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Seven laboratories decided to compare their molecular diagnostic techniques to identify Mediterranean theileriosis caused by Theileria annulata. Each laboratory used either PCR or PCR and reverse line blot hybridization (RLB) to identify T. annulata. Five laboratories sent their own samples to laboratory 4 to be recoded and passed on to at least two other laboratories. A total of 120 blood samples were analyzed during this study, generating 540 results. Laboratory 1 sent only T. annulata-infected samples (positive control batch), and all the laboratories testing this batch found 100% infection. Laboratory 2 sent only negative samples from a Mediterranean area where T. annulata was unknown, and two laboratories out of three found a few positive samples in these negative samples. For the remaining samples, detection performance was variable. Agreement between laboratories ranged from 21.4 to 91.3%. The overall mismatch between laboratories was around 30% by whatever technique used. This paper describes the methodological parameters that could explain the variation of results.

A reverse line blot hybridisation (RLB) of 21 oligonucleotides with polymerase chain reaction (PCR) amplified regions of 16S rRNA (Ehrlichia/Anaplasma group) or 18S rRNA (Babesia/Theileria group) genes of haemoparasites detected Theileria annulata, T. buffeli/orientalis, Babesia bovis, B. bigemina, B. divergens, Ehrlichia bovis, Anaplasma marginale, A. centrale and unknown species within the Rickettsia tribe.A very high prevalence of mixed infections was detected, which indicated that animals infected with Babesia spp. were also infected with Theileria spp. and/or Anaplasma spp. The tick distribution appeared to be seasonal with Hyalomma marginatum as the most frequently observed tick and Boophilus annulatus and Ixodes ricinus as the least frequently observed ticks. Other species identified in the 818 ticks collected during the five sampling periods between April 1998 and November 1999 included H. lusitanicum, Rhipicephalus sanguineus group, R. bursa, Dermacentor marginatus, Haemaphysalis punctata, B. annulatus and I. ricinus.

A reverse line blot hybridization (RLB) test was developed to specifically identify six Theileria spp. (T. annulata, T. parva, T. mutans, T. velifera, T. taurotragi, and T. buffeli/orientalis) and three Babesia spp. (B. bovis, B. bigemina, and B. divergens). No cross reaction was observed with other livestock pathogens (such as Anaplasma marginale, A. centrale, A. ovis, Cowdria ruminantium, Trypanosoma brucei, T. congolense, and T. vivax). This method was used to test bovine blood samples collected in Sicily in April and November, 1998. Preliminary results indicated that T. annulata and T. buffeli/orientalis were the main species observed in cattle blood. Babesia species represented 1.8% and 23.5% in April and November, respectively.

Theileriosis is an infectious disease in tropical countries and in the Mediterranean area. It is caused by Theileria, a haemoprotozoan, transmitted by vectors belonging to the Ixodidae. In Southern Italy and in Sicily the infection is due mainly to T. annulata, but in some cases other species are involved in the disease. The authors describe a method to identify theileriosis in cattle blood samples, using PCR and hybridization techniques. Different primer sets were used to amplify different DNA target sequences, both genus and species specific. Blood samples from cattle were collected in Sicily. The DNA extracted from blood samples was employed first to detect the presence of the 18S ribosomal subunit gene specific for Theileria genus. Successively the positive samples were analysed to identify the species, T. annulata or T. buffeli/orientalis, using as target sequences for amplification respectively a fragment of the TAMS-1 and p33/34 antigens gene. Here the authors describe for the first time the presence of T. buffeli/orientalis infection in Sicilian herds. In fact 66% of positive blood samples were T. buffeli/orientalis infected.

A total of 153 blood samples were collected all over the Sicilian island in 15 farms. 29.4% and 25.5% were PCR positive for Theileria annulata and T. buffeli/orientalis respectively. 15.7% of cattle blood samples showed a mixed infection with both Theileria species. Each farm showed an infection rate ranging from 0% to 100%. Hyalomma marginatum and Boophilus annulatus were the most common ticks collected in April and November 1998 respectively. The aim of the present work was to investigate on haemoparasite species spread in Sicily. Ticks vectors involved in the transmission, distribution, risk areas and seasons, state of healthy carrier, are also discussed in this paper.