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.

Effect of Bacillus Subtilis on Immune Function of Hd11 Chicken Macrophages

Poultry is frequently contaminated by Salmonella, a pathogen leading to human health concern worldwide. This study aimed to evaluate the effect of Bacillus subtilis (BS)strain 048 (BS048) on the activation, phagocytosis, sterilization, cytokine secretion, and nitrogen oxide synthesis of HD11 chicken macrophages subjected to Salmonella enteritidis challenge, using lipopolysaccharide treatment as a negative control. The results showed: (1) BS048 had no significant effect on extracellular lactate dehydrogenase activity (p>0.05), while lipopolysaccharide treatment significantly increased extracellular lactate dehydrogenase activity (p >0.05), while lipopolysaccharide treatment significantly increased extracellular lactate dehydrogenase activity (p 0.05);(2)BS048 significantly upregulated the expression levels of pro-inflammatory cytokines (interleukin (IL)- 1â and IL-6), anti-inflammatory cytokines(IL-10 and transforming growth factor-â1), and anti-viral cytokine, interferon-â (p<0.01); ; (3) BS048 significantly upregulated the mRNA expression level of the inducible nitric oxide synthase and its activity as well as extracellular nitrogen oxide level (p <0.01). In conclusion, BS048 could improve antiinflammatory and immune functions of HD11 chicken macrophages, without cytotoxic effects on these cells.(AU)

Effect of flunixin or ketoprofen in caudectomy by elastration in lambs: pain and neutrophil function

ABSTRACT: Painful procedures can affect the function of innate immune cells, such as neutrophils and macrophages, increasing the risk of infectious diseases. The present work aimed to verify if the analgesics flunixin meglumine or ketoprofen can attenuate the pain/discomfort of newborn lambs submitted by elastration tail docking and thereby avoid the impairment of blood granulocytes function. Twenty-one neonate lambs were divided into three treatments: the control group (n=7), not subjected to caudectomy; the flunixin group (n=7), subjected to caudectomy under local anesthesia and analgesia with two doses of flunixin meglumine; and the ketoprofen group (n=7), subjected to caudectomy under local anesthesia and two doses of ketoprofen. Pain indicators were observed by pain posture score (PS), the number of vocalizations (V), frequency of the movement of the ears (EF), and respiratory rates (RR), observed by a 10 minutes videos for each time points: -15min, 6h, 48h, and 144h. At the same time points, the reactive oxygen species (ROS) production and phagocytosis of blood granulocytes were measured by flow cytometry. At 6h after caudectomy, there was a pain indicator increase (RR, V, and PS), a blood granulocyte percentage increase, and a granulocytes phagocytosis reduction for both groups. At 48h, the ketoprofen group spend more time in pain posture and, at 144h, they exhibited a ROS production granulocyte reduction without signs of pain. We conclude the flunixin meglumine and ketoprofen did not prevent the acute pain/discomfort caused by caudectomy, because the groups showed a pain behavior and impaired of the innate immune response however, the flunixin meglumine was effective in controlling the chronic pain and their effects on blood granulocytes function in compare ketoprofen.

RESUMO: Procedimentos dolorosos podem afetar a função das células imunes inatas como neutrófilos e macrófagos, aumentando o risco de ocorrer doenças infeciosas. Desta maneira, o presente trabalho pretendeu verificar se os analgésicos flunixin meglumine ou cetoprofeno conseguem atenuar a dor/desconforto de cordeiros neonatos submetidos a caudectomia por elastração, evitando assim o comprometimento da função dos granulócitos sanguíneos. Para tanto 21 cordeiras foram aleatoriamente divididas em três tratamentos: grupo controle (n=7) não submetido a caudectomia, grupo flunixin (n=7) submetido a caudectomia precedida por anestesia local e duas doses de flunixin meglumine, e cetoprofeno (n=7) submetido a caudectomia precedida por anestesia local e duas doses de cetoprofeno. Mensurou-se os indicadores de dor/desconforto após caudectomia por observações de escore de postura de dor (EP), número de vocalizações (V), frequência de movimentar as orelhas (FO) e frequência respiratória (FR), observados em vídeos de 10 minutos nos momentos -15 min e 6, 48 e 144h. Nos mesmos momentos, avaliou-se a as funções de produção de espécies reativas de oxigênio (ERO) e de fagocitose por granulócitos sanguineos em citometria de fluxo. Notou-se aumento dos indicadores de dor (FR, de V e de EP), da porcentagem dos granulócitos sanguíneos e redução da eficiência de fagocitose em ambos os grupos as 6h. As 48h, os animais do grupo cetoprofeno ainda apresentava mais tempo em postura de dor que os demais grupos e as 144h, apresentou redução da produção ERO por granulócitos. Tais achados permitem concluir que tanto o flunexin meglumine como o cetoprofeno não preveniram dor/desconforto agudo promovido pela elastração, pois os dois grupos manifestaram comportamento de dor e redução da resposta imune inata. Ainda, o analgésico flunixin meglumine foi efetivo em controlar a dor mais tardia e seus efeitos na função de granulócitos sanguíneos em comparação ao cetoprofeno.

Effect of flunixin or ketoprofen in caudectomy by elastration in lambs: pain and neutrophil function / Efeito de flunixin ou cetoprofeno em cordeiras submetidas a caudectomia por elastração: dor e função neutrofílica

Painful procedures can affect the function of innate immune cells, such as neutrophils and macrophages, increasing the risk of infectious diseases. The present work aimed to verify if the analgesics flunixin meglumine or ketoprofen can attenuate the pain/discomfort of newborn lambs submitted by elastration tail docking and thereby avoid the impairment of blood granulocytes function. Twenty-one neonate lambs were divided into three treatments: the control group (n=7), not subjected to caudectomy; the flunixin group (n=7), subjected to caudectomy under local anesthesia and analgesia with two doses of flunixin meglumine; and the ketoprofen group (n=7), subjected to caudectomy under local anesthesia and two doses of ketoprofen. Pain indicators were observed by pain posture score (PS), the number of vocalizations (V), frequency of the movement of the ears (EF), and respiratory rates (RR), observed by a 10 minutes videos for each time points: -15min, 6h, 48h, and 144h. At the same time points, the reactive oxygen species (ROS) production and phagocytosis of blood granulocytes were measured by flow cytometry. At 6h after caudectomy, there was a pain indicator increase (RR, V, and PS), a blood granulocyte percentage increase, and a granulocytes phagocytosis reduction for both groups. At 48h, the ketoprofen group spend more time in pain posture and, at 144h, they exhibited a ROS production granulocyte reduction without signs of pain. We conclude the flunixin meglumine and ketoprofen did not prevent the acute pain/discomfort caused by caudectomy, because the groups showed a pain behavior and impaired of the innate immune response however, the flunixin meglumine was effective in controlling the chronic pain and their effects on blood granulocytes function in compare ketoprofen.(AU)

Procedimentos dolorosos podem afetar a função das células imunes inatas como neutrófilos e macrófagos, aumentando o risco de ocorrer doenças infeciosas. Desta maneira, o presente trabalho pretendeu verificar se os analgésicos flunixin meglumine ou cetoprofeno conseguem atenuar a dor/desconforto de cordeiros neonatos submetidos a caudectomia por elastração, evitando assim o comprometimento da função dos granulócitos sanguíneos. Para tanto 21 cordeiras foram aleatoriamente divididas em três tratamentos: grupo controle (n=7) não submetido a caudectomia, grupo flunixin (n=7) submetido a caudectomia precedida por anestesia local e duas doses de flunixin meglumine, e cetoprofeno (n=7) submetido a caudectomia precedida por anestesia local e duas doses de cetoprofeno. Mensurou-se os indicadores de dor/desconforto após caudectomia por observações de escore de postura de dor (EP), número de vocalizações (V), frequência de movimentar as orelhas (FO) e frequência respiratória (FR), observados em vídeos de 10 minutos nos momentos -15 min e 6, 48 e 144h. Nos mesmos momentos, avaliou-se a as funções de produção de espécies reativas de oxigênio (ERO) e de fagocitose por granulócitos sanguineos em citometria de fluxo. Notou-se aumento dos indicadores de dor (FR, de V e de EP), da porcentagem dos granulócitos sanguíneos e redução da eficiência de fagocitose em ambos os grupos as 6h. As 48h, os animais do grupo cetoprofeno ainda apresentava mais tempo em postura de dor que os demais grupos e as 144h, apresentou redução da produção ERO por granulócitos. Tais achados permitem concluir que tanto o flunexin meglumine como o cetoprofeno não preveniram dor/desconforto agudo promovido pela elastração, pois os dois grupos manifestaram comportamento de dor e redução da resposta imune inata. Ainda, o analgésico flunixin meglumine foi efetivo em controlar a dor mais tardia e seus efeitos na função de granulócitos sanguíneos em comparação ao cetoprofeno.(AU)

Effect of flunixin or ketoprofen in caudectomy by elastration in lambs: pain and neutrophil function / Efeito de flunixin ou cetoprofeno em cordeiras submetidas a caudectomia por elastração: dor e função neutrofílica

Painful procedures can affect the function of innate immune cells, such as neutrophils and macrophages, increasing the risk of infectious diseases. The present work aimed to verify if the analgesics flunixin meglumine or ketoprofen can attenuate the pain/discomfort of newborn lambs submitted by elastration tail docking and thereby avoid the impairment of blood granulocytes function. Twenty-one neonate lambs were divided into three treatments: the control group (n=7), not subjected to caudectomy; the flunixin group (n=7), subjected to caudectomy under local anesthesia and analgesia with two doses of flunixin meglumine; and the ketoprofen group (n=7), subjected to caudectomy under local anesthesia and two doses of ketoprofen. Pain indicators were observed by pain posture score (PS), the number of vocalizations (V), frequency of the movement of the ears (EF), and respiratory rates (RR), observed by a 10 minutes videos for each time points: -15min, 6h, 48h, and 144h. At the same time points, the reactive oxygen species (ROS) production and phagocytosis of blood granulocytes were measured by flow cytometry. At 6h after caudectomy, there was a pain indicator increase (RR, V, and PS), a blood granulocyte percentage increase, and a granulocytes phagocytosis reduction for both groups. At 48h, the ketoprofen group spend more time in pain posture and, at 144h, they exhibited a ROS production granulocyte reduction without signs of pain. We conclude the flunixin meglumine and ketoprofen did not prevent the acute pain/discomfort caused by caudectomy, because the groups showed a pain behavior and impaired of the innate immune response however, the flunixin meglumine was effective in controlling the chronic pain and their effects on blood granulocytes function in compare ketoprofen.(AU)

Procedimentos dolorosos podem afetar a função das células imunes inatas como neutrófilos e macrófagos, aumentando o risco de ocorrer doenças infeciosas. Desta maneira, o presente trabalho pretendeu verificar se os analgésicos flunixin meglumine ou cetoprofeno conseguem atenuar a dor/desconforto de cordeiros neonatos submetidos a caudectomia por elastração, evitando assim o comprometimento da função dos granulócitos sanguíneos. Para tanto 21 cordeiras foram aleatoriamente divididas em três tratamentos: grupo controle (n=7) não submetido a caudectomia, grupo flunixin (n=7) submetido a caudectomia precedida por anestesia local e duas doses de flunixin meglumine, e cetoprofeno (n=7) submetido a caudectomia precedida por anestesia local e duas doses de cetoprofeno. Mensurou-se os indicadores de dor/desconforto após caudectomia por observações de escore de postura de dor (EP), número de vocalizações (V), frequência de movimentar as orelhas (FO) e frequência respiratória (FR), observados em vídeos de 10 minutos nos momentos -15 min e 6, 48 e 144h. Nos mesmos momentos, avaliou-se a as funções de produção de espécies reativas de oxigênio (ERO) e de fagocitose por granulócitos sanguineos em citometria de fluxo. Notou-se aumento dos indicadores de dor (FR, de V e de EP), da porcentagem dos granulócitos sanguíneos e redução da eficiência de fagocitose em ambos os grupos as 6h. As 48h, os animais do grupo cetoprofeno ainda apresentava mais tempo em postura de dor que os demais grupos e as 144h, apresentou redução da produção ERO por granulócitos. Tais achados permitem concluir que tanto o flunexin meglumine como o cetoprofeno não preveniram dor/desconforto agudo promovido pela elastração, pois os dois grupos manifestaram comportamento de dor e redução da resposta imune inata. Ainda, o analgésico flunixin meglumine foi efetivo em controlar a dor mais tardia e seus efeitos na função de granulócitos sanguíneos em comparação ao cetoprofeno.(AU)

Once upon a time, inflammation

Inflammation has accompanied humans since their first ancestors appeared on Earth. Aulus Cornelius Celsus (25 BC-50 AD), a Roman encyclopedist, offered a still valid statement about inflammation: "Notae vero inflammationis sunt quatuor: rubor et tumor cum calore and dolore", defining the four cardinal signs of inflammation as redness and swelling with heat and pain. While inflammation has long been considered as a morbid phenomenon, John Hunter (18th century) and Elie Metchnikoff (19th century) understood that it was a natural and beneficial event that aims to address a sterile or an infectious insult. Many other famous scientists and some forgotten ones have identified the different cellular and molecular players, and deciphered the different mechanisms of inflammation. This review pays tribute to some of the giants who made major contributions, from Hippocrates to the late 19th and first half of the 20th century. We particularly address the discoveries related to phagocytes, diapedesis, chemotactism, and fever. We also mention the findings of the various inflammatory mediators and the different approaches designed to treat inflammatory disorders.(AU)

Primary culture macrophages from fish as potential experimental model in toxicity studies with multiwalled carbon nanotubes

Multiwalled carbon nanotube (MWCNT) has been broadly used in several sectors of society. This material when exposed to the environment might reach the aquatic animals and cause toxic effects. Here, it was evaluated the MWCNTs toxicity in melanomacrophages primary culture that was submitted to 1 µ gm L-1 MWCNTs for 24 hours. After exposition to MWCNT, 48 and 59% liver and spleen melanomacrophages were healthy, respectively. The control group presented 85% viability. Phagocytosis activity of melanomacrophages was observed by presence of black inclusions in cytoplasm. The findings indicate MWCNT was cytotoxic to melanomacrophages, where its release and effect into aquatic environment must be more studied. Finally, the melanomacrophages present large potential as experimental model for evaluation of carbon-based nanomaterial toxicity.

Primary culture macrophages from fish as potential experimental model in toxicity studies with multiwalled carbon nanotubes

Multiwalled carbon nanotube (MWCNT) has been broadly used in several sectors of society. This material when exposed to the environment might reach the aquatic animals and cause toxic effects. Here, it was evaluated the MWCNTs toxicity in melanomacrophages primary culture that was submitted to 1 µ gm L-1 MWCNTs for 24 hours. After exposition to MWCNT, 48 and 59% liver and spleen melanomacrophages were healthy, respectively. The control group presented 85% viability. Phagocytosis activity of melanomacrophages was observed by presence of black inclusions in cytoplasm. The findings indicate MWCNT was cytotoxic to melanomacrophages, where its release and effect into aquatic environment must be more studied. Finally, the melanomacrophages present large potential as experimental model for evaluation of carbon-based nanomaterial toxicity.(AU)

Once upon a time, inflammation

Inflammation has accompanied humans since their first ancestors appeared on Earth. Aulus Cornelius Celsus (25 BC-50 AD), a Roman encyclopedist, offered a still valid statement about inflammation: "Notae vero inflammationis sunt quatuor: rubor et tumor cum calore and dolore", defining the four cardinal signs of inflammation as redness and swelling with heat and pain. While inflammation has long been considered as a morbid phenomenon, John Hunter (18th century) and Elie Metchnikoff (19th century) understood that it was a natural and beneficial event that aims to address a sterile or an infectious insult. Many other famous scientists and some forgotten ones have identified the different cellular and molecular players, and deciphered the different mechanisms of inflammation. This review pays tribute to some of the giants who made major contributions, from Hippocrates to the late 19th and first half of the 20th century. We particularly address the discoveries related to phagocytes, diapedesis, chemotactism, and fever. We also mention the findings of the various inflammatory mediators and the different approaches designed to treat inflammatory disorders.(AU)

Salinomycin intoxication in pigs associated with the use of tiamulin in the State of Santa Catarina, Brazil / Intoxicação por salinomicina em suínos associado ao uso de tiamulina no Estado de Santa Catarina

This paper describes a spontaneous outbreak of toxic myopathy in finishing pigs due to the ingestion of feed with salinomycin associated with tiamulin and addresses the control methods performed during the outbreak to minimize economic losses resulting from the intoxication. A lot of 940 pigs fed feed containing 30 ppm salinomycin received 100 ppm tiamulin (via water) to control recurrent respiratory diseases on the farm. After ingesting tiamulin, some animals manifested clinical signs of motor incoordination, stiff gait, reluctance to move, muscle weakness and tremors, dyspnea, depression, and decubitus, remaining in "sitting-dog position" or with the abduction of the pelvic limbs, and rested on tip-toes when in a standing position. Two animals were euthanized for macro-and microscopic evaluation. The other sick animals received supportive anti-inflammatory treatment. The most relevant macroscopic finding observed during necropsy was the slight pallor of the pelvic limb muscles. The main histopathological findings consisted of multifocal areas of hyaline degeneration and marked necrosis of skeletal myofibers, with macrophage infiltrate associated with cell regeneration and skeletal fiber phagocytosis. These lesions were more intense in the longissimus dorsi, diaphragm, and masseter muscles. The definitive diagnosis was based on epidemiological aspects and clinical lesional conditions compatible with toxic myopathy secondary to ionophore intoxication.(AU)

Este trabalho descreve um surto espontâneo de miopatia tóxica em suínos na fase de terminação, decorrente da ingestão de ração contendo salinomicina associada a tiamulina e aborda os métodos de controle realizados durante o surto, para minimizar os prejuízos econômicos decorrentes da intoxicação. Um lote de 940 suínos alimentados com ração contendo 30 ppm de salinomicina recebeu tiamulina na dose de 100 ppm (via água) para controle de doenças respiratórias recorrentes na granja. Após a ingestão da tiamulina, alguns animais manifestaram sinais clínicos de incoordenação motora, andar rígido, relutância ao movimento, fraqueza e tremores musculares, dispneia, depressão e decúbito, permanecendo em "posição de cão sentado" ou com abdução dos membros pélvicos, e quando em estação, apoiavam-se nas pinças dos cascos. Dois animais foram eutanasiados para avaliação macro e microscópica. Os demais animais doentes receberam tratamento suporte com anti-inflamatório. O achado macroscópico mais relevante observado durante a necropsia foi palidez leve dos músculos dos membros pélvicos. Os principais achados histopatológicos foram áreas multifocais de degeneração hialina e necrose acentuada de miofibras esqueléticas, com infiltrado de macrófagos associado às figuras de regeneração celular e à fagocitose de fibras esqueléticas. Essas lesões foram mais intensas nos músculos longissimus dorsi, diafragma e masseter. O diagnóstico definitivo baseou-se nos aspectos epidemiológicos e no quadro clínico-lesional compatíveis com miopatia tóxica secundária a intoxicação por ionóforo.(AU)

Innate immunity in chicken lines developed by EMBRAPA Suínos e Aves: antimicrobial activity of macrophages and serum

The objective of this study was to compare the antimicrobial activity of macrophages and serum in laying hen (MM, CC, and CCc) and broiler chicken lineages (TT and LL). Macrophages were evaluated for phagocytic and antimicrobial activity. Microbicidal serum activity was evaluated by the resistance test for serum and the agar test. The results showed that phagocytic activity was higher in males of the MM strain, with 13% of macrophages presenting phagocytosis, while the other lineages studied, and even female MM, presented a rate of 6% of phagocytic cells. However, antimicrobial activity in macrophages from males of CCc lineage and females of TT lineage were higher, eliminating more than 30% of the Salmonella enterica inoculum, while in the other strains, the results were similar, with inoculum reduction below 30%. In the serum resistance assay, female laying lines presented higher antibacterial activity than female broiler lines. In the trials to evaluate the microbicide activity of the serum, females of both broiler and laying lineages presented higher performance when compared with males of the same lineage. Females of laying hen lines (MM and CC) present a greater antibacterium activity than males. These results can contribute to a better understanding of the immune response in broiler chicken and laying hen lineages, to aid development of lineages of birds more resistant to pathogens.

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.

Peritoneal cryptococcosis in a dog

Background: Cryptococcosis is an opportunistic, systemic mycotic disease caused by yeasts of genus Cryptococcus.Cryptococcus neoformans might cause serious public health problems, as they can be transmitted to humans, domesticand wild animals. In cats and dogs, the main site of infection is the upper lower respiratory tract, however, the infectioncan extend and affect other sites in the organism, however, the peritoneal manifestation of the disease is rare. Thus, thepresent work aims to report a case of cryptococcosis in a canine, mixed breed, female, adult, from the Metropolitan Regionof Belém, PA, Brazil.Case: A mixed breed dog, female, adult was admitted in a veterinary hospital at Belém, presenting abdominal discomfortand, after the clinical examination, emergency exploratory laparotomy was indicated, and it were observed disseminatedlesions in the abdominal cavity, with multiple nodules attached to the peritoneum and intestinal serosa and bladder, uterus,among other organs. Fragments from the surgical biopsy were fixed, and processed routinely according to the techniquesfor histological tissue processing. Histological examination revealed a predominance of granulomatous lesions in peritoneum and mesentery organs. It was observed a discrete inflammatory reaction of macrophages, epithelioid cells andgiant cells, many in phagocytosis of spherical or ovoid organisms, with a thick capsule and a clear perinuclear halo. Inthe PAS staining, the Cryptococcus conidia presented eosinophilic characteristics and some of them had the capsule welldemarcated, which was shown to be radiated. Also it was possible to see the budding yeast and in the Grocott stain, theCryptococcus stained in black.Discussion: Cryptococcosis with peritoneal involvement in animals is rarely reported in the literature. Cryptococcus is afungus that presents tropism by the...

Peritoneal cryptococcosis in a dog

Background: Cryptococcosis is an opportunistic, systemic mycotic disease caused by yeasts of genus Cryptococcus.Cryptococcus neoformans might cause serious public health problems, as they can be transmitted to humans, domesticand wild animals. In cats and dogs, the main site of infection is the upper lower respiratory tract, however, the infectioncan extend and affect other sites in the organism, however, the peritoneal manifestation of the disease is rare. Thus, thepresent work aims to report a case of cryptococcosis in a canine, mixed breed, female, adult, from the Metropolitan Regionof Belém, PA, Brazil.Case: A mixed breed dog, female, adult was admitted in a veterinary hospital at Belém, presenting abdominal discomfortand, after the clinical examination, emergency exploratory laparotomy was indicated, and it were observed disseminatedlesions in the abdominal cavity, with multiple nodules attached to the peritoneum and intestinal serosa and bladder, uterus,among other organs. Fragments from the surgical biopsy were fixed, and processed routinely according to the techniquesfor histological tissue processing. Histological examination revealed a predominance of granulomatous lesions in peritoneum and mesentery organs. It was observed a discrete inflammatory reaction of macrophages, epithelioid cells andgiant cells, many in phagocytosis of spherical or ovoid organisms, with a thick capsule and a clear perinuclear halo. Inthe PAS staining, the Cryptococcus conidia presented eosinophilic characteristics and some of them had the capsule welldemarcated, which was shown to be radiated. Also it was possible to see the budding yeast and in the Grocott stain, theCryptococcus stained in black.Discussion: Cryptococcosis with peritoneal involvement in animals is rarely reported in the literature. Cryptococcus is afungus that presents tropism by the...(AU)

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology

Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.(AU)

Apesar do potencial apresentado pela tecnologia de propagação mediada para a aquicultura e conservação de peixes Neotropicais, o pobre entendimento do sistema imune do hospedeiro pode resultar na rejeição e destruição do material do doador. Com isso, se fazem necessários o estudo e o desenvolvimento de métodos para análise tanto dos efeitos de drogas imunossupressoras quanto para a avaliação da imunocompatibilidade entre doadores e receptores. Logo, o presente estudo teve como objetivo aperfeiçoar um método para analisar a fagocitose in vivo em Astyanax altiparanae usando Saccharomyces cerevisiae marcado e avaliar seu perfil hematológico resultante da indução inflamatória. Para isso, S. cerevisiae foram marcados com vermelho Congo e injetados na cavidade celomática dos A. altiparanae na concentração de 2,5 x 106 células.mL-1. Peixes injetados com PBS e peixes não injetados foram mantidos como controle. Sangue foi colhido e a capacidade fagocítica e o índice fagocítico foram determinados após 1, 2, 3 e 6 horas após à injeção (hpi). A injeção de levedura estimulou a fagocitose com sucesso, com o melhor resultado atingido após 2 hpi. Ainda, foi observada uma alta rastreabilidade das leveduras fagocitadas e não fagocitadas sob microscopia óptica devido à marcação com vermelho Congo. O perfil hematológico foi similar ao observado usualmente em infecções recém-induzidas, indicando migração de linfócitos ao sítio inflamatório e aumento no número de fagócitos circulantes devido à resposta natural ao estímulo inflamatório. Como conclusão, nosso método foi eficiente para analisar a fagocitose in vivo em A. altiparanae e será uma ferramenta importante para a avaliação de eficácia de drogas imunossopressoras para esta espécie. Em adição, estes resultados podem contribuir para futuros estudos em imunocompetência em peixes, tanto em âmbito laboratorial quanto a campo.(AU)

Avaliação da atividade funcional dos neutrófilos sanguíneos em ovelhas primíparas e pluríparas da raça Lacaune / [Functional evaluation of blood neutrophils in primiparous and pluriparous Lacaune ewes]

This study aimed to evaluate the neutrophil oxidative metabolism and phagocytosis of Zymonsan particles of primiparous and pluriparous Lacaune ewes during the first 30 days after lambing. A total of 20 ewes were evaluated, 10 primiparous (GPR) and 10 pluriparous (GPL). Evaluation of basal oxidative metabolism was performed using the nitroblue tetrazolium (NBT) technique, stimulating neutrophil phagocytosis with Zymosan particles. Blood samples were collected at parturition day (M1) and 1, 3, 7, 15 and 30 days after parturition, corresponding to M2, M3, M4, M5 and M6, respectively. In relation to the groups, GPR presented lower oxidative basal metabolism neutrophils in M1 compared to M4, in M3 with M1, M2, M4 and M5. In M4 and M5 differences were found at all times and in M6 with M4 and M5. Higher percentage of neutrophils than phagocytes were found in M4, M5 and M6 than in M1 and M2 in GPL animals. At all times GPR presented a lower percentage of phagocytosis than GPL. Thus it is concluded that the immune response in pluriparous sheep was more efficient than in primiparous sheep.(AU)

Influência do vírus da leucemia bovina sobre a atividade imunológica por meio da função neutrofílica / Influence of the bovine leukemia virus on the immunological activity by the neutrophilic function

Background: Bovine leukemia virus (VLB) is an oncogenic deltaretrovirus associated with the development of persistent lymphocytosis (LP) and lymphosarcomas in cattle. LP is characterized by chronic elevation of the number of circulating lymphocytes, in the case of B lymphocytes. Several studies have described functional changes in various leukocyte populations in both blood and milk in VLB-infected animals. The impact of some chronic diseases of low lethality is aggravated by the emergence of comorbidities.The objective of the present study was to evaluate the oxidative metabolism and neutrophil phagocytosis of bovines of the Holtein breed naturally infected with the bovine leukemia virus (VLB). Materials, Methods & Results: In this study, 20 cows were divided into three groups: (NG) seven non-seroreagent animals for VLB and without hematological alterations; (GAL) eight seroreagent animals for VLB and without hematological alterations; and (GLP) five seroreagent animals for VLB with persistent lymphocytosis (LP). The oxidative metabolism of neutrophils was determined by the tetrazolium nitroblast reduction test stimulated or not with Zymosan particles. The percentage of neutrophils that phagocytosed Zymosan particle (s) was also evaluated. The data were initially evaluated for normality and homoscedasticity by the Shapiro-Wilk test. Then the ANOVA test followed by the Student-Newman-Keuls test was applied for the comparison between the NG, GAL and GLP animals. Comparison between the NG animals and the seroreagent animals for the VLB (GVLB) was also performed through the unpaired Student’s t-test. The value of P < 0.05 was considered significant. No significant differences were observed in oxidative neutrophil metabolism in stimulated and non-stimulated samples with Zymosan particles nor in the percentage...

Marbofloxacin induces leishmanicidal activity and less inflammatory response in Leishmania chagasi infected macrophages / Marbofloxacina induz atividade leishmanicida e menos resposta inflamatória em macrófagos infectados por Leishmania chagasi / Marbofloxacino induce actividad leishmanicida y menor respuesta inflamatoria en macrófagos infectados con Leishmania chagasi

Marbofloxacin has promising leishmanicidal activity due to the direct action on the Leishmania chagasi amastigotes. It was developed only for veterinary medicine uses, and it could be used as an drug for the treatment of Canine Visceral Leishmaniasis (CVL).In the present study, we evaluated the leishmanicidal effect and macrophage modulation of marbofloxacin in macrophages infected with amastigotes of L. chagasi. Macrophages were collected from peritoneum of BALB/c mice and infected with promastigotes of L. chagasi. After internalization and transformation into amastigotes forms, cells were treated with marbofloxacinin concentration of100, 500 and 750 μg/mL for 18 hours. The leishmanicidal effect was evaluated by morphological spects of amastigotes inside of macrophages by phagocytosis assay and detection of death amastigotes and macrophages by TUNEL assay. Macrophage modulation was evaluated by release of cytotoxic metabolites and cytokine production. The results showed that L. chagasi-infected macrophages treated with the highest concentration of the drug showed lower amount of amastigotes into the macrophages (p<0,0632) than untreated infected cells. There was pronounced presence of cellular vacuoles in the treated infected-cells, and more apoptotic amastigotes in alive macrophages. It was observed decreased levels of H2O2, IL-1β, IL-6, and TNF-αdose-dependent of marbofloxacin in infected macrophages. The results indicate the leishmanicidal effect of marbofloxacin in infected macrophages and immunomodulation actions in these cells.

A marbofloxacina tem promissora atividade leishmanicida devido à ação direta sobre as amastigotas de Leishmania chagasi. Foi desenvolvido apenas para uso em medicina veterinária, podendo ser utilizado no tratamento da Leishmaniose Visceral Canina (LVC). No presente estudo, avaliamos o efeito leishmanicida e a modulação macrofágica da marbofloxacina em macrófagos infectados com amastigotas de L. chagasi. Os macrófagos foram coletados do peritônio de camundongos BALB/c infectados com promastigotas de L. chagasi. Após internalização e transformação em formas amastigotas, as células foram tratadas com marbofloxacina nas concentrações 100, 500 e 750μg / mL por 18 horas. O efeito leishmanicida foi avaliado pelos aspectos morfológicos de amastigotas dentro de macrófagos, por ensaio de fagocitose e detecção de amastigotas e macrófagos mortos por ensaio TUNEL. A modulação dos macrófagos foi avaliada pela liberação de metabólitos citotóxicos e produção de citocinas. Os resultados mostraram que macrófagos infectados com L. chagasi tratados com a maior concentração da droga apresentaram menor quantidade de amastigotas nos macrófagos (p <0,0632) do que células infectadas não tratadas. Houve a presença pronunciada de vacúolos celulares nas células infectadas tratadas, e mais amastigotas apoptóticas em macrófagos vivos. Observou-se diminuição dos níveis de H2O2, IL-1β, IL-6 e TNF-αdependente da dose de marbofloxacina em macrófagos infectados. Os resultados indicam o efeito leishmanicida da marbofloxacina em macrófagos infectados e ações imunomoduladoras nessas células.

El marbofloxacino tiene una actividad leishmanicida prometedora debido a la acción directa sobre amastigotes de Leishmania chagasi. Fue desarrollado solo para uso en medicina veterinaria y puede usarse en el tratamiento de la leishmaniasis visceral canina (LVC). En el presente estudio, evaluamos el efecto leishmanicida y la modulación de macrófagosde marbofloxacino en macrófagos infectados con amastigotes de L. chagasi. Se recogieron macrófagos del peritoneo de ratones BALB/c infectados con promastigotes de L. chagasi. Despuésde la internalizacion y transformacion en formas amastigotes, las células se trataron con marbofloxacina a concentraciones de 100, 500 y 750 μg / ml durante 18 horas. El efecto leishmanicida se evaluó mediante los aspectos morfológicosde amastigotes dentro de macrófagos, mediante ensayo de fagocitosis y detección de amastigotes y macrófagos muertos mediante ensayo TUNEL. La modulación de los macrófagos se evaluó mediante la liberación de metabolitos citotóxicos y la producción de citocinas. Los resultados mostraron que los macrófagos infectados con L. chagasi tratados con la concentración más altas del fármaco tenían una menor cantidad de amastigotes en los macrófagos (p <0,0632) que las células infectadas no tratadas. Hubo una presencia pronunciada de vacuolas celulares en las células infectadas tratadas y más amastigotes apoptóticas en macrófagos vivos. Se observó una disminuicón en los niveles de H2O2, IL-1β, IL-6 y TNF-α dependiendo de la dosis de marbofloxacino en macrófagos infectados. Los resultados indican el efecto leishmanicida de el marbofloxacino en macrófagos infectados y acciones inmunomoduladoras en estas células.

Marbofloxacin induces leishmanicidal activity and less inflammatory response in Leishmania chagasi infected macrophages / Marbofloxacina induz atividade leishmanicida e menos resposta inflamatória em macrófagos infectados por Leishmania chagasi / Marbofloxacino induce actividad leishmanicida y menor respuesta inflamatoria en macrófagos infectados con Leishmania chagasi

Marbofloxacin has promising leishmanicidal activity due to the direct action on the Leishmania chagasi amastigotes. It was developed only for veterinary medicine uses, and it could be used as an drug for the treatment of Canine Visceral Leishmaniasis (CVL).In the present study, we evaluated the leishmanicidal effect and macrophage modulation of marbofloxacin in macrophages infected with amastigotes of L. chagasi. Macrophages were collected from peritoneum of BALB/c mice and infected with promastigotes of L. chagasi. After internalization and transformation into amastigotes forms, cells were treated with marbofloxacinin concentration of100, 500 and 750 μg/mL for 18 hours. The leishmanicidal effect was evaluated by morphological spects of amastigotes inside of macrophages by phagocytosis assay and detection of death amastigotes and macrophages by TUNEL assay. Macrophage modulation was evaluated by release of cytotoxic metabolites and cytokine production. The results showed that L. chagasi-infected macrophages treated with the highest concentration of the drug showed lower amount of amastigotes into the macrophages (p<0,0632) than untreated infected cells. There was pronounced presence of cellular vacuoles in the treated infected-cells, and more apoptotic amastigotes in alive macrophages. It was observed decreased levels of H2O2, IL-1β, IL-6, and TNF-αdose-dependent of marbofloxacin in infected macrophages. The results indicate the leishmanicidal effect of marbofloxacin in infected macrophages and immunomodulation actions in these cells.(AU)

A marbofloxacina tem promissora atividade leishmanicida devido à ação direta sobre as amastigotas de Leishmania chagasi. Foi desenvolvido apenas para uso em medicina veterinária, podendo ser utilizado no tratamento da Leishmaniose Visceral Canina (LVC). No presente estudo, avaliamos o efeito leishmanicida e a modulação macrofágica da marbofloxacina em macrófagos infectados com amastigotas de L. chagasi. Os macrófagos foram coletados do peritônio de camundongos BALB/c infectados com promastigotas de L. chagasi. Após internalização e transformação em formas amastigotas, as células foram tratadas com marbofloxacina nas concentrações 100, 500 e 750μg / mL por 18 horas. O efeito leishmanicida foi avaliado pelos aspectos morfológicos de amastigotas dentro de macrófagos, por ensaio de fagocitose e detecção de amastigotas e macrófagos mortos por ensaio TUNEL. A modulação dos macrófagos foi avaliada pela liberação de metabólitos citotóxicos e produção de citocinas. Os resultados mostraram que macrófagos infectados com L. chagasi tratados com a maior concentração da droga apresentaram menor quantidade de amastigotas nos macrófagos (p <0,0632) do que células infectadas não tratadas. Houve a presença pronunciada de vacúolos celulares nas células infectadas tratadas, e mais amastigotas apoptóticas em macrófagos vivos. Observou-se diminuição dos níveis de H2O2, IL-1β, IL-6 e TNF-αdependente da dose de marbofloxacina em macrófagos infectados. Os resultados indicam o efeito leishmanicida da marbofloxacina em macrófagos infectados e ações imunomoduladoras nessas células.(AU)

El marbofloxacino tiene una actividad leishmanicida prometedora debido a la acción directa sobre amastigotes de Leishmania chagasi. Fue desarrollado solo para uso en medicina veterinaria y puede usarse en el tratamiento de la leishmaniasis visceral canina (LVC). En el presente estudio, evaluamos el efecto leishmanicida y la modulación de macrófagosde marbofloxacino en macrófagos infectados con amastigotes de L. chagasi. Se recogieron macrófagos del peritoneo de ratones BALB/c infectados con promastigotes de L. chagasi. Despuésde la internalizacion y transformacion en formas amastigotes, las células se trataron con marbofloxacina a concentraciones de 100, 500 y 750 μg / ml durante 18 horas. El efecto leishmanicida se evaluó mediante los aspectos morfológicosde amastigotes dentro de macrófagos, mediante ensayo de fagocitosis y detección de amastigotes y macrófagos muertos mediante ensayo TUNEL. La modulación de los macrófagos se evaluó mediante la liberación de metabolitos citotóxicos y la producción de citocinas. Los resultados mostraron que los macrófagos infectados con L. chagasi tratados con la concentración más altas del fármaco tenían una menor cantidad de amastigotes en los macrófagos (p <0,0632) que las células infectadas no tratadas. Hubo una presencia pronunciada de vacuolas celulares en las células infectadas tratadas y más amastigotes apoptóticas en macrófagos vivos. Se observó una disminuicón en los niveles de H2O2, IL-1β, IL-6 y TNF-α dependiendo de la dosis de marbofloxacino en macrófagos infectados. Los resultados indican el efecto leishmanicida de el marbofloxacino en macrófagos infectados y acciones inmunomoduladoras en estas células.(AU)

In vivo phagocytosis and hematology in Astyanax altiparanae, a potential model for surrogate technology

Abstract Although the potential of surrogate propagation technology for aquaculture and conservation of Neotropical fish, the poor understanding of the host immune system may results in rejection and destruction of the donor material. Thus, it is necessary to study and to develop methods to evaluate the effects of immunosuppressive drugs employment and to evaluate the immunocompatibility between donor and receptor. Thus, the present study aimed to optimize a methodology to assess in vivo phagocytosis in Astyanax altiparanae using Saccharomyces cerevisiae and to evaluate their hematological response resultant from the inflammatory induction. To this, S. cerevisiae were labeled with Congo red and injected in the coelomic cavity of A. altiparanae at the concentration of 2.5 x 106 cells mL-1. A PBS solution and a non-injected group were kept as control. Fish blood was sampled and the phagocytic capacity and index were determined at 1, 2, 3 and 6 h post-injection (hpi). The yeast injection successfully stimulated phagocytosis, with the best result for phagocytosis assessment after 2 hpi. Moreover, it was achieved a high traceability of phagocytized and non-phagocytized yeast under optic microscopy analysis due to the Congo red labeling. The hematological profile was similar to usually observed in early infections, indicating lymphocyte migration to inflammatory site and increase in number of circulating phagocytes due to natural response to inflammatory stimulus. In conclusion, our method was efficient to assess in vivo phagocytosis in A. altiparanae and will be an important tool to evaluate the efficacy of immunosuppressive drugs in this species. Additionally, these results may serve as support for further studies in fish immunocompetence, both in laboratory and in field conditions.

Resumo Apesar do potencial apresentado pela tecnologia de propagação mediada para a aquicultura e conservação de peixes Neotropicais, o pobre entendimento do sistema imune do hospedeiro pode resultar na rejeição e destruição do material do doador. Com isso, se fazem necessários o estudo e o desenvolvimento de métodos para análise tanto dos efeitos de drogas imunossupressoras quanto para a avaliação da imunocompatibilidade entre doadores e receptores. Logo, o presente estudo teve como objetivo aperfeiçoar um método para analisar a fagocitose in vivo em Astyanax altiparanae usando Saccharomyces cerevisiae marcado e avaliar seu perfil hematológico resultante da indução inflamatória. Para isso, S. cerevisiae foram marcados com vermelho Congo e injetados na cavidade celomática dos A. altiparanae na concentração de 2,5 x 106 células.mL-1. Peixes injetados com PBS e peixes não injetados foram mantidos como controle. Sangue foi colhido e a capacidade fagocítica e o índice fagocítico foram determinados após 1, 2, 3 e 6 horas após à injeção (hpi). A injeção de levedura estimulou a fagocitose com sucesso, com o melhor resultado atingido após 2 hpi. Ainda, foi observada uma alta rastreabilidade das leveduras fagocitadas e não fagocitadas sob microscopia óptica devido à marcação com vermelho Congo. O perfil hematológico foi similar ao observado usualmente em infecções recém-induzidas, indicando migração de linfócitos ao sítio inflamatório e aumento no número de fagócitos circulantes devido à resposta natural ao estímulo inflamatório. Como conclusão, nosso método foi eficiente para analisar a fagocitose in vivo em A. altiparanae e será uma ferramenta importante para a avaliação de eficácia de drogas imunossopressoras para esta espécie. Em adição, estes resultados podem contribuir para futuros estudos em imunocompetência em peixes, tanto em âmbito laboratorial quanto a campo.