Morphological and molecular characterization of Cystoisospora laidlawi oocysts (Apicomplexa: Eimeriidae) in farmed American mink (Neovison vison) in Denmark.

From a longitudinal survey conducted on 30 Danish mink farms in 2016, 11.0% of faecal samples (456/4140) were positive for Cystoisospora laidlawi oocysts by microscopy, with 60% (189/315) of mink being positive at least once during the study period. Morphological analysis of sporulated oocysts identified Cystoisospora oocysts measuring 34.3 × 29.5 µm with an oocyst length/width (L/W) ratio of 1.2. The morphological features of the oocysts were identical to Isospora laidlawi previously morphological identified in farmed mink from Denmark and elsewhere. Phylogenetic analysis of 18S rDNA sequences (1221 bp) from three positive mink indicated that Cystoisospora from mink shared the highest genetic similarity to C. canis from a Canadian dog (99.6%). The phylogenetic analysis placed Cystoisospora from mink in a clade with other Cystoisospora isolates.

Morphological and molecular characterisation of Eimeria vison-like oocysts (Apicomplexa:Eimeriidae) in farmed mink (Neovison vison) in Denmark.

A survey was conducted on 30 Danish mink farms from April to October 2016 to determine the prevalence and species of Eimeria in Danish farmed mink. In total, 2.6% of mink faecal samples (108/4140) were positive for Eimeria vison-like oocysts by microscopy, with 24.8% (78/315) of mink being positive at least once during the study period. Morphological analysis of sporulated oocysts (n = 20) identified Eimeria vison-like oocysts measuring 21.0 × 13.8 µm with a length/width (L/W) ratio of 1.5. Phylogenetic analysis of 18S rRNA sequences (1221 bp) from three positive mink indicated that Eimeria vison-like shared the highest genetic similarity to Eimeria sp. ex Apodemus agrarius from a Striped field mouse (A. agrarius) from the Czech Republic (99.6%). Analysis of a shorter region of 18S (531 bp) revealed that the E. vison-like genotype sequences grouped in the same clade and shared 97.7% similarity with E. furonis. At the cytochrome c oxidase subunit I (COI) locus, mink-derived sequences were not available from GenBank and phylogenetic analysis placed the novel E. vison-like in a clade with E. cf. ictidea (99.4% similarity) from a black footed ferret (Mustela nigripes) from Canada.

Target validation of the inosine monophosphate dehydrogenase (IMPDH) gene in Cryptosporidium using Phylomer(®) peptides.

Cryptosporidiosis, 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 nitazoxanide, only partly efficacious in immunocompetent 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 Cryptosporidium parvum (IMPcp) and Cryptosporidium 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.

Cryptosporidium species in sheep and goats from Papua New Guinea.

Species of Cryptosporidium are extensively recognised as pathogens of domesticated livestock and poultry, companion animals, wildlife, and are a threat to public health. Little is known of the prevalence of Cryptosporidium spp. in humans, domesticated animals or wildlife in Papua New Guinea (PNG). The aim of the present study was to screen sheep and goats for Cryptosporidium using molecular tools. A total of 504 faecal samples were collected from sheep (n=276) and goats (n=228) in village, government and institutional farms in PNG. Samples were screened by nested PCR and genotyped at the 18S rRNA and at the 60kDa glycoprotein (gp60) loci. The overall prevalences were 2.2% for sheep (6/278) and 4.4% (10/228) for goats. The species/genotypes identified were Cryptosporidium hominis (subtype IdA15G1) in goats (n=6), Cryptosporidium parvum (subtypes IIaA15G2R1and IIaA19G4R1) in sheep (n=4) and in goats (n=2), Cryptosporidium andersoni (n=1) and Cryptosporidium scrofarum (n=1) in sheep, Cryptosporidium xiao (n=1) and Cryptosporidium rat genotype II (n=1) in goats. This is the first report of Cryptosporidium spp. identified in sheep and goats in PNG. Identification of Cryptosporidium in livestock warrants better care of farm animals to avoid contamination and illness in vulnerable population. The detection of zoonotic Cryptosporidium in livestock suggests these animals may serve as reservoirs for human infection.

Further characterisation of two Eimeria species (Eimeria quokka and Eimeria setonicis) in quokkas (Setonix brachyurus).

The identification and characterisation of novel Eimeria species has largely been based on sporulated oocyst and sporocyst morphology, the host species and the geographical range. Variation in the size and shape of Eimeria oocysts across their host range however, make the identification and characterisation of novel species using traditional methodologies alone problematic. The use of molecular markers and phylogenetic analysis has greatly advanced our ability to characterise Eimeria species and has recently been applied to understand evolutionary relationships among Eimeria species from Australian marsupials. In the present study, Eimeria species isolated from quokkas (Setonix brachyurus) captured from Two Peoples Bay, Bald Island and Rottnest Island, Western Australia, were morphologically identified as Eimeria quokka and Eimeria setonicis. Both Eimeria species were identified as being polymorphic in nature with regards to sporulated oocyst and sporocyst morphometrics. Phylogenetic analysis using 18S rRNA and COI (cytochrome c oxidase subunit 1) genes, grouped E. quokka and E. setonicis within the Eimeria marsupial clade together with Eimeria trichosuri from brushtail possums, Eimeria macropodis from tammar wallabies (Macropus eugenii) and several unidentified macropod Eimeria species from western grey kangaroos (Macropus fuliginosus). This study is the first to characterise E. quokka and E. setonicis by molecular analysis, enabling more extensive resolution of evolutionary relationships among marsupial-derived Eimeria species.

Identification of novel and zoonotic Cryptosporidium species in fish from Papua New Guinea.

There is still limited information on the distribution of Cryptosporidium species and genotypes in fish. The present study investigated the prevalence of Cryptosporidium species in cultured freshwater (n=132), wild freshwater (n=206) and wild marine (n=276) fish in Papua New Guinea (PNG) by PCR screening at the 18S rRNA locus. A total of seven fish (2 cultured freshwater, 1 wild freshwater and 4 wild marine fish) were identified as positive for Cryptosporidium. Specifically, Cryptosporidium was found in four different host species (Nile tilapia, Oreochromis niloticus; silver barb, Puntius gonionotus; mackerel scad, Decapterus maracellus and oblong silver biddy, Gerres oblongus), giving an overall prevalence of 1.14% (95% CI: 0.3-2%, n=7/614). Of the seven positive isolates, five were identified as C. parvum and two were a novel piscine genotype, which we have named piscine genotype 8. Piscine genotype 8 was identified in two marine oblong silver biddies and exhibited 4.3% genetic distance from piscine genotype 3 at the 18S locus. Further subtyping of C. parvum isolates at the 60 kDa glycoprotein (gp60) locus identified 3 C. parvum subtypes (IIaA14G2R1, IIaA15G2R1 and IIaA19G4R1) all of which are zoonotic and a C. hominis subtype (IdA15G1). The zoonotic Cryptosporidium were identified in fish samples from all three groups; cultured and wild freshwater and wild marine fish. Detection of Cryptosporidium among aquaculture fingerlings warrants further research to gain a better understanding of the epidemiology of Cryptosporidium infection in cultured fish. The identification of zoonotic Cryptosporidium genotypes in fish from PNG has important public health implications and should be investigated further.

Additional novel Cryptosporidium genotypes in ornamental fishes.

Current knowledge on the prevalence and genotypes of Cryptosporidium in fishes is still limited. This study investigated the prevalence of Cryptosporidium species in 171 ornamental fishes, belonging to 33 species, collected from 8 commercial aquariums around Perth, Western Australia. All samples were screened by nested PCR targeting the 18S rRNA locus. A total of 6 positives were identified by PCR at the 18S locus from 4 different species of fishes (red eye tetra, Moenkhausia sanctaefilomenae; gold gourami, Trichogaster trichopterus; neon tetra, Paracheirodon innesi; goldfish, Carassius auratus auratus), giving an overall prevalence of 3.5% (6/171). Four different genotypes were identified, only one of which has been previously reported in fish; piscine genotype 4 in a neon tetra isolate, a rat genotype III-like isolate in a goldfish, a novel genotype in three isolates from red eye (piscine genotype 7) which exhibited a 3.5% genetic distance from piscine genotype 1 and a piscine genotype 6-like from a gold gourami (1% genetic distance). Further biological and genetic characterisation is required to determine the relationship of these genotypes to established species and strains of Cryptosporidium.

Microsatellite typing and population structuring of Trypanosoma evansi in Mindanao, Philippines.

Trypanosoma evansi, a blood-borne protozoan parasite with an extensive geographical range is the causative agent of the livestock disease known as surra. A total of 140 out of 179 T. evansi isolates collected between 2006 and 2007 from 44 villages (comprising of 16 reported surra outbreaks) in 3 provinces (Agusan del Sur (ADS), Surigao del Sur (SDS) and Agusan del Norte (ADN)) in Mindanao, Philippines were each successfully genotyped using a suite of 7 polymorphic microsatellites. The study identified 16 multi locus genotypes (MLG) within the T. evansi isolates and evidence of the spread of surra outbreaks from one village to another, most likely due to the movement of infected animals. Genotyping provided evidence of population sub-structuring with 3 populations (I, II and III (only 1 isolate)) identified. The most abundant population was II, which was the predominant population in ADS and SDS (p=0.022). In addition, buffalo mortality was statistically higher in outbreak areas associated with isolates from population I (13.6%) than with isolates from population II (6.9%) (p=0.047). The present study has highlighted the utility of microsatellite loci to improve understanding of the epidemiology of T. evansi and in tracking surra outbreaks.

Prevalence and on-farm risk factors for diarrhoea in meat lamb flocks in Western Australia.

Diarrhoea is a widespread problem for sheep enterprises worldwide. A cross-sectional epidemiological study was conducted using a questionnaire to determine the prevalence of diarrhoea and associated risk factors where there was evidence of recent diarrhoea (active diarrhoea or fresh faecal soiling of breech fleece) for meat lambs on farms in southern Western Australia during 2010. The response rate was 41.4% (139/336). Evidence of recent diarrhoea was reported on 64.8% of farms, with a mean of 6.9% lambs affected per farm. Location of a farm and a higher annual rainfall were associated with an increased diarrhoea prevalence. Binary logistic regression analysis suggested that the drinking water source was associated with the incidence of diarrhoea, since lamb flocks supplied with dam water were 117 times (95% CI: 18.2, 754.8) more likely to have observed diarrhoea or fresh breech fleece faecal soiling than lamb flocks supplied with other sources of water. Faecal worm egg counts were used by 65% of respondents to determine whether an anthelmintic treatment was warranted and 74% of respondents administered a treatment to their meat lambs. In response to a range of diarrhoea scenarios presented to respondents (5%, 25% and 50% of the flock with evidence of recent diarrhoea), 15.1% would have elected to administer an anthelmintic treatment regardless of differences in prevalence.

Longitudinal investigation of protozoan parasites in meat lamb farms in southern Western Australia.

In this study, 96 faecal samples were collected from pregnant Merino ewes, at two broad-acre, commercial sheep farms in southern Western Australia, on two separate occasions (16 and 2 weeks prior to lambing). Following lambing, 111 (Farm A) and 124 (Farm B) female crossbred lambs (2-6 weeks old), were individually identified using ear tags (a numbered tag and a radio-frequency tag). A total of 1155 faecal samples were collected only from these individually identified lambs on five separate sampling occasions. All samples were screened using PCR to detect Cryptosporidium (18S rRNA and actin loci) and Giardia duodenalis (glutamate dehydrogenase and triosephosphate isomerise loci). The overall prevalences (lambs positive for a parasite on at least one of the five samplings) at Farm A and B were 81.3% and 71.4%, respectively for Cryptosporidium and similarly 67.3% and 60.5% for Giardia, respectively. Cryptosporidium and Giardia prevalences at individual samplings ranged between 18.5 and 42.6% in lambs and were <10% in the ewes. Cryptosporidium xiaoi was the most prevalent species detected at all five samplings and was also isolated from lamb dam water on Farm B. Cryptosporidium ubiquitum was most commonly detected in younger lambs and Cryptosporidium parvum was detected in lambs at all five samplings, typically in older lambs and as part of a mixed species infection with C. xiaoi. A novel, possibly new genotype (sheep genotype I), was identified in six Cryptosporidium isolates from Farm B. Giardia duodenalis assemblage E was the most common genotype detected at all five samplings, with greater proportions of assemblage A and mixed assemblage A and E infections identified in older lambs. This longitudinal study identified high overall prevalences of Cryptosporidium and Giardia in lambs grazed extensively on pastures, while reinforcing that sampling a random selection of animals from a flock/herd on one occasion (point prevalence), underestimates the overall prevalence of these parasites in the flock/herd across an extended time period. Based on these findings, grazing lambs were identified as a low risk source of zoonotic Cryptosporidium and Giardia species/genotypes, with these protozoa detected at all five samplings in some lambs, indicating that these individuals were either unable to clear the naturally acquired protozoan infections or were repeatedly re-infected from their environment or other flock members.

Cryptosporidium and Giardia associated with reduced lamb carcase productivity.

On two extensive sheep farms in southern Western Australia, 111 (Farm A) and 124 (Farm B) female crossbred lambs (2-6 weeks old) were randomly selected and individually identified using ear tags (a numbered tag and radio-frequency tag) at marking. On five separate occasions, faecal samples were collected and live weight, body condition score (BCS), faecal consistency score (FCS), breech fleece faecal soiling score and faecal dry matter percentage (DM%) were recorded. Lamb hot carcase weight (HCW) and dressing percentage were measured at slaughter. Faecal samples were screened by PCR for Cryptosporidium (18S rRNA, actin and 60 kDa glycoprotein [gp60] loci), Giardia duodenalis (glutamate dehydrogenase [gdh] and triosephosphate isomerise [tpi]) and Campylobacter jejuni (16S rRNA). Observation of Eimeria oocysts and faecal worm egg counts (WECs) were performed using a modified McMaster technique. The WECs were adjusted for FCS for analyses. Faecal samples were screened for patent strongylid infections using PCR (specifically ITS-2 nuclear ribosomal DNA for Teladorsagia circumcincta, Trichostrongylus spp. and Haemonchus contortus). Lambs positive for Cryptosporidium at least once had lighter HCWs by 1.25 kg (6.6%) (P=0.029) and 1.46 kg (9.7%) (P<0.001) compared to lambs never positive for Cryptosporidium for Farms A and B respectively. Similarly, dressing percentages were 1.7% (P=0.022) and 1.9% (P<0.001) lower in Cryptosporidium-positive lambs on Farms A and B respectively. Lambs positive for Giardia at least once had 0.69 kg (P<0.001) lighter HCWs and 1.7% (P<0.001) lower dressing percentages compared to lambs never positive for Giardia on Farm B only. Cryptosporidium-positive lambs at the second sampling were 4.72 (P=0.010) and 3.84 (P=0.002) times more likely to have non-pelleted faeces compared to Cryptosporidium-negative lambs for Farms A and B respectively. Breech fleece faecal soiling scores of Cryptosporidium-positive lambs were 3.36 (P=0.026) and 2.96 (P=0.047) times more likely to be moderate to severe (scores 3-5), compared to negative lambs at the second sampling for Farms A and B respectively. Live weight, growth rate and BCS were inconsistently associated with protozoa detection across different samplings and farms. Adjusted WEC was correlated positively with FCS and negatively with faecal DM%, differing between sampling occasions and farms. Campylobacter jejuni prevalence was very low (<1%). Adjusted WEC were not correlated with carcase attributes, growth rates or live weights. This study is the first to quantify productivity consequences of naturally acquired protozoa infections in lambs managed under extensive farming conditions.

Vector of Trypanosoma copemani identified as Ixodes sp.

A total of 41 ticks were collected from 15 quokkas on Bald Island and 2 ticks from a Gilbert's potoroo from Two Peoples Bay. Three species of Ixodid ticks Ixodes australiensis, Ixodes hirsti and Ixodes myrmecobii were identified on the quokkas known to have a high prevalence of Trypanosoma copemani. Tick faeces from ticks isolated from 8 individual quokkas and a Gilbert's potoroo were examined with one identified as positive for trypanosomes. Faecal examination revealed trypanosomes similar to in vitro life-cycle stages of T. copemani. In total 12 ticks were dissected and trypanosomes found in sections of their midgut and haemolymph, 49 and 117 days after collection. Tick faeces, salivary glands and midguts from I. australiensis were screened using an 18S rRNA PCR with amplification seen only from the midguts. Sequencing showed 100% homology to T. copemani (genotype A) and 99.9% homology to the wombat (AII) isolate of T. copemani. Trypanosomes were only detected in I. australiensis as neither I. hirsti nor I. myrmecobii survived the initial 30-day storage conditions. We therefore identify a vector for T. copemani as I. australiensis and, given the detection of trypanosomes in the faeces, suggest that transmission is via the faecal-oral route.

The potential impact of native Australian trypanosome infections on the health of koalas (Phascolarctos cinereus).

Whole blood collected from koalas admitted to the Australian Zoo Wildlife Hospital (AZWH), Beerwah, QLd, Australia, during late 2006-2009 was tested using trypanosome species-specific 18S rDNA PCRs designed to amplify DNA from Trypanosoma irwini, T. gilletti and T. copemani. Clinical records for each koala sampled were reviewed and age, sex, blood packed cell volume (PCV), body condition, signs of illness, blood loss, trauma, chlamydiosis, bone marrow disease, koala AIDS and hospital admission outcome ('survival'/ 'non-survival') were correlated with PCR results. Overall 73.8% (439/595) of the koalas were infected with at least 1 species of trypanosome. Trypanosoma irwini was detected in 423/595 (71.1%), T. gilletti in 128/595 (21.5%) and T. copemani in 26/595 (4.4%) of koalas. Mixed infections were detected in 125/595 (21%) with co-infections of T. irwini and T. gilletti (101/595, 17%) being most common. There was a statistical association between infection with T. gilletti with lower PCV values and body condition scores in koalas with signs of chlamydiosis, bone marrow disease or koala AIDS. No association between T. gilletti infection and any indicator of health was observed in koalas without signs of concurrent disease. This raises the possibility that T. gilletti may be potentiating other disease syndromes affecting koalas.

Novel trypanosome Trypanosoma gilletti sp. (Euglenozoa: Trypanosomatidae) and the extension of the host range of Trypanosoma copemani to include the koala ( Phascolarctos cinereus).

Trypanosoma irwini was previously described from koalas and we now report the finding of a second novel species, T. gilletti, as well as the extension of the host range of Trypanosoma copemani to include koalas. Phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated that T. gilletti was genetically distinct with a genetic distance (± s.e.) at the 18S rDNA locus of 2.7 ± 0.5% from T. copemani (wombat). At the gGAPDH locus, the genetic distance (± s.e.) of T. gilletti was 8.7 ± 1.1% from T. copemani (wombat). Trypanosoma gilletti was detected using a nested trypanosome 18S rDNA PCR in 3/139 (∼2%) blood samples and in 2/29 (∼7%) spleen tissue samples from koalas whilst T. irwini was detected in 72/139 (∼52%) blood samples and T. copemani in 4/139 (∼3%) blood samples from koalas. In addition, naturally occurring mixed infections were noted in 2/139 (∼1.5%) of the koalas tested.

Transient transfection of Cryptosporidium parvum using green fluorescent protein (GFP) as a marker.

Cryptosporidium parvum is a protozoan parasite that infects a variety of mammals. The parasite has been shown to harbor a dsRNA virus (CPV) and in the present study, we have developed a CPV transient transfection system for this parasite by using green fluorescent protein (GFP) to replace the partial gene encoding region of the larger dsRNA (CPV-L) and the smaller dsRNA (CPV-S) virus. Two viral RNA-mediated transfection vectors: pCPVL-GFP and pCPVS-GFP were successfully constructed and both in vitro transcripts were electroporated into oocysts and sporozoites. Transient expression of GFP was detected in C. parvum oocysts and excysted sporozoites by fluorescence microscopy and by RT-PCR detection of GFP mRNA and antisense RNA in transfected C. parvum oocysts. Our study provides a new approach for studying gene expression and regulation in C. parvum and will hopefully lead to the construction of a stable CPV transfection system in the future.

Trypanosoma irwini n. sp (Sarcomastigophora: Trypanosomatidae) from the koala ( Phascolarctos cinereus).

The morphology and genetic characterization of a new species of trypanosome infecting koalas (Phascolarctos cinereus) are described. Morphological analysis of bloodstream forms and phylogenetic analysis at the 18S rDNA and gGAPDH loci demonstrated this trypanosome species to be genetically distinct and most similar to Trypanosoma bennetti, an avian trypanosome with a genetic distance of 0.9% at the 18S rDNA and 10.7% at the gGAPDH locus. The trypanosome was detected by 18S rDNA PCR in the blood samples of 26 out of 68 (38.2%) koalas studied. The aetiological role of trypanosomes in koala disease is currently poorly defined, although infection with these parasites has been associated with severe clinical signs in a number of koalas. Based on biological and genetic characterization data, this trypanosome species infecting koalas is proposed to be a new species Trypanosome irwini n. sp.

Blood, Bull Terriers and Babesiosis: further evidence for direct transmission of Babesia gibsoni in dogs.

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.

Babesia gibsoni: detection during experimental infections and after combined atovaquone and azithromycin therapy.

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.

PCR-RFLP for the detection and differentiation of the canine piroplasm species and its use with filter paper-based technologies.

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 2.7x10(-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 2.7x10(-5)% using blood applied to both Flinders Technology Associates (FTA) cards and IsoCodetrade mark 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.

In vitro isolation and characterisation of the first canine Neospora caninum isolate in Australia.

Neospora caninum was isolated and established in vitro from the skin lesion of a naturally infected dog. The identity of the parasite was evaluated by immunofluorescent antibody test (IFAT), microscopy, Western blotting and polymerase chain reaction (PCR). N. caninum DNA was detected in the whole blood, serum, skin lesion, rectal scrapings and faeces of the infected dog utilising a nested PCR targeting the Nc-5 gene of N. caninum. Antigenic and genetic characterisation of the isolate, designated WA-K9, at a number of loci including the Nc-5 gene, heat shock protein 70 (HSP-70) gene, alpha-tubulin and beta-tubulin genes revealed no variation between this isolate and two N. caninum isolates from different geographic areas. Clinical aspects of this case, which included cutaneous and neurological disease, are also discussed.