Phagocytotic activity and gene expression of leukocytes isolated from Astyanax lacustris / Atividade fagocítica e expressão gênica de leucócitos isolados de Astyanax lacustris

The constant intensification of aquaculture has considerable increased the stress levels of farmed fish and, consequently, the number and intensity of diseases outbreaks. Thus, studies on fish immune response, especially regarding the interaction of fish leukocytes with potential pathogens and xenobiotics are of great importance in order to develop new prophylactic and curative strategies. We isolated leukocytes from the head kidney of Astyanax lacustris­an important Neotropical fish species for aquaculture and a potential model for Neotropical aquaculture research­using a Percoll centrifugation protocol. The isolated leukocytes were incubated with lipopolysaccharide (LPS), and the expression of genes IL-1ß, IL-8, LysC, and LysG were measured. We assessed the phagocytotic activity of leukocytes using Congo red-dyed yeast, a novel and cost-effective protocol that has been developed in this study. The isolated leukocytes responded to LPS induction, exhibiting strong IL-1ß and IL-8 upregulation, two of the most important pro-inflammatory interleukins for vertebrates immune reponse. The optimal concentration of yeast for the phagocytic assay was 106 cells mL-1, resulting in acceptable phagocytic capacity (PC) but without excess of yeasts during the counting process, ensuring a high precision and accuracy of the method. To the best of our knowledge, the present study is the first to investigate the in vitro gene expression and phagocytic activity of leukocytes isolated from A. lacustris. Our findings will serve as a reference for future studies on the immunology and toxicology of Neotropical fish.

A constante intensificação da aquicultura tem aumentado consideravelmente os níveis de estresse dos animais cultivados e, consequentemente, o número e a intensidade dos surtos de doenças. Logo, estudos sobre a resposta imune dos peixes, especialmente relacionados com a interação dos leucócitos de peixes com potenciais patógenos e xenobióticos, são de grande importância para o desenvolvimento de novas estratégias profiláticas e curativas. No presente trabalho, nós obtivemos sucesso ao isolar leucócitos oriundos do rim cranial de Astyanax lacustris ­ uma importante espécie de peixe Neotropical para a aquicultura e um modelo em potencial para pesquisas em aquicultura Neotropical ­ usando um protocolo de centrifugação com Percoll. Os leucócitos isolados foram incubados com lipossacarídeo (LPS) e, a expressão dos genes IL-1ß, IL-8, LysC, e LysG foi avaliada. Ainda, um novo protocolo para avaliação da atividade fagocítica dos leucócitos utilizando leveduras coradas com Vermelho Congo foi estabelecido. Os leucócitos isolados responderam à indução com LPS, exibindo up regulation dos genes IL-1ß e IL-8, duas das interleucinas pró-inflamatórias mais importantes para a resposta imune de vertebrados. Além do mais, a concentração ótima de leveduras para a avaliação da fagocitose foi de 106 células mL-1, resultando em uma capacidade fagocítica (PC) aceitável, mas sem excesso de leveduras durante o processo de contagem, garantindo maior precisão e eficácia do método. Até o presente momento, o presente estudo é o primeiro a investigar a expressão gênica e atividade fagocítica de leucócitos isolados de A. lacustris através da abordagem in vitro. Ainda, nossos resultados servirão de referência para futuros estudos em imunologia e toxicologia de peixes Neotropicais.

Performance of the tetra-primer PCR technique compared to PCR-RFLP in the search for rs12979860 (C/T) and rs8099917 (T/G) single nucleotide polymorphisms (SNPs) in the IFNL4 gene / Desempenho da técnica tetra-primer PCR em relação a PCR-RFLP na pesquisa de polimorfismos de nucleotídeos únicos (SNPs) rs12979860 (C/T) e rs8099917 (T/G) no gene IFNL4

Single nucleotide polymorphisms (SNPs, rs12979860 e rs8099917) in the Interferon Lambda 4 gene (IFNL4, formerly IFNL3and/or IL28B) has been associated with failure in the innate immune response, sustained virological response in hepatitis C, and HTLV-1-associated myelopathy (HAM) development. To search for these polymorphisms several methodologies can be employed, such as sequencing, real-time or quantitative polymerase chain reaction (qPCR), restriction fragment length polymorphism analysis in PCR products (PCR-RFLP), and tetra-primer PCR. The present study compared the performance of the tetra-primer PCR in relation to the PCR-RFLP, both optimized in the Research HTLV Laboratory of the Center of Immunology of Instituto Adolfo Lutz in São Paulo. One hundred DNA samples obtained from patients of STD/Aids Reference Centre in São Paulo, previously analyzed for IL28B SNPs by PCR-RFLP were selected for analysis, after confirming that they represent all IL28B SNPs patterns described in the literature. The results obtained showed concordance between the PCR-RFLP and the tetra-primer PCR SNPs results, and because of the low cost, easy to perform, and minor employment of biological specimen and reagents, the tetra-primer PCR is of choice to be used in routine. (AU)

Polimorfismos de nucleotídeos únicos (single nucleotide polymorphisms, SNPs rs12979860 e rs8099917) no gene que codifica o Interferon Lambda 4 (IFNL4, antigamente IFNL3 e/ou IL28B) têm sido associados às falhas na resposta imune inata e resposta virológica sustentada na hepatite C, e a mielopatia associada ao HTLV-1 (HTLV-1-associated myelopathy, HAM). A pesquisa destes polimorfismos pode empregar diversas metodologias: sequenciamento, reação em cadeia da polimerase em tempo real ou quantitativa (quantitative polymerase chain reaction, qPCR), análise de fragmentos de restrição enzimática em produtos de PCR (restriction fragment length polymorphism in PCR products, PCR-RFLP) e a tetra-primer PCR. Este estudo comparou o desempenho da tetra-primer PCR em relação a PCR-RFLP, ambas otimizadas no Laboratório de Pesquisa em HTLV do Centro de Imunologia do Instituto Adolfo Lutz de São Paulo. Foram selecionadas 100 amostras de DNA obtidas de pacientes do Centro de Referência e Treinamento em DST/Aids de São Paulo cujos SNPs na IL28B foram anteriormente determinados por PCR-RFLP e representaram todos os perfis descritos em literatura. Os resultados obtidos mostraram concordância entre elas, e pelo fato da tetra-primer PCR ter menor custo, ser de fácil execução, empregar menos tempo, insumos e material biológico, é a técnica de escolha para uso em rotina. (AU)

Sheep naturally infected with Anaplasma ovis - evaluation of cardiac and inflammatory biomarkers

Background: Anaplasmosis, also called gall sickness or tropical bovine ehrlichiosis, is an infectious disease caused by species belonging to the genus Anaplasma in domestic and wild animals in tropical and subtropical regions. Anaplasma ovis and A. phagocytophilum are important pathogens of sheep. A. ovis is considered the most common species affecting sheep. The infection is usually subclinical and progresses with high fever, anaemia, icterus, weight loss and abortions. This study aimed to investigate changes in cardiac damage markers, oxidative stress and antioxidant status, cytokines, and acute phase proteins in sheep naturally infected with A. ovis. Materials, Methods & Results: For this purpose, a total of 40 animals, including 20 healthy sheep and 20 sheep infected with anaplasmosis, were used. A. ovis was diagnosed based on clinical findings and peripheral blood smear. Blood smears were prepared from the ear vein. The smears were stained with Giemsa and examined for the presence of Anaplasma spp. Infection was also confirmed by polymerase chain reaction (PCR) analysis. The genomic DNA was isolated from blood, and the MSP-4 gene region was amplified as A. ovis specific target gene. Twenty clinically healthy sheep of the same age group, reared under the same conditions and testing negative in the molecular assessment were used as controls. Blood samples were collected from the cephalic vein and and centrifuged to obtain serum. The serum stored at -20°C until the analysis stage. Serum samples were used for the analysis of cardiac damage markers [troponin I (cTnI), creatine kinase MB (CK-MB), lactate dehydrogenase (LDH) and aspartate transaminase (AST)], oxidative stress parameters [malondialdehyde (MDA), total antioxidant status (TAS), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)], cytokines [interleukins IL-6, IL-1ß and IL-10, tumour necrosis factor α (TNF-α), and interferon-γ (IFN-γ)] and acute phase proteins [C-reactive protein (CRP), serum amyloid A (SAA) and haptoglobin (Hp)]. cTnI and CK-MB levels were measured using a chemiluminescent immunoassay. MDA, TAS, SOD, CAT, GPx, TNF-α, IL-1ß, IL-6, IL-10, IFN-γ, SAA and Hp levels were measured by an ELISA reader. LDH, AST and CRP levels were measured in an autoanalyzer. cTnI and LDH levels were significantly increased in the infected animals compared to the healthy ones (P < 0.05). The concentration of AST was decreased in infected animals. MDA, TAS, SOD, CAT and GPx levels were significantly increased in the infected animals compared to the healthy ones (P < 0.05). The levels of the inflammatory parameters such as TNF-α, IL-1ß, IL-10 and IFN-γ were significantly increased in the infected animals compared to the healthy ones (P < 0.05). Hp level were significantly increased in the infected group compared with the control group (P < 0.05). However, there was no significant change in CK-MB, SAA and CRP concentrations in the infected animals (P > 0.05). Discussion: Ovine anaplasmosis is an obligate intracellular arthropod disease that causes widespread changes in haematobiochemical, immune response and oxidative stress parameters. Cardiac damage is often overlooked in field conditions due to the lack of adequate knowledge about the pathophysiology of the disease. Our results showed that A. ovis infection leads to significant changes in cardiac biomarkers and that the parasite can cause cardiac dysfunction. This is the first report on cardiac damage markers in Anaplasma-infected sheep. Additionally, the levels of proinflammatory and oxidative stress markers that may cause functional disorders were also found to be increased. Thus, measuring markers of cardiac function, oxidative stress and inflammation can be a useful tool in the early diagnosis of ovine anaplasmosis.

Bisacurone gel ameliorated burn wounds in experimental rats via its anti-inflammatory, antioxidant, and angiogenic properties

Purpose: To investigate putative mechanism of wound healing for chitosan-based bisacurone gel against secondary burn wounds in rats. Methods: A second-degree burn wound with an open flame using mixed fuel (2 mL, 20 seconds) was induced in Sprague Dawley rats (male, 180-220 g, n = 15, each) followed by topical treatments with either vehicle control (white petroleum gel, 1%), silver sulfadiazine (1%) or bisacurone gel (2.5, 5, or 10%) for 20 days. Wound contraction rate and paw withdrawal threshold were monitored on various days. Oxidative stress (superoxide dismutase, glutathione, malondialdehyde, and nitric oxide), pro-inflammatory cytokines (tumour necrosis factor-alpha, interleukins by enzyme-linked immunosorbent assay), growth factors (transforming growth factor-ß, vascular endothelial growth factor C using real time polymerase chain reaction and Western blot assay) levels, and histology of wound skin were assessed at the end. Results: Bisacurone gel showed 98.72% drug release with a 420.90­442.70 cps viscosity. Bisacurone gel (5 and 10%) significantly (p < 0.05) improved wound contraction rate and paw withdrawal threshold. Bisacurone gel attenuated oxidative stress, pro-inflammatory cytokines, and water content. It also enhanced angiogenesis (hydroxyproline and growth factor) and granulation in wound tissue than vehicle control. Conclusions: These findings suggested that bisacurone gel can be a potential candidate to treat burn wounds via its anti-inflammatory, antioxidant, and angiogenic properties.

Immunological indices of Giant African Land snails (Archachatina marginata) improved with fixed dose of vitamin C during acute heat stress

The study determined immunological indices of Giant African Land snail (Archachatina marginata) improved with fixed dose of vitamin C under acute heat stress (AHS). Prior to the AHS, vitamin C was administered for four weeks to two treatment groups, while other two treatment groups were not. Each treatment was monitored, haemolymph collected at 0, 30 and 60 minutes exposure times. Immunological cytokines: interferon gamma (IFN-γ) and interleukin 2 (IL-2); and total haemocyte counts (THC) were determined. Under AHS, vitamin C elevated (p <0.05) IFN-γ production (606.33 ± 302.86) compared to other groups with or without vitamin C administration (7.20 ± 1.58 vs. 73.20 ± 32.23 vs. 7.80 ± 1.36). IL-2 was not affected (p >0.05) by vitamin C under AHS. Highest (p <0.05) THC values was obtained with vitamin C administration under AHS, but reduced under no AHS. Exposure time affected (p <0.05) IFN-γ production and THC values, but not IL-2 (p >0.05). With fixed dose of vitamin C and exposure time, highest (p <0.05) IFN-γ values were obtained under AHS with vitamin C administration at 30 minutes and at 60 minutes in THC, compared to other groups. The study concluded that fixed dose of vitamin C at 150 mg kg-1 of feed was appropriate under AHS to boost the immune system of the animals.(AU)

Cyclosporine A increases the intensity of Toxocara canis infection in swiss mice / Ciclosporina A aumenta a intensidade da infecção por Toxocara canis em camundongos suíços

Toxocariasis is a zoonotic disease of worldwide distribution. The connection between parasitic diseases and conditions that depress the immune system, such as the use of immunosuppressive drugs, has been studied. The purpose of this study was to evaluate the effect of Cyclosporine A (CsA) on the intensity of infection, humoral response and gene transcription of interleukins IL-4, IL-10 and IL-12 in mice experimentally infected with Toxocara canis. To this end, mice were divided into two groups treated with CsA (G1: 10 mg/Kg and G2: 50 mg/kg), the G3 and G4 group received PBS. After the last administration of the drug or PBS (orally every 48 hours for 15 days), groups G1, G2 and G3 were inoculated with 1200 eggs of T. canis. Was collected blood samples on days zero, 15 and 30 days post-inoculation (PI), for ELISA test and the mice were euthanized 30 days PI. The organs and striated muscle tissue were collected for the recovery of larvae. The splenocytes were analyzed by RT-PCR. The intensity of infection in the mice treated with 50 mg/kg of CsA was 65.5% higher than in the control group (p=0.001). An analysis of the kinetics of anti-Toxocara antibody revealed that the groups treated with CsA showed significantly higher mean levels of antibodies on day 15 PI. The transcription of the three tested interleukins showed no statistical difference between G2 and G3 (control). It was concluded that the immunosuppression triggered by CsA (50 mg/Kg) favored the establishment of a larger number of T. canis larvae without, however, altering immunoglobulin production and IL-4, IL-10 and IL-12 transcription on day 30 PI.

A toxocaríase é uma zoonose de distribuição mundial. A conexão entre doenças parasitárias e condições que deprimem o sistema imunológico, como o uso de drogas imunossupressoras, tem sido estudada. O objetivo deste estudo foi avaliar o efeito da Ciclosporina A (CsA) na intensidade da infecção, resposta humoral e transcrição gênica das interleucinas IL-4, IL-10 e IL-12 em camundongos experimentalmente infectados com Toxocara canis. Para tanto, os camundongos foram divididos em dois grupos tratados com CsA (G1: 10 mg/Kg e G2: 50 mg/kg), os grupos G3 e G4 receberam PBS. Após a última administração da droga ou PBS (via oral a cada 48 horas por 15 dias), os grupos G1, G2 e G3 foram inoculados com 1200 ovos de T. canis. Foram coletadas amostras de sangue nos dias zero, 15 e 30 dias pós-inoculação (PI), para teste de ELISA e os camundongos foram eutanasiados 30 dias PI. Os órgãos e tecido muscular estriado foram coletados para a recuperação das larvas. Os esplenócitos foram analisados ​​por RT-PCR. A intensidade da infecção nos camundongos tratados com 50 mg/kg de CsA foi 65,5% maior do que no grupo controle (p=0,001). Uma análise da cinética do anticorpo anti-Toxocara revelou que os grupos tratados com CsA apresentaram níveis médios de anticorpos significativamente maiores no dia 15 PI. A transcrição das três interleucinas testadas não apresentou diferença estatística entre G2 e G3 (controle). Concluiu-se que a imunossupressão desencadeada pela CsA (50 mg/Kg) favoreceu o estabelecimento de um maior número de larvas de T. canis sem, no entanto, alterar a produção de imunoglobulinas e a transcrição de IL-4, IL-10 e IL-12 no dia 30 PI.

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)

Encefalitozoonose em coelho (Oryctolagus cuniculus) / Encephalitozoonosis in rabbit (Oryctolagus cuniculus)

Background: Encephalitozoonosis is caused by the protozoan Encephalitozoon cuniculi, in rabbits, and can affect humans. The disease can be fatal and difficult to diagnose. It can be asymptomatic or cause vestibular neurological disease, paralysis, uveitis in addition to chronic kidney disease in rabbits. The transmission of the microorganism's spores occurs by ingestion, inhalation, or by the transplacental route. The aim of this work is to report a case of encephalitozoonosis in a pet rabbit (Oryctolagus cuniculus). Case: An Oryctolagus cuniculus with a history of paraparesis of the thoracic and pelvic limbs was referred for necropsy, the evolution of the clinical picture happened in one day. After death, a necropsy was performed. Organ fragments were collected, fixed, and processed routinely for histology. Macroscopically, there was evidence of hepatic lobes, without injury to the other organs. Microscopically it was observed in the white and gray substance of the telencephalon multiple circumscribed granulomas composed of a necrotic center surrounded by macrophages, giant multinucleated cells in addition to lymphocytes and plasmocytes in the periphery, delimited by fibrous connective tissue. Around the vessels, perivascular cuffs with 2 to 4 layers of lymphocytic infiltrate were observed. Besides, special staining of Schiff's Periodic Acid (PAS) and Ziehl-Neelsen was performed, in which numerous cylindrical, eosinophilic structures of approximately 2.5 x 1.0 µm were observed, compatible with E. cuniculi spores. Besides, histiocytic lymphoblasts pericoronitis was noted in the liver. There were no relevant changes in the kidney. Discussion: The diagnosis of encephalitozoonosis in rabbits was based on clinical and anatomopathological findings. Tetraparesis was the predominant sign in the present case and was justified by telencephalic lesions. This clinical sign is included in the literature but is less common than the syndrome such as head tilt and paralysis. The diagnosis of the disease is usually made by post mortem examination when it is possible to identify the spores in the lesions. Multifocal granulomatous encephalitis was the most significant finding in this case, which is also consistent with other studies. The pathogenesis of granulomatous lesions is still controversial. It is known that spores allow phagocytosis by macrophages, which induce the production of interleukins and other cytokines by TCD4 + lymphocytes, thereby activating the action of TCD8 + (cytotoxic) lymphocytes. Natural killer cells, granulocytes, other macrophages, and B lymphocytes are also recruited. Although there is such an inflammatory response, the antibodies produced are not efficient to eliminate the agent from the host organism, however, they contribute to the process of opsonization and consequent phagocytosis, facilitating the destruction of the microsporidium by macrophages. The neurological form was predominant in this case, with no chronic or ocular renal forms, possibly due to the rapid clinical evolution. Special stains were useful for visualizing intralesional spores. Although PAS staining is considered to be of little use, it was relevant in this case. The visualization of the agent made it possible to distinguish differential diagnoses, among them vestibular syndrome secondary to otitis due to pasteurellosis, toxoplasmosis, neoplasms, traumas, or diseases of the spine. Thus, a diagnosis of encephalitozoonosis was made in a rabbit through clinical and anatomopathological correlation using Ziehl-Neelsen and PAS stains.

Analysis of inflammatory and metabolic biomarkers in patients submitted to abdominoplasty after bariatric surgery

Purpose: To evaluate the serum variations of Interleukins (Il) and CPR of abdominoplasties in post-bariatric patients and, to equate the homeostasis (HOMA) from the variations of glycemia and insulin to evolute the metabolic modifications.Methods: Fourteen women were submitted to abdominoplasties with weight loss after a gastroplasty. Levels of IL4, IL6, IL10, CRP, glycemia and insulin were obtained during the pre-operative, trans-operative, 24 hours post, 7th and 14th postoperative days.Results: The IL4 was higher at 24 hours post-surgery, and after a moderate decrease, it remained high until the 14th day. The IL6 and CRP had an expressive increase during the trans-operative period. The CRP remained high, and the IL6 decreased on the 7th and 14th days. The IL10 increased during the transoperative period, and it posteriorly decreased to lower levels in comparison to the pre-operative period. The already increased glycemia during the pre-operative period was even higher during the trans-operative and then, returned to preliminary values on the 7th and 14th days after surgery. The HOMA accompanied the insulin. Conclusion: The inflammatory and glycemic serum levels decrease after abdominiplasty in obese post-bariatric patients.(AU)

Salmonella typhimurium vaccine to control a brazilian Salmonella heidelberg strain in broiler chickens / Vacina de Salmonella typhimurium para controlar cepa brasileira de Salmonella heidelberg em frangos de corte

Foodborne Salmonella infections in humans, which results from the consumption of contaminated poultry meat and eggs, are a major public health concern. Vaccination of animals against Salmonella is one strategy to prevent these infections and reduce the risks to public health. Live attenuated Salmonella enterica vaccines can confer protection against salmonellosis by inducing both cell-mediated and mucosal immune responses. This study assessed a live, attenuated Salmonella enterica Typhimurium (ST) vaccine in broiler chickens against a heterologous challenge with Salmonella Heidelberg (SH) by evaluating bacterial quantification, immune cells infiltration, and cytokine gene expression in the cecum. The treatments were: T1, non-vaccinated, non-challenged; T2, non-vaccinated, SH-challenged; T3, ST-vaccinated and SH-challenged. At 28 days of age, the ST-vaccinated group had significantly recovered reduction of SH in the crop (P<0,01) and cecum (P = 0,021) compared to the non-vaccinated SH-challenged group, with no significant changes (P˃0,05) in macrophages, T CD4+, or T CD8+ cells dynamics during the same period. Aerosol vaccination on the first day promoted greater interleukin-12 expression in the liver (P<0,05) and interleukin-10 expression and T CD8+ cells in the ileum 16 hours after housing. After prime-boosted oral immunization on the 13th day, the vaccinated group had greater expression of macrophages and T CD4+ cells in the liver (P<0,05) than the control group. Two doses of a live ST-attenuated vaccine promoted a partial cross-protective effect against SH strain UFPR1 challenge in broilers.

Infecções por Salmonella transmitidas por alimentos como consumo de carne de frango e ovos contaminados em seres humanos constituem um importante problema de saúde pública. A vacinação de animais contra Salmonella é uma estratégia para prevenir essas infecções e reduzir o risco para a saúde pública. As vacinas vivas atenuadas de Salmonella enterica podem conferir proteção contra a salmonelose, induzindo respostas imunológicas mediadas por células e em mucosas. Este estudo avaliou uma vacina viva e atenuada de Salmonella enterica Typhimurium (ST) em frangos de corte contra um desafio heterólogo com Salmonella Heidelberg (SH), avaliando a quantificação de Salmonella, infiltração de células imunes e a expressão de genes de citocinas no ceco. Os tratamentos foram: T1, não vacinado, não desafiado; T2, não vacinado, desafiado com SH; T3, ST-vacinado, desafiado com SH. Aos 28 dias de idade, o grupo vacinado com ST apresentou significativa redução de SH no papo (P<0,01) e no ceco (P = 0,021) comparado ao grupo T2-não vacinado SH-desafiado, sem alterações significativas na dinâmica celular de macrófagos, T CD4+ ou T CD8+ (P˃0,05) durante o mesmo período. A vacinação por aerossol no primeiro dia promoveu maior expressão de IL-12 no fígado (P<0,05), maior expressão de IL-10 e células T CD8+ no íleo, 16 horas após o alojamento. Após o reforço de imunização oral ao 13º dia, o grupo vacinado apresentou maior expressão de macrófagos e células T CD4+ no fígado (P<0,05) do que o grupo controle. Duas doses de uma vacina viva atenuada de ST promoveram um efeito de proteção cruzada parcial contra o desafio da cepa de Salmonella Heidelberg cepa UFPR1 em frangos de corte.

Salmonella typhimurium vaccine to control a brazilian Salmonella heidelberg strain in broiler chickens / Vacina de Salmonella typhimurium para controlar cepa brasileira de Salmonella heidelberg em frangos de corte

Foodborne Salmonella infections in humans, which results from the consumption of contaminated poultry meat and eggs, are a major public health concern. Vaccination of animals against Salmonella is one strategy to prevent these infections and reduce the risks to public health. Live attenuated Salmonella enterica vaccines can confer protection against salmonellosis by inducing both cell-mediated and mucosal immune responses. This study assessed a live, attenuated Salmonella enterica Typhimurium (ST) vaccine in broiler chickens against a heterologous challenge with Salmonella Heidelberg (SH) by evaluating bacterial quantification, immune cells infiltration, and cytokine gene expression in the cecum. The treatments were: T1, non-vaccinated, non-challenged; T2, non-vaccinated, SH-challenged; T3, ST-vaccinated and SH-challenged. At 28 days of age, the ST-vaccinated group had significantly recovered reduction of SH in the crop (P<0,01) and cecum (P = 0,021) compared to the non-vaccinated SH-challenged group, with no significant changes (P˃0,05) in macrophages, T CD4+, or T CD8+ cells dynamics during the same period. Aerosol vaccination on the first day promoted greater interleukin-12 expression in the liver (P<0,05) and interleukin-10 expression and T CD8+ cells in the ileum 16 hours after housing. After prime-boosted oral immunization on the 13th day, the vaccinated group had greater expression of macrophages and T CD4+ cells in the liver (P<0,05) than the control group. Two doses of a live ST-attenuated vaccine promoted a partial cross-protective effect against SH strain UFPR1 challenge in broilers.(AU)

Infecções por Salmonella transmitidas por alimentos como consumo de carne de frango e ovos contaminados em seres humanos constituem um importante problema de saúde pública. A vacinação de animais contra Salmonella é uma estratégia para prevenir essas infecções e reduzir o risco para a saúde pública. As vacinas vivas atenuadas de Salmonella enterica podem conferir proteção contra a salmonelose, induzindo respostas imunológicas mediadas por células e em mucosas. Este estudo avaliou uma vacina viva e atenuada de Salmonella enterica Typhimurium (ST) em frangos de corte contra um desafio heterólogo com Salmonella Heidelberg (SH), avaliando a quantificação de Salmonella, infiltração de células imunes e a expressão de genes de citocinas no ceco. Os tratamentos foram: T1, não vacinado, não desafiado; T2, não vacinado, desafiado com SH; T3, ST-vacinado, desafiado com SH. Aos 28 dias de idade, o grupo vacinado com ST apresentou significativa redução de SH no papo (P<0,01) e no ceco (P = 0,021) comparado ao grupo T2-não vacinado SH-desafiado, sem alterações significativas na dinâmica celular de macrófagos, T CD4+ ou T CD8+ (P˃0,05) durante o mesmo período. A vacinação por aerossol no primeiro dia promoveu maior expressão de IL-12 no fígado (P<0,05), maior expressão de IL-10 e células T CD8+ no íleo, 16 horas após o alojamento. Após o reforço de imunização oral ao 13º dia, o grupo vacinado apresentou maior expressão de macrófagos e células T CD4+ no fígado (P<0,05) do que o grupo controle. Duas doses de uma vacina viva atenuada de ST promoveram um efeito de proteção cruzada parcial contra o desafio da cepa de Salmonella Heidelberg cepa UFPR1 em frangos de corte.(AU)

Correlation between clinical findings, mast cell count and interleukin 31 immunostaining in the skin of dogs with atopic dermatitis / Correlação entre achados clínicos, contagem de mastócitos e imunomarcação de interleucina 31 na pele de cães com dermatite atópica

In this study the correlation between the clinical score, mast cell count and interleukin 31 (IL-31) immunostaining in the skin of dogs with atopic dermatitis was determined. A total of 31 dogs of different breeds, from one to eight years of age, were chosen for the study. The 20 females and 11 males were categorized based on the CADESI-4 system, as having discrete, moderate or marked atopic dermatitis. Skin samples were collected from the axillary and interdigital regions and stained with hematoxylin and eosin for cytohistomorphological analyses and toluidine blue to evaluate the mast cell counts, and immunohistochemistry for the IL-31 immunostaining. Animals revealing higher atopic dermatitis scores had greater numbers of mast cells and IL-31 immunolabeled cells. More numbers of cells immunolabeled for IL-31 were evident in the axillary skin compared with the interdigital skin in dogs having this condition. A correlation was identified between the clinical scores and mast cell numbers in the interdigital region, as well as between the clinical scores and number of cells immunolabeled for IL-31 in the axillary area. A correlation was also reported between the mast cell numbers and IL-31 immunolabeled cells only in the axillary skin, and none in the interdigital regions. It was thus concluded that the mast cells and IL-31 are involved in the pathogenesis of the canine atopic dermatitis (CAD), as well as lymphocytes and plasma cells. It was also observed that the higher the degree of clinical severity of the disease, the more the numbers of mast cells and IL-31 in the skin of those animals suffering from CAD, which implies the influence of these immunological constituents on the genesis of pruritus and disease progression.(AU)

Este estudo avaliou a correlação entre o escore clínico, a contagem de mastócitos e a imunomarcação de interleucina 31 (IL-31) na pele de cães com dermatite atópica. Foram selecionados 31 cães de diferentes raças, com idade entre um e oito anos, sendo 20 fêmeas e 11 machos, divididos em discretamente, moderadamente e acentuadamente acometidos por dermatite atópica segundo o sistema CADESI-4. Amostras da pele das regiões axilar e interdigital foram colhidas e submetidas às colorações de hematoxilina e eosina para a avaliação cito-histomorfológica e azul de toluidina para a contagem de mastócitos, bem como a técnica de imunoistoquímica para a imunomarcação de IL-31. Os animais com maior escore de dermatite atópica apresentaram maior número de mastócitos e de células imunomarcadas para IL-31. Houve maior número de células imunomarcadas para IL-31 na pele da axila em relação à interdigital nos cães com a doença. Foi constatada correlação entre o escore clínico e a quantidade de mastócitos no interdígito, bem como entre o escore clínico e a quantidade de células imunomarcadas para IL-31 na axila. Também foi verificada correlação entre a quantidade de mastócitos e células imunomarcadas para IL-31 na pele da região axilar, mas não da interdigital. Conclui-se que mastócitos e a IL-31 estão envolvidos na patogenia da DAC, assim como linfócitos e plasmócitos. Também, quanto maior o grau de severidade clínica da doença, maior a quantidade de mastócitos e IL-31 na pele dos animais com DAC, o que remete à influência desses componentes imunológicos na gênese do prurido e progressão da doença.(AU)

Pathogenesis of uterine diseases in dairy cattle and implications for fertility

Uterine diseases in cattle occur at all stages of the reproduction cycle but the majority of cases is found in the postpartum period. The inflammation of the uterus is generally defined as metritis or endometritis, with several graduations, e.g. puerperal metritis, clinical metritis, clinical or subclinical endometritis. Whether uterine diseases have a negligible, moderate or detrimental effect on fertility is still under discussion and depends on definitions and classification. In the past, it was assumed that the pregnant uterus is free of pathogens, but recent studies found several species including pathogens in the uterus and endometrium of pregnant cows. After parturition, a broad diversity of bacteria with >200 different species has been found in the early postpartum period. Not all of these bacteria, however, are considered as pathogens. Furthermore, bacteriological findings provide only evidence for infection but not for inflammation. For some bacteria, particularly Escherichia coli and Trueperella pyogenes pathogenic mechanism resulting in metritis and endometritis have been elucidated in detail. The role of bacteria that can be regarded as opportunistic or potential pathogens, e.g. Bacillus pumilus, is still under investigation. The understanding of the uterine microbiota and its interactions is increasing with the use of modern high-resolution techniques such as Fouriertransform infrared spectroscopy. Endometrial cytology provides additional information about alterations in the endometrium. Knowledge of innate uterine defense mechanism in cattle has increased a lot in the recent past. It can be speculated that improving or modulating uterine defense mechanism will be part of future prevention and treatment approaches beyond the use of antimicrobials. In this context, cellular and molecular defense mechanisms have been in the focus of interest, e.g. the role of interleukins or mucins. This review gives a short overview on some aspects of recent research on uterine diseases in cattle.

Pathogenesis of uterine diseases in dairy cattle and implications for fertility

Uterine diseases in cattle occur at all stages of the reproduction cycle but the majority of cases is found in the postpartum period. The inflammation of the uterus is generally defined as metritis or endometritis, with several graduations, e.g. puerperal metritis, clinical metritis, clinical or subclinical endometritis. Whether uterine diseases have a negligible, moderate or detrimental effect on fertility is still under discussion and depends on definitions and classification. In the past, it was assumed that the pregnant uterus is free of pathogens, but recent studies found several species including pathogens in the uterus and endometrium of pregnant cows. After parturition, a broad diversity of bacteria with >200 different species has been found in the early postpartum period. Not all of these bacteria, however, are considered as pathogens. Furthermore, bacteriological findings provide only evidence for infection but not for inflammation. For some bacteria, particularly Escherichia coli and Trueperella pyogenes pathogenic mechanism resulting in metritis and endometritis have been elucidated in detail. The role of bacteria that can be regarded as opportunistic or potential pathogens, e.g. Bacillus pumilus, is still under investigation. The understanding of the uterine microbiota and its interactions is increasing with the use of modern high-resolution techniques such as Fouriertransform infrared spectroscopy. Endometrial cytology provides additional information about alterations in the endometrium. Knowledge of innate uterine defense mechanism in cattle has increased a lot in the recent past. It can be speculated that improving or modulating uterine defense mechanism will be part of future prevention and treatment approaches beyond the use of antimicrobials. In this context, cellular and molecular defense mechanisms have been in the focus of interest, e.g. the role of interleukins or mucins. This review gives a short overview on some aspects of recent research on uterine diseases in cattle.(AU)

Immune response of sheep naturally infected with Haemonchus spp. on pastures with two different nutritional conditions / Resposta imune de ovinos naturalmente infectados com Haemonchus spp. em pastagens com duas condições nutricionais diferentes

Parasitic nematodes in the gastrointestinal tract of small ruminants are considered one of the main causes of economical and productive losses. Haemonchus contortus is characterized as the major parasite affecting sheep worldwide. This study was designed to evaluate immunological responses in sheep naturally infected with Haemonchus spp. in two different feed grounds: native and cultivated pasture. Fecal and blood samples were collected weekly from fifteen Corriedale sheep, ages 8 to 12 months, to determine the counting of eggs per gram of feces (EPG), and the concentration of haematocrit, eosinophils, and cytokines during an 84-day experiment. Bromatological analysis of the two pasture treatments (types) showed levels of crude protein and non-fibrous carbohydrates of 9.7% and 12.3% in the native pasture and 14.3% and 23.7% for the cultivated pasture, respectively. No significant difference in haematocrit concentration was found during the experiment between different pastures (p 0.05). However, significant differences were found in EPG, eosinophils, interleukins, and immunoglobulins levels after 21 days in the cultivated (nutritionally-enhanced) pasture. These data suggest that immune response was characterized in sheep by significantly higher peripheral eosinophilia and an increase in serum concentrations of IgE, IgA, IgG, TNF-?, IFN-?, and IL-6. Likewise, the improvement in forage condition resulted in a reduction of EPG and an increase of elements that improve a sheeps immune system response against Haemonchus spp. infection.(AU)

Parasitas nematódeos do trato gastrointestinal de pequenos ruminantes são considerados uma das principais causas de perdas econômicas e produtivas. Haemonchus contortus é caracterizado como o parasita mais importante afetando ovinos em todo o mundo. Este estudo foi delineado para avaliar a resposta imunológica de ovinos naturalmente infectados com Haemonchus spp. em dois grupos alimentares diferentes: pastagem nativa e cultivada. Amostras de fezes e de sangue foram coletadas semanalmente de quinze ovinos Corriedale, com de 8 a 12 meses de idade, para determinar a contagem de ovos por grama de fezes (OPG), e a concentração do hematócrito, eosinófilos e citocinas durante 84 dias de experimento. Análises bromatológicas dos dois tratamentos de pastagens (tipos) demonstraram níveis de proteína bruta e carboidratos não-fibrosos de 9.7% e 12.3% na pastagem nativa e 14.3% e 23.7% para pastagem cultivada, respectivamente. Não foi encontrada diferença significativa na concentração do hematócrito durante o experimento entre as diferentes pastagens (p 0,05). Entretanto, foram encontradas diferenças significativas no OPG, eosinófilos, interleucinas e níveis de imunoglobulinas depois de 21 na pastagem cultivada (melhorada nutricionalmente). Estes dados sugerem que a resposta imune em ovinos foi caracterizada por significativa elevação da eosinofilia periférica e aumento das concentrações séricas de IgE, IgA, IgG, TNF-β, IFN-γ, e IL-6. Do mesmo modo, a melhoria nas condições de forragem resultou na redução do OPG e aumento dos elementos que melhoram a respostado sistema imune de ovinos contra infecção pelo Haemonchus spp. (AU)

Postpartum uterine infection and endometritis in dairy cattle

After parturition, uterine involution, regeneration of the endometrium, return of ovarian cyclic activity, and the control of pathogenic bacteria in the uterus is required before cows are likely to conceive again. However, pathogenic bacteria often cause uterine disease in modern dairy cattle, leading to decreased productivity and reduced fertility. This review aims to provide an overview of postpartum uterine infection and disease in dairy cattle. Metritis and endometritis are the main postpartum clinical conditions; although, subclinical endometritis is an emerging issue. Postpartum uterine disease is associated with the isolation of Escherichia coli, Trueperella pyogenes, and anaerobic pathogenic bacteria. Sensing of bacteria or their pathogen-associated molecules, such as lipopolysaccharide, by the innate immune system generates inflammatory responses. Endometrial inflammation includes increased expression of complement, calgranulins, interleukins and acute phase proteins, as well as the chemotaxis of neutrophils and macrophages to the site of infection. Uterine disease is also characterised by tissue damage, including endometrial cytolysis caused by the cholesterol-dependent cytolysin, pyolysin. The responses to pathogens are energetically expensive, and depletion of the key cellular nutrients, glucose or glutamine, impairs inflammatory responses by endometrial tissues. For sustainable intensification of the dairy industry over the next 50 years, it is vital to understand why high-milk-yield cows are so susceptible to uterine pathology and develop new ways to prevent uterine disease.

Postpartum uterine infection and endometritis in dairy cattle

After parturition, uterine involution, regeneration of the endometrium, return of ovarian cyclic activity, and the control of pathogenic bacteria in the uterus is required before cows are likely to conceive again. However, pathogenic bacteria often cause uterine disease in modern dairy cattle, leading to decreased productivity and reduced fertility. This review aims to provide an overview of postpartum uterine infection and disease in dairy cattle. Metritis and endometritis are the main postpartum clinical conditions; although, subclinical endometritis is an emerging issue. Postpartum uterine disease is associated with the isolation of Escherichia coli, Trueperella pyogenes, and anaerobic pathogenic bacteria. Sensing of bacteria or their pathogen-associated molecules, such as lipopolysaccharide, by the innate immune system generates inflammatory responses. Endometrial inflammation includes increased expression of complement, calgranulins, interleukins and acute phase proteins, as well as the chemotaxis of neutrophils and macrophages to the site of infection. Uterine disease is also characterised by tissue damage, including endometrial cytolysis caused by the cholesterol-dependent cytolysin, pyolysin. The responses to pathogens are energetically expensive, and depletion of the key cellular nutrients, glucose or glutamine, impairs inflammatory responses by endometrial tissues. For sustainable intensification of the dairy industry over the next 50 years, it is vital to understand why high-milk-yield cows are so susceptible to uterine pathology and develop new ways to prevent uterine disease.(AU)

Post-ride inflammatory markers in endurance horses / Marcadores inflamatórios em cavalos após enduro

The acute phase reaction occurs in response to diseases, surgical trauma and can happen after strenuous exercise, like long distance events. This study aimed to evaluate the Serum Amyloid A (SAA), pro-inflammatory interleukins (IL-1β and TNFα) and creatine phosphokinase (CK) after free speed races like physical stress markers in endurance horses. Blood of 21 trained horses that competed in tests of 80, 120 and 160km, was taken before and after race. There was a significant increase in CK and SAA after the race in all horses, as well as the anti-inflammatory interleukin IL-10. But when results were compared between the groups, only the SAA was different, being higher in 160km horses. There was no change in pro-inflammatory interleukins IL-1β and TNFα before and after the event; however, the TNFα was higher in 160km horses after the race. Based on data obtained in this study it was concluded that stress is more dependent on the distance than speed imposed and that SAA is a good marker of physical effort, since it is accompanied by other markers well established as CK. Interleukins are not a good markers since they did not change significantly.(AU)

A reação de fase aguda da inflamação ocorre como resposta a doenças, traumas cirúrgicos e pode acontecer após exercício físico extenuante, como provas de longa distância. O presente estudo teve por objetivo avaliar o Amiloide A Sérico (SAA), interleucinas pró-inflamatórias e a enzima creatinafosfoquinase (CK) após provas de velocidade livre como marcadores de esforço físico em cavalos de enduro. Utilizou-se sangue de 21 cavalos devidamente treinados, que competiram em provas de 80, 120 e 160km, colhido antes e depois da prova. Houve aumento significativo em CK e SAA após a prova em todos os cavalos, assim como da interleucina anti-inflamatória IL-10. Porém, quando os resultados foram comparados entre os grupos, somente a SAA apresentou diferença, sendo maior nos cavalos de 160km. Não foi observada alteração nas interleucinas pró-inflamatórias IL-1β e TNFα antes e depois da prova, todavia, o TNFα foi maior nos cavalos de 160km após a prova. Baseado nos dados obtidos neste estudo, conclui-se que o esforço é dependente da distância percorrida e não da velocidade imposta e que a SAA é um bom marcador de esforço físico, desde que acompanhada de outros marcadores já consagrados, como a CK. As interleucinas não se apresentaram bons marcadores, já que não sofreram alterações significativas.(AU)

Influência do esforço físico sobre o grau de inflamação no sistema respiratório de equinos atletas

O sistema respiratório apresenta a segunda maior prevalência de lesões responsáveis por queda de rendimento em equinos atletas e é considerado o fator limitante de performance dos animais hígidos nas provas de alto rendimento. Essas informações apontam para a alta exigência e estresse sobre as vias aéreas de cavalos que participam de competições. Hipotetizou-se que o esforço físico realizado em modalidades esportivas provoca resposta inflamatória nas vias aéreas de equinos atletas. Portanto, esse trabalho tem como objetivo avaliar marcadores da inflamação nas vias aéreas de equinos atletas saudáveis frente ao esforço físico realizado nas modalidades esportivas de Três Tambores e Marcha. Foram utilizados dezesseis equinos saudáveis e sem histórico de afecções respiratórias, das raças Mangalarga Marchador e Quarto-de-milha, distribuídos uniformemente entre as modalidades de Marcha (MA) e Três Tambores (TT), respectivamente. Todos os animais foram submetidos a uma simulação de prova da modalidade em que participam, sendo realizada uma avaliação do sistema respiratório prévia (M0) e outra 24 horas após (M1) a realização do exercício. Em ambos os momentos foram realizados exame físico, endoscopia das vias aéreas e lavado traqueal, a partir do qual foi realizada a citologia e quantificação das interleucinas IL-4, IL-10, IL-17, IFN-a e IFN-y. Não foi observada variação nos parâmetros do exame físico nos dois momentos. Já na avaliação endoscópica, foi constatado um aumento significativo no escore de acúmulo de muco na traqueia após o exercício em ambos os grupos (p<0.001). Em dois animais do grupo TT foi observada a presença de sangue na traqueia no momento M1 (p<0,07). Na citologia do lavado traqueal observou-se um aumento significativo no número de neutrófilos em ambos os grupos após o exercício (p<0,001), mas que ocorreu de maneira mais acentuada nos animais de marcha (p=0,01). Também observou-se aumento significativo no número de hemossiderófagos no momento M1 (p<0,01), que nesse caso, foi mais intenso no grupo de Três Tambores (p=0,008). Dentre as Interleucinas avaliadas, o principal resultado observado foi o aumento na concentração de IL-17 em ambos os grupos, mas com significância estatística apenas no grupo de Marcha (p=0,02). Esses resultados apontam em concordância com a hipótese, permitindo a conclusão de que o exercício gera resposta inflamatória nas vias aéreas de equinos.

The respiratory system has the second highest prevalence of injuries responsible for decreased performance in equine athletes and it is considered a limiting factor for the performance of healthy animals in high performance tests. This information points to the high airways demand and stress of athetic horses. It was hypothesized that the physical effort performed in competition causes an inflammatory response in equine athletes airways. Therefore, the aim of this study was to evaluate markers of respiratory inflammation of healthy equine athletes under physical effort performed in Barrel Racing and Marcha modalities. Sixteen healthy horses of the Mangalarga Marchador and Quarter-horses breeds with no history of respiratory disorders were used and evenly distributed between the Marcha (MA) and Barrel Racing (TT) modalities, respectively. All animals were submitted to a simulation of the modality they participate, with a previous respiratory system examination (M0) and another 24 hours after (M1) the exercise performance. At both sampling times, physical examination, airways endoscopy and tracheal wash were performed, cytology and interleukins IL-4, IL-10, IL-17, IFN-a and IFN-y quantification were done. There was no parameters variation of the physical examination at both sampling times. In the endoscopic evaluation, there was a significant increase in the tracheal mucus accumulation score after exercise in both groups (p <0.001). In two horses of TT group, blood was observed in the trachea at M1 (p <0.07). In cytology tracheal wash there was a significant increase in the number of neutrophils in both groups after exercise (p <0.001), but Marcha horses had the highest result (p = 0.01). There was also a significant increase in the number of hemosiderophages at M1 (p <0.01), which in this case it was more intense in the Barrels Racing group (p = 0.008). The main result among the interleukins evaluated, the expressive increase observed was of the IL-17 concentration in both groups, but only the Marcha group had statistical significance (p = 0.02). These results agrees with the present hypothesis, which leads to the conclusion that exercise generates an inflammatory response in horse airways.