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.

Genotypic profile of benzimidazole resistance associated with SNP F167Y and F200Y beta-tubulin gene in Brazilian populations of Haemonchus contortus of goats.

Benzimidazoles are the most common anthelminthic used for control of gastrointestinal nematodes of goats in the Brazilian semi-arid region. Resistance to these compounds in the nematode Haemonchus contortus has been associated with single nucleotide polymorphisms (SNP) in codons 167 (F167Y) and 200 (F200Y) on the ß-tubulin isotype 1 gene. To determine the resistance profile to benzimidazoles of populations of H. contortus of goats of Brazilian semi-arid region, larvae of 29 populations of these nematodes were individually genotyped by real time PCR using a Taqman assay. The percentage of larvae homozygous (RR) for SNP F200Y was relatively low (18.9%), particularly when compared to SNP F167Y (32.7%), indicating that the latter has more relevance in this region. However, the associations between these two SNP demonstrate percentages of resistance ranging from 34.7% to 100% between populations, being the highest percentages for homozygous individuals resistant for the mutation 167 and susceptible to mutation 200 (RR-F167Y/F200Y-SS: 26.7%), followed by combination of heterozygous for both mutations (F167Y-SR/F200Y-SR: 22.8%). These results indicate high levels of resistance in populations of H. contortus of goats in the Brazilian semi-arid region, and thus ineffective antiparasitic control with the use of benzimidazoles in the region.

Galectins and collectinis expression are increased in Haemonchus contortus-infected corriedale sheep.

Galectins and collectins are proteins classified in the lectin family that have the ability to recognize molecular patterns associated with pathogens. Studies on cattle have demonstrated high expression of these proteins during infection with gastrointestinal nematodes. The aim of this study was to investigate whether the level of Haemonchus contortus infection would alter the expression of galectins (Gal11 and Gal14) and collectins (SPA and CGN) in sheep. Twelve Corriedale sheep exposed to natural infection with nematodes were divided into two groups: group 1 (G1, n = 7) and group 2 (G2, n = 5), with low and high parasite burdens, respectively, based on fecal egg counts and abomasal parasite counts. The fecal egg counts and abomasal parasite counts were significantly different (p < 0.05) between the groups. Galectin and collectin gene expression was observed in all sheep abomasal samples. However, animals with lower infection levels showed lower expression of the genes Gal14, SPA and CGN (p < 0.05). Expression of lectins was associated with the abomasal H. contortus burden, thus suggesting that these proteins may have a role in controlling of this infection.

Galectins and collectinis expression are increased in Haemonchus contortus-infected corriedale sheep / Aumento da expressão gênica de colectinas e galectinas em ovinos corriedale infectados por Haemonchus contortus

Galectins and collectins are proteins classified in the lectin family that have the ability to recognize molecular patterns associated with pathogens. Studies on cattle have demonstrated high expression of these proteins during infection with gastrointestinal nematodes. The aim of this study was to investigate whether the level of Haemonchus contortus infection would alter the expression of galectins (Gal11 and Gal14) and collectins (SPA and CGN) in sheep. Twelve Corriedale sheep exposed to natural infection with nematodes were divided into two groups: group 1 (G1, n = 7) and group 2 (G2, n = 5), with low and high parasite burdens, respectively, based on fecal egg counts and abomasal parasite counts. The fecal egg counts and abomasal parasite counts were significantly different (p < 0.05) between the groups. Galectin and collectin gene expression was observed in all sheep abomasal samples. However, animals with lower infection levels showed lower expression of the genes Gal14, SPA and CGN (p < 0.05). Expression of lectins was associated with the abomasal H. contortus burden, thus suggesting that these proteins may have a role in controlling of this infection.

Colectinas e galectinas são proteínas da família das lectinas que possuem a capacidade de reconhecer padrões moleculares associados aos patógenos. Estudos em bovinos têm demonstrado a alta expressão dessas proteínas durante a infecção por nematoides gastrintestinais. O objetivo deste estudo foi investigar se o nível de infecção de Haemonchus contortus altera a expressão de colectinas (SPA e CGN) e galectinas (Gal11 e Gal14) de ovinos. Doze ovinos da raça Corriedale expostos a infecção natural com nematoides foram separados em dois grupos: grupo 1 (G1, n=7) com menor grau de parasitismo; e grupo 2 (G2, n=5) com maior grau, a partir da contagem do número de parasitos recuperados do abomaso e OPG. A contagem de OPG e de parasitos recuperados do abomaso dos grupos G1 e G2 apresentaram diferença estatística (p<0,05). A expressão dos genes de colectinas e galectina foi observada em todas as amostras de abomaso dos ovinos, porém animais com menor grau de infecção apresentaram menor expressão dos genes de Gal14, SPA e CGN (p<0,05). A expressão de lectinas foi associada ao número de H. contortus encontrados no abomaso de ovinos, indicando um possível papel dessas proteínas no controle da infecção.

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...

Experimental infection with Neospora caninum in pregnant bitches.

In this study, transplacental transmission of Neospora caninum in bitches at different stages of pregnancy was evaluated. Three bitches were inoculated in the 3rd week and three in the 6th week of gestation with 10(8) tachyzoites of N. caninum (Nc-1 strain). All the infected bitches and at least one of their offspring presented anti-N. caninum antibodies according to the indirect fluorescent antibody test (IFAT > 400). The pups and their mothers were sacrificed and tissues from the central nervous system (CNS), popliteal lymph nodes, skeletal muscle, brain, lungs, heart and liver were analyzed for the presence of N. caninum using the nested polymerase chain reaction (nested PCR), restriction fragment length polymorphism (RFLP) and immunohistochemistry (IHC). The parasite was found in the pups in lymph node, CNS, heart and liver tissues using nested PCR. There was no difference in perinatal mortality between the offspring from bitches infected in the 3rd week of gestation (60%) and in the 6th week (53.8%).

Experimental infection with Neospora caninum in pregnant bitches / Infecção experimental com Neospora caninum em cadelas prenhes

In this study, transplacental transmission of Neospora caninum in bitches at different stages of pregnancy was evaluated. Three bitches were inoculated in the 3rd week and three in the 6th week of gestation with 10(8) tachyzoites of N. caninum (Nc-1 strain). All the infected bitches and at least one of their offspring presented anti-N. caninum antibodies according to the indirect fluorescent antibody test (IFAT > 400). The pups and their mothers were sacrificed and tissues from the central nervous system (CNS), popliteal lymph nodes, skeletal muscle, brain, lungs, heart and liver were analyzed for the presence of N. caninum using the nested polymerase chain reaction (nested PCR), restriction fragment length polymorphism (RFLP) and immunohistochemistry (IHC). The parasite was found in the pups in lymph node, CNS, heart and liver tissues using nested PCR. There was no difference in perinatal mortality between the offspring from bitches infected in the 3rd week of gestation (60%) and in the 6th week (53.8%).

Neste estudo a transmissão transplacentária de Neospora caninum foi avaliada em fêmeas em diferentes estágios de gestação. Três cadelas foram inoculadas na 3ª semana e três na 6ª semana de gestação com 10(8) taquizoítos de N. caninum (cepa Nc-1). Todas as cadelas infectadas, e pelo menos um de seus filhotes, apresentaram anticorpos anti-N. caninum por imunofluorescência indireta (RIFI > 400). Os filhotes e suas mães foram sacrificados e tecidos de sistema nervoso central (SNC), linfonodo poplíteo, músculo esquelético, cérebro, pulmões, coração e fígado foram analisados para a presença de N. caninum pela reação em cadeia da polimerase (nested PCR), polimorfismo de comprimento de fragmentos de restrição (RFLP) e imunoistoquímica (IHQ). O parasita foi encontrado em filhotes em linfonodo, SNC, coração e fígado pela nested PCR. Mortalidade perinatal não apresentou diferença entre os filhotes das cadelas infectadas na 3ª semana (60%) ou na 6ª semana de gestação (53,8%).

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.

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, five (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.

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 percent) 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, five (41.7 percent) 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 por cento) 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 por cento) foram positivos ao T. gondii. Apenas um pombo veio a óbito no 23º 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.

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.

[Use of the real time RT-PCR for immune related gene expression quantitation in experimentally infected Neospora caninum bovine calves]. / Emgrego da RT-PCR em tempo real para a quantificação da expressão de genes associados a resposta imune em bezerros bovinos experimentalmente infectados por Neospora caninum.

Neospora caninum is one of the main causes of abortion and natimortality in cattle. Host immune defense is capable to inhibit tachyzoite activity during acute infection, but there is no action against bradyzoites in tissue cysts. Activation and modulation of this response is controlled by cell mediators. The real-time RT-PCR technique was employed to detect some of those mediators during N. caninum infection. Holstein and Nelore calves intramuscularly infected with tachyzoites and uninfected controls were slaughtered at the sixth day post-infection and popliteal lymph node, liver and brain cortex samples were analyzed. Real-time RT-PCR detected gene expression in all tissues. No significant variation of GAPDH gene expression was detected among groups, its amplification efficiency was similar to the other genes tested and it was used as the endogenous control for the analysis. Comparisons between infected and uninfected groups allowed the relative gene expression quantification. IFN-gamma and TNF-alpha genes showed increased expression in some samples. iNOS and TGF-beta1 genes had some non-significant variations and IL-4 and IL-10 stayed practically unaltered.

Emprego da RT-PCR em tempo real para a quantificação da expressão de genes associados à resposta imune em bezerros bovinos experimentalmente infectados por Neospora caninum / Use of the real time RT-PCR for immune related gene expression quantitation in experimentally infected Neospora caninum bovine calves

Neospora caninum é um dos principais agentes causadores de abortamentos e natimortalidade em bovinos. A defesa imune do hospedeiro é capaz de inibir a atividade dos taquizoítos na fase aguda da infecção, mas não age sobre os bradizoítos nos cistos teciduais. A ativação e a modulação dessa resposta de defesa são controladas por mediadores celulares. A técnica do RT-PCR em tempo real foi empregada para a detecção de alguns desses mediadores durante a infecção pelo N. caninum. Foram analisadas amostras de linfonodos poplíteos, fígado e córtex cerebral de bezerros Holandeses e Nelores infectados com taquizoítos por via intramuscular e controles não-infectados, abatidos no sexto dia pós-inoculação. A RT-PCR em tempo real detectou a expressão dos genes em todos os tecidos analisados. Não houve variação significativa na expressão do gene GADPH entre os grupos, a eficiência de amplificação desse foi similar aos demais genes testados e foi empregado como controle endógeno na análise. A comparação entre infectados e não-infectados permitiu a quantificação relativa da expressão gênica. A expressão dos genes IFN-γ e TNF-α apresentou elevação significante em algumas amostras. Os genes iNOS e TGF-β1 apresentaram algumas variações não-significativas e os valores de IL-4 e IL-10 permaneceram praticamente inalterados.

Neospora caninum is one of the main causes of abortion and natimortality in cattle. Host immune defense is capable to inhibit tachyzoite activity during acute infection, but there is no action against bradyzoites in tissue cysts. Activation and modulation of this response is controlled by cell mediators. The real-time RT-PCR technique was employed to detect some of those mediators during N. caninum infection. Holstein and Nelore calves intramuscularly infected with tachyzoites and uninfected controls were slaughtered at the sixth day post-infection and popliteal lymph node, liver and brain cortex samples were analyzed. Real-time RT-PCR detected gene expression in all tissues. No significant variation of GAPDH gene expression was detected among groups, its amplification efficiency was similar to the other genes tested and it was used as the endogenous control for the analysis. Comparisons between infected and uninfected groups allowed the relative gene expression quantification. IFN-γ and TNF-α genes showed increased expression in some samples. iNOS and TGF-β1 genes had some non-significant variations and IL-4 and IL-10 stayed pratically inaltered.

Antibody dynamics during gestation in cows naturally infected with Neospora caninum from four dairy herds in Brazil / Dinâmica de anticorpos durante a gestação em vacas naturalmente infectadas com Neospora caninum de quatro rebanhos leiteiros no Brasil

Neospora caninum has been described as an important cause of abortion in bovine worldwide. The objective of the present study was to characterize patterns of antibody dynamics during gestation in dairy cows naturally infected with N. caninum. Twelve N. caninum naturally infected cows were selected from four dairy herds from Brazil and blood samples were monthly collected during pregnancy. Serum were tested for antibodies against N. caninum by Indirect Fluorescent Antibody Test (IFAT). During this period, all cows remained clinically normal and gave birth to healthy calves. The cows remained seropositives during the study and N. caninum IFAT titers ranged from 100 to 12,800; only animal 234 presented one negative result in the first month of pregnancy. Significant differences of N. caninum IFAT titers were found between months from 1 to 9 of pregnancy bythe Friedman Test (P<0.001). The statistical analysis showed an increase of N. caninum antibody titers from second and third trimester of pregnancy in relation to first trimester. High titers were observed in few cows after month fifth of pregnancy. This study showed a variation of specific antibody levels in seropositive cows during different gestational periods. The highest values were observed during the second and third trimester. The antibody increase after the fifth month of gestation was not associated to abortion.

Neospora caninum é descrito como uma importante causa de abortamento em bovinos por todo o mundo. O objetivo do presente estudo foi caracterizar o padrão da dinâmica de anticorpos durante a gestação em vacas leiteiras infectadas naturalmente por N. caninum. Doze vacas gestantes infectadas naturalmente com N.caninum foram selecionadas de quatro rebanhos leiteiros do Brasil e amostras de sangue foram mensalmente colhidas da concepção até o parto das vacas. Soros foram testados para anticorpos contra N. caninum pela reação de imunofluorescência indireta (RIFI). Durante a gestação, todas as vacas permaneceram clinicamente normais e geraram bezerros saudáveis. As vacas permaneceram soropositivas durante o estudo e títulos de anticorpos anti-N.caninum variaram de 100 a 12.800; somente o animal 234 apresentou um resultado negativo no primeiro mês de gestação. Diferenças significativas dos títulos da RIFI para N. caninum foram encontradas entre os meses de 1 a 9 de gestação pelo Teste de Friedman (P<0,001). As análises estatísticas mostraram um aumento dos títulos de anticorpos anti-N. caninum no segundo e terceiro trimestre de gestação em relação ao primeiro trimestre. Altos títulos de anticorpos foram observados em algumas vacas após o mês cinco de gestação. Este estudo mostrou variação dos níveis de anticorpos em vacas soropositivas durante diferentes períodos gestacionais. Altos títulos foram observados durante o segundo e terceiro trimestre e o aumento dos títulos de anticorpos após o quinto mês de gestação não foi associado a abortamentos.

Localized multigene expression patterns support an evolving Th1/Th2-like paradigm in response to infections with Toxoplasma gondii and Ascaris suum.

Human infectious diseases have been studied in pigs because the two species have common microbial, parasitic, and zoonotic organisms, but there has been no systematic evaluation of cytokine gene expression in response to infectious agents in porcine species. In this study, pigs were inoculated with two clinically and economically important parasites, Toxoplasma gondii and Ascaris suum, and gene expression in 11 different tissues for 20 different swine Th1/Th2-related cytokines, cytokine receptors, and markers of immune activation were evaluated by real-time PCR. A generalized Th1-like pattern of gene expression was evident in pigs infected with T. gondii, along with an increased anti-inflammatory gene expression pattern during the recovery phase of the infection. In contrast, an elevated Th2-like pattern was expressed during the period of expulsion of A. suum fourth-stage larvae from the small intestine of pigs, along with low-level Th1-like and anti-inflammatory cytokine gene expression. Prototypical immune and physiological markers of infection were observed in bronchial alveolar lavage cells, small intestinal smooth muscle, and epithelial cells. This study validated the use of a robust quantitative gene expression assay to detect immune and inflammatory markers at multiple host tissue sites, enhanced the definition of two important swine diseases, and supported the use of swine as an experimental model for the study of immunity to infectious agents relevant to humans.

Identification of key immune mediators regulating T helper 1 responses in swine.

This publication describes the cloning of full or partial length sequences for pig TBX21 (T-bet), MYD88, ICSBP1, CD8A (CD8alpha), CD8B (CD8beta), and CD28 cDNAs. Real-time PCR assays have been developed for the relative quantitation of these products as well as previously characterized transcripts that encode exon A-containing CD45, HLX1, IRF1, STAT1 and RPL32. When used for examining temporal immune gene expression in the liver of Toxoplasma gondii infected pigs, the positive regulators of Th1 responses, IRF1, MYD88, and STAT1, were found to be expressed prior to the simultaneous upregulation of interferon gamma (IFNG), HLX1 and TBX21 gene expression. In contrast, in the mesenteric lymph node (MLN), only expression of IRF1 and IFNG was significantly upregulated. Based on their demonstrated utility in establishing an immune response pathway, these PCR assays should be valuable additions to our swine immune toolkit.

Níveis de IgG séricos em bezerros experimentalmente infectados pelo Haemonchus placei / Serum IgG level in calves experimentally infected with Haemonchus placei

Dois experimentos foram realizados para se conhecer a dinâmica dos anticorpos séricos (IgG) em bezerros após infecçäo com Haemonchus placei. No Experimento 1 estudou-se os níveis de IgG ao longo da primeira e segunda infecçäo. No Experimento 2 a influência de diferentes níveis protéicos da dieta e a resposta à infecçäo, medida através dos níveis de IgG séricos, foi observada em bezerros imunizados e näo imunizados, através de infecções sucessivas e experimentalmente desafiados com L3 de H. placei. Observou-se que os bezerros infectados desenvolveram resistência a reinfecções, com acentuado decréscimo nos valores de ovos por grama de fezes (OPG) na segunda infecçäo. Entretanto, nesse mesmo período, os níveis de IgG séricos mantiveram-se elevados, independentes do decréscimo do OPG. Observou-se também que bezerros submetidos a dieta protéica adequada apresentaram maior produçäo de IgG sérico, sugerindo uma melhor resposta às infecções quando comparados aos submetidos a dietas com níveis protéicos mais baixos

Efficacy and persistence of cypermethrin 4.5% + DDVP 50% (Ectofarma®) in the control of Boophilus microplus and larvae of Dermatobia hominis on naturally infested crossbred dairy cattle / Eficácia e persistência da associação de cipermetrina 4,5 % + DDVP 50% (Ectofarma®) no controle do Boophilus microplus e de larvas de Dermatobia hominis em bovinos leiteiros, mestiços, naturalmente infestados

The effectiveness and persistence of Ectofarma* in the control of B. microplus and D. hominis larvae were evaluated. Initially, the effect on estimated reproduction and percentage of reduction was determined by dipping female ticks in 1:400 diluted solution of the product. The percentage of reduction was 95.34, and the reduction on hatchability of larvae was 100%. Then, in a field trial the product, in the same dilution, was handsprayed over a group of nine bovines. The number of ticks on treated and untreated animals were counted on days 0 ,1 ,2 ,3 ,7 ,1 4 ,2 1 , 28, 35 and 42 after treatment. D. hominis larvae were counted on day 3 and further, weekly, until the sixth week. The efficacy in vitro of Ectofarma" on the hatchability of B. microplus eggs was 100%. In the field trial this product was highly effective against ticks during 3 weeks, and ticks mean number on the treated group was lower than on control one, until thefourth week after treatment. Reduction of D.hominis larvae reached 65% on the third day post-treatment, and mean number of larvae in treated animals was always lower than the amount found in untreated ones.

Testou-se a sensibilidade da cepa de B. microplus do campus de Pirassununga da USP ao Ectofarma®, na diluição 1:400 através dobiocarrapaticidograma. A ovipostura no grupo tratado (A) foi reduzida em 95,34%, em relação ao grupo-controle (B) e a eclosão de larvas provenientes dos ovos do grupo B foi de 100%, enquanto no grupo A não ocorreu eclosão. Diante desse resultado, efetuou-se o teste a campo, utilizando dois grupos de nove bovinos mestiços. No dia 0 os números médios de B. microplus a 5 mm e de bernes nos animais dos dois grupos eram estatisticamente iguais. Os bovinos do grupo A foram aspergidos com o produto na mesma diluição utilizada no teste in vitro. Os carrapatos foram contados nos dias 1, 2, 3, 7, 14, 21, 28, 35 e 42 e os bernes no 3fi dia pós-tratamento (dpt) e depois, semanalmente, até a 68 semana. Houve redução drástica no número médio de carrapatos do grupo A do primeiro ao 210 dpt, aumentando a partir de 28® dpt. A redução do número médio de larvas de D. hominis chegou a 65% no 35 dpt, nos animais do grupo tratado, e embora a eficácia do produto tenha sido baixa, o número médio de bernes no grupo A foi sempre menor do que no grupo B, após o tratamento.