Search

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% Iambs 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. [ABSTRACT FROM AUTHOR]

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

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 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.

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.

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.

The present study describes the complete in vitro development of Cryptosporidium parvum (cattle genotype) in RPMI-1640 maintenance medium devoid of host cells. This represents the first report in which Cryptosporidium is shown to multiply, develop and complete its life cycle without the need for host cells. Furthermore, cultivation of Cryptosporidium in diphasic medium consisting of a coagulated new born calf serum base overlaid with maintenance medium greatly increased the total number of Cryptosporidium stages. Type I and II meronts were detected giving rise to two morphologically different merozoites. Type I meronts, which appear as grape-like clusters as early as 48 h post culture inoculation, release merozoites, which are actively motile, and circular to oval in shape. Type II meronts group in a rosette-like pattern and could not be detected until day 3 of culturing. Most of the merozoites released from type II meronts are generally spindle-shaped with pointed ends, while others are rounded or pleomorphic. In contrast to type I, merozoites from type II meronts are less active and larger in size. Sexual stages (micro and macrogamonts) were observed within 6-7 days of culturing. Microgamonts were darker than macrogamonts, with developing microgametes, which could be seen accumulating at the periphery. Macrogamonts have a characteristic peripheral nucleus and smooth outer surface. Oocysts at different levels of sporulation were seen 8 days post culture inoculation. Cultures were terminated after 4 months when the C. parvum life cycle was still being perpetuated with the presence of large numbers of excysting and intact oocysts. Culture-derived oocysts obtained after 46 days p.i. were infective to 7- to 8-day-old ARC/Swiss mice. The impact of C. parvum developing in cell-free culture is very significant and will facilitate many aspects of Cryptosporidium research.

Cryptosporidiosis is an enteric disease of animals and humans that can be fatal in immunocompromised individuals. There is no known effective treatment for cryptosporidiosis. Bilbies are threatened marsupials and are bred in captivity as part of a recovery program to re-introduce this species to the southwest of Western Australia. Cryptosporidium muris infection was detected in the faeces of bilbies at a captive breeding colony. Stress associated with a high density of bilbies in enclosures may have predisposed some of the bilbies to infection with C. muris. C. muris has been described in mice and was found in the faeces of one mouse trapped in the breeding enclosures. It is likely the bilbies acquired the infection from mice by faecal contamination of food and water. The infection cleared within 2 months from some bilbies, however others remained infected for 6 months and treatment was attempted with dimetridazole. Subsequently the parasite was no longer be detectable in the faeces.

A longitudinal survey of gastro-intestinal parasites was conducted over a 3-year period in remote communities in the north-west of Western Australia where, based on diagnosis by microscopy of faecal samples, Rodentolepis (=Hymenolepis) nana was found to be the most common enteric parasite. In the present study, using molecular tools, we describe the unexpected discovery, of a mixed infection with a second hymenolepidid species, Rodentolepis (=Hymenolepis) microstoma in four of the surveyed individuals. In the absence of any reliable earlier reports we believe this is to be the first instance of the detection of R. microstoma from human hosts. The development of a diagnostic restriction fragment polymorphism has enabled the study of R. microstoma in human populations and will greatly facilitate a more thorough understanding of the epidemiology of this parasite in the future.

The present study describes the complete development of all life cycle stages of Cryptosporidium andersoni in the HCT-8 cell line. The in vitro cultivation protocols were the same as those used for the successful growth of all life cycle stages of Cryptosporidium parvum (Int. J. Parasitol. 31 (2001) 1048). Under these culture conditions, C. andersoni grew and proliferated rapidly with the completion of the entire life cycle within 72h post-infection. The developmental stages of C. andersoni are larger than those of C. parvum enabling easier identification of life cycle stages including a previously unrecognised extracellular stage. The presence of this extracellular stage was further confirmed following its isolation from the faeces of infected cattle using a laser microdissection technique. This stage was present in large numbers and some of them were seen undergoing syzgy. Extraction of DNA from the extracellular stage, followed by polymerase chain reaction-restriction fragment length polymorphism and sequencing of the 18S rDNA confirmed that this is a stage in the life cycle of C. andersoni. In vitro, extracellular stages were always observed moving over the HCT-8 cells infected with C. andersoni. Comparative observations with C. parvum also confirmed the presence of extracellular stages. Extracellular stages were recovered from in vitro culture after 5 days post-infection with the cattle genotype of C. parvum and from infected mice. At least two morphologically different stages (stages one and two) were purified from mice after 72h of infection. The presence and morphological characterisation of extracellular developmental stages in the life cycle of Cryptosporidium confirms its relationship to gregarines and provides important implications for our understanding of the taxonomic and phylogenetic affinities of the genus Cryptosporidium. The growth of C. andersoni in cell culture now provides a means of studying its development, metabolism, and behaviour as well as testing its response to different therapeutic agents.

Small intraerythrocytic parasites were observed in the blood of three related male American Pit Bull Terriers. Two of the dogs, both less than 1-year-old, were anaemic at the time of initial examination and the third, an adult and sire of the two younger dogs, had a normal haemogram and low parasitaemia. The morphological appearance of the erythrocyte inclusions, analysis of a 450-bp region of the 18S rRNA gene and antibody titres provided evidence that this parasite was Babesia gibsoni, a species not previously reported in Australia.

Iran is an important endemic focus of cystic hydatid disease (CHD) where several species of intermediate host are commonly infected with Echinococcus granulosus. Isolates of E. granulosus were collected from humans and other animals from different geographical areas of Iran and characterized using both DNA (PCR-RFLP of ITS1) and morphological criteria (metacestode rostellar hook dimensions). The sheep and camel strains/genotypes were shown to occur in Iran. The sheep strain was shown to be the most common genotype of E. granulosus affecting sheep, cattle, goats and occasionally camels. The majority of camels were infected with the camel genotype as were 3 of 33 human cases. This is the first time that cases of CHD in humans have been identified in an area where a transmission cycle for the camel genotype exists. In addition, the camel genotype was found to cause infection in both sheep and cattle. Results also demonstrated that both sheep and camel strains can be readily differentiated on the basis of hook morphology alone.

We have cloned and sequenced the ribosomal internal transcribed spacer-1 (ITS1) of Cryptosporidium wrairi. Phylogenetic analysis of this region provided further support to the validity of C. wrairi as a distinct species and also to the concept that many of the genotypes recently identified within C. parvum are in fact separate species. Analysis placed the "cattle" and "mouse" genotypes of C. parvum as each other's closest relatives and C. wrairi as a sister group to these two genotypes, followed by the "human" genotype.