The nematode parasite Angiostrongylus vasorum is an increasingly important cause of respiratory and other diseases in dogs. Geographical spread from previously limited endemic foci has occurred rapidly. This paper investigates parasite epidemiology around the location of the first reported case in Scotland in 2009: by detection of A vasorum-specific DNA in gastropod intermediate hosts, and in dogs circulating DNA and specific antibodies, and first stage larvae in faeces. Overall prevalence in gastropods was 6.7 per cent (16/240), with parasite DNA found in slugs in the Arion ater and Arion hortensis species aggregates and the snail Helix aspersa (syn. Cornu aspersum). Of 60 dogs presenting with clinical signs compatible with angiostrongylosis, none tested positive using PCR on peripheral blood or Baermann test on faeces, and none of 35 tested for circulating anti-A vasorum antibodies were positive. PCR prevalence in gastropods was highest (11 per cent) in the park frequented by the canine angiostrongylosis index case. Molecular survey for infection in gastropods is a potentially informative and efficient method for characterising the distribution of A vasorum and therefore local risk of canine infection. However, there appears to be a complex relationship between prevalence in gastropods and emergence of canine clinical disease, which requires further work to advance understanding of parasite transmission and geographical disease spread.

SUMMARY Timely diagnosis of the nematode Angiostrongylus vasorum in dogs is important in view of severe and permanent lung and cardiovascular lesions that may occur. The performance of the classical Baermann coprological method was compared with ELISAs for the serological detection of circulating antigen and specific antibodies and with Polymerase chain reaction (PCR) performed on EDTA blood, feces and tracheal swabs of serial samples from experimentally inoculated dogs over 13 weeks post inoculation (wpi) ( n = 16) and following anthelmintic treatment ( n = 6). Patency was observed from 6·7 to 7·6 wpi in all dogs, Baermann results were then mostly positive (116/119, 97%) during the patent period, with wide variations in the numbers of first stage larvae numbers. Blood PCR was tested positive on 1–2 occasions in 11/16 dogs in the pre-patent period, while all tested positive by antibody-detection ELISA by 6 wpi. The proportion of dogs testing positive by fecal PCR and antigen-detection ELISA rose early in the patent period. Tracheal swabs were occasionally DNA-positive in 3/16 dogs starting from 10 wpi. Following treatment, larval excretion stopped within 3 weeks and blood PCR results became negative within 1 week (5/6 dogs), while 4/6 dogs were positive for parasite DNA in tracheal swabs. Parasite antigen and specific antibodies both persisted in the blood for 3–9 weeks after treatment, with average optical densities and the proportion of positive dogs falling gradually, while results using other tests were much more variable. Results indicate that the earliest and most consistent results are obtained by the ELISAs, which can also be used for monitoring dogs after anthelmintic treatment.

Cryptcisporidiosis, a gastroenteric disease characterised mainly by diarrheal illnesses in humans and mammals is caused by infection with the protozoan parasite Cryptosporidium. Treatment options for cryptosporidiosis are limited, with the current therapeutic nitizoxanide, only partly efficacious inlmmunocompetent individuals. The parasite lacks de novo purine synthesis, and is exclusively dependant on purine salvage from its host. Inhibition of the inosine 5' monophosphate dehydrogenase (IMPDH), a purine salvage enzyme that is essential for DNA synthesis, thereby offers a potential:drug target against this parasite. In the present study, a yeast-two-hybrid system was used to identify Phylomer peptides within a library constructed from the genomes of 25 phylogenetically diverse bacteria that targeted the IMPDH of Oyptosporidium parvum (IMPcp) and Gyptosporidium hominis (IMPch). We identified 38 unique interacting Phylomers, of which, 12 were synthesised and screened against C parvum in vitro. Two Phylomers exhibited significant growth inhibition (81.2-83.8% inhibition; P < 0.05), one of which consistently exhibited positive interactions with IMPcp and IMPch during primary and recapitulation yeast two-hybrid screening and did not interact with either of the human IMPDH proteins. The present study highlightsthe potential of Phylomer peptides as target validation tools for Cryptosporidium and other organisms and diseases because of their ability to bind with high affinity to target proteins and disrupt function. (C) 2014 Elsevier Inc. All rights reserved.

Severe infestation with Aelurostrongylus abstrusus was identified in the lungs and small intestine of a 2-month-old kitten that died due to verminous pneumonia and enteritis. On clinical examination, the kitten had dyspnoea, pneumonia, pleural effusion, ascites and diarrhoea. An interstitial pattern was evident radiographically in the lungs. The kitten died before treatment could be instituted. On gross and histopathological examination, there was severe interstitial pneumonia and large numbers of A. abstrusus eggs and larvae were present in alveoli, together with fewer adult nematodes in small bronchioles. The mucosa of the small intestine was invaded by large numbers of A. abstrusus larvae. The findings were consistent with a hyperinfection syndrome due to A. abstrusus.

SUMMARY Molecular phylogeography has revolutionised our ability to infer past biogeographic events from cross-sectional data on current parasite populations. In ecological parasitology, this approach has been used to address fundamental questions concerning host-parasite co-evolution and geographic patterns of spread, and has raised many technical issues and problems of interpretation. For applied parasitologists, the added complexity inherent in adding population genetic structure to perceived parasite distributions can sometimes seem to cloud rather than clarify approaches to control. In this paper, we use case studies firstly to illustrate the potential extent of cryptic diversity in parasite and parasitoid populations, secondly to consider how anthropogenic influences including movement of domestic animals affect the geographic distribution and host associations of parasite genotypes, and thirdly to explore the applied relevance of these processes to parasites of socio-economic importance. The contribution of phylogeographic approaches to deeper understanding of parasite biology in these cases is assessed. Thus, molecular data on the emerging parasites Angiostrongylus vasorum in dogs and wild canids, and the myiasis-causing flies Lucilia spp. in sheep and Cochliomyia hominovorax in humans, lead to clear implications for control efforts to limit global spread. Broader applications of molecular phylogeography to understanding parasite distributions in an era of rapid global change are also discussed.

Angiostrongylus vasorum is an emerging parasite that is currently distributed through Western Europe and parts of South America. An isolated population is also present in Newfoundland, Canada. This presents a risk of onward spread into North America, but its origin is unknown. To ascertain the phylogeographic relationships and genetic diversity of A. vasorum within the western Palaearctic and eastern Nearctic ecozones, a total of 143 adult and larval nematode specimens were collected from foxes (Vulpes vulpes) and dogs (Canis lupus familiaris) in Canada, Denmark, France, Germany, Ireland, the Netherlands, Portugal and the United Kingdom, and a coyote (Canis latrans) in Canada. DNA was extracted and the second internal transcribed spacer and two mitochondrial loci were amplified and sequenced. Multiple haplotypes (n = 35) based on combined mitochondrial sequences (1078 bp) of the partial cytochrome oxidase subunit I (COI), large subunit ribosomal RNA (rrnL) and the complete nicotinamide adenine dinucleotide dehydrogenase 3 (NADH3) sequences, were observed throughout the Palaearctic countries sampled; however, only a single haplotype was observed for the Canadian A. vasorum population. The likely origin of A. vasorum in Newfoundland is therefore inferred to be within the western Palaearctic. There was no evidence of genetic segregation of parasites in dogs, foxes and coyotes, supporting the hypothesis that transmission occurs between wild and domestic canids. The transmission dynamics and population structure of this nematode are further discussed.

Infection with the nematode Angiostrongylus vasorum is an emerging cause of canine disease in Europe and part of North America, yet published data on its epidemiology in endemic areas are lacking. This study tested faecal samples from 897 dogs attending veterinary practices in the southern part of Great Britain, a long standing endemic focus. Among 790 dogs presenting with respiratory or other signs broadly suggestive of angiostrongylosis, 16% tested positive on a single Baermann's examination, compared with 2% of healthy dogs in the same catchment areas. Risk factors for positive tests included age (higher risk in younger dogs), season (more cases earlier in the calendar year), and worming history (lower risk if given milbemycin oxime in the past 12 weeks). Sex, neutering status and breed were not significant in terms of risk of testing positive. The most common clinical signs in infected dogs were respiratory, along with non-specific signs such as lethargy and exercise intolerance, while bleeding, neurological and gastrointestinal signs were also recorded. Around half the dogs sampled that showed signs of extra-pulmonary disease also had respiratory signs. Direct faecal smears and Baermann's tests read after one hour detected 56% and 83% of diagnosed cases respectively. The data confirm that A. vasorum is commonly associated with disease in endemic areas, which manifests with a broad range of signs at primary care level. Information on risk factors is useful in diagnosis and control, and forms a basis for further epidemiological investigation.

The parasitic nematode Angiostrongylus vasorum is an emerging challenge for companion animal and wildlife health, with reported increases in both distribution and incidence in Europe. To facilitate improved detection of this parasite, a SYBR green real-time polymerase chain reaction (PCR) was developed to amplify a region of the second internal transcribed spacer (ITS-2) of A. vasorum from both definitive and intermediate host samples. The PCR assay was capable of detecting a single of plasmid DNA containing the entire ITS-2 region, a single first stage larva (L1) in 200μl canine EDTA blood, a single L1 in 200mg of canine faeces and a single L3 in 10mg of Biomphalaria glabrata tissue. The assay exhibited a high level of specificity to A. vasorum and whilst it potentially amplifies DNA of other Angiostrongylus species, it did not amplify DNA from a range of other common canine parasitic nematodes. Field evaluation of the PCR assay was conducted by screening canine EDTA blood and faecal samples from suspected cases of A. vasorum infection and compared with Baermann's detection, and also by screening a range of gastropod species from an endemic area. Real-time quantitative PCR offers a more efficient means of detecting A. vasorum infection with a lower limit of detection than traditional diagnostic tests, and it therefore has important clinical and epidemiological applications.

Little is known of the prevalence and life-cycle of trypanosomes in mammals native to Australia. Native Australian trypanosomes have previously been identified in marsupials in the eastern states of Australia, with one recent report in brush-tailed bettongs (Bettongia penicillata), or woylie in Western Australia in 2008. This study reports a novel Trypanosoma sp. identified in blood smears, from 7 critically endangered Gilbert's potoroos (Potorous gilbertii) and 3 quokkas (Setonix brachyurus) in Western Australia. Trypanosomes were successfully cultured in vitro and showed morphological characteristics similar to members of the subgenus Herpetosoma. Phylogenetic analysis of 18S rRNA gene sequences identified 2 different novel genotypes A and B that are closely related to trypanosomes previously isolated from a common wombat (Vombatus ursinus) in Victoria, Australia. The new species is proposed to be named Trypanosoma copemani n. sp.

SUMMARYAngiostrongylus vasorumis a nematode parasite of sylvan and domestic species of the family Canidae. It has a broad but patchy distribution worldwide, and there is evidence for geographical spread and increasing incidence of infection in recent years. While historicallyAngiostrongylus-like nematodes identified in dogs and foxes have been described asA. vasorumin Europe andAngiocaulus raillietiin South America, more recent taxonomic revision has amalgamated these into a single species,A. vasorum. Here we report, for the first time, the molecular characterization of isolates ofA. vasorumfrom Germany, Portugal, Denmark and the United Kingdom on the basis of the mitochondrial COI gene and the second ribosomal internal transcribed spacer. When compared with isolates from Brazil, sequence analysis revealed 2 distinct genotypes. Estimated rates of evolution based on COI sequences for both nematode and host are consistent with the hypothesis that the presence ofA. vasorumin South America is a result of an ancient evolutionary event.Angiostrongylus vasorumin South America potentially represents a separate species to that observed in Europe.

To further investigate the recently described avian piroplasm, Babesia kiwiensis, blood samples were collected from 13 wild-caught and 8 zoo-captive brown kiwi (Apteryx mantelli) and screened for the presence of piroplasm DNA using a nested-polymerase chain reaction (PCR) targeting the 18S rRNA gene of most members of Piroplasmida. All captive birds gave a negative PCR result, while 12 wild-caught birds were PCR positive. The nearly full-length 18S rRNA gene for B. kiwiensis was sequenced. Upon phylogenetic analysis, it was found to belong to the babesid group of piroplasms and was ancestral, yet genetically similar, to the Babesia canis-related species. An insight into the current taxonomy of the avian piroplasms is also given. An Ixodes anatis tick collected from 1 of the North Island brown kiwi was also screened using PCR and was found to be positive for B. kiwiensis DNA.

This study reports on the epidemiology of Babesia gibsoni in American Pit Bull Terriers living in a region of western Victoria in southern Australia. Both American Pit Bull Terriers (n = 100) and other dog breeds (n = 51) were screened for B gibsoni using immunofluorescent antibody testing (IFAT) and/or polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). A questionnaire was also completed by each dog owner, ascertaining the husbandry and habits of the dogs sampled. Fourteen dogs were positive for B gibsoni using IFAT and/or PCR-RFLP and all were American Pit Bull Terriers. Dogs that were male and/or had been bitten by or were biters of other American Pit Bull Terriers were more likely to be B gibsoni positive, thus suggesting that blood-to-blood transmission contributes to the spread of this disease between dogs.

The prevalence of hematozoan infections (Hepatozoon canis and Babesia sp., particularly Babesia canis vogeli) in canids from Venezuela, Thailand and Spain was studied by amplification and sequencing of the 18S rRNA gene. H. canis infections caused simultaneously by two different isolates were confirmed by RFLP analysis in samples from all the geographic regions studied.In Venezuela, blood samples from 134 dogs were surveyed. Babesia infections were found in 2.24% of the dogs. Comparison of sequences of the 18S rRNA gene indicated that protozoan isolates were genetically identical to B. canis vogeli from Japan and Brazil. H. canis infected 44.77% of the dogs. A representative sample of Venezuelan H. canis isolates (21.6% of PCR-positives) was sequenced. Many of them showed 18S rRNA gene sequences identical to H. canis Spain 2, albeit two less frequent genotypes were found in the sample studied.In Thailand, 20 dogs were analyzed. No infections caused by Babesia were diagnosed, whereas 30% of the dogs were positive to hematozoan infection. Two protozoa isolates showing 99.7-100% identity to H. canis Spain 2 were found.In Spain, 250 dogs were studied. B. canis vogeli infected 0.01% of the animals. The sequence of the 18S rRNA gene in Spanish isolates of this protozoa was closely related to those previously deposited in GenBank() (> 99% identity). Finally, 20 red foxes were screened for hematozoans employing semi-nested PCR and primers designed to detect Babesia/Theileria. Fifty percent of the foxes were positive to Theileria annae. In addition, it was found that the PCR assay was able as well to detect Hepatozoon infections. Thirty five percent of the foxes were infected with two different H. canis isolates showing 99.8-100% identity to Curupira I from Brazil. (c) 2006 Elsevier B.V. All rights reserved.

Babesia gibsoni is a protozoan parasite of dogs worldwide yet both an effective treatment and a reliable method for detecting subclinical cases of this emerging infection remain elusive. Experimental B. gibsoni infections were established in vivo to investigate the efficacy of combined atovaquone and azithromycin drug therapy and to determine the detection limits of a nested-PCR, IFAT and microscopy during various stages of infection. While atovaquone and azithromycin produced a reduction in parasitaemia, it did not eliminate the parasite and drug resistance appeared to develop in one dog. Polymerase chain reaction was found to be most useful in detecting infection in the pre-acute and acute stages, while IFAT was most reliable during chronic infections. Microscopy is suggested to be only effective for detecting acute stage infections. This study also describes the detection of B. gibsoni in tissue samples during chronic infections for the first time, suggesting possible sequestration of this parasite.

As a continuation of our studies on molecular epizootiology of piroplasmosis in Spain and other countries, we present in this contribution the finding of new hosts for some piroplasms, as well as information on their 18S rRNA gene sequences. Genetic data were complemented with sequences of apocytochrome b gene (whenever possible). The following conclusions were drawn from these molecular studies: Theileria annulata is capable of infecting dogs, since it was diagnosed in a symptomatic animal. According to cytochrome b sequences, isolates from cows and dog present slight differences. The same isolates showed, however, identical sequence in the 18S rRNA gene. This exemplifies well the usefulness of the mitochondrial gene for examining infra-specific variation. Babesia bovis is an occasional parasite of equines, since it was detected in two symptomatic horses. We found evidence of genetic polymorphism occurring in the 18S rRNA gene of Spanish T. equi-like and B. ovis isolates. B. bennetti from Spanish seagull is loosely related to B. ovis, and might represent a genetically distinct branch of babesids. A partial sequence of a cytochrome b pseudogene was obtained for the first time in Babesia canis rossi from South Africa. The pseudogene is distantly related to B. bigemina cytochrome b gene. These new findings confirm the ability of some piroplasms to infect multiple hosts, as well as the existence of a relatively wide genetic polymorphisms with respect to the cytochrome b gene. On the other hand, the existence of mtDNA-like pseudogenes of possible nuclear location in piroplasms is interesting due to their possible impact on molecular phylogeny studies.

Canine piroplasmosis is an emerging disease worldwide, with multiple species of piroplasm now recognised to infect dogs. A nested polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay was developed for the detection and differentiation of each of the piroplasm species currently known to infect dogs on the basis of the 18S ribosomal RNA gene. The assay can potentially amplify and discriminate between Theileria annae, Theileria equi, Babesia conradae, Babesia gibsoni, Babesia sp. (Coco) and each of the Babesia canis subspecies. Non-canine piroplasm species can also potentially be detected using the described assay, however amplification of Neospora caninum was also observed. The PCR was found to have a high detection limit, capable of detecting a 2.7×10−7% parasitaemia or the equivalent of 1.2 molecules of target DNA when using DNA extracted from whole EDTA blood and detected a parasitaemia of 2.7×10−5% using blood applied to both Flinders Technology Associates (FTA®) cards and IsoCode™ Stix. The application of blood samples to filter paper may greatly assist in piroplasm identification in regions of the world where local technologies for molecular characterisation are limited. The assay reported here has the potential to be standardised for routine screening of dogs for piroplasmosis.

Southeast Asia remains one of the few relatively uncharted regions of the world with respect to parasitic diseases of companion animals. The combination of tropical climate, large populations of stray dogs and cats, and increasing popularity of pet ownership in Southeast Asia provides an ideal environment for vector-borne disease transmission. Early reports suggested that arthropod-transmitted infections associated with protozoa, filariae, rickettsiae and bacteria are prevalent in the region. However, more recent investigation into the epidemiology and zoonotic potential of these pathogens has been neglected, and further study is essential if we are to develop a better understanding of their diversity and significance in both dogs and cats.

The haemoprotozoan Babesia canis has been recognized in Australia for many years, and a second, smaller species has recently been discovered. Amplification and sequencing of a partial region of the 18S small subunit ribosomal RNA (rRNA) gene enabled detection and characterization of the large and small canine babesiae of Australia for the first time. Isolates from northern Australia were genetically characterized to be 99% homologous to Babesia canis vogeli, confirming previous speculation about the subspecies of B. canis endemic to Australia. The partial 18S rRNA gene sequence amplified from isolates obtained in southeastern Australia was genetically identical to Babesia gibsoni, a species not previously known in Australia. The polymerase chain reaction (PCR) used was shown to be specific to Babesia and had a high sensitivity, detecting DNA at a parasitemia of approximately 0.0000027%. This study also reports the first known detection and characterization of B. canis DNA in Rhipicephalus sanguineus ticks using PCR.