Ramon Marc Eichenberger

RNA Seq analysis of the Eimeria tenella gametocyte transcriptome reveals clues about the molecular basis for sexual reproduction and oocyst biogenesis

BackgroundThe protozoan Eimeria tenella is a common parasite of chickens, causing avian coccidiosis, a disease of on-going concern to agricultural industries. The high prevalence of E. tenella can be attributed to the resilient oocyst stage, which is transmitted between hosts in the environment. As in related Coccidia, development of the eimerian oocyst appears to be dependent on completion of the parasite’s sexual cycle. RNA Seq transcriptome profiling offers insights into the mechanisms governing the biology of E. tenella sexual stages (gametocytes) and the potential to identify targets for blocking parasite transmission.ResultsComparisons between the sequenced transcriptomes of E. tenella gametocytes and two asexual developmental stages, merozoites and sporozoites, revealed upregulated gametocyte transcription of 863 genes. Many of these genes code for proteins involved in coccidian sexual biology, such as oocyst wall biosynthesis and fertilisation, and some of these were characterised in more depth. Thus, macrogametocyte-specific expression and localisation was confirmed for two proteins destined for incorporation into the oocyst wall, as well as for a subtilisin protease and an oxidoreductase. Homologues of an oocyst wall protein and oxidoreductase were found in the related coccidian, Toxoplasma gondii, and shown to be macrogametocyte-specific. In addition, a microgametocyte gamete fusion protein, EtHAP2, was discovered.ConclusionsThe need for novel vaccine candidates capable of controlling coccidiosis is rising and this panel of gametocyte targets represents an invaluable resource for development of future strategies to interrupt parasite transmission, not just in Eimeria but in other Coccidia, including Toxoplasma, where transmission blocking is a relatively unexplored strategy.

Severe Taenia ovis outbreak in a sheep flock in south-west England

SMALL ruminants are the intermediate host of several canine tapeworm species, including Taenia ovis . In sheep, the larval stages of this cestode (previously called Cysticercus ovis ) manifest as cystic lesions. T ovis infection in sheep has been reported to be endemic in parts of Great Britain (Jones and Walters 1992, Green and others 1995), but up-to-date epidemiological data are scarce. The English sheep industry suffers annual economic losses of over £7 million due to the presence of T ovis cysts and subsequent condemnation of the carcases at the abattoir (English Beef and Lamb Executive [EBLEX] 2011). The adult stage of T ovis is found in the intestine of canids, particularly dogs, foxes and wolves. Sheep become infected with eggs on contaminated pastures, and the larval stages develop in skeletal and heart muscle. The parasitic cycle is completed when the definitive host ingests viable cysts. This short communication describes an extended severe outbreak of cysticercosis in a large sheep flock in Somerset, south-west England. In 2009, 7 per cent (600 of 9000) of the slaughtered sheep from a farm were rejected at abattoir inspection due to cysticercosis. In April 2010, lesions from six hearts of condemned carcases were analysed morphologically (Fig 1) and by a Taenia -specific PCR followed by sequence analysis (Trachsel and others 2007), resulting in the diagnosis of T ovis infection. Fig 1 Taenia ovis lesions …

Ticks on dogs and cats: a pet owner-based survey in a rural town in northeastern Switzerland.

Changes in the endemic foci of tick populations and invasions of tick species to new areas have become evident in Europe, leading to changes in the epidemiology of tick-transmitted diseases. However, data about tick infestations of pet animals are limited. Following the recent identification of a new focus of canine babesiosis in northeastern Switzerland, we investigated the occurrence of tick vectors in this region by using a pet owner-based sampling strategy. All dog owners in a rural town were sent postal requests to send ticks from their dogs and cats over two consecutive years, beginning in April 2012. In total 3003 ticks were submitted for identification from 249 dogs (approximately 20% of the resident dog population) and from 117 cats. Ixodes ricinus was the most abundant species identified in 96.8% (n=2124) and 74.3% (n=601) of the individual samples submitted from dogs and cats, respectively. Two other tick species, I. hexagonus and Dermacentor reticulatus, were recorded on both host species, with host infestation prevalences below 2%. On cats (but not on dogs), as many as 24.0% (n=194) of the specimens were identified as a fourth tick species, I. trianguliceps. Overall, 93.5% of the ticks were adults (93.8% and 93.0% in dogs and cats), 4.4% nymphs (5.7% in dogs and 1% in cats) and 2% larvae (0.5% and 6.0% in dogs and cats), respectively. The highest infestation intensity was 49 I. ricinus ticks from an individual dog. However, 55.6% of the submissions from dogs and 24.8% from cats contained only one tick. This survey demonstrated that pet owners can contribute to a cost-effective tick surveillance and identified a new tick focus of D. reticulatus. The finding of I. trianguliceps exclusively on cats might be related to behavioural traits of the cats or to a more readily detection of these very small ticks during petting by their owners.

Prognostic Markers in Acute Babesia canis Infections.

Background Canine babesiosis, caused by Babesia canis, is a prevalent and clinically relevant disease in Europe. Severe acute babesiosis is characterized by a high mortality but prognosis is not always correlated with clinical signs nor with the level of parasitemia. Objective This study evaluated prognostic markers associated with poor outcomes in acute Babesia canis infections. Animals and Methods We compared the results of routine laboratory profiles, hand‐held lactate and glucose analyzer, and the acute phase response in 2 groups of naturally infected dogs (7 survivors and 8 nonsurvivors). Samples were collected at the time of first admission and before any treatment. Subsequently, the course of prognostic markers was followed in 3 dogs experimentally inoculated with B. canis. Results Nonsurvivors showed significantly higher concentrations of lactate, triglycerides and phosphate and lower hematocrit, leukocyte counts, total serum protein concentrations, and thrombocyte counts when compared to survivors. All nonsurvivors (8/8) had hyperlactatemia, whereas most survivors (6/7) had values within the reference range. All survivors had leucocyte counts within the reference range, unlike the nonsurvivors, which showed leukopenia. During the course of acute babesiosis, the variables serum lactate, triglyceride, and phosphate concentrations, and thrombocyte count only exceeded a prognostic threshold during acute crisis. Conclusions and clinical importance Poor outcome in acute B. canis infection is indicated by changes in the laboratory profile. Intensive care should be considered for dogs presenting with moderate anemia, severe thrombocytopenia, mild to moderate leukopenia, hyperlactatemia, moderately increased serum phosphate, and triglyceride concentrations, and moderately decreased total serum protein concentrations.

High prevalence of bovine cysticercosis found during evaluation of different post-mortem detection techniques in Belgian slaughterhouses

Bovine cysticercosis (BCC), caused by the helminth Taenia saginata, is currently diagnosed solely by official meat inspection (MI) based on macroscopic detection of viable cysticerci or typical lesions of degenerated larvae. MI has a known low sensitivity (<16%), leading to a large proportion of infected cattle carcasses entering the human food chain and posing a risk to public health. Prevalence in Belgium based on MI results is estimated at around 0.22%. Due to the low sensitivity of MI, alternative techniques to detect BCC should be considered. This study evaluates MI, MI with additional incisions in the heart, specific antibody detection against excretory/secretory (E/S) in the Ab-ELISA and circulating antigens in the B158/B60 Ag-ELISA on 715 (101 MI-positive and 614 MI-negative) samples collected from carcasses at slaughterhouses in Belgium. Full dissection of the predilection sites was considered the reference test. During the study, mostly carcasses with (very) light infections were detected containing predominantly degenerated or calcified cysticerci and only few viable cysticerci. Dissection of the predilection sites detected 144 (23%) additional infections in the 614 MI-negative carcasses. When sequentially performing first the dissection of the predilection sites, followed by the Ag-ELISA and the Ab-ELISA, an additional 36% of MI-negative carcasses were found positive for BCC, resulting in a prevalence very much higher than the above mentioned 0.22%. The B158/B60 Ag-ELISA showed a sensitivity of 40% for the detection of carcasses containing viable cysticerci and a specificity of 100%, and detected 70 positive carcasses of which only 14 had been identified as positive during MI. If Ag-ELISA were implemented as a detection technique for BCC in the slaughterhouses, many infected carcasses would still not be detected due to the sensitivity of 40%. But as sensitivity increases with increasing number of cysticerci in the carcass, the infected carcasses passing inspection will be the ones containing only a few viable cysticerci and thus posing a smaller food safety problem. Ag-ELISA is preferred over the ES Ab-ELISA in this study, which had a sensitivity of 13.3% and a specificity of 91.7% in a population with overall low infection burdens.

Successful intestinal Echinococcus multilocularis oncosphere invasion and subsequent hepatic metacestode establishment in resistant RccHan™:WIST rats after pharmacological immunosuppression.

Susceptibility/resistance to larval Echinococcus multilocularis infection varies greatly depending on host species and strains. Whereas several mice strains and non-human primates are highly susceptible to alveolar echinococcosis, rats and most of humans are considered as more resistant. In this study, we aimed to elucidate factors responsible for host resistance in rats (Experiments A-D). (A) The parasite establishment was not observed in immunocompetent Wistar rats orally inoculated with sodium hypochlorite resistant eggs with/without pig bile, or activated/non-activated oncospheres (NAO). Peritoneal inoculation with NAO or metacestode tissue allowed the parasite establishment in rats. (B) T-cell-deficient athymic nude rats showed complete resistance against the metacestode establishment after oral inoculation with parasite eggs. This finding suggests that T-cell-independent parasite clearance occurred in the animals during early phase of the parasite invasion. Finally, Wistar rats that received pharmacological immunosuppression using either dexamethasone (DMS) alone or methotrexate (MTX) i.p. alone or a combination of these compounds were orally inoculated with the parasites eggs. As a result (D), successful establishment of metacestode with protoscoleces was observed in all 3 rats treated with DMS (s.c.) alone or in all 6 rats treated with DMS (s.c.) plus MTX but not in 8 rats with MTX alone, suggesting that factors affected by DMS treatment are responsible to regulate the parasite invasion and establishment.

Genome-wide analysis of gene expression and protein secretion of Babesia canis during virulent infection identifies potential pathogenicity factors

Infections of dogs with virulent strains of Babesia canis are characterized by rapid onset and high mortality, comparable to complicated human malaria. As in other apicomplexan parasites, most Babesia virulence factors responsible for survival and pathogenicity are secreted to the host cell surface and beyond where they remodel and biochemically modify the infected cell interacting with host proteins in a very specific manner. Here, we investigated factors secreted by B. canis during acute infections in dogs and report on in silico predictions and experimental analysis of the parasite’s exportome. As a backdrop, we generated a fully annotated B. canis genome sequence of a virulent Hungarian field isolate (strain BcH-CHIPZ) underpinned by extensive genome-wide RNA-seq analysis. We find evidence for conserved factors in apicomplexan hemoparasites involved in immune-evasion (e.g. VESA-protein family), proteins secreted across the iRBC membrane into the host bloodstream (e.g. SA- and Bc28 protein families), potential moonlighting proteins (e.g. profilin and histones), and uncharacterized antigens present during acute crisis in dogs. The combined data provides a first predicted and partially validated set of potential virulence factors exported during fatal infections, which can be exploited for urgently needed innovative intervention strategies aimed at facilitating diagnosis and management of canine babesiosis.

The merozoite-specific protein, TgGRA11B, identified as a component of the Toxoplasma gondii parasitophorous vacuole in a tachyzoite expression model

The apicomplexan, Toxoplasma gondii, infects all warm-blooded animals as intermediate hosts but only felids as definitive hosts. Dense granule proteins are critical for the survival of Toxoplasma within host cells but, whilst these proteins have been studied intensively in tachyzoites, little is known about their expression in the coccidian stages in the cat intestine. Transcriptomic profiling indicates that two putative dense granule proteins, TgGRA11A and TgGRA11B, are expressed uniquely in merozoites. Immunofluorescent microscopy of Toxoplasma-infected cat intestine and tachyzoites engineered to express TgGRA11B, reveals that it is a dense granule protein that traffics into the parasitophorous vacuole and its membrane.

Estimating prevalence and diagnostic test characteristics of bovine cysticercosis in Belgium in the absence of a ‘gold standard’ reference test using a Bayesian approach

A Bayesian model was developed to estimate values for the prevalence and diagnostic test characteristics of bovine cysticercosis (Taenia saginata) by combining results of four imperfect tests. Samples of 612 bovine carcases that were found negative for cysticercosis during routine meat inspection collected at three Belgian slaughterhouses, underwent enhanced meat inspection (additional incisions in the heart), dissection of the predilection sites, B158/B60 Ag-ELISA and ES Ab-ELISA. This Bayesian approach allows for the combination of prior expert opinion with experimental data to estimate the true prevalence of bovine cysticercosis in the absence of a gold standard test. A first model (based on a multinomial distribution and including all possible interactions between the individual tests) required estimation of 31 parameters, while only allowing for 15 parameters to be estimated. Including prior expert information about specificity and sensitivity resulted in an optimal model with a reduction of the number of parameters to be estimated to 8. The estimated bovine cysticercosis prevalence was 33.9% (95% credibility interval: 27.7-44.4%), while apparent prevalence based on meat inspection is only 0.23%. The test performances were estimated as follows (sensitivity (Se) - specificity (Sp)): enhanced meat inspection (Se 2.87% - Sp 100%), dissection of predilection sites (Se 69.8% - Sp 100%), Ag-ELISA (Se 26.9% - Sp 99.4%), Ab-ELISA (Se 13.8% - Sp 92.9%).