Taenia solium: Development of an Experimental Model of Porcine Neurocysticercosis.

Human neurocysticercosis (NC) is caused by the establishment of Taenia solium larvae in the central nervous system. NC is a severe disease still affecting the population in developing countries of Latin America, Asia, and Africa. While great improvements have been made on NC diagnosis, treatment, and prevention, the management of patients affected by extraparenchymal parasites remains a challenge. The development of a T. solium NC experimental model in pigs that will allow the evaluation of new therapeutic alternatives is herein presented. Activated oncospheres (either 500 or 1000) were surgically implanted in the cerebral subarachnoid space of piglets. The clinical status and the level of serum antibodies in the animals were evaluated for a 4-month period after implantation. The animals were sacrificed, cysticerci were counted during necropsy, and both the macroscopic and microscopic characteristics of cysts were described. Based on the number of established cysticerci, infection efficiency ranged from 3.6% (1000 oncospheres) to 5.4% (500 oncospheres). Most parasites were caseous or calcified (38/63, 60.3%) and were surrounded by an exacerbated inflammatory response with lymphocyte infiltration and increased inflammatory markers. The infection elicited specific antibodies but no neurological signs. This novel experimental model of NC provides a useful tool to evaluate new cysticidal and anti-inflammatory approaches and it should improve the management of severe NC patients, refractory to the current treatments.

[Evaluation of the impact of a control program against taeniasis-cysticercosis (Taenia solium)]. / Evaluación del impacto de un programa de control de la teniasis-cisticercosis (Taenia solium).

OBJETIVE: The impact of a control program is evaluated to eventually eradicate taeniasis-cysticercosis (Taenia solium) based on education and vaccination of pigs. MATERIALS AND METHODS: The prevalence of porcine cysticercosis was estimated using tongue inspection, ultrasound and determination of antibodies, before and three years after the application in three regions of the state of Guerrero. RESULTS: A significant reduction in the prevalence of porcine cysticercosis of 7 to 0.5% and 3.6 to 0.3% estimated by tongue examination or ultrasound respectively (p<0.01) and a no significant decrease in seroprevalence from 17.7 to 13.3% were observed. CONCLUSIONS: The reduction of the prevalence of taeniasis-cysticercosis establishes the program's effectiveness in preventing infection. The sustained presence of antibodies, compatible with contact of Taenia solium or other related helminths, underlines the importance of maintaining interventions to achieve eradication.

Evaluación del impacto de un programa de control de la teniasis-cisticercosis (Taenia solium) / Evaluation of the impact of a control program against taeniasis-cysticercosis (Taenia solium)

Objetivo. Evaluar el impacto de un programa de control de la teniasis-cisticercosis por Taenia solium con fines de erradicación, basado en educación de la comunidad y vacunación de cerdos. Material y métodos. Se estimó la prevalencia de cisticercosis porcina por medio de la palpación de lengua, ultrasonido y presencia de anticuerpos en suero, antes de iniciar el programa y tres años después, en tres regiones del estado de Guerrero. Resultados. Se observó una reducción significativa en la prevalencia de cisticercosis porcina de 7 a 0.5% y de 3.6 a 0.3%, estimadas por examen de lengua y ultrasonido, respectivamente (p<0.01), y una disminución no significativa de la seroprevalencia de 17.7 a 13.3%. Conclusiones. La reducción de la prevalencia de teniasis-cisticercosis comprueba la efectividad del programa para prevenir la infección. La presencia sostenida de anticuerpos es compatible con continuos contactos con Taenia solium u otros helmintos relacionados, y señala la necesidad de mantener las intervenciones para lograr su erradicación.

Objetive. The impact of a control program is evaluated to eventually eradicate taeniasis-cysticercosis (Taenia solium) based on education and vaccination of pigs. Materials and methods. The prevalence of porcine cysticercosis was estimated using tongue inspection, ultrasound and determination of antibodies, before and three years after the application in three regions of the state of Guerrero. Results. A significant reduction in the prevalence of porcine cysticercosis of 7 to 0.5% and 3.6 to 0.3% estimated by tongue examination or ultrasound respectively (p<0.01) and a no significant decrease in seroprevalence from 17.7 to 13.3% were observed. Conclusions. The reduction of the prevalence of taeniasis-cysticercosis establishes the program's effectiveness in preventing infection. The sustained presence of antibodies, compatible with contact of Taenia solium or other related helminths, underlines the importance of maintaining interventions to achieve eradication.

Adaptation of ostriches to transport-induced stress: physiometabolic response.

This study assessed the effects of five different transport periods on physiometabolic responses and gas exchange in ostriches. It included 138 ostriches that were assigned to five experimental groups. G1 included 78 birds that were set aside as a reference group (RG). Each one of the four remaining experimental groups included 20 ostriches, which were transported in the following manner: G1 on one occasion for a period of 1 h; G2 on one occasion for 2 h; and G3 on one occasion for 3 h; G4a was made up of the ostriches from G1 but they were shipped on a second occasion, for 2 h (making a total of 3 h); finally, G4b was that same group, but after a third transport period, on this occasion during 3 h (for a total of 6 h). Groups G2 and G3 presented the most marked blood alterations (P < 0.05), including an increase in pH, hypocapnia (27.8 ± 0.80 mmHg), hypernatremia (171.75 ± 1.84 mmol/L), hypocalcemia (0.95 ± 0.03 mmol/L), and hyperglycemia (224.05 ± 3.94 mg/dL). Also, group G3 presented the lowest hematocrit values (26.5 ± 0.47 HTC%). Therefore, transporting ostriches with no prior experience for 3 h caused the most pronounced physiometabolic changes.

Effective protection induced by three different versions of the porcine S3Pvac anticysticercosis vaccine against rabbit experimental Taenia pisiformis cysticercosis.

In an effort to develop an effective and affordable oral vaccine against porcine Taenia solium cysticercosis, the S3Pvac anti-cysticercosis vaccine was expressed in papaya calli. Taenia pisiformis experimental rabbit cysticercosis was used as a model to compare the efficacy of the oral vaccine vs. the injectable S3Pvac-synthetic and S3Pvac-phage versions. Oral S3Pvac-papaya significantly reduced the expected number of hepatic lesions and peritoneal cysticerci to a similar extent than the injectable vaccines. This study reports for the first time an effective oral vaccine against T. pisiformis cysticercosis, possibly useful against porcine T. solium cysticercosis.

Recombinant S3Pvac-phage anticysticercosis vaccine: Simultaneous protection against cysticercosis and hydatid disease in rural pigs.

This paper provides macroscopic and histological evidence on the statistically significant protective effects of S3Pvac-phage vaccination against porcine cysticercosis and hydatidosis. The study included 391 rustically bred pigs (187 vaccinated and 204 controls). Vaccination significantly reduced the prevalence of cysticercosis by 61.7%. Vaccination also significantly reduced by 56.1% the prevalence of hydatidosis caused by Echinococcus granulosus in pigs. The presence of the vaccine epitopes in both cestodes is probably involved in the cross-protection observed. Increased inflammation was found in 5% of cysticerci recovered from controls, versus 24% from vaccinated pigs (P<0.01). Hydatid cysts were non-inflammatory in either group. Vaccination was effective to prevent one single disease, but it failed to prevent the simultaneous infections with both parasites in a same pig. The widening of the S3Pvac-phage vaccine protective repertoire to include hydatidosis is a convenient feature that should reduce the prevalence of two frequent zoonoses that affect rustic porcine breading with a single action. Thus, the costs of two different vaccination programs would be reduced to a single one with significant reduction in both zoonoses.

Characterization of S3Pvac anti-cysticercosis vaccine components: implications for the development of an anti-cestodiasis vaccine.

BACKGROUND: Cysticercosis and hydatidosis seriously affect human health and are responsible for considerable economic loss in animal husbandry in non-developed and developed countries. S3Pvac and EG95 are the only field trial-tested vaccine candidates against cysticercosis and hydatidosis, respectively. S3Pvac is composed of three peptides (KETc1, GK1 and KETc12), originally identified in a Taenia crassiceps cDNA library. S3Pvac synthetically and recombinantly expressed is effective against experimentally and naturally acquired cysticercosis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the homologous sequences of two of the S3Pvac peptides, GK1 and KETc1, were identified and further characterized in Taenia crassiceps WFU, Taenia solium, Taenia saginata, Echinococcus granulosus and Echinococcus multilocularis. Comparisons of the nucleotide and amino acid sequences coding for KETc1 and GK1 revealed significant homologies in these species. The predicted secondary structure of GK1 is almost identical between the species, while some differences were observed in the C terminal region of KETc1 according to 3D modeling. A KETc1 variant with a deletion of three C-terminal amino acids protected to the same extent against experimental murine cysticercosis as the entire peptide. On the contrary, immunization with the truncated GK1 failed to induce protection. Immunolocalization studies revealed the non stage-specificity of the two S3Pvac epitopes and their persistence in the larval tegument of all species and in Taenia adult tapeworms. CONCLUSIONS/SIGNIFICANCE: These results indicate that GK1 and KETc1 may be considered candidates to be included in the formulation of a multivalent and multistage vaccine against these cestodiases because of their enhancing effects on other available vaccine candidates.

Genetic diversity of Taenia solium cysticerci from naturally infected pigs of central Mexico.

This study was designed to explore if each individual case of naturally acquired porcine cysticercosis, living in different geographic rural areas of central Mexico, is caused by one or more different specimens of Taenia solium tapeworm. The genetic variability among cysticerci from the same pig and that from different pigs was assessed by random amplified polymorphic DNA markers (RAPDs), through the percentage of polymorphic loci, the number of effective alleles, the expected heterozygosity and the Shannon index. The parasite population's reproductive structure was estimated through the association index (I(A)), and the degree of genetic differentiation and variation was determined using AMOVA. Using six different random primers, and a total of 181 cysticerci from 14 pigs, 88 different loci were amplified: 85% were polymorphic between pigs and 24% within pigs. The phenogram grouped the cysticerci into eight major clusters, with differences in the genetic distances among all cysticerci from 14 pigs ranging from 0.78 to 1. Most of the cysticerci grouped in accord with their different geographical origin and with their pig of origin. The similarity matrix produced from the phenogram (obtained by UPGMA) and the original similarity matrix yielded a good cophenetic correlation (r=0.82317, P=0.0004), which suggests that the phenogram accurately represents the original genetic similarities between isolates. The combination of I(A) (0.0-0.089) with the genetic diversity index (0.009-0.073) supports the idea that DNA diversity in T. solium cysticerci of naturally infected pigs is within the range expected from a recombination process occurring during sexual reproduction. The small genetic diversity found within the cysticerci of each pig (33.81%), when compared with that between pigs (66.19%), indicates that pigs are rarely infected by different tapeworms. It would then appear that porcine cysticercosis courses with effective concomitant immunity, as occurs in ovine cysticercosis.

Spatial distribution of Taenia solium porcine cysticercosis within a rural area of Mexico.

Cysticercosis is caused by Taenia solium, a parasitic disease that affects humans and rurally bred pigs in developing countries. The cysticercus may localize in the central nervous system of the human, causing neurocysticercosis, the most severe and frequent form of the disease. There appears to be an association between the prevalence of porcine cysticercosis and domestic pigs that wander freely and have access to human feces. In order to assess whether the risk of cysticercosis infection is clustered or widely dispersed in a limited rural area, a spatial analysis of rural porcine cysticercosis was applied to 13 villages of the Sierra de Huautla in Central Mexico. Clustering of cases in specific households would indicate tapeworm carriers in the vicinity, whereas their dispersal would suggest that the ambulatory habits of both humans and pigs contribute to the spread of cysticercosis. A total of 562 pigs were included in this study (August-December 2003). A global positioning system was employed in order to plot the geographic distribution of both cysticercotic pigs and risk factors for infection within the villages. Prevalence of pig tongue cysticercosis varied significantly in sampled villages (p = 0.003), ranging from 0% to 33.3% and averaging 13.3%. Pigs were clustered in households, but no differences in the clustering of cysticercotic and healthy pigs were found. In contrast, the presence of pigs roaming freely and drinking stagnant water correlated significantly with porcine cysticercosis (p = 0.07), as did the absence of latrines (p = 0.0008). High prevalence of porcine cysticercosis proves that transmission is still quite common in rural Mexico. The lack of significant differentiation in the geographical clustering of healthy and cysticercotic pigs weakens the argument that focal factors (e.g., household location of putative tapeworm carriers) play an important role in increasing the risk of cysticercosis transmission in pigs. Instead, it would appear that other wide-ranging biological, physical, and cultural factors determine the geographic spread of the disease. Extensive geographic dispersal of the risk of cysticercosis makes it imperative that control measures be applied indiscriminately to all pigs and humans living in this endemic area.

Vaccines against cysticercosis.

Taenia solium cysticercosis is a major parasitic disease that seriously and frequently affects human health and economy in undeveloped countries. Since pigs are an indispensable intermediate host, it is conceivable to curb transmission by reducing pig cysticercosis through their effective vaccination. This article reviews current knowledge on the development vaccines against porcine cysticercosis. It highlights the development of several versions of S3Pvac aimed to increase effectiveness, reduce costs and increase feasibility by novel delivery systems and alternative routes of administration.

Inexpensive anti-cysticercosis vaccine: S3Pvac expressed in heat inactivated M13 filamentous phage proves effective against naturally acquired Taenia solium porcine cysticercosis.

In search of reducing vaccine production costs', a recombinant M13 phage version of the anti-cysticercosis tripeptide vaccine (S3Pvac) was developed. The efficacy of S3Pvac-Phage vs. placebo was evaluated in a randomized trial that included 1,047 rural pigs in 16 villages of Central Mexico. Three to five months after vaccination 530 pigs were examined by tongue inspection. At 5-27 months of age, 331 pigs (197 vaccinated/134 controls) were inspected at necropsy. Vaccination reduced 70% the frequency of tongue cysticercosis and, based on necropsy, 54% of muscle-cysticercosis and by 87% the number of cysticerci.

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.

Early stages of development of the Taenia solium metacestode in pigs.

In order to identify the early stages of Taenia solium metacestodes, 12 pigs were each fed 100,000 viable eggs and later killed and necropsied at different times after infection. Hematoxylin-eosin (HE) and immunohistochemical techniques (IHCs) were used to identify onchospheres and cysticerci in different tissues. At 2 days postinfection (dpi) structures compatible with onchospheres were found in the lumen of the small intestine, and in the mesenteric blood vessels and lymph nodes. At 4 dpi, these same structures were observed in the small intestine, the liver, and skeletal muscles. Between 6 and 39 dpi, they were found only in skeletal muscles. Between 2 and 6 dpi the postonchospheres were circular and oval shaped and measured between 6 and 34 x 27 microm. From 14 to 39 dpi, well-developed metacestodes 550 x 750 microm were observed. IHCs support the identification of early stages of T. solium.

Improvement of the synthetic tri-peptide vaccine (S3Pvac) against porcine Taenia solium cysticercosis in search of a more effective, inexpensive and manageable vaccine.

Vaccination of pigs may curtail Taenia solium transmission by reducing the number of cysticerci, the precursors of adult intestinal tapeworms in humans. Several antigen preparations induce protection against porcine cysticercosis in experimental settings but only one subunit vaccine (S3Pvac) has been tested and proved effective in the field against naturally acquired disease. Besides improving of the vaccine's effectiveness, significant reductions in production costs and in the logistics of its administration are necessary for the feasibility of nationwide control programs. This review highlights the development of several versions of S3Pvac aimed to increase effectiveness, reduce costs and increase feasibility by novel delivery systems and alternative routes of administration.

Therapeutic capacity of the synthetic peptide-based vaccine against Taenia solium cysticercosis in pigs.

The S3Pvac synthetic vaccine composed of three peptides (GK1, KETc1 and KETc12) effectively protect against pig cysticercosis. Preliminary results point to an additional cysticidal capacity induced by S3Pvac or GK1 immunization. Herein, clear evidences of the cysticidal effect of S3Pvac but not of GK1 are presented. S3Pvac immunization of already experimentally infected pigs induced a reduction in the parasite load, in the vesicular cysticerci and in their viability. It also substantially increases the percent of histological damaged cysticerci more importantly in muscles than in brains, with a concomitant reduction in the antibody levels. Thus, S3Pvac represents a powerful means of controlling cysticercosis infection in pigs.

Apoptosis induced by gamma irradiation of Taenia solium 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.

[Taeniasis and detection of antibodies against cysticercus among inhabitants of a rural community in Guerrero State, Mexico]. / Teniosis y detección de anticuerpos anticisticerco en personas de una comunidad rural del estado de Guerrero.

OBJECTIVE: To assess the frequency of Taenia solium carriers and its relationship with human cysticercosis in a Mexican locality. MATERIAL AND METHODS: A cross-sectional study was conducted in 1998, in a locality of Guerrero State, Mexico. Four hundred and three fecal samples were analyzed by enzyme-linked immunosorbent assay (ELISA) to detect Taenia sp coproantigen. Ninety two serum samples were also analyzed for antibodies against cisticerci using the immunoelectrotransfer blot assay (IET). Data were analyzed using descriptive statistics and odds ratios. RESULTS: Five of the 403 fecal samples were positive (1.2%). The adult cestode was recovered in only two people. Three (3.26%) out of the 92 serum samples that were analyzed for anticysticercus antibodies were positive. Seventeen serum samples corresponded to people living with a person positive to the coproantigen test (first group), the remaining 75 were obtained from people without a history of releasing taenia proglottids (second group). In the first group, 2 positive sera were detected (11.8%), while in the second only I was positive (1.3%) (odds ratio = 9.87, 95% confidence interval 0.64 to 295.56, p = 0.08). CONCLUSIONS: The difficulty to obtain the adult parasite in persons positive to coproantigens, may be due to difficult expulsion, a shorter permanence of the parasite in the host, insufficient dosage of treatment, or to lack of specificity of the diagnostic test. Further studies are needed to evaluate these possibilities; a better knowledge of parasite transmission dynamics will allow the implementation of prevention and control measures and a better assessment of diagnostic tests under field conditions. The English version of this paper is available at: http://www.insp.mx/salud/index.html.

[Laboratory animals and official Mexican norms (NOM-062-ZOO-1999)]. / Animales de laboratorio y la Norma Oficial Mexicana (NOM-062-ZOO-1999).

This article concerns animal experimentation and official Mexican norm Nom 0062-Zoo-1999 entitled Technical specifications for the production, care and use of laboratory animals. The history of animal experimentation is briefly resumed. During the nineteenth century, doubts arose as to the right to expose animals to experimental procedures that frequently cause pain and suffering. The first law which protected animals against cruelty was passed in Great Britain in 1876; subsequently, other nations approved similar legislation. During the second part of the twentieth century, opposition to animal experimentation grew. Other groups, mainly scientists and pharmaceutical concerns, defended the right to use animals in research. New knowledge concerning the neurophysiology, cognitive capacity, and the animal faculty to experience pain is briefly mentioned. Guidelines on care and use of animals used in research published in several countries are listed. Finally, the recently published Mexican legislation (Norm) referring to production, care and use of laboratory animals is discussed and its benefits are stressed.

The experimental infection of pigs with different numbers of Taenia solium eggs: immune response and efficiency of establishment.

Three of 4 pigs inoculated with 10 eggs of Taenia solium became infected. In those pigs infected with larger numbers of eggs, all became infected. Specific antibodies against the metacestodes were found in serum at day 30 postinoculation (PI) in animals that received 1,000 or more eggs and at day 60 in those that received 10 or 100 eggs. The concentration and diversity of antibodies increased up to the day of death in pigs that received 10,000 or 100,000 eggs. All pigs infected with 1,000 or more eggs developed antibodies, but only 40% and 75% of pigs that received 10 and 100 eggs, respectively, developed antibodies. Metacestodes were found in the muscles of 23 of the 27 infected animals. In 35.7% of the pigs that received 1,000 or more eggs, metacestodes were also found in the brain. Most of the metacestodes found in pigs infected with 10 or 100 eggs were caseous, whereas in pigs infected with 1,000 or more eggs the majority of metacestodes were vesicular. This study shows that the severity of T. solium infection and the possible regulation of the immune system-evasion mechanisms depend on the number of metacestodes that succeed in establishing themselves and remain vesicular.

Taenia solium cysticercosis: lymphocytes in the inflammatory reaction in naturally infected pigs.

Immunohistochemistry was used to examine the type of lymphocytes in muscle taeniosis-cysticercosis in naturally infected pigs. The inflammatory response studied was classified into lesions of grades 1, 3, and 5. In grade 1, with a minimal inflammatory infiltrate consisting of eosinophils and a few mononuclear cells, the immunostaining showed more CD4+ cells than CD8+ cells and IgM cells. In grade 3, when the granulomatous reaction was not yet well developed and the destruction of the parasite began, CD4+ and Ig M+ were the predominant cells, although CD8+ cells showed a notable increase. In grade 5, with a few parasitic structures surrounded by an extensive granulomatous infiltrate, lymphocyte subsets were decreased in number and did not show differences from grade 1 except for IgM+ cells, which remained increased. The organization of an active inflammatory response against the metacestode of Taenia solium in pigs includes the sequential participation of CD4+, CD8+ and IgM+ lymphocytes.