Identification of Avian Toll-Like Receptor 3 and 7 and Analysis of Gene Variation Sites

Toll-like receptors 3 and 7 (TLR3 and 7) mediate immune responses through the recognition of viral single-stranded RNA and double-stranded RNA and therefore play important roles in host defense. Differences in TLR3 or 7 may affect host resistance to RNA viral infection. To illuminate these differences, the partial coding sequence (CDS) of TLR3 and 7 genes were cloned and amplified from the Phasianus colchicus and Numida meleagris, total 64 avian species of TLR3 and 7 sequences were later analyzed. Based on the results, 315 non-synonymous mutation sites and 202 synonymous mutation sites were also observed in the avian TLR3, and 227 non-synonymous mutation sites and 174 synonymous mutation sites were observed in the avian TLR7. Among these sites, 44 and 45 sites were observed in functional regions of TLR3 and 7, used common variation of amino acids in most avian species. A number of these different sites appeared to affect the recognition and were also visualized. H59Y, E60K, G64R, E93K, L112S, K117E, N118K, R120H, V123M, L163F, R443Q, R459K, E460D, C485H, and F511L for TLR3, and I432V, M437V, and T732S for TLR7 were considered. It is possible that these sites bind to ligands and play crucial roles in viral recognition. These data indicated that the positive selection has occurred in the avian TLR3 and 7 genes.(AU)

Early pregnancy affects expression of Toll-like receptor signaling members in ovine spleen

Toll-like receptors (TLRs) are involved to the maternal immune tolerance. The spleen is essential for adaptive immune reactions. However, it is unclear that early pregnancy regulates TLR-mediated signalings in the maternal spleen. The purpose of this study was to investigate the effects of early pregnancy on expression of TLR signaling members in the ovine spleen. Ovine spleens were collected at day 16 of the estrous cycle, and at days 13, 16 and 25 of pregnancy (n = 6 for each group). Real-time quantitative PCR, western blot and immunohistochemistry analysis were used to detect TLR signaling members, including TLR2, TLR3, TLR4, TLR5, TLR7, TLR9, myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6) and interleukin-1-receptor-associated kinase 1 (IRAK1). The results showed that expression levels of TLR2, TLR4 and IRAK1 were downregulated, but expression levels of TLR3, TLR5, TLR7, TLR9, TRAF6 and MyD88 were increased during early pregnancy. In addition, MyD88 protein was located in the capsule, trabeculae and splenic cords of the maternal spleen. This paper reports for the first time that early pregnancy has effects on TLR signaling pathways in the ovine spleen, which is beneficial for understanding the maternal immune tolerance during early pregnancy.(AU)

Early pregnancy affects expression of Toll-like receptor signaling members in ovine spleen

Abstract Toll-like receptors (TLRs) are involved to the maternal immune tolerance. The spleen is essential for adaptive immune reactions. However, it is unclear that early pregnancy regulates TLR-mediated signalings in the maternal spleen. The purpose of this study was to investigate the effects of early pregnancy on expression of TLR signaling members in the ovine spleen. Ovine spleens were collected at day 16 of the estrous cycle, and at days 13, 16 and 25 of pregnancy (n = 6 for each group). Real-time quantitative PCR, western blot and immunohistochemistry analysis were used to detect TLR signaling members, including TLR2, TLR3, TLR4, TLR5, TLR7, TLR9, myeloid differentiation primary-response protein 88 (MyD88), tumor necrosis factor receptor associated factor 6 (TRAF6) and interleukin-1-receptor-associated kinase 1 (IRAK1). The results showed that expression levels of TLR2, TLR4 and IRAK1 were downregulated, but expression levels of TLR3, TLR5, TLR7, TLR9, TRAF6 and MyD88 were increased during early pregnancy. In addition, MyD88 protein was located in the capsule, trabeculae and splenic cords of the maternal spleen. This paper reports for the first time that early pregnancy has effects on TLR signaling pathways in the ovine spleen, which is beneficial for understanding the maternal immune tolerance during early pregnancy.

Conceptus-modulated innate immune function during early pregnancy in ruminants: a review

Abstract This review focuses on the innate immune events modulated by conceptus signaling during early pregnancy in ruminants. Interferon-tau (IFN-τ) plays a role in the recognition of pregnancy in ruminants, which involves more than the inhibition of luteolytic pulses of PGF2α to maintain corpus luteum function. For successful pregnancy establishment, the allogenic conceptus needs to prevent rejection by the female. Therefore, IFN-τ exerts paracrine and endocrine actions to regulate the innate immune system and prevent conceptus rejection. Additionally, other immune regulators work in parallel with IFN-τ, such as the pattern recognition receptors (PRR). These receptors are activated during viral and bacterial infections and in early pregnancy, but it remains unknown whether PPR expression and function are controlled by IFN-τ. Therefore, this review focuses on the main components of the innate immune response that are involved with early pregnancy and their importance to avoid conceptus rejection.

Conceptus-modulated innate immune function during early pregnancy in ruminants: a review

This review focuses on the innate immune events modulated by conceptus signaling during early pregnancy in ruminants. Interferon-tau (IFN-) plays a role in the recognition of pregnancy in ruminants, which involves more than the inhibition of luteolytic pulses of PGF2 to maintain corpus luteum function. For successful pregnancy establishment, the allogenic conceptus needs to prevent rejection by the female. Therefore, IFN- exerts paracrine and endocrine actions to regulate the innate immune system and prevent conceptus rejection. Additionally, other immune regulators work in parallel with IFN-, such as the pattern recognition receptors (PRR). These receptors are activated during viral and bacterial infections and in early pregnancy, but it remains unknown whether PPR expression and function are controlled by IFN-. Therefore, this review focuses on the main components of the innate immune response that are involved with early pregnancy and their importance to avoid conceptus rejection.(AU)

Quantitative analysis of expression patterns of different cell surface Toll-like receptor genes imparting heat stress resilience to indigenous Osmanabadi and Salem Black goat breeds

The study was conducted to establish the differences in the expression pattern of different cell surface toll-likereceptors (TLRs) between indigenous Osmanabadi and Salem Black goats subjected to summer season induced heat stress. The primary objective of the study is to assess the thermotolerance ability of these two breeds based on maintaining the immune status during exposure to heat stress. The results indicated that the expression of TLR1, TLR4, TLR5, TLR6, and TLR10 genes in both the breeds showed different expression pattern. The expression pattern of all these genes did not differed between the control (OC) and heat stress (OHS) group in Osmanabadi breed but significantly higher (P<0.05) expression of these genes were reported in heat stress group (SBHS) as compared to control group (SBC) in Salem Black breed. However, the striking difference was established in the expression pattern of TLR2 between these breeds. The TLR expression was significantly higher in heat stress group of Osmanabadi breed. But TLR2 did not express in Salem Back breed. The higher expression of most of the cell surface TLRs in the SBHS group as compared to OHS group indicates the superior resilient capacity of Salem Black goats to maintain immune status even during exposure to adverse environmental condition. Further, these TLRs could serve as indicators to reflect better thermo-tolerance of Salem Black breed over Osmanabadi breed.(AU)

Quantitative analysis of expression patterns of different cell surface Toll-like receptor genes imparting heat stress resilience to indigenous Osmanabadi and Salem Black goat breeds

The study was conducted to establish the differences in the expression pattern of different cell surface toll-likereceptors (TLRs) between indigenous Osmanabadi and Salem Black goats subjected to summer season induced heat stress. The primary objective of the study is to assess the thermotolerance ability of these two breeds based on maintaining the immune status during exposure to heat stress. The results indicated that the expression of TLR1, TLR4, TLR5, TLR6, and TLR10 genes in both the breeds showed different expression pattern. The expression pattern of all these genes did not differed between the control (OC) and heat stress (OHS) group in Osmanabadi breed but significantly higher (P<0.05) expression of these genes were reported in heat stress group (SBHS) as compared to control group (SBC) in Salem Black breed. However, the striking difference was established in the expression pattern of TLR2 between these breeds. The TLR expression was significantly higher in heat stress group of Osmanabadi breed. But TLR2 did not express in Salem Back breed. The higher expression of most of the cell surface TLRs in the SBHS group as compared to OHS group indicates the superior resilient capacity of Salem Black goats to maintain immune status even during exposure to adverse environmental condition. Further, these TLRs could serve as indicators to reflect better thermo-tolerance of Salem Black breed over Osmanabadi breed.

Toll-like receptor 2 and 4 expression in the bovine corpus luteum during the different stages of the estrous cycle

The aim of this study was to elucidate the presence of components of the innate immune system inthe bovine corpus luteum (CL) by detecting the expression and cell-specific localization of TLR2 and TLR4 during different stages of the estrous cycle in a control study design. Bovine CL samples were collected from a local slaughterhouse and assigned to three groups as follows: developing CL (dCL; n = 6, approx. days 3–6), mature CL (mCL; n = 5, approx. days 8–12), and regressing CL (rCL; n = 5, approx. days 17–19). An upregulation of TLR2 mRNA was detected only in rCL(P < 0.05). Localization of the TLR2 protein was particularly apparent in luteal cells and a prominent immunofluorescent signal corresponding to TLR2 was detected only in rCL. TLR4 mRNA were higher in mCLand rCL compared to dCL(P < 0.05). The presence of the TLR4 protein in bovine CL was clearly detected in the luteal cells of both mCL and rCL. The results of this study suggest a role for TLRs in the development, maintenance, and regression of bovine CL. TLR signaling mediated pathway in luteal cells may involve in the regression of CL via regulation of TLR2 and TLR4.(AU)

Agaricus brasiliensis polysaccharides stimulate human monocytes to capture Candida albicans, express toll-like receptors 2 and 4, and produce pro-inflammatory cytokines

Agaricus brasiliensis é um cogumelo medicinal com atividades imunomoduladoras e antitumorais atribuídas aos ß-glucanos presentes na fração polissacarídica de seu corpo de frutificação. Uma vez que os ß-glucanos aumentam a imunorresponsividade celular, neste estudo objetivamos avaliar o efeito de uma fração rica em polissacarídeos tratados com ácido (ATF) de A. brasiliensis sobre a capacidade de monócitos humanos de aderir / fagocitar células de levedura C. albicans . expressão de receptores de reconhecimento de padrões e sua capacidade de produzir citocinas. Métodos: A adesão / fagocitose de C. albicans marcada com FITC foi avaliada por citometria de fluxo. As células foram incubadas com anticorpos marcados com fluorocromo específicos para TLR2 e 4, ßGR e MR e também avaliadas por citometria de fluxo. Os monócitos foram cultivados com ATF, e os sobrenadantes da cultura foram coletados para análise da produção de citocinas in vitro por ELISA (TNF-α, IL-1ß, IL-12 e IL-10). Resultados: ATF aumentou significativamente a aderência / fagocitose de C. albicans por monócitos e isso foi associado com expressão aumentada de TLR2 e TLR4, enquanto nenhum efeito foi observado em ßGR ou MR. Além disso, a expressão de TLR4 e TLR2 foi associada a níveis mais elevados de produção in vitro de TNF-α e IL-1, respectivamente. A produção de IL-10 também foi aumentada pelo tratamento com ATF, mas não encontramos associação entre sua produção e a expressão de receptores Toll-like. Conclusão: Nossos resultados nos forneceram evidências de que polissacarídeos de A. brasiliensis afetam monócitos humanos provavelmente através da modulação de receptores Toll-like.(AU)

Agaricus brasiliensis polysaccharides stimulate human monocytes to capture Candida albicans, express toll-like receptors 2 and 4, and produce pro-inflammatory cytokines

Agaricus brasiliensis é um cogumelo medicinal com atividades imunomoduladoras e antitumorais atribuídas aos ß-glucanos presentes na fração polissacarídica de seu corpo de frutificação. Uma vez que os ß-glucanos aumentam a imunorresponsividade celular, neste estudo objetivamos avaliar o efeito de uma fração rica em polissacarídeos tratados com ácido (ATF) de A. brasiliensis sobre a capacidade de monócitos humanos de aderir / fagocitar células de levedura C. albicans . expressão de receptores de reconhecimento de padrões e sua capacidade de produzir citocinas. Métodos: A adesão / fagocitose de C. albicans marcada com FITC foi avaliada por citometria de fluxo. As células foram incubadas com anticorpos marcados com fluorocromo específicos para TLR2 e 4, ßGR e MR e também avaliadas por citometria de fluxo. Os monócitos foram cultivados com ATF, e os sobrenadantes da cultura foram coletados para análise da produção de citocinas in vitro por ELISA (TNF-α, IL-1ß, IL-12 e IL-10). Resultados: ATF aumentou significativamente a aderência / fagocitose de C. albicans por monócitos e isso foi associado com expressão aumentada de TLR2 e TLR4, enquanto nenhum efeito foi observado em ßGR ou MR. Além disso, a expressão de TLR4 e TLR2 foi associada a níveis mais elevados de produção in vitro de TNF-α e IL-1, respectivamente. A produção de IL-10 também foi aumentada pelo tratamento com ATF, mas não encontramos associação entre sua produção e a expressão de receptores Toll-like. Conclusão: Nossos resultados nos forneceram evidências de que polissacarídeos de A. brasiliensis afetam monócitos humanos provavelmente através da modulação de receptores Toll-like.(AU)

Endometrial transcription of microbial molecular patterns receptors in Gyr and F1 Holstein x Gyr postpartum cows / Transcrição endometrial de receptores de padrões moleculares microbianos em vacas Gir e F1 Holandês x Gir após o parto

Zebu and Holstein x Zebu crossbred have low incidence of uterine infection when compared to Holstein cows. Resistance to uterine infections may be associated with the ability to recognize invading microorganisms. Endometrial transcription of microbial molecular patterns receptors has been investigated in the postpartum period of Holstein cows, but it is completely unknown in Zebu or Holstein x Zebu cows. In this study, 9 Gyr and 12 F1 Holstein x Gyr cows were submitted to endometrial biopsies at the first and seventh days postpartum, with the objective to measure transcription levels of toll-like receptors (TLRs) 1/6, 2, 4, 5, and 9; nucleotide-binding oligomerization domain (NOD)-like receptors 1 and 2; and coreceptors cluster of differentiation 14 (CD14) and myeloid differentiation protein-2 (MD-2). There was a significant (P<0.05) decrease in transcription of TLR5 in Gyr, and an increase in transcription of TLR9 in F1 cows, between the first and seventh day postpartum. Both groups had low incidences of uterine infections up to 42 days postpartum. Uterine involution completed at 27.7 ± 10.1 and 25.1 ± 4.7 days postpartum for Gyr and F1 cows, respectively. In Gyr cows, higher transcription levels of TLR1/6 and NOD1 correlated to a longer period required for uterine involution. In F1 cows, lower levels of TLR1/6, TLR2 and NOD2 correlated to a longer period required for uterine involution. In conclusion, some pathogen recognition receptors associated significantly with the time required for uterine involution in Gyr and F1 cows.(AU)

Vacas Zebu e mestiças Holandês x Zebu apresentam baixas incidências de infecções uterinas quando comparadas às Holandesas. A resistência às infecções uterinas pode estar relacionada com a capacidade de reconhecimento dos microrganismos invasores. A transcrição endometrial de receptores de padrões moleculares microbianos tem sido investigada em vacas Holandesas recém-paridas, porém ainda é desconhecida em vacas Zebu e mestiças Holandês x Zebu. No presente estudo, nove vacas Gir e 12 F1 Holandês x Gir foram submetidas a biópsias endometriais no primeiro e no sétimo dia após o parto, com o objetivo de mensurar os níveis de transcrição gênica dos receptores tipo Toll (TLRs) 1/6, 2, 4, 5 e 9; receptores tipo NOD 1 e 2; e dos coreceptores CD14 e MD-2. Houve diminuição significativa (P < 0,05) do nível de transcrição de TLR5 em vacas Gir e aumento da transcrição de TLR9 em vacas F1, entre o primeiro e o sétimo dia após o parto. Os dois grupos apresentaram baixas incidências de infecções uterinas até 42 dias pós-parto. O período de involução uterina foi de 27,7 ± 10,1 e 25,1 ± 4,7 dias pós-parto, para vacas Gir e F1, respectivamente. No grupo de vacas Gir, altos níveis de transcrição de TLR1/6 e NOD1 tiveram correlação significativa com o prolongamento do período de involução uterina. No grupo de vacas F1, baixos níveis de transcrição de TLR1/6, TLR2 e NOD2 foram associados a maiores períodos de involução uterina. Portanto, os níveis de transcrição endometrial de alguns receptores de padrões moleculares microbianos na primeira semana após o parto podem estar relacionados com o tempo requerido para ocorrência da involução uterina em vacas Gir e F1.(AU)

Endometrial transcription of microbial molecular patterns receptors in Gyr and F1 Holstein x Gyr postpartum cows / Transcrição endometrial de receptores de padrões moleculares microbianos em vacas Gir e F1 Holandês x Gir após o parto

Zebu and Holstein x Zebu crossbred have low incidence of uterine infection when compared to Holstein cows. Resistance to uterine infections may be associated with the ability to recognize invading microorganisms. Endometrial transcription of microbial molecular patterns receptors has been investigated in the postpartum period of Holstein cows, but it is completely unknown in Zebu or Holstein x Zebu cows. In this study, 9 Gyr and 12 F1 Holstein x Gyr cows were submitted to endometrial biopsies at the first and seventh days postpartum, with the objective to measure transcription levels of toll-like receptors (TLRs) 1/6, 2, 4, 5, and 9; nucleotide-binding oligomerization domain (NOD)-like receptors 1 and 2; and coreceptors cluster of differentiation 14 (CD14) and myeloid differentiation protein-2 (MD-2). There was a significant (P<0.05) decrease in transcription of TLR5 in Gyr, and an increase in transcription of TLR9 in F1 cows, between the first and seventh day postpartum. Both groups had low incidences of uterine infections up to 42 days postpartum. Uterine involution completed at 27.7 ± 10.1 and 25.1 ± 4.7 days postpartum for Gyr and F1 cows, respectively. In Gyr cows, higher transcription levels of TLR1/6 and NOD1 correlated to a longer period required for uterine involution. In F1 cows, lower levels of TLR1/6, TLR2 and NOD2 correlated to a longer period required for uterine involution. In conclusion, some pathogen recognition receptors associated significantly with the time required for uterine involution in Gyr and F1 cows.(AU)

Vacas Zebu e mestiças Holandês x Zebu apresentam baixas incidências de infecções uterinas quando comparadas às Holandesas. A resistência às infecções uterinas pode estar relacionada com a capacidade de reconhecimento dos microrganismos invasores. A transcrição endometrial de receptores de padrões moleculares microbianos tem sido investigada em vacas Holandesas recém-paridas, porém ainda é desconhecida em vacas Zebu e mestiças Holandês x Zebu. No presente estudo, nove vacas Gir e 12 F1 Holandês x Gir foram submetidas a biópsias endometriais no primeiro e no sétimo dia após o parto, com o objetivo de mensurar os níveis de transcrição gênica dos receptores tipo Toll (TLRs) 1/6, 2, 4, 5 e 9; receptores tipo NOD 1 e 2; e dos coreceptores CD14 e MD-2. Houve diminuição significativa (P < 0,05) do nível de transcrição de TLR5 em vacas Gir e aumento da transcrição de TLR9 em vacas F1, entre o primeiro e o sétimo dia após o parto. Os dois grupos apresentaram baixas incidências de infecções uterinas até 42 dias pós-parto. O período de involução uterina foi de 27,7 ± 10,1 e 25,1 ± 4,7 dias pós-parto, para vacas Gir e F1, respectivamente. No grupo de vacas Gir, altos níveis de transcrição de TLR1/6 e NOD1 tiveram correlação significativa com o prolongamento do período de involução uterina. No grupo de vacas F1, baixos níveis de transcrição de TLR1/6, TLR2 e NOD2 foram associados a maiores períodos de involução uterina. Portanto, os níveis de transcrição endometrial de alguns receptores de padrões moleculares microbianos na primeira semana após o parto podem estar relacionados com o tempo requerido para ocorrência da involução uterina em vacas Gir e F1.(AU)

Agaricus brasiliensis polysaccharides stimulate human monocytes to capture Candida albicans, express toll-like receptors 2 and 4, and produce pro-inflammatory cytokines

Abstract Background Agaricus brasiliensis is a medicinal mushroom with immunomodulatory and antitumor activities attributed to the -glucans presented in the polysaccharide fraction of its fruiting body. Since -glucans enhance cellular immunoresponsiveness, in this study we aimed to evaluate the effect of an acid-treated polysaccharide-rich fraction (ATF) of A. brasiliensis on the ability of human monocytes to adhere/phagocyte C. albicans yeast cells, their expression of pattern recognition receptors and their ability to produce cytokines. Methods Adhesion/phagocytosis of FITC-labeled C. albicans was evaluated by flow cytometry. Cells were incubated with specific fluorochrome-labeled antibodies for TLR2 and 4, GR and MR and also evaluated by flow cytometry. Monocytes were cultured with ATF, and culture supernatants were collected for analysis of in vitro cytokine production by ELISA (TNF-, IL-1, IL-12 and IL-10). Results ATF significantly increased the adherence/phagocytosis of C. albicans by monocytes and this was associated with enhanced expression of TLR2 and TLR4, while no effect was observed on GR or MR. Moreover, expression of TLR4 and TLR2 was associated with higher levels of in vitro production of TNF- and IL-1, respectively. Production of IL-10 was also increased by ATF treatment, but we found no association between its production and the expression of Toll-like receptors. Conclusion Our results provided us with evidence that A. brasiliensis polysaccharides affect human monocytes probably through the modulation of Toll-like receptors.

Upregulation of tlr4 mrna expression levels in broiler chickens under acute heat stress

Toll-like receptors (TLRs) are well-characterized in mice and rats, but little is known on their role in broiler chickens during acute heat stress (AHS). The aim of the present study was to estimate the change in TLR4 mRNA expression in the liver, kidney's pleen, heart, and small intestine of broiler chickens under AHS. A total of 240 healthy Arbor Acres (AA) broiler chickens were randomly divided into four groups: control group (22±1°C; 0h), HS2, HS5 and HS10 (38±1°C; 2h, 5h and 10h of heat stress, respectively). As AHS duration increased, TLR4 mRNA expression slightly decreased at HS2 and HS5, but was dramatically elevated in HS10 (p 0.001) compared with the control group in the small intestine, as well as in the spleen at HS2 (p=0.001) and HS10 (p 0.001). The mRNA expression levels of TLR4 significantly increased in the liver, heart, and kidneys (p 0.001) at HS10, and in the kidneys at HS5 (p=0.003). It is found that TLR4 mRNA expression at HS10 in different organs was significantly higher (p 0.001) compared with HS2 and HS5. The results of the present study suggest that AHS may modulate the functional responses of the liver, kidney, spleen, heart, and small intestine of broilers by regulating TLR4 mRNA expression.(AU)

Toll-like receptor 2 and 4 expression in the bovine corpus luteum during the different stages of the estrous cycle

The aim of this study was to elucidate the presence of components of the innate immune system inthe bovine corpus luteum (CL) by detecting the expression and cell-specific localization of TLR2 and TLR4 during different stages of the estrous cycle in a control study design. Bovine CL samples were collected from a local slaughterhouse and assigned to three groups as follows: developing CL (dCL; n = 6, approx. days 3–6), mature CL (mCL; n = 5, approx. days 8–12), and regressing CL (rCL; n = 5, approx. days 17–19). An upregulation of TLR2 mRNA was detected only in rCL(P < 0.05). Localization of the TLR2 protein was particularly apparent in luteal cells and a prominent immunofluorescent signal corresponding to TLR2 was detected only in rCL. TLR4 mRNA were higher in mCLand rCL compared to dCL(P < 0.05). The presence of the TLR4 protein in bovine CL was clearly detected in the luteal cells of both mCL and rCL. The results of this study suggest a role for TLRs in the development, maintenance, and regression of bovine CL. TLR signaling mediated pathway in luteal cells may involve in the regression of CL via regulation of TLR2 and TLR4.

Upregulation of tlr4 mrna expression levels in broiler chickens under acute heat stress

Toll-like receptors (TLRs) are well-characterized in mice and rats, but little is known on their role in broiler chickens during acute heat stress (AHS). The aim of the present study was to estimate the change in TLR4 mRNA expression in the liver, kidney's pleen, heart, and small intestine of broiler chickens under AHS. A total of 240 healthy Arbor Acres (AA) broiler chickens were randomly divided into four groups: control group (22±1°C; 0h), HS2, HS5 and HS10 (38±1°C; 2h, 5h and 10h of heat stress, respectively). As AHS duration increased, TLR4 mRNA expression slightly decreased at HS2 and HS5, but was dramatically elevated in HS10 (p 0.001) compared with the control group in the small intestine, as well as in the spleen at HS2 (p=0.001) and HS10 (p 0.001). The mRNA expression levels of TLR4 significantly increased in the liver, heart, and kidneys (p 0.001) at HS10, and in the kidneys at HS5 (p=0.003). It is found that TLR4 mRNA expression at HS10 in different organs was significantly higher (p 0.001) compared with HS2 and HS5. The results of the present study suggest that AHS may modulate the functional responses of the liver, kidney, spleen, heart, and small intestine of broilers by regulating TLR4 mRNA expression.

EXPRESSÃO GÊNICA DE RECEPTORES TOLL-LIKE 2 E 4 E RECEPTORES DE CÉLULAS T WC1 ASSOCIADA AOS NÍVEIS DE INFECÇÃO EM BOVINOS NATURALMENTE INFECTADOS PELOS AGENTES DA TRISTEZA PARASITÁRIA

A babesiose bovina é uma doença infecciosa causada pelos protozoários Babesia bovis e Babesia bigemina, parasitas intraeritrocitários que induzem à anemia hemolítica intravascular. A anaplasmose, por sua vez, é causada pela rickettsia Anaplasma marginale, e envolve, principalmente, a manifestação de anemia seguida de hemólise massiva. A ocorrência dessas infecções segue a dispersão do carrapato Rhipicephalus microplus e, quando em ação concomitante, forma o complexo de doenças intitulado Tristeza Parasitária Bovina (TPB), reconhecido por acarretar perdas econômicas na pecuária devido, por exemplo, à redução na produção de leite e carne, e à infertilidade temporária. Neste cenário, estudos de expressão gênica focados na avaliação da resposta imune se mostram uma alternativa na determinação de bases genéticas voltadas para a seleção de bovinos resistentes a essas enfermidades e, por consequência, implicam na melhoria do fenótipo quanto à qualidade e à produtividade do rebanho. Neste trabalho objetivou-se avaliar as flutuações de expressão gênica de receptores toll-like 2 e 4, receptores de células T WC1 e nível de infecção pelos hemoparasitas em amostras de sangue de 46 bezerros da raça Canchim, desde o nascimento até os quatro meses e meio de idade, totalizando dez colheitas, por meio da Reação em Cadeia da Polimerase Quantitativa (qRT-PCR). Os dados obtidos mostraram variações na expressão gênica ao longo do tempo e associações entre estas variações e os níveis de infecção pelos agentes da tristeza parasitária bovina apresentaram efeitos de colheita e de classe de precedência significativos. Este estudo apresenta flutuação dos níveis de expressão desses genes frente às infecções estudadas e sugere que, apesar das oscilações de expressão ao longo do estudo, há participação desses receptores durante o desenvolvimento da imunidade contra as infecções pelos hemoparasitas. Conclui-se que este trabalho pode ser o ponto de partida para os estudos dos genes TLR2, TLR4 e WC1 como marcadores genéticos de resistência/suscetibilidade em bovinos infectados com os agentes da tristeza parasitária.

Bovine babesiosis is an infectious disease caused by protozoa Babesia bovis and Babesia bigemina, intra-erythrocyte parasites that induce intravascular hemolytic anemia. On the other hand, anaplasmosis is caused by Anaplasma marginale bacterium and mainly involves the manifestation of anemia followed by massive hemolysis. These infections occur with the Rhipicephalus microplus tick dispersion and when in concomitant action, form the disease complex called tickborne diseases, recognized for causing economic losses in livestock due, for example, to the reduction in the production of milk and meat and temporary infertility. In this scenario, studies of gene expression focused on the immune response evaluation are shown as an alternative in determining genetic bases aimed at selecting cattle resistant to these diseases and, consequently, imply improving the phenotype regarding the quality and productivity of the herd. This study aimed to evaluate the variation in gene expression of toll-like receptors 4 and T cells WC1, and infection level by hemoparasites in blood samples from 46 Canchim calves, from birth to four and a half months of age, totaling ten collections, using Real-Time Quantitative Reverse Transcription Quantitative Polymerase Chain Reaction (qRT-PCR). The data obtained showed variations in gene expression over time, and associations between these results and the infection level by the tick-borne diseases agents showed significant effects of collections and class of precedence. This study presents fluctuations in the expression levels of these genes against infections studied and suggests that, despite the oscillations of expression throughout the study, there is participation of these receptors during the development of immunity against infections by hemoparasites. We conclude that this work can be the starting point for studies of TLR2, TLR4, and WC1 genes as genetic markers of resistance/susceptibility in cattle infected with the agents of tick-borne diseases.

PERFIS DE EXPRESSÕES DE GENES DA IMUNIDADE INATA QUANTO AO TECIDO LINFOIDE E CARACTERIZAÇÃO DA REGIÃO 3-UTR DO GENE DO INTERFERON-GAMA ASSOCIADO AO SEU PADRÃO DE EXPRESSÃO GÊNICA NA MEMBRANA CONJUNTIVAL DE BUBALINOS DA AMAZÔNIA

A bubalinocultura vem apresentando elevado desempenho nos últimos anos, especialmente na Amazônia Brasileira, embora esta apresente condições ambientais adversas que influenciam no surgimento de doenças, incluindo infecções oculares. Assim, a determinação de genes relacionados à imunidade inata ocular é necessária em populações bubalinas. Buscamos determinar os perfis de expressão de genes relacionados ao sistema imune inato na membrana conjuntival de búfalos da Amazônia e caracterizar a região 3-UTR do gene Interferon gama determinando o seu perfil de expressão nessa população. Para isso, amostras de tecido conjuntival ocular e sangue foram coletadas de búfalos abatidos no Estado do Amapá. Os animais foram classificados em três grupos (G1, G2 e G3) de acordo com o grau quantitativo de tecido linfoide associado à conjuntiva por técnicas histológicas das amostras conjuntivais as quais foram submetidas a quantificação da expressão de citocinas inflamatórias, receptores tolllike 4 e -defensina-110 relativa ao gene endógeno. Amostras de sangue foram submetidas a PCR convencional usando iniciadores da região 3 UTR do gene Interferon gama. Os produtos foram purificados, sequenciados e as sequências editadas com outras correspondentes de búfalos e de espécies mamíferas. As mesmas foram alinhadas e comparadas a partir da análise de similaridade genética e análise filogenética. Na conjuntiva dos animais do G1 houve expressão apenas para IFNG e TLR4. Enquanto o G2 houve alta expressão para os genes IL6, IL10, IFNG, TLR4 e os animais do G3 apresentaram elevada expressão para todos os genes estudados. O grau de similaridade genética formou os grupos dos ruminantes, primatas, carnívoros, quirópteros, cetáceos e indivíduos isolados como suiformes e rinocerontes. Ruminantes e cetáceos apresentam alta relação filogenética enquanto houveram diferentes distâncias evolutivas de búfalos com as demais espécies de mamíferos. Nossos estudos serviram para definir os processos imunológicos inatos que ocorrem na membrana conjuntival de uma população de búfalos e determinar o perfil evolutivo em relação a outras espécies mamíferas.

Buffalo production has shown high performance in recent years, especially in the Brazilian Amazon, although it presents adverse environmental conditions that influence the emergence of diseases, including eye infections. Thus, the determination of genes related to ocular innate immunity is necessary in buffalo populations. We sought to determine the expression profiles of genes related to the innate immune system in the conjunctival membrane of Amazon buffaloes and to characterize the 3-UTR region of the Interferon gamma gene, determining its expression profile in this population. For this purpose, samples of ocular conjunctival tissue and blood were collected from buffaloes slaughtered in the State of Amapá. The animals were classified into three groups (G1, G2 and G3) according to the quantitative degree of lymphoid tissue associated with the conjunctiva by histological techniques of the conjunctival samples which were also subjected to quantification of the expression of inflammatory cytokines, tolllike receptors 4 and -defensin-110 relative to the endogenous gene. Blood samples were subjected to conventional PCR using primers from the 3' UTR region of the Interferon gamma gene. The products were purified, sequenced and the sequences edited with corresponding ones from buffalo and mammalian species. They were aligned and compared based on genetic similarity and phylogenetic analysis. In the conjunctiva of animals from G1 there was expression only for IFNG and TLR4. While G2 there was high expression for genes IL6, IL10, IFNG, TLR4 and animals from G3 showed high expression for all genes studied. The degree of genetic similarity formed the groups of ruminants, primates, carnivores, bats, cetaceans and isolated individuals such as suiformes and rhinos. Ruminants and cetaceans have a high phylogenetic relationship while there were different evolutionary distances between buffaloes and other mammal species. Our studies served to define the innate immunological processes that occur in the conjunctival membrane of a buffalo population and to determine the evolutionary profile in relation to other mammalian species.

Expression of CD14 and toll-like receptors 2 and 4 by milk neutrophils in bovine mammary glands infected with Corynebacterium bovis / Expressão de CD14 e dos receptores do tipo toll 2 e 4 por neutrófilos lácteos provenientes de glândulas infectadas por Corynebacterium bovis

This study evaluated the expression of CD14, toll-like receptor (TLR) 2 and TLR4 on the surface of milk neutrophils in bovine mammary glands infected with Corynebacterium bovis. Here, we used 23 culture-negative control quarters with no abnormal secretion on the strip cup test and milk somatic cell count lower than 1x105 cells/mL, and 14 C. bovis infected quarters. The identification of neutrophils, as well as, the percentage of neutrophils that expressed CD14, TLR2 and TLR4 were analyzed by flow cytometry using monoclonal antibodies. The present study encountered no significant difference in the percentages of milk neutrophils that expressed TLR2 and TLR4 or in the expression of TLR4 by milk neutrophils. Conversely, a lower median fluorescence intensity of TLR2 in milk neutrophils was observed in C. bovis-infected quarters. The percentage of neutrophils that expressed CD14 and the median fluorescence intensity of CD14 in milk neutrophils was also lower in C. bovis-infected quarters.(AU)

O presente estudo objetivou avaliar alterações na expressão de CD14, e dos receptores do tipo toll (TLR) 2 e 4 na superfície de neutrófilos lácteos provenientes de glândulas mamárias infectadas por Corynebacterium bovis. O presente estudo utilizou 23 quartos negativos no exame bacteriológico, sem alterações na prova de fundo escuro e com contagem automática de células somáticas menor que 1 x105 células/mL, e 14 quartos mamários infectados por C. bovis A identificação de neutrófilos, assim como a porcentagem de neutrófilos lácteos que expressaram CD14, TLR2 e 4 foram avaliadas por citometria de fluxo utilizando anticorpos monoclonais. A porcentagem de neutrófilos que expressaram TLR2 e TLR4 nos quartos mamários infectados por C. bovis não diferiu dos quartos mamários sadios, assim como na expressão de TLR4. No entanto, a intensidade de fluorescência do TLR2 na superfície dos neutrófilos foi menor nos quartos mamários infectados por C. bovis. A porcentagem de neutrófilos que expressaram CD14 e a intensidade de fluorescência da molécula de CD14 foi menor na superfície dos neutrófilos lácteos dos quartos infectados por C. bovis.(AU)

Imunidade inata uterina em vacas após o parto / Uterine innate immunity in postpartum cows

A imunidade inata é o principal mecanismo de defesa responsável pelo controle da contaminaçãouterina em vacas recém-paridas. O reconhecimento inicial de determinados componentes estruturais demicrorganismos invasores do ambiente uterino é realizado por receptores específicos, localizados nas célulasendometriais. Estudos sobre a atuação desses receptores são importantes para elucidar os mecanismos queregulam a resposta endometrial contra os patógenos, a qual pode evoluir para a proteção imunológica ou para oestabelecimento de infecções uterinas nos casos de falhas na ativação da resposta inflamatória. O objetivo destarevisão é relatar as principais informações disponíveis sobre imunidade inata uterina em vacas após o parto.(AU)

Innate immunity is the main defense mechanism responsible for controlling uterine contamination inpostpartum cows. The initial recognition of certain structural components of microorganisms that invade theuterine environment is mediated by specific receptors located in endometrial cells. Studies about the role ofthese receptors are important for elucidating the mechanisms that regulate endometrial response againstpathogens, which can progress to immune protection or establishment of uterine infections in cases of failures inthe activation of the inflammatory response. The objective of this review is to describe the most relevantknowledge about uterine innate immunity in postpartum cows.(AU)