Guillermina Avila

Induction of Protection against Porcine Cysticercosis by Vaccination with Recombinant Oncosphere Antigens

ABSTRACT Two recombinant Taenia solium oncosphere antigens, designated TSOL18 and TSOL45-1A, were investigated as vaccines to prevent transmission of the zoonotic disease cysticercosis through pigs. Both antigens were effective in inducing very high levels of protection (up to 100%) in three independent vaccine trials in pigs against experimental challenge infection with T. solium eggs, which were undertaken in Mexico and Cameroon. This is the highest level of protection that has been achieved against T. solium infection in pigs by vaccination with a defined antigen. TSOL18 and TSOL45-1A provide the basis for development of a highly effective practical vaccine that could assist in the control and, potentially, the eradication of human neurocysticercosis.

Comparative development of Taenia solium in experimental models

Various mammals were evaluated as experimental models of adult Taenia solium. Suppressed and nonsuppressed hosts were used as experimental models. Infections were performed per os with cysticerci obtained from pigs; immunosuppression was induced with methyl prednisolone acetate at intervals of 10-14 days after infection. Tapeworms developed in hamsters, gerbils, and chinchillas but failed to develop in rabbits, cats, pigs, and rhesus monkeys. In infectable animals, treatment with the steroid facilitated maintenance and development of the parasite, and more tapeworms were obtained. Mature and some pregravid proglottids were recovered from hamsters and gerbils, whereas a gravid T. solium was obtained from a chinchilla at 12 wk postinfection. Eggs recovered from the chinchilla transformed into cysticerci in a pig 12 wk after oral infection. The T. solium-chinchilla experimental system seems to be an alternative definitive host for this parasite and thus the basis for a great diversity of studies.

Effect of parasite burden on the detection of Fasciola hepatica antigens in sera and feces of experimentally infected sheep.

The effect of Fasciola hepatica parasite burden on the detection of excretory/secretory (E/S) antigens in sera and feces of experimentally infected sheep was evaluated using a double antibody-based capture enzyme-linked immunosorbent assay (ELISA). Four groups of five sheep each were used. The first three groups were infected with 50, 100 and 200 metacercariae of F. hepatica, and the fourth group remained as non-infected control. On the day of infection and weekly thereafter, serum and fecal samples were taken. ELISA detected F. hepatica E/S antigen levels in serum from the first week post-infection (wpi) and in fecal supernatant from the fourth wpi, which were significantly (p<0.05) higher than controls. F. hepatica eggs were not detected until after the eighth wpi. The correlation between absorbance of E/S antigens in serum with the fluke burden was 0.77 (p<0.0001) and in feces 0.76 (p<0.0001) at 12th wpi. The sensitivity of the assay to detect E/S antigens in serum was 86.6% and in feces 93.3%. It is concluded that the ELISA technique used in this study offers a diagnostic alternative for detecting early infections of F. hepatica in sheep.

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.

TAENIA SOLIUM: DESCRIPTION OF THE INTESTINAL IMPLANTATION SITES IN EXPERIMENTAL HAMSTER INFECTIONS

Experimental infections in golden hamsters with viable Taenia solium metacestodes were used to study by light and electron microscopy the implantation site of the adult tapeworm in the intestinal wall. Implantation sites from 3-, 4-, 10-, and 40-day infections were located in the upper third of the duodenum, excised and fixed in Zenkers or Karnovskys solution, embedded in Polybed resin, and sectioned longitudinally to observe the position of the worm on the intestinal wall. The scolex of the tapeworm was situated between host villi, with the rostellum penetrating the intestinal wall and the suckers entrapping adjacent villi. Serial sections through several whole implantation sites revealed that the worm was anchored to the host by all 4 suckers simultaneously, each of which was located at a different level and had entrapped intestinal villi in its cavity. Host tissue within the suckers was damaged, exhibiting various degrees of cell lysis and necrosis of epithelial and submucosal cells. The tegumentary surface and microtriches of the scolex were well preserved, with occasional coalescence of tegumentary microvesicles in 10- and 40-day-old infections; microtriches were in direct contact with the damaged host tissue. This study is the first morphological and ultrastructural description of the attachment of T. solium to the intestinal wall employing an experimental model, the results of which may contribute to a better understanding of the biology of human tapeworm infections.

Portrait of Human Tapeworms

The tapeworms of the genus Taenia that infect human beings are T. solium, T. saginata and T. saginata asiatica. Taenia solium and T. saginata exhibit unequivocal features that characterize them; in contrast, only recent DNA studies, morphological characteristics, and epidemiological and sanitary aspects indicate that T. saginata asiatica is a subspecies of T. saginata. These 3 tapeworms occur in humans in their adult stage, and the intermediate hosts are pigs for T. solium and T. saginata asiatica and cows for T. saginata. Their identification is crucial considering the migratory increase from Asia to the Western Hemisphere and the fact that these tapeworms coexist in the same environment in Asia; furthermore, it is estimated that movement in both directions across the United States–Mexico border exceeds 200 million persons per yr, and thus, opportunities for acquiring and transporting T. solium infections are multiplied. It is not easy to distinguish among these tapeworms; therefore, a comparative diagram of the 3 parasites is shown in this article, which will facilitate their identification. All morphological features, some of which allow for identification, are clear and can be easily distinguished among the 3 tapeworms.

Taenia solium: current understanding of laboratory animal models of taeniosis.

Neurocysticercosis is a public health problem in many developing countries and is the most frequent parasitic disease of the brain. The human tapeworm carrier is the main risk factor for acquiring neurocysticercosis. Since the parasite lodges only in the human intestine, experimental models of Taenia solium taeniosis have been explored. Macaques, pigs, dogs, cats and rabbits are unsuccessful hosts even in immunodepressed status. By contrast, rodents are adequate hosts since tapeworms with mature, pregravid and, in some cases, gravid proglottids develop after infection. In this review, information that has been generated with experimental models of taeniosis due to T. solium is discussed. Initially, the use of the model for immunodiagnosis of human taeniosis and evaluation of intervention measures is summarized. Next, descriptions of tapeworms and comparison of hamsters, gerbils and other mammals as experimental models are discussed, as well as data on the humoral immune response, the inflammatory reaction and the production of cytokines associated to Th1 and Th2 responses in the intestinal mucosa. Finally, evaluation of protection induced against the development of tapeworms by recombinant T. solium calreticulin in hamsters is summarized and compared to other studies.

Cytokine Response in the Intestinal Mucosa of Hamsters Infected with Taenia solium

Taenia solium grows in experimentally infected hamsters. An inflammatory reaction in the intestinal mucosa surrounding the scolex of the worms is produced. We searched for mRNA of Th1 and Th2 cytokines by in situ hybridization in intestinal biopsies. Hamsters were infected with T. solium cysticerci and necropsied on different days post infection (d.p.i.). Tissue from the small intestine was taken from the area surrounding the tapeworm scolex, fixed, and processed for histology. Antisense probes for the detection of interferon (IFN)‐γ, interleukin (IL)‐4, IL‐5, and IL‐13 were used. Kinetics of each cytokine was defined through detection on specific mRNA by counting the number of positive infected hamsters and of positive cells per 100 enterocytes on different d.p.i. IFN‐γ was detected as of d.p.i. 2; all animals were positive on d.p.i. 4 and 8; and on d.p.i. 16, only 20% were still positive. IL‐13 had a pattern similar to IFN‐γ, but all hamsters remained positive until d.p.i. 16 when the experiment was terminated. IL‐4 was positive in 40% of infected hamsters on d.p.i. 6. On d.p.i. 8, IL‐5 was only detected in 20% but increased to 100% by d.p.i. 16. These data suggest that tapeworms induce a mixed Th1/Th2 response with a polarization toward Th2 at 2 weeks post infection, which may influence the expulsion of worms.

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.

Immunological mechanisms involved in the protection against intestinal taeniosis elicited by oral immunization with Taenia solium calreticulin

Oral immunization with functional recombinant Taenia solium calreticulin (rTsCRT) induces 37% reduction in tapeworm burden in the experimental model of intestinal taeniosis in hamsters. Furthermore, tapeworms recovered from vaccinated animals exhibit diminished length, being frequently found in more posterior parts of the small intestine. The aim of this study was to analyze the immunological mechanisms involved in protection in response to rTsCRT oral immunization. Hamsters were orally immunized with rTsCRT using cholera toxin (CT) as adjuvant, weekly for 4 weeks. Fifteen days after the last boost animals were challenged with four T. solium cysticerci. Reduction in the adult worm recovery and increased transcription of mRNA for IL-4 and IFN-γ in the mucosa of rTsCRT+CT immunized animals were observed. Immunization also induced goblet cell hyperplasia in the mucosa surrounding the implantation site of the parasite. Specific IgG and IgA antibodies in serum and fecal supernatants were detected after the second immunization, being more pronounced after challenge. Our data suggest that oral vaccination with rTsCRT+CT regulates a local expression of IL-4 and IFN-γ, stimulating secretion of IgA that, together with the increase of goblet cells and mucin production, could result in an unfavorable environment for T. solium promoting an impaired tapeworm development.