Occurrence of Hemogregarine in Two Species of Brazilian Freshwater Turtles

Background: Hemogregarines are the most common intraerythrocytic parasites found in reptiles. The genus Haemogregarina has aquatic vertebrates as intermediate hosts, and as definitive hosts the leeches. The genus Hepatozoon can be found parasitizing amphibians, reptiles, birds and mammals and its main vectors invertebrates are mosquitoes, tsetse flies, lice, fleas and mites. The diagnosis of these parasites is done by the technique of blood smear, but modern diagnoses include evaluation of blood by polymerase chain reaction (PCR).  The aim this study was to determine the occurrence of infection by hemogregarine in freshwater turtles, through PCR.Materials, Methods & Results: Samples from 99 freshwater turtles of species P. expansa and P. geoffroanus of Fundação Zoológico de Brasília,Distrito Federal, Brazil, were used. The animals was captured using a hand net, and were immediately individually identified. The blood samples was collected by puncture of the occipital sinus, and placed into tubes containing sodium heparin anticoagulant for hematologic and molecular analysis. Two different sets of oligonucleotides were used, one to detect hemogregarines and other to detect Hepatozoon sp. infection. The presence of hemogregarine was detected in 20 samples analyzed (n = 99), these eleven samples were positive for hemogregarine, 5 were Hepatozoon sp. and 4  were positive for both oligonucleotides. Laboratory abnormalities were observed in the concentration of total plasma proteins, total serum proteins and globulin, and in the number of thrombocytesin animals positive for hemogregarines and only alterations in the number of thrombocytes were observed in Hepatozoon sp. positive animals of both species.Discussion: This study showed that there is a high occurrence of infection by hemogregarines in the freshwater turtles samples examinated. The remarkable difficulty of identifying morphological differences, combined with the development of universal oligonucleotides, make further assessments infections hematozoa to be performed using molecular tools and specially  sequencing of the 18S rRNA gene for hemogregarines.  Protein levels in animals depends on the management, diets and normal physiological variations of each species. Hypoproteinemia is commonly observed in reptiles with chronic malnutrition and gastrointestinal parasitism. Therefore it is suggested that the cause of this low level in the measurement of proteins could be directly linked to the presence of hemoparasites because with the infection more animals feeding unless the negative, causing malnutrition, or even the concomitant presence of gastrointestinal parasites, because the study did not evaluate this level of parasitism. Thrombocytes in reptiles participate in blood clotting and some studies have shown that they also have phagocytic capacity. Thrombocytopenia in reptiles is usually a result of excessive use or deficient production. Thus, thrombocytopenia observed in hemogregarines positive animals of this study may suggest that the presence of this group of parasites can cause thrombocytopenia that may be associated with a deficient production of thrombocytes or even greater consumption of these blood cells. The increase of thrombocytes observed in animals positive for Hepatozoon sp. may be related to the defense of the animal, since thrombocytes also have phagocytic activity. No changes were observed in laboratorial tests of P. expansa, which may be due to the small number of this animal species analyzed

Concordancia entre los métodos de flotación con sulfato de zinc y sedimentación centrífuga para el diagnóstico de parásitos intestinales

Introduction: The diagnosis of intestinal parasitic infections depends on the parasite load, the specific gravity density of the parasite eggs, oocysts or cysts, and the density and viscosity of flotation or sedimentation medium where faeces are processed.Objective: To evaluate the concordance between zinc sulphate flotation and centrifugal sedimentation in the recovery of parasites in faecal samples of children.Materials and methods: Faecal samples of 330 children from day care centers were evaluated by zinc sulphate flotation and centrifugal sedimentation techniques. The frequencies of detection of parasites by each method were determined and the agreement between the diagnostic techniques was evaluated using the kappa index, with 95% confidence intervals.Results: The faecal flotation in zinc sulphate diagnosed significantly more cases of Trichuris trichiura infection when compared to centrifugal sedimentation (39/330; 11.8% vs. 13/330; 3.9%, p<0.001), with low diagnostic concordance between methods (kappa=0.264; 95% CI: 0.102-0.427). Moreover, all positive samples for Enterobius vermicularis eggs (n=5) and Strongyloides stercoralis larvae (n=3) were diagnosed only by zinc sulphate. No statistical differences were observed between methods for protozoa identification.Conclusions: The results showed that centrifugal flotation in zinc sulphate solution was significantly more likely to detect light helminths eggs such as those of T. trichiura and E. vermicularis in faeces than the centrifugal sedimentation process.Introducción. El diagnóstico de infecciones parasitarias intestinales depende de la carga de parásitos, la densidad de la gravedad específica de los huevos, ooquistes o quistes de parásitos, y de la densidad y viscosidad de los reactivos de flotación o sedimentación usados para procesar las heces.Objetivo. Evaluar la concordancia entre el método de flotación de sulfato de zinc y la sedimentación por centrifugación en la recuperación de parásitos en muestras fecales de niños.Materiales y métodos. Se evaluaron las muestras fecales de 330 niños de guarderías mediante las técnicas de flotación con sulfato de zinc y de sedimentación por centrifugación. Se determinó la frecuencia de detección de parásitos con cada método y se evaluó la concordancia entre las técnicas de diagnóstico mediante el índice kappa, con intervalos de confianza del 95 %.Resultados. Mediante la flotación fecal con sulfato de zinc, se diagnosticó un número significativamente mayor de casos de infección por Trichuris trichiura que con la sedimentación por centrifugación (39/330; 11,8 % Vs. 13/330; 3,9 %) (p<0,001), con poco acuerdo entre los métodos (kappa=0,264; IC95% 0,102-0,427). Además, todas las muestras positivas para huevos de Enterobius vermicularis (n=5) y larvas de Strongyloides stercoralis (n=3) se diagnosticaron solamente por sulfato de zinc. No se observaron diferencias estadísticamente significativas entre los métodos para la identificación de protozoos.Conclusiones. La flotación centrífuga en una solución de sulfato de zinc presentó una probabilidad significativamente mayor de detectar los huevos livianos de helmintos como T. trichiura y E. vermicularis en heces, que el proceso de sedimentación por centrifugación

Exposição de capivaras (Hydrochoerus hydrochaeris) à Rickettsia no Distrito Federal, área não endêmica para febre maculosa brasileira

In this study, whole blood samples and ticks were collected from 57 capybaras in recreational areas in the Federal District, Brazil, aiming to investigate the presence of Rickettsia spp. using polymerase chain reaction (PCR) and indirect immunofluorescence (IFAT) assays. None of the capybara blood samples yielded rickettsial DNA by PCR. Among 55 capybara serum samples tested by IFAT, 53 (96.3%) reacted to Rickettsia spp. Among these, 21 (39.6%) identified the R. bellii antigen as the probable antigen involved in a homologous reaction (PAIHR), whereas 2 (3.8%) identified the R. parkeri antigen. Ticks collected from capybaras were identified as 173 Amblyomma sculptum and 410 A. dubitatum, in addition to nine Amblyomma spp. larvae. A sample of 231 ticks was subjected to DNA extraction and PCR for Rickettsia species. None of 122 A. sculptum yielded rickettsial DNA. Molecular evidence of R. bellii was found in 25/108 (23.1%) and of Rickettsia sp. strain Cooperi (R. parkeri-like agent) in 2/108 (1.9%) of the A. dubitatum samples. These results suggest a greater exposure to R. bellii in these capybara populations, in addition to a more significant number of A. dubitatum, which might characterize the Federal District region as not endemic for Brazilian spotted fever

Use of PCR in identification of polycystic kidney disease in cats of the Distrito Federal and its disorders laboratory

Monografia (graduação)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, 2013.A doença renal policística (DRP) felina, também conhecida como PKD (policystic kidney disease), é a doença de herança genética mais predominante em gatos, principalmente da raça Persa ou mestiços dessa raça. Caracteriza-se pela presença de cistos de vários tamanhos que podem ocorrer no córtex ou na medula renal. O presente trabalho propôs a utilização de novas ferramentas moleculares (PCR) para o diagnóstico precoce da DRP em felinos. Foram utilizadas amostras de sangue total de 229 gatos domésticos, sendo 188 amostras de animais atendidos no Hospital Veterinário de Pequenos Animais da UnB e 41 amostras provenientes de um laboratório de Patologia Clínica Veterinária do Gama. No laboratório de Patologia Clínica do Hospital Veterinário da UnB, parte das amostras foram submetidas a hemograma completo, testes bioquímicos e o restante na PCR para a identificação dos animais portadores de DRP (mutação). Das amostras colhidas, 48,5% eram fêmeas, 48,5% machos e em 3% o sexo não foi informado. A idade média foi de 6,3 anos, oscilando entre 2 meses e 20 anos. Desses 229 animais, 11,8% foram positivos para PKD1, desses 88,9% provenientes do Hospital Veterinário da UnB, e 11,1% proveniente do Laboratório do Gama. De acordo com as proporções, nenhum parâmetro hematológico e bioquímico diferiu entre os gatos com a mutação e sem a mutação. Os resultados obtidos durante a pesquisa permitiram concluir nos animais avaliados que: a mutação genética para DRP não foi observada somente na raça persa, mas também em raças que podem ter sido intercruzadas com a linhagem persa, os animais positivos nem sempre apresentam sintomas clínicos e alterações nos exames laboratoriais o que caracteriza o estado de portador da doença e a PCR é uma importante ferramenta diagnóstica precoce para DRP. __________________________________________________________________________ ABSTRACTFeline polycystic kidney disease (PKD) is a genetic disease prevalent in cats, especially the Persian breed or crossbreed of that race, characterized by the presence of cysts of various sizes, which can occur in the renal cortex or medulla. This paper proposed the use of new molecular tools (PCR) for early diagnosis of PKD in cats. We used blood samples from 229 cats, 188 samples of animals treated at the Small Animal Veterinary Hospital of UnB and 41 samples came from a Veterinary Clinical Pathology Laboratory at Gama. At the Clinical Pathology Laboratory in the Small Animal Hospital of UnB, part of the samples was subjected to complete blood count (CBC), biochemical tests and the remainder in PCR for the identification of animals with PKD (mutation). From the samples, 48.5% were females, 48.5% males and 3% the gender was not informed. The mean age was 6.3 years, ranging from 2 months to 20 years. Of these 229 animals, 11.8% were positive for PKD1, 88.9% of them from the Veterinary Hospital of UnB, and 11.1 % from the Laboratory at Gama. Accordingly to the proportions, no biochemical and hematological parameters differed among cats with and without the mutation. The results obtained during the research led to the following conclusions about the evaluated animals: the genetic mutation for PKD was not only observed in the Persian breed, but also in races that may have been intercrossed with Persian lineage; the positive animals do not always present clinical signs and changes in laboratory tests, characterizing the carrier state of the disease; and the PCR is an important early diagnostic tool for PKD

Ocurrence of leptospira spp. and its laboratory alterations in the capybaras (Hydrochoerus hydrochaeris) population in Distrito Federal

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, 2020.A capivara (Hydrochoerus hydrochaeris) é considerada o maior roedor do mundo, com excelente capacidade de adaptação em ambientes urbanizados e, por ser um animal sinantrópico, atua como transmissora de diferentes doenças. A leptospirose é uma doença contagiosa que atinge animais domésticos, silvestres e humanos, causada pela infecção por algumas espécies patogênicas do gênero Leptospira. Estudos sobre leptospirose em capivaras são escassos, e não existem pesquisas sobre essa doença nessa espécie no Distrito Federal e entorno. O objetivo desse estudo foi analisar a presença do DNA do agente e/ou de anticorpos anti-Leptospira spp em capivaras do Distrito Federal. Para isso, 56 capivaras de vida livre foram capturadas e o sangue coletado. As colheitas foram realizadas em dois locais distintos do Distrito Federal. As amostras foram utilizadas para a realização da análises hematológicas, bioquímicas, para detecção do DNA da Leptospira spp. pela reação da polimerase em cadeia (PCR) e para a realização da soroaglutinação microscópica (SAM). O ponto de corte utilizado no SAM foi 1:100. Nenhum animal apresentou amplificação na PCR para Leptospira spp., mas 41,1% (23/56) dos animais apresentaram anticorpos anti-Leptospira spp. no SAM. Os sorovares presentes foram hardjo, icterohaemorrhagiae, copenhageni e grippotyphosa. Nos exames laboratoriais foram observadas diferenças significativas (p<0,05) nas análises bioquímicas de creatinina, albumina e globulina, que poderiam indicar comprometimentos renais e hepáticos nos animais reagentes. Alterações no leucograma dos três animais com as maiores titulações (1:800 e 1:1600), podem denotar processos inflamatórios decorrentes de fases agudas da infecção pela Leptospira spp. A PCR utilizando amostras de sangue total para avaliação da infecção por Leptospira spp das capivaras de vida livre não se mostrou uma boa ferramenta. Mais estudos são necessários para a comprovação das alterações na hematologia e na bioquímica sérica. A presença de animais reagentes na sorologia mostra que a bactéria está circulante no ambiente urbano do Distrito Federal.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).The capybara (Hydrochoerus hydrochaeris) is considered the largest rodent in the world, with excellent adaptability in urbanized environments, and being a synanthropic animal, they can serve as a transmitter of different diseases. Leptospirosis is a contagious disease that affects domestic, wild and human animals, and it is caused by infection by some pathogenic species of the genus Leptospira. Studies on leptospirosis in capybaras are scarce, and there are no research on this disease in this species in the Federal District and its surroundings. The aim of this study was to analyze the presence of the agent's DNA and/or anti-Leptospira spp antibodies in capybaras in the Federal District. For this, 56 free-living capybaras were captured and the blood collected. The samples were taken in two different locations in the Federal District. The samples were used to carry out hematological and biochemical analyzes, whole blood was used to detect the DNA of Leptospira spp. by polymerase chain reaction (PCR) and the serum was used for microscopic agglutination test (MAT). No animals showed amplification in the PCR test for Leptospira spp., however 41.1% (23/56) of the animals showed anti-Leptospira spp. in MAT, the cutoff point used was 100. The serovars present were hardjo, icterohaemorrhagiae, copenhageni and grippotyphosa. In general, the animals showed a significant difference (p <0.05) in the biochemical analyzes of creatinine, albumin and globulin, this could represent that the reactive animals would have their renal and hepatic systems affected. When comparing the 3 animals with the highest titers (1: 800 and 1: 1600) with the non-reactive animals, changes in the leukogram were observed, and the inflammatory process caused by the leptospires in the acute phase of the disease could justify these changes. The PCR test for health evaluation of free-living capybaras was not a good tool when using only whole blood samples. Further studies are needed to prove changes in hematology and serum biochemistry. The presence of reactive animals in serology shows that the bacterium is circulating in the urban environment of the Federal District

Occurrence of Hemogregarine in Two Species of Brazilian Freshwater Turtles

Background: Hemogregarines are the most common intraerythrocytic parasites found in reptiles. The genus Haemogregarina has aquatic vertebrates as intermediate hosts, and as definitive hosts the leeches. The genus Hepatozoon can be found parasitizing amphibians, reptiles, birds and mammals and its main vectors invertebrates are mosquitoes, tsetse flies, lice, fleas and mites. The diagnosis of these parasites is done by the technique of blood smear, but modern diagnoses include evaluation of blood by polymerase chain reaction (PCR).  The aim this study was to determine the occurrence of infection by hemogregarine in freshwater turtles, through PCR.Materials, Methods & Results: Samples from 99 freshwater turtles of species P. expansa and P. geoffroanus of Fundação Zoológico de Brasília,Distrito Federal, Brazil, were used. The animals was captured using a hand net, and were immediately individually identified. The blood samples was collected by puncture of the occipital sinus, and placed into tubes containing sodium heparin anticoagulant for hematologic and molecular analysis. Two different sets of oligonucleotides were used, one to detect hemogregarines and other to detect Hepatozoon sp. infection. The presence of hemogregarine was detected in 20 samples analyzed (n = 99), these eleven samples were positive for hemogregarine, 5 were Hepatozoon sp. and 4  were positive for both oligonucleotides. Laboratory abnormalities were observed in the concentration of total plasma proteins, total serum proteins and globulin, and in the number of thrombocytesin animals positive for hemogregarines and only alterations in the number of thrombocytes were observed in Hepatozoon sp. positive animals of both species.Discussion: This study showed that there is a high occurrence of infection by hemogregarines in the freshwater turtles samples examinated. The remarkable difficulty of identifying morphological differences, combined with the development of universal oligonucleotides, make further assessments infections hematozoa to be performed using molecular tools and specially  sequencing of the 18S rRNA gene for hemogregarines.  Protein levels in animals depends on the management, diets and normal physiological variations of each species. Hypoproteinemia is commonly observed in reptiles with chronic malnutrition and gastrointestinal parasitism. Therefore it is suggested that the cause of this low level in the measurement of proteins could be directly linked to the presence of hemoparasites because with the infection more animals feeding unless the negative, causing malnutrition, or even the concomitant presence of gastrointestinal parasites, because the study did not evaluate this level of parasitism. Thrombocytes in reptiles participate in blood clotting and some studies have shown that they also have phagocytic capacity. Thrombocytopenia in reptiles is usually a result of excessive use or deficient production. Thus, thrombocytopenia observed in hemogregarines positive animals of this study may suggest that the presence of this group of parasites can cause thrombocytopenia that may be associated with a deficient production of thrombocytes or even greater consumption of these blood cells. The increase of thrombocytes observed in animals positive for Hepatozoon sp. may be related to the defense of the animal, since thrombocytes also have phagocytic activity. No changes were observed in laboratorial tests of P. expansa, which may be due to the small number of this animal species analyzed

Predominio del subconjunto AII de Giardia duodenalis en niños pequeños de Salvador, Bahía, Brasil

Introduction. Giardia duodenalis is an intestinal protozoan with a high prevalence in children of developing countries. Molecular studies revealed a great genetic diversity of G. duodenalis, with assemblages A and B found mainly in humans. Despite its importance, the information on the molecular epidemiology of human giardiasis is still limited in Brazil.Objective. To characterize G. duodenalis molecular isolates in children from Salvador, Bahia, Brazil.Materials and methods. Giardia duodenalis positive fecal samples were obtained from 71 children from two day care centers and 39 users of a clinical analysis laboratory. Samples were analyzed by PCR-RFLP of the glutamate dehydrogenase (gdh) and beta-giardin genes and by the sequencing of beta-giardin.Results. Of the 110 G. duodenalis samples, 80 (72.7%) amplified one or both target genes. Of these, 62 (77.5 %) were identified as assemblage A and 18 (22.5%) as assemblage B. The subassemblage AII was identified in 58.8% (n=47) of isolates followed by the sub-assemblage AI (18.8%, n=15), BIV (11.2%, n=9), and BIII (5.0%, n=4). The AII sub-assemblage was the most frequent in children of both day care centers whereas AI was found only in the group attended at the clinical laboratory. Sub-assemblage AII predominated in children under two years.Conclusions. The higher frequency of AII sub-assemblage suggests that anthroponotic transmission is more common in Salvador, but that zoonotic transmission pathways are also present and a change in susceptibility to different molecular patterns of Giardia may occur during child growth.Introducción. Giardia duodenalis es un protozoo intestinal de gran prevalencia en los niños de los países en desarrollo. En estudios moleculares se ha evidenciado la gran diversidad genética de G. duodenalis y se han identificado los conjuntos A y B, principalmente en humanos. A pesar de su importancia, el conocimiento de la epidemiología molecular de la giardiasis humana aún es limitado en Brasil.Objetivo. Caracterizar los aislamientos moleculares de G. duodenalis de muestras tomadas a niños de Salvador, Bahía, Brasil.Materiales y métodos. Las muestras fecales positivas para G. duodenalis se obtuvieron de 71 niños de dos guarderías y de 39 usuarios de un laboratorio de análisis clínicos. Las muestras se analizaron mediante PCR-RFLP de los genes gdh y beta-giardin, y secuenciación de beta-giardin.Resultados. De las 110 muestras de G. duodenalis, en 80 (72,7 %) se amplificaron uno o ambos genes. De estos, 62 (77,5 %) se identificaron como pertenecientes al conjunto A y 18 (22,5 %) al B. El subconjunto AII se identificó en el 58,8 % (n=47) de los aislamientos, seguido del AI en el 18,8% (n=15), el BIV en el 11,2% (n=9) y el BIII en el 5,0% (n=4).El subconjunto AII fue el más frecuente en los niños de ambas guarderías, en tanto que el AI solo se encontró en el grupo atendido en el laboratorio clínico. El subconjunto AII predominó en los niños menores de dos años.Conclusiones. La mayor frecuencia del subconjunto AII sugiere que la transmisión antroponótica es más común en Salvador, pero también existen vías de transmisión zoonóticas, y que pueden ocurrir cambios en la sensibilidad frente a diferentes patrones moleculares de Giardia durante el crecimiento infantil