Prevalence of bovine theileriosis in North Central region of Algeria by real-time polymerase chain reaction with a note on its distribution.

To determine the presence and distribution of bovine theileriosis in the North Central region of Algeria, 358 DNA samples and 359 blood smears were analyzed from nine provinces. Theileria DNA extracted from cattle blood was amplified by fluorescence resonance energy transfer polymerase chain reaction (FRET-PCR). Blood smears were examined for Theileria piroplasms by microscopical examination (ME) of Giemsa-stained slides. While microscopical identification revealed only 42 animals being infected with Theileria piroplasms, PCR-positive amplification using Theileria genus-specific primers was obtained from 132 Theileria spp. (P < 0.0001). Among the 132 positives, 108 animals (81.8 %) were found positive of Theileria annulata, while 24 (18.2 %) were found positive for Theileria sp. (P < 0.0001). However, melting curve analysis of these latter samples revealed the presence of two different peaks, 51.5 ± 0.5 °C corresponding to Theileria sp1 and 52.5 ± 0.5 °C for Theileria sp2. Cloning and sequencing of Theileria sp1 and Theileria sp2 using the Cox primers indicated that these species are very closely related to Theileria buffeli. There is a highly significant difference in the distribution of theileriosis between different provinces (P < 0.0001). This disparity between provinces is probably due to differences in tick contact, influenced by the subhumid bioclimatic gradient and differences in agricultural land use.

Outcome of acute East African trypanosomiasis in a Polish traveller treated with pentamidine.

BACKGROUND: African trypanosomiasis is a parasitic infection sporadically imported to Europe by tourists or immigrants returning from endemic areas. We present the first and an unusual case of East African trypanosomiasis imported to Poland by a patient returning from a tourist trip to Uganda and Rwanda, which was successfully treated with pentamidine. CASE PRESENTATION: A 61-year-old Polish man was admitted to the Department because of high-grade fever and multi-organ dysfunction after a tourist trip to East Africa. He experienced a single tsetse fly bite during a safari trip to the Queen Elizabeth National Park in Uganda. On admission, his clinical status was severe, with high fever of 41ºC, preceded by chills, bleeding from the gums and oral mucosa, haemorrhages at the sites of venipuncture, numerous ecchymoses, fine-spotted skin rash, tachycardia, hepatosplenomegaly, dehydration, jaundice, dyspnoea, hypoxaemia, generalised oedema and oliguria. There was a typical non-painful trypanosomal chancre with central necrosis and peripheral erythema on his left arm. Laboratory investigations showed leucopenia, thrombocytopenia, haemolytic anaemia, hyperbilirubinaemia, hypoglycaemia, elevated creatinine and urea, high activity of aminotransferases, elevated levels of inflammatory markers, hypoproteinaemia, proteinuria, abnormal clotting and bleeding times, low fibrinogen level, metabolic acidosis, and electrolyte disturbances. A peripheral blood smear showed numerous Trypanosoma brucei trypomastigotes with a massive parasitaemia of 100,000/µl. T. brucei rhodesiense subspecies was finally identified on the basis of the characteristic serum resistance-associated gene using a polymerase chain reaction, and a seroconversion of specific immunoglobulin M and G antibodies in the peripheral blood by enzyme-linked immunosorbent assay. Serological tests for T. brucei gambiense subspecies were negative. A severe clinical course of acute rhodesiense trypanosomiasis with renal failure, respiratory distress, disseminated intravascular coagulation syndrome, haemolysis, liver insufficiency and myocarditis was confirmed. Intensive anti-parasitic and symptomatic treatment was immediately instituted, including intravenous pentamidine, plasmaphereses, oxygen therapy, blood transfusion, catecholamine administration, and fluid infusions, as well as haemostatic, hepatoprotective, anti-inflammatory, antipyretic and diuretic drugs. The final outcome was a full recovery with no late sequelae. CONCLUSION: Sleeping sickness should always be considered in the differential diagnosis of fever in people returning from safari trips to the national parks or nature reserves of sub-Saharan Africa.

A Theileria parva isolate of low virulence infects a subpopulation of lymphocytes.

Theileria parva is a tick-transmitted protozoan parasite that infects and transforms bovine lymphocytes. We have previously shown that Theileria parva Chitongo is an isolate with a lower virulence than that of T. parva Muguga. Lower virulence appeared to be correlated with a delayed onset of the logarithmic growth phase of T. parva Chitongo-transformed peripheral blood mononuclear cells after in vitro infection. In the current study, infection experiments with WC1(+) γδ T cells revealed that only T. parva Muguga could infect these cells and that no transformed cells could be obtained with T. parva Chitongo sporozoites. Subsequent analysis of the susceptibility of different cell lines and purified populations of lymphocytes to infection and transformation by both isolates showed that T. parva Muguga sporozoites could attach to and infect CD4(+), CD8(+), and WC1(+) T lymphocytes, but T. parva Chitongo sporozoites were observed to bind only to the CD8(+) T cell population. Flow cytometry analysis of established, transformed clones confirmed this bias in target cells. T. parva Muguga-transformed clones consisted of different cell surface phenotypes, suggesting that they were derived from either host CD4(+), CD8(+), or WC1(+) T cells. In contrast, all in vitro and in vivo T. parva Chitongo-transformed clones expressed CD8 but not CD4 or WC1, suggesting that the T. parva Chitongo-transformed target cells were exclusively infected CD8(+) lymphocytes. Thus, a role of cell tropism in virulence is likely. Since the adhesion molecule p67 is 100% identical between the two strains, a second, high-affinity adhesin that determines target cell specificity appears to exist.

Development of loop-mediated isothermal amplification (LAMP) assays for rapid detection of Ehrlichia ruminantium.

BACKGROUND: The 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. RESULTS: Two 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. CONCLUSIONS: Due 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.

Folding properties of the hepatitis B core as a carrier protein for vaccination research.

The hepatitis B core (HBc) protein has been used successfully in numerous experiments as a carrier for heterologous peptides. Folding and capsid formation of the chimeric proteins is not always achieved easily. In silico analyses were performed to provide further comprehension of the feasibility for predicting successful capsid formation. In contrast to previous work, we show that common in silico predictions do not ensure assembly into particles. We included new considerations regarding capsid formation of HBc fusion proteins. Not only the primary sequence and the length of the inserts seem important, also the rigidity, the distance between the N and the C-terminus and the presence of cysteines, which could form disulphide bonds, could influence proper capsid formation. Furthermore, new conformational insights were formulated when linkers were added to create extra flexibility of the chimeric particles. Different hypotheses were suggested to clarify the obtained results. To this extent, the addition of glycine-rich linkers could lower high rigidity of the insert, removal of the strain of the core protein or ease interaction between the HBc and the insert. Finally, we observed specific changes in capsid formation properties when longer linkers were used. These findings have not been reported before in this and other virus-like particle carriers. In this study, we also propose a new high-yield purification protocol for fusion proteins to be used in vaccination experiments with the carrier protein or in comparative studies of particulate or non-particulate HBc fusion proteins.

Bovine piroplasmosis-anaplasmosis and clinical signs of tropical theileriosis in the plains of Djurdjura (north Algeria).

The study was conducted during tick activity season over a period of 5 years in the Djurdjura Plains, Algeria. A total of 299 cattle (Holstein, Montbeliard, Fleckvieh and crossbred animals) with clinical signs were included in this study. A total of 171 animals were found positive for at least one pathogen by Giemsa-stained blood smears examination Theileria annulata (136/299, 45.5%), Babesia bovis (14/299, 4.7%), B. bigemina (3/299, 1.0%) and Anaplasma marginale (12/299, 4.0%) were identified. Six animals were co-infected by T. annulata and A. marginale. Although no ticks were collected from diseased animals, clinical signs in cattle were hyperthermia (120/136, 88.3%), gluttony followed by anorexia (113/136, 83.1%), lymph node enlargement (99/136, 72.8%), anaemia (82/136, 60.3%), icterus (58/136, 42.6%) and haemoglobinuria (36/136, 26.5%). Gluttony followed by anorexia was considered highly suggestive of an incubation of tropical theileriosis as shown by a higher receptivity index (IR = 0.89-1). This clinical sign is evident in young Montbeliard and young Holstein males with anaemia (IR = 1) and icterus (IR = 0.78-0.81) which is earlier than haemoglobinuria (IR = 0.51-0.54). The incidence of T. annulata was maximum in July (n = 57), as well as B. bovis (n = 6) and A. marginale (n = 13). These results highlight the preponderance of tropical theileriosis in north-central Algeria, where gluttony followed by anorexia is probably a prodromal symptom during the incubation period of the disease.

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.

Genetic diversity of Ehrlichia ruminantium in Amblyomma variegatum ticks and small ruminants in The Gambia determined by restriction fragment profile analysis.

Understanding genetic diversity of Ehrlichia ruminantium in host and vector populations is an important prerequisite to controlling heartwater by vaccination in traditional livestock systems in sub-Saharan Africa. We carried out a study in two phases: (i) evaluating the usefulness of the PCR-RFLP assay based on the map1 coding sequence of E. ruminantium as a discriminatory tool to characterise genetic diversity, (ii) applying the technique to field samples from Amblyomma variegatum ticks and small ruminants to characterise genotypic diversity of the organism in three main agroecological zones of The Gambia, Sudano-Guinean (SG), Western Sudano-Sahelian (WSS) and Eastern Sudano-Sahelian (ESS). Restriction fragment length polymorphisms were observed among different strains of E. ruminantium supporting the usefulness of the PCR-RFLP technique for studying genetic diversity of the organism. Restriction enzyme map1 profile analysis indicated the presence in The Gambia of multiple genotypes (at least 11) of E. ruminantium with sites in the WSS and SG zones showing comparatively high number of diverse genotypes. Profiles similar to the Kerr Seringe genotype (DQ333230) showed the highest distribution frequency, being present at sites in all three agroecological zones, thereby making the strain a suitable candidate for further characterisation in cross-protection studies. An additional three genotypes showed relatively high distribution frequency and were present in all three zones making them equally important for isolation and subsequent characterisation. The study demonstrated the occurrence of mixed infections with E. ruminantium genotypes in ruminants and ticks.

Orientation of bovine CTL responses towards PIM, an antibody-inducing surface molecule of Theileria parva, by DNA subunit immunization.

East Coast fever, an acute lymphoproliferative disease of cattle, is caused by the apicomplexan parasite Theileria parva. Protective immunity is mediated by CD8(+) cytotoxic T lymphocytes directed against schizont-infected cells. The polymorphic immunodominant molecule, although an antibody-inducing surface molecule of the schizont, has been hypothesized to play a role in protective immunity. In order to evaluate the immunogenicity of PIM for inducing CTL, cattle were immunized with PIM in isolation from other T. parva antigens, forcing the presentation of PIM-derived epitopes on the MHC class I molecules. Although parasite-specific cytotoxicity was induced in both vaccinated animals, their immune response was clearly different. One animal generated MHC-restricted parasite-specific CTL against PIM while the other calf exhibited a strong PIM-specific proliferative response but non-MHC-restricted parasite-specific cytotoxicity. Only calf 1 survived a lethal sporozoite challenge. This DNA immunization technique with an antigen in isolation of CTL-immunodominant antigens might open possibilities for directing CTL responses against predefined antigens, such as strain cross-reacting CTL antigens.

The sensitivity of PCR and serology in different Theileria parva epidemiological situations in Rwanda.

Theileria parva is the causative agent of a lethal tick-borne disease of cattle occurring in eastern, central and southern Africa. Variations in the sensitivity of the serological and molecular tests with seasonal vector occurrence and discrepancies between low PCR prevalence and high T. parva vector density are a setback to estimate true prevalences. Therefore, the objectives of the present studies were to evaluate (1) the sensitivity of three serological tests (IFAT, ELISA and SELISA) and one molecular test (PCR) in the diagnosis of chronic T. parva infections in four different agro-ecological zones of Rwanda and (2) the effect of tick challenge and animal's age on the sensitivity of PCR. Blood samples from 635 bovines were collected in four agro-ecological zones of Rwanda. All sera were screened using the IFAT, ELISA, SELISA and PCR. The binary results of the four diagnostic tests were introduced separately for each agro-ecological zone in a Bayesian model to estimate the prevalence of T. parva infections and the sensitivity of the four diagnostic tests. All test specificities were set to 100%. The estimated T. parva prevalence was much higher (83-85%) than estimations based on single diagnostic tests. The estimated sensitivity of serological tests was relatively constant and ranged from 57 to 75% in the various areas. The sensitivity of PCR showed more pronounced variations, ranging from 66% in the low T. parva transmission (high land) zones compared to 24% in the highly vector infested (low land) zones. Calves and adult cattle (n=194) were also sampled in regularly and irregularly dipped herds in the low land region. The apparent T. parva prevalence detected by PCR was significantly higher in calves than in adult cattle and in herds regularly treated with acaricides, while no significant differences were found with IFAT. The conditional probability that a sample was positive at PCR while it was positive at IFAT was significantly lower in adults. The implication of these findings in the use of diagnostic assays for epidemiological studies is discussed.

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)%.

Survey of Anaplasma phagocytophilum and Anaplasma sp. 'Omatjenne' infection in cattle in Africa with special reference to Ethiopia.

BACKGROUND: As evidence of the infection of domestic animals by Anaplasma phagocytophilum and Anaplasma sp. 'Omatjenne' is presently becoming available, understanding the epidemiological and ecological significance of infection is important to quantify the clinical and socio-economic impact of the diseases they cause. METHODS: The first aim of this study was to analyse the occurrence of A. phagocytophilum and Anaplasma sp. 'Omatjenne' in cattle samples collected from selected African countries using a polymerase chain reaction and restriction enzyme fragment length polymorphism. Secondly, this study was aimed at the molecular identification of Ehrlichia spp. and Anaplasma spp. infection in ruminants raised under different production systems in selected sites in central Ethiopia. RESULTS: In total, 695 samples from cattle in six African countries were analysed. Overall, 45 positive results were obtained for Anaplasma sp. 'Omatjenne' (6.47%) and 19 for A. phagocytophilum (2.73%). Anaplasma sp. 'Omatjenne' was detected in all countries except Tanzania while A. phagocytophilum was detected only in samples from Ethiopia. The proportion of samples tested positive for Anaplasma sp. 'Omatjenne' ranged from 1.2% in Morocco to 16% in Rwanda. The occurrence of both agents is now confirmed in African cattle. For the survey in Ethiopia a semi-nested 16S rDNA polymerase chain reaction followed by restriction fragment length polymorphism was used for the identification of Ehrlichia spp. and Anaplasma spp. in blood samples. Randomly selected samples were also analysed by pCS20 polymerase chain reaction for the detection of E. ruminantium. Positive results were obtained for E. ruminantium and five species of Anaplasma including a zoonotic species. To our knowledge, this is the first report of infection of domestic ruminants with A. phagocytophilum, A. ovis and Anaplasma sp. 'Omatjenne' in Ethiopia. CONCLUSION: The present study showed widespread occurrence of Anaplasma sp. 'Omatijenne' in African cattle and five Anaplasma species in Ethiopia.

Human tapeworms in north Vietnam.

Sixty-five Taenia samples were collected from patients in a referral hospital in Hanoi, north Vietnam, for species identification by morphological and molecular techniques. PCR-RFLP of a mitochondrial 12S rDNA fragment, developed for this study, allowed direct differentiation between all Taenia spp., overcoming the disadvantages of classical morphological examination, which failed on disintegrated samples. Taenia saginata asiatica was the most common species (55.4%) followed by T. saginata (38.5%) and T. solium (6.2%). This report demonstrates the complexity of the epidemiology of Taenia spp. in Vietnam and the need for further work to reveal transmission patterns of these species.

Longitudinal monitoring of Ehrlichia ruminantium infection in Gambian lambs and kids by pCS20 PCR and MAP1-B ELISA.

BACKGROUND: The epidemiology of E. ruminantium infection in extensively managed young animals is not adequately understood. Thus in this study, we monitored the onset (age at first infection) and kinetics of E. ruminantium infection and antibody response in extensively managed newborn lambs and kids at three sites in The Gambia. METHODS: We used a nested pCS20 PCR and MAP1-B ELISA in a longitudinal study to monitor the onset (age at first infection) and kinetics of E. ruminantium infection and antibody response respectively, in 77 newborn lambs and kids under a traditional husbandry system at three sites (Kerr Seringe, Keneba, Bansang) in The Gambia where heartwater is known to occur. The animals were monitored for field tick infestation and the comparative performance of the two assays in detecting E. ruminantium infection was also assessed. RESULTS: The infection rate detected by pCS20 PCR varied between 8.6% and 54.8% over the 162-day study period. Nineteen per cent of the animals in week 1 post-partum tested positive by pCS20 PCR with half of these infections (7/14) detected in the first 3 days after birth, suggesting that transmission other than by tick feeding had played a role. The earliest detectable A. variegatum infestation in the animals occurred in week 16 after birth. Antibodies detected by MAP1-B ELISA also varied, between 11.5% and 90%. Although there is considerable evidence that this assay can detect false positives and due to this and other reasons serology is not a reliable predictor of infection at least for heartwater. In contrast to the pCS20 PCR, the serological assay detected the highest proportion of positive animals in week 1 with a gradual decline in seropositivity with increasing age. The pCS20 PCR detected higher E. ruminantium prevalence in the animals with increasing age and both the Spearman's rank test (rs = -0.1512; P = 0.003) and kappa statistic (-0.091 to 0.223) showed a low degree of agreement between the two assays. CONCLUSION: The use of pCS20 PCR supported by transmission studies and clinical data could provide more accurate information on heartwater epidemiology in endemic areas and single-occasion testing of an animal may not reveal its true infection status. The view is supported because both the vector and vertical transmission may play a vital role in the epidemiology of heartwater in young small ruminants; the age range of 4 and 12 weeks corresponds to the period of increased susceptibility to heartwater in traditionally managed small ruminants.

A DNA sequence capture extraction method for detection of Mycobacterium avium subspecies paratuberculosis in feces and tissue samples.

Culturing of Mycobacterium avium subspecies paratuberculosis (Map) remains difficult and is time consuming. An alternative for the rapid detection of Map in samples is PCR. We have developed a sensitive DNA-extraction method based on sequence capture for the rapid detection of M. avium subspecies paratuberculosis by PCR in fecal and tissue samples. The method detected 10(2)Map/g feces using spiked samples, and reached a diagnostic sensitivity of 33,7% compared to 22% for culture. Analysis of tissue samples gave 65 polymerase chain reaction (PCR)-positive (42.2%) and 49 culture-positive samples (31.8%). Therefore, the detection limit of the DNA-extraction is the same as previously reported for culture, the PCR assay could detect more positive samples than the culture method.

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 inspector's 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.

Genetic variation in Rhipicephalus appendiculatus (Acari: Ixodidae) from Zambia: correlating genetic and ecological variation with Rhipicephalus appendiculatus from eastern and southern Africa.

Based on their ecology, Rhipicephalus appendiculatus ticks from eastern and southern Africa have been divided into three groups. We investigated how two geographic genetically differentiated stocks of R. appendiculatus from the southern and the eastern provinces of Zambia, representing two ecological groups, i.e., southern African and transition groups, respectively, genetically compare to stocks from east Africa (Rwanda) and southern Africa (Zimbabwe and South Africa). The ITS2 and two mitochondrial genes segments, 12s rDNA and COI, were used in the investigations. The ITS2 tree did not show support for differentiation into any groups, while the two mitochondrial genes trees (12s rDNA and COI) showed two genetically differentiated groups: an east African genetic group which included specimens from Rwanda and the plateau area of the eastern province of Zambia, and a southern African genetic group represented by specimens from South Africa, Zimbabwe and specimens collected on the fringes of the eastern province plateau in the Nyimba district of Zambia. This suggests that the two geographically differentiated stocks of the southern and eastern provinces of Zambia might be part of two wider geographic genetically differentiated R. appendiculatus groups that extend beyond Zambia. Stocks of "transition" ecology (eastern province) belong to the east African genetic group and the differences in ecology within this genetic grouping may be due to genetic polymorphism, phenotypic plasticity, and other local factors.

Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva.

Theileria parva is an important veterinary protozoan causing the tick-borne disease East Coast fever. Transfection of Theileria parasites will facilitate the investigation of many aspects of this apicomplexan infection and its unique host-parasite interaction. The pathogen has the extraordinary capacity of transforming B and T cells into clonally dividing cancerous cell lines in a reversible way. Sequence data of the entire T. parva genome are available and in vitro infected cell lines can easily be generated, thereby eliminating the use of animals in the evaluation of the evolution of the transfected sporozoites. Here we report, for the first time, on experiments towards successful transient transfection of T. parva sporozoites, making use of a new generation transfection device. Plasmid DNA containing the strong EF-1α promoter and an Azami Green reporter gene were integrated by nucleofection into freshly purified T. parva sporozoites. Expression of Azami Green was detected with a fluorescence microscope and confirmed by counter staining with a monoclonal directed against a sporozoite protein. Despite the fact that transfection efficiencies are still low, this is the first step towards a stable infection method of T. parva parasites. In the long run, transfected parasites might become an alternative way to induce immunity without clinical signs.

An unusual mosaic structure of the PIM gene of Theileria parva and its relationship to allelic diversity.

Genetic diversity and structural organisation of the polymorphic immunodominant molecule (PIM) gene of the protozoan parasite Theileria parva was studied in isolates from sympatric and allopatric areas. The analyses revealed a mosaic structure consisting of highly conserved regions shared among some of the isolates from geographically different areas and homologous sequence runs shared among isolates from one area. The specific pattern of diversity in which large insertions and deletions were observed, giving a mosaic structure to the PIM locus, is quite exceptional for single-locus genes. The polymorphic middle region of the gene was characterised by large deletions or insertions in many isolates. There was no correlation between the copy number of the tetrapeptide repeats in this region and the total length of the sequence. The gene was highly polymorphic when compared with sequences from other known T. parva antigenic regions. The findings support the concept that as yet unidentified mechanisms are generating extensive diversity and shaping the PIM locus. The relevance of this finding for diagnosis and the relationship between these mechanisms and the possible role of this protein in host immune responses is discussed.

Newly developed primers for the detection of Mycobacterium avium subspecies paratuberculosis.

Recent publications reported the existence of IS900 like sequences in mycobacteria different from Mycobacterium avium subspecies paratuberculosis (Map). The primers used for IS900 detection of Map have amplified these sequences causing false positive results. In this study, we have developed two new PCR assays for the detection of Map. The first assay is based on the IS900 sequence using primers different from the ones previously reported, the second assay on the f57 sequence. The specificity of the tests was checked by analysis of 190 mycobacterial isolates (74 Map and 116 non-Map isolates). All Map strains were positive and all non-Map strains were negative. Serial dilutions of Map bacteria were used to assess the sensitivity of the assays. We achieved a sensitivity of 1CFU per PCR for both assays. In addition, a PCR-simulating computer programme was used to evaluate the specificity of the new IS900 primers. The combination of the two PCR assays has proven to be useful for the identification of Map but validation on a large range of clinical samples still needs to be done.