Surface plasmon resonance-based inhibition assay for real-time detection of Cryptosporidium parvum oocyst

A surface plasmon resonance (SPR)-based inhibition assay method using a polyclonal anti-mouse IgM arrayed Cryptosporidium sensor chip was developed for the real-time detection of Cryptosporidium parvum oocysts. The Cryptosporidium sensor chip was fabricated by subsequent immobilization of streptavidin and polyclonal anti-mouse IgM (secondary antibody) onto heterogeneous self-assembled monolayers (SAMs). The assay consisted of the immunoreaction step between monoclonal anti-C. parvum oocyst (primary antibody) and oocysts, followed by the binding step of the unbound primary antibody onto the secondary antibody surface. It enhanced not only the immunoreaction yield of the oocysts by batch reaction but also the accessibility of analytes to the chip surface by antibody–antibody interaction. Furthermore, the use of optimum concentration of the primary antibody maximized its binding response on the chip. An inversely linear calibration curve for the oocyst concentration versus SPR signal was obtained in the range of 1×106–1×102 oocysts ml-1. The oocyst detection was also successfully achieved in natural water systems. These results indicate that the SPR-based inhibition assay using the Cryptosporidium sensor chip has high application potential for the real-time analysis of C. parvum oocyst in laboratory and field water monitoring.

Use of live oocyst vaccine in the control of turkey coccidiosis: Effect on performance and intestinal morphology

An experiment was conducted to determine the effects of different coccidiosis-preventing programs on performance and intestinal morphology of commercial turkeys. Three hundred fifteen1-d-old female commercial cross turkey poults (British United Turkeys, BUT Big 9) were distributed into 3 treatments with 5 replicates of 21 birds each. Three programs were evaluated from 1 to 70 d of age, where program 1 had no anticoccidial drug and no vaccination against coccidiosis; program 2 had an anticoccidial drug (maduramycin 1%, 5 ppm); and program 3 had a vaccination (commercial vaccine, 4 species of Eimeria). All the groups were challenged with a dose of oocysts sporulated (20,000/bird) of 2 species of Eimeria at 21 d of age. In the growing phase (d 0-28), BW, BW gain, and FCR were significantly greater in treated groups compared with control group. In the fattening phase, the performance was not affected by treatments. Treatments and coccidiosis challenge had no significant effects on intestinal villus height. These observations support other reports that confirm live oocyst vaccination can be used effectively as a preventive against avian coccidiosis in commercially reared turkeys.

Selection and characterisation of two attenuated vaccine lines of Eimeria tenella in Australia

Objective To attenuate two strains of Eimeria tenella by selecting for precocious development and evaluate the strains in characterisation trials and by field evaluation, to choose one precocious line for incorporation into an Australian live coccidiosis vaccine for poultry. Design Two strains from non-commercial flocks were passaged through chickens while selecting for precocious development. Each strain was characterised for drug sensitivity, pathogenicity, protection against homologous and heterologous challenge, and oocyst output in replicated experiments in which the experimental unit was a cage of three birds. Oocyst output and/or body weight gain data collected over a 10 to 12 day period following final inoculation were measured. Feed conversion ratios were also calculated where possible. Results Fifteen passages resulted in prepatent periods reduced by 24 h for the Redlands strain (from 144 h to 120 h)and 23 h for the Darryl strain (from 139 h to 116 h). Characterisation trials demonstrated that each precocious line was significantly less pathogenic than its parent strain and each effectively induced immunity that protected chickens against challenge with both the parent strain and other virulent field strains. Both lines had oocyst outputs that, although significantly reduced relative to the parent strains, remained sufficiently high for commercial vaccine production, and both showed susceptibility to coccidiostats. Conclusion Two attenuated lines have been produced that exhibit the appropriate characteristics for use in an Australian live coccidiosis vaccine.

Antibody response against endogenous stages of an attenuated strain of Eimeria tenella

The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite lifecycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.

Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, d-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using 4-(14) C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.

Role of plasmodium translationally repressed gene products in malaria transmission

Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2015

Caractérisation de l’infection naturelle à Cryptosporidium spp. chez le chien et le chat vus en établissement vétérinaire

Cryptosporidium spp. est un protozoaire parasite du système gastro-intestinal largement répandu chez les vertébrés et causant la cryptosporidiose, une zoonose occasionnant des troubles digestifs sévères pouvant entrainer la mort chez les individus immunodéficients. Au Canada, la déclaration de cette maladie est obligatoire depuis l’an 2000. Ainsi, il est pertinent de mieux comprendre l’infection chez les animaux de compagnie, puisqu’ils sont potentiellement un réservoir du parasite. Durant l’année 2008, des échantillons fécaux provenant de 1 202 chats (n = 371) et chiens (n = 831) de la province du Québec ont été analysés par comptage des ookystes de Cryptosporidium spp. au moyen de la technique de centrifugation en solution de sulfate de zinc. Dans cette étude,la prévalence de Cryptosporidium spp. chez les chats (28/371 : 7,55 %) et chez les chiens(88/831 : 10,59 %) de compagnie confirme leur potentiel en tant que réservoir du parasite. Au Québec, de par leur nombre, les chats sont potentiellement un réservoir zoonotique du parasite plus important que celui des chiens, bien qu’il n’existe pas de différence significative entre la prévalence du parasite chez le chat et le chien pour l’année 2008. L’âge (p = 0,0001) et l’infection concomitante par Giardia spp. (p = 0,0001) se sont avérés être des facteurs associés avec la présence de Cryptosporidium spp. chez le chien. Parmi l’ensemble des variables testées chez le chat (l’âge, le sexe, la saison et l’infection concomitante par Giardia spp.), aucune n’a été associée de manière significative à la présence du parasite chez le chat. Ceci peut être dû au nombre limité d’individus testés pour cette espèce. Un suivi de l’excrétion des ookystes de Cryptosporidium spp. chez deux chats suggère que l’excrétion des ookystes peut se faire sur une période de sept mois et que le taux d’excrétion varie dans le temps. Le diagnostic moléculaire des espèces et génotypes de Cryptosporidium spp. isolés à partir des échantillons de matières fécales devait être réalisé par la technique de PCR emboîtée des fragments des gènes ARNr 18S et HSP70 et du séquençage des produits de PCR. Aucun résultat positif n’a toutefois été obtenu. Afin d’augmenter la puissance statistique des analyses épidémiologiques sur la prévalence de Cryptosporidium spp., il serait nécessaire à l’avenir de travailler sur un nombre d’animaux beaucoup plus important.

The role of osmiophilic bodies and Pfg377 expression in female gametocyte emergence and mosquito infectivity in the human malaria parasite plasmodium falciparum

Osmiophilic bodies are membrane-bound vesicles, found predominantly in Plasmodium female gametocytes, that become progressively more abundant as the gametocyte reaches full maturity. These vesicles lie beneath the subpellicular membrane of the gametocyte, and the release of their contents into the parasitophorous vacuole has been postulated to aid in the escape of gametocytes from the erythrocyte after ingestion by the mosquito. Currently, the only protein known to be associated with osmiophilic bodies in Plasmodium falciparum is Pfg377, a gametocyte-specific protein expressed at the onset of osmiophilic body development. Here we show by targeted gene disruption that Pfg377 plays a fundamental role in the formation of these organelles, and that female gametocytes lacking the full complement of osmiophilic bodies are significantly less efficient both in vitro and in vivo in their emergence from the erythrocytes upon induction of gametogenesis, a process whose timing is critical for fertilization with the short-lived male gamete. This reduced efficiency of emergence explains the significant defect in oocyst formation in mosquitoes fed blood meals containing Pfg377-negative gametocytes, resulting in an almost complete blockade of infection.

Partial protection against tissue cysts formation in pigs vaccinated with crude rhoptry proteins of Toxoplasma gondii

A vaccine containing crude Toxoplasma gondii rhoptry proteins incorporated in the immunostimulating complexes (ISCOM) adjuvant was tested in pigs for protecting against tissue cyst formation. For this, 38 mixed breed pigs were divided into four groups, G1 (vaccinated challenged, n = 10) received two doses (100 mu g/dose) of the rhoptry vaccine at days 0 and 21, G2 (vaccinated challenged, n = 10) received viable tachyzoites (7 x 10(7)) of the RH strain at day 0, G3 (unvaccinated challenged, n = 10) and G4 (unvaccinated unchallenged, n = 8). Pigs were challenged with 4 x 10(4) VEG strain oocysts 57 days later. The G1 pigs produced high IgG antibody levels in the indirect enzyme-linked immunosorbent assay (ELISA) after the second dose of rhoptry vaccine, but were not clinically protected against a high dose oocyst challenge. Partial protection was observed in G1 at the chronic phase of infection, when compared with G3. Pigs in group 2 developed high antibody levels and were protected against clinic signs. T gondii was not detected in two (G1) and three (G2) pigs by mouse bioassay. The results indicate partial protection in pigs vaccinated with a rhoptry vaccine. (c) 2005 Elsevier B.V. All rights reserved.

Eimeria Spp. in Brazilian Water Buffalo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Occurrence of gastrointestinal parasites in goat kids

Amostras fecais de cabritos machos e fêmeas, de diferentes raças e com até uma ano de idade, foram examinadas para determinação do número de ovos e oocistos por grama de fezes (OPG e OoPG, respectivamente) e coprocultura para identificação genérica dos nematódeos. Ovos de helmintos e oocistos de Eimeria spp. foram observados em 93,06% (188/202) e 77,22% (156/202) das amostras, respectivamente. Pelas coproculturas, foram identificados os gêneros Cooperia em 11,88% (24/202), Haemonchus em 51,98% (105/202), Oesophagostomum em 9,4% (19/202), Strongyloides em 5,94 (12/202) e Trichostrongylus em 20,79% (42/202) das amostras. As espécies de Eimeria encontradas foram E. alijevi em 25,24% (51/202), E. arloingi em 7,42% (15/202), E. caprina em 2,97% (6/202), E. caprovina em 10,39% (21/202), E. christenseni em 4,45% (9/202), E. joklchijevi em 11,38% (23/202), E. hirci em 9,4% (19/202) e E. ninakohlyakimovae em 28,71% (58/202) das amostras. Dentre os parasitas gastrintestinais, houve predominância do gênero Haemonchus e de duas espécies de Eimeria: E. ninakohlyakimovae e E. alijevi.

SPECIES OF COCCIDIA OCCURRING IN LAMBS IN SAO-PAULO STATE, BRAZIL

The purpose of this experiment was to characterize the species of Eimeria affecting lambs and their infection pattern. Faecal samples were collected from each animal at 14-day intervals, beginning when lambs were 2 weeks old and ending when they were 32 weeks old. The oocysts were counted and identified as E. intricata, E. parva, E. pallida, E. crandallis, E. bakuensis, E. weybridgensis, E. ahsata and E. ovinoidalis. The highest oocyst counts were observed when the lambs were 4-8 weeks old.

Real-time PCR assay targeting the actin gene for the detection of Cryptosporidium parvum in calf fecal samples

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anticoccidial effects of coumestans from Eclipta alba for sustainable control of Eimeria tenella parasitosis in poultry production

The aim of this study was to evaluate the anticoccidial efficacy of a product containing coumestans from Eclipta alba. Experimental conditions were set up as to reproduce the environment conditions for husbandry adopted in commercial broiler farms. Broilers were raised in broiler chicken shed provided with feeders, drinkers, illumination and temperature control systems and floor covering to afford an adequate nourishing environment. Male Cobb broilers (240) were assigned to four experimental groups being each experimental group set apart in rice straw-covered shed isolated with wire mesh. One-day-old broilers were reared in a coccidian-free environment with ad libitum supply of filtered water and freely available standard feed, from the 1st to the 35th day of life. The T1 group received standard feed (negative control); T2 was treated with standard feed supplemented with 66 ppm of salinomycin (positive control); groups T3 and T4 had standard feed supplemented with the ethyl acetate fraction from methanolic extract of E. alba aerial parts, which contains the coumestans WL and DWL (120 and 180 ppm, respectively). The chicken broilers were individually infected with 2 x 104 oocysts of Eimeria tenella when they were 14 days old and were monitored weekly to evaluate zootechnical parameters such as weight gain and food conversion ratio. Counting of coccidial oocyst in chiken feces was assessed from random samples, from the 21st to 28th days of life, which corresponded to 7-14 days after the infection. Five chickens selected at random from each experimental group were subsequently euthanized at 21, 28 or 35 days of life to determine the lesion score in the cecal region and to excise a cecum portion for histopathological evaluation. The group treated with coumestans from E. alba presented an average weight gain and food conversion ratio higher than the negative control group and similar to the mean value of the positive control group. Coumestan-treated groups showed a significant decrease in the oocyst counting since the 21th day of life and displayed a reduced number of macroscopic lesions. Histopathological evaluations of cecum fragments showed that both treatments induced the migration of defense cells at the site of infection. A severe destruction of the cecal lining was found in the intestinal tract of broilers fed with a coumestans dose of 180 ppm. Overall, our results validate the use of a phytotherapy containing E. alba coumestans at a dose of 120 ppm as a therapeutic or prophylactic agent against avian coccidiosis. (C) 2010 Elsevier B.V. All rights reserved.

Experimental infection of bovines with oocysts of Toxoplasma gondii

Calves aged 3 mth were readily infected with oocysts and cysts of Toxoplasma gondii administered by the oral route. Fever, respiratory distress, nasal discharge, and hyperemia of the conjunctivas were the most significant clinical signs noted in the infected animals. Parasitemia was demonstrated in all infected calves. It occurred on different days and up to 62 days after the infection. Toxoplasma was demonstrated in tissues of all infected calves, and the organ most frequently parasitized was the lymph node. Parasitism of the retina was demonstrated in 2 calves. All infected animals had antibody against T. gondii in their serum. The Sabin-Feldman dye test and the indirect immunofluorescent test were both useful in detecting antitoxoplasma antibody.