PCR detection of Babesia ovata from cattle reared in Japan and clinical significance of coinfection with Theileria orientalis

We describe here the clinical significance of coinfection with Theileria orientalis and Babesia ovata in cattle. Anemia status in a herd of dairy cattle in Japan was investigated in relation to infection with these parasites. Our findings indicate that while B. ovata infection might not be the primary cause of anemia in the cattle, it may contribute to the clinical development of anemia in animals coinfected with both B. ovata and T. orientalis.     

Citrate synthase gene sequence: a new tool for phylogenetic analysis and identification of Ehrlichia

The sequence of the citrate synthase gene (gltA) of 13 ehrlichial species (Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia muris, an Ehrlichia species recently detected from Ixodes ovatus, Cowdria ruminantium, Ehrlichia phagocytophila, Ehrlichia equi, the human granulocytic ehrlichiosis [HGE] agent, Anaplasma marginale, Anaplasma centrale, Ehrlichia sennetsu, Ehrlichia risticii, and Neorickettsia helminthoeca) have been determined by degenerate PCR and the Genome Walker method. The ehrlichial gltA genes are 1,197 bp (E. sennetsu and E. risticii) to 1,254 bp (A. marginale and A. centrale) long, and GC contents of the gene vary from 30.5% (Ehrlichia sp. detected from I. ovatus) to 51.0% (A. centrale). The percent identities of the gltA nucleotide sequences among ehrlichial species were 49.7% (E. risticii versus A. centrale) to 99.8% (HGE agent versus E. equi). The percent identities of deduced amino acid sequences were 44.4% (E. sennetsu versus E. muris) to 99.5% (HGE agent versus E. equi), whereas the homology range of 16S rRNA genes was 83.5% (E. risticii versus the Ehrlichia sp. detected from I. ovatus) to 99.9% (HGE agent, E. equi, and E. phagocytophila). The architecture of the phylogenetic trees constructed by gltA nucleotide sequences or amino acid sequences was similar to that derived from the 16S rRNA gene sequences but showed more-significant bootstrap values. Based upon the alignment analysis of the ehrlichial gltA sequences, two sets of primers were designed to amplify tick-borne Ehrlichia and Neorickettsia genogroup Ehrlichia (N. helminthoeca, E. sennetsu, and E. risticii), respectively. Tick-borne Ehrlichia species were specifically identified by restriction fragment length polymorphism (RFLP) patterns of AcsI and XhoI with the exception of E. muris and the very closely related ehrlichia derived from I. ovatus for which sequence analysis of the PCR product is needed. Similarly, Neorickettsia genogroup Ehrlichia species were specifically identified by RFLP patterns of RcaI digestion. If confirmed this technique will be useful in rapidly identifying Ehrlichia spp.     

Detection of Anaplasma phagocytophilum and Ehrlichia sp. HF strains in Ixodes ricinus ticks in Brittany, France

DNA extracts from 156 tick pools, 18 blood specimens and 17 spleens from European woodmice (Apodemus sylvaticus) collected in Brittany, France were tested by PCR for the 16S rRNA gene of Anaplasmataceae. Positive amplicons were sequenced and confirmed, either by amplification and sequencing of a second gene, or by a second PCR specific for the P44 and gltA genes of Anaplasma phagocytophilum and the gltA gene of Ehrlichia sp. HF. In addition to A. phagocytophilum, the study detected Ehrlichia sp. HF for the first time in Ixodes ricinus ticks. This organism has only been detected previously in Ixodes ovatus ticks from Japan.     

The prognosis and immunohistochemical evaluation of five perianal Paget's disease cases

Perianal Paget's disease is categorized as Paget's disease, which is epidermotropic neoplasm arising from the apocrine glands of perianal region, or Pagetoid spread invaded from rectal or anal canal cancer. It has been reported that the immunohistochemical staining of GCDFP15, the marker of the apocrine epithelium, and CK20 is used to be distinguished between Paget's disease and Pagetoid spread. Five patients with perianal Paget's disease who underwent a surgical operation had been treated in our department between 1997 and 2006. We analyzed the clinical findings and the treatment of these patients and investigated the expression of GCDFP15 and CK20 by immunohistochemical staining. All cases presented the redness around perianal regions, and 2 cases were recognized a tumor at the anal canal. We preoperatively diagnosed these cases as Pagetoid spread and others without tumor regions as Paget's disease. Surgical treatment was performed for all patients. As a result of immunohistochemical staining, 2 cases of Pagetoid spread were negative for GCDFP15, and positive for CK20. It was compatible with the preoperative diagnosis. Only one of 3 Paget's disease cases was positive for GCDFP15 and negative for CK20 resulting in the diagnosis of perianal Paget's disease. Based on the expression of negative for GCDFP15 and positive for CK20, others were seemed to be Pagetoid spread. A treatment strategy including surgical operation and chemotherapy is different between patients with Paget's disease and those with Pagetoid spread. Therefore, it is essential to investigate the expression pattern of GCDFP15 and CK20 using the tissue from the biopsy to identify the disease for appropriate treatment.     

Analysis of the 16S rRNA Gene Sequence of Anaplasma centrale and Its Phylogenetic Relatedness to Other Ehrlichiae

The nucleotide sequence of the Anaplasma centrale 16S rRNA gene was determined and compared with the sequences of ehrlichial bacteria. The sequence of A. centrale was closely related to Anaplasma marginale by both level-of-similarity (98.08% identical) and distance analysis. A species-specific PCR was developed based upon the alignment data. The PCR can detect A. centrale DNA extracted from 10 infected bovine red blood cells in a reaction mixture. A. centrale DNA was amplified in the reaction, but not other related ehrlichial species.