The application of PCR and reverse line blot hybridization to detect arthropod-borne hemopathogens of dogs and cats in Trinidad.

Arthropod-borne diseases are important causes of morbidity and mortality of companion animals in Trinidad. As clinical signs are vague, more sensitive methods to diagnose these diseases based on the polymerase chain reaction (PCR) followed by reverse line blot hybridization (RLB) of amplified products are being developed. An RLB of 14 oligonucleotide probes coupled with polymerase chain-amplified regions of 16S rRNA or 18S rRNA genes of hemoparasites from cats and dogs detected Ehrlichia canis, Anaplasma platys, Babesia canis vogeli, feline mycoplasmas ("Candidatus Mycoplasma haemominutum,"Mycoplasma haemofelis), and some unknown species within the Babesia/Theileria group and the Anaplasma/Ehrlichia tribe. Amplified products were obtained from blood samples collected from 348 dogs and 15 cats. Overall, hemopathogen DNA was detected in 92 (26.4 per cent) dogs and six (40.0 per cent) cats. E. canis (49, 14.1 per cent) and feline mycoplasma (5, 33.3 per cent) DNA were most frequently identified in dogs and cats, respectively. B. canis vogeli (1, 6.7 per cent) and E. canis (1, 6.7 per cent) were also detected in cats. Mixed infections of Anaplasma/Ehrlichia sp. and Babesia sp. were present in five (1.4 per cent) dogs, while mixed infections of the feline mycoplasmas were present in two (13.3 per cent) cats, one of which was also positive for E. canis. Pyrexia was significantly associated with a positive RLB result in dogs (P= 0.00, chi(2), 1 df). This is the first reported application of macro-arraying techniques to detect arthropod-borne hemopathogens of companion animals in the Americas and the first detection of DNA of B. canis vogeli and E. canis in cats in Trinidad.

Application of PCR and Reverse Line Blot Hybridization to Detect Arthropod 1 Transmitted Haemopathogens in Horses Presented at a Referral Veterinary Facility in 2 New Jersey, USA

Equine granulocytic ehrlichiosis is a tick-borne zoonotic disease caused by the 36 intracellular bacterium Anaplasma phagocytophilum. This disease has been well 37 documented in California, USA and sporadic cases have been recorded in New Jersey 38 and other states. Equine piroplasmosis is a reportable disease in the United States with 39 the most recent outbreak reported in Florida in 2008. In order to detect these diseases in 40 an area of low prevalence, two PCRs followed by a reverse line blot (RLB) hybridization 41 process were performed on DNA extracted from blood of 133 horses presented at a large 42 equine referral clinic in New Jersey. One PCR amplified the 16S rRNA gene for 43 Anaplasma/ Ehrlichia and gene and the other the 18S rRNA gene for Theileria/Babesia. 44 Amplified products were applied to a membrane to which oligonucleotide probes for equine granulocytic ehrlichiosis (Anaplasma phagocytophilum) and equine piroplasmosis 46 (Theileria equi and Babesia caballi) were covalently attached. Two horses were positive 47 for Anaplasma phagocytophilum .This is the first published record of A phagocytophilum 48 DNA to be detected in horses in the United States using the reverse line blot 49 hybridization process. This technique is useful for large scale epidemiological screening 50 and detection of several tick-transmitted haemopathogens simultaneously in animals with 51 vague clinical signs of disease.