Kapel, C.M.O.

Petrakos, M.

Styliara, M.

Theodoropoulos, G.

English

Copenhagen University Research Information System

u n i ve r s i t y  o f  co pe n h ag e n  Københavns UniversitetEffect of fox, pig, sheep, and poultry bile on the establishment of domestic and sylvaticspecies of Trichinella in ratsTheodoropoulos, G.; Styliara, M.; Petrakos, M.; Kapel, C.M.O.Published in:ParazitologiyaDOI:10.1017/S003118200300307XPublication date:2003Document versionPublisher's PDF, also known as Version of recordCitation for published version (APA):Theodoropoulos, G., Styliara, M., Petrakos, M., & Kapel, C. M. O. (2003). Effect of fox, pig, sheep, and poultrybile on the establishment of domestic and sylvatic species of Trichinella in rats. Parazitologiya, 126, 461-464.https://doi.org/10.1017/S003118200300307XDownload date: 02. Feb. 2020Eﬀect of fox, pig, sheep, and poultry bile on theestablishment of domestic and sylvatic species ofTrichinella in ratsG. THEODOROPOULOS1*, M. STYLIARA1, M. PETRAKOS1and C. M. O. KAPEL21Department of Anatomy and Physiology of Farm Animals, Faculty of Animal Science, Agricultural University of Athens,75 Iera Odos,Votanikos, Athens 11855, Greece2Danish Centre for Experimental Parasitology, The Royal Veterinary and Agricultural University, Dyrlaegevej 100,DK-1870 Frederiksberg C, Denmark(Received 14 September 2002; revised 13 December 2002; accepted 16 December 2002)SUMMARYMost sylvatic species of Trichinella are known to have poor infectivity to rats, but in the present study oral administrationof bile from other hosts appeared to modify this infectivity. A total of 75 rats were inoculated in groups of 25 with 3 speciesof Trichinella (T. spiralis, T. nativa, and T. nelsoni) and each group of rats was given per os daily doses of bile from pig,sheep, chicken and fox respectively (4r5 rats). As a control 1 group of 5 rats was given daily doses of water. Whereas, theaddition of bile did not increase the establishment of T. spiralis, fox bile had a signiﬁcant positive eﬀect on the establish-ment of muscle larvae of T. nativa and T. nelsoni. Addition of bile to cultures of the same Trichinella species had an overallnegative eﬀect on the in vitro survival of larvae. The present observation that carnivore bile favours the establishmentof sylvatic Trichinella may explain why carnivores are equally receptive to all Trichinella species.Key words: Trichinella spiralis, Trichinella nativa, Trichinella nelsoni, bile, rats.INTRODUCTIONFoxes have a high susceptibility to all species ofTrichinella spp. (Kapel, 2000) and as amain reservoirin Europe (Pozio, 1998) they are often used in therole of indicator population for monitoring wildlifetrichinellosis. This uniform high infectivity of theTrichinella species is markedly diﬀerent from whatis found in rodents and herbivorous animals wheredomesticTrichinella spiralis has a signiﬁcantly higherinfectivity than sylvatic species (Kapel, 2000). Themechanisms underlying these diﬀerences have notbeen investigated in detail up to now. It is knownthat chickens are resistant to infective T. spiralis lar-vae and this resistance has been linked to the del-eterious eﬀects that extracts from the small intestine,pancreas, and bile of chickens have on the larvae(Barriga, 1981). Other authors (Smyth, Gemmell &Smyth, 1969) have found that diﬀerences in the com-position of bile between deﬁnitive host species mayinﬂuence host speciﬁcity in Echinococcus granulosus.The objective of the present study was to evaluatethe eﬀect of bile from fox, pig, sheep, and poultryon the infectivity to rats of domestic and sylvaticTrichinella species. In addition, the in vitro survivalof muscle larvae was monitored in cultures wherebile from the same animals was added.MATERIALS AND METHODSSpecies and source of muscle larvae and bileThe Trichinella species used were T. spiralis(ISS004), T. nativa (ISS042), and T. nelsoni(ISS037), which have beenmaintained by serial pass-ages in mice. Larvae were released from thoroughlyminced mouse muscle tissue by digestion (1 L H2O,10 g 1 : 10 000 IU Pepsin, 10 ml of 37% HCl) for 1 hat 37 xC.Bile was collected from foxes, pigs, sheep, andchicken and stored at x80 xC prior to experimen-tation.In vivo studyA total of 75 female inbred 10-week-old Wistar ratswere inoculated per os through a feeding needle with1000 larvae in 3 groups of 25 rats with respectivelyT. spiralis, T. nativa and T. nelsoni and divided fur-ther in 5 groups of 5 rats. Four groups received di-luted bile from fox, pig, sheep or chicken respectivelyand 1 control group received water. For 21 days, ratswere administered daily portions (0.5 ml) of bile(diluted 1 : 2.6 in tap water) or water respectively.All inoculations during the experiments were doneafter anaesthesia with CO2. The rats were sacriﬁced* Corresponding author: Department of Anatomy andPhysiology of Farm Animals, Faculty of Animal Science,Agricultural University of Athens, 75 Iera Odos,Votani-kos, Athens 11855, Greece. Tel : +30 210 5294387. Fax:+30 210 5294388. E-mail : gtheo@aua.gr461Parasitology (2003), 126, 461–464. f 2003 Cambridge University PressDOI: 10.1017/S003118200300307X Printed in the United Kingdom5 weeks p.i. and the reproductive capacity index(RCI: larvae recovered/larvae inoculated) was cal-culated for each rat.In vitro studyLarvae of each Trichinella species were culturedin vitro by using the method described by Kapel &Gamble (2000). The culturemediumwasDulbecco’smodiﬁed Eagle’s medium (DMEM) supplementedwith 2 mM L-glutamine, 4500 mg glucose/l, 25 mMHEPES and antibiotics (50 U penicillin/ml+50 mgstreptomycin). For each species of Trichinella six30 ml culture ﬂasks were used. Each ﬂask contained500 larvae in 5 ml of medium (concentration: 100larvae/ml). Bile from each of the study animals wasadded at an amount of 0.5 ml in each of the 3 ﬂasksat a ﬁnal concentration of 1 : 10 while the remaining3 ﬂasks served as controls. The ﬂasks were kept at37 xC. The number of motile (live) and non-motile(dead) larvae in sample optical areas was counteddaily under a dissecting microscope at 30 magniﬁ-cations, and the percentage of motile (live) larvae foreach ﬂask was calculated. Counts were done until alllarvae were immotile (dead).Statistical analysisProportions of motile (live) larvae of eachTrichinellaspecies cultured in vitro in the presence of each bileor not (control) were analysed using ANOVA. Themethod is appropriate since the binominal distri-bution can be approximated by the normal for largesamples as in this case (n=100). Since interactionterms were found signiﬁcant the eﬀect of each com-bination was tested separately.Multiple comparisonswere used to identify signiﬁcant diﬀerences and testswere performed using Tukey’s honest signiﬁcantdiﬀerence (HSD) test (Miller, 1981). The infectivity(RCI) of each Trichinella species to rats adminis-tered with bile or water (control) was analysed usingANOVA as well. The Analysis was performed usingSPSS v. 11.RESULTSIn vivo studyThe infectivity (RCI) of the 3 Trichinella species torats administered with bile or water is presentedin Table 1. Infection was established in all groupsof rats inoculated with T. spiralis, but no signiﬁcantdiﬀerences in RCI were observed between the vari-ous groups of rats administered with bile or water.Infection with T. nativa was established only in thegroup of rats administered with fox bile. Infectionwith T. nelsoni was established in the groups of ratsadministered with fox, pig, and sheep bile whereasRCI was signiﬁcantly higher (P<0.001) only in thegroup of rats administered with fox bile.Table 1. Reproductive capacity index (RCI: larvae recovered/larvae inoculated) in ratsInoculumT. spiralis T. nativa T. nelsoniRCI Mean¡S.D. RCI Mean¡S.D. RCI Mean¡S.D.Fox bile 13.770 66.963¡93.84 0.027 0.045¡0.03 0.014 0.0186¡0.0097*15.300 0.016 0.01333.525 0.093 0.01038.520 0.065 0.034233.700 0.024 0.022Pig bile 18.150 9.950¡8.41 0.000 0.000¡0.00 0.004 0.0038¡0.001310.350 0.000 0.0051.350 0.0030.0020.005Sheep bile 5.400 15.510¡8.35 0.000 0.000¡0.00 0.005 0.0024¡0.001817.400 0.000 0.0038.250 0.000 0.00222.650 0.000 0.00023.850 0.000 0.002Chicken bile 12.825 13.894¡4.24 0.000 0.000¡0.00 0.000 0.000¡0.0018.000 0.000 0.0007.538 0.000 0.00017.213 0.000 0.000Water (Control) 12.150 12.435¡10.00 0.000 0.000¡0.00 0.000 0.000¡0.003.375 0.000 0.0003.300 0.000 0.00027.300 0.000 0.00016.050 0.000 0.0000.000* Signiﬁcant diﬀerence (P<0.001) with other RCI values in the same column.G. Theodoropoulos and others 462In vitro studyThe percentages of motile (live) larvae of the 3Trichinella species cultured in the presence of bilefrom any animal were signiﬁcantly lower (P<0.001)compared to their respective control cultures (Fig. 1).On the other hand, the percentage of motile (live)T. nelsoni larvae cultured in the presence of fox bilewas signiﬁcantly higher (P<0.01) than in the pres-ence of pig bile (P<0.01) or sheep bile.DISCUSSIONThe mechanisms that are responsible for diﬀerencesin host susceptibility to various Trichinella speciesare only poorly understood, but the enteric phaseof the life-cycle is a potential point at which theinteraction between host and parasite can be stopped(Kapel, 2000). During the intestinal phase of infec-tion both larvae and adults come in contact with bile,and the present study suggests that the compositionof the bile inﬂuences the infectivity as well as thein vitro survival of larvae.Several authors have demonstrated that T. nelsoniand T. nativa have indeed very low infectivity in ratsand that T. spiralis is highly infective (Pozio et al.1992; Kapel, 2000; Malakauskas, Kapel & Webster,2001). Whereas the present study did not demon-strate a further increase in the infectivity ofT. spiralis with addition of bile, the reproduction ofT. nativa and T. nelsoni was obviously improved byaddition of the fox bile. Although preliminary, thisobservation may reﬂect that most sylvatic species ofTrichinella initially have been adapted to the carni-vore gut environment, and that the domestic T.spiralis has developed an ability to reproduce with-out speciﬁc carnivore stimulation.The results of the in vitro study show that bile,regardless of its origin, had a deleterious eﬀect on themotility of the 3Trichinella species. Although severalfactors could be responsible for the increased mor-tality, the bile may stimulate the initial moultingprocesses leading to incomplete cuticle transform-ations that increase mortality.Themechanism explaining the action of bile on theinfectivity of Trichinella sp. is not known. Possibleactions of bile on Trichinella larvae are, the digestionof larvae as it has been shown in the case of chickenbile (Barriga, 1981), the inhibition of larvae byFig. 1. Percentages of motile (live) Trichinella spiralis, Trichinella nativa and Trichinella nelsoni larvae cultured in thepresence of bile from study animals.Eﬀect of fox bile on establishment of Trichinella in rats 463immune secretory IgA from bile (Jacqueline et al.1981), the alteration of the parasite’s behavioural andnutritional status by exposure to bile (Stewart et al.1987), the structural reorganization of the surfacecoat of larvae by exposure to bile (Smith, Selkirk &Gounaris, 2000), and the modiﬁcation of tyrosinephosphorylated proteins on the larval cuticle by incu-bation of the larvae with bile (Allegretti et al. 2001).Nevertheless, all the above mechanism explain thedetrimental eﬀect of bile on Trichinella larvae. Theexact mechanism explaining the favorable eﬀect ofthe feeding of carnivore bile on the reproductivecapacity of sylvatic Trichinella adult worms in situin the small intestine of the rats is not known. Appar-ently, the carnivore bile could survive the digestionby stomach enzymes and the absorption in the smallintestine in order to impose its favorable eﬀect on theparasite as observed in the present study.In conclusion, carnivore bile appears to favour theestablishment of sylvatic Trichinella spp.REFERENCESALLEGRETTI, S., BEAUMONT, V., ROBERT-GANGNEUX, F.,CREUZET, C., ROISIN, M. P. & DUPOUY-CAMET, J. (2001).Detection of tyrosine phosphorylated proteins inTrichinella spiralis L1 larvae. Parasite-Journal de laSocie´te´ Franc¸aise de Parasitologie 8, S59–S61.BARRIGA, O. O. (1981). Evidence, nature, and implicationsof the constitutive resistance to Trichinella spiralis ingallinaceous birds. American Journal of VeterinaryResearch 42, 1963–1965.JACQUELINE, E., CRINQUETTE, J., BOUT, D., BARROIS, J. &VERMES, A. (1981). Trichinella spiralis in rats: in vivoeffects of the bile and in vitro action of secretory IgAfrom bile.Annales de Parasitologie Humaine et Compare´e(Paris) 56, 395–400.KAPEL, C. M. O. (2000). Host diversity and biologicalcharacteristics of the Trichinella genotypes and theireffect on transmission. Veterinary Parasitology 93,263–278.KAPEL, C. M. O. & GAMBLE, H. R. (2000). Infectivity,persistence, and antibody response to domestic andsylvatic Trichinella spp. in experimentally infectedpigs. International Journal for Parasitology 30,215–221.MILLER, R. G. JR. (1981). Simultaneous Statistical Inference,2nd Edn. Springer-Verlag, NY.MALAKAUSKAS, A., KAPEL, C. M. O. & WEBSTER, P. (2001).Infectivity, persistence and serological response of nineTrichinella genotypes in rats. Parasite-Journal de laSocie´te´ Franc¸aise de Parasitologie 8, 216–222.POZIO, E. (1998). Trichinellosis in the European Union:epidemiology, ecology and economic impact.Parasitology Today 14, 35–38.POZIO, E., LA ROSA, G., ROSSI, P. & MURRELL, K. D. (1992).Biological characterisation of Trichinella isolatesfrom various host species and geographical regions.Journal of Parasitology 78, 647–653.SMITH, V. P., SELKIRK, M. E. & GOUNARIS, K. (2000).A reversible protein phosphorylation system is present atthe surface of infective larvae of the parasitic nematodeTrichinella spiralis. FEBS Letters 483, 104–108.SMYTH, J. D., GEMMELL, M. & SMYTH, M. M. (1969).Establishment of Echinococcus granulosus in the intestineof normal and vaccinated dogs. Indian Journal ofHelminthology Srivastava Commemorative volume:167–178.STEWART, G. I., DESPOMMIER, D. D., BURNHAM, J. & RAINES,K. M. (1987). Trichinella spiralis : behavior, structure,and biochemistry of larvae following exposure tocomponents of the host enteric environment.Experimental Parasitology 63, 195–204.G. Theodoropoulos and others 464

Effect of fox, pig, sheep, and poultry bile on the establishment of domestic and sylvatic species of Trichinella in rats

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Effect of fox, pig, sheep, and poultry bile on the establishment of domestic and sylvatic species of Trichinella in rats

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