First report of bovine anaplasmosis by Anaplasma centrale in Europe

Anaplasma centrale is an intraerytrocytic tick-born rickettsia (Rickettsiales: Anaplasmataceae), which is responsible for mild infections in cattle (Dumler et al., 2001). In contrast, the closely related species Anaplasma marginale is the aetiological agent of acute anaplasmosis, a bovine syndrome characterised by a progressive haemolytic anaemia associated with fever, weight loss, abortion, decreased milk production and in some cases death of the infected cattle (Palmer, 1989). Since cross-protective immunity to A. marginale can be acquired after A. centrale infection, the latter is used as a live vaccine for routine vaccination of cattle in Israel, Australia, Africa and South America (Bock et al., 2003). Molecular epidemiology of A. centrale is poorly studied and only few strains have been characterised at molecular level so far (Inokuma et al., 2001; Liu et al., 2005). In this note, we report a clinical case of bovine anaplasmosis associated with A. centrale infection in a 4-year-old dairy cow in Italy. The causative agent together with 4 additional A. centrale strains detected in southern Italy were subjected to sequence and phylogenetic analyses. To our knowledge, this is the first report on detection and molecular characterisation of A. centrale in continental Europe. The clinical case, observed during an epidemiological cross-sectional study on bovine tick-borne diseases (TBDs) (Carelli et al., 2001), occurred in 2000 in a dairy herd of the province of Bari, Apulia region, Italy. Clinical signs of acute anaplasmosis, consisting of hypogalactia, mucosal paleness, depression, temperature (40-40.5°C), were observed in a 4-year-old Holstein cow out of 25 dairy cows. EDTA-blood samples collected during the acute stage of infection were used for haematological investigations and detection of TBD agents. Giemsa-stained blood smears showed the intraerythrocytic presence of dense, rounding, basophilic inclusions with the central localisation typical of A. centrale. DNA extracted was subjected to reverse line blot (RLB) assay for simultaneous detection and identification of tick-born haemoparasites (Gubbels et al., 1999; Bekker et al., 2002). A. centrale was detected in blood samples, whereas the presence of A. marginale DNA, even at low titres, was definitively ruled out by a real-time PCR assay recently developed (Carelli et al., 2007). Standardised molecular methods carried out on blood samples, as well as on nasal, ocular and rectal swabs, failed to identify viral pathogens. In order to confirm the circulation of A. centrale in Italy, blood samples collected from 51 cattle belonging to 18 different herds of three regions of southern Italy (Apulia, Calabria, Basilicata) were selected from a previous study (Carelli et al., 2001) and analysed by RLB hybridisation. A. centrale DNA was detected in 20 animals. Single infection by A. centrale were only observed in 4 samples, whereas in the remaining 16 samples multiple TBDs pathogens were found. The genetic variation between the A. centrale strain recovered from the clinical case (strain CC) and the 4 strains responsible for single infections in herds of southern Italy, as well as their relatedness to reference A. centrale strains, was evaluated by sequence and phylogenetic analyses on 16S rDNA and groEL (HSP60) sequences, as previously reported (Lew et al., 2003). In the 16S rDNA sequences, strain CC showed a 100% nt identity to the other Italian A. centrale strains, as well as to the South-African isolate LT, and a high genetic relatedness was also found with strain Vaccine (99.92% nt identity). In contrast, Japanese A. centrale strains Aomori and SS40C-L were more distantly related (nt identity of 98.28% and 95.21%, respectively) than A. marginale (99.33-99.55%) or Anaplasma ovis (99.40-99.63%) reference isolates. This pattern of segregation was also evident in the phylogenetic tree where Italian A. centrale strains segregated together with LT and Vaccine reference strains but separately from the Japanese isolates. A higher degree of variation was observed when groEL sequences were analysed. In this gene, the Italian A. centrale strains displayed a high genetic relatedness to each other (99.58100% nt identity), but they were less related to LT and Vaccine isolates (98.40-98.82% and 97.7298.06, respectively). Comparison with Japanese isolates was not possible, as groEL sequences are not available in the GenBank database. However, both Italian and reference strains were distantly related to A. marginale (94.61-95.45 %) and A. ovis (85.69-86.66%) reference isolates. The phylogenetic tree constructed on the groEL sequences showed that the Italian A. centrale strains are grouped within a unique cluster which is separate from reference isolates of A. centrale, as well as from A. marginale and A. ovis. A. centrale is widespread in tropical and subtropical areas of the worlds, and recently it has been also reported in Sicily (Georges et al., 2001). However, in that study, the detection was carried out by means of RLB assay without providing any sequence and evolutionary data. Moreover, to date only few A. centrale isolates have been analysed at the molecular level, so that our study contributes to better understand the genetic variability of A. centrale strains from different geographic areas. The Italian strains, including that associated to acute disease, were found to be related to A. centrale isolates of African origin. Some questions arise about the origin of A. centrale circulating in Italy. A limited experiment using the A. centrale vaccine imported from South Africa was carried out in 1982 in cattle herds of Sicily (Gallo et al., 1982). As a consequence of the vaccination trial, the employed live-vaccine strain could have spread widely in cattle herds of Sicily and then imported in other regions of southern Italy. However, the limited data obtained from the present study do not support this hypothesis, as the Italian strains diverged from the vaccine in both 16S rDNA and groEL sequences, showing a higher relatedness to the field isolate LT. Alternatively, autochthonous strains may have been circulating in Italy or repeated introductions of A. centrale from the African continent may have occurred. Extensive molecular investigation and genetic analysis of A. centrale strains circulating in Italy should clarify definitively these epidemiological aspects.