Dirk Geysen

PCR-RFLP using Ssu-rDNA amplification as an easy method for species-specific diagnosis of Trypanosoma species in cattle

A single polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay was used to characterise all important bovine trypanosome species. This is the first report of a sensitive pan-trypanosome PCR assay amplifying all species including T. vivax to a comparable extent using a single primer pair. A semi-nested PCR approach resulted in the detection of one T. congolense trypanosome genome/40 microl of blood, applied as buffy coat on filter paper. Restriction enzyme analysis using Msp1 and Eco571 gave a clear distinction between T. congolense, T. brucei, T. vivax and T. theileri. Several subgroups within the T. congolense group could be distinguished but no differences between the species belonging to the subgenus Trypanozoon or between T. simiae and T. theileri could be found. The use of MboII restriction enzyme allowed differentiation between T. simiae and T. theileri. The potential of the essay to be used as a suitable diagnostic tool is discussed.

Molecular tools for the rapid detection of drug resistance in animal trypanosomes

There are currently 17 African countries in which animal trypanocidal drug resistance has been reported. Large-scale surveys were carried out in only ten of them. The lack of baseline information is mainly due to the fact that the methods currently available for the detection of drug resistance are laborious, expensive and time consuming. In this review the mechanisms involved in resistance to isometamidium and diminazene will be discussed, together with some new molecular detection tools that have been developed recently enabling faster diagnosis of drug resistance than conventional laboratory or field tests.

Development and evaluation of a real-time polymerase chain reaction test for the detection of Theileria parva infections in Cape buffalo (Syncerus caffer) and cattle.

Corridor disease, caused by the tick-borne protozoan parasite Theileria parva, is a controlled disease in South Africa. The Cape buffalo is the reservoir host and uninfected buffalo have become sought-after by the game industry in South Africa, particularly for introduction into Corridor disease-free areas. A real-time polymerase chain reaction (PCR) test for detection of T. parva DNA in buffalo and cattle was developed to improve the sensitivity and specificity of the official diagnostic test package in South Africa. Oligonucleotide primers and hybridization probes were designed based on the 18S ribosomal RNA (rRNA) gene. Amplification of control DNA using Theileria genus-specific primers resulted in detection of T. taurotragi and T. annulata, in addition to T. parva. A T. parva-specific forward primer was designed which eliminated amplification of all other Theileria species, except for Theileria sp. (buffalo); however only the T. parva product was detected by the T. parva-specific hybridization probe set. The real-time PCR assay requires less time to perform, is more sensitive than the other molecular assays previously used in T. parva diagnostics and can reliably detect the parasite in carrier animals with a piroplasm parasitaemia as low as 8.79 x 10(-4)%.

Development of Loop-Mediated Isothermal Amplification (LAMP) assays for rapid detection of Ehrlichia ruminantium

BackgroundThe rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a potential zoonotic disease of ruminants transmitted by ticks of the genus Amblyomma. The disease is distributed in nearly all of sub-Saharan Africa and some islands of the Caribbean, from where it threatens the American mainland. This report describes the development of two different loop-mediated isothermal amplification (LAMP) assays for sensitive and specific detection of E. ruminantium.ResultsTwo sets of LAMP primers were designed from the pCS20 and sodB genes. The detection limits for each assay were 10 copies for pCS20 and 5 copies for sodB, which is at least 10 times higher than that of the conventional pCS20 PCR assay. DNA amplification was completed within 60 min. The assays detected 16 different isolates of E. ruminantium from geographically distinct countries as well as two attenuated vaccine isolates. No cross-reaction was observed with genetically related Rickettsiales, including zoonotic Ehrlichia species from the USA. LAMP detected more positive samples than conventional PCR but less than real-time PCR, when tested with field samples collected in sub-Saharan countries.ConclusionsDue to its simplicity and specificity, LAMP has the potential for use in resource-poor settings and also for active screening of E. ruminantium in both heartwater-endemic areas and regions that are at risk of contracting the disease.

Molecular epidemiology of Theileria parva in the field

Summary Molecular tools based on seminested RFLP‐PCR techniques to characterize field parasites in bloodspots dried on filter paper permitted investigation of the extent and the dynamics of diversity of Theileria parva populations in the field. Parallel molecular studies explored the long‐term genome stability of various isolates by probing Southern blots of EcoRI digested total genomic DNA with four different reference nucleic acid probes. Three polymorphic single copy loci encoding for antigen genes were developed for seminested PCR detection in order to apply them for a multilocus approach in population genetic studies. Seven alleles were identified for the polymorphic immunodominant molecule (PIM) locus by using restriction enzymes, and 4 alleles each for the p150 and p104 loci. A simple DNA extraction method gave good results in amplifying these loci from carrier animals using samples of blood dried on filter papers. Results from probing Southern blots of cultures taken at sequential timepoints indicate relative genome stability in T. parva in comparison to other parasitic protozoa such as Plasmodium. Comparatively homogeneous profiles in sympatric isolates from Zambia were identified using all four probes and PCR amplified products which contrasted with the variety found amongst Kenyan stocks. Preliminary characterization of T. parva field samples from the Southern Province of Zambia strongly suggest clonal expansion of one of the components of a non‐Zambian trivalent vaccine used on a limited scale in the Province from 1985 until 1992.

Boophilus microplus ticks found in West Africa

Early in 2007, during a small-scale survey of the ticks infesting cattle in Azaguie about 50 km north of Abidjan, Ivory Coast, ticks, belonging to a species which to our knowledge had never been reported in West Africa before, were encountered. Boophilus microplus was the only member of this genus collected from cattle in the area. No Boophilus annulatus or Boophilus geigyi were recovered although they had previously been recorded in this region (Aeschlimann 1967). The collection site at Azaguie (5°37 15.63 N and 4°05 12.76 W) is situated at an altitude of 85 m above sea level and is best characterised as dense humid forest. The ticks were initially morphologically identiWed at the Institute of Tropical Medicine in Antwerp, Belgium, using the identiWcation manual of Walker et al. (2003), after which some were sent to the Faculty of Veterinary Science, University of Pretoria, South Africa for conWrmation. At the same time sequencing of the ITS2 region of some of the remaining specimens conWrmed the initial identiWcation. The exact means or route of introduction of B. microplus into the Ivory Coast has not been determined. It has, however, been documented that N’dama bulls were introduced from Lower Congo (Democratic Republic of Congo) in 1985, principally on to the Marahoue Ranch for genetic improvement and subsequently for the establishment of herds of improved cattle for local farmers (Shaw and Hoste 1987). As far as we know B. microplus does not occur in the Congo, nor has it ever been documented in the countries bordering the Ivory Coast. But it has been reported from Zambia immediately to the south (Berkvens et al. 1998).

Validation of meat inspection results for Taenia saginata cysticercosis by PCR-restriction fragment length polymorphism.

Bovine cysticercosis is a zoonosis caused by the larval stage (cysticercus) of the human tapeworm Taenia saginata. Infected cattle is an important food safety issue besides an economic concern. Humans get infected by eating raw or undercooked meat containing viable cysticerci. Visual meat inspection of bovines is the only public health measure implemented to control transmission to humans, but it lacks sensitivity and objectivity. It may underestimate the prevalence of the disease by a factor 3 to 10. Furthermore, the success of the method depends on the expertise of the meat inspector as well as on the stage of development of the cysticerci. The focus of this study was to develop and explore the usefulness of a PCR assay as an objective alternative to evaluate the meat inspectors visual inspection results. Hereto, a PCR was developed for the detection of T. saginata DNA in muscle lesions. Based on the laboratory classification of lesions, almost 97% of viable cysts were confirmed by PCR, while for dead cysts, the percentage was approximately 73%. Taken together, these data demonstrate the difficulties of visual meat inspection and their objective interpretation, emphasizing the need to improve current assays to strengthen the control of bovine cysticercosis.

Development and validation of a PCR-RFLP test to identify African Rhipicephalus (Boophilus) ticks

The cattle tick Rhipicephalus (Boophilus) microplus has recently invaded West Africa and caused anxiety amongst farmers in Ivory Coast, as livestock production was severely affected. The introduction of this tick species has remained unnoticed for several years, as all the members of this genus are very similar in appearance. To overcome the cumbersome morphological identification of the four closely related R. (Boophilus) spp. in the region, a PCR-RFLP test, based on a part of the second internal transcribed spacer ribosomal DNA (ITS2), was developed. The molecular tool was successfully validated with a large number of ticks recently collected from West Africa and that were identified both morphologically and genetically. The tool developed is simple, fast, reliable and reproducible; hence it can be routinely applied for species identification.

Molecular detection of Ehrlichia ruminantium infection in Amblyomma variegatum ticks in The Gambia

In West Africa, losses due to heartwater disease are not known because the incidence/prevalence has not been well studied or documented. To develop a diagnostic tool for molecular epidemiology, three PCR-based diagnostic assays, a nested pCS20 PCR, a nested map1 PCR and a nested reverse line blot (RLB) hybridization assay, were evaluated to determine their ability to detect infection in vector ticks, by applying them simultaneously to A. variegatum field ticks to detect Ehrlichia ruminantium, the causative agent of heartwater. The nested pCS20 PCR assay which amplified the pCS20 gene fragment showed the highest detection performance with a detection rate of 16.6%; the nested map1 PCR, which amplified the gene encoding the major antigenic protein1 (map1 gene) showed a detection rate of 11% and the RLB, based on the 16S rDNA sequence of anaplasma and ehrlichial species, detected 6.2%. The RLB, in addition, demonstrated molecular evidence of Ehrlichia ovina, Anaplasma marginale and Anaplasma ovis infections in The Gambia. Subsequently, the pCS20 assay was applied to study the prevalence and distribution of E. ruminantium tick infection rates at different sites in five divisions of The Gambia. The rates of infection in the country ranged from 1.6% to 15.1% with higher prevalences detected at sites in the westerly divisions (Western, Lower River and North Bank; range 8.3–15.1%) than in the easterly divisions (Central River and Upper River; range 1.6–7.5%). This study demonstrated a gradient in the distribution of heartwater disease risk for susceptible livestock in The Gambia which factor must be considered in the overall design of future upgrading programmes.

Transmission of Theileria parva in the traditional farming sector in the Southern Province of Zambia during 1997-1998

The incidence of first contact with the protozoan Theileria parva was determined in two traditional cattle herds in the Southern Province of Zambia during a period of average rainfall in 1997 and 1998, following a drought in the previous two years. Compared to that period, there was a marked increase in the number of rainy season first contacts attributable to transmission by Rhipicephalus appendiculatus adults. However, there were still more dry season contacts that resulted from nymphal transmission. These results highlight the important role that climate plays in the transmission of theileriosis in the Southern Province of Zambia.