Annetta Zintl

Zoonotic babesiosis: Overview of the disease and novel aspects of pathogen identity

Babesiosis is a zoonosis caused by tick-transmitted intraerythrocytic protozoa of the Phylum Apicomplexa. The disease mostly occurs in the USA, but cases have also been reported in several European countries, in Egypt, India, Japan, Korea, Taiwan, and South Africa. The main pathological event is lysis of erythrocytes resulting in haemolytic anaemia, which in severe cases may lead to organ failure and death, particularly in immunocompromised patients. The 2 groups of parasites involved, Babesia microti-like and Babesia sensu stricto (s.s.) species, differ in their life cycle characteristics and susceptibility to antibabesial drugs. Molecular taxonomy is now making a major contribution to the identification of novel pathogens within both groups. Effective treatment of severe cases was initially hampered by the lack of specific antibabesial drugs for human use, but increased use of supportive measures and of the recently developed antimalarial, atovaquone, particularly in combination with azithromycin, has improved the prospects for management of acute disease especially when caused by Babesia s.s. species. Prevention should be based primarily on increasing the awareness of physicians and the public to the risks, but infection from blood transfusions is particularly difficult to prevent. Expanding deer populations, resulting in wider distribution and greater abundance of ticks, heightened medical awareness, and growing numbers of immunocompromised patients are likely to result in a continuing rise of reported cases.

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.

Possible mechanisms underlying age-related resistance to bovine babesiosis.

Calves infected with the tick‐borne parasites Babesia spp. do not develop severe clinical babesiosis. Instead they display persistent low parasitaemias without any apparent ill‐effects. This age‐related resistance not only benefits the host, but also furthers parasite transmission. Both calves and adult animals respond with a Th I immune response to primary infection. Here we hypothesize that the difference in the outcome of infection may at least partly be explained by the localization and timing of the inflammatory response: in calves NO production occurs early and appears to be concentrated in the spleen. On the other hand, there is evidence that a delayed and systemic inflammatory response occurs in adult animals that is ineffectual and probably contributes to the pathogenesis. An improved understanding of the possible mechanisms that underlie this phenomenon may lead to new approaches for the treatment and immune prophylaxis of the disease.

Babesias of red deer (Cervus elaphus) in Ireland

Blood samples were obtained from 38 wild red deer (Cervus elaphus) at two sites in Ireland and subjected to PCR analysis of the 18S rRNA gene followed by sequencing. Two fragments of the 18S rRNA gene were generated by two different PCR protocols and subsequent sequencing suggested that at least six of the deer were infected by a babesia that, in those loci, is indistinguishable from Babesia divergens, an important tick-borne pathogen of cattle and of zoonotic significance. Additionally, a B. odocoilei-like parasite was detected in three samples and a babesia that did not match any sequences in the GenBank database was found in five samples. Neither B. capreoli nor B. venatorum (EU1) were found. There have been several reports of B. divergens occurring in deer species, including red deer, roe deer (Capreolus capreolus) and reindeer (Rangifer tarandus). However, in view of recent re-sequencing of bovine-origin samples deposited previously in GenBank, it is unlikely that any of these sequences from deer are B. divergens. The present study describes the only deer piroplasm detected so far that shows complete identity with B. divergens, in just over half of the 18S rRNA gene. The entire gene of this deer parasite should be analysed and transmission experiments undertaken before the infectivity of B. divergens for red deer can be confirmed.

Bulk milk ELISA and the diagnosis of parasite infections in dairy herds: a review

The bulk milk enzyme-linked immune sorbent assay (ELISA) is a rapid and inexpensive method of assessing herd exposure to pathogens that is increasingly being used for the diagnosis of parasite infections in dairy herds. In this paper, with the dairy herd health veterinarian in mind, we review the principles of the assay and the recent literature on the potential role of bulk milk ELISA for the diagnosis of ostertagiosis, fasciolosis, parasitic bronchitis due to cattle lung worm and neosporosis. It is generally accepted that assay results reflect exposure to the parasite rather than the presence of active infection. Bulk milk ELISA can be a useful tool for the veterinary practitioner as a component of a herd health monitoring programme or in the context of a herd health investigation. It can also play a role in regional or national surveillance programmes. However, the results need to be interpreted within the context of the herd-specific health management, the milk production pattern and the parasite life cycle.

Bovine paramphistomes in Ireland

Paramphistome infections have been associated with significant morbidity, caused chiefly by the activity of juvenile flukes in the intestine of the ruminant final host. Most cases have been reported in tropical and sub-tropical areas. However, recent reports of an apparent increase in the incidence of rumen fluke and its geographical range in Europe have renewed interest in a parasite previously thought to be of little significance in temperate regions. Moreover, the identity of rumen flukes present in the British Isles is currently being revised. As a result, work is underway throughout Europe to review and re-assess the clinical and economic significance of rumen flukes. During the present study, historical diagnostic laboratory records were interrogated for recent changes in the incidence of rumen fluke in Ireland. Three cattle herds were monitored for the presence of paramphistome eggs using coprological analysis over a period of 2 months (in the case of a group of housed steers) and 14 months (in the case of two extensively operated farms), respectively. Adult rumen fluke collected following slaughter were weighed and typed in two loci. We found that Calicophoron daubneyi is the most common if not only paramphistome species present in Ireland and that infections in cattle are now much more prevalent than was the case five or six years ago. The pylogenetic relationship of our isolates to the only published sequence and to C. daubneyi isolates from Northern Ireland was analysed. Genetic heterogeneity was similar all over the island and comparable to that of Fasciola hepatica, a fact that may have implications for the parasites ability to develop resistance to the very limited number of drugs currently available for treatment. The same haplotypes predominated throughout the island. Although the clinical significance of C. daubneyi is still uncertain, considering the apparent pervasiveness of the parasite, rumen fluke should be considered a differential diagnosis when treating scour or ill-thrift in young calves, and goats and sheep of any age.

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.

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.

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 parasites 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 Giemsas 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.