Presence and identity of Babesia microti in Ireland.

Babesia microti is a tick-transmitted protozoan parasite of wildlife that can also cause serious disease in humans. It is now well established that B. microti represents an assemblage of different strains or species, only some of which are important zoonotic pathogens. Therefore, in order to assess the potential public health risk associated with B. microti in any given location, it is important to determine the strains that are present. This is the first study on the presence and identity of B. microti in Ireland. Overall, 314 wood mice (Apodemus sylvaticus), 243 bank voles (Myodes glareolus) and 634 questing Ixodes ricinus nymphs collected in various locations across Ireland were screened for the presence of B. microti by metabarcoding and nested PCR, respectively. Overall 8 rodent spleen samples (1.4%) were positive for B. microti, while all tick samples tested negative. Rodent isolates were identified as the 'Munich' strain which rarely causes human disease and is chiefly transmitted by the mouse tick, Ixodes trianguliceps. Together with reports from the UK these results suggest that B. microti does not represent a significant public health risk in Britain or Ireland.

Meta-analysis: A useful tool to assess infection prevalence and disease ecology of Borrelia burgdorferi sensu lato in nymphal ticks in North-Western Europe with recommendations for a standardised approach to future studies.

Lyme borreliosis is a vector-borne disease of concern in Europe. While neuroborreliosis data are reportable at EU level, it can nevertheless be difficult to make comparisons of disease risk between neighbouring countries. This study used proportion meta-analyses to compare environmental markers of disease risk between woodland sites in two countries in North-Western Europe (Ireland, Scotland). 73 site-visits from 12 publications were analysed, resulting in a significantly higher pooled nymphal infection prevalence (NIP) in Ireland (8.2% (95% CI: 5.9-11.4%)) than Scotland (1.7%(95% CI 1.1-2.5%)). All other analysed parameters of disease risk were also higher in Ireland than Scotland. Subgroup-meta-analyses and meta-regressions were used to assess the influence of environmental variables on NIP. NIP increased significantly with increasing woodland size in Ireland, but not Scotland, which may be accounted for by Ireland's highly fragmented landscape. Assuming the application of strict inclusion/exclusion criteria and control of variables, proportion meta-analysis can provide useful insights in disease ecology, as it allows for the achievement of high study powers incorporating samples collected across multiple sites, which is otherwise often a prohibitively difficult and resource-heavy feat in environmental studies in disease ecology. A standardised approach to data collection is recommended to achieve more robust meta-analyses in future in conjunction with additional research on environmental factors affecting Lyme borreliosis risk in Europe, particularly pertaining to the impact of host species on NIP.

The prevalence and distribution of Anaplasma phagocytophilum genotypes in Ixodes ricinus nymphs collected from farm- and woodland sites in Ireland.

Despite the economic importance of grass-based livestock production in Ireland and the fact that many veterinarians and farmers regard tickborne fever (TBF) as an increasingly important disease, especially in sheep, little is currently known about the prevalence and genetic diversity of its causative agent, Anaplasma phagocytophilum. In the present study, 1376 nymphal Ixodes ricinus ticks collected from woodland, farmland, bog and limestone pavement habitats were screened for A. phagocytophilum using TaqMan PCR. Positive samples were further analysed by conventional nested PCR targeting the 16S rRNA, msp4 and groEL loci. Overall 4.5% I. ricinus nymphs were found to be infected. The genetic heterogeneity was comparable to that reported elsewhere in Europe, with greater genetic diversity of 16S variants in ticks collected from farmland than from woodland. All isolates belonged to groEL ecotype I indicating that rodents and birds do not contribute to the epidemiology of tickborne fever in Ireland. In the 16S and groEL loci, a number of the Irish isolates matched European sequences from humans, horses and dogs. The 16S sequences that were identical to human isolates from Europe also matched Ap-ha, the most common human pathogenic strain in the USA. Three isolates also matched published sequences from horses in the msp4 locus. No isolate matched human, equine and canine sequences in all 3 loci. Our results represent the first molecular characterization of Irish A. phagocytophilum isolates.

Comparison of three methods for the detection of Angiostrongylus vasorum in the final host.

Angiostrongylosis is potentially fatal parasitic nematode infection affecting dogs which can be difficult to diagnose. In recent years several microscopical, serological and molecular detection methods have been developed, however there are few studies that have compared the relative performance of these methods. Screening necropsy material from an opportunistic sample of 140 foxes (82 of which were considered to be infected with Angiostrongylus vasorum), indicated sensitivities of 84.1% for dissection and visual examination of plucks, 69.5% for nested PCR of an 18S rRNA fragment and 76.8% for a canine A. vasorum antigen detection test (IDEXX Angio Detect) of tissue fluid samples respectively. Agreement between the tests ranged from 45.6 to 79.7%. A novel nested PCR-RFLP for the detection and identification of canid lungworm spp. is described.

Geographical distribution of Angiostrongylus vasorum in foxes (Vulpes vulpes) in the Republic of Ireland.

The reported incidence of the metastrongylid nematode Angiostrongylus vasorum, that infects dogs and other canids, is increasing worldwide outside recognized endemic foci. This apparent expansion of the parasite's range is causing concern to veterinary clinicians as the disease caused in dogs can be life threatening and its treatment is not straightforward. The red fox is thought to be a reservoir host for dogs. To investigate the spatial distribution of infection in foxes in Ireland, the hearts and lungs of 542 foxes from all over Ireland were examined. The incidence of infection was found to be 39·9% [95% confidence interval (CI) 35·7-44·1] with positive samples occurring in each of the country's 26 counties. This report confirms that the parasite is endemic in Ireland and the overall prevalence is the second highest in Europe. This is the first survey of A. vasorum infection in Irish foxes and highlights the potential exposure of the Irish dog population to high risk of cross-infection. Additionally, Crenosoma vulpis was found in seven of the foxes, a parasite not previously reported in the Irish fox.

The worm turns: trematodes steering the course of co-infections.

A reductionist approach to the study of infection does not lend itself to an appraisal of the interactions that occur between 2 or more organisms that infect a host simultaneously. In reality, hosts are subject to multiple simultaneous influences from multiple pathogens along the spectrum from symbiotic microflora to virulent pathogen. In this review, we draw from our own work on Fasciola hepatica and that of others studying helminth co-infection to give examples of how such interactions can influence not only the outcome of infection but also its diagnosis and control. The new tools of systems biology, including both the "omics" approaches and mathematical biology, have significant promise in unraveling the as yet largely unexplored complexities of co-infection.

Longitudinal and spatial distribution of GP60 subtypes in human cryptosporidiosis cases in Ireland.

Within Europe, Ireland has one of the highest reported infection rates with the diarrhoeal protozoan pathogen Cryptosporidium. In this study 249 Cryptosporidium parvum isolates collected from Irish patients between 2000 and 2009 were subtyped by sequence analysis of the GP60 locus. A subsample of 127 isolates was also typed at the MS1 and ML1 loci. GP60 subtype IIaA18G3R1 was the predominant subtype in every year and every season throughout the country. Over the 10-year period there was no evidence that host immunity to the predominant subtype caused a shift in its prevalence. Length frequency distributions of the GP60 TCA/TCG repeats compiled from published data, showed distinct patterns for countries with predominantly zoonotic or anthroponotic transmission cycles, respectively. Although considered to be mostly affected by zoonotic cryptosporidiosis, the GP60 fragment length of Irish C. parvum isolates mirrored that of countries with predominantly human-to-human transmission, indicating more complex routes of infection between livestock and humans. Due to their homogeneity, ML1 and MS1 were not considered useful loci for subtyping C. parvum strains in Ireland.

Survival of Cryptosporidium parvum oocysts in the presence of hydrated lime.

To investigate the effects of hydrated lime on the survival of Cryptosporidium oocysts, the percentage viability of oocysts was assessed using fluorescent in situ hybridisation. In the absence of lime and with lime at a concentration of 1 per cent, there was a gradual decline in oocyst viability during the 10-day trial. Although the addition of 5 or 10 per cent lime caused the total number of oocysts to decrease, there appeared to be an increase in the proportion of potentially viable oocysts.

In vitro culture combined with quantitative TaqMan PCR for the assessment of Toxoplasma gondii tissue cyst viability.

Toxoplasma gondii is a serious food-borne pathogen with a worldwide distribution. In order to assess the risk of contracting toxoplasmosis from certain foods, many studies rely on the molecular detection of T. gondii DNA. However, determining the viability of parasites in positive samples is much more problematic. In this paper we describe a novel viability assay that relies on semi-quantitative comparison of the amount of parasite DNA present in samples used to infect host cell monolayers in vitro, and the amount of DNA detected in the same monolayers after 23 days incubation. Our assay is robust, easy to perform and interpret and offers a viable alternative to bioassays, for use in epidemiological studies, or the evaluation of specific food safety treatments.

The prevalence of Cryptosporidium species and subtypes in human faecal samples in Ireland.

Cryptosporidium is an important cause of diarrhoeal disease worldwide and, as several recent waterborne outbreaks have shown, poses a significant threat to public health in Ireland. We identified the Cryptosporidium spp. in 199 positive human stool samples by PCR-RFLP of the 18S rRNA and COWP gene loci. Subspecies were identified in 104 samples by sequence analysis of the 60 kDa glycoprotein (gp60) gene fragment. Overall C. parvum was identified in 80%, and C. hominis in 20% of cases. No other Cryptosporidium spp. were detected. C. parvum was by far the most common species in the rural, more sparsely populated west of Ireland and exhibited a pronounced spring peak coincident with a peak in the national cryptosporidiosis incidence rate. Our data indicated a trend towards higher proportions of C. hominis in older age groups. Ninety-nine per cent of all subtyped C. parvum isolates belonged to allele family IIa, of which allele IIaA18G3R1 was by far the most common (63%). According to a recent study by Thompson and colleagues [Parasitology Research (2007), 100, 619-624] this allele is also the most common in Irish cattle. Subtyping of the C. hominis isolates indicated that they belonged to a geographically widely distributed allele (IbA10G2) known to have caused several water- and foodborne outbreaks around the world. The predominance of C. parvum, its geographic and seasonal distribution and the IIaA18G3R1 subtype underlines the importance of zoonotic Cryptosporidium transmission in Ireland.

Prevalence of Cryptosporidium species in intensively farmed pigs in Ireland.

Natural Cryptosporidium infections in pigs are widespread but generally apathogenic. This study was undertaken to determine the prevalence of zoonotic Cryptosporidium spp. in piggeries in Ireland, where the drinking water supply is particularly vulnerable to contamination with zoonotic species. Overall, infections were detected in 39 out of 342 animals (11.4%), with highest infection rates among weaners (15%) and sows (13.3%). Twenty-nine positive samples were genotyped based on SSU rRNA sequence analysis. Infections with Cryptosporidium parvum, the most important zoonotic species were rare and are likely to be of greater concern to animal handlers than suppliers of drinking water. In addition to C. parvum, Cryptosporidium suis, Cryptosporidium pig genotype II, Cryptosporidium muris and a previously undescribed genotype were identified. ABI-profiles indicated the presence of different alleles in at least 40% of all genotyped isolates. This was confirmed in 3 isolates by cloning of the PCR products. Since chronic mixed infections appear to be quite common in pigs they could be considered as models for mixed infections in immunocompromised humans.

Babesia divergens (Phylum Apicomplexa) in vitro growth in the presence of calf serum.

Resistance to severe babesiosis in young calves has frequently been ascribed to an unknown serum factor(s) which inhibits growth of Babesia bovis in vitro. Our experiments show that young calf sera are as suitable as adult bovine sera for the in vitro culture of Babesia divergens, indicating that in this species at least inverse age resistance is due to alternative mechanisms. The suitability of commercial foetal calf sera for B. divergens cultures seems highly variable.

Chymotrypsin and neuraminidase treatment inhibits host cell invasion by Babesia divergens (Phylum Apicomplexa).

The process of host cell invasion by Babesia divergens is poorly understood and improved knowledge of the mechanism involved could lead to development of measures effective in disease prevention. The investigate parasite ligands on the erythrocyte surface, B. divergens cultures in bovine erythrocytes were transferred into enzyme-treated bovine, human, ovine and equine erythrocytes. Parasite invasion of bovine erythrocytes was not affected by trypsin treatment while treatment with alpha-chymotrypsin led to a reduction in parasite growth of 20-40%. Treatment of bovine and non-bovine erythrocytes with neuraminidase decreased their susceptibility to invasion by up to 97% implicating sialic acid as an important erythrocyte ligand for babesia, but the addition of either bovine or human N-acetylneuraminyl-lactose to B. divergens cultures in bovine erythrocytes had no inhibitory effect.

Invasion, and short- and long-term survival of Babesia divergens (Phylum Apicomplexa) cultures in non-bovine sera and erythrocytes.

In order to explore the feasibility of producing a Babesia divergens live vaccine free of bovine material contaminants the parasite's ability to grow in human, sheep and horse erythrocytes and serum and serum-free medium was investigated. B. divergens was successfully maintained in bovine erythrocytes overlaid with serum-free HL-1 medium. Supplementation of the culture medium with bovine or sheep serum improved parasite growth (monitored by measuring parasitaemia and uptake of tritiated hypoxanthine) whereas horse and human sera reduced parasite growth. As assessed by Giemsa's stained and FITC-labelled blood smears, the parasite invaded all erythrocyte types. Polyparasitism was less common in sheep and horse erythrocytes than in bovine and human erythrocytes. Accole stages were observed in bovine, human and sheep but not in horse erythrocytes. Proliferation following invasion was higher in human but lower in horse and sheep erythrocytes compared with bovine erythrocytes. Long-term cultures of B. divergens reached similar peak parasitaemias in human, sheep and bovine erythrocytes. Attempts to establish long-term cultures in horse erythrocytes failed. These results suggest that B. divergens is not host specific at the level of host cell attachment and invasion. Instead, parasite survival appears to be decided once the organism has gained access into the cell.

Experimental infections of farmed eels with different Trypanosoma granulosum life-cycle stages and investigation of pleomorphism.

Trypanosoma granulosum, a flagellate protozoon commonly found in the blood of the European eel Anguilla anguilla, was injected experimentally into uninfected eels purchased from a local farm. In order to investigate the infectivity of different stages in the life cycle, trypanosomes from various sources were used for inoculation. Infectivity was greatly reduced in in vitro culture stages inoculated at 20 C. Isolated bloodstream stages injected into groups of animals held at 12 and 20 C could be detected for over 70 days but did not appear to multiply. Naturally infected Hemiclepsis marginata, a piscivorous leech known to serve as vector, produced detectable, single-peak infections in eels held at 20 C. Infections were characterized by a prepatent stage and a phase of rising parasitemia. Peak infection intensities ranged between 1 and 7 x 10(4) trypanosomes/ml. Trypanosomes in the bloodstream of eels experimentally infected with leeches, divided at a very low rate during the early stages of infection. Small morphs present during the early phase of rising parasitemia were gradually replaced by larger trypanosomes. The overall length frequency distribution of trypanosomes was unimodal.