Validation of in vitro-produced and freeze-dried whole cell lysate antigens for ELISA Trypanosoma evansi antibody detection in camels.

Trypanosoma evansi is a blood parasite responsible for surra in mammals, with a high impact in camels and horses. The WOAH-recommended reference method for detecting immunoglobulin G directed against T. evansi is ELISA, using whole cell lysate antigens (WCLAs). WCLAs are prepared with T. evansi produced in laboratory rodents, separated from blood cells using DE-cellulose anion exchange chromatography. As parasite lysates are fragile, antigens are preserved frozen pending use. For these reasons and others, T. evansi WCLAs are not commercially available. They are produced in small quantities, in a limited number of specialized laboratories, and they require a reliable and expensive cold chain for their shipment. In this study, we assessed and validated in vitro production of T. evansi and lyophilization of WCLAs in comparison with the reference method using frozen WCLAs prepared with parasites produced in rodents. Using a set of 400 samples monthly collected from 12 naturally infected camels followed-up for 1384 days, and two batches of referenced serum samples (infected, n = 12; non-infected, n = 15), statistical studies on qualitative and semi-quantitative results of the ELISAs did not show any significant difference when comparing the four combinations of parasites produced in vivo or in vitro, and frozen or freeze-dried WCLSAs. A repeatability study (28 repeats in 9 serum samples) was fully satisfying (p-value = 0.055). With the more convenient in vitro-produced freeze-dried WCLAs it was possible to: (i) avoid the ethical concern of in vivo production, (ii) improve the standardization of antigen production, (iii) secure antigen preservation during shipment and (iv) save a considerable amount of money (DE52-cellulose and dry-ice cold chain being avoided). Additional studies with other Trypanosoma spp are required for further extending ELISA to regional laboratories in enzootic areas, especially in view of the current progress in the "Progressive Control Pathway" (PCP) for trypanosomes in Africa.

No more cold-chain failures, using dehydrated reagents in ELISA antibody-detection against animal trypanosomes of African origin.

Animal trypanosomoses due to trypanosomes of African origin (ATAO), mainly caused by Trypanosoma congolense type Savannah (TCS), T. brucei brucei (TBB), T. vivax (TV), and T. evansi, are widespread diseases that affect domestic and wild mammals and have a huge economic impact. ATAO clinical suspicions are usually confirmed by parasitological and molecular methods, while sero-epidemiological surveys are generally carried out using the OIE-recommended ELISA method based on whole cell lysate soluble antigens (WCLSA) from purified trypanosomes; this reagent is usually stored frozen. With a view to expanding this ELISA test, we assessed, standardized, and validated the use of dehydrated rather than frozen WCLSA and serum samples. For the three ELISA assays (TV, TCS & TBB), a repeatability study revealed no significant difference between repeats. The results obtained using frozen rather than freeze-dried antigen and serum strongly correlated for Pearson's correlation values (>0.93) and Lin's measure ("very good" to "excellent"). Reproducibility was robust, with Pearson's correlation values >0.97 for inter technician effects, and 0.87 (TV) to 0.97 (TBB & TCS) for inter-laboratory tests; their combination was "very satisfactory" to "excellent" according to Lin's measure and there was no impact on qualitative test results. Dehydrated reagents offer the advantage of shipment at room temperature, allowing the secured provision of reagents to regional laboratories. Together with a compendium of standard diagnostic protocols for ATAO (/OIE), dehydrated reagents will enable the serological diagnosis of ATAO at regional level in endemic countries. This very welcome improvement in the context of the Progressive Control Pathway for trypanosomes, recently launched by African countries, will possibly be extended to Latin America in the near future.

A review and illustrated description of Musca crassirostris, one of the most neglected haematophagous livestock flies.

Tabanids, stomoxyine flies, hippoboscids and tsetse flies are the most well-known brachyceran biting flies of livestock. Only a few other higher Diptera have developed the unique mouthparts required for blood feeding. These neglected blood feeders can also have direct effects on hosts through blood loss, and are likely to contribute to the transmission of pathogens. Musca crassirostris (Diptera: Muscidae) is one of the most abundant of the muscid flies with this haematophagous lifestyle; it is widespread in the Palaearctic, Afrotropical and Oriental regions. The present study reviews and summarizes the biology and morphology of this species, and its potential for impact on animals and humans. The study also provides a fully illustrated description of the fly to facilitate its identification, and reviews information on abundance, with a focus on recent trapping surveys in Thailand. When sampled using traps designed for other biting flies, M. crassirostris appears to be four and 45 times more abundant than stomoxyines and tabanids, respectively. High numbers of M. crassirostris in the vicinity of livestock have also been associated with outbreaks of disease, such as that of a fatal plague in bovine farms in Egypt. This calls for a reconsideration of its potential impacts on livestock economics and health, and thus the development of suitable control methods.

[Atypical human trypanosomoses]. / Les trypanosomoses humaines atypiques.

Trypanosomes are principally responsible for two human diseases: human African trypanosomiasis (HAT) or sleeping sickness (caused by Trypanosoma brucei gambiense and T. b. rhodesiense), and Chagas disease, also called South American trypanosomiasis (T. cruzi). However, some trypanosomes that are natural parasites only of animals can sometimes infect humans and cause the so-called "atypical human trypanosomiases" (aHT). T. evansi, the agent causing surra in camels, horses, dogs, and bovines, and T. lewisi, a cosmopolite rat parasite, are the most frequently involved. These atypical infections involve no or only minor symptoms, but major symptoms are sometimes present. Parasite elimination is generally spontaneous, but can require treatment. Molecular tools, such as polymerase chain reaction, have improved the accuracy of parasite identification. Immunological techniques, mainly immunoenzymatic assays, can detect asymptomatic subjects. Several causes, most often concomitant, have been hypothesized, including immune immaturity, immunodeficiency, and close contact with infected animals. Innate immunity to animal trypanosomes depends on a trypanolytic factor called apolipoprotein L-I, present in human serum. A deficit in both apolipoprotein L-I alleles has been reported in an Indian patient infected by T. evansi. The prevalence of aHT is probably underestimated. Moreover, these trypanosomes might become potential emerging zoonotic pathogens, due to their ability to invade new hosts. An international network has been set up to survey these aHT (NAHIAT: Network on Atypical Human Infections by Animal Trypanosomes).

Molecular prevalence of Trypanosoma spp. in wild rodents of Southeast Asia: influence of human settlement habitat.

SUMMARY: This study investigated the molecular prevalence of Trypanosoma lewisi and T. evansi in wild rodents from Cambodia, Lao PDR and Thailand. Between 2008 and 2012, rodents (and shrews) were trapped in nine locations and 616 of these were tested using three sets of primers: TRYP1 (amplifying ITS1 of ribosomal DNA of all trypanosomes), TBR (amplifying satellite genomic DNA of Trypanozoon parasites) and LEW1 (amplifying ITS1 of ribosomal DNA of T. lewisi). Based on the size of the PCR products using TRYP1, 17% were positive for T. lewisi and 1·0% positive for Trypanozoon. Results were confirmed by sequencing PCR products and by using more specific primers (LEW1 and TBR). The specificity of TRYP1 primers, however, failed as rodent DNA was amplified in some instances, giving unexpected product sizes. Using LEW1 primers, 13·3% of the samples were confirmed positive for T. lewisi, both by PCR and sequencing. In Thailand, T. lewisi was found in Rattus tanezumi, R. exulans and Berylmys; in Lao PDR, in R. tanezumi and R. exulans, and in Cambodia in R. tanezumi, R. exulans and R. norvegicus. Using TBR, 1·3% of the samples tested positive for Trypanozoon by PCR and sequencing; T. evansi is the only species of the Trypanozoon subgenus possibly present in wild Asian rodents. These results confirmed its presence in rodents from Thailand (R. tanezumi), Lao PDR (R. tanezumi, R. nitidus) and Cambodia (R. tanezumi, Niviventer fulvescens, Maxomys surifer). Based on the information related to rodent trapping, it was found that rodent species trapped in and around human dwellings had a higher prevalence of T. lewisi infection. R. tanezumi and R. exulans, two synanthropic species, were mainly found infected in this habitat suggesting a role as a reservoir and thus a potential source of T. lewisi for human infection.

Molecular demonstration of Trypanosoma evansi and Trypanosoma lewisi DNA in wild rodents from Cambodia, Lao PDR and Thailand.

In this study, we investigated the molecular evidence of Trypanosoma evansi in wild rodents from Cambodia, Lao PDR and Thailand. Between November 2007 and June 2009, 1664 rodents were trapped at eight sites representative of various ecological habitats. Of those animals, 94 were tested by direct microscopic blood examination, 633 using the Card Agglutination Test for Trypanosomes (CATT/T. evansi) and 145 by Polymerase Chain Reaction (PCR) with two sets of primers: TRYP1 (amplifying ITS1 of ribosomal DNA of all trypanosomes) and TBR (amplifying satellite genomic DNA of Trypanozoon parasites). Using TRYP1, based on the size of the PCR products, 15 samples from the three countries were positive for Trypanosoma lewisi (two were confirmed by sequencing), and three were positive for Trypanozoon (one was confirmed by sequencing and three by TBR primers); the specificity of the primers failed as rodent DNA was amplified in some cases. Using TBR, six samples were positive for Trypanozoon (one was confirmed by sequencing); as T. evansi is the only species of the Trypanozoon sub-genus possibly present in Asian rodents, these results confirmed its presence in rodents from Thailand (Rattus tanezumi) and Cambodia (R. tanezumi, Niviventer fulvescens & Maxomys surifer). Further investigations are necessary to establish the situation in Lao PDR. None of the 16 samples most strongly positive to the CATT proved to be positive for Trypanozoon by PCR. The merits of the CATT for such studies were not confirmed. Studying the urban and rural circulation of these parasites in rodents will enable an evaluation of human exposure and infection risk, as human infections by T. evansi were recently described in India and by T. lewisi in India and Thailand. As sequencing PCR products is expensive, the development of new molecular and serological tools for rodents would be very useful.

Atypical hyperpachymorph Trypanosoma (Nannomonas) congolense forest-type in a dog returning from Senegal.

Trypanosoma congolense forest-type was identified by PCR in France, in a dog returning from Senegal. This paper describes the morphological features of the parasite on Giemsa-stained smears. Slender forms and "latent bodies" represent 30.4% and 20.4%, respectively. Some rosettes have been observed (0.8%). The predominant form (48.4%) is stumpy, close to "montgomeryi-form", but it is unusually broad, with a width/length ratio (WLr) of 0.40-0.55, while that of "montgomeryi-forms" is close to 0.3. To the best of our knowledge, this is the first description of such a form of T. (Nannomonas). Also unusual, the shape of the cytoplasm appears to be tightened by an "S-" or "C-" shaped flagellum. We propose naming this peculiar morphotype "hyperpachymorph", and adding its description to that of T. congolense forest-type. Thus T. (Nannomonas) forms would include: sphaeromorph or "latent body-form" (globular), hyperleptomorph (rodhaini-form, very long and slender, with a free flagellum); leptomorph (simiae-form, slender, with a free flagellum); isomorph (congolense-form, short, generally without a free flagellum); pachymorph (montgomeryi-form, short and stout; 0.25 < WLr < 0.34, without a free flagellum), and hyperpachymorph ("hyper montgomeryi-form", short and very stout; 0.35 < WLr < 0.7, without a free flagellum).

[Abundance and species diversity of tabanids (Diptera) in the biosphere reserve Ipassa-Makokou (Gabon) during the rainy season]. / Chorologie des Tabanidae (Diptera) dans la réserve de biosphère Ipassa-Makokou (Gabon) en saison des pluies.

The abundance and species diversity of tabanids were evaluated by trapping of insects using Vavoua traps, during the rainy season, from October 4 to November 30, 2009, in three different habitats: primary forest, secondary forest and village, in the biosphere reserve Ipassa-IRET Makokou in Gabon. Eight species belonging to three genera of tabanids have been identified for a total of 402 specimens caught. The tabanid species numerically the most abundant were: Tabanus secedens Walker, 1854 (55.2%), Tabanus obscurehirtus Ricardo, 1908 (13.9%), Chrysops dimidiatus Wulp, 1885 (11.2%) and Chrysops silaceus Austen, 1907 (10.7%). The less abundant species were Tabanus par Walker, 1854 (3.2%), Tabanus besti arbucklei Austen, 1912 (3%), Tabanus marmorosus congoicola Bequaert, 1930 (1%) and Ancala fasciata fasciata (Fabricius, 1775) (0.5%). Specimens of the genera Tabanus and Chrysops could not be identified, these insects represented respectively 0.7% and 0.5% of the insects trapped. The highest proportion of tabanids was trapped in secondary forest (75.1%) and the lower in primary forest (4.5%).

First outbreak of Trypanosoma evansi in camels in metropolitan France.

The first outbreak of trypanosomosis caused by Trypanosoma evansi in camels in France was reported on a farm in the Aveyron Department. Five camels were imported from the Canary Islands to the farm in early July 2006, and trypanosomes were observed on a stained blood smear from one of them, which died in October. On further investigations, trypanosomes were observed in the blood of five camels, three of them indigenous to the farm and two that had been imported. On the basis of microscopical examination (morphological criteria and measurements) and serological results based on the card agglutination T evansi test and PCR typing, the parasites were identified as T evansi. After treatment with melarsomine, the infected camels rapidly became negative by parasitological tests and were negative two to four months later by serological tests. The parasite was probably transmitted by tabanids and Stomoxys calcitrans, which were abundant in July to September 2006. No parasites were observed in other animals on the farm or on neighbouring farms, but some of the sheep on these farms were positive by PCR or serology.

First phenotypic description of Fasciola hepatica/Fasciola gigantica intermediate forms from the human endemic area of the Nile Delta, Egypt.

Fasciola gigantica is the main fasciolid species in Africa; however, F. hepatica and F. gigantica overlap in some countries. Egypt deserves mentioning because of the emerging situation of human fascioliasis in the Nile Delta area. The morphometric characteristics of fasciolid adults infecting the main livestock species present in the Nile Delta human endemic area are analyzed through a computer image analysis system (CIAS) on the basis of standardized measurements known to be useful for the differentiation of both fasciolid species. This is the first time that such a study is performed in an African country and, therefore, the results are compared to (i) F. hepatica (European Mediterranean area) and F. gigantica (Burkina Faso) standard populations, i.e. geographical areas where both species do not co-exist, and (ii) F. hepatica and F. gigantica populations from geographical areas where both species do co-exist, including the presence of intermediate forms (Iran). Results indicate the presence of F. hepatica, F. gigantica and intermediate forms (Fasciola sp.) in Egypt for the first time, and demonstrate the usefulness of CIAS for the phenotypic characterization of liver fluke adults from a concrete fascioliasis endemic area. Body roundness, body length over body width, and distance between the ventral sucker and the posterior end of the body provide useful tools for studying inter- and intraspecific morphological diversity in Fasciola adults. The application of these markers to specimens from geographical areas where F. hepatica and F. gigantica co-exist, such as in Egypt and Iran, suggest a strong population-level variation in Fasciola adult morphology.

The application of PCR-ELISA to the detection of Trypanosoma congolense type savannah (TCS) in bovine blood samples.

PCR-ELISA was set up to detect strains of Trypanosoma congolense type savannah (TCS) in field samples of buffy coats. Results of PCR-ELISA and PCR were compared and the effectiveness of both techniques was also compared with the Murray's method for the detection of TCS in 257 bovine buffy coats. The PCR products were labelled with digoxigenin (DIG-dUTP) during amplification cycles of the repetitive satellite DNA. A biotinylated DNA capture probe was used to detect the PCR products by ELISA in streptavidin coated microplates. Both the PCR-ELISA and PCR were more sensitive and more specific than the Murray's method. Of the 257 buffy coats analysed by the three techniques, PCR-ELISA and PCR detected TCS in 98 and 97 buffy coats respectively, whereas the Murray's method detected only 39 samples. PCR-ELISA and PCR had almost the same sensitivity and specificity. PCR-ELISA and PCR respectively detected TCS in 39.2% and 38.6% in all the 334 samples analysed by both techniques in this study.

Biting flies and Trypanosoma vivax infection in three highland districts bordering lake Tana, Ethiopia.

An epidemiological study was conducted to determine the prevalence of trypanosomosis in cattle, small ruminants and Equidae, and to identify biting flies; potential mechanical vectors of trypanosomes in the three districts of Bahir Dar Zuria, Dembia and Fogera, bordering lake Tana, Ethiopia. About 1509 cattle, 798 small ruminants and 749 Equidae were bled for the prevalence study using the buffy-coat method and the measurement of the hematocrit value. Sixty-six NGU and 20 monoconical traps were deployed for the fly survey. The results indicated the presence of trypanosomes in 6.1% (92/1509) of the cattle with a maximum during the late rainy season (9.6%) than the early dry season (3.6%) at Fogera district. Prevalence at the district level varied from 4% to 9.6%. Only one sheep (1/122) and one goat (1/676) were found positive for T. vivax-like trypanosomes and none of the Equidae was positive. All the trypanosomes encountered in cattle belong to the single species of T. vivax. The PCV was negatively associated with detection of T. vivax (21.6% in infected versus 25.4% in non-infected cattle). A total of 55,398 biting flies were caught of which 49,353 (89.08%) belong to Stomoxys, 4715 (8.51%) to horse flies and 1330 (2.4%) to Chrysops species. There was no tsetse fly. Species identification has indicated the presence of Atylotus agrestis, Chrysops streptobalia, Stomoxys calcitrans, S. nigra, S. pulla, S. pallida, S. sitiens, S. taeniata, S. uruma, Haematopota lasiops and Hippobosca variegata. The overall apparent density was 214.7flies/trap/day. Seasonal comparison showed higher fly catches in the late rainy season than the early dry season. This study indicated that T. vivax infections culminate in cattle at the same time as mechanical vectors such as Stomoxys sp. and Atylotus agrestis. Therefore, attention towards T. vivax infection in cattle is essential to control the impact of the disease on productivity. A further study on biting flies is recommended.

A longitudinal epidemiological survey of bovine trypanosomosis and its vectors in the White Volta river basin of Northern Ghana.

A longitudinal epidemiological survey of bovine trypanosomosis and its vectors was carried out in the Volta river basin of Northern Ghana to determine the relationship between cattle management and the incidence of bovine trypanosomosis. Two groups of sentinel cattle under different systems of management, classified as "fully-sedentary" and "partially-sedentary" (depending on the type of management) were followed over a 1-year period starting from March 2003 onwards. Cattle were screened at intervals of 3 months using the buffy coat technique (BCT). Buffy coat specimen from animals that were positive for the BCT and those that were negative, but with a packed cell volume (PCV) of less than 21% were further tested using the polymerase chain reaction (PCR). Plasma from all animals were tested for antibody using the indirect antibody enzyme-linked immunosorbent assay (ELISA). Trypanosomosis challenge was determined in tandem with the epidemiological survey with watering sites of sentinel cattle being the foci of interest. The parasitological prevalence at the start of the survey was higher in the fully-sedentary group (9%) than in the partially-sedentary group (3%). In subsequent visits, however, the parasitological incidence was consistently higher in the partially-sedentary group than in the fully-sedentary group. The mean seroprevalence (ELISA) of both groups increased from 3% in March to 54% in December. Statistical analysis of the serological results using a random effect logistic regression, showed a significant difference in incidence of bovine trypanosomosis between the two groups. There was also a significant effect of time. The influence of cattle herding on host-vector-parasite interface and its consequence on the incidence of trypanosomosis are discussed.

A cross-sectional epidemiological survey of bovine trypanosomosis and its vectors in the Savelugu and West Mamprusi districts of northern Ghana.

The epidemiology of bovine trypanosomosis was investigated in two districts (Savelugu and West Mamprusi) of Northern Ghana with different land use and environmental characteristics. The land use intensity and environmental change was suspected to be higher in the Savelugu District. A cross-sectional entomological survey conducted along the White Volta river and its tributaries confirmed the presence of only Glossina palpalis gambiensis and G. tachinoides. The challenge index as measured by the product of tsetse density and tsetse infection rate was much higher in the West Mamprusi (19.6) than in the Savelugu district (4.7). A total of 1013 cattle (508 in Savelugu and 505 in West Mamprusi) were bled from a random selection of 16 villages in the Savelugu District and 13 villages in the West Mamprusi District. Blood samples were examined for trypanosomes by the buffy coat technique (BCT). Blood samples that were positive in the BCT or negative in the BCT but with packed cell volume (PCV) values below 21 were further tested with a polymerase chain reaction for trypanosomal DNA. Plasma samples of all cattle were serologically tested with an indirect ELISA for trypanosomal antibodies. The parasitological and serological prevalence of bovine trypanosomoses was significantly higher in West Mamprusi (16 and 53%, respectively) than in Savelugu District (8 and 24%, respectively). An evaluation of animal health at the village herd level, using PCV as an index of anaemia, provided various epidemiological scenarios prevalent in the entire study area.

Applications of PCR-based tools for detection and identification of animal trypanosomes: a review and perspectives.

This paper aims to review the applications of the polymerase chain reaction (PCR) for the detection and identification of trypanosomes in animals. The diagnosis of trypanosomes, initially based on microscopic observations and the host range of the parasites, has been improved, since the 1980s, by DNA-based identification. These diagnostic techniques evolved successively through DNA probing, PCR associated to DNA probing, and currently to PCR alone. Several DNA sequences have been investigated as possible targets for diagnosis, especially multi-copy genes such as mini-exon, kinetoplastid mini-circles, etc., but the most favoured target is the nuclear satellite DNA of mini-chromosomes, which presents the advantages, and the drawbacks, of highly repetitive short sequences (120-600 bp). Several levels of specificity have been achieved from sub-genus to species, sub-species and even types. Random priming of trypanosome DNA has even allowed "isolate specific" identification. Other work based on microsatellite sequences has provided markers for population genetic studies. For regular diagnosis, the sensitivity of PCR has increased with the advancement of technologies for sample preparation, to reach a level of 1 trypanosome/ml of blood, which has brought to field samples a sensitivity two to three times higher than microscopic observation of the buffy coat. Similarly, PCR has allowed an increase in the specificity and sensitivity of diagnosis in vectors such as tsetse flies. However, because of the diversity of Trypanosoma species potentially present in a single host, PCR diagnosis carried out on host material requires several PCR reactions; for example, in cattle, up to five reactions per sample may be required. Research is now focusing on a diagnosis based on the amplification of the internal transcribed spacer-1 (ITS-1) of ribosomal DNA which presents the advantages of being a multi-copy locus (100-200), having a small size (300-800 bp), which varies from one taxon to another but is conserved in size in a given taxon. This may lead to the development of a multi-species-specific diagnostic protocol using a single PCR. By reducing the cost of the PCR diagnosis, this technique would allow a greater number of field samples to be tested in epidemiological studies and/or would increase the variety of Trypanosoma species that could be detected. Further investigations are required to develop and optimise multi-species-specific diagnostic tools for trypanosomes, which could also serve as a model for such tools in other pathogens.

Comparative pathogenicity of three genetically distinct types of Trypanosoma congolense in cattle: clinical observations and haematological changes.

The pathology of African bovine trypanosomosis was compared in Zebu cattle subcutaneously inoculated with three clones of trypanosomes corresponding to the three genetically distinct types of Trypanosoma congolense; savannah-type, west African riverine/forest-type and kilifi-type. All inoculated animals became parasitaemic between 7 and 11 days post-infection (dpi). The savannah-type showed consistently higher levels of parasitaemia and lower packed red cell volume percentages and leukocyte counts than the other two types. The syndrome was also more severe in the savannah-type and led inexorably to death between 29 and 54 dpi while animals with the forest or the kilifi-types recovered from earlier symptoms and haematological alterations after 3 months of infection. By the end of the experiment, the animals self-cured from the forest-type infection and the kilifi-type passed under control. The results of the present study indicated clear difference in pathogenicity between the three types of T. congolense; the savannah-type was virulent while the forest-type was of low pathogenicity and the kilifi-type was non-pathogenic.

Comparative pathogenicity of three genetically distinct Trypanosoma congolense-types in inbred Balb/c mice.

Inbred Balb/c mice were infected with three clones of Trypanosoma congolense (Sam.28.1, Dind.3.1 and K60.1A) corresponding, respectively, to the three genetically distinct types (savannah, forest and kilifi) defined within this species, for the purpose of comparing their pathogenicity for a better understanding of the epidemiology of African trypanosomosis. Another clone of savannah type, IL 3000, was also tested simultaneously to study a probable strain variation. Both the clones of savannah type were found of extreme virulence with loss of appetite, rough hair, rapid respiration, lethargy, and all mice died within a week. Parasitaemias evolved rapidly to the first peak by day 3-5 post-inoculation without any remission and the course of disease was correlated positively with the prepatent period. The clones of the forest type and the kilifi type were of low virulence with chronic infection and symptoms progressively less patent throughout the infection; only one mouse died in each experimental group.

Detection and identification of Trypanosoma of African livestock through a single PCR based on internal transcribed spacer 1 of rDNA.

Primers hybridising with the rDNA cistron have previously been evaluated for PCR diagnosis specific for kinetoplastids, and shown to detect and differentiate the Trypanosoma brucei complex and Trypanosoma cruzi. Kin1 and Kin2 primers, amplifying internal transcribed spacer 1, were subsequently evaluated for the diagnosis of African livestock trypanosomosis. Based on the size of the PCR products obtained, Kin primers allowed detection and identification of three Trypanosoma congolense types (savannah, forest and Kenya Coast), with distinction among themselves and from the subgenus Trypanozoon (T. brucei spp., Trypanosoma evansi and Trypanosoma equiperdum), Trypanosoma vivax, Trypanosoma simiae and Trypanosoma theileri. These primers were shown to be suitable for the sensitive and type-specific diagnosis of African livestock trypanosome isolates through a single PCR even in the case of multi-taxa samples. With field samples (buffy-coat from cattle blood) sensitivity was close to the sensitivity observed in single reactions with the classical specific primers for the Trypanozoon subgenus and T. congolense-type savannah, but was lower for detection of T. vivax. Additional reaction, improvement of DNA preparation, and/or new primers design are necessary to improve the sensitivity for detection of T. vivax in field samples. However, these primers are suitable for isolate typing through a single PCR.

The analysis of the cross-reactions occurring in antibody-ELISA for the detection of trypanosomes can improve identification of the parasite species involved.

In Africa, the main pathogenic trypanosomes of livestock are Trypanosoma vivax, T. congolense and T. brucei. The geographical distributions and hosts of these three species are very similar. As they differ markedly in pathogenicity and epidemiology, however, a species-specific serological test for infection would be very useful for epidemiological studies. The antibody-ELISA (Ab-ELISA) that have been developed for detecting the Trypanosoma spp. most commonly infecting livestock give satisfactory sensitivity and genus specificity. Unfortunately, they are not species-specific because of strong cross-reactions between the pathogenic Trypanosoma spp. In the present study, carried out in Burkina Faso, the results of standardized Ab-ELISA for T. vivax, T. brucei or T. congolense were compared using 1288 plasma samples from sheep experimentally infected with T. vivax, T. evansi and/or T. congolense. If the results were interpreted, as usual, only using a positivity threshold (PT), the strong cross-reactions observed led to a mean species-specificity of < 30%. However, analysis of the reactions observed in the three types of Ab-ELISA revealed that the homologous reactions were stronger than the heterologous for almost all of the single and mixed infections (98.3% and 99.0%, respectively). In monospecific infections exceeding the PT study of the positivity score produced in each of the three types of Ab-ELISA increased species-specificity to > 96%. It therefore appears that comparison of the strengths of the reactions seen in Ab-ELISA could greatly improve sero-epidemiological surveys of trypanosome infections in domestic ruminants, although the technique remains to be evaluated in experimentally infected cattle.