Experimental and immunological comparison of Trichinella spiralis and Trichinella nativa

Trichinella is a widely spread zoonotic nematode parasite. Human infection occurs after eating under-cooked meat, typically pork, wild boar or horse, containing infective Trichinella-larvae. Heavy infection may be fatal. Eleven genotypes of Trichinella have been differentiated by PCR-based methods. In Finland, four species have been confirmed: Trichinella spiralis, T. nativa, T. britovi and T. pseudospiralis. Trichinella spiralis occurs in the domestic cycle, whereas T. nativa is the most common species infecting sylvatic animals in Finland. These two species have differences in their infectivity in different hosts and also in their resistance to freezing; T. spiralis does not survive at -20ºC, but in certain host species T. nativa does. To learn more about the two most common Trichinella species in Finland, T. spiralis and T. nativa, these species were compared in this thesis both in vivo and in vitro. Raccoon dogs (Nyctereutes procyonoides), common Trichinella hosts in the Finnish fauna, and complement factor C6-deficient rats, in which the complement membrane attack complex was inactivated, were used as in vivo experimental models. Raccoon dogs are favourable hosts for Trichinellae - they do not suffer from any clinical signs of trichinellosis even when the infection doses are fairly high. No significant differences in the course of infection were noted between T. spiralis and T. nativa. A peak of eosinophilic granulocytes was observed on the second week of infection, and weight loss and anaemia were more common in the infected group than in controls. Clear morphological differences between the species were observed in the tissue capsules. Trichinella spiralis capsules were lemon-shaped, whereas the capsules of T. nativa were more spherical and had more intense inflammation around them. Specific antibodies were recognized after two weeks of infection in both ELISA and Western blot analysis. The role of the complement system in the host's defence against Trichinella was evaluated in an experimental infection of normal and complement factor C6-deficient rats with both T. spiralis and T. nativa. Trichinella nativa has a lower infectivity in rats. The survival of larvae in normal and C6-deficient serum was also observed in vitro. No effect of C6 deficiency was noted in either species in vivo or in vitro. When exposed to human serum, no binding of complement factors C1q, C3, C8 or C9 to the outermost layer of the cuticle of adults, newborn larvae or muscle larvae was observed. This suggests that Trichinellae have mechanisms for evasion of complement, and that membrane attack complex does not explain the different infectivity of these two species in rats. To investigate differences between T. spiralis and T. nativa, soluble proteins of their crude larval extracts were analysed by two-dimensional electrophoresis. Clearly different protein patterns were seen. Freezing was also shown to cause some changes in protein patterns. After MALDI-TOF analysis, we were not, however, able to identify the different proteins in database searches. Immunological differences were observed in two-dimensional Western blot analysis.Trichinella-sukkulamato on laajalle levinnyt loinen niin isäntäeläinkirjoltaan kuin maantieteellisestikin. Se on zoonoottinen, se voi tarttua sekä ihmisiin, että eläimiin. Voimakkaat infektiot voivat aiheuttaa kuoleman. Tartunnan voi saada syömällä raakaa tai huonosti kypsennettyä, tyypillisesti sian, villisian tai hevosen, lihaa. Trichinella-genotyyppejä tunnetaan tällä hetkellä 11. Ne voidaan erottaa toisistaan PCR-menetelmin. Suomesta on löydetty tähän mennessä neljä lajia: Trichinella spiralis, T. nativa, T. britovi ja T. pseudospiralis. Näistä T. spiralis esiintyy kotieläimillä, kun taas T. nativa on tavallisin laji villieläinten infektioissa. Nämä kaksi lajia eroavat tartuntakyvyltään eri isäntäeläimiin. Myös pakastuksen kestävyydessä on eroja: T. spiralis ei selviydy kauan -20ºC:ssa, kun taas eräiden isäntäeläinten lihaksistossa T. nativa selviytyy useita kuukausia, tai jopa vuosia. Tässä tutkimuksessa vertailtiin kahta Suomen yleisintä Trichinella-lajia, T. spiralis ja T. nativa, in vivo ja in vitro. Supikoira (Nyctereutes procyonoides), yleinen Trichinella-loisten isäntälaji Suomen luonnossa, ja rotta toimivat in vivo koemalleina. Supikoirat ovat suotuisia isäntiä Trichinella-lajeille - niillä ei esiinny kliinisiä trikinelloosi-oireita suurehkoillakaan infektioannoksilla. Trichinella spiralis ja T. nativa infektioiden välillä ei havaittu merkittävää eroa tällä isännällä. Toisen infektioviikon lopulla todettiin piikkimäinen nousu veren eosinofiilisten granulosyyttien määrässä. Painon väheneminen ja anemia olivat yleisempiä infektoidulla eläimillä verrattuna kontrollieläimiin. Histologiselta morfologialtaan T. spiralis -kudoskapselit olivat sitruunan muotoisia, kun taas T. nativa -kapselit olivat pyöreämpiä, ja niiden ympärillä oli voimakas tulehdusreaktio. Spesifejä vasta-aineita todettiin kahden infektioviikon jälkeen sekä ELISA-, että Western blot -menetelmillä. Elimistön puolustusmekanismeihin kuuluvan komplementtijärjestelmän merkitystä Trichinella-infektiossa tutkittiin infektoimalla normaaleja ja komplementtitekijä C6-puutteisia rottia T. spiralis ja T. nativa -loisilla. Trichinella nativa infektoi rottia heikommin kuin T. spiralis. Trichinella-toukkien elävyyttä tutkittiin normaalissa ja C6-puutteisessa seerumissa myös in vitro. C6-puute ei vaikuttanut kummankaan Trichinella-lajin selviytymiseen in vivo tai in vitro. Seerumin komplementtitekijöiden C1q, C3, C8 tai C9 sitoutumista ei havaittu loisen uloimmassa kerroksessa aikuisilla, vastasyntyneillä tai lihasvaiheen loisilla. Trichinella-loiset pystyvät estämään komplementin sitoutumista pinnalleen. Komplementtijärjestelmän membraaneja tuhoava kompleksi ei selitä lajien erilaista infektiivisyyttä rottiin. Trichinella spiralis ja T. nativa lihastoukkien liukoisten proteiinien eroja analysoitiin kaksiulotteisella geelielektroforeesilla. Elektroforeesikuviot erosivat toisistaan selvästi. Myös loisten pakastuskäsittelyllä oli vaikutusta geelikuvioihin. Tietokantahauissa, MALDI-TOF -massaspektrometrian jälkeen, eroavia proteiineja ei kuitenkaan voitu tunnistaa. Immunologisia eroja havaittiin kaksiulotteisella Western blot -menetelmällä

Comparison of commercial ELISA tests for the detection of Toxoplasma antibodies in the meat juice of naturally infected pigs

Toxoplasmosis is a globally distributed protozoal zoonosis. Pigs are considered an important reservoir of Toxoplasma gondii and pork a major infection source of human toxoplasmosis. ELISA methods are commonly used diagnostic tools for detecting Toxoplasma infections. They are also used for slaughterhouse-based serological monitoring of toxoplasmosis in pigs to identify positive farms. The methods used are non-standardised with varying sensitivity and specificity. In our study, four commercial ELISA tests for the detection of Toxoplasma antibodies in the meat juice of slaughter pigs were compared with a modified agglutination test (MAT) as a reference. The cut-off values of the ELISA tests provided by the manufacturer varied between 0.20 and 0.50, and clearly influenced prevalence. The sensitivity of tests I, II and III varied between 96.4 and 78.6. Sensitivity was unacceptably low (3.6) for test IV (cut-off=0.30). Tests I, II and III had the highest accuracy and the best agreement with the reference test when a cut-off of 0.30 was used. Test II and III showed very good agreement (K=0.92 and 0.84, respectively) with the MAT. A very strong correlation (Pearson correlation >0.89) was observed between the S/P values of tests I, II and III. Our results demonstrate that the test and cut-off value used influence the results of the apparent seroprevalence studies. (C) 2017 Elsevier B.V. All rights reserved.Peer reviewe

Dispersal of taeniid eggs : Experimental faecal contamination of forest environment followed by DNA detection in wild berries

Publisher Copyright: © 2022 The Author(s)To understand Taeniidae epidemiology, the principles of egg-dispersion dynamics under natural conditions must be known. In this study, non-zoonotic Taenia laticollis was used as a model parasite for the family Taeniidae (including Echinococcus spp.). An experiment to investigate dispersion from contaminated faeces to the surroundings was performed both with bilberries (Vaccinium myrtillus) and lingonberries (Vaccinium vitis-idaea), both of which are commercially harvested wild berries in Finland. For this experiment, 30 g of fox faeces was inoculated with 30,000 T. laticollis eggs for the bilberry experiment and 100,000 eggs for the lingonberry experiment. The faecal material was placed in the middle of good berry growth areas in four locations for bilberries and eight locations for lingonberries. After 41-42 days, berries at different distances (0-15 m) from the original contamination spot were collected and delivered to our laboratory. DNA was extracted from washed and sieved material and analysed using T. laticollis-specific semi-quantitative SYBR Green real-time polymerase chain reaction (qPCR). Taenia laticonis-specific DNA was recovered from 67% (8/12) of bilberry samples but not reliably from any of the lingonberry samples 0% (0/24), although the exposure dose was higher for those. The qPCR results suggest that under natural conditions, taeniid egg dispersion from the contamination spot is demonstrated but attachment is berry specific. The surface of bilberries may be more adhesive for taeniid eggs than the waxier and harder pericarp of the lingonberries or there might be a difference in the dispersal mechanism caused by different biotopes.Peer reviewe

The first Linguatula serrata case in an imported dog in Finland

Non peer reviewe

Toxoplasma gondii in the Subarctic and Arctic

Volume: 52Non peer reviewe

Seroprevalence of Encephalitozoon cuniculi and Toxoplasma gondii antibodies and risk-factor assessment for Encephalitozoon cuniculi seroprevalence in Finnish pet rabbits (Oryctolagus cuniculus)

Background Neurological signs, such as head tilt, torticollis, paralysis, and seizures, are common in rabbits. Differential diagnoses include two zoonotic infections caused by the microsporidial fungi Encephalitozoon cuniculi and the apicomplexan protozoa Toxoplasma gondii. Both infections are mainly latent in rabbits but may cause severe or even fatal disease. Although several international studies have reported the seroprevalence of these pathogens in different commercial rabbit populations, similar prevalence studies and risk-factor analyses among family-owned pet rabbits are uncommon and lacking in Scandinavia. We sought to estimate the seroprevalence and possible risk factors for E. cuniculi and T. gondii among Finnish pet rabbits. We used ELISA to measure E. cuniculi IgG seroprevalence of 247 rabbits and modified direct agglutination test for T. gondii seroprevalence of 270 rabbits. Samples were collected as part of the Finnish Pet Rabbit Health Research project. Internet-based questionnaires (n = 231) completed by the rabbit owners were used for risk-factor analysis. Results The apparent seroprevalence of E. cuniculi was 29.2% and true seroprevalence of T. gondii 3.9%. Risk factors were analysed only for E. cuniculi due to the low T. gondii seroprevalence. The final multivariable logistic regression model revealed that rabbits spending the whole summer outdoors had a higher risk of being E. cuniculi seropositive than rabbits with limited outdoor access. Additionally, rabbits living in households with only one or two rabbits had higher risk of being E. cuniculi seropositive than those in multi-rabbit households. Conclusions Nearly one third of Finnish pet rabbits participating in this study had E. cuniculi IgG antibodies, indicating previous exposure to this pathogen. The prevalence is similar to that reported previously in clinically healthy rabbit populations in UK and Korea. While the seroprevalence of T. gondii was low (3.9%), antibodies were detected. Therefore, these zoonotic parasitic infections should be considered as differential diagnoses when treating rabbits.Peer reviewe

Risk factors for equine intestinal parasite infections and reduced efficacy of pyrantel embonate against Parascaris sp.

Gastrointestinal parasites, Parascaris sp. and strongyles, are common in young horses worldwide and control of these parasites is challenged by increasing anthelmintic resistance. Our aim was to identify risk factors for these infections as well as to assess the efficacy of fenbendazole (dose 7.5 mg/kg) and pyrantel embonate (dose 19 mg/kg) against Parascaris sp. We also evaluated association between owner observed symptoms and patent infections with these parasites. Fecal samples were collected from 367 young horses in Finland and a questionnaire study was conducted. Fecal egg counts were performed by Mini-FLOTAC (R) method. Univariable logistic regression models using patent infection status (Yes/No), separately for Parascaris sp. and strongyle infections as an outcome were run initially to screen potential risk factors collected by the questionnaire. After the initial screening, multiple logistic regression models were constructed and run to account for correlated data structure, risk factors and potential confounders simultaneously. Two significant risk factors for a patent Parascaris sp. infection were found: breeding farm size (p = 0.028) and frequency of horse movements (p = 0.010). Horses originating from large breeding farms were more likely (OR = 2.47, 95% confidence interval (CI) 1.10-5.51) to shed Parascaris sp. eggs upon relocation to training stables compared to horses originating from small breeding farms. Horses living in farms with frequent horse movements to other premises had higher odds (OR = 3.56, 95% CI: 1.35-9.39) of a patent Parascaris sp. infection compared to farms with less frequent horse movements. Risk factors for patent strongyle infection included age (p <0.001) and season (p = 0.017). Horses were less likely (OR = 0.27, 95% CI: 0.10-0.66) to shed strongylid eggs during the spring compared to the winter. Horses excreting over 200 ascarid eggs per gram were included in the anthelmintic efficacy trial. A mean FECR less than 90% was interpreted as presence of anthelmintic resistance. The mean FECR was 98.5% (95% CI: 95.8-100) and 68.0% (95% CI: 52.7-83.3) in the fenbendazole (n = 31) and pyrantel (n = 26) treatment groups, respectively. In conclusion, we identified two new risk factors for patent Parascaris sp. infection; breeding farm size and frequency of horse movements. Reduced efficacy of pyrantel against Parascaris sp. was observed for the second time in Europe. A relatively high Parascaris sp. prevalence in yearlings (34%) and two-year-olds (20%) was observed, which has not been reported earlier. An association between symptoms and a patent Parascaris sp. infection was observed in foals.Peer reviewe

The first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the Nordic countries

Abstract Background Leishmania spp. are zoonotic protozoans that infect humans and other mammals such as dogs. The most significant causative species in dogs is L. infantum. In dogs, leishmaniosis is a potentially progressive, chronic disease with varying clinical outcomes. Autochthonous cases of canine leishmaniosis have not previously been reported in the Nordic countries. Results In this report we describe the first diagnosed autochthonous cases of canine leishmaniosis in Finland, in which transmission via a suitable arthropod vector was absent. Two Finnish boxers that had never been in endemic areas of Leishmania spp., had never received blood transfusions, nor were infested by ectoparasites were diagnosed with leishmaniosis. Another dog was found with elevated Leishmania antibodies. A fourth boxer dog that had been in Spain was considered to be the source of these infections. Transmission occurred through biting wounds and semen, however, transplacental infection in one of the dogs could not be ruled out. Two of the infected dogs developed a serious disease and were euthanized and sent for necropsy. The first one suffered from membranoproliferative glomerulonephritis and the second one had a chronic systemic disease. Leishmania sp. was detected from tissues by PCR and/or IHC in both dogs. The third infected dog was serologically positive for Leishmania sp. but remained free of clinical signs. Conclusions This case report shows that imported Leishmania-infected dogs may pose a risk for domestic dogs, even without suitable local arthropod vectors