Collectins and galectins in the abomasum of goats susceptible and resistant to gastrointestinal nematode infection.

Originally described in cattle, conglutinin belongs to the collectin family and is involved in innate immune defense. It is thought that conglutinin provides the first line of defense by maintaining a symbiotic relationship with the microbes in the rumen while inhibiting inflammatory reactions caused by antibodies leaking into the bloodstream. Due to the lack of information on the similar lectins and sequence detection in goats, we characterized the goat conglutinin gene using RACE and evaluated the differences in its gene expression profile, as well as in the gene expression profiles for surfactant protein A, galectins 14 and 11, interleukin 4 and interferon-gamma in goats. We used Saanen and Anglo Nubian F2 crossbred goats monitored over a period of four months and characterized them as resistant (R) or susceptible (S) based on the average values of EPG counts. Goat conglutinin was similar to bovine conglutinin, but its gene expression varied among different tissues. However, as with bovine conglutinin, it was most highly expressed in the liver. Variation in conglutinin (R=24.3±3.9; S=23.5±2.6, p=0.059), protein surfactant A (R=23.8±5.2, S=24.4±2.3, p=0.16), galectin 14 (R=15.9±3.5, S=14.7±6.2, p=0.49) and galectin l1 gene expression (R=25.4±2.6, S=25.8±3.7, p=0.53) was not significant between groups. However, there were weak correlations between interleukin 4 and the protein surfactant A gene (r=0.459, p=0.02) and between interleukin 4 and galectin 11 (r=0.498, p=0.01). Strong correlation between interferon-gamma and galectin 14 (r=0.744, p=0.00) was observed. Galectin 14 was negatively correlated with the number of nematodes in the goat (r=-0.416, p=0.04) as well as the EPG count (r=-0.408, p=0.04). This is the first study to date that identifies the gene expression of conglutinin, surfactant protein A and galectins 14 and 11 in the goat abomasum. In conclusion, we present evidence that lectin is involved in the immune response to gastrointestinal nematodes, which suggests that collectins and galectins are involved in the molecular recognition of helminths.

Resistência anti-helmíntica em rebanhos caprinos nos biomas Caatinga e Mata Atlântica / Anthelmintic resistance in goat herds in the Caatinga and Mata Atlântica biomes

A utilização de anti-helmínticos por longos períodos como principal medida de controle das parasitoses gastrintestinais de ruminantes levou a ineficácia aos levamisol, benzimidazóis e avermectinas. Este estudo descreve a atividade anti-helmíntica in vivo em populações naturais de nematoides trichostrongilídeos de caprinos. Foram selecionados 18 rebanhos provenientes dos biomas Caatinga (n=12) e Mata Atlântica (n=6), do Estado da Bahia, Brasil, criados em pastagens comunais em região semiárida. Grupos de oito a 10 animais foram tratados com albendazol (ABZ), ivermectina (IVM), levamisol (LEV), moxidectina (MOX) e closantel (CLOS). Os resultados do Teste de Redução da Contagem de Ovos nas Fezes indicaram resistência simultânea dos gêneros Haemonchus sp. e Trichostrongylus spp. para o ABZ, IVM, LEV, MOX e CLOS. As percentagens de eficácia variaram de 0-92%, 0-75%, 0-91%, 69-97% e 0-85% para o ABZ, IVM, LEV, MXD e CLOS, respectivamente, no bioma Caatinga e 0-59% para o ABZ e 9-59% para o IVM no bioma Mata Atlântica. Verificou-se nos rebanhos eficácia inferior a 95% para estes anti-helmínticos, com exceção de um único rebanho no qual a eficácia para MOX foi de 97%, o que sugere a presença de NGIs resistentes aos principais classes de anti-helmínticos em rebanhos caprinos destes biomas...

The use of anthelmintic drugs for long periods as the main measure control of gastrointestinal nematodes (GINs) has led to the inefficacy of levamisole, benzimidazoles and macrocyclic lactones. This study describes the in vivo anthelmintic activity against natural trichostrongyle nematodes populations in goats. We selected 18 herds from the Caatinga (n=12) and Mata Atlântica (n=6) biomes, Bahia State, Brazil, raised in communal pastures in semiarid region. Groups of 8 to 10 goats were treated with albendazole (ABZ), ivermectin (IVM), levamisole (LEV), moxidectin (MOX), and closantel (CLOS). The results of the Fecal Egg Count Reduction Test indicated simultaneous resistance of Haemonchus sp. and Trichostrongylus spp. genera against albendazole (ABZ), ivermectin (IVM), levamisole (LEV), moxidectin (MOX), and closantel (CLOS). The efficacy percentages ranged from 0 to 92%, 0 to 75%, 0 to 91%, 69 to 97%, and 0 to 85% for ABZ, IVM, LEV, MXD and CLOS respectively in the Caatinga bioma, and 0 to 59% for ABZ and 9 to 59% for IVM in the Mata Atlântica biome. Most herds showed efficacy lower than 95% for anthelmintics, with the exception of one herd in which the efficacy for MOX was 97%. The results indicated the presence of GINs resistant to main anthelmintics classes in goat herds in these biomes...

Viability of sporulated oocysts of Neospora caninum after exposure to different physical and chemical treatments.

The aim of the present study was to evaluate the viability of Neospora caninum sporulated oocysts after various chemical and physical treatments. Bioassays in gerbils and molecular techniques (PCR-RFLP) were used for identification of the oocysts shed by experimentally infected dogs. Sporulated oocysts were purified and divided into 11 treatment groups as follows: absolute ethanol for 1 hr; 20 C for 6 hr; 4 C for 6 hr; 60 C for 1 min; 100 C for 1 min; 10% formaldehyde for 1 hr; 10% ammonia for 1 hr; 2% iodine for 1 hr; 10% sodium hypochlorite for 1 hr; 70% ethanol for 1 hr; and one group was left untreated and kept as a positive control. All chemical treatments were performed at room temperature (37 C). A total of 33 gerbils, or 3 gerbils per treatment, were used for bioassays. After treatment, the oocysts were divided into aliquots of 1,000 oocysts and orally administered to gerbils. After 63 days, the gerbils were anesthetized and killed with 0.2 ml of T61; blood and tissue samples were collected for serological (IFAT and western blotting), molecular (real-time PCR), histopathology, and immunohistochemical tests. Treatments were considered effective only if all 5 detection techniques tested negative. High temperatures at 100 C for 1 min and 10% sodium hypochlorite for 1 hr were the only treatments that met this condition, effectively inactivating all oocysts.

Experimental infection of dogs (Canis familiaris) with sporulated oocysts of Neospora caninum.

Neospora caninum is widely distributed in the world and this parasite is one of the major causes of abortion in cattle. Dogs and coyotes are definitive hosts of N. caninum and several species of domestic and wild animals are intermediate hosts. Dogs can become infected by the ingestion of tissues containing cysts and then excrete oocysts. It is not yet known whether sporulated oocysts are able to induce a patent infection in dogs, i.e. a shedding of N. caninum oocysts in feces. The objective of this study was to experimentally examine the infection of dogs by sporulated oocysts. The oocysts used in the experiment were obtained by feeding dogs with brain of buffaloes (Bubalus bubalis) positive for anti-N. caninum antibodies by indirect fluorescent antibody test (IFAT ≥200). Oocysts shed by these dogs were confirmed to be N. caninum by molecular methods and by bioassay in gerbils, and sporulated N. caninum oocysts were used for the oral infection of four dogs. The dogs were 8 weeks old and negative for antibodies to N. caninum and Toxoplasma gondii. Dogs 1 and 4 received an inoculum of 10,000 sporulated oocysts each; dog 2 an inoculum of 5000 sporulated oocysts and dog 3 received 1000 sporulated oocysts of N. caninum. The total feces excreted by these dogs were collected and examined daily for a period of 30 days. No oocysts were found in their feces. The dogs were monitored monthly for a 6-month period to observe a possible seroconversion and when this occurred the animals were eliminated from the experiment. Dogs 1 and 4 seroconverted 1 month after the infection with titer, in the IFAT, of 1600 and 800, respectively; the other two dogs presented no seroconvertion during the 6-month period. Dogs 1 and 2 were euthanized 180 days after infection and were examined for the detection of N. caninum in tissues (brain, muscle, lymph node, liver, lung, heart and bone marrow) by immunohistochemistry and PCR with negative results in both techniques. Bioassay in gerbils with brain of these dogs was also performed and again the results were negative. In conclusion, dogs infected with sporulated oocysts of N. caninum were not able to shed oocysts in feces. However, a higher dose of infection stimulated the production of antibodies against N. caninum in the dogs.

Toxoplasma gondii: diagnosis of experimental and natural infection in pigeons (Columba livia) by serological, biological and molecular techniques / Toxoplasma gondii: diagnóstico da infecção experimental e natural em pombos (Columba livia) por métodos sorológicos, biológicos e moleculares

This study aimed to diagnose experimental and natural Toxoplasma gondii infection in pigeons (Columba livia) by serological, biological and molecular techniques. Twelve pigeons, free of infection, were inoculated with 50 sporulated oocysts of T. gondii (VEG sample) and four remained uninfected controls. Four birds (three infected and one control) were euthanized at 15, 30, 45 and 60 days post-infection (dpi), and their tissues were used to perform a bioassay in mice and nested-PCR using B1 gene as target. Blood was obtained weekly and it was tested for the presence of anti-T. gondii antibodies by the indirect Fluorescent antibody test (IFAT) and modified agglutination test (MAT). Seven (58.3%) out of 12 inoculated pigeons were positive by serological techniques and titers ranged between 1:40 and 1:5120 by MAT and between 1:512 and 1:4096 by IFAT. Complete agreement was seen between the results obtained by serological techniques and nested-PCR in seven positive birds. In the bioassay in mice, ive (41.7%) out of 12 pigeons inoculated were positive to T. gondii. Only one pigeon died at 23 dpi due to toxoplasmosis. A second study with free-living pigeons was performed for detection of anti-T. gondii antibodies. Birds were captured in the municipalities of São Paulo, Ibiúna and Sorocaba, São Paulo State, Southeastern Brazil. All 126 free-living birds were negative to anti-T. gondii antibodies by MAT (titer < 1:5). Bioassays were performed in mice with tissues from all captured birds and T. gondii was not isolated in any pigeon.

O presente estudo teve por objetivo diagnosticar a infecção experimental e natural pelo Toxoplasma gondii em pombos (Columba livia) por técnicas sorológicas, biológicas e moleculares. Doze pombos, livres de infecção, foram inoculados com 50 oocistos esporulados de T. gondii (amostra VEG), e quatro permaneceram como controles não infectados. Aos 15, 30, 45 e 60 dias pós-infecção (dpi), quatro aves (três infectadas e uma controle) foram sacrificadas e com seus tecidos realizou-se bioensaio em camundongos e nested-PCR, utilizando-se B1 como gene alvo. Sangue, para a pesquisa de anticorpos anti-T. gondii, foi obtido semanalmente, e a presença de anticorpos foi determinada pela reação de imunofluorescência indireta (RIFI) e pela técnica de aglutinação modificada (MAT). Dos 12 pombos inoculados, sete (58,3%) foram positivos pelas técnicas sorológicas, apresentando títulos que variaram de 40 a 5.120 no MAT e de 512 a 4.096 na RIFI. Concordância total foi observada entre os resultados obtidos pelas técnicas sorológicas e pela nested-PCR com sete animais positivos. No bioensaio em camundongos, dos 12 pombos inoculados, cinco (41,7%) foram positivos ao T. gondii. Apenas um pombo veio a óbito no 23° dpi, devido à toxoplasmose. Um segundo estudo, com pombos de vida livre, foi realizado para a pesquisa de anticorpos anti-T. gondii. As aves foram capturadas nos municípios de São Paulo, Ibiúna e Sorocaba, Estado de São Paulo. Todos os 126 pombos de vida livre foram negativos a anticorpos anti-T. gondii, testados pelo MAT (título < 5). Foram realizados bioensaios em camundongos com tecidos de todas as aves capturadas e também, por esta técnica, T. gondii não foi isolado em nenhuma ave.