Comparison between immunofluorescence and immunohistochemistry for Listeria monocytogenes detection in formalin-fixed paraffin-embedded tissues / Comparação entre imunofluorescência e imuno-histoquímica para detecção de Listeria monocytogenes em tecidos fixados e parafinizados

Listeria monocytogenes is a bacterium that infect humans and animals and causes a zoonotic disease characterized by encephalitis, septicemia or abortion. In addition, listeriosis leads to significant economic losses due to animal death and sacrifice. This research compared the technique of immunofluorescence (IF) and immunohistochemistry (IHC) for the diagnosis of L. monocytogenes in formalin-fixed and paraffin-embedded (FFPE) tissues. A total of 30 tissue blocks from 15 animals with history and/or lesions compatible with listeriosis were selected. For both IHC and IF, the same diluted (1:200) polyclonal primary antibody was used against L. monocytogenes serotypes 1 and 4. For IHC, a polymer secondary antibody conjugated to peroxidase (HRP) was used. For IF, samples were incubated with a fluorescein-labeled anti-rabbit IgG secondary antibody. Each sample was classified according to the presence and percentage of immunolabeling area. From 30 samples, 10 were positive at least for one technique, whereas eight samples were positive for both IHC and IF with similar score. There was strong immunolabeling in tissue samples from bovines experimentally infected with L. monocytogenes ATCC 7644, as well as in nervous tissues from naturally infected ruminants. Additionally, IF did not show any difference in sensitivity when compared to IHC. Using processed biological materials for IF, instead of fresh tissues, is a quite unique technique, since there are few protocols described. Therefore, this study demonstrated that both techniques are efficient to detect L. monocytogenes in FFPE tissues.

Listeria monocytogenes é uma bactéria que infecta humanos e animais, podendo causar uma doença zoonótica caracterizada por encefalite, septicemia e abortos. Além disso, a listeriose resulta em perdas econômicas significativas pela morte e sacrifício de animais. O objetivo deste trabalho foi comparar a técnica de imunofluorescência (IF) e imuno-histoquímica (IHQ) para detecção de L. monocytogenes em tecidos fixados e parafinizados. Foram selecionados 30 blocos de tecidos de 15 animais com histórico e/ou lesões compatíveis com listeriose. Para ambas as técnicas, foi utilizado o mesmo anticorpo primário policlonal diluído (1:200) para detecção de L. monocytogenes sorotipos 1 e 4. Para a IHQ foi utilizado anticorpo secundário em polímero acoplado a peroxidase (HRP). Para a IF, as amostras foram incubadas com anticorpo secundário anti-IgG de coelho marcado com fluoresceína. Cada amostra foi classificada quanto à presença de imunomarcação e porcentagem de área positiva. Das 30 amostras, 10 foram positivas em pelo menos uma das técnicas, sendo oito amostras positivas em ambas IHQ e IF com o mesmo escore. Houve forte imunomarcação tanto em amostras de bovino experimentalmente infectado com L. monocytogenes ATCC 7644, como em amostras de tecido nervoso de ruminantes naturalmente infectados. Além disso, a IF não apresentou diferença na sensibilidade quando comparada com a IHQ. O uso de materiais biológicos processados para IF, ao invés de tecidos frescos, é algo inovador, uma vez que existem poucos protocolos descritos. Portanto, este estudo demonstrou que as duas técnicas foram eficientes para detectar L. monocytogenes em tecidos fixados em formol e parafinizados.

Eurytrema coelomaticum infection: correlation between parasite burden and impairment of pancreatic exocrine enzyme secretion / Eurytrema coelomaticum: correlação entre carga parasitária e insuficiência pancreática exócrina

Eurytrema coelomaticum is a trematode reported in the pancreatic ducts of ruminants. It is conjectured that may cause disorders in the pancreas, as well as digestive and metabolic processes dependent on them. This study, determined if there is an impairment of exocrine pancreatic function, and correlated it with parasite burden. Pancreas, blood, and fecal samples were collected from 119 bovines at a abattoir. Stool samples were subjected to the gelatin and x-ray film digestion tests (to detect the presence of trypsin in feces). Using blood samples, the following biochemical tests were performed: amylase, lipase, glucose, fructosamine, cholesterol, triglycerides, total protein, albumin, and globulins. Analyses were correlated with pancreatic parasite burden. Cattle with a high parasitic load presented higher incidence of negative tests in both gelatin digestion and x-ray film digestion tests (P < 0.001) when compared to non-parasitized animals and those with a low parasitic load. Changes in those tests only occurred if the parasitemia was moderate or severe. The activity of the amylase and lipase enzymes was significantly higher in animals with low parasitemia (P < 0.05), compared to non-parasitized animals and with a high parasitic burden. In this study, in cases of high parasitemia, negative results were observed in both gelatin and x-ray film in the feces digestion tests. However, the low infection of E. coelomaticum, higher levels of serum amylase and lipase that also indicated loss of pancreatic exocrine functions were reported.

Eurytrema coelomaticum, um trematódeo de ductos pancreáticos de ruminantes. Conjectura-se que possa ocasionar transtornos nas funções pancreáticas, mais especificamente nos processos digestivos e metabólicos dependentes destas. Neste estudo, o objetivo foi determinar se há comprometimento da função pancreática exócrina, correlacionado-a a carga parasitária. Foram utilizados pâncreas e respectivas amostras de sangue e fezes de 119 bovinos. As amostras de fezes foram submetidas aos testes de digestão da gelatina em tubo e digestão de filme radiográfico, ambos para detecção de tripsina nas fezes. Foram realizados os seguintes exames bioquímicos em amostras de sangue: amilase, lipase, glicemia, frutosamina, colesterol, triglicerídeos, proteínas totais, albumina e globulinas. Após isto, as análises bioquímicas foram correlacionadas com a quantidade numérica de parasitas encontrados no pâncreas (post-mortem). Houve maior quantidade de testes negativos (digestão do filme radiográfico e prova de digestão da gelatina) nos animais com alta carga parasitária (P < 0.001), quando comparados aos animais não parasitados e com baixa carga parasitária. Portanto, os exames supracitados se alteram somente se a quantidade de parasitas for moderada ou severa. As atividades das enzimas amilase e lipase foram significativamente maiores nos animais que apresentavam baixa parasitemia (P < 0.05), em comparação com os animais com alta carga parasitária e não parasitados. Conclui-se que em quadros de alta parasitemia há alteração significativa nos testes de digestão nas fezes, e que em quadros de baixa parasitemia há alterações significativas nos valores de amilase e lipase séricas, ambos comprovando alterações pancreáticas importantes, de acordo com o quadro de parasitemia.

Plant poisoning containing hydrocyanic acid in cattle in Southern Brazil

Background: Cyanogenic plants accumulate cyanogenic glycosides and release hydrocyanic acid (HCN). In Brazil, therehave been reports of several plants that cause HCN poisoning in animals and lead to a fast death with few clinical signs andlesions on post mortem examination. Some cultivars of Cynodon spp. grasses cause HCN poisoning in cattle in Brazil. Theobjectives of this work were to report the occurrence of deaths by HCN poisoning in cattle as diagnosed by the veterinarypathology laboratory, describe the quantity of HCN in some cultivars of Cynodon spp., as well as, to describe one cultivarof genus Cynodon never reported as poisonous.Materials, Methods & Results: The archives of the Veterinary Pathology Laboratory (LPV) at the Concórdia Campus ofthe Instituto Federal Catarinense (IFC) were reviewed, seeking cases with a diagnosis of hydrocyanic acid poisoning incattle after post mortem examination. The amount of HCN present in some cultivars of the Cynodon genus was quantified due to the high frequency of poisoning cases. From the 1,235 post mortem examinations of cattle 28 (2.27%) werediagnosed with spontaneous hydrocyanic acid poisoning, 17 cases (60.7%) due to ingestion of Prunus sp. or Manihotsp., and 11 cases (39.3%) of Cynodon dactylon ingestion. Most animals were found dead, normally having presented noclinical signs. Macroscopic evaluation mainly showed a severe amount of unchewed and undigested leaves or grass mixedin the ruminal content presenting a bitter almond odor. It was possible to infer that, among cultivars of the Cynodon genus, Florakirk showed the highest levels of HCN compared (P < 0.05) with Star of Puerto Rico, Tifton 68, Tifton 44, andCoast-Cross. Furthermore, Tifton 85 and Jiggs showed undetected levels of HCN...

Plant poisoning containing hydrocyanic acid in cattle in Southern Brazil

Background: Cyanogenic plants accumulate cyanogenic glycosides and release hydrocyanic acid (HCN). In Brazil, therehave been reports of several plants that cause HCN poisoning in animals and lead to a fast death with few clinical signs andlesions on post mortem examination. Some cultivars of Cynodon spp. grasses cause HCN poisoning in cattle in Brazil. Theobjectives of this work were to report the occurrence of deaths by HCN poisoning in cattle as diagnosed by the veterinarypathology laboratory, describe the quantity of HCN in some cultivars of Cynodon spp., as well as, to describe one cultivarof genus Cynodon never reported as poisonous.Materials, Methods & Results: The archives of the Veterinary Pathology Laboratory (LPV) at the Concórdia Campus ofthe Instituto Federal Catarinense (IFC) were reviewed, seeking cases with a diagnosis of hydrocyanic acid poisoning incattle after post mortem examination. The amount of HCN present in some cultivars of the Cynodon genus was quantified due to the high frequency of poisoning cases. From the 1,235 post mortem examinations of cattle 28 (2.27%) werediagnosed with spontaneous hydrocyanic acid poisoning, 17 cases (60.7%) due to ingestion of Prunus sp. or Manihotsp., and 11 cases (39.3%) of Cynodon dactylon ingestion. Most animals were found dead, normally having presented noclinical signs. Macroscopic evaluation mainly showed a severe amount of unchewed and undigested leaves or grass mixedin the ruminal content presenting a bitter almond odor. It was possible to infer that, among cultivars of the Cynodon genus, Florakirk showed the highest levels of HCN compared (P < 0.05) with Star of Puerto Rico, Tifton 68, Tifton 44, andCoast-Cross. Furthermore, Tifton 85 and Jiggs showed undetected levels of HCN...(AU)

Outbreak of rickets in pigs in the west of Santa Catarina

Background: Rickets is a deficiency pathology that occurs in young and growing animals, leading to deficient bone mineralization. Rickets has been reported in several species producing numerous economic losses. The disease is caused by nutritional imbalance of calcium (Ca), phosphorus (P) and vitamin D. The aim of this work was to report two outbreaks of rickets in commercial weaning farms in the Seara city, in the western region of Santa Catarina State (SC), Brazil. Case: In August 2016, the Veterinary Pathology Laboratory (LPV) at the Concórdia Campus of the Catarinense Federal Institute (IFC) diagnosed two outbreaks of rickets in pigs in the weaning phase in Seara, SC. The clinical history was obtained by interviewing the field veterinarian and the farmers. In the anamnesis, both pig farmers stated having used a feed premix product from the same company, starting three months ago, and after that the pigs presented clinical signs of posterior paralysis and progressive weight loss. Six animals were submitted to necropsy and organs were collected from the abdominal and thoracic cavities; central nervous system and bones; fixed in 10% buffered formalin, routinely processed, paraffin embedded and stained with hematoxylin and eosin (HE) for histopathological analysis. Bone specimens were decalcified in nitric acid working solution 20 times their volume, during 5 days. In addition, samples of the premix product containing minerals and vitamins were sent to a specialized laboratory to analyze macroelements levels through the atomic absorption methodology. In property 1, there was a batch of 100 animals, the morbidity rate was 15% and lethality was 5%. In property 2, among 30 animals, the morbidity and lethality rate were 33%. Five animals from property 1 and one...

Outbreak of rickets in pigs in the west of Santa Catarina

Background: Rickets is a deficiency pathology that occurs in young and growing animals, leading to deficient bone mineralization. Rickets has been reported in several species producing numerous economic losses. The disease is caused by nutritional imbalance of calcium (Ca), phosphorus (P) and vitamin D. The aim of this work was to report two outbreaks of rickets in commercial weaning farms in the Seara city, in the western region of Santa Catarina State (SC), Brazil. Case: In August 2016, the Veterinary Pathology Laboratory (LPV) at the Concórdia Campus of the Catarinense Federal Institute (IFC) diagnosed two outbreaks of rickets in pigs in the weaning phase in Seara, SC. The clinical history was obtained by interviewing the field veterinarian and the farmers. In the anamnesis, both pig farmers stated having used a feed premix product from the same company, starting three months ago, and after that the pigs presented clinical signs of posterior paralysis and progressive weight loss. Six animals were submitted to necropsy and organs were collected from the abdominal and thoracic cavities; central nervous system and bones; fixed in 10% buffered formalin, routinely processed, paraffin embedded and stained with hematoxylin and eosin (HE) for histopathological analysis. Bone specimens were decalcified in nitric acid working solution 20 times their volume, during 5 days. In addition, samples of the premix product containing minerals and vitamins were sent to a specialized laboratory to analyze macroelements levels through the atomic absorption methodology. In property 1, there was a batch of 100 animals, the morbidity rate was 15% and lethality was 5%. In property 2, among 30 animals, the morbidity and lethality rate were 33%. Five animals from property 1 and one...(AU)

Experimental infection by Brucella ovis: changes in NTPDase, 5’-nucleotidase and acetylcholinesterase associated cerebral oxidative stress

Background: Changes in purinergic and cholinergic signaling have been demonstrated in various pathologies associatedwith inflammation; however, the changes in brucellosis caused by the Gram-negative coccobacillus Brucella ovis are notknown. B. ovis is generally asymptomatic in ewes. Hepatosplenomegaly has been described in B. ovis, a non-zoonoticspecies, characterized by an extravascular inflammatory response. Purinergic system enzymes are closely involved withthe modulation of the immune system, pro- and anti-inflammatory events. The objective of this study was to investigatethe role of ectonucleotidases and cholinesterase’s in the brains of mice experimentally infected with B. ovis.Materials, Methods & Results: Forty-eight animals were divided into two groups: control (n = 24) and infected (n = 24).In group infected, 100 µL containing 1.3 x 107 UFC B. ovis /mL via intraperitoneal was used in inoculation. The brainswere collected from the animals on days 7, 15, 30 and 60 post-infection (PI). We measured levels of TBARS (substancesreactive to thiobarbituric acid) and ROS (reactive oxygen species) in the brain. The activity of NTPDase (using ATP andADP as substrate) and 5’-nucleotidase (using AMP as substrate) were evaluated in brain in addition to histopathologicalanalysis. No histopathological lesions were observed in the control group nor the infected group at days 7, 15, and 30 PI.However, multifocal areas with moderate microgliosis in the cerebral cortex were observed at day 60 PI in the infectedanimals. B. ovis DNA was detected in brain. During the course of infection, B. ovis caused greater lipid peroxidation inthe brains of infected animals than in the control group at day 60 PI. No significant results were observed at 7, 15 or day30 PI. Similarly, there was significantly more reactive oxygen species at day 60 PI in brains of infected animals than inthe control group. NTPDase activity (using ATP ...

Typhlocolitis by Edwardsiella tarda in a cow

Background: Edwardsiella tarda has been report as etiology of gastroenteritis in both human and veterinary medicine,usually associated with exposure to aquatic environments in immunocompromised individuals. The present report describesa case of typhlocolitis in a cow in the west region of Santa Catarina state, Brazil.Case: After clinically evaluation of animal and proceeding euthanasia and realized the necropsy. Tissue samples were collected, and routinely processed for histological examination. Histopathological lesions were described as mild, moderateand severe. Tissue and swabs samples of small and large intestine were sent to standard microbiological culture processing.At necropsy, cattle presented severe dehydration and emaciation. Eye and vaginal mucosa were severely congested. Theopening of the abdominal cavity revealed a great amount of greenish fluid and a large amount of fecal material, associatedwith diffuse severe peritonitis, evidenced by hyperemia and severe deposition of fibrin in the visceral and parietal peritonealsurface. In the serosa of the cecum, there were two points of rupture, observed in the proximal colon. The mucosa of cecumand colon were severely edematous, hyperemic, and presented diffusely distributed pinpoint round hemorrhages, as wellas fibrinonecrotic material adhered to the surface. Histologically, in the mucosa of cecum and colon, moderate to severediffuse inflammatory infiltrate of neutrophils, lymphocytes and plasma cells associated with multifocal severe necrosiswere observed. Moderate diffuse fibrin deposition was evidenced in the submucosa and muscular, as well as multifocalmoderate necrosis in the muscular layer. In the serous, severe diffuse inflammatory infiltrate of neutrophils associated withfibrin deposition and innumerous coccoid bacterial colonies were observed. The samples subjected to bacterial isolationshowed growth of Edwardsiella tarda. All samples were negative for Salmonella spp. and...

Typhlocolitis by Edwardsiella tarda in a cow

Background: Edwardsiella tarda has been report as etiology of gastroenteritis in both human and veterinary medicine,usually associated with exposure to aquatic environments in immunocompromised individuals. The present report describesa case of typhlocolitis in a cow in the west region of Santa Catarina state, Brazil.Case: After clinically evaluation of animal and proceeding euthanasia and realized the necropsy. Tissue samples were collected, and routinely processed for histological examination. Histopathological lesions were described as mild, moderateand severe. Tissue and swabs samples of small and large intestine were sent to standard microbiological culture processing.At necropsy, cattle presented severe dehydration and emaciation. Eye and vaginal mucosa were severely congested. Theopening of the abdominal cavity revealed a great amount of greenish fluid and a large amount of fecal material, associatedwith diffuse severe peritonitis, evidenced by hyperemia and severe deposition of fibrin in the visceral and parietal peritonealsurface. In the serosa of the cecum, there were two points of rupture, observed in the proximal colon. The mucosa of cecumand colon were severely edematous, hyperemic, and presented diffusely distributed pinpoint round hemorrhages, as wellas fibrinonecrotic material adhered to the surface. Histologically, in the mucosa of cecum and colon, moderate to severediffuse inflammatory infiltrate of neutrophils, lymphocytes and plasma cells associated with multifocal severe necrosiswere observed. Moderate diffuse fibrin deposition was evidenced in the submucosa and muscular, as well as multifocalmoderate necrosis in the muscular layer. In the serous, severe diffuse inflammatory infiltrate of neutrophils associated withfibrin deposition and innumerous coccoid bacterial colonies were observed. The samples subjected to bacterial isolationshowed growth of Edwardsiella tarda. All samples were negative for Salmonella spp. and...(AU)

Experimental infection by Brucella ovis: changes in NTPDase, 5-nucleotidase and acetylcholinesterase associated cerebral oxidative stress

Background: Changes in purinergic and cholinergic signaling have been demonstrated in various pathologies associatedwith inflammation; however, the changes in brucellosis caused by the Gram-negative coccobacillus Brucella ovis are notknown. B. ovis is generally asymptomatic in ewes. Hepatosplenomegaly has been described in B. ovis, a non-zoonoticspecies, characterized by an extravascular inflammatory response. Purinergic system enzymes are closely involved withthe modulation of the immune system, pro- and anti-inflammatory events. The objective of this study was to investigatethe role of ectonucleotidases and cholinesterases in the brains of mice experimentally infected with B. ovis.Materials, Methods & Results: Forty-eight animals were divided into two groups: control (n = 24) and infected (n = 24).In group infected, 100 µL containing 1.3 x 107 UFC B. ovis /mL via intraperitoneal was used in inoculation. The brainswere collected from the animals on days 7, 15, 30 and 60 post-infection (PI). We measured levels of TBARS (substancesreactive to thiobarbituric acid) and ROS (reactive oxygen species) in the brain. The activity of NTPDase (using ATP andADP as substrate) and 5-nucleotidase (using AMP as substrate) were evaluated in brain in addition to histopathologicalanalysis. No histopathological lesions were observed in the control group nor the infected group at days 7, 15, and 30 PI.However, multifocal areas with moderate microgliosis in the cerebral cortex were observed at day 60 PI in the infectedanimals. B. ovis DNA was detected in brain. During the course of infection, B. ovis caused greater lipid peroxidation inthe brains of infected animals than in the control group at day 60 PI. No significant results were observed at 7, 15 or day30 PI. Similarly, there was significantly more reactive oxygen species at day 60 PI in brains of infected animals than inthe control group. NTPDase activity (using ATP ...(AU)