Animal models for Opisthorchis viverrini infection

We investigated the utility of various animal models for the study of opisthorchiasis in humans and its common sequel of cholangiocarcinoma (CCA). Rats, mice, gerbils, and hamsters were infected with Opisthorchis viverrini metacercariae. Worms from the infected animal hosts were recovered from livers and counts made of eggs per gram of feces. Worms were observed in and recovered from hamsters and gerbils but not rats and mice. The recovered worms from the infected gerbils were larger and more physiologically developed than those from the infected hamsters. The results suggest that gerbils are more susceptible to infection by Opisthorchis viverrini and thus more suitable for modeling opisthorchiasis and its connection to CCA.     

Growth of a Japanese isolate ofTaenia crassiceps in intermediate and definitive hosts

Higher infection rates were observed in gerbils and voles than in ICR mice after oral inoculation with eggs of a Japanese isolate ofTaenia crassiceps. Asexual reproduction ofT. crassiceps cysticerci was observed in all gerbils and voles infected i.p. with the cysticerci. However, ICR mice and Wistar rats were not suitable for the asexual proliferation ofT. crassiceps. The hooks of cysticerci from mice were smaller than those from gerbils. In experimentally infected puppies, parasite development was noted as follows: strobilation and initial differentiation of the genital primordia on day 7 postinoculation (p.i.), appearance of the testes on day 9, observation of the ovaries on day 10, and development of the lateral branches of the uterus on day 15. The prepatent period was 27–31 days. After day 15 p.i., most of the worms were recovered from the middle third of the small intestine. The number of proglottids shed per day by each strobila was about 1. The number of eggs contained in a gravid segment was about 13000.     

Establishment and survival of the strobilar stage ofTaenia crassiceps in hamsters,gerbils, and mice,with reference to different helminth isolates

Following the oral administration of metacestodes of two isolates ofTaenia crassiceps, the enteral establishment and survival of the strobilar stage were examined in golden hamsters (Mesocricetus auratus), Mongolian gerbils (Meriones unguiculatus) and laboratory mice. The origin of the isolates wasMicrotus montebelli caught in Japan in 1985 orClethrionomys rutilus captured on St. Lawrence Island, Bering Sea, in 1988 (abbreviated as JPN and SLI isolates), respectively. The enteral establishment of the SLI isolate was distinctly higher than that of the JPN isolate in golden hamsters and mice, whereas the difference was marginal in Mongolian gerbils. All initially-established parasites survived to become gravid adults in prednisolone-treated golden hamsters and Mongolian gerbils; the average recovery of cestodes of the SLI and JPN isolates were 55.8%–76.7% vs 11.7%–35.0% in the former and 28.0%–52.7% vs 25.8%–32.2% in the latter. The distinctly higher level of enteral establishment of the SLI isolate in golden hamsters makes available a model for quantitative studies on parasite-host relationships in experimental taeniasis.This study was supported by grants 01790490, 02044083 and 03044016 from the Ministry of Education, Science and Culture, Japan     

Homologous and heterologous immunization against the metacestodes ofTaenia saginata andTaenia taeniaeformis in cattle and mice

Summary Cattle and mice were immunized against infection withTaenia saginata andTaenia taeniaeformis, respectively, using antigens obtained from both homologous and heterologous species of cestodes. Mice were protected against infection withT. taeniaeformis when they were immunized intramuscularly or orally with either a somatic antigen extracted from the metacestodes or an excretory/secretory (E/S) antigen collected during the in vitro culture of oncospheres ofT. taeniaeformis. Also, the intramuscular or oral immunization of mice with the E/S antigens from the oncospheres ofT. saginata was highly effective in inducing protection against infection withT. taeniaeformis, as was intramuscular immunization with a somatic antigen extracted from the metacestodes ofTaenia crassiceps and prior infection with viable metacestodes ofT. crassiceps. Furthermore, three-month-old calves developed a protective immunity against infection withT. saginata when they were immunized intramuscularly with the E/S products of oncospheres of the homologous parasite or a heterologous parasite,T. taeniaeformis. In addition, the E/S products of the heterologous parasite,T. taeniaeformis, as well as the homologous parasite,T. saginata, were highly effective when used to immunize pregnant heifers, either intramuscularly or via the intramammary route, resulting in a passive transfer of immunity againstT. saginata to newborn calves.     

Comparative studies on animal models for <Emphasis Type="Italic">Opisthorchis viverrini</Emphasis> infection: host interaction through susceptibility and pathology

Syrian hamsters and gerbils are animal models for Opisthorchis viverrini infection. In both models, the parasites develop into adults with different pathologies of the hepatobiliary system. However, no comparative pathological studies have yet been completed. We therefore investigated host interaction through the susceptibility and pathological changes of Syrian hamsters and gerbils infected with 50 O. viverrini metacercariae for 30, 60, and 90 days post-infection. Animals were sacrificed at each time point for comparative study. Susceptibility and infectivity were investigated through worm burden. Parasite morphology and reproductive organs were stained with carmine and observed under light microscopy. Reproductive organs and eggs per worm were counted to confirm worm maturity. Bile acid components of both animal groups were analyzed by thin-layer chromatography. The results showed that infection in gerbils was of greater severity than in Syrian hamsters by observation of bile obstruction, enlargement of the gallbladder and common bile duct, and generation of fibrosis and cirrhosis. The worm burden of infected gerbils was lower than that observed in Syrian hamsters. Infectivity in both Syrian hamsters and gerbils was 100% with infection by 50 metacercariae; whereas with 10 metacercariae, the infectivity in gerbils was zero to very low, but still 100% in Syrian hamsters. The largest body size of worms, and the largest ovary and testes areas, was correlated with eggs per gram of feces and eggs per worm. The bile acid components cholic acid and chenodeoxycholic acid were undetectable in gerbils. The present study suggests that although Syrian hamsters, usually the host selection for an animal model, are susceptible to O. viverrini infection, infected gerbils produce worms that mature more rapidly, have larger body sizes, and more fully developed reproductive organs; this may be caused by the difference in bile acid components.     

Comparison of infectivities of six tick-derived isolates of Borrelia burgdorferi for rodents and ticks.

The infectivity and dissemination to the skin of six isolates of Borrelia burgdorferi were evaluated by inoculating them into groups of deer mice (Peromyscus maniculatus), hamsters, and Swiss Webster mice. Rodent infection was assayed by culture of ear punch biopsy specimens taken at 4, 8, and 12 weeks postinoculation (p.i.). Spirochetes were detected in biopsy specimens from individuals of all three host species that had been inoculated with four isolates (CA3, CA4, CA7, and CA8). Ear punch biopsy specimens taken from Swiss Webster mice at 12 weeks p.i. yielded an additional reisolate (CA2), even though these animals did not seroconvert. The remaining isolate (CA9) was not recovered from any host. However, two deer mice and all hamsters and Swiss Webster mice inoculated with CA9 seroconverted. All six isolates were of low infectivity to ticks when inoculated intramuscularly into hosts. Only 4 (1.6%) of 250 Ixodes pacificus larvae acquired and transstadially maintained infection from hosts inoculated intramuscularly. Infectivity of three isolates for ticks also was tested in Swiss Webster mice injected intradermally. The mean prevalences of infection in xenodiagnostic ticks fed on these mice at 4 weeks p.i. were 47.9, 1.2, and 2.2% for isolates CA4, CA7, and CA8, respectively. The mean prevalences of infection for ticks fed on the same mice at 12 weeks p.i. were 36.4, 11.8, and 20.4%, respectively. Such differences in the infectivity and rate of dissemination of individual isolates of B. burgdorferi should be considered during studies of reservoir and vector competence.     

Immunology and morphology studies on the proliferation of in vitro cultivatedEchinococcus multilocularis metacestodes

The larval stage ofEchinococcus multilocularis causes alveolar echinococcosis (AE) in various mammals, including humans. Traditionally metacestodes are maintained in the laboratory by serial transplantation passages into susceptible animals such as mice or gerbils. However, in animal models it has always been difficult to draw definite conclusions about the factors modulating metacestode differentiation, and investigations on gene expression and respective regulation have been hampered by the complexicity of the host-parasite interplay. This paper describes the maintenance and proliferation ofE. multilocularis metacestodes as well as the formation of protoscolices in a chemically defined medium devoid of host influence. The interactive role of a heterologous human cell line (CACO2) in the in vitro development of metacestodes was also assessed. The morphology and ultrastructure of in vitro-generated metacestodes was studied using scanning (SEM) and transmission electron microscopy (TEM). Different cultivation procedures were analyzed in terms of expression of B-and T-cell epitopes and of the relevant laminated layer-antigen Em2; the exact localization of this antigen was further demonstrated by immunogold electron microscopy.     

Opportunistic nature of the mammalian microsporidia: experimental transmission of Trachipleistophora extenrec (Fungi: Microsporidia) between mammalian and insect hosts

Spores of Trachipleistophora extenrec, originally isolated from the muscles of the Madagascan insectivore Hemicentetes semispinosus and maintained by serial passage in severe combined immunodeficiency (SCID) mice, were fed to larvae of the Egyptian cotton leafworm Spodoptera littoralis. Extensive infection of larval tissues ensued and caused larval and pupal mortality. The development of T. extenrec in the insect host, studied both by light and electron microscopy, followed generally the same life cycle as in the mammalian host. However, some differences in the fine structure of the parasite grown in both types of hosts were found. Spores isolated from the insect host caused infection of SCID mice when injected intramuscularly. Our results suggest that T. extenrec may be originally an insect microsporidian. This likelihood is corroborated by its structural similarity and phylogenetic relationship to two other microsporidia having insects either as unique hosts (Vavraia culicis) or being able to infect both mammalian and insect host (Trachipleistophora hominis).     

Apoptosis induced by gamma irradiation of<Emphasis Type="Italic"> Taenia solium</Emphasis> metacestodes

Gamma irradiation of food is considered a possible approach to control food-borne diseases. In cysticercosis, previous studies have shown that irradiating (with 0.3 kGy) pork infected with Taenia solium larvae completely inhibits growth of the parasite. This study was conducted to evaluate the mechanisms that induce the effect of gamma irradiation on metacestodes of T. solium. Metacestodes were obtained from several infected pigs and irradiated with a dose of 0.3 kGy. The viability of the metacestodes was evaluated by their capacity to evaginate in vitro and in vivo development to tapeworms after they were orally infected into prednisolone-treated golden hamsters. Using the typical ladder pattern of fragmented DNA and the TdT-mediated DUTP-nick-end labeling assay, apoptosis was evaluated in metacestodes after irradiation and in the scolices and tapeworms recovered from infected hamsters at 21 days post-infection. Apoptosis was observed in the structure of scolices obtained from hamsters at 21 days post-infection with irradiated metacestodes, This study provides evidence of the existence of apoptosis in the irradiated metacestodes of T. solium and helps elucidate the possible mechanisms that are involved when gamma irradiation inhibits the normal development of the T. solium metacestode into the adult worm.     

Apoptosis patterns in experimental Taenia solium and Taenia crassiceps strobilae from golden hamsters

Apoptosis or programmed cell death (PCD) patterns of two taeniid species, Taenia solium and Taenia crassiceps, were explored in adult tapeworms grown in golden hamsters. Animals were fed either ten viable T. solium cysticerci from naturally infected pigs or from T. crassiceps WFU strain maintained in Balb/c mice. Adult strobilae were recovered from the intestine at different times after infection and either frozen at –70°C or fixed in paraformaldehyde–glutaraldehyde. Frozen sections were processed using the DNA fragmentation fluorescent TUNEL reagents and examined in an epifluorescent microscope. Fixed tissues were processed for light and electron microscopy. Typical apoptotic cells were found in the central core of scolex and strobilar tissues, mainly in the germinal tissue and subtegumentary areas. By the TUNEL technique, cells exhibited the characteristic fluorescent images of condensed nuclear chromatin. By light microscopy of thick sections stained with toluidine blue, we found a number of small rounded cells which had lost their cytoplasmic bridges and had shrunken nuclei with aggregated chromatin, cells which were found interspersed with normal syncytial cells. Similar cell morphology was confirmed by electron microscopy. Stunted viable worms, recovered with longer mature specimens, had very short strobilae and exhibited a large number of apoptotic cells in the germinal neck tissues. The results are consistent with the syncytial nature of these parasites, and strongly suggest that cell proliferation and PCD in these adult cestodes are continuous processes of the germinal tissue and tegumentary cytons.     

Hammondia heydorni-like oocysts shed by a naturally infected dog and Neospora caninum NC-1 cannot be distinguished

This study describes transmission experiments using Hammondia heydorni-like oocysts isolated in 1996 from a naturally infected dog. The isolate was designated as H. heydorni-Berlin-1996. Examination of sera from infected intermediate hosts showed immunoblot reactions that resembled patterns observed after Neospora caninum NC-1 infection. Furthermore, N. caninum DNA could be demonstrated in tissue samples (e.g. heart, brain) of experimentally infected intermediate hosts and in oocyst preparations from H. heydorni-Berlin-1996. The isolated oocysts did not induce any detectable disease in any of the inoculated adult intermediate hosts (goats, sheep, gerbils, guinea pigs, multimammate rats, BALB/c mice, SCID mice), even upon immunosuppression. Furthermore, neither histological lesions nor parasite stages could be identified in the tissues of all fetuses recovered from two multimammate rats that had been infected prior to pregnancy. An experiment with one dog fed a second time on infected intermediate host tissue indicated that immunity may prevent repeated oocyst shedding in N. caninum-infected dogs. In addition, the study clearly demonstrates that N. caninum can be readily transmitted by dogs that have ingested exclusively skeletal muscles of infected intermediate hosts. Therefore, the study has consequences for the recommendations for farmers to prevent postnatal transmission of N. caninum to cattle. It indicates that feeding of any tissues of potential intermediate hosts (including sheep, goats, rodents) to final hosts may induce the shedding of oocysts in these hosts and thus pose a risk for post-natal infection of cattle. With respect to oocyst morphology and the infectivity of muscle tissues for final hosts, no differences were seen in comparison with observations made in the past on Isospora bigemina/I. heydorni/H. heydorni. Therefore, earlier studies made on I. bigemina/I. heydorni/H. heydorni have to be re-evaluated critically to determine whether they may have included N. caninum or other protozoan parasites that use dogs as final hosts and have an oocyst morphology resembling that of I. bigemina/I. heydorni/H. heydorni.     

Ultrastructure of spermiogenesis and the spermatozoon in<Emphasis Type="Italic"> Taenia crassiceps</Emphasis> strobilae WFU strain (Cestoda,Cyclophyllidea, Taeniidae) from golden hamsters

Strobilae from Taenia crassiceps (WFU strain) were obtained from outbred hamsters (Mesocricetus auratus) by feeding them viable metacestodes maintained by intraperitoneal passage in female Balb/c mice. Mature and gravid proglottids from strobilae were recovered from hamster intestines and fixed for light and electron microscopy. By light microscopy, the expected structure of taeniid proglottids was observed. Ultrastructural analysis of ten proglottids showed that testicular follicles and vas deferens contained filiform spermatids, with a single axoneme, and an elongated helicoidal nucleus inserted between the axoneme and the spiraled cortical microtubules. At the apical cone, a single crest-like body was found and mature spermatids also exhibited transverse intracytoplasmic walls. The morphology and characters of the spermatids in T. crassiceps conform to type III spermiogenesis, which has been described in other taeniids.     

Host specificity ofGiardia muris isolates from mouse and golden hamster

One isolate ofGiardia muris from a naturally infected laboratory mouse (Mus musculus) and one from a naturally infected golden hamster (Mesocricetus auratus) were passaged three times by the inoculation of ten cysts (the minimal infectious dose) into barrier-maintained homologous hosts. Both of the resultant isolates were tested for infectivity by intragastric inoculation of 3–5×10⁵ cysts into 40 mice (2 inbred strains), 40 rats (2 inbred strains), and 19 golden hamsters (1 outbred strain). Rats were not susceptible to infection with either isolate. Mice and golden hamsters did develop infections following their inoculation with the heterologous isolates. The mean intensity of heterologous infections with the hamster isolates was significantly lower than that of homologous infections. The mouse isolate induced a higher mean intensity of infection in hamsters as compared with homologous recipients. The mean intensity of infections induced by both isolates was greater in male hamsters than in females.     

Saline extract of Taenia saginata metacestodes as an alternative antigen for the immunodiagnosis of neurocysticercosis in human cerebrospinal fluid

The aim of the present research was to test the application of Taenia saginata metacestodes as an alternative antigen for use in enzyme-linked immunosorbent assay (ELISA) and Western Blotting (WB) tests compared with the use of metacestodes antigen of Taenia solium in cerebrospinal fluid (CSF) samples. The samples were obtained from 35 patients with definitive neurocysticercosis (NCC)—group 1—and 44 patients with other neurological disorders (control)—group 2. The sensitivity and specificity of ELISA, using antigen obtained from T. solium, applied to the patients of group 1 yielded results of 100%. When the tests were conducted using T. saginata metacestodes, results were 100% and 93.2%, respectively. The 47–52-, 64–68-, and 70-kDa antigens showed high frequencies in CSF samples from patients with NCC when WB was conducted with both antigens. The results indicate that T. saginata metacestodes can be used as an alternative antigen for the diagnosis of human NCC in CSF samples.     

Application of <Emphasis Type="Italic">Taenia saginata</Emphasis> metacestodes as an alternative antigen for the serological diagnosis of human neurocysticercosis

Serological tests are an important tool for the diagnosis of neurocysticercosis (NCC), the disease caused by Taenia solium metacestodes. The aim of the present research was to test the application of Taenia saginata metacestodes as an alternative antigen for use in the immunofluorescence antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA), and Western blotting (WB) tests compared with the metacestodes antigen of T. solium in serum samples. The samples were obtained from 130 individuals: 20 from patients with definitive NCC, Group 1; 18 from individuals infected by Taenia sp., Group 2; 40 from individuals infected by various parasites, Group 3; and 40 from healthy individuals, Group 4. The sensitivity of IFAT, ELISA, and WB using antigen obtained from T. solium applied to the patients of Group 1 yielded results of 85, 95, and 95%, respectively, for the three tests. When the tests were conducted using T. saginata metacestodes, results were 75, 80, and 85%, respectively. The specificity of IFAT, ELISA, and WB using antigen obtained from T. solium yielded results of 94.9, 88.8, and 93.9%. When the tests were conducted using T. saginata metacestodes, results were 95.9, 88.8, and 93.6%, respectively. No statistical differences for sensitivity or specificity among the antigens were found. In conclusion, the results indicated that T. saginata metacestodes can be used as an alternative antigen for NCC diagnosis.     

Transmission dynamics of two strains of <Emphasis Type="Italic">Schistosoma mansoni</Emphasis> utilizing novel intermediate and definitive hosts

The intimate host–parasite relationship mandates adaptation to the genetic and phenotypic variability of their counterparts. Here, inbred and outcrossed strains of Schistosoma mansoni were challenged with “local” and “novel” intermediate and definitive hosts to examine effects of genetic variability and novelty on infection success and dynamics. Genetically distinct lines of Biomphalaria glabrata intermediate hosts exposed to inbred and outcrossed S. mansoni larvae were assessed for differences in both snail and parasite life-history parameters. Cercariae from each parasite–snail treatment were used to infect “local” and “novel” Mus musculus definitive hosts to assess parasite infectivity and fitness. Outcrossed parasites significantly reduced snail growth, were more productive, and induced greater host mortality than inbred parasites. Mouse strain did not influence parasite infectivity or reproduction, but parasite and snail host genetic background did, affecting both sex-specific infectivity and parasite productivity. Overall, genetic background of S. mansoni and its intermediate snail host altered life history traits and transmission dynamics of the parasite throughout its life cycle.     

Multiplex PCR identification of <Emphasis Type="Italic">Taenia</Emphasis> spp. in rodents and carnivores

The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.     

The role of IgA immunoglobulins in the passive transfer of protection to Taenia taeniaeformis in the mouse.

Normal mice were protected against infection with metacestodes of Taenia taeniaeformis when administered intestinal, colostral or serum immunoglobulins obtained from adult mice previously orally infected with the parasitie. The protective capacity of these preparations was found to be associated mainly with IgA of colostrum and intestinal secretions and IgG of serum. The removal of IgA and IgG from immune colostrum and serum, respectively, abolished the protective effect. Neonatal mice were protected against infection with T. taeniaeformis when fed purified colostral IgA and serum IgG from immune donors. The intraduodenal injection of intestinal IgA from immune donors into 4-week-old mice passively protected the recipients against infection with T. taeniaeformis, but intestinal IgG from immune donors had no protective effect when given in this manner. The protective capacity of IgA and IgG was largely eliminated by prior absorption with T. taeniaeformis antigen or hatched, activated oncospheres of T. taeniaeformis.     

Hepatic tissue culture model for study of host-parasite interactions in alveolar echinococcosis.

An in vitro model for growth and differentiation of the metacestode tissue of the tapeworm Echinococcus multilocularis is described. This model simulates the organotropism of the parasite toward the liver of the intermediate host. In the presence of collagen-embedded primary hepatocytes from rats and humans, which can be kept in culture for 2 to 3 months, the parasitic vesicles grew by exogenous budding and multiplied about 12-fold within 3 weeks. In contrast, without the hepatocytes, the metacestodes rapidly degenerated. Development of protoscolices was seen only in the presence of rat hepatocytes but not in coculture of the metacestodes with hepatocytes of human origin, thus reflecting the in vivo situation during infection of rodents and in alveolar echinococcosis in humans. The experiments indicated that growth of the metacestodes and development of protoscolices depended on soluble low-molecular-weight factors released by the hepatocytes. The in vitro-grown metacestodes did not differ morphologically from the larvae found in infected intermediate hosts, and their infectivity was completely maintained. This report describes the first in vitro model of alveolar echinococcosis and will be the basis for future studies on host-parasite interactions of this important zoonosis.     

Transmission experiments on the host specificity ofHeterophyes species in 16 potential definitive hosts

Sixteen different species of piscivorous mammals and birds were tried as experimental definitive hosts forHeterophyes heterophyes, H. aequalis andH. dispar. The hosts were classified in four categories, by fluke longevity, recovery and size, and the number of uterine eggs (embryonated/unembryonated): (1) Canidae and the cat were highly susceptible hosts for all three species ofHeterophyes; (2) several mammals and herons showed a reduced susceptibility to infection (H. aequalis, 6 species;H. dispar, 1 species;H. heterophyes, 0 species); (3) In a group of hosts specific to each trematode, flukes were recovered up to 14 days post infection, but their uterine eggs did not become embryonated; (4) In a fourth category of hosts, chiefly Mustelidae, flukes could not be recovered. Taking also the literature into account it is concluded that man is a highly susceptible host forH. heterophyes, and that probablyH. aequalis andH. dispar may reach reproductive maturity in humans also. The described wide host range ofH. aequalis appears to be more typical for Heterophyidae than the comparably narrow host range ofH. heterophyes.