Immunisation with Neospora caninum subunits rsNcSAG4 and rsNcGRA1 (NcSAG4 and NcGRA1 epitopes construct) in BALB/c mice: the profile of the immune response and controlling the vertical transmission.

Neospora caninum is an apicomplexan protozoan that causes neosporosis, which has a high economic impact on cattle herds with no available vaccine. During infection, the secretion of dense granules and the expression of surface antigens play an important role in hosting immunomodulation. However, some epitopes of those antigens are immunogenic, and using these fractions could improve the subunit antigens in vaccine design. This study evaluates the recombinant peptides rsNcGRA1 and rsNcSAG4 derived from NcGRA1 and NcSAG4 native antigens as vaccine candidates produced by a fermentative process in the yeast culture system of Komagataella phaffii strain Km71, confirmed by colony PCR, SDS-PAGE, and western blotting. The assay was conducted in BALB/c mice using the peptides at low (25 µg) and standard (50 µg) dosages in monovalent and combined administrations at three time points with saponin as an adjuvant assessing the immunogenicity by antibodies response and cytokine production. We challenge the females after pregnancy confirmation using 2 × 105 NC-1 tachyzoites previously propagated in Vero cells. We assessed the chronic infection in dams and vertical transmission in the offspring by PCR and histopathology. Mice, especially those immunised with combined peptides and monovalent rsNcGRA1 at a standard dose, controlling the chronic infection in dams with the absence of clinical manifestations, showed an immune response with induction of IgG1, a proper balance between Th1/Th2 cytokines and reduced vertical transmission in the pups. In contrast, dams inoculated with a placebo vaccine showed clinical signs, low-scored brain lesions, augmented chronic infection with 80% positivity, 31% mortality in pups, and 81% vertical transmission. These findings indicate that rsNcGRA1 peptides in monovalent and combined with rsNCSAG4 at standard dose are potential vaccine candidates and improve the protective immune response against neosporosis in mice.

Intestinal changes and performance parameters in ticks feeding on calves immunized with subunits of immunogens against Rhipicephalus microplus.

We describe the intestinal changes and biological parameters of the tick species Rhipicephalus microplus exposed to the immune response of calves vaccinated with two subunits of immunogens. The first group of Bos taurus calves was immunized with a synthetic peptide (SBm7462), whereas the second group received an inoculum for synthetic control. The third group was immunized with a recombinant peptide (rSBm7462); an inoculum was injected into a fourth group of calves for recombinant control. Each formulation was administered to these calves during three times at intervals of 30 days. At 21 days after the last immunization, the calves were challenged using a total of 4500 larvae per animal. Indirect ELISA was realized to identify the kinetics of IgGs from samples of calves studied. Naturally detaching ticks were collected for analyses of biological performance and histological changes in the midgut. We dissected randomly detached ticks. The midgut of each of these ticks was removed and processed routinely for histology, stained with hematoxylin-eosin (H&E) and slow Giemsa. Slides were also subjected to immunohistochemistry. The antibody response showed significant induction of high-affinity IgGs in calves immunized with both peptides in comparison to calves of the control groups. Histological changes included damage of the intestinal epithelium in ticks fed on immunized hosts and intense immunostaining in midgut cells, using the serum of calves immunized with recombinant peptide. There were significant differences in all biological performing parameters of ticks detached from vaccinated calves in comparison with ticks of the control groups. We identified reductions of 87.7 and 93.5% in engorged ticks detached from calves immunized with a synthetic and recombinant peptides, respectively, a 28 and 8.60% lower egg mass in groups immunized with synthetic and recombinant peptides, respectively, and a 38.4% reduction of the value of nutrient index/tick in the group immunized with the recombinant peptide. Our findings show that the immune response induced by small peptides in cattle can modify the digestion and metabolism of ticks fed on vaccinated animals, resulting in changes in tick performance.

Pythiosis cutaneous in horses treated with triamcinolone acetonide. Part 3. Histomorphometric analysis / Pythiosis cutánea en equinos tratados con acetonida de triamcinolona. Parte 3. Análisis histomorfométrico

ABSTRACT Objective. The objective of the study was to analyze Histomorphometrically of the healing process with cutaneous pythiosis in horses treated with triamcinolone acetonide. Materials and methods. 24 horses with pythiosis were used, to a group 50 mg of intramuscular triamcinolone acetonide (GT) was applied, while the other group was not applied treatment (GC). They were collected tissue biopsies, processed, sliced and stained with hematoxylin and eosin (H & E), Gomori trichrome (TG), picrosirius red / polarization (PR / P) and Grocott methenamine silver (GMS). Photomicrographs were selected and 10 histological changes, analyzed with BioEstat 5.0 software, obtaining quantities of tissue cells such as eosinophils, neutrophils, macrophages, fibroblasts and collagen through planimetric evaluation point count. Results. In GSM staining was observed decrease in the presence of intralesional hyphae of P. insidiosum to 16 days (p<0.05). Staining H&E, we observed a decrease of the inflammatory process, shown in eosinophils (p=0.0001), neutrophils (p=0.0001), and macrophage (p=0.00001). In the staining of GT and PR/P increase the amount of fibroblasts and collagen fibers were observed, also the gradual exchange of type III collagen to type I, increased fibroblast show significant (p=0.0001) from day 16 until day 40, the expression of collagen was significant (p=0.0001) from day 16 until the end of the study. It was statistically significant correlation between neutrophils and macrophages (p=0.00018), collagen and eosinophil (p=0.03) and fibroblasts and collagen (p=0.02). The animals in the CG do not present histomorphometric improvement during the study. Conclusions. We conclude that the cell produces triamcinolone acetonide and histomorphometric tecidual recovery in horses with pythiosis.

RESUMEN Objetivo. El objetivo del estudio fue analizar histomorfométricamente el proceso de cicatrización en equinos con pythiosis tratados con acetonida de triamcinolona. Materiales y métodos. Se utilizaron 24 equinos con pythiosis, a un grupo se aplicó 50 mg de acetonida de triamcinolona intramuscular (GT), mientras que al otro grupo no fue aplicado tratamiento (GC). Fueron colectadas biopsias de tejido, procesadas, laminadas y coloreadas con hematoxilina eosina (H&E), tricrómico de Gómori (TG), picrosirius red/polarization (PR/P) y plata metanamina de Grocott (GMS). Fueron seleccionados y fotomicrografiados 10 campos histológicos, analizados con el software BioEstat 5.0, obteniéndose las cantidades tisulares de células como eosinófilos, neutrófilos, macrófagos, fibroblastos y colágeno a través de evaluación planimétrica por conteo de puntos. Resultados. En coloración de GSM, se observó disminución de hifas intralesionales del Pythium a los 16 días (p<0.05). En coloración de H&E, hubo disminución del proceso inflamatorio, evidenciado en eosinófilos (p=0.0001), neutrófilos (p=0,0001) y macrófagos (p=0.00001). En coloraciones de TG y PR/P, hubo aumento de fibroblastos, fibras colágenas y cambio progresivo de colágeno tipo III a tipo I, el aumento de fibroblastos fue significativo (p=0.0001) del día 16 al 40, la expresión de colágeno fue altamente significativa (p=0.0001) del día 16 hasta el final del estudio. Hubo correlación significativa entre neutrófilos y macrófagos (p=0.00018), colágeno y eosinófilos (p=0.03) y colágeno y fibroblastos (p=0.02). Los animales del GC no presentaron mejoría histomorfométrica durante el estudio. Conclusiones. Es posible concluir que la acetonida de triamcinolona produce recuperación histomorfométrica celular y tecidual en los equinos con pythiosis.

Pythiosis cutaneous in horses treated with triamcinolone acetonide. Part 2. Histological and histochemical description / Pythiosis cutánea en equinos tratados con acetonida de triamcinolona. Parte 2. Descripción histológica e histoquímica

ABSTRACT Objective. The study aimed to evaluate the histomorphometry tissue recovery process of the skin granuloma of skin pythiosis in horses treated with triamcinolone acetonide. Materials and methods. We conducted a descriptive study, not probabilistic in convenience animals with cutaneous pythiosis. 24 horses were used with cutaneous pythiosis, a group of 12 animals was administered 50 mg of intramuscular injection of triamcinolone acetonide (TG) and the other group was not applied any treatment (CG). Are tissue biopsies performed for histological and histochemical evaluation and stained with hematoxylin eosin (HE), Gomori trichrome (GT), picrosirius red polarization (PR/P), Grocott methenamine silver (GMS) and periodic acid-Schiff (PAS). Results. It is noted that in TG inflammation was gradually decreasing, as evidenced in decreased fibrin layer leukocyte, PMN and phenomena Splendore Hoepli, also in increased angiogenesis, epiteliogénesis, and increasing the overall amount of fibroblasts and collagen fibers, anyway in the progressive replacement of collagen type III to type I collagen at the end of the process, and that the presence of intralesional pseudo-hyphae of Pythium insidiosum reduces it to the second week. Neither of the animals in the CG showed improvement in histological and histochemical characteristics of pythiosis and maintained equal to the first day throughout the study. Conclusions. The use of triamcinolone acetonide is a good therapeutic alternative for the treatment of granulomatous pythiosis wounds in horses with 100% clinical recovery and demonstrated with histological and histochemical findings.

RESUMEN Objetivo. El objetivo del estudio fue realizar una descripción histológica e histoquímica del proceso de cicatrización de la pythiosis cutánea en equinos tratados con acetonida de triamcinolona. Materiales y métodos. Se Realizó un estudio de tipo descriptivo, no probabilístico en animales de conveniencia con pythiosis cutánea. Fueron utilizados 24 equinos con pythiosis, siendo aplicado 50 mg de acetonida de triamcinolona vía intramuscular a un grupo de 12 animales (GT) y en otro grupo no fue aplicado tratamiento (GC). Se realizaron biopsias de tejidos para evaluación histológica e histoquímica, en las coloraciones de hematoxilina eosina (H&E), tricrómico de Gómori (TG), picrosirius red/polarization (PR/P) y plata metanamina de Grocott (GMS). Resultados. Se observó que en el GT el proceso inflamatorio fue disminuyendo progresivamente, evidenciado en la disminución de la capa fibrino leucocitaria, PMN y de los fenómenos de Splendore Hoepli, así como mayor angiogénesis, epiteliogénesis y aumento de la cantidad de fibroblastos y fibras colágenas generales, así mismo en el cambio progresivo de colágeno tipo III a colágeno tipo I al final del proceso, además de que la presencia de hifas intralesionales de Pythium insidiosum disminuyó a la segunda semana. Ninguno de los animales del GC presentó mejoría en las características histológicas e histoquímicas de la pythiosis y se mantuvieron iguales al primer día durante todo el estudio. Conclusiones. La acetonida de triamcinolona es una buena alternativa terapéutica en el tratamiento de las heridas granulomatosas por pythiosis en equinos con 100% de recuperación clínica y demostrada con las constataciones histológicas e histoquímicas.

Cutaneous pythiosis in horses treated with triamcinolone acetonide. Part 1. Clinical characterization / Pythiosis cutánea en equinos tratados con acetonida de triamcinolona. Parte 1. Caracterización clínica

ABSTRACT Objective. The objective of the present study was to characterizer clinically of the healing process of cutaneous pythiosis in horses treated with triamcinolone acetonide. Materials and methods. We conducted a descriptive study, not probabilistic, of convenience. 24 horses were used with cutaneous pythiosis confirmed, a group of 12 animals was administered 50 mg of intramuscular injection of triamcinolone acetonide (TG) and the other group was not applied any treatment (CG). They are undergoing clinical and microscopic characterization of the lesion every 4 days until the complete recovery of granuloma in the GT. Being verified decreased fibrin bloody exudate, pruritus, presence of fistulae and output kunkers, likewise radiographic projections were performed to assess the degree of compromise bone and tissue fibrosis. Results. Macroscopically animals GT present progressive decrease in the clinical features of the disease at 16±1.4 days post treatment application. The wound contraction was 100% at 60±3.4 days after application of the treatment in all granulomas of GT, being highly significant (p<0.0001). Radiological monitoring of chronic skin lesions, met invasion to underlying tissues compromising bone structures, characterized by tissue fibrosis, moderate exostosis, bake as osteolysis and osteomyelitis. Neither of the animals in the CG showed improvement in clinical characteristics, and macroscopic of pythiosis and maintained equal to the first day throughout the study. Conclusions. The use of triamcinolone acetonide is a good therapeutic alternative for the treatment of granulomatous Pythiosis wounds in horses with 100% clinical recovery.

RESUMEN Objetivo. El objetivo del estudio fue caracterizar clínicamente el proceso de cicatrización de la pythiosis cutánea en equinos tratados con acetonida de triamcinolona. Materiales y métodos. Se realizó un estudio descriptivo, no probabilístico, de conveniencia. Fueron utilizados 24 equinos con pythiosis cutánea. A un grupo le fue aplicado 50 mg de acetonida de triamcinolona vía intramuscular (GT) y al otro grupo no fue aplicado tratamiento (GC). Se realizó caracterización clínica y macroscópica de la lesión cada 4 días hasta la recuperación completa del granuloma en el GT. Siendo verificada la disminución del exudado fibrinosanguinolento, prurito, presencia de trayectos fistulosos y salida de kunkers, también, fueron realizadas proyecciones radiológicas para evaluar el grado de compromiso óseo y fibrosis tecidual. Resultados. Macroscópicamente, los animales del GT presentaron diminución progresiva en las características clínicas de la enfermedad a los 16±1,4 días post aplicación del tratamiento. La contracción de la herida fue del 100% a los 60±3.4 días post aplicación del tratamiento en todos los granulomas del GT, siendo altamente significativa (p<0.0001). Radiológicamente, se encontró invasión a los tejidos subyacentes comprometiendo estructuras óseas, caracterizándose por fibrosis tisular, exostosis moderada, osteólisis y osteomielitis. Los animales del GC no presentaron mejoría en las características clínicas y macroscópicas de la pythiosis y se mantuvieron iguales durante todo el estudio. Conclusiones. La acetonida de triamcinolona es una buena alternativa terapéutica en el tratamiento de las heridas granulomatosas de pythiosis en equinos con 100% de recuperación clínica.

FREQUENCY OF THE ANTI- Toxoplasma Gondii ANTIBODY IN GOAT AND SHEEP FARMS IN THE STATE OF PERNAMBUCO BRAZIL / FREQUÊNCIA DE ANTICORPOS ANTI- Toxoplasma gondii EM PROPRIEDADES DE CRIAÇÃO DE CAPRINOS E OVINOS NO ESTADO DE PERNAMBUCO

The present study was carried out in order to verify the presence of anti-Toxoplasma gondii antibodies in goat and sheep farms located in different mesoregions within the State of Pernambuco Brazil. Blood samples were collected from 164 goats and 124 sheep and submitted to the indirect immunofluorescence technique. The results showed positive rates of 47.6% (78/164) and 48.4% (60/124) for goats and sheep, respectively. A significant difference of infection rates in the different regions was observed in both goats and sheep.KEYWORDS: goat; immunofluorescence; Pernambuco; sheep; toxoplasmosis.

Desenvolveu-se o presente estudo com o objetivo de averiguar a frequência de anticorpos contra T. gondii em propriedades de criação caprina e ovina de diferentes mesorregiões do Estado de Pernambuco. Foram coletadas amostras de sangue de 164 caprinos e 124 ovinos, as quais foram submetidas à técnica de Imunofluorescência Indireta. Foram obtidas taxas de 47,6% (78/164) e 48,4% (60/124) de positividade para caprinos e ovinos, respectivamente. Diferenças significativas foram obtidas entre as taxas de infecção das diferentes mesorregiões tanto em caprinos como em ovinos.PALAVRAS-CHAVE: caprino; imunofluorescência; ovino; Pernambuco; toxoplasmose.

FREQUENCY OF THE ANTI- Toxoplasma Gondii ANTIBODY IN GOAT AND SHEEP FARMS IN THE STATE OF PERNAMBUCO BRAZIL / FREQUÊNCIA DE ANTICORPOS ANTI- Toxoplasma gondii EM PROPRIEDADES DE CRIAÇÃO DE CAPRINOS E OVINOS NO ESTADO DE PERNAMBUCO

The present study was carried out in order to verify the presence of anti-Toxoplasma gondii antibodies in goat and sheep farms located in different mesoregions within the State of Pernambuco Brazil. Blood samples were collected from 164 goats and 124 sheep and submitted to the indirect immunofluorescence technique. The results showed positive rates of 47.6% (78/164) and 48.4% (60/124) for goats and sheep, respectively. A significant difference of infection rates in the different regions was observed in both goats and sheep.KEYWORDS: goat; immunofluorescence; Pernambuco; sheep; toxoplasmosis.

Desenvolveu-se o presente estudo com o objetivo de averiguar a frequência de anticorpos contra T. gondii em propriedades de criação caprina e ovina de diferentes mesorregiões do Estado de Pernambuco. Foram coletadas amostras de sangue de 164 caprinos e 124 ovinos, as quais foram submetidas à técnica de Imunofluorescência Indireta. Foram obtidas taxas de 47,6% (78/164) e 48,4% (60/124) de positividade para caprinos e ovinos, respectivamente. Diferenças significativas foram obtidas entre as taxas de infecção das diferentes mesorregiões tanto em caprinos como em ovinos.PALAVRAS-CHAVE: caprino; imunofluorescência; ovino; Pernambuco; toxoplasmose.

Concentrações de imunoglobulinas G em colostro de vacas mestiças holandês-zebu

Eighty-eight healthy crossbred holstein-zebu (HZ) dairy cows were distributed in four groups as follow: Group 1 = less than 70% HZ (19 cows); Group 2 = 75% HZ animals, corresponding to ¾ HZ (46 cows); Group 3 = crossbred animals between 80 to 90% HZ, corresponding to 13/16 HZ and 7/8 HZ (11 cows); Group 4 = cows more than 90% HZ (12 animals). After birth, colostrum samples were obtained from each cow and than were analyzed for immunoglobulin G (IgG) concentration by single radial immunodifusion assay. IgG colostral concentrations were not influenced by group of crossbred HZ and its mean concentration (145.97mg/m img src="http:/img/fbpe/cr/v31n6/a19img01.gif">) were higher than that related for purebreds Bos taurus dairy cows. Considering these results, it was concluded that artificial methods of feeding colostrum to crossbred HZ calves are not necessary. Probably, assisted natural sucking as soon after birth as possible is enough to get passive immune transfer.

Oitenta e oito vacas leiteiras mestiças holandês-zebu (HZ) foram distribuídas de acordo com a percentagem de grau de sangue holandês em quatro grupos: Grupo 1 = animais com menos de 70% de grau de sangue holandês (19 vacas); Grupo 2 = animais com 75% de grau de sangue holandês, que equivale a ¾ HZ (46 vacas); Grupo 3 = mestiços entre 80 e 90% de grau de sangue holandês, que corresponderam a 13/16 HZ e 7/8 HZ (11 vacas); Grupo 4 = vacas com mais de 90% de grau de sangue holandês (12 vacas). Após o parto, amostras de colostro da primeira ordenha foram colhidas e, posteriormente, submetidas à imunodifusão radial simples para determinações dos níveis de imunoglobulinas G (IgG). Os resultados obtidos permitiram concluir que as concentrações de IgG não são influenciadas pelos graus de sangue estudados e seus valores médios (145,94mg/m img src="http:/img/fbpe/cr/v31n6/a19img01.gif">) são muito superiores aos relatados para animais de raças taurinas puras. Por esse motivo, não se justifica o emprego de métodos artificiais de fornecimento do colostro em se tratando de mestiços HZ. Ademais, o nível elevado de IgG observado no colostro sugere que a adoção da prática da mamada natural assistida seja adequada para assegurar que, na maioria das vezes, os neonatos não venham a apresentar falhas de transferência de imunidade passiva.

Concentrações de imunoglobulinas G em colostro de vacas mestiças holandês-zebu

Eighty-eight healthy crossbred holstein-zebu (HZ) dairy cows were distributed in four groups as follow: Group 1 = less than 70% HZ (19 cows); Group 2 = 75% HZ animals, corresponding to ¾ HZ (46 cows); Group 3 = crossbred animals between 80 to 90% HZ, corresponding to 13/16 HZ and 7/8 HZ (11 cows); Group 4 = cows more than 90% HZ (12 animals). After birth, colostrum samples were obtained from each cow and than were analyzed for immunoglobulin G (IgG) concentration by single radial immunodifusion assay. IgG colostral concentrations were not influenced by group of crossbred HZ and its mean concentration (145.97mg/m img src="http:/img/fbpe/cr/v31n6/a19img01.gif">) were higher than that related for purebreds Bos taurus dairy cows. Considering these results, it was concluded that artificial methods of feeding colostrum to crossbred HZ calves are not necessary. Probably, assisted natural sucking as soon after birth as possible is enough to get passive immune transfer.

Oitenta e oito vacas leiteiras mestiças holandês-zebu (HZ) foram distribuídas de acordo com a percentagem de grau de sangue holandês em quatro grupos: Grupo 1 = animais com menos de 70% de grau de sangue holandês (19 vacas); Grupo 2 = animais com 75% de grau de sangue holandês, que equivale a ¾ HZ (46 vacas); Grupo 3 = mestiços entre 80 e 90% de grau de sangue holandês, que corresponderam a 13/16 HZ e 7/8 HZ (11 vacas); Grupo 4 = vacas com mais de 90% de grau de sangue holandês (12 vacas). Após o parto, amostras de colostro da primeira ordenha foram colhidas e, posteriormente, submetidas à imunodifusão radial simples para determinações dos níveis de imunoglobulinas G (IgG). Os resultados obtidos permitiram concluir que as concentrações de IgG não são influenciadas pelos graus de sangue estudados e seus valores médios (145,94mg/m img src="http:/img/fbpe/cr/v31n6/a19img01.gif">) são muito superiores aos relatados para animais de raças taurinas puras. Por esse motivo, não se justifica o emprego de métodos artificiais de fornecimento do colostro em se tratando de mestiços HZ. Ademais, o nível elevado de IgG observado no colostro sugere que a adoção da prática da mamada natural assistida seja adequada para assegurar que, na maioria das vezes, os neonatos não venham a apresentar falhas de transferência de imunidade passiva.