sensitive and well-validated diagnostics tests, coupled with an understanding of the genetics of virulence in the different strains of T. evansi. To date there were no such effective tests, and drugs used in treatment had not been systematically evaluated.
Both Indonesia and the Philippines wanted to establish control mechanisms to reduce livestock infection and improve productivity. This project therefore involved both countries, along with Papua New Guinea and Australia, building on earlier research into the parasite carried out at James Cook University, Townsville (ACIAR project AS1/1996/150) that demonstrated the inadequacy of existing diagnostic tests.
2. Executive Summary
2(i) Purpose and context of the project
2. Executive Summary
2(i) Purpose and context of the project
Animal trypanosomiasis (Surra) caused by Trypanosoma evansi is endemic throughout southeast Asia where it is an important constraint to the productivity of smallholder livestock. Surra does not occur in PNG and Australia, however, there is strong serological evidence that it has spread to Irian Jaya. If Surra were to enter PNG and Australia it would have a devastating effect on livestock and native marsupials that are highly susceptible to infection. Current control efforts for Surra are based on the treatment of individual animals with a limited repertoire of drugs the efficacy of which has not been systematically determined.
The main purpose of this project is to enhance the capability for surveillance of T. evansi in eastern Indonesia, PNG and Australia and to improve knowledge of the epidemiology of, and control measures for surra. To achieve this the project will transfer diagnostic technology and provide specific training to staff in diagnostic laboratories in Sulawesi, Papua Province, PNG and the Philippines. The development of rational control programs for surra and models to predict the spread of T. evansi and occurrence of disease require accurate data on the determinants of infection and disease. In the Philippines, epidemiological studies will be undertaken to gather the data necessary to identify these key epidemiological factors so that the effectiveness of the National Control Program, initiated by the Philippines government in 2001, can be improved. In addition, the project will evaluate the efficacy of existing trypanocidal drugs and conditions for their effective use in horses and buffalo in Indonesia and the Philippines to enable the selection of effective and sustainable treatments for surra.
2(ii) Collaborating research institutions
Division of Veterinary and Biomedical Sciences, Murdoch University, Perth, Western Australia
Research Institute for Veterinary Science (Balitvet), Bogor, Indonesia
Disease Investigation Centre, Maros, South Sulawesi, Indonesia
Dinas Peternakan, Jayapura, Indonesia
University of Southern Mindanao, Philippines
Department of Agriculture, Region XI, Philippines
National Agriculture and Quarantine Inspection Authority, Papua New Guinea
2(iii) Results or expected results
There were two main facets of work undertaken in 2001: Development of a laboratory model for chronic T. evansi infection and serological surveys of livestock in four provinces of Mindanao.
Groups of rats were infected with a non-pathogenic isolate of T. b. brucei that was reported to induce a chronic cyclical parasitaemia. T. b. brucei were detectable within 3 days of infection in all infected rats. However, infected rats did not develop a cyclical parasitaemia. A PCR was successfully used to amplify a sequence of the tubulin gene of T. brucei. These results showed that rats infected with T. b. brucei did not develop a suitable parasitaemia for the study of chronic T. evansi infection. Results from testing tissue samples with a PCR showed it is possible to detect trypanosomal DNA in host tissues and work will continues to further develop this procedure.
Surveys of livestock in eastern Indonesia and Mindanao, Philippines show that the epidemiology of infection differs in each country. The results of the survey in Mindanao have identified three municipalities, Mtalam, Mlang and Kabacan that differ in the prevalence of T. evansi infection and the history of outbreaks of clinical disease. A longitudinal study will be undertaken in these areas to determine the incidence and outcome of infection in local livestock and to determine other factors involved in the development of disease.
Results from an outbreak of surra in Region 9 (Zamboanga) in Mindanao were used to develop a model to assist with the selection of the most appropriate control strategy in the face of an outbreak. The output of this simple model indicated that the prevalence of disease and efficacy of treatment are important factors in the decision-making process and that cost (cost of treatment and value of livestock) should not influence the choice of control option. For example, a policy of treatment of all livestock in an affected area should be considered when the prevalence of disease exceeds 30-40%. Further refinement of this model is required to ensure that it can be used in the field.
2(iv) Direction of future research activities.
Future research includes:
Extension of pathogenicity study in mice to compare isolates of T. evansi from the Philippines with Indonesia isolates. This is required to determine if the pathogenicity of isolates of T. evansi is a factor in the emergence of the large number of outbreaks of surra in the Philippines. If differences are discovered in the pathogenicity of T. evansi from the Philippines and Indonesia genetic comparison will be required to characterise differences in the isolates to provide information or a tool that can be used to determine the spread/distribution of pathogenic isolates.
The transfer of diagnostic technology. This will be achieved in the first instance at a workshop at Balitvet (held April 2002) to transfer diagnostic technology to participating institutions. The workshop will be expanded to include additional participants from the Philippines and Indonesian DIC’s (approximately 20 in total) to improve the effectiveness of the transfer of knowledge and techniques.
Determination of key factors involved in the transmission of T. evansi infection and the development of clinical disease. This information is required for the development of rational control programs and to permit an understanding of the factors that will lead to the geographical spread of T. evansi.
Continuation of efforts to develop a model for chronic T. evansi infection and to use this model to investigate hypotheses drawn from data generated by the epidemiological investigations in Mindanao
Evaluation of existing trypanocidal compounds. The most effective drug for the treatment of T. evansi infection is Suramin. Suramin is no longer manufactured and will soon be unavailable in Indonesia where it is the mainstay of treatment of surra. Most other drugs have been poorly evaluated for use against T. evansi in horses and buffalo (the main host species affected in southeast Asia). The selection of alternatives must be based on the best available information on the effective dose and toxicity in the host. Experimental studies will be conducted at Balitvet to provide data that will be used to make these decisions.
Transfer of existing technologies/training workshop at Balitvet
A workshop on the diagnosis and epidemiology of T. evansi that was originally planned in 2001 was postponed and conducted at Balitvet in April 2002. Additional funding from the Crawford Fund ($15,000) allowed the inclusion of additional scientists from Indonesia and the Philippines. A total of 16 scientists from Indonesia, the Philippines and PNG participated in the workshop.
Participants at the workshop were given ‘hands on’ training in the use of improved diagnostic techniques for the detection of T. evansi and on the epidemiological principles required to design and interpret disease surveys.
Project participants at University of Southern Mindanao (USM), Department of Agriculture, Southern Mindanao, Region XI (DA Xi) in the Philippines, and the District Investigation Centre, (DIC) of the Department of Livestock Services, Maros in Indonesia, were supplied with reagents and a rotator necessary to perform the Card Agglutination Test for T. evansi (CATT/T. evansi). Each laboratory has undertaken prevalence surveys using this serological assay.
In February 2002 Dr Erlinda Lim (DA XI), Dr Buenviaje (USM) and Dr Carlito Sanchez (USM) visited Murdoch University for 4 weeks to receive training in epidemiological principles and the use of diagnostic tests so that they are able to undertake longitudinal studies of T. evansi infection in Mindanao.
Application of molecular techniques to improve the accuracy of diagnosis of T. evansi
Good progress has been made in the development of molecular techniques to detect T. evansi DNA in blood and tissues of infected animals. The sensitivity of a PCR using 6 different published primer sets, and primers designed for rDNA ITS and cysteine protease gene were compared. The most sensitive PCR primer pair was able to detect 1 trypanosome per ml of blood. This is a significant improvement over previously published reports. The sensitivity of the PCR is higher than the theoretical sensitivity of 5 trypanosomes per ml of blood. It is possible that the sensitivity of this assay has been increased because each samples was stored frozen before DNA extraction. This may have lysed the trypanosomes in each sample releasing the DNA into solution thus increasing sensitivity compared to fresh blood that would have contained intact parasites. This work is ongoing and the sensitivity of PCR using fresh and frozen blood will be compared further.
The use of alternative methods of samples storage and DNA extraction are being evaluated. These include dried blood stored on filter papers (plain paper and FTA cards (Whatman)) and the use of other commercial DNA extraction kits. The use of dried blood is particularly important because it will allow continuous surveillance in remote areas.
Determine genetic basis for intraspecific variation in pathogenicity
Two experiments were conducted to compare the pathogenicity (measured as the length of survival of infected mice) of different isolates of T. evansi from Indonesia and the Philippines. Results show that the T. evansi isolates can be ranked in order of increasing pathogenicity; Sudan (Africa)>Indonesia>the Philippines. In addition, one isolate of T. evansi collected from an apparently healthy buffalo was also less pathogenic (significantly longer survival time) compared to the 9 isolates collected from animals suffering clinical disease. These results are important because they may explain to some extent the differences observed in the epidemiology of surra in Africa, Indonesia and the Philippines.
Three minisatellite loci (Biteau, Bringaud et al. 2000, Mol Biochem Parasitol 105(2):187-202) revealed a genetic difference between an Indonesian isolate and 5 Philippine isolates (which were all identical) that differed in their pathogenicity for mice (see section on pathogenicity). The internal transcribed spacer region (ITS1) for T. evansi isolates from Africa (4), Indonesia (7) and the Philippines (3) was sequenced in an attempt to further define differences in these isolates. Differences were observed but no strict geographic grouping could be determined.
Evaluate the efficacy of existing trypanocidal drugs for the treatment of T. evansi
Preliminary studies at Central Mindanao University (CMU) show that Diminazene aceturate (the standard treatment for surra in the Philippines) does not cure T. evansi infection in goats when a single dose of 7 mg/kg is given. This is important because there are anecdotal reports of treatment failures have been received from Mindanao. Quinapyramine is probably the most suitable drug for routine use in the Philippines (and perhaps in Indonesia) because it is cheap, easily obtainable from manufacturers in India, efficacious and it has a prophylactic effect. Previous studies in mice at Balitvet have shown that is effective against 10 Indonesian isolates.
Further work will be undertaken at Balitvet in 2003 to evaluate the variation in sensitivity of T. evansi isolates to Quinapyramine and to determine effective doses rates for livestock. Stocks of breeding BALB/c mice have been provided and Amir Husein is in the process of developing a nucleus of breeding mice for use in this experiment and future work at Balitvet.
Epidemiology of infection with T. evansi in Mindanao
A survey was conducted by USM to determine the prevalence of T. evansi infection in cattle and carabao (buffalo) owned by 50 farmers in the municipalities of Mlang and Matalam. These locations were chosen because previous surveys had shown that the prevalence of infection differed in the two municipalities. Results from the survey in 2002 show that the prevalence of T. evansi infection in each Municipality is now the same. Data were also collected on a range of management factors and further analysis of this data will be completed in 2003.
Transfer of existing technologies
The final project workshop was attended by project staff from Indonesia, the Philippines, Papua New Guinea and Australia as well as scientists from the Northern Australian Quarantine Strategy (NAQS) and the Northern Territory State Government. Project outputs were presented and discussed in the context of the priorities of each partner country. Information suitable for dissemination at the smallholder level was identified and data suitable for publication were also identified.
Application of molecular techniques to improve the accuracy of diagnosis of T. evansi
This aspect of the project has progressed well and a prototype sample collection kit using Whatman FTA Cards has been developed and provided to project participants in Mindanao for evaluation. In addition, veterinary staff members from NAQS have purchased FTA cards and preliminary evaluation in their program in East Timor has confirmed their suitability for use to collect blood samples for surveillance for surra and other blood-borne infections. The use of FTA cards to collect blood samples in remote areas will significantly enhance surveillance for T. evansi in Australia and PNG.
Results of testing tissue samples from animals experimentally infected with T. evansi showed that lung and heart, in particular the heart valve, were positive on more occasions than other tissues and blood. This information has been provided to veterinary staff at NAQS because it will ensure the correct samples are collected during surveys of wild or feral animals in Australia and PNG where individual animals are killed and samples taken post mortem.
Determine genetic basis for intraspecific variation in pathogenicity There is insufficient data to determine the genetic basis for differences observed in the pathogenicity of T. evansi in mice. Genetic differences have been identified between isolates of T. evansi from Indonesia, the Philippines and Kenya (as part of a related project), showing correlation between differences in the epidemiology of infection in camels and geographic separation in Kenya, while differences in Indonesia correlate with geographic separation. It is difficult to interpret results from the Philippines because there are only a limited number of isolates from two regions of Mindanao
Evaluate the efficacy of existing trypanocidal drugs for the treatment of T. evansi. Four separate experiments were conducted at Balitvet to determine the comparative sensitivity of 5 isolates of T. evansi from different geographic locations in Indonesia to three trypanocidal drugs (diminazene aceturate, quinapyramine, cymelarsan). Only cymelarsan was deemed suitable for further evaluation in large animals because both diminazene and quinapyramine failed to cure all animals, even at high potentially toxic dose rates, and quinapyramine caused overt signs of toxicity at sub-curative doses. No signs of toxicity were observed with cymelarsan.
Epidemiology of infection with T. evansi in Mindanao Good progress was made in collecting data to determine some of the risk factors associated with T. evansi infection in Mindanao. A large data set was collected from longitudinal surveys of 50 cattle owners and 50 buffalo owners from Matalam and Kabacan. The set contained information on the prevalence of infection with T. evansi, factors relating to the individual livestock and their management by farmers, and information about social factors such as household income and education levels. Results show that the prevalence of infection in Kabacan has decreased markedly from 2001 to 2002.
Data analysis showed that carabao (buffalo) owners who also own goats are twice as likely to have animals that register positive to the Card Agglutination Trypanosoma Test (CATT), and that farmers with only elementary education are five times more likely to have CATT-positive animals compared to farmers who have secondary level education. There is also a weak association between increasing income and increasing likelihood of having CATT-positive buffaloes. These results show that communication packages must focus on farmers with elementary-level education to ensure the greatest impact. More data on other management factors are required to interpret the significance of goats and household income in the epidemiology of infection with T. evansi.
A Microsoft Access database has been developed to store information on the frozen isolates of T. evansi held at Balitvet, allowing this valuable resource to be used more effectively to study T. evansi.
Project researchers at Murdoch University have developed a mouse model with similar features to ruminant surra in Mindanao. This research tool will be used to develop tools and methodologies for the study of the pathogenesis of surra as well as tests to detect infection.
Determine the socio-economic profile of surra in Mindanao
A workshop was held in Davao in September 2004 to provide training for project staff as well as veterinary staff from other regional offices who form part of the Mindanao Unified Surra Control Approach (MUSCA) program. Preliminary results from a field visit during the workshop showed that carabao are the most important livestock species and that their value is measured by their use as draft animals. If an outbreak of surra occurred mortality in carabao would significantly reduce the capacity of farmers to generate income and provide food from the production of cash crops.
Two preliminary surveys to determine the prevalence of infection with T. evansi in carabao have been performed in a region of Mindanao where there is no evidence of clinical surra (Bukidnon) and in a region that has experienced a recent outbreak of surra (Surigao del Norte). Results showed that there is a significant difference in the serological prevalence of infection between the two regions determined. The prevalence of parasitaemia with T. evansi in Surigao del Norte was high (12.5%), which suggests that transmission of T. evansi is still occurring despite intensive use of trypanocidal drugs in the area with Diminazine aceturate (Trypan).
The next phase of this work will be to design a longitudinal survey of carabao in these two areas. This will be completed during a site visit in May 2005.
Determine the efficacy of cymelarsan for the treatment of chronic T. evansi infection
An experiment was conducted at Balitvet to re-evaluate if different isolates of T. evansi from Indonesia vary in their susceptibility to cymelarsan. A series of experiments conducted as part of the original AS1/2000/009 was unable to unequivocally prove this mainly because of the need to use small experimental groups (5). The results show that there is no significant difference between the sensitivity of each of three isolates of T. evansi to cymelarsan. This is important because it suggests that Indonesian isolates of T. evansi are likely to be reasonably homogeneous in their susceptibility to treatment with cymelarsan.
A larger experimental infection study is now underway at Balitvet to determine the efficacy of cymelarsan for the treatment of buffalo chronically infected with T. evansi.
Develop control programs and extension packages
The development of a training manual and extension material for surra in the Philippines will proceed once data from surveys conducted late in 2004/early 2005 is available.
Consolidate technical capacity in Australia and PNG for detection of T. evansi
Field validation of the Whatman FTA cards for the collection of blood samples in PNG and northern Australia proceeded as planned. The sample collection protocol proved to be achievable under field conditions and has now been adopted by staff working in the NAQS program.
The project’s main scientific achievement is a considerably enhanced capability for effective surveillance of Surra in Irian Jaya, PNG and the Philippines. A network of researchers on T. evansi from Indonesia, the Philippines, Australia, PNG and external agencies such as ILRI is now firmly established.
The successful outcomes were achieved by the transfer of current diagnostic technologies which were then improved by identifying more specific and sensitive tests for serological diagnosis. The scientists developed molecular techniques for low-level detection of organisms in blood samples, identified genetic markers for pathogenicity of T. evansi and tested their usefulness in predicting outbreaks of clinical disease. They also investigated the efficacy of existing trypanocidal drugs for the treatment of T. evansi. The elucidation of the epidemiology of infection with T. evansi in Mindanao and Sulawesi was a significant and important contribution to understanding the disease in Asia. A secondary, vital achievement was development of techniques to collect and send blood and tissue samples to Australia in compliance with Australian quarantine standards.
The research to date suggests there are multiple factors involved in the development of outbreaks of Surra in the Philippines. The severity of outbreaks of Surra in Mindanao could be due in part to genetic differences compared to Indonesian isolates, and preliminary results using the mouse pathogenicity test suggest this. Pathogenic strains of T. evansi were identified in specific areas, indicating that control programs must focus on regions where acute clinical disease is most likely and that the response to notifications of clinical disease must be rapid to prevent death due to infection.
Further technology transfers to participating countries should improve the effectiveness of surveillance for T. evansi. The information gathered can then be incorporated into guidelines on the most cost-effective treatments of infection with T. evansi. This should improve the success of treatment and control programs based on treatment of infected animals.