Georgios Theodoropoulos

The role of mucins in host–parasite interactions: Part II – helminth parasites

Some parasites express mucin-like molecules. These have possible roles in attachment and invasion of host cells and in the avoidance of host immune processes. Enzymes of parasite origin might also facilitate infection, either by degrading host mucus barriers or by generating binding sites on host cells. Host mucins have roles in preventing parasite establishment or in parasite expulsion. They, in turn, might be exploited by parasites, either as sources of fuel or binding sites, or as host-finding targets. Here, we describe the biochemical properties of mucins and mucin-like molecules in relation to interactions (established and putative) between helminth parasites and their hosts.

Determination of the within and between flock prevalence and identification of risk factors for Salmonella infections in laying hen flocks housed in conventional and alternative systems

Salmonella outbreaks in humans are often linked with the consumption of contaminated eggs. Therefore a profound knowledge of the actual prevalence of Salmonella spp. in laying hens and the factors that influence the presence and persistence of Salmonella on a farm is of utmost importance. The housing of laying hens in conventional battery cages will be forbidden in the European Union (EU) from 2012 onwards. There is an urgent need to evaluate whether this move to alternative housing systems will influence the prevalence of Salmonella in laying hens. Therefore, a cross-sectional study was performed in 5 European countries (Belgium, Germany, Greece, Italy and Switzerland) to determine the between and within flock prevalence of hens shedding Salmonella and to investigate whether there is an effect of the housing type on Salmonella prevalence. In total 292 laying hen farms were sampled in the month prior to depopulation. An on-farm questionnaire was used to collect information on general management practices and specific characteristics of the sampled flock. Twenty-nine flocks were found positive for at least 1 Salmonella-serotype. In these flocks the within flock prevalence of shedding hens, determined by individual sampling of 40 hens, varied between 0% and 27.50%. A wide variety of serotypes was isolated with Salmonella Enteritidis as the most common. Housing in conventional battery cages, the absence of dry cleaning in between production rounds and sampling in winter turned out to be risk factors for the shedding of Salmonella Enteritidis or Typhimurium (P<0.05).

Seroprevalence of equine piroplasms and host-related factors associated with infection in Greece.

Serum samples were collected from a total of 544 equids that included 524 horses, 13 mules, and 7 ponies from various regions of mainland Greece and were examined by competitive-inhibition ELISA (cELISA) to evaluate the level of exposure of Greek equids to Theileria (Babesia) equi and/or Babesia caballi, the causative agents of piroplasmosis. Association between seropositivity and host-related factors of species, gender, age, origin, activity and location were investigated. The overall seroprevalence was 11.6% (9.1-14.6%) with 95% confidence limit. The seroprevalence for T. equi and B. caballi was found to be 11% (8.6-14%) and 2.2% (1.2-3.9%), respectively. The animal-related factors significantly linked with seropositivity were the species, activities of farming, racing, recreation, and geographic location in Attica, Macedonia, Peloponnese and Thessaly region (p<0.05). The relative risks for the presence of T. equi, B. caballi and mixed infection in mules compared to horses was 8.39, 33.58 and 40.31, respectively. The infection level for T. equi, B. caballi and mixed infection were significantly higher in farm equids than in racing equids (p<0.05). Also, the rate of infection of T. equi was higher in farm equids than recreational equids (p<0.05). The relative risk of T. equi infection between farming equids and equids used only for recreation activity was 3.25-1, while the relative risk of B. caballi infection was 0.14-1 for racing animals relative to recreation animals. The region with the highest level of infection to both parasites was Thessaly (38.8% T. equi and 6.1% B. caballi), followed by Peloponnese (10.4% T. equi and 3.9% B. caballi), Attica region (8.3% T. equi and 0.6% B. caballi) and finally Macedonia the region with the lowest prevalence (6.6% T. equi and 4.4% B. caballi). A higher seroprevalence rate was found among local animals compared to imported equids, indicating that equine piroplasm infection is enzootic in Greece. T. equi seroprevalence was significantly different and higher among increasing age groups of equids, suggesting persistent infections or lower transmission levels whereby animals may need to be exposed longer before acquiring the infection. Competent tick vectors Rhipicephalus bursa and Rhipicephalus sanguineus for the transmission of equine piroplasmosis were recovered from horses and dogs, respectively.

Epidemiological survey of cestode-larva disease in Greek sheep flocks

The epidemiological status of hydatidosis, cysticercosis, and gid in sheep and the production practices of sheep farmers that increase the risk of exposure of sheep to infecting agents of these diseases were investigated in Thessaly, central Greece during 2002-2006. A total of 700 hoggets (sheep up to the age of 1 year) and 1500 adult sheep were examined randomly at an abattoir survey and the prevalences of hydatidosis and cysticercosis were found 39.32% and 29.41%, respectively. Hoggets had significantly lower prevalences of hydatidosis and cysticercosis compared to adult sheep (p<0.001). The distribution of parasitic cysts showed an overdispersion pattern mostly in the hogget population. The proportion of fertile hydatid cysts was significantly higher in hoggets compared to adult sheep (p<0.001). A cohort of 74 sheep flocks was observed during the same period in a prospective survey to assess the incidence of gid. A total of 57 sheep belonging to 15 flocks (20.27% of investigated flocks) developed clinical signs of gid at an annual rate of 11.40+/-4.77 (95% C.I.). In the infected flocks there were 3.80+/-0.92 (95% C.I.) gid cases per flock. The mean age of onset of clinical signs for all sheep was 11.86+/-1.33 months (95% C.I.) (range 5-22 months) while for hoggets was 8.48+/-0.73 (95% C.I.) (range 5-12 months) and for adult sheep 17.23+/-1.42 (95% C.I.) (range 13-22 months). A significant difference was observed in the prevalence of unilateral blindness between hoggets (31.42%) and adult sheep (4.54%) (p<0.05). The most common production practices identified in a questionnaire survey of sheep farmers that may increase the risk of exposure of sheep to hydatidosis, cysticercosis, and gid were the improper disposal of dead animals, the access of farm dogs to offal of slaughtered sheep, the carelessness of farmers to treat farm dogs with anthelmintics, and the grazing of flocks in fields where stray dogs have free access.

Towards a standardised surveillance for Trichinella in the European Union.

Each year, more than 167 million pigs in the European Union (EU) are tested for Trichinella spp. under the current meat hygiene regulations. This imposes large economic costs on countries, yet the vast majority of these pigs test negative and the public health risk in many countries is therefore considered very low. This work reviewed the current Trichinella status across the EU as well as the national level of monitoring and reporting. It also reviewed which animal species were affected by Trichinella and in which species it should be surveyed. This information was used to design a cost-effective surveillance programme that enables a standardised monitoring approach within the EU. The proposed surveillance programme relies on identifying sub-populations of animals with a distinct risk. Low-risk pigs are finisher pigs that originate from so-called controlled housing. All other pigs are considered high-risk pigs. Controlled housing is identified by the application of a specific list of management and husbandry practices. We suggest that member states (MS) be categorised into three classes based on the confidence that Trichinella can be considered absent, in the specified sub-population of pigs above a specified design prevalence which we set to 1 per million pigs. A simple and transparent method is proposed to estimate this confidence, based on the sensitivity of the surveillance system, taking into account the sensitivity of testing and the design prevalence. The probability of detecting a positive case, if present, must be high (>95 or >99%) to ensure that there is a low or negligible risk of transmission to humans through the food chain. In MS where the probability of a positive pig is demonstrated to be negligible, testing of fattening pigs from a sub-population consisting of pigs from controlled housing can be considered unnecessary. Furthermore, reduced testing of finishers from the sub-population consisting of pigs from non-controlled housing might even be considered, if conducted in conjunction with a proportionate sampling scheme and a risk-based wildlife surveillance programme where applicable. The proposed surveillance programme specifies the required number of samples to be taken and found negative, in a MS. A MS with no data or positive findings will initially be allocated to class 1, in which all pigs should be tested. When a MS is able to demonstrate a 95% or 99% confidence that Trichinella is absent, the MS will be allocated to class 2 or 3, in which the testing requirement is lower than in class 1.

A seroepidemiological study of exposure to Toxoplasma, Leishmania, Echinococcus and Trichinella in equids in Greece and analysis of risk factors.

The role of horses in the transmission of parasitic zoonoses either as a source of infection to vectors or through contamination of definitive hosts is gaining importance worldwide. For this reason sera from 773 equids including 753 horses, 13 mules and seven ponies in four regions of Greece were investigated by ELISA for the presence of IgG antibodies against Toxoplasma, Leishmania, Echinococcus and Trichinella. Anti-Toxoplasma antibodies were detected in all regions with an overall prevalence of 1.8%. In contrast, antibodies to Leishmania, Echinococcus, and Trichinella were present only in horses from the equestrian centre located in Attica region, but the status of Trichinella could not be confirmed. The seroprevalence of infection was 0.3% for Leishmania, 0.1% for Echinococcus and 0.1% for Trichinella. Only one horse was positive with a mixed infection of Toxoplasma, Leishmania and Trichinella. The following host characteristics were investigated for any significant effects on the prevalence of Toxoplasma infection: gender, age, species, origin of birth, activity, and location. The type of activity (p<0.05) and location (p<0.01) of the animals were found to be significant risk factors for Toxoplasma infection. The relative risk (RR) for Toxoplasma infection comparing the regions of Peloponnese and Thessaly to Attica were 6.92 and 6.78, respectively. Due to the very low prevalence of Echinococcus, Leishmania, and Trichinella infections, the associated risk factors were not analysed. The low seroprevalences observed suggest that the risk of infection from equids to people is very low, especially when consumption of horse meat is uncommon in this country.

A novel non-amplification assay for the detection of Leishmania spp. in clinical samples using gold nanoparticles

Leishmaniosis is a zoonose caused by protozoans of the genus Leishmania. The need for accurate diagnostic investigation of cases of leishmaniosis has rendered today the use of molecular biology techniques broadly applicable. However, the reliable application of these methods requires highly-specialised personnel, dedicated equipment and space. The aim of this study was the design and construction of functionalized gold nanoparticles (AuNPs) that would be incorporated into an easily applicable DNA detection methodology for the identification of Leishmania spp. in clinical samples. AuNPs 20nm in diameter were conjugated with four oligonucleotide probes, targeting kinetoplastid minicircle DNA of Leishmania spp. In the absence of complimentary DNA, AuNPs-probes precipitate under acid environment causing a change of color from red to purple, which can be detected by visual observation. In the presence of target DNA the color of the solution remains red. The specific methodology was applied to positive and negative control samples and whole blood collected from dogs with suspected canine leishmaniosis. The methods minimum detection limit was defined to 11.5ng of target DNA per μl of sample. Repeatability and reproducibility were 100%. Relative sensitivity and specificity referenced to PCR were calculated to 92% and 100% regarding collectively control and clinical samples. The proposed approach can be considered an appealing diagnostic solution especially for screening purposes in enzootic areas, where detection of very small amounts of the targeted analyte is not top priority.

Surveillance of feral swine for Trichinella spp. and Toxoplasma gondii in the USA and host-related factors associated with infection

Trichinella spp. and Toxoplasma gondii are important zoonotic parasites that infect warm blooded animals and humans worldwide. Among domesticated food animals, pigs are the main host for Trichinella spiralis. Pigs, chickens, sheep, and goats are known to be infected with T. gondii at varying rates, depending on husbandry. Infections in wildlife with these parasites are generally higher than in domesticated species. Feral swine act as reservoirs of infection in the sylvatic ecosystem for Trichinella spp. and T. gondii, acting as sources of infection for peridomestic carnivores whose home ranges overlap with domestic pigs. Feral swine can have direct contact with non-biosecure domestic pigs, presenting opportunity for direct disease transmission through cannibalistic behavior. Determination of the prevalence of Trichinella spp. and T. gondii infection in feral swine is needed to understand the risk of transmission of these parasites to domestic pigs. A cross-sectional serological survey was conducted between 2006 and 2010 to estimate the antibody prevalence of Trichinella spp. and T. gondii and risk factors associated with infection in feral swine in the USA. Serum samples were tested from 3247 feral pigs from 32 states; results are reported from 26 states. Maximum entropy ecological niche modeling and spatial scan statistic were utilized to predict the geographic range and to examine clusters of infection of Trichinella spp. and T. gondii in feral pigs. The seroprevalence of antibodies to Trichinella spp. and T. gondii was 3.0% and 17.7%, respectively. Species distribution modeling indicated that the most probable distribution areas for both parasites was similar, concentrated primarily in the South and the Midwest regions of the USA. A follow up survey conducted during 2012-2013 revealed that 2.9% of 984 sampled feral swine were seropositive for Trichinella spp., and 28.4% were seropositive for T. gondii. Three hundred and thirty (330) tongues were collected from the 984 sampled animals during 2012-2013; 1.81% were tissue positive for T. spiralis muscle larvae; no other genotypes were found. The potential exists for introduction of these pathogens into domestic herds of non-biosecure domestic pigs as a result of increasing overlap of the range of feral pigs with non-biosecure domestic pigs production facilities in the USA.

Geographic distribution modelling for ruminant liver flukes (Fasciola hepatica) in south-eastern Europe.

Maximum entropy ecological niche modelling was utilised to predict the geographic range for fluke genotypes and haplotypes in south-eastern Europe, using the Maxent program. The lowest (0.832) and the highest (0.947) area under the curve values were observed in the models for the haplotypes CtCmt1 and CtCmt2.2, respectively. Precipitation and temperature contribute equally to model building of the genotypes based on the 28S rDNA gene. In regard to the mtDNA gene region, precipitation is the most important factor in modelling the CtCmt1 haplotype range, while temperature appears to be the most important factor in modelling the CtCmt2.1 and CtCmt2.2 haplotype ranges. The highest level of probability for the geographic distribution of Fasciola hepatica genotypes and haplotypes covered the regions of southern Bulgaria and central and northern Greece which contain a high concentration of potential ruminant hosts.

Seroprevalence and risk factors associated with zoonotic parasitic infections in small ruminants in the Greek temperate environment

A cross-sectional serological study was carried out to screen the sheep and goat population of Thessaly, Greece for evidence of infection with Toxoplasma, Toxocara, Leishmania, and Echinococcus and to determine the risk factors related to herd characteristics, herd management practices, farmer status, and the bioclimatic variables associated with these zoonotic parasitic infections. A total of 540 sheep and goat serum samples were examined. The seroprevalence of infection in all examined animals was 24.5% for Toxoplasma, 32% for Toxocara, 0% for Leishmania and 85.9% for Echinococcus. The final logistic regression model showed that the species of small ruminant, herd size, anthelmintic treatment, class of anthelmintic treatment, grazing with other herds, educational level of farmer, elevation of farm location, and generalized land cover were associated with Toxoplasma gondii infections, while the species of small ruminant, farm type, anthelmintic treatment, class of anthelmintic treatment, rotation of grazing, age of farmer, elevation of farm location, and generalized land cover were associated with Toxocara canis infections. Antibodies to T. gondii were detected in 102 (28.3%) of 360 sheep and in 30 (16.8%) of 179 goats. Animals in small flocks (150-300 animals) had an approximately 0.42-fold lower risk of having positive cases of T. gondii among animals compared with large flocks (>300 animals). Antibodies to T. canis were found in 155 (42.9%) of 361 sheep and 18 (10.1%) of 179 goats. The later finding constitutes the first report of seropositive goats to Toxocara. The risk of positivity for T. canis was 7.71-fold higher in sheep than in goats. Geographically, animals from plain areas had 2.9 and 2.01-fold higher risk of having positive cases of T. gondii and T. canis respectively. The significant bioclimatic variables (p<0.05) associated with the occurrence locations of T. gondii infection were related to higher temperature, lower precipitation, and lower elevation compared to the absence locations of T. gondii. The significant bioclimatic variables (p<0.05) associated with occurrence locations of T. canis infection were related to lower temperature and higher precipitation compared to absence locations of T. canis. These findings are useful to formulate appropriate control strategies for zoonotic parasites of sheep and goats in Greece and other areas with similar climatic conditions.