Taenia solium: cell reactions to the larva (Cysticercus cellulosae) in naturally parasitized, immunized hogs

In hogs naturally infected with Taenia solium larvae (i.e., Cysticercus cellulosae), we studied the host response induced by antigens obtained from the larvae. Histopathological studies of cysticerci removed after 4 and 8 weeks of immunization showed an intense inflammatory reaction surrounding the larvae. The response was greater in the 8-week specimens. A dense layer of eosinophils was in close contact with the external membrane of the bladder wall and, in several cases, the eosinophils had infiltrated this tegument. Many eosinophils were seen in the spiral canal of larvae. This infiltration by eosinophils increased with time. Preparations from the 8-week samples showed many degenerated and disrupted eosinophils whose granules were found in close contact with the outer membrane of the larval tegument and, in some cases, had entered through the broken surface of this structure. More than 90% of the larvae were found in various stages of degeneration; the rest were completely destroyed and surrounded by a mass of eosinophils. After immunization, peripheral blood eosinophilia increased to 17%, whereas the eosinophilia of the control hog was 4% throughout the study. The larval worms removed from control hogs showed intact structures, with a low degree of infiltration by eosinophils and a discrete inflammatory reaction surrounding the bladder wall of the larvae.     

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.     

Ascaris suum: aldolase I purification and immunologic study

Fructose-1,6-diphosphate d-glyceraldehyde-3-phosphate lyase (aldolase) (EC 4.1.2.13) from the body wall of Ascaris suum was purified 80-fold by a combination of salt precipitation and ion-exchange chromatography. A group of pigs was immunized with the purified aldolase preparation and was subsequently challenged with infective Ascaris larvae. The immunized animals showed clinical and histopathologic symptoms of acute sensitization reaction. Thrice as many larvae were found in the nonimmunized control pigs as compared to the immunized animals.     

Comparative immunopotentiating properties of saponin and incomplete Freund's adjuvant coupled to affinity purified larval antigen of Hyalomma anatolicum anatolicum

Comparative immunostimulatory properties of saponin and incomplete Freund's adjuvant (IFA) were studied using affinity purified 39 kDa larval antigen of Hyalomma anatolicum anatolicum. Significant antibody response to 39 kDa antigen was detected in the sera of rabbits immunized with both 39 kDa plus saponin and 39 kDa plus IFA in comparison to their corresponding control animals. Insignificant differences were noted in the antibody response between the animals of two immunized groups. Upon challenge, the animals immunized with 39 kDa plus IFA rejected 76.2 +/- 9.7% of larvae and 45.8 +/- 4.1% of adults while in group of animals injected with 39 kDa plus saponin rejected 80.9 +/- 11.2% of larvae and 47.2 +/- 5.7% of adults.     

Humoral response to the injection of acellular Pertussis vaccine

The humoral response of mice and rabbits to the injection of whole-cell pertussis vaccine (PV) and acellular pertussis vaccine (APV), developed at the Mechnikov Research Institute for Vaccines and Sera (Russian Acad. Med. Sci.) in Moscow, was studied. In the sera of immunized animals antibodies to the antigenic complex were determined in the direct hemagglutination (DHA) test, specific antibodies to filamentous hemagglutinin (FHA) and pertussis toxin (PT)--in the enzyme immunoassay (EIA) and antibodies neutralizing PT in a cytopathogenic dose (CPD)--in neutralization test on Chinese hamster ovary (CHO) cells. In mice and rabbits immunized with APV the antibody titers determined in the DHA test were higher than those in the animals immunized with PV. Specific antibodies titers to FHA and PT in the sera of rabbits immunized with APV were also higher than those in the sera of rabbits immunized with PV. High dilutions of sera taken from the animals immunized with APC neutralized 4-16 doses of PT in the neutralization test on CHO cells. The most important result of this study was the detection of a more pronounced immune response in the animals immunized with APV in comparison with that induced by PV according to the results obtained in EIA and in the test of PT CPD neutralization on CHO cells.     

Antibodies in the intestinal secretions of rats. Primary and secondary responses to polymeric flagellin.

The antibody responses in serum and secretions obtained from the mucosal surfaces of the small intestine of rats immunized by a parenteral and intestinal route have been compared. Though no significant differences in the mean serum titres were found, the responses of animals immunized via the latter route to large doses of antigen were far less uniform. Apart from the first few days of the primary response, antibody activity was found in three major immunoglobulin classes (IgG2, IgA and IgM), irrespective of the route of immunization. Significant antibody activity appeared in the intestinal surface secretions only after two injections of antigen. In rats immunized parenterally the activity was found only in the IgG2 component. Whilst activity was found in both IgG2 and IgA fractions of the secretions obtained from intestinally immunized rats, it was predominantly of the IgG2 class. The possible significance of this observation is discussed.     

Induction of protection against Brugia malayi infection in jirds by microfilarial antigens

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.     

Immunosuppression in experimental cryptococcosis in rats

The presence of the microorganism, cortical hyperplasia and germinal centers was detected in the thymus of rats infected with 10⁷ viable Cryptococcus neoformans cells and immunized at 7 days afterwards with 2.5 mg (0.1 ml) of human serum albumin (HSA) incorporated to complete Freund's adjuvant (CFA) (Group 2). There was no modification of the glandular structure in the thymus of the animals only immunized with HSA-CFA (Group 1). The weight of the thymus of group 2, animals infected and immunized, was increased compared with the weight of the thymus of group 1 animals, this became evident by the increase of the thymic index (TI) (p <0.005). This rate was obtained calculating the thymus weight/total body weight ratio × 1000.Thymic cells (10⁷ cells in 1 ml) obtained from both groups of animals were transferred to normal syngeneic rats of the same sex. The recipient rats were immunized with HSA-CFA 24 h later and 14 days after the transference, the response of delayed-type hypersensitivity (DTH) was studied in them. It could be observed that the thymic cells coming from group 2 animals were capable of suppressing significatively the afferent pathway of the DTH response to HSA when compared with the response of the animals that received cells coming from group 1 rats (p<0.0001).     

Morphologic and genetic identification of Taenia tapeworms in Tanzania and DNA genotyping of Taenia solium

Species identification of Taenia tapeworms was performed using morphologic observations and multiplex PCR and DNA sequencing of the mitochondrial cox1 gene. In 2008 and 2009, a total of 1,057 fecal samples were collected from residents of Kongwa district of Dodoma region, Tanzania, and examined microscopically for helminth eggs and proglottids. Of these, 4 Taenia egg positive cases were identified, and the eggs were subjected to DNA analysis. Several proglottids of Taenia solium were recovered from 1 of the 4 cases. This established that the species were T. solium (n = 1) and T. saginata (n = 3). One further T. solium specimen was found among 128 fecal samples collected from Mbulu district in Arusha, and this had an intact strobila with the scolex. Phylegenetic analysis of the mtDNA cox1 gene sequences of these 5 isolates showed that T. saginata was basal to the T. solium clade. The mitochondrial cox1 gene sequences of 3 of these Tanzanian isolates showed 99% similarity to T. saginata, and the other 2 isolates showed 100% similarity to T. solium. The present study has shown that Taenia tapeworms are endemic in Kongwa district of Tanzania, as well as in a previously identified Mbulu district. Both T. solium isolates were found to have an "African/Latin American" genotype (cox1).     

Protective immunity against Taenia crassiceps murine cysticercosis induced by DNA vaccination with a Taenia saginata tegument antigen

This study investigated the protective capacity of the recombinant Taenia saginata Tso18 antigen administered as a DNA vaccine in the Taenia crassiceps murine model of cysticercosis. This Tso18 DNA sequence, isolated from a T. saginata oncosphere cDNA library, has homologies with Taenia solium and Echinococcus sp. It was cloned in the pcDNA3.1 plasmid and injected once intramuscularly into mice. Compared to saline-vaccinated control mice, immunization reduced the parasite burden by 57.3-81.4%, while lower levels of non-specific protection were induced in control mice injected with the plasmid pcDNA3.1 (18.8-33.1%) or a plasmid with irrelevant construct, pcDNA3.1/3D15 (33.4-38.8%). Importantly, significant levels of protection were observed between the pcDNA3.1/Tso18 plasmid and pcDNA3.1/3D15 plasmid immunized mice. Mice immunized with pTso18 synthesized low levels of, primarily IgG1 sub-class, antibodies. These antibodies were shown to recognize a 66 kDa antigen fraction of T. crassiceps and T. solium. Splenocytes enriched in both CD4+CD8- and CD4-CD8+ T cells from these vaccinated mice proliferated in vitro when exposed to antigens from both T. solium and T. crassiceps cestodes. Immunolocalization studies revealed the Tso18 antigen in oncospheres of T. saginata and T. solium, in the adult tapeworm and in the tegument of T. solium cysticerci. The protective capacity of this antigen and its extensive distribution in different stages, species and genera of cestodes points to the potential of Tso18 antigen for the possible design of a vaccine against cestodes.     

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.     

Can Taenia solium latent post-oncospheral stages be found in muscle tissue of cysticercosis-infected pigs (Sus scrofa)?

The existence of latent Taenia solium post-oncospheral stages in the tissues of infected pigs has been postulated. To assess whether such structures exist and can be detected, we examined muscle samples from cysticercosis-infected and uninfected pigs. Pork samples were homogenized, centrifuged, and resuspended in saline solution. Round microscopic structures of approximately 10 microm with variable refringence were found in the pellets of all samples from both infected and uninfected pigs. These became homogeneously red after staining with Sudan IV and disappeared after ether extraction. The only difference between samples from infected and uninfected pigs was the presence of inflammatory cells and tissue necrosis debris in the former group. Taenia solium oncospheres were stained and observed for comparative purposes, before and after inoculation into pork. Control oncospheres were ellipsoidal, had nucleated basophile cells in their interior, and showed red aggregates on their surfaces when stained with 3% Sudan IV. While rounded microscopical structures similar to those previously reported were found, these differed morphologically from oncospheres, were of a lipid nature, and occurred in both infected and uninfected animals. No evidence supporting the presence of latent post-oncospheral stages of Taenia solium was generated in this series of experiments.     

Onchocerca volvulus: immunization of chimpanzees with X-irradiated third-stage (L3) larvae.

To provide a theoretical basis for the potential development of vaccines against Onchocerca volvulus (Ov) a trial has been conducted to assess the protective efficacy of immunization of chimpanzees with X-irradiated L3 larvae. Approximately 1000 larvae were injected at 0, 1, and 7 months. The immunized animals, and unimmunized controls, were then challenged with 250 live L3. In order to provide possibly protective exposure to the immunologically distinct L4 epicuticle, a radiation dose (45 krad) was chosen which preserved about 50% of the molting ability of unirradiated larvae. Despite the presence of a strong immune response to crude adult worm extracts, and to cloned Ov antigens, at the time of challenge little or no significant protection against patent infection was observed: three of four immunized animals developed patent infection as compared to four of four controls. One immunized animal failed to become patent or to manifest the late antibody response to adult worm antigens seen in both subpatent and patent infections in this model, and may have been protected from infection. The implications of these studies for future attempts to immunize against O. volvulus are discussed.     

Synthesis of biologically active influenza virus hemagglutinin in insect larvae.

The hemagglutinin of influenza (fowl plague) virus was expressed in larvae of Heliothis virescens by using recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector. Animals were infected with the recombinant virus either by parenteral injection or by feeding. For oral uptake, recombinant virus occluded in polyhedra obtained from cultured Spodoptera frugiperda cells after coinfection with authentic AcNPV was used. Immunohistological analyses of infected animals revealed that the hemagglutinin was expressed only in those tissues that are also permissive for the replication of authentic AcNPV. These tissues included hypodermis, fat body, and tracheal matrix. After oral infection, hemagglutinin was also detected in individual gut cells. The amount of hemagglutinin synthesized in larvae after parenteral infection was 0.3% of the total protein, compared with 5% obtained in cultured insect cells. The hemagglutinin was transported to the cell surface and expressed in polarized cells only at the apical plasma membrane. It was processed by posttranslational proteolysis into the cleavage products HA1 and HA2. Oligosaccharides were attached by N-glycosidic linkages and were smaller than those found on hemagglutinin obtained from vertebrate cells. Hemagglutinin from larvae expressed receptor binding and cell fusion activities, but quantitation of the hemolytic capacity revealed that it was only about half as active as hemagglutinin from vertebrate or insect cell cultures. Chickens immunized with larval tissues containing hemagglutinin were protected from infection with fowl plague virus. These observations demonstrate that live insects are able to produce a recombinant membrane protein of vertebrate origin in biologically active form.     

Effect of Nippostrongylus brasiliensis infection on anti-hapten IgE antibody response in the mouse: I. Induction of carrier specific helper cells

Inoculation of infective larvae of Nippostrongylus brasiliensis into A/J, BALB/c, and SJL mice primed intraperitoneally (ip) 3 weeks before infection with 1 μg of dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) mixed with 1 mg Al(OH)₃ induced a carrier effect on anti-DNP IgE and IgG₁ antibody responses when the experimental mice were secondarily immunized with an ip injection of 1 μg of DNP-coupled N. brasiliensis extract (DNP-Nb) plus alum 2 weeks after infection. The magnitude of the hapten specific antibody response did not correlate rigidly with the number of larvae in the inoculum. Thus, a dose of 100 larvae was as effective in inducing the carrier effect as a dose of 800 larvae. Kinetic studies in A/J and BALB/c mice revealed that the anti-DNP IgE antibody response reached a maximum titer 7 days after the secondary immunization. These studies also showed that the enhanced IgE antibody response persisted for more than 40 days, while the response in all control groups terminated prior to that time. Using the adoptive transfer system, it was demonstrated that lymphoid cells obtained from the spleens or the mesenteric lymph nodes of infected mice cooperated with DNP-KLH primed cells to produce hapten specific IgE and IgG, antibodies when the challenge was made with DNP-Nb but not when it was made with 1 μg DNP-ovalbumin, clearly indicating carrier specificity. The helper activity of the cells obtained from infected mice was completely abolished or greatly reduced by the in vitro treatment with anti-θ serum and complement. The helper cells with maximum activity were present as early as 14 days after inoculation. The level of helper activity gradually decreased after 14 days. The results indicate that N. brasiliensis infection is effective in inducing carrier specific helper cells of thymic origin (T cells) in anti-DNP antibody responses. These results confirm those obtained by other investigators and add the new observation that N. brasiliensis infection elicits special helper T cells which induce an enhancement as well as a prolongation of anti-DNP IgE antibody response.     

Ancylostoma ceylanicum: immunization with soluble worm extract and responses to challenge infection of dogs

When dogs were immunized with soluble extract of adult Ancylostoma ceylanicum antigen, they were partially resistant to challenge infection in this model of human hookworm infection. Two immunizing doses, each of 1 mg protein suspended in Freund's complete adjuvant, were administered to one group of animals 1 and 3 weeks prior to infection with 5000 larvae. When compared with control dogs given the same infective dose, fecal egg excretion and intestinal adult worm burden in the immunized animals were reduced by 59 and 74%, respectively. Infection had no significant effect on hemoglobin concentrations, mean red cell volumes, total white cell counts, platelet levels, or spontaneous and phytohemagglutinin-induced lymphocyte transformations in both control and immunized animals. Both groups developed an eosinophilia, and lymphocytes from the immunized dogs responded transiently to stimulation with both larval and adult worm antigens. Specific IgM antibodies were transitory in both groups of dogs following infection. IgG antibodies developed significantly 2 weeks after infection in the immunized group; however, they did not appear until 4 weeks after infection in the control group. Both groups developed IgA antibodies 1 week after infection. They were maintained in the control dogs, in contrast to the levels in immunized animals which subsided rapidly 4 weeks after infection. Therefore, when animals are injected with soluble adult worm antigen prior to infection, specific protective immunity is acquired.     

Leukocytes and interferon in the host response to viral infections. II. Enhanced interferon response of leukocytes from immune animals

Glasgow, Lowell A. (University of Rochester School of Medicine and Dentistry, Rochester, N.Y.). Leukocytes and interferon in the host response to viral infections. II. Enhanced interferon response of leukocytes from immune animals. J. Bacteriol. 91:2185-2191. 1966.-The production of interferon was studied under in vitro conditions in peritoneal leukocytes or macrophages from mice immunized with Chikungunya virus (CV). Cultures of leukocytes obtained from animals immune to CV produced 2- to 10-fold greater amounts of interferon when exposed to an inoculum of CV than similar cell preparations from nonimmune, control animals. The viral inhibitor produced in increased quantity by CV-immune leukocytes had the biological and biochemical properties of interferon. The enhanced interferon production was inhibited by actinomycin D. This response of immune leukocytes was specific, and was initiated only by CV; it was not observed in leukocytes from animals immunized against other viruses which were challenged with CV. The presence of neutralizing antibody could not be related to this response. The observed increase in interferon production was not dependent upon an enhanced virus uptake. The data are presented as a possible new dimension of the "immune response" and may suggest a mechanism for the phenomenon of "tissue immunity."     

Oesophagostomum columbianum: gamma-irradiated third-stage larvae for immunization in lambs

40-kR gamma-irradiated third-stage larvae of Oesophagostomum columbianum were used for the immunization of Kashmir Merino lambs. Male lambs (aged from 8 to 12 weeks) were immunized in two separate experiments by two doses of irradiated larvae, given 21 days apart, and subsequently challenged with normal larvae. Judging by the establishment of worms resulting from the challenge infections in the immunized and control groups of lambs in the two experiments, a high degree of immunity was shown to develop in young lambs vaccinated with 500, followed 21 days later with 2000, 40-kR irradiated larvae. Lambs from the immunized groups showed more nodules in the intestine, a high percentage of which were positive for histotrophic stages of O. columbianum, than did controls. The importance of this finding in relation to the possible use of a vaccine for the immuno-prophylaxis of oesophagostomiasis in sheep and other animals is discussed.     

Trichinella spiralis: Generation in the presence of rat serum of factors chemotactic for rat cells

Chemotaxis of rat peritoneal cells, of which the eosinophil was the predominant migratory cell type, toward incubates of Trichinella spiralis was studied using a modified Boyden chamber. Excysted muscle larvae, preadults, and adults were incubated in a buffered medium for 20 hr at 37 C. Worms were incubated alone or with serum or spleen cells, or both, from immune and nonimmune rats. Incubates of worm stages alone possessed no chemotactic activity as compared with incubation medium as a negative control and zymosan-activated serum as a positive control. Both normal and immune sera tested alone stimulated cell migration to the same degree. Incubates of spleen cells from either normal or immunized hosts did not show chemotactic activity. Chemotaxis caused by normal and immune sera were not altered by incubation with homologous spleen cells. Addition of larva, preadults, and adult worms to sera, however, enhanced chemotactic activity over sera alone. Chemotaxis caused by larvae plus immune sera was significantly greater than that stimulated by larvae plus normal sera. This difference decreased when preadults were substituted for larvae and was not observed when adult worms were used. Reversal of the chemical gradients showed that active cell migration caused by various incubates was due to Chemotaxis.     

Suppression of Brugia malayi (sub-periodic) larval development in Aedes aegypti (Liverpool strain) fed on blood of animals immunized with microfilariae

Preliminary studies were carried out to investigate the role of filarial specific antibodies, raised in an animal model against the filarial parasite, Brugia malayi (sub-periodic), in blocking their early development in an experimental mosquito host, Aedes aegypti (Liverpool strain). In order to generate filarial specific antibodies, Mongolian gerbils, Meriones unguiculatus, were immunized either with live microfilariae (mf) of B. malayi or their homogenate. Mf were harvested from the peritoneal cavity of Mongolian gerbils with patent infection of B. malayi and fed to A. aegypti along with the blood from immunized animals. Development of the parasite in infected mosquitoes was monitored until they reached infective stage larvae (L3). Fewer number of parasites developed to first stage (L1) and subsequently to L2 and L3 in mosquitoes fed with blood of immunized animals, when compared to those fed with blood of control animals. The results thus indicated that filarial parasite specific antibodies present in the blood of the immunized animals resulted in the reduction of number of larvae of B. malayi developing in the mosquito host.