A Comparison of Peripheral Blood Smears, Autologous Cell Cultures, and Reverse Line Blot Hybridisation in Screening for Anaplasma/Ehrlichia in Roaming Dogs and Symptomatic Dogs in Trinidad

This study compared two methods to detect cases of canine ehrlichiosis in a field setting. One method was a polymerase chain reaction for the 16S rRNA gene followed by reverse line blot hybridisation with genera and species-specific probes for Anaplasma/Ehrlichia. The second method was an autologous cell culture of peripheral leucocytes isolated from heparinised blood and maintained in a homologous canine serum in Dulbecco’s Modified Eagle medium without antibiotics. The cultures were examined under light microscopy for inclusion bodies after 48 h. Leucocytes were successfully propagated for 20 of the 34 samples submitted for autologous cell culture. Inclusion bodies were observed after cell culture in leucocytes of eight dogs. Two dogs were positive to the Anaplasma/Ehrlichia genera probe and six dogs were positive to the E. canis probe after reverse line blot hybridisation. There was acceptable agreement between reverse line blot hybridisation and cell culture results. Both reverse line blot hybridisation and autologous cell cultures can be used to detect E. canis in subclinical and clinical cases of disease. A definitive diagnosis of E. canis is best achieved by a combination of clinical signs, positive autologous cell culture, and reverse line blot hybridisation results.

First broad-range molecular screening of tick-borne pathogens in Ixodes (Pholeoixodes) kaiseri, with special emphasis on piroplasms

Recently, the occurrence of Ixodes (Pholeoixodes) kaiseri has been reported for the first time in several European countries, but data on the molecular analysis of this hard tick species are still lacking. Therefore, in this study DNA extracts of 28 I. kaiseri (collected from dogs and red foxes in Germany, Hungary and Romania) were screened with reverse line blot hybridisation (RLB), PCR and sequencing for the presence of 43 tick-borne pathogens or other members of their families from the categories of Anaplasmataceae, piroplasms, rickettsiae and borreliae. Rickettsia helvetica DNA was detected in one I. kaiseri female (from a red fox, Romania), for the first time in this tick species. Six ticks (from red foxes, Romania) contained the DNA of Babesia vulpes, also for the first time in the case of I. kaiseri. Molecular evidence of R. helvetica and B. vulpes in engorged I. kaiseri does not prove that this tick species is a vector of the above two pathogens, because they might have been taken up by the ticks from the blood of foxes. In addition, one I. kaiseri female (from a dog, Hungary) harboured Babesia sp. badger type-B, identified for the first time in Hungary and Central Europe (i.e. it has been reported previously from Western Europe and China). The latter finding can be explained by either the susceptibility of dogs to Babesia sp. badger type-B, or by transstadial survival of this piroplasm in I. kaiseri.

Detection of viruses in the exotic shrimp Penaeus vannamei Boone, 1931 cultured in India

Pacific white shrimp Penaeus vannamei Boone, 1931, is a recently introduced species in India. P. vannamei samples, collected from various shrimp farms of Karnataka, India were subjected to polymerase chain reaction (PCR) based detection of whitespot syndrome virus (WSSV), hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), taura syndrome virus (TSV) and infectious myonecrosis virus (IMNV). Out of the 81 shrimp samples analysed, 41 samples (50.6%) were found positive for WSSV and four (4.9%) were positive for IHHNV. Among 41 WSSV positive samples, 20 (48.7%) samples were found positive for WSSV by 1st step PCR, while the remaining 21 (51.2%) samples were positive by nested PCR. WSSV positive samples were further confirmed by dot blot hybridisation assay. However, clinical signs/disease symptoms were not observed in any of the shrimp samples tested positive for the viruses.

Confirmation of occurrence of Babesia vogeli in a dog in Windhoek, central Namibia

Although there is evidence of high seroprevalence of antibodies to Babesia spp. in dogs in central Namibia, clinical babesiosis is rarely diagnosed. Rhipicephalus sanguineus sensu lato, the vector of Babesia vogeli, is common in Namibia while Haemaphysalis elliptica, the vector of the highly virulent but morphologically indistinguishable Babesia rossi, has rarely been recorded, mainly in northern Namibia. On the basis of vector occurrence, clinical cases of canine babesiosis in Windhoek, central Namibia, have been ascribed to B. vogeli. DNA extracted from a blood smear made from a sick dog was subjected to the reverse line blot hybridisation assay. The polymerase chain reaction amplicons hybridised with the B. vogeli–specific probe, but not with the Babesia canis– and B. rossi–specific probes. Although attempts at cloning and sequencing of the full-length 18S rRNA gene were unsuccessful, we can confirm that B. vogeli occurs in central Namibia.

Molecular evidence of Ehrlichia canis and Rickettsia massiliae in ixodid ticks of carnivores from South Hungary

To monitor the emergence of thermophilic, Mediterranean ixodid tick species and tick-borne pathogens in southern Hungary, 348 ticks were collected from shepherd dogs, red foxes and golden jackals during the summer of 2011. Golden jackals shared tick species with both the dog and the red fox in the region.Dermacentornymphs were collected exclusively from dogs, and the sequence identification of these ticks indicated that dogs are preferred hosts of bothD. reticulatusandD. marginatusnymphs, unlike previously reported. Subadults of three ixodid species were selected for reverse line blot hybridisation (RLB) analysis to screen their vector potential for 40 pathogens/groups. Results were negative forAnaplasma,BabesiaandTheileriaspp. Investigation ofD. marginatusnymphs revealed the presence ofEhrlichia canis,Rickettsia massiliaeandBorrelia afzeliifor the first time in this tick species. These findings broaden the range of those tick-borne agents, which are typically transmitted byRhipicephalus sanguineus, but may also haveDermacentorspp. as potential or alternative vectors.Ehrlichiacaniswas also newly detected inIxodes canisugalarvae from red foxes. In absence of transovarial transmission in ticks this implies that Eurasian red foxes may play a reservoir role in the epidemiology of canine ehrlichiosis.