Effective protection against experimental Taenia solium tapeworm infection in hamsters by primo-infection and by vaccination with recombinant or synthetic heterologous antigens

The disease caused by Taenia solium is progressively being recognized as a growing global threat for public human health and pig husbandry that requires the development of effective control measures. A central participant in the taeniasis/cysticercosis transmission network is the human carrier of the adult tapeworm because of its great potential in spreading the infection. Herein, evidence is presented that a primary infection of golden hamsters with orally administered T. solium cysticerci improved the host's resistance against a secondary infection. Likewise, previous vaccination increased the hamster's resistance. Similar high levels of protection (> 78%) were induced by systemic or oral vaccination with the S3Pvac anticysticercosis synthetic peptide vaccine or the highly immunogenic recombinant chimera based on the protective peptide KETc1 bound to Brucella spp. lumazine synthase (BLS-KETc1). Increased resistance after primo-infection and vaccination possibly results from changes in the immune conditions prevailing in the host's intestine. The contribution to protection from the KETc1 and BLS epitopes in a chimeric vaccine is under study. Preventive vaccination of definitive hosts of T. solium against the tapeworm, the most relevant step in the taeniasis/cysticercosis transmission, may greatly impact the dynamics of endemic disease and has not been studied or tried previously.     

Cytokine,Antibody and Proliferative Cellular Responses Elicited by Taenia solium Calreticulin upon Experimental Infection in Hamsters

Taenia solium causes two diseases in humans, cysticercosis and taeniosis. Tapeworm carriers are the main risk factor for neurocysticercosis. Limited information is available about the immune response elicited by the adult parasite, particularly the induction of Th2 responses, frequently associated to helminth infections. Calreticulin is a ubiquitous, multifunctional protein involved in cellular calcium homeostasis, which has been suggested to play a role in the regulation of immune responses. In this work, we assessed the effect of recombinant T. solium calreticulin (rTsCRT) on the cytokine, humoral and cellular responses upon experimental infection in Syrian Golden hamsters (Mesocricetus auratus). Animals were infected with T. solium cysticerci and euthanized at different times after infection. Specific serum antibodies, proliferative responses in mesenteric lymph nodes and spleen cells, as well as cytokines messenger RNA (mRNA) were analyzed. The results showed that one third of the infected animals elicited anti-rTsCRT IgG antibodies. Interestingly, mesenteric lymph node (MLN) cells from either infected or non-infected animals did not proliferate upon in vitro stimulation with rTsCRT. Additionally, stimulation with a tapeworm crude extract resulted in increased expression of IL-4 and IL-5 mRNA. Upon stimulation, rTsCRT increased the expression levels of IL-10 in spleen and MLN cells from uninfected and infected hamsters. The results showed that rTsCRT favors a Th2-biased immune response characterized by the induction of IL-10 in mucosal and systemic lymphoid organs. Here we provide the first data on the cytokine, antibody and cellular responses to rTsCRT upon in vitro stimulation during taeniasis.     

Renewed hope for a vaccine against the intestinal adult Taenia solium

Review of experimental and observational evidence about various cestode infections of mammalian hosts revives hope for the development of an effective vaccine against adult intestinal tapeworms, the central protagonists in their transmission dynamics. As for Taenia solium, there are abundant immunological data regarding cysticercosis in humans and pigs, but information about human taeniasis is scarce. A single publication reporting protection against T. solium taeniasis by experimental primo infection and by vaccination of an experimental foster host, the immunocompetent female hamster, kindles the hope of a vaccine against the tapeworm to be used in humans, its only natural definitive host.     

Eradication of Taenia solium cysticercosis: a role for vaccination of pigs.

Neurocysticercosis due to Taenia solium is an important cause of human morbidity and mortality, particularly in Latin America and parts of Africa and Asia. The disease has been recognised as potentially eradicable. Emphasis has been placed on control of the parasite through mass chemotherapy of human populations to remove tapeworm carriers. This strategy does not control the source of tapeworm infections, cysticercosis in pigs, and parasite transmission may continue due to incomplete chemotherapy coverage of human tapeworm carriers or because of immigration of tapeworm carriers into control areas. Exceptionally effective, practical vaccines have been developed against cysticercosis in sheep and cattle and a recent trial has proved recombinant antigens to be effective against Taenia solium cysticercosis in pigs. A new strategy for eradication of Taenia solium is proposed, based principally on a combined approach of chemotherapy of human tapeworm carriers and vaccination of all pigs at risk of infection.     

Inflammatory responses in the intestinal mucosa of gerbils and hamsters experimentally infected with the adult stage of Taenia solium

The inflammatory response in gerbils and hamsters harbouring experimental infections with Taenia solium adult parasites as well as worm burden and duration of infections were examined. For this purpose, non-suppressed or immunosuppressed rodents were infected with eight cysticerci and necropsied at different times up to 35 days post-infection. Cells in the mucosa surrounding the implantation site of T. solium scolices (duodenum-jejunum) and in ileum were counted in stained sections. A competitive enzyme linked immunosorbent assay was used to determine histamine concentration in intestinal fluid. In non-suppressed hosts, an inflammatory reaction developed with scarce macrophages, a slight increase of plasma cells, lymphocytes and fibroblasts, a moderate increase of eosinophils and neutrophils, and high numbers of goblet and mast cells. Goblet cells began to increase at 6 days post-infection and peaked at 13 days post-infection with a four-fold increase with respect to the control group. Mast cells only increased in gerbils starting at 9 days post-infection with an eight-fold increase when cells peaked between 11 and 19 days post-infection. Histamine concentration in intestinal fluid of gerbils had a similar behaviour to mast cells. Minimal increase of mast cells was seen in hamsters. The recovery of tapeworms was inversely related to the number of both cell types, which decreased when tapeworms were eliminated. Infections lasted up to 25 days in gerbils and up to 46 days in hamsters. Worms measured only 1-2 cm in gerbils and up to 40 cm in hamsters. When gerbils were suppressed with the steroid methyl predinisolone, tapeworms could be recovered up to 35 days post-infection and tapeworms measured up to 22 cm, a minor increase of goblet and mast cells was observed and histamine concentration was similar to that in non-infected animals. Our results suggest that expulsion of T. solium in gerbils and hamsters may be related to the increase of goblet cells and mast cells, but these cells may have different roles in each rodent model of taeniosis.     

Vaccination against Taenia solium cysticercosis

Taenia solium is a parasite that causes human cysticercosis. Its life cycle includes the adult stage, the egg and the larval stage. Human cysticercosis is a disease related to underdevelopment, the main clinical manifestation is neurocysticercosis. Control measures include mass cestocidal treatment aimed to cure possible taeniosis cases. Although useful it has certain disadvantages, such as the generation of symptomatology in occult neurocysticercosis. Alternatively, health education has been shown to be highly effective since people become aware of the importance of human and porcine cysticercosis and the possibility of eliminating it. Nevertheless it has to be implemented by knowledgeable people. On the other hand, the life cycle can be controlled by avoiding swine cysticercosis. This review describes the studies performed to vaccinate pigs against T. solium and indicate that short time perspectives are very encouraging for the production of an optimal vaccine.     

The Asian Taenia and the possibility of cysticercosis

In certain Asian countries, a third form of human Taenia, also known as the Asian Taenia, has been discovered. This Asian Taenia seems to be an intermediate between Taenia solium and T. saginata since in morphological terms it is similar to T. saginata, yet biologically, as it uses the same intermediate host (pigs), it is more akin to T. solium. Taenia solium causes human cysticercosis, while T. saginata does not. It is not known whether the Asian taeniid is able to develop to the larval stage in humans or not. The arguments proposed by those authors who consider it unlikely that the Asian Taenia causes human cysticercosis are: (a) its molecular similarities with T. saginata; (b) the absence of cases of human cysticercosis in populations where the Asian adult is highly prevalent; and (c) the unsupporting results derived from an experimental infestation study. These three arguments are debated, although bearing in mind that at present there is still no clear scientific data to support that human cysticercosis can be caused by the Asian Taenia.     

ELISA and immunoblot using purified glycoproteins for serodiagnosis of cysticercosis in pigs naturally infected with Taenia solium

The establishment of reliable serological methods for cysticercosis in pigs is important for the surveillance, control and prevention of taeniosis/cysticercosis in humans as well as in pigs to prevent economic loss. Both ELISA and immunoblot using glycoproteins (GPs) purified by a single step of preparative iso-electric focusing, which are highly useful for human cysticercosis, have been applied for a serological study in pigs naturally infected with Taenia solium. All sera from pigs showed similar responses to those in human cysticercosis. Therefore, it is expected that both ELISA and immunoblots using GPs would be useful in differentiating infected pigs from uninfected ones.     

A review of taeniasis and cysticercosis in the Lao People's Democratic Republic

Taeniasis and cysticercosis are important but underreported parasitic zoonoses in the Lao People's Democratic Republic (Lao PDR). Reports of human and pig cysticercosis are rather limited and based largely on anecdotal evidence. To date, no structured surveys of disease prevalence or incidence have been reported. However, one unpublished pilot survey of pig cysticercosis in a slaughterhouse in northern Laos estimated prevalence to be 1.7%, without speciation of parasite cysts. Over the past 20 years, nine surveys of intestinal helminthic infection have been conducted; the prevalence of human taeniasis ranged from 0 to 14.0%. The study designs and sample sizes varied greatly, however a high degree of spatial and age variation in taeniasis prevalence was evident. These results are however inconclusive as the species of tapeworm infecting the people was not determined. To further our knowledge of taeniasis and cysticercosis in Lao PDR, structured community-based surveys in high-risk areas are required in combination with the use of sensitive and specific diagnostic tests capable of identifying the pork tapeworm, Taenia solium. This will enable the development and implementation of control measures that are both appropriate and sustainable if T. solium is shown to be a public health threat.     

A seroepidemiological survey of Taenia solium cysticercosis in Nabo, Guangxi Zhuang Autonomous Region, China

We have observed the seropositive rate of Taenia solium cysticercosis in residents at Nabo Village, Tiandong County, Guangxi Zhuang Autonomous Region, China by enzyme linked immunosorbent assay. The village had been found to be a relatively high endemic area of porcine cysticercosis among roaming pigs. Of 202 persons examined four males aged 15, 25, 35 and 41 year-old exhibited absorbance (abs) at 0.18, 0.20, 0.35 and 0.55, respectively. In addition, two females whose ages were 35 and 39 years revealed specific antibody levels of abs 0.26 and 0.41 in their sera. Overall positive rate among the people was 2.97%. All of these persons agreed that they had ingested the pork infected with T. solium metacestode (TsM), while history of proglottid discharge was not noticed from all of them. Three males and one female complained of intermittent headache. Our findings reinforced not only that the prevalence of cysticercosis might be related with roaming pigs infected with TsM but also that behavioral and environmental practices in local community constituted risk factors for transmission of the infection.     

Taeniasis-cysticercosis in Southern Ecuador: assessment of infection status using multiple laboratory diagnostic tools

Taenia solium-taeniasis and cysticercosis were studied in the human and porcine populations of a rural community in the Southern Ecuadorian Andes. From the 1059 inhabitants, 800 serum samples and 958 stool samples could be collected. In addition, 646 from the estimated 1148 pigs were tongue inspected. Circulating antigen was detected by enzyme linked immunosorbent assay (Ag-ELISA) in 2.25% of the human population, whereas intestinal taeniasis was detected in 1.46% by the formalin-ether technique. Following treatment and recovery of tapeworm fragments these were all identified as T. solium. Porcine cysticercosis was diagnosed in 3.56% of the pigs by tongue inspection. In addition, enzyme linked immunoelectrotransfer blot (EITB) was performed on a subset group of 100 humans to confirm the results of the Ag-ELISA. One hundred serum samples from pigs were also analysed by EITB. It appeared that 43 and 74% of humans and pigs had antibodies against T. solium cysticerci, respectively. It is concluded that contrary to the high exposure of the human population to T. solium that is suggested by EITB, the number of active cysticercosis cases, diagnosed by Ag-ELISA, was low, which may indicate endemic stability. The further use of complementary diagnostic methods for a better understanding of the epidemiology of T. solium is suggested.     

Taenia solium Infections in a rural area of Eastern Zambia-a community based study

Background

Taenia solium taeniosis/cysticercosis is a parasitic infection occurring in many developing countries. Data on the status of human infections in Zambia is largely lacking. We conducted a community-based study in Eastern Zambia to determine the prevalence of human taeniosis and cysticercosis in a rural community.

Methods and Findings

Stool and serum samples were collected from willing participants. Geographical references of the participants'' households were determined and household questionnaires administered. Taeniosis was diagnosed in stool samples by coprology and by the polyclonal antibody-based copro-antigen enzyme-linked immunosorbent assay (copro-Ag ELISA), while cysticercosis was diagnosed in serum by the B158/B60 monoclonal antibody-based antigen ELISA (sero-Ag ELISA). Identification of the collected tapeworm after niclosamide treatment and purgation was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A total of 255 households from 20 villages participated in the study, 718 stool and 708 serum samples were collected and examined. Forty-five faecal samples (6.3%) were found positive for taeniosis on copro-Ag ELISA while circulating cysticercus antigen was detected in 5.8% (41/708) individuals. The tapeworm recovered from one of the cases was confirmed to be T. solium on PCR-RFLP. Seropositivity (cysticercosis) was significantly positively related to age (p = 0.00) and to copro-Ag positivity (taeniosis) (p = 0.03) but not to gender. Change point analysis revealed that the frequency of cysticercus antigens increased significantly in individuals above the age of 30. Copro-Ag positivity was not related to age or gender. The following risk factors were noted to be present in the study community: free-range pig husbandry system and poor sanitation with 47.8% of the households visited lacking latrines.

Conclusions

This study has recorded high taeniosis and cysticercosis prevalences and identified the need for further studies on transmission dynamics and impact of the disease on the local people.     

Absence of the SP/SP receptor circuitry in the substance P-precursor knockout mice or SP receptor, neurokinin (NK)1 knockout mice leads to an inhibited cytokine response in granulomas associated with murine Taenia crassiceps infection

Neurocysticercosis, caused by the cestode Taenia solium, is the most common parasitic infection of the human central nervous system that leads to seizures. Taenia crassiceps cysticercosis in mice is an experimental model for Taenia solium cysticercosis. Similar to the human infection, live parasites cause little or no granulomatous inflammation. Dying parasites initiate a granulomatous reaction. The neuropeptide, substance P (SP), stimulates T-helper (TH) 1 cytokine production. In the current studies, we determined whether absence of SP/SP receptor circuitry in the SP-precursor, preprotachykinin, knockout or SP-receptor, neurokinin (NK) 1, knockout mice affected granuloma cytokine production. We hence compared the levels of Th1 cytokines interleukin (IL)-2 and interferon (IFN)-gamma, and levels of Th2/immunoregulatory cytokines IL-4 and IL-10, by enzyme-linked immunosorbent assay in T. crassiceps-induced granulomas derived from infected C57BL/6 wild type (WT) versus SP-precursor knockout and NK1 knockout mice. We found that mean levels of IL-2, IFN-gamma, IL-4, and IL-10 in infected WT-derived granulomas were significantly higher than those of granulomas derived from infected SP-precursor knockout or the NK1 receptor (NKIR)knockout mice. Levels of Th2/immunoregulatory cytokines, IL-4 and IL-10 were higher in early stage granulomas (histologically-staged on basis of evidence of parasite remnants) versus late stage granulomas (no parasite-remnants) of both knockouts, whereas the reverse was noted in WT-derived granulomas. These study established that the absence of an SP/SP receptor circuitry in the SP precursor knockout mice or NK1 receptor knockout mice led to an inhibited cytokine response.     

Sexual development of Taenia solium in hamsters from rodent-derived cysticerci

In order to determine whether Taenia solium can be maintained in the laboratory using rodents as definitive hosts, six nude rats, 20 immunosuppressed Mongolian gerbils and 20 immunosuppressed Syrian hamsters were each inoculated through a stomach tube with three cysticerci recovered from SCID mice. No adult worms of T. solium were found in the intestinal tract of any of these 46 rodents. In addition, five immunosuppressed Syrian hamsters were fed with the same number of cysticerci enclosed in rodent muscles from SCID mice. Two of these hamsters were found to be infected 40 days post-infection, each harbouring a sexually developed worm in the intestinal tract. Although no eggs were produced, prepatent infections may be possible if a longer time was allowed for worm development. Moreover, the maintenance of the life cycle of T. solium in the laboratory using the rodent model can be established.     

Regulation of the immune response to cestode infection by progesterone is due to its metabolism to estradiol

The aim of this work was to investigate the role of progesterone during Taenia crassiceps cysticercosis, and the immunological mechanisms involved in its effects, by relating progesterone treatment to whole parasite counts, to host humoral and cellular immune response, to the presence or absence of nuclear receptors to sex steroids in splenocytes, and to serum sex steroid levels in infected mice of both genders. Progesterone treatment increased parasite loads two-fold in females and three-fold in males compared with control mice. The expression of the Th2 cytokine profile (IL-4, IL-6 and IL-10) was markedly increased in infected mice of both genders, while progesterone treatment returned this expression to basal levels. However, the Th1 cytokine profile (IFN-gamma and TNF-alpha) was not affected by infection, whilst progesterone treatment increased the expression of both cytokines two-fold compared to uninfected, infected and placebo-treated mice. Testosterone serum levels decreased in infected male mice by 95%, and treatment with progesterone did not affect them. In females, no change in testosterone levels was observed. Progesterone levels increased three-fold only in progesterone-treated infected mice of both sexes, while estradiol levels in female and male progesterone-treated infected mice increased two-fold compared to infected control mice. The infection markedly induced the expression of progesterone receptor (PR) isoforms A and B in splenocytes of infected mice of both genders (five-fold). Metabolism of progesterone to estradiol was demonstrated by the use of the anti-estrogen tamoxifen, which reduced parasite loads 100% in infected mice of both sexes treated with progesterone. These results suggest that progesterone, possibly through its metabolism to estradiol, affects establishment, growth and reproduction of the helminth parasite T. crassiceps.     

Differential expression of calreticulin in developmental stages of Taenia solium

Taenia solium, a cestode that causes neurocysticercosis and taeniasis in humans, has a complex life cycle. The adult tapeworm develops in the intestine of human beings and is also responsible for neurocysticercosis, which is caused by the metacestode or cysticercus that develops in the brain. Recently, we have cloned the coding region for T. solium calreticulin (TsCRT) as a functional Ca(2+)-binding protein. Calreticulin is a ubiquitous protein involved in cellular Ca2+ homeostasis and protein folding. These important functions affect several aspects of cell physiology. To explore the expression of TsCRT during the T. solium life cycle, we used a specific polyclonal antibody raised against recombinant TsCRT to localize this protein by immunolabeling techniques. In sections of cysticerci obtained from swine muscle, as well as of adult tapeworms obtained after infection of hamsters with cysticerci, TsCRT was preferentially localized in tegumentary and muscle cytons of the suckers and rostellum. In mature proglottids obtained from infected humans, positive staining was observed in spermatogonia, ovogonia, uterine epithelium, and cells of the vas deferens. In the gravid uterus, the morula and early stage embryos were highly positive to TsCRT. However, expression diminished as embryonic development progressed and was absent in fully developed oncospheres that were surrounded by an embryophore. A similar down regulation was observed during spermatogenesis. Although early spermatocytes showed a high expression of TsCRT, mature spermatozoa present in the vas deferens were completely negative. These data indicate that calreticulin expression is spatially and temporally regulated during development of T. solium, especially during germ cell development and embryogenesis. In addition, these original images illustrate, for the first time, these processes at a histological level.     

Different effects of chorionic gonadotropin on Taenia crassiceps and Taenia solium cysticerci cultured in vitro

Hormones play a significant role in murine Taenia crassiceps cysticercosis, and they may also participate in the susceptibility to Taenia solium cysticercosis. In the present study, in vitro effects are reported for human chorionic gonadotropin (hCG) on the larval stages of T. crassiceps (WFU strain) and T. solium. hCG effectively promotes parasite reproduction, i.e., it increases the number of buds on T. crassiceps cysticerci and the percentage of evagination and parasite length in T. solium. This is the first report in which a direct effect of hCG is reported for a parasite. hCG or mouse luteinizing hormone could be recognized by the cysticerci as mitogenic factors and contribute to the female and pregnancy bias toward susceptibility to T. crassiceps and T. solium cysticercosis, respectively.     

Chinchilla laniger can be used as an experimental model for Taenia solium taeniasis

Chinchilla laniger has been reported as an experimental definitive host for Taenia solium; however no information about its suitability and yield of gravid tapeworm proglottids containing viable and infective eggs has been published. In total 55 outbred female chinchillas were infected with 4 cysticerci each; hosts were immunodeppressed with 6 or 8 mg of methyl-prednisolone acetate every 14 days starting the day of infection and their discomfort was followed. Kinetics of coproantigen ELISA or expelled proglottids was used to define the infection status. Efficiency of tapeworm establishment was 21% and of parasite gravidity was 8%; chinchillas showed some degree of suffering along the infection. Viability of eggs obtained from gravid proglottids was tested comparing methods previously published, our results showed 62% viability with propidium iodide, 54% with trypan blue, 34% with neutral red, 30% by oncosphere activation and 7% with bromide 3-(4,5-dimetil-tiazol-2-il)-2,5-difenil-tetrazolio (MTT) reduction; no statistical differences were obtained between most techniques, except activation. Four piglets were infected with 50,000 eggs each, necropsy was performed 3 months later and, after counting the number of cysticerci recovered, the percentage of infection was similar to data obtained with T. solium eggs recovered from humans. Our results demonstrate that the experimental model of T. solium taeniasis in C. laniger is a good alternative for providing eggs and adult tapeworms to be used in different types of experiments; optimization of the model probably depends on the use of inbred hosts and on the reduction of infected animals' suffering.     

Immune humoral response in pigs infected with eggs and posoncospheres of Taenia solium

Due to the importance of cysticercosis in Mexico and Latin America and to the fact that in the last years another mechanism of infection for this disease has been proposed, i.e. through postoncospheres and immunosuppression of the host, we have considered relevant to perform the present work, which consisted in assessing the immune response induced by dexamethasone as well as that produced by parasites in pigs infected with T. solium eggs, or postoncosphere-infected, and in postoncosphere-infected and dexamethasone-treated animals. We used 10 recently weaned pigs, three were used as controls, two of them without the drug and one with it; two were infected with T. solium eggs; five with postoncospheres receiving also dexamethasone three of them. We evaluated the humoral response against parasite antigen using indirect haemagglutination (IH) and ELISA methods. Results of the immune humoral response revealed titres of up to 1:128 in T. solium eggs infected animals, of 1:16 in postoncosphere infected animals, and of 1:32 towards the end of the experiment in postoncosphere plus dexamethasone animals. Absorbance titres with ELISA confirmed these findings. Data obtained by IH show that the antibody titres of the pigs challenged with postoncospheres and postoncospheres plus dexamethasone are positive as compared to the titres obtained in the pigs infected with T. solium eggs. Results from the ELISA confirmed this finding, since, from weeks 14 to 17, the pigs became positive, behaving as those pigs that developed cysticercosis. This is relevant as it indicates that the antiposcosphere antibodies recognized antigens of T. solium larvae.