Orientador: Valéria Marçal Felix de Lima / Banca: Flávia Lombardi Lopes / Banca: Alexandra Ivo Medeiros / Resumo: A Leishmaniose Visceral (LV) no homem é uma doença crônica e frequentemente fatal se não tratada. A doença possui alta taxa de mortalidade e a região de Araçatuba concentra grande número de casos no estado de São Paulo. A Leishmaniose Visceral Canina (LVC) constitui um grave problema de Saúde Pública, pois os animais infectados são potentes transmissores do parasita para humanos pelo vetor flebotomínio. O cão é, portanto, um alvo importante nas medidas de controle. A progressão da infecção canina é acompanhada por falha na imunidade celular com redução de linfócitos circulantes e citocinas que suprimem a função dos macrófagos. A função da célula T na indução da resposta celular é determinante para a eliminação do parasita no interior dos macrófagos. Embora a supressão imunológica já esteja caracterizada, os fatores determinantes são pouco conhecidos. Recentes estudos mostraram que a regulação da função efetora dos macrófagos e células T parece depender de microRNAs (miRNAs). Existem muitas evidências da função dos miRNAs na regulação da expressão de proteínas que são fundamentais para o desenvolvimento, função e diferenciação de vários tipos celulares do sistema imunológico. Uma desregulação da atividade dos miRNAs está envolvida em diversas doenças, incluindo a LV. Na LVC, devido a supressão imunológica celular ser determinante para a progressão da doença, o conhecimento de miRNAs associados a regulação imunológica pode ser importante para a mudança do padrão de resposta. / Abstract: Visceral Leishmaniasis (VL) in humans is a chronic and often fatal disease if left untreated. The disease has high mortality rate, and the region of Araçatuba concentrates a large number of cases in São Paulo state. Canine VL (CVL) is a serious public health problem, because infected animals are potent transmitters of the parasite to humans by the vector phlebotomine. Therefore, dogs are important targets in the control measures. The progression of canine infection is accompanied by a failure of cellular immunity with reducing of circulating lymphocytes and cytokines that suppress the macrophage function. The role of T cells in the induction of cellular response is crucial to the elimination of the parasite in macrophages. Although immunosuppression is already characterized, the determining factors are not well known. In the last decade studies have shown that regulation of effector function of macrophages and T cells appears to depend on microRNAs (miRNAs). There are many evidences of function of miRNA in regulating of the expression of proteins that are primordial for the development, function and differentiation of various cell types of the immune system. A deregulation of miRNA expression is involved in a variety of disorders including VL. In CVL, due to cell immune suppression be determinant of disease progression, knowledge of miRNAs associated with immune regulation may be important for change the response pattern. / Mestre

Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias / Cryptosporidium spp. (Familia Cryptosporidiidae), es un grupo de protozoos y parásitos obligados intracelulares que afectan a un amplio rango de vertebrados. En diversos estudios se han descrito a los roedores como hospederos de Cryptosporidium spp., señalando que estos mamíferos presentan la infección de manera natural. Esto los convierte en un riesgo potencial para la salud pública, sobretodo porque muchos roedores contribuyen a la contaminación de agua, suelo y alimentos, al dejar sus deposiciones en todo lugar en donde se desplazan y alimentan. En nuestro conocimiento no existe en Chile ningún registro sobre infección de Cryptosporidium en roedores silvestres. Dado que los cambios en el uso de suelo pueden modificar la estructura y composición de especies, y a su vez, modificar la transmisión de patógenos, en este trabajo se evaluó la presencia y la tasa de infección de Cryptosporidium spp. en heces de roedores silvestres en un área de la zona central de Chile donde existen extensas superficies de plantaciones forestales de pino Monterrey (Pinus radiata). Se analizó si la tasa de infección de Cryptosporidium spp. mostraba variación según distintas variables ambientales (tipo de hábitat, estación del año) y propias de los hospederos (e.g. diversidad de especies, especie de roedor, sexo). Se hipotetizó que las variaciones estructurales en poblaciones y comunidades de roedores silvestres modulan la tasa de infección de Cryptosporidium spp. Se muestrearon roedores silvestres en la Reserva Nacional Los Queules (Región del Maule) y zonas aledañas de plantaciones de pino Monterrey (Pinus radiata) durante el año 2016. Los sitios de muestreo correspondieron a bosque nativo, plantaciones de pino adulta y plantaciones de pino jóvenes. La detección 2 de ooquistes de Cryptosporidium spp. en las heces de los animales se realizó mediante la técnica modificada de tinción de Ziehl-Neelsen. De las 614 muestras analizadas, se detectaron un total de 29 positivas (4,7%) a Cryptosporidium spp., pertenecientes a heces de 4 especies nativas de roedores (5,5% Abrothrix longipilis; 4% Abrothrix olivaceous; 6,6% Oligoryzomys longicaudatus y 3,9% Phyllotis darwini). No se encontraron diferencias en la tasa de infección entre las especies de roedores, tampoco entre los distintos tipos de hábitats ni entre las distintas estaciones del año muestreadas. Se rechazó la hipótesis de esta tesis dado que no hubo evidencia clara en los análisis estadísticos de que la abundancia, riqueza y diversidad de especies puedan modular la tasa de infección de Cryptosporidium spp. Este hallazgo es el primer reporte de Cryptosporidium spp. en roedores silvestres en Chile. La tasa de infección fue comparativamente baja a otros estudios en roedores de otros países, los que pueden llegar a tasas de infección de hasta 68,5%. Las plantaciones de pino no estarían aumentando la tasa de infección de este parásito en roedores, ya que ésta se encontró en porcentajes parecidos en roedores del bosque nativo del área de estudio. Posiblemente otras variables no consideradas en este estudio podrían estar modulando la tasa de infección. Dada la presencia de Cryptosporidium spp. en roedores chilenos, futuros estudios sobre este protozoo en roedores deberían enfocarse en zonas más cercanas a asentamientos humanos para determinar posibles riesgos de infección en la interfaz humano-animales domésticos-animales silvestres / Cryptosporidium spp. (Family Cryptosporidiidae), is a group of protozoa and obligate intracellular parasite, which affect a wide range of vertebrates. Several studies sustain that Cryptosporidium spp. is the most prevalent gastrointestinal parasite in domestic animals and humans. In many studies, rodents have been described as hosts for Cryptosporidium spp., pointing that these mammals present the infection in a natural way. This turns them into a potential risk for public health, mainly because many rodents contribute to water, food and soil contamination as they leave their feces at every place they move around and feed. To our knowledge, there is no record of Cryptosporidium infection in wild rodents in Chile. Due to the fact that land use change can modify species structure and composition and also modify pathogen transmission, this work aimed to evaluate the presence and infection rate of Cryptosporidium spp. in wild rodents from an area of central Chile were vast plantations of Monterrey pine (Pinus radiata) exist. Cryptosporidium spp. infection rate was analyzed from faeces aiming to detect variation according to different environmental variables (habitat type and season) and host variables (e.g. species diversity, rodent species, gender). The hypothesis was that structural variations in rodent populations and communities modulate Cryptosporidium spp. infection rates. Wild rodents from Reserva Nacional Los Queules (Región del Maule) and surrounding areas of Monterrey pine plantations were sampled during 2016. Sampling sites were in native forest, adult pine plantations and young pine plantations. Cryptosporidium spp. oocyst detection in feces was performed by acid-fast method (Ziehl Neelsen). 4 Out of the 614 analyzed samples, 29 were positive to Cryptosporidium spp. (4.7%), belonging to 4 different native rodent species (5,5% Abrothrix longipilis, 4% Abrothrix olivaceous, 6,6% Oligoryzomys longicaudatus y 3,9% Phyllotis darwini). No differences were found on infection rates between rodent species, habitat type, or season. Thus, the hypothesis was rejected, because there was no clear evidence from statistic results that species’ abundance, richness, and diversity can modulate Cryptosporidium spp. infection rates. This finding is the first report of Cryptosporidium spp. in wild rodents in Chile. Infection rate was low compared to other studies in rodents from other different parts of the world, in which it can reach up to 68.5%. Pine plantations are not increasing the infection rates of this parasite in rodents as infection rate in these habitats were similar than the ones from native forest. Presumably other variables not considered in this work could be modulating the infection rate. Given the presence of Cryptosporidium spp. in Chilean wild rodents, future studies about this protozoa in wild rodents should focus in areas closer to human settlements, in order to determine possible risks of infection at the human-domestic animals-wildlife interface / Financiamiento: Proyecto FONDECYT Postdoctorado 3160037

El presente trabajo de tesis doctoral pretende indagar las siguientes hipótesis de trabajo: los estadios larvales de Contracaecum spp. e Hysterothylacium spp. experimentan migración hacia la musculatura estriada en las especies de peces estudiadas; los estadios larvales de Contracaecum spp. obtenidas de peces naturalmente infectados provocan lesiones en el tracto digestivo de mamíferos, provocando una respuesta inmune humoral y celular que puede caracterizarse según criterios hematológicos e histopatológicos. Para poner a prueba estas hipótesis se planteó como objetivo general determinar el rol que cumplen las larvas de Contracaecum spp. e Hysterothylacium spp. presentes en recursos pesqueros del sudoeste bonaerense, en el desarrollo de la anisakidosis humana. Los objetivos específicos del trabajo fueron: determinar el rango de hospedadores naturales de Contracaecum spp. e Hysterothylacium spp. en peces marinos y continentales, precisar el status sistemático de estos estadios, evaluar la capacidad miotrópica de los mismos en peces destinados al consumo humano y tipificar el daño tisular de los estadios larvales invasores determinando el poder infectivo en mamíferos, mediante el empleo de un modelo murino y analizando el efecto de esta parasitosis sobre los parámetros hemáticos en el mismo. Para el estudio se tomaron especies de peces de importancia comercial en la región: Cynoscion guatucupa, Odontesthes argentinensis y Odontesthes bonariensis. De un total de 316 ejemplares de peces examinados entre los años 2009-2011, 111 correspondieron a la especie C. guatucupa, 105 correspondieron a O. argentinensis y 100 a O. bonariensis. Se recolectaron larvas de Contracaecum sp. e Hysterothylacium sp. de la cavidad celómica de dichas especies. Las mismas fueron estudiadas histológica y morfológicamente. Se realizaron estudios moleculares de las larvas halladas en C. guatucupa, en los cuales se evidenció una asociación con especies de Contracaecum que parasitan mamíferos. Se tomaron muestras de filete de cada especie de pez, 3.316,85 g de C. guatucupa, 1.754,9 g de O. bonariensis y 1.327,1 g. de O. argentinensis, que fueron analizados mediante digestión enzimática. En el total de filetes analizados solo se encontraron 4 nematodes en O. bonariensis. Para tipificar el daño tisular de los estadios larvales encontrados y determinar el poder infestivo en los mamíferos se inocularon, siguiendo un protocolo de infestación, ratones de cepa Balb/cAnN de 30 g. de peso promedio. Se utilizaron técnicas histológicas de rutina y coloración de hematoxilina-eosina. Los resultados mostraron dos infestaciones positivas con larvas L3 pertenecientes a C. guatucupa. La histopatología evidenció la larva fijada al tubo digestivo, principalmente en la región fúndica del estómago, la cual penetró el epitelio de la mucosa gástrica llegando hasta la capa muscular. Se observó una compresión total del epitelio, con pérdida de la estructura glandular y adoptando un aspecto aplanado entorno a la larva. Un infiltrado leucocitario con presencia de monocitos, neutrófilos, eosinófilos y linfocitos, rodeó los tejidos próximos al sitio de penetración de la larva. En uno de los casos positivos de infestación se observó variación en la composición leucocitaria de la sangre de los ratones. Parte del material infestado fue fijado en glutaraldehído 2,5%, para la observación mediante microscopia electrónica de barrido. La identificación a nivel especifico empleando técnicas moleculares se realizó solo para las larvas de Contracaecum sp. provenientes de la pescadilla, C. guatucupa, debido a que fue la única especie de parasito de las ensayadas que produjo resultados positivos de infestación. Los análisis de las secuencias genómicas indican que éstas larvas se ubicaron en el mismo cluster junto a C. margolisi y C. ogmorhini, ambos parásitos de mamíferos marinos. Hasta tanto se disponga de mayor información a nivel genético del género Contracaecum se concluye que las larvas que produjeron lesión en la mucosa de ratones BALB /cAnN pertenecen al complejo Contracaecum ogmorhini s.l. De acuerdo con los resultados alcanzados hasta el momento, las larvas de Contracaecum ogmorhini s.l. infestivas para el modelo murino utilizado, potencialmente inducirían lesión en otros mamíferos, incluido el hombre. No obstante la exigua prevalencia e intensidad larval registrada en la carne de los pescados estudiados, sumado al hábito de cocción completa de los platos a base de filetes, permiten considerar de bajo riesgo epidemiológico a las especies estudiadas. / The aim of the present work is to test the following hypotheses: the larval stage of Contracaecum spp. and Hysterothylacium spp. migrate towards the musculature in the hosts fishes; the larval stage of Contracaecum spp. and Hysterothylacium spp. obtained of infected fishes caused injuries in the digestive tract of mammals, provoking humoral and cellular immune responses that can be characterized according to hematological and histophatological criteria. To test these hypotheses it was considered as a general objective to determine the role of Contracaecum spp. and Hysterothylacium spp. present in the southwest of Buenos Aires fishery resources in the development of human anisakidosis. The following specific purposes were added: to determine the range of natural hosts of Contracaecum spp. and Hysterothylacium spp. in marine and continental fishes, to confirm the systematic status of these stages, to evaluate the myotropic behavior of the helminths in fish used as human food and to assess the tissue damage of the larval stages invading the mucosa of the digestive tract and to analyzes the effect of this parasitosis on hematological parameters. Some fish species of commercial importance in the region Cynoscion guatucupa, Odontesthes argentinensis and Odontesthes bonariensis were considered to the study. A total of 316 fish individuals were analyzed between 2009 and 2011, 111 corresponded to C. guatucupa, 105 specimens to O. argentinensis and 100 to O. bonariensis. Contracaecum spp. and Hysterothylacium spp. larvae were collected from the visceral cavity of these fishes. They were studied histologically and morphologically. Fillet samples of each host were prepared and 3.316,85 grs were taken from C. guatucupa, 1.754,9 grs. From O. bonariensis and 1.327,1 grs from O. argentinensis, further analyzed by enzymatic digestion. Only four larval nematodes were found from all of the flesh examined. To assess the tissue damage and determine the infective power of the larval nematodes, they were inoculated per oss in mammals, following a protocol of infestation, using a strain of BALB / cAnN mice about 30 grs. in mean weight. There were performed histological sections stained with hematoxylin-eosin. The results showed two positive infestations with Contracaecum sp. L3 from C. guatucupa. Histopathology showed the larvae attached to the gastrointestinal tract, mainly in the fundic region of the stomach, penetrating the gastric mucosal epithelium and reaching the muscle layer. A total compression of the epithelium was observed, with loss of glandular structure and adopting a flattened appearance around the larva. A leukocyte infiltration with monocytes, neutrophils, eosinophils and lymphocytes surrounding the penetration site next larval tissues was observed. In the positive cases it was noted a variation in the blood leukocyte counts. Part of the infected material, fixed in glutaraldehide 2,5 %, was prepared for the observation under scanning electron microscopy. The identification at the specific level employing molecular technics was performed only on larvae of Contracaecum sp. proceeding from the stripped weakfish, Cynoscion guatucupa, because of these were the only that brought positive results in the experimental infections. The comparative analysis of the genomic sequences shows that these parasites cluster near the marine mammals parasites C. margolisi and C. ogmorhini. Until more information to the genetic composition of Contracaecum could be available, the larvae which provoking injuries in the mucosal tissue of BALB /cAnN mice belong to the taxonomical complex Contracaecum ogmorhini s.l. According to the results reached up to the moment using a murine model, the infective C. ogmorhini s.l. L3, could potentially induce damage to other mammals, including man. Nevertheless the exiguous prevalence and larval intensity found in the flesh, along with the habit of well done cooking food based on filet, allow consideras low epidemiological risk the anisakid species studied.

Hepatitis E virus (HEV) is an important human pathogen, with pigs and likely other animal species serving as natural reservoirs. There are currently four recognized HEV genotypes that infect humans within the genus Hepevirus of the family Hepeviridae. Genotypes 1 and 2 are human viruses that are associated with waterborne and fecal-oral transmission in developing countries, while genotypes 3 and 4 have been identified in humans and other animal species and are zoonotic and endemic in both industrialized and developing countries. In my dissertation research, we identified the first strain of HEV from rabbits in the United States. We subsequently determined the complete genome sequence of the virus. Phylogenetic analyses of the full-length sequence indicated that U.S. rabbit HEV is a distant member of the zoonotic genotype 3, thus raising a potential concern for zoonotic infection. In order to investigate the cross-species potential of rabbit HEV, we then determined its antigenic cross-reactivity with other animal strains of HEV. Additionally, we demonstrated that the novel rabbit HEV could cross species barriers and infect pigs under experimental conditions. Finally, we attempted to determine the risk factors and sources of foodborne HEV infection in the United States. We detected HEV for the first time from non-liver pork commercial products in the United States and demonstrated consumption of undercooked meat a risk factor for HEV infection. HEV sequences of genotype 3 origin were detected from pork products purchased from grocery stores in Southwest Virginia. Approximately 6.3% (21/335) of university students tested seropositive for HEV antibodies and, importantly, those with a history of consuming undercooked meats were 13 times more likely to be seropositive. These results further underscore the importance of cooking pork thoroughly and using proper hygiene when preparing meals. / Ph. D.

Hepatitis E virus (HEV) is the major cause of enterically transmitted non-A, non-B hepatitis in many developing countries, and is also endemic in many industrialized countries. Due to the lack of an effective cell culture system and a practical animal model, the mechanisms of HEV pathogenesis and replication are poorly understood. It has been speculated that HEV replicates in sites other than the liver. Since HEV is presumably fecal-orally transmitted it is unclear how the virus reaches the liver and extrahepatic replication could be a possible explanation. The recent identification of swine HEV from pigs affords us an opportunity to systematically study HEV replication in a swine model. We experimentally infected specific-pathogen-free (SPF) pigs with two strains of HEV: swine HEV and the US-2 strain of human HEV. Eighteen pigs (group 1) were each inoculated intravenously with swine HEV, nineteen pigs (group 2) with the US-2 strain of human HEV, and seventeen pigs (group 3) as uninoculated controls. To identify the potential extrahepatic sites of HEV replication using the swine model, two pigs from each group were necropsied at 3, 7, 14, 20, 27, and 55 days post inoculation (DPI). Thirteen different types of tissues and organs were collected from each necropsied animal. Reverse transcriptase PCR (RT-PCR) was used to detect the presence of positive strand HEV RNA in each tissue collected during necropsy at different DPIs. A negative strand-specific RT-PCR was standardized and used to detect the replicative, negative-strand of HEV RNA from tissues that tested positive for the positive strand RNA. As expected, positive strand HEV RNA was detected in almost every type of tissue at some time point during viremic period between 3 and 27 DPI. Positive-strand HEV RNA was still detectable in some tissues in the absence of serum HEV RNA from both swine and human HEV inoculated pigs. However, replicative, negative strand of HEV RNA was detected primarily in the small intestine, lymph nodes, colon, and liver. Our results demonstrate for the first time that HEV replicates in tissues other than the liver and that the gastrointestinal tract is also the target of virus infection. The data from this study may have important implications for HEV pathogenesis, xenotransplantation, and the development of an in vitro cell culture system for HEV. / Master of Science

Unravelling the complexities of a disease with multiple wildlife host and multiple tsetse vector species is no easy task. After over a century of field observations, experimental studies, anecdotal evidence and conjecture, the role of wildlife in the transmission of trypanosomes is still unclear. Recently, however, frameworks used in the studies of other vector-borne diseases with wildlife reservoirs showed that not only is it possible to understand transmission, but that spatio-temporal predictions of human disease risk and targeted control are realistic aims, even in such complex systems. This thesis explores the epidemiology of human African trypanosomiasis (HAT) in the Serengeti-Mara ecosystem in Northern Tanzania, where recent cases in tourists have highlighted the disease as a public health and economic concern. Assessment of the prevalence of trypanosome infections in different wildlife species is the first step in investigating the relative importance of different species in disease transmission. Identification of trypanosomes relies on sensitive and specific diagnostic tests. Polymerase chain reaction (PCR) protocols based on interspecies differences in the length of the ribosomal internal transcribed spacer (ITS) regions have been widely used in livestock to identify multiple trypanosome species in one PCR reaction. This study represents the first assessment of these protocols on blood samples collected from wildlife. Clonal sequence analysis of PCR products revealed a large range of trypanosomes circulating in wildlife, including Trypanosoma congolense, Trypanosoma brucei, Trypanosoma simiae Tsavo, Trypanosoma godfreyi and Trypanosoma vivax. In addition sequences similar to known sequences, termed Trypanosoma simiae-like and T. vivax-like trypanosomes, may reflect further diversity. However, further characterisation is needed before ITS protocols can be used widely for epidemiological studies in wildlife. The prevalence of T. brucei s.l. and T. congolense varied widely between species. This variation was predominantly explained by taxonomic classification, suggesting intrinsic differences in response to trypanosomes. Trypanosoma brucei rhodesiense, the subspecies responsible for HAT, was identified in lion, hyaena and reedbuck. Age significantly affected the prevalence of T. congolense in lion and hyaena, with the highest prevalence in subadults. The lack of statistically significant differences in prevalence between animals sampled live or after death confirmed that post-mortem sampling provides a method for increasing sample sizes in wildlife studies. The complex relationship between tsetse density and prevalence of trypanosome infections illustrated the difficulties of assessing data from diverse ecosystems with many potential confounding factors. A cross-sectional study of Glossina swynnertoni and Glossina pallidipes, the main tsetse species in Serengeti, highlighted the difficulties of integrating the results of microscopy and PCR to generate meaningful measures of the prevalence of transmissible T. brucei infections for epidemiological studies. However, PCR results suggested that G. pallidipes may be more important as a vector of T. brucei s.l. than has been previously recognised. Spatial variation in both tsetse density and the prevalence of trypanosome infections suggests human disease risk is heterogeneous. The results of this study, along with relevant literature, are considered within the context of frameworks used for other vector-borne diseases and the implications for disease management discussed.

Estandariza y evalúa una prueba ELISA casera para la detección de anticuerpos IgG anti T. gondii en población peruana. Se sometió a evaluación mediante un estuche referencial de ELISA mexicano, un total de 178 sueros de personas de Perú, los cuales fueron catalogados como positivos o negativos a la presencia de anticuerpos IgG anti T. gondii. Para la determinación del punto de corte del ELISA casero se emplearon curvas ROC y se determinaron los parámetros diagnósticos de la técnica; así como la correlación inter e intra ensayo. / Tesis

Ehrlichia chaffeensis es una bacteria intracelular obligatoria que pertenece a la familia Anaplasmataceae y es el agente causal de la Ehrlichiosis Monocítica Humana (EMH), una enfermedad con signos inespecíficos que se transmite en su mayoría mediante la mordedura de garrapatas de los géneros Amblyomma, Dermacentor y Rhipicephalus y mediante transfusiones sanguíneas de un individuo afectado a otro susceptible. En el presente estudio se detectó a través de PCR anidada, la frecuencia relativa de E. chaffeensis en propietarios de caninos domésticos con Ehrlichiosis. La toma de muestra se realizó en 20 distritos de la provincia de Lima y se encontraron muestras positivas en los distritos de Lurín, San Juan de Lurigancho, Comas, Chorrillos, Carabayllo y Ate, por lo que se concluye que E. chaffeensis está distribuida en pobladores de al menos seis distritos de la provincia de Lima. Se analizaron 60 muestras de sangre periférica y se encontraron diez positivos (10/60), lo que proporcionó una frecuencia relativa de 16.66%. El 80% (8/10) de las muestras positivas pertenecieron a individuos del sexo femenino y sólo el 20% (2/10) al sexo masculino. Palabras clave: Ehrlichia chaffeensis, Ehrlichiosis Monocítica Humana, PCR, PCR anidada. / --- E. chaffeensis is an obligately intracelullar bacteria in the family Anaplasmataceae and is the agent of the Human Monocytic Ehrlichiosis (HME), a disease with unspecific symptoms that is mainly transmitted through tick bites of the genera Amblyomma, Dermacentor and Rhipicephalus, and through blood transfusions from an infected to a sensitive person. In this study we detected a relative frecuency of E. chaffeensis in owners of domestic canines with Ehrlichiosis using Nested PCR. The sampling was done in 20 districts of the province of Lima and we found positive samples in the districts of Lurín, San Juan de Lurigancho, Comas, Chorrillos, Carabayllo and Ate, so we came to the conclusion that E. chaffeensis is distributed in at least six districts of the province of Lima. We tested 60 peripheral blood samples and we found ten positive samples, this gives us a 16.66% relative frecuency. 80% (8/10) of the positive samples belonged to females and 20% (2/10) belonged to males. Key words: Ehrlichia chaffeensis, Human Monocytic Ehrlichiosis, PCR, Nested PCR.

El objetivo del presente estudio fue determinar la presencia de seropositividad frente a Ehrlichia canis (E. canis) y Ehrlichia chaffeensis (E. chaffeensis) en médicos veterinarios o individuos que realizaban actividades veterinarias y que hayan estado en contacto con animales con Ehrlichiosis canina en el distrito de Lima Metropolitana, para lo cual se utilizó muestras de suero sanguíneo de 90 individuos con las características anteriormente mencionadas, 55 varones y 35 mujeres. Los 90 sueros fueron evaluados utilizando inmunofluorescencia indirecta (IFI), utilizando placas que contenían células DH82 (monocitos caninos) infectados con E. canis y E. chaffeensis además de sueros controles positivo y negativo. Se encontró que 21 (23.33 %) y 18 (20 %) sueros fueron positivos a E. canis y a E. chaffeensis, respectivamente. Además la seropositividad hallada para E. canis en hombres y mujeres fue de 21.8 % (12/55) y 25.7 % (9/35), respectivamente. Asimismo, la seropositividad hallada para E. chaffeensis en hombres y mujeres fue de 18.2 % (10/55) y 22.86 % (8/35), respectivamente; encontrando una ligera diferencia entre ambos, pero al igual que para E. canis, al realizar la prueba estadística Chi cuadrado, se llegó a determinar que no existe diferencia estadísticamente significativa, por lo tanto la seropositividad hallada es indistinta al sexo. Teniendo en cuenta que es una enfermedad zoonótica emergente y los resultados obtenidos, es recomendable iniciar estudios epidemiológicos y de vigilancia de la Ehrlichiosis en el Perú. Palabras claves: Ehrlichia canis, Ehrlichia chaffeensis, inmunofluorescencia indirecta, humanos. / --- The goal of the present research was to determinate the presence of seropositivity of Ehrlichia canis (E. canis) and Ehrlichia chaffeensis (E. chaffeensis) in veterinaries or subjects involved in veterinary activities and had been exposed to animals with canine ehrlichiosis in Lima, to which it was used 90 samples of serum blood of subjects with the previous characteristics, 55 males and 35 females. The 90 serums were evaluated using indirect immunofluorescence (IIF) using plates with OH82 cells (canine monocites) infected with E. canis and E. chaffeensis, and positive and negative controls. It was found that 21 (23,33%) and 18 (20%) serums were positive to E. canis and E. chaffeensis, respectively. The seropositivity of E. canis in males and females was 21,8% (12/55) and 25.7% (9/35), respectively. Moreover, the seropositivity found for E. chaffeensis in men and women was 18,2 % (10/55) and 22,86% (8/35), respectively; founding a smooth difference between both, but the same as for E. canis, when using Chi square test, no statistic difference was found, therefore the seropositivity found is indistinct to sex. Having in consideration that ehrlichiosis is a emergent zoonotic disease and the results found, is advisable to begin epidemiologic and control researches of ehrlichiosis in Peru. Key words: Ehrlichia canis, Ehrlichia chaffeensis, indirect immunofluorescence

Enterocytozoon bieneusi y Giardia duodenalis son considerados como los parásitos intestinales más comunes de seres humanos, animales domésticos, silvestres y en cautiverio. Estos patógenos son responsables de causar diarrea en individuos inmunocomprometidos e inmunocompetentes. El presente estudio tuvo por objetivo identificar los genotipos de E. bieneusi y G. duodenalis en muestras de heces de crías de alpaca. Las muestras de heces usadas en el estudio correspondieron a 126 crías de alpaca de 1 a 30 días de edad, de tres regiones geográficas en los andes del Perú (Huancavelica, Cuzco y Puno). Para el diagnostico se amplifico mediante PCR anidado la región espaciador transcrito interno (ITS) del gen ARNr de E. bieneusi, así como el locus triosafosfato isomerasa (TPI) de G. duodenalis. Todas las muestras positivas (E. bieneusi = 65; G. duodenalis = 42) fueron secuenciadas. Sesenta y cinco crías (51.6%) resultaron ser positivas en el PCR para E. bieneusi. Se encontró diez distintos genotipos de E. bieneusi en los aislamientos de crías de alpaca, seis de los aislamientos pertenecen a nuevos genotipos (ALP1, ALP2, ALP3, ALP4, ALP5 y ALP6). Cinco crías (7.7%) fueron positivos para el genotipo P, cuatro crías (6.2%) fueron positivos para el genotipo Tipo IV, dos (3.1%) al genotipo D, una (1.5%) al genotipo Beb6, 48 (73,8%) al genotipo ALP1, y cinco crías fueron positivos para cada genotipo nuevo (ALP2, ALP3, ALP4, ALP5 y ALP6). Así mismo, cuarenta y dos muestras fueron positivas para G. duodenalis. Los ensamblajes A (n = 33) y E (n = 9) fueron detectados en las crías. El ensamblaje A fue el más frecuentemente en crías de Puno y Huancavelica, mientras que el ensamblaje E se encontró en 8 crías en Cuzco y uno de Huancavelica. El papel potencial de E. bieneusi y G. duodenalis en el síndrome diarreico neonatal de la alpaca necesita ser seriamente considerada y evaluada, a pesar de no haber asociación entre la presencia de los parásitos y la presentación de diarrea. Palabras claves: Enterocytozoon bieneusi, Giardia duodenalis, alpaca, diarrea, zoonosis / --- Enterocytozoon bieneusi and Giardia duodenalis are considered the most common intestinal parasites found in humans and domestic, captive and wild animals. These pathogens are responsible for causing diarrhea in immunocompromised and immunocompetent individuals. The aim of the present study was to identify E. bieneusi and G. duodenalis genotypes in fecal samples from alpaca crias. Fecal samples were collected from 126 alpaca crias up to 30 days of age from three geographic regions in the highland of Peru (Huancavelica, Cuzco and Puno). The internal transcribed spacer (ITS) region of the rRNA gene of E. bieneusi, and the amplification of the triosephosphate isomerase (TPI) locus of G. duodenalis, were amplified for a nestedPCR. All positives samples were sequenced. Sixty-five crias (51.6%) were found to be PCR positive for E. bieneusi. Ten distinct genotypes of E. bieneusi were found in alpaca cria isolates, six of all isolates belong to novel genotypes (ALP1, ALP2, ALP3, ALP4, ALP5, and ALP6). Five (7.7%) crias were positive to genotype P, four (6.2%) crias were positive to genotype Type IV, two (3.1%) to genotype D, one (1.5%) to genotype Beb6, 48 (73.8%) to genotype ALP1, and five crias were positive each to new genotype (ALP2, ALP3, ALP4, ALP5, and ALP6). Forty-two samples were positive for G. duodenalis. Assemblages A (n=33) and E (n=9) were detected in crias, Assemblage A was the most frequently found in Puno and Huancavelica, while Assemblage E was found in 8 crias in Cuzco and one from Huancavelica. The potential role of E. bieneusi and G. duodenalis in the neonatal diarrhea syndrome of alpaca needs to be seriously considered and further evaluated, although had not association between the presence of parasites and diarrhea. Key words: Enterocytozoon bieneusi, Giardia duodenalis, alpaca, diarrhea, zoonosis.