Onchocerca lupi in imported dogs in the UK: implications for animal and public health

Abstract Background Onchocerca lupi is a filarial nematode affecting dogs, and occasionally cats and humans, in continental Europe, North Africa, the Middle East, and the USA. Adult worms are usually found in periocular nodules and enucleation is sometimes required if the infection fails to respond to other treatment options. Case presentation Here, we report the presence of O. lupi in the UK for the first time. Of two dogs re-homed from continental Europe, one developed an ocular nodule seven years after arrival from Portugal. The conjunctival perilimbal mass in its left eye was surgically removed but despite anthelminthic treatment, a further nodule developed in the same eye six months later. In the second case - a dog imported from Romania 12 months earlier - a perilimbal mass was excised from the left eye and prior anthelminthic treatment was supplemented with oral prednisolone and doxycycline. However, nodules recurred, and the left globe was subsequently enucleated. Conjunctival hyperaemia then appeared in the right eye and neither additional anthelminthic treatment nor removal of worm masses failed to prevent the further development of lesions. Excised adult worms were identified in both cases as O. lupi based on morphological characteristics, as well as PCR and sequencing of cytochrome c oxidase subunit I and 12S rRNA gene fragments. Conclusion O. lupi parasitosis can apparently remain cryptic in dogs for several years before any clinical signs manifest. Moreover, the progression of infection can be highly aggressive and recalcitrant to both surgical intervention and anthelminthic treatment. Increasingly, former stray dogs of unknown infection status are entering the UK, raising both veterinary and public health concerns. </jats:sec

Diagnosis of Hepatozoon canis in young dogs by cytology and PCR

<p>Abstract</p> <p>Background</p> <p><it>Hepatozoon canis </it>is a widespread tick-borne protozoan affecting dogs. The diagnosis of <it>H. canis </it>infection is usually performed by cytology of blood or buffy coat smears, but this method may not be sensitive. Our study aimed to evaluate the best method to achieve a parasitological diagnosis of <it>H. canis </it>infection in a population of receptive young dogs, previously negative by cytology and exposed to tick infestation for one summer season.</p> <p>Results</p> <p>A total of 73 mongrel dogs and ten beagles younger than 18 months of age, living in an animal shelter in southern Italy where dogs are highly infested by <it>Rhipicephalus sanguineus</it>, were included in this study. In March-April 2009 and in October 2009, blood and bone marrow were sampled from each dog. Blood, buffy coat and bone marrow were examined by cytology only (at the first sampling) and also by PCR for <it>H. canis </it>(second sampling). In March-April 2009, only one dog was positive for <it>H. canis </it>by cytological examination, whereas in October 2009 (after the summer season), the overall incidence of <it>H. canis </it>infection by cytological examinations was 43.9%. Molecular tests carried out on samples taken in October 2009 showed a considerably higher number of dogs positive by PCR (from 27.7% up to 51.2% on skin and buffy coat tissues, respectively), with an overall positivity of 57.8%. All animals, but one, which were positive by cytology were also PCR-positive. PCR on blood or buffy coat detected the highest number of <it>H. canis</it>-positive dogs displaying a sensitivity of 85.7% for both tissues that increased up to 98% when used in parallel. Twenty-six (74.8%) out of the 28 <it>H. canis</it>-positive dogs presented hematological abnormalities, eosinophilia being the commonest alteration observed.</p> <p>Conclusions</p> <p>The results suggest that PCR on buffy coat and blood is the best diagnostic assay for detecting <it>H. canis </it>infection in dogs, although when PCR is not available, cytology on buffy coat should be preferred to blood smear evaluation. This study has also demonstrated that <it>H. canis </it>infection can spread among young dogs infested by <it>R. sanguineus </it>and be present in the majority of the exposed population within 6 months.</p

Canine and ovine tick-borne pathogens in camels, Nigeria

AbstractIn April 2008, whole blood samples were collected from 36 dromedary camels in Sokoto, North-western Nigeria. Following PCR and reverse line blotting, twenty-two samples (61%) resulted positive for Ehrlichia/Anaplasma spp. and three (8%) for Theileria/Babesia spp., with three (8%) cases of co-infections being found. Both sequence and BLAST analyses identified Ehrlichia/Anaplasma spp. and Theileria/Babesia spp. positive cases as Anaplasma platys and Theileria ovis, respectively.This is the first report of the detection of A. platys and T. ovis in camels from sub-Saharan Africa. The epidemiological relevance of this finding is enhanced by the close living of these animals with both dogs and small ruminants. The high prevalence detected for A. platys suggests a possible role of camels as carriers of this infection

Comparative analyses of mitochondrial and nuclear genetic markers for the molecular identification of Rhipicephalus spp.

The genus Rhipicephalus (Acari: Ixodidae) comprises a large number of vectors of pathogens of substantial medical and veterinary concern; however, species identification based solely on morphological features is often challenging. In the present study, genetic distance within selected Rhipicephalus species (i.e., Rhipicephalus bursa, Rhipicephalus guilhoni, Rhipicephalus muhsamae, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus), were investigated based on molecular and phylogenetic analyses of fragments of the mitochondrial 16S, 12S and cytochrome c oxidase subunit 1 (cox1) genes, as well as of the whole sequences of the ribosomal internal transcribed spacer-2 (ITS-2) region. Mean values of inter-specific genetic distance (e.g., up to 12.6%, 11.1% and 15.2%), as well as of intra-specific genetic distance (e.g., 0.9%, 0.9% and 1%), calculated using the Kimura-2 parameter substitution model with uniform rates among sites for 16S, 12S and cox1 genes, respectively, confirmed the differentiation of the rhipicephaline species herein examined. The molecular identification was also supported by the distinct separation of species-specific clades inferred from the phylogenetic analyses of all mitochondrial sequences. Conversely, little interspecific divergence was detected amongst ribosomal ITS-2 sequences (i.e., up to 2.8%) for species belonging to the R. sanguineus complex, which resulted in the ambiguous placement of selected R. sanguineus s.l. and R. turanicus sequences in the corresponding phylogenetic tree. Results from this study confirm the suitability of mtDNA markers for the reliable identification of ticks within the Rhipicephalus genus and provide a framework for future studies of taxonomy, speciation history and evolution of this group of ticks

A duplex real-time PCR assay for the detection and differentiation of Leishmania infantum and Leishmania tarentolae in vectors and potential reservoir hosts

Leishmanioses are vector-borne diseases, some of zoonotic concern, transmitted by phlebotomine sand flies (Diptera, Psychodidae). Reports of reptile-associated Leishmania tarentolae in humans, and of Leishmania infantum in Sergentomyia minuta sand flies prompted the development of an internal transcribed spacer 1-based duplex quantitative real-time PCR (dqPCR) to detect and differentiate these Leishmania spp. The specificity of dqPCR was assessed by processing DNA samples from Phlebotomus spp. (n = 188) and Se. minuta (n = 171) and from tissues (i.e., heart, liver, muscle, lungs, spleen, kidney, eggs) of Podarcis siculus (n = 4) and Tarentola mauritanica (n = 3). The analytical sensitivity of the dqPCR, assessed using 10-fold serial dilutions of DNA from Leishmania spp. and spiked DNA samples from lizards, was 2.3 × 10-7 ng/2 µl for L. infantum and 2.1 × 10-7 ng/2 µl for L. tarentolae. The dqPCR detected up to 4.3 × 10-6 ng/2 µl of L. infantum and up to 4.9 × 10-7 ng/2 µl of L. tarentolae. Of the 359 phlebotomine sand flies tested, five (3.6%) and two (1.4%) Ph. perniciosus scored positive for L. infantum and L. tarentolae, respectively. Similarly, of 171 Se. minuta, 56 (32.7%) and six (3.5%) scored positive for L. tarentolae and L. infantum, respectively. Co-infection was detected in two Se. minuta (1.2%). Out of seven reptiles tested, four P. siculus were positive for L. tarentolae. This new dqPCR may improve the diagnosis of L. infantum and L. tarentolae and aid to assess the role of lizards as reservoirs and of Se. minuta as vector, for these Leishmania spp

Molecular xenomonitoring of Dirofilaria immitis and Dirofilaria repens in mosquitoes from north-eastern Italy by real-time PCR coupled with melting curve analysis.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are transmitted by bloodsucking culicid mosquitoes belonging to Culex, Aedes, Ochlerotatus, Anopheles and Mansonia genera. The detection of filariae in mosquitoes for assessing distribution of vectors and/or of pathogens in a given area (also known as "xenomonitoring"), when based on individual dissection of wild-caught female mosquitoes is time consuming and hardly applicable in large epidemiological surveys. Our study aimed to evaluate the recently developed duplex real-time PCR for screening large number of culicids and to assess their positivity for D. immitis and D. repens in an area where both species are endemic. METHODS: A duplex real-time PCR was used to detect and differentiate D. immitis and D. repens in mosquitoes collected in six provinces of the Veneto region using 43 carbon dioxide-baited traps under the frame of an entomological surveillance program to monitor the vectors of West Nile disease. From early May till October 2010, unfed female mosquitoes (n = 40,892) were captured in 20 selected sites. RESULTS: Mosquitoes were identified as Culex pipiens, Ochlerotatus caspius, Aedes vexans and Culex modestus and grouped into 995 pools according to species, day and site of collection (from minimum of 1 to maximum of 57). Out of 955 pools, 23 (2.41 %) scored positive for Dirofilaria spp. of which, 21 (2.2 %) for D. immitis and two (0.21 %) for D. repens. An overall Estimated Rate of Infection (ERI) of 0.06 % was recorded, being higher in Och. caspius and Ae. vexans (i.e., 0.18 % and 0.14 %, respectively). At least one mosquito pool was positive for Dirofilaria spp. in each province with the highest ERI recorded in Vicenza and Padova provinces (i.e., 0.42% and 0.16 %, respectively). Mosquitoes collected in all provinces were positive for D. immitis whereas, only two (i.e., Padova and Rovigo) provinces scored positive for D. repens. All mosquito species, except for Cx. modestus, were positive for D. immitis, whereas D. repens was only found in Cx. pipiens. CONCLUSIONS: The results suggest that both Dirofilaria species are endemic and may occur in sympatry in the examined area. The molecular approach herein used represents a powerful tool for surveillance programs of D. immitis and D. repens in the culicid vectors towards a better understanding of the epidemiology of the infections they cause and their seasonal transmission patterns