Pre-clinical evaluation of the effect of co-medication with antibiotics and oral steroids in Göttingen Minipigs on the biological activity of the probiotic medicinal product TSO (Trichuris suis ova).

The probiotic medicinal product TSO (Trichuris suis ova) is administered to patients with active ulcerative colitis in an ongoing clinical phase IIb trial where the typical co-medications are steroids (prednisolone or budesonide) and antibiotics (e.g., phenoxymethylpenicillin). The present pre-clinical study evaluates the effects of these co-medications on the biological activity of TSO in Göttingen Minipigs. This translationally relevant pre-clinical model allows administration of TSO with and without oral steroids or antibiotics in a manner similar to the administration to patients, followed by quantification of the biological activity of TSO. The biological activity of TSO was not affected by oral steroids but was reduced by oral antibiotics. Fecal calprotectin, the common marker of intestinal inflammation in patients with UC, did not differ between groups.

Echinococcus multilocularis in Denmark 2012-2015: high local prevalence in red foxes.

In Western Europe, the Echinococcus multilocularis lifecycle is predominantly sylvatic, typically involving red foxes (Vulpes vulpes) as the main definitive hosts with Microtus spp. and Arvicola spp. as intermediate hosts. During a 4-year surveillance study (2012-2015), Danish red foxes and raccoon dogs (n = 1345) were examined for E. multilocularis. Moreover, 134 insectivores and rodents collected in South Jutland during spring and summer 2016 were examined for the presence of metacestodes. The sedimentation and counting technique and molecular typing were used to identify E. multilocularis infections in the carnivores, while the rodent livers were examined macro- and microscopically for parasite lesions. Following morphological identification of E. multilocularis adult worms, the identity was verified by sequence analysis of the 12S rRNA gene in most cases (n = 13). Echinococcus multilocularis infection was demonstrated in 19 red foxes (Vulpes vulpes) originating from only two specific areas of South Jutland, namely Højer and Grindsted, and in two raccoon dogs (Nyctereutes procyonoides), originating from Højer. In Højer, 28.5% (CI 95% 11.7-45.3) of the examined red foxes were E. multilocularis positive per year. Moreover, positive red foxes were identified each year from 2012 to 2015, while E. multilocularis positive red foxes were only identified in Grindsted in 2013 (4.0%) and 2014 (6.4%). In contrast, all collected rodents were negative for E. multilocularis. We conclude that E. multilocularis is locally endemic in South Jutland with a high local prevalence in Højer.

Host-specific serological response to Angiostrongylus vasorum infection in red foxes (Vulpes vulpes): implications for parasite epidemiology.

Angiostrongylus vasorum is a cardiovascular nematode increasingly found in dogs and foxes in endemic foci throughout Europe. The present study evaluates ELISAs for detection of circulating antigens and specific antibodies against A. vasorum in foxes. Blood and worm burdens (WBs) from carcasses of 215 Swiss wild red foxes (Vulpes vulpes) and from 75 farmed foxes of different age groups experimentally inoculated once or repeatedly with infective doses of 50, 100 or 200 third-stage larvae were obtained. Antigen detection in the naturally infected Swiss foxes had 91·2% sensitivity and 89·4% specificity, whereas the corresponding figures for antibody detection were 42·2 and 92·0%. The experimentally infected foxes became positive for circulating antigens 5-10 weeks post-inoculation (wpi) and remained highly positive up to 22 wpi, irrespectively of further challenge inoculation. The antibody responses in the same foxes were highly variable: high optical density (OD) values were reached 5-7 wpi in all animals, followed by a decrease in over half of the animals despite accumulating and consequently high WBs resulting in persistent infections. After each challenge, a slight increase of OD values was observed 7 weeks later. We hypothesize that infected foxes develop a variable and non-protective immunity. Such parasite tolerance allows long-term survival of A. vasorum in the animals, and may explain why the parasite appears to spread rapidly within a fox population, an epidemiological dynamic that is evident in many parts of Europe where A. vasorum has been found over the last decades.

Trichuris suis ova therapy in relapsing multiple sclerosis is safe but without signals of beneficial effect.

BACKGROUND: An observational study has suggested that relapsing-remitting multiple sclerosis patients with helminth infections have lower disease activity and progression than uninfected multiple sclerosis patients. OBJECTIVE: To evaluate the safety and efficacy on MRI activity of treatment with TSO in relapsing MS. METHODS: The study was an open-label, magnetic resonance imaging assessor-blinded, baseline-to-treatment study including ten patients with relapsing forms of multiple sclerosis. Median (range) age was 41 (24-55) years, disease duration 9 (4-34) years, Expanded Disability Status Scale score 2.5 (1-5.0), and number of relapses within the last two years 3 (2-5). Four patients received no disease modifying therapy, while six patients received IFN-ß. After an observational period of 8 weeks, patients received 2500 ova from the helminth Trichuris suis orally every second week for 12 weeks. Patients were followed with serial magnetic resonance imaging, neurological examinations, laboratory safety tests and expression of immunological biomarker genes. RESULTS: Treatment with Trichuris suis orally was well-tolerated apart from some gastrointestinal symptoms. Magnetic resonance imaging revealed 6 new or enlarged T2 lesions in the run-in period, 7 lesions in the early period and 21 lesions in the late treatment period. Two patients suffered a relapse before treatment and two during treatment. Eight patients developed eosinophilia. The expression of cytokines and transcription factors did not change. CONCLUSIONS: In a small group of relapsing multiple sclerosis patients, Trichuris suis oral therapy was well tolerated but without beneficial effect.

Morphological and molecular analyses of larval taeniid species in small mammals from contrasting habitats in Denmark.

Taeniid infections in intermediate hosts manifest themselves as extraintestinal larval stages which, in early development, lack species-specific characteristics. The inability to distinguish infections of zoonotic importance such as Echinococcus multilocularis from other taeniid infections that have mainly veterinary significance stimulated the development of species-specific molecular diagnostics. In this study, the prevalence of taeniid infections in potential intermediate hosts was evaluated using both morphological diagnosis and a newly described multiplex polymerase chain reaction (PCR) for species determination. Small mammals (N= 719) were trapped in three different types of habitats in north-east Zealand, Denmark. The sensitivity of the multiplex PCR (90.5%) exceeded that of morphological examination (57.9%) for identifying 95 taeniid infections. The use of the multiplex PCR resulted in higher prevalence rates due to improved detection of immature liver infections with Hydatigera taeniaeformis and Versteria mustelae, but did not affect the observed prevalence rates of peritoneal metacestodes of Taenia polyacantha. The prevalence of taeniid infections showed a significant difference according to habitat type, potentially identifying a 'sylvatic' transmission and an 'urban' transmission, with marked variation among different taeniid species. Versteria mustelae and T. polyacantha were more prevalent in rural forests, while infections with H. taeniaeformis were dominant in urban parks/forests and in residential and farm gardens. The multiplex PCR facilitated a better utilization of wildlife samples by yielding a higher number of definitive diagnoses of ambiguous taeniid infections in liver lesions, allowing for more accurate epidemiological data and, hence, a more accurate risk assessment.

Trichuris suis ova therapy for allergic rhinitis does not affect allergen-specific cytokine responses despite a parasite-specific cytokine response.

BACKGROUND: Parasitic helminths have been shown to reduce inflammation in most experimental models of allergic disease, and this effect is mediated via cytokine responses. However, in humans, the effects of controlled helminth infection on cytokine responses during allergy have not been studied. OBJECTIVE: The aim was to investigate whether infection with the nematode parasite Trichuris suis alters systemic cytokine levels, cellular cytokine responses to parasite antigens and pollen allergens and/or the cytokine profile of allergic individuals. METHODS: In a randomized double-blinded placebo-controlled clinical trial (UMIN trial registry, Registration no. R000001298, Trial ID UMIN000001070, URL: http://www.umin.ac.jp/map/english), adults with grass pollen-induced allergic rhinitis received three weekly doses of 2500 Trichuris suis ova (n = 45) or placebo (n = 44) over 6 months. IFN-γ, TNF-α, IL-4, IL-5, IL-10 and IL-13 were quantified via cytometric bead array in plasma. Cytokines, including active TGF-ß, were also quantified in supernatants from peripheral blood mononuclear cells cultured with parasite antigens or pollen allergens before, during and after the grass pollen season for a sub-cohort of randomized participants (T. suis ova-treated, n = 12, Placebo-treated, n = 10). RESULTS: Helminth infection induced a Th2-polarized cytokine response comprising elevated plasma IL-5 and parasite-specific IL-4, IL-5 and IL-13, and a global shift in the profile of systemic cytokine responses. Infection also elicited high levels of the regulatory cytokine IL-10 in response to T. suis antigens. Despite increased production of T. suis-specific cytokines in T. suis ova-treated participants, allergen-specific cytokine responses during the grass pollen season and the global profile of PBMC cytokine responses were not affected by T. suis ova treatment. CONCLUSIONS AND CLINICAL RELEVANCE: This study suggests that cytokines induced by Trichuris suis ova treatment do not alter allergic reactivity to pollen during the peak of allergic rhinitis symptoms.

Trichinella infection in a hunting community in East Greenland.

Trichinella nativa infection (trichinellosis) is highly prevalent in Arctic wildlife, but the human burden of trichinellosis in present-day Greenland is unknown. The study aimed to determine Trichinella seroprevalence in an eastern Greenlandic hunting community and to evaluate risk factors for seropositivity. Overall, 998 inhabitants aged 10 years in the Ammassalik municipality were tested for Trichinella-specific IgG antibodies. Background information was obtained from questionnaires. Seropositivity was 1.4% in persons aged <40 years and increased to >12% in those aged 60 years. Older age, occupation as hunter or fisherman, and consumption of polar bear meat significantly increased the risk of Trichinella seropositivity. The seropositivity age pattern probably reflects changes in dietary preferences, but could also reflect mandatory meat inspection since 1966. However, preventive measures against Trichinella infection should be strengthened in Greenland.

The occurrence of Angiostrongylus vasorum in terrestrial slugs from forests and parks in the Copenhagen area, Denmark.

A total of 298 slugs belonging to four species, Arion lusitanicus, A. ater, A. ater rufus and Limax maximus, were collected from six different localities within a radius of 30 km from Copenhagen and examined for naturally acquired Angiostrongylus vasorum infection. Overall, 28 slugs (9%) were infected, but the prevalence varied among the studied localities: Rude Forest (26%), West Amager Forest (18%), Jaegersborg Forest and Deer Park (8%), Frederiksberg Park (4%), Assistens Cemetery Park (0%) and Frederiksberg Botanical Garden (0%). Only third-stage larvae (L3) were recovered from the slugs, in numbers ranging from 1 to 392 per slug. Overall 82% of the infected slugs harboured fewer than 10 larvae and only 14% harboured over 100 larvae.

Human antibody recognition of Anisakidae and Trichinella spp. in Greenland.

High levels of total IgE are observed among children in Greenland. To evaluate the extent to which Anisakidae and Trichinella spp. contribute to the high total IgE level, an ELISA and a western blot were developed for the detection of IgG antibodies to Anisakidae, based on excretory/secretory antigens from Anisakidae larvae. Western blots with Anisakidae and Trichinella antigens discriminated between Anisakidae and Trichinella infections, enabling cross-reactivity between the two parasite infections to be eliminated. Serum samples from 1012 children in Greenland were analysed for specific antibodies to Anisakidae and Trichinella. Eleven children were IgG-positive for Trichinella and nine were IgG-positive for Anisakidae, indicating a relatively low prevalence of both infections among children in Greenland. Faecal samples from 320 children were also examined for other intestinal parasites. Enterobius vermicularis was found in one sample and Blastocystis hominis in 32 samples, but no other intestinal parasites were identified. In total, 304 children had elevated total IgE levels. There was a significant association between Trichinella seropositivity and high levels of total IgE, but not between Anisakidae seropositivity and total IgE. The data indicate that parasitic infections alone do not explain the high level of total IgE observed among children in Greenland.

Repeated inoculations with the lung and heartworm nematode Angiostrongylus vasorum result in increasing larval excretion and worm burden in the red fox (Vulpes vulpes).

The French heartworm Angiostongylus vasorum is found in European red fox (Vulpes vulpes) and dog populations, where it appears to be spreading geographically. Once introduced into new areas, it establishes in local fox populations, typically to over 50% prevalence in a few years. High susceptibility and constant excretion of first stage larvae (L1) by the definitive hosts are prerequisites for sustaining high parasite biomass in a particular habitat. The present study explores the hypothesis that repeated ingestion of gastropods in nature will result in accumulation of adult worms and elevated excretion of L1 in feces. Experimentally infected foxes were subsequently inoculated via stomach tube once (9 weeks post initial inoculation) or twice (9 and 13 weeks post inoculation (wpi)) with 100 third stage A. vasorum larvae (L3) previously isolated from aquatic snails infected with L1 from a naturally infected dog. Despite large variation in fecal larval excretion for the individual animals within the groups, excretion of L1 was significantly higher in foxes twice inoculated as compared to foxes inoculated only once. With an outlier in the once inoculated group removed, excretion became significantly higher in the three times inoculated group. Establishment of adult worms varied and only a trend to higher worm burdens was found in the group of foxes inoculated three times. However, this became significant with the same single outlier removed. Overall, it appears that protective immunity to A. vasorum does not appear to occur in V. vulpes with animals exhibiting high infection intensities without obvious clinical signs. The increasing larval excretion in foxes being repeatedly exposed to A. vasorum L3 support the hypothesis that foxes under natural conditions may repeatedly ingest infected gastropods and remain a source of environmental contamination for several months, potentially contributing to the establishment of endemic foci through increasing L1 excretion.

Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats.

A total of 15 red foxes, 15 raccoon dogs, 15 domestic dogs and 15 domestic cats were each infected with 20,000 protoscolices of Echinococcus multilocularis. At 35, 63, and 90 days post inoculation (dpi), five animals from each group were necropsied and the worm burdens determined. The highest worm burdens in foxes (mean of 16,792) and raccoon dogs (mean of 7930) were found at 35 dpi. These declined to a mean of just 331 worms in foxes and 3213 worms in raccoon dogs by day 63 with a further decline to 134 worms in foxes and 67 worms in raccoon dogs by day 90. In dogs, there was no significant difference between worm burdens recovered at days 35 (mean of 2466) and day 90 (mean of 1563), although reduced numbers were recovered on day 63 (mean of 899). In cats, worms were found in four animals 35 dpi (mean of 642), in three at 63 dpi (mean of 28) and in two at 90 dpi (mean of 57). Faecal egg counts were determined at 3 day intervals from 25 dpi. A mathematical model of egg excretion dynamics suggested that the mean biotic potential per infected animal was high in foxes (346,473 eggs); raccoon dogs (335,361 eggs) and dogs (279,910 eggs) but very low for cats (573 eggs). It also indicated that approximately 114, 42 and 27 eggs per worm were excreted in the faeces of dogs, raccoon dogs and foxes, respectively. The fecundity of worms in cats was low with an average of less than one egg per worm. The peak levels of coproantigen were detected earlier in foxes and raccoon dogs than in dogs. Eggs recovered from foxes, raccoon dogs and dogs resulted in massive infections in experimental mice. However, metacestodes did not develop from eggs originating from infected cats. It is concluded that foxes, raccoon dogs and dogs are good hosts of E. multilocularis. In contrast, the low worm establishment, the very few excreted eggs and the lack of infectivity of eggs strongly indicate that cats play an insignificant role in parasite transmission.

Helminths of red foxes (Vulpes vulpes) in Denmark.

An epidemiological study of helminths in 1040 red foxes collected from various localities in Denmark during 1997-2002, revealed 21 helminth species at autopsy, including nine nematode species: Capillaria plica (prevalence 80.5%), Capillaria aerophila (74.1%), Crenosoma vulpis (17.4%), Angiostrongylus vasorum (48.6% from Northern Zealand (endemic area)), Toxocara canis (59.4%), Toxascaris leonina (0.6%), Uncinaria stenocephala (68.6%), Ancylostoma caninum (0.6%), and Trichuris vulpis (0.5%); seven cestodes: Mesocestoides sp. (35.6%), a number of Taeniid species (Taenia pisiformis, T. hydatigena, T. taeniaeformis, T. crassiceps, and unidentified Taenia spp.) (22.8%), and Echinococcus multilocularis (0.3%); four trematodes: Alaria alata (15.4%), Cryptocotyle lingua (23.8%), Pseudamphystomum truncatum (3.6% from Northern Zealand), and Echinochasmus perfoliatus (2.4% from Northern Zealand); one acanthocephalan: Polymorphus sp. (1.2%). Significant difference in prevalence was found for T. canis and A. vasorum according to host sex, and for T. canis, U. stenocephala, Mesocestoides sp., Taenia spp., A. alata, A. vasorum, and Capillaria spp. according to age groups (adult, young or cub). Prevalence and average worm intensity for each helminth species varied considerably according to geographical locality, season, and year. Aggregated distribution was found for several helminth species. The two species E. multilocularis and E. perfoliatus are first records for Denmark.

Meat inspection for Trichinella in pork, horsemeat and game within the EU: available technology and its present implementation.

A new EU directive relating to meat inspection for Trichinella, expected to come into force in 2006, imposes important modifications to current legislation. Nevertheless, several issues need more attention. Optimisation of methods, especially concerning sensitivity and digestibility of the meat to be inspected, along with further simplification of the legislation with regard to the number of techniques accepted, is recommended to guarantee that all member states of the EU will be given tools to perform inspection of consumer meat at the same high level. Additionally, there is a need for guidelines and protocols regarding optimal proficiency testing procedures. This paper presents an overview of the current methods for Trichinella meat inspection and their implementation in the EU, listing advantages and disadvantages for each method, including some suggestions for specific points of improvement.

Meat inspection for Trichinella in pork, horsemeat and game within the EU: available technology and its present implementation.

A new EU directive relating to meat inspection for Trichinella, expected to come into force in 2006, imposes important modifications to current legislation. Nevertheless, several issues need more attention. Optimisation of methods, especially concerning sensitivity and digestibility of the meat to be inspected, along with further simplification of the legislation with regard to the number of techniques accepted, is recommended to guarantee that all member states of the EU will be given tools to perform inspection of consumer meat at the same high level. Additionally, there is a need for guidelines and protocols regarding optimal proficiency testing procedures.

Changes in the EU legislation on Trichinella inspection--new challenges in the epidemiology.

The European Union (EU) countries are searching for new ways to certify meat free of Trichinella; however, with the expansion of the EU, the acceptance of a unilateral method is complicated by the variability of pig and human trichinellosis among EU countries, where significantly higher prevalence rates have been observed in the newly added eastern countries. Several attempts have been made to define Trichinella-free areas, but certification of Trichinella-free pig production farms appears to be the only feasible approach. The increasing prevalence of the non-encapsulating species, Trichinella pseudospiralis, in game, domestic pigs and humans has eliminated the compression technique from the new EU legislation to be enacted in 2006. Also, the observation that several species of Trichinella tolerate freezing in horse meat for up to 4 weeks has forced a change in legislation as well where freezing is no longer an option for certifying horse meat. Because current serological detection methods are not suited for meat inspection, classical direct detection methods and inactivation by freezing remain the methods of choice for pork. It has been proposed, therefore, to automate direct inspection methods as a cost effective alternative to certify pig farms free of Trichinella.

Studies on vertical transmission of Trichinella spp. in experimentally infected ferrets (Mustela putorius furo), foxes (Vulpes vulpes), pigs, guinea pigs and mice.

Vertical transmission of Trichinella spiralis was evaluated in ferrets (n=21), foxes (n=11), pigs (n=12), guinea pigs (n=16), and mice (n=41). The placental barrier to be crossed by migratory Trichinella larvae varies structurally in different animal species. Ferrets and foxes have an endotheliochorial placenta structure, guinea pigs and mice a haemochorial, and pigs an epitheliochorial placenta. The non-encapsulating Trichinella pseudospiralis larvae have an extended muscle migration prior to entering a muscle cell. To evaluate if T. pseudospiralis was more likely to be transmitted to offspring, an additional group of foxes (n=11) infected with T. pseudospiralis was included. Two different dose levels were used for ferrets, pigs, guinea pigs, and mice. In pigs and guinea pigs, infection was given at different times of the gestation period. Vertical transmission, measured as recovery of muscle larvae in the offspring, was demonstrated in both ferrets groups, in all four guinea pig groups, and in the high dose mouse group, but not in any fox or pig groups.

Muscle distribution of sylvatic and domestic Trichinella larvae in production animals and wildlife.

Only a few studies have compared the muscle distribution of the different Trichinella genotypes. In this study, data were obtained from a series of experimental infections in pigs, wild boars, foxes and horses, with the aim of evaluating the predilection sites of nine well-defined genotypes of Trichinella. Necropsy was performed at 5, 10, 20 and 40 weeks post inoculation. From all host species, corresponding muscles/muscle groups were examined by artificial digestion. In foxes where all Trichinella species established in high numbers, the encapsulating species were found primarily in the tongue, extremities and diaphragm, whereas the non-encapsulating species were found primarily in the diaphragm. In pigs and wild boars, only Trichinella spiralis, Trichinella pseudospiralis and Trichinella nelsoni showed extended persistency of muscle larvae (ML), but for all genotypes the tongue and the diaphragm were found to be predilection sites. This tendency was most obvious in light infections. In the horses, T. spiralis, Trichinella britovi, and T. pseudospiralis all established at high levels with predilection sites in the tongue, the masseter and the diaphragm. For all host species, high ML burdens appeared to be more evenly distributed with less obvious predilection than in light infections; predilection site muscles harbored a relatively higher percent of the larval burden in light infections than in heavy infections. This probably reflects increasing occupation of available muscle fibers as larger numbers of worms accumulate. Predilection sites appear to be influenced primarily by host species and secondarily by the age and level of infection.

Serological evidence of Trichinellosis in local pigs of Nepal.

In Nepal, animal husbandry is a major source of income. Pig husbandry is practiced in rural, peri-urban, and urban communities. Free ranging "back yard" pigs and the practice of feeding offal is a very common management practice which potentially allows for the transmission of trichinellosis; however, this zoonosis has never been reported from this region. A total of 425 serum samples were collected from local pigs. These were initially screened by ELISA after which positive samples were examined by Western blot. This procedure identified two samples which had clear specific bands for Trichinella; however, muscle samples tested by HCL-pepsin digestion were found to be negative. If these highly specific serological analyses are confirmed, this would be the first report of trichinellosis in Nepal and a prevention program should be initiated to limit the access of pigs to open garbage dumps which exist both in towns and on farms.

Effects of raw biles and their non-protein fractions from fox, pig, sheep and chicken on the survival of Trichinella spp. in vitro.

The in vitro differential effect of fox, pig, sheep and chicken bile and corresponding non-protein fractions at various concentrations on the motility of released muscle larvae of Trichinella spiralis, Trichinella nativa and Trichinella nelsoni was examined. In many cases, the percentages of motile (live) larvae of the three Trichinella species cultured in the presence of the non-protein fractions of bile from the study animals were significantly higher (p<0.001) compared to their respective control cultures. In addition, the percentages of motile (live) larvae of all Trichinella species cultured in the presence of the non-protein fraction of bile at every concentration from all study animals were significantly higher (p<0.001) compared to their respective cultures in the presence of raw bile. Not only did results with non-protein fractions of bile differ from those with raw bile, but also the non-protein fraction, with increased dilution, showed a decrease in the percentages of motile larvae while the opposite was true with the raw bile (p<0.001). These observations indicate that the non-protein fraction of bile prolongs the in vitro survival of larvae.

Experimental studies in pigs on Trichinella detection in different diagnostic matrices.

A total of 72 specific pathogen-free (SPF) and Iberian pigs (three animals per group) were inoculated with 200, 1000 or 20,000 muscle larvae of T. spiralis, T. nativa, T. britovi and T. pseudospiralis. For each animal, the muscle larva burden was evaluated in nine muscle samples by digestion. The anti-Trichinella IgG kinetics in blood samples, taken twice prior and at days 5, 10, 15, 20, 25, 30, 40, 50 and 60 post-inoculation, and in muscle juice, obtained at necropsy, was evaluated by an ELISA using an excretory/secretory antigen. The mean larval recovery rate in SPF/Iberian pigs corresponded with the level of inoculum dose, and tongue, diaphragm and masseter were identified as predilection muscles. In SPF and Iberian pigs receiving 20,000 larvae of T. spiralis, an earlier seroconversion was detected from day 25 post-inoculation. At a 10-fold dilution, the muscle juice showed a good test agreement with blood serum.