Piroplasma la Necunoscuta Brazilia

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Veterinary Parasitology 134 (2005) 193213 www.elsevier.com/locate/vetpar

Hemorrhagic disease in dogs infected with an unclassied intraendothelial piroplasm in southern BrazilAlexandre Paulino Loretti a,*, Severo Sales Barros ba

Section of Veterinary Pathology, Department of Veterinary Clinical Pathology, Faculty of Veterinary Medicine, Federal University of Rio Grande do Sul (UFRGS), CEP 91540-000, Porto Alegre, RS, Brazil b Department of Animal Pathology, Faculty of Veterinary Medicine, Federal University of Pelotas (UFPel), CEP 96010-900, Pelotas, RS, Brazil Received 2 May 2005; received in revised form 4 July 2005; accepted 6 July 2005

Abstract A hemorrhagic disease affecting dogs in Brazil, referred to popularly as nambiuvu (bloody ears) and believed to be transmitted by ticks, has been observed in animals infected with an organism described originally in 1910 as a piroplasm, and known locally as Rangelia vitalli. In this series of 10 cases, the disease was characterized by anaemia, jaundice, fever, splenoand lymphadenomegaly, hemorrhage in the gastrointestinal tract, and persistent bleeding from the nose, oral cavity and tips, margins and outer surface of the pinnae. The ixodid ticks Rhipicephalus sanguineus and Amblyomma aureolatum infested affected dogs from suburban and rural areas, respectively. Laboratory ndings included regenerative anaemia, spherocytosis, icteric plasma and bilirubinuria. Those intracellular organisms were found in bone marrow smears but not in blood smears. Microscopically, zoites were seen within the cytoplasm of blood capillary endothelial cells. Parasitized and non-parasitized endothelial cells were positive immunohistochemically for von Willebrand factor (vWF). Langhans-type multinucleate giant cells were observed in the lymph nodes and choroid plexus. There was prominent erythrophagocytosis by macrophages in the lymph node sinuses and inltration of the medullary cords by numerous plasma cells. Ultrastructurally, this organism had an apical complex that included a polar ring and rhoptries but no conoid. This parasite was contained within a parasitophorous vacuole that had a trilaminar membrane with villar protrusions and was situated in the cytoplasm of capillary endothelial cells. This organism tested positive by immunohistochemistry for Babesia microti. This pathogen was also positive by in situ hybridization for B. microti. Tentative clinical diagnosis in these cases was based on the history, clinical picture, haemogram and favorable response to therapy, and conrmed through microscopic examination of smears from the bone marrow or histological sections of multiple tissues, especially lymph nodes where zoites were most frequently found. The disease was reproduced by intravenous inoculation of blood from a naturally infected dog into an experimental dog. The authors demonstrate in this study that this organism is a protozoa of the phylum Apicomplexa, order Piroplasmorida. This piroplasm seems to be different from

* Corresponding author. Present address: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1. Tel.: +1 519 824 4120x54674; fax: +1 519 824 5930. E-mail address: aloretti@uoguelph.ca (A.P. Loretti). 0304-4017/$ see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.07.011


A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193213

Babesia since it has an intraendothelial stage. Molecular phylogenetic analysis is necessary to better characterize this parasite and clarify its taxonomic status. # 2005 Elsevier B.V. All rights reserved.Keywords: Rangelia vitalli; Protozoa; Apicomplexa; Piroplasmorida; Dog; Brazil

1. Introduction Nambiuvu (bloody ears), peste de sangue (bleeding plague) or febre amarela dos caes (yellow fever of dogs) is a disease that commonly affects dogs from rural and suburban areas in Brazil. Over the years, this malady has been associated with an unclassied organism that occurs within endothelial cells and erythrocytes. The original reference to this disease comes from 1908. In a short communication published by Antonio Carini about the most common infectious and parasitic diseases of domestic animals in Brazil at that time, he mentioned observing a disease of dogs called nambiuvu. It was suspected to be caused by a piroplasm since it was clinically similar to malignant jaundice (canine babesiosis), a disease that hadnt been described yet in Brazil at that time (Carini, 1908). Two years later, in 1910, Bruno Rangel Pestana published two scientic papers characterizing the morphology of this unusual protozoan parasite as seen under the light microscope, and describing the epidemiological, clinical and pathological aspects of the disease caused by this atypical piroplasm. He proposed the new taxonomic name Piroplasma vitalli since this was a hitherto undescribed piroplasm. He also used this parasite species name to pay a tribute of respect to his mentor, the brazilian biomedical scientist Vital Brazil, internationally renowned for the discovery of the polyvalent anti-ophidic serum used to treat bites of venomous snakes (Pestana, 1910a, 1910b). In 1914, Antonio Carini and Jesuno Maciel published a paper together about this disease in which they proposed that the name of this previously unidentied canine piroplasm should be changed to Rangelia vitalli to honor the investigator Bruno Rangel Pestana, who rst observed the presence of this organism within endothelial cells and red blood cells of Brazilian dogs affected by nambiuvu. In this publication, they reemphasized that this was a new species of canine piroplasm, and that it should be included in a separate genus (Carini and Maciel, 1914).

The popular name nambiuvu was coined in the past by Brazilian aboriginal inhabitants in reference to blood dripping continuously from the tips, margins and outer surface of both pinnae, a clinical sign usually observed in this illness. Categories of dogs affected by this pathogen include hunting dogs, herding dogs, search dogs, police dogs, guard dogs and companion dogs. Ixodid ticks have been implicated as the natural vectors of this organism since cases of infection by this protozoa have been consistently associated with the presence of these ectoparasites on the host or in the environment (or both). However, there are no published studies to date showing that ticks can transmit this protozoal pathogen to dogs. The disease may occur at any time of the year, although peak occurrence is usually observed during the summer and is associated with the presence of large populations of ticks. Recovered patients develop a strong immunity against this protozoal parasite but still the organism can persist for months in these clinically normal, cured animals which can act as healthy carriers of the pathogen. The host range of this protozoa seems to be restricted to domestic dogs since other mammals, birds or laboratory animals cannot be infected experimentally by this parasite (Pestana, 1910a, 1910b; Carini and Maciel, 1914). There is no consensus about the life cycle and taxonomic status of this organism at this time. It is described that its life cycle consists of an intraerythrocytic developmental phase (blood stage), and an extraerythrocytic phase occurring in the cytoplasm of endothelial cells (tissue stage). It is uncommon to nd this protozoa in blood smears in both natural and experimental cases. According to the literature, the intraerythrocytic form of this parasite is most often seen in very low numbers in blood smears if blood is drawn during an episode of high fever in the acute stage of the disease (Pestana, 1910a, 1910b; Carini and Maciel, 1914). Some researchers have found this organism only within parasitophorous vacuoles in the

A.P. Loretti, S.S. Barros / Veterinary Parasitology 134 (2005) 193213


cytoplasm of endothelial cells from blood capillaries but not within red blood cells (Krauspenhar et al., 2003). This unclassied organism has been misidentied as Toxoplasma gondii (Wenyon, 1926; Moreira, 1938) and as Leishmania donovani (Pocai et al., 1998) in histological sections, and misdiagnosed as Babesia canis (Wenyon, 1926; Moreira, 1938; Levine, 1973) in blood lms. Some authors claim that cases of nambiuvu of dogs described by other investigators as being caused by R. vitalli (Pestana, 1910a, 1910b; Carini and Maciel, 1914) were, in fact, dual infections by B. canis and T. gondii, but provide no substantial data to support this statement (Wenyon, 1926; Moreira, 1938; Paraense and Vianna, 1948; Levine, 1973). Recently, this issue was revisited by a group of investigators (Krauspenhar et al., 2003) as a retrospective study of cases of infection with this unclassied organism that, during the 1980s and 1990s, were mistaken for cases of canine visceral leishmaniasis (Pocai et al., 1998). In areas where this disease is common, such as in the State of Rio Grande do Sul (RS), southern Brazil, any dog with high fever, anaemia, jaundice and hemorrhages and infested by ticks is suspected of being infected by this unclassied pathogen. There may be sufcient justication for treatment even without blood examination being made or in the absence of demonstrable organisms in the peripheral blood since this organism is very difcult to recover in blood smears, especially in the chronic form of the disease. In many cases, the diagnosis may need to be based on the animals positive response to antiprotozoal therapy. Supportive evidence for a diagnosis of nambiuvu in dogs include lymphadenopathy, splenomegaly, bleeding tendencies (Pestana, 1910a, 1910b; Carini and Maciel, 1914; Braga, 1935; Carini, 1948), a haemogram consistent with immunemediated haemolytic anaemia (IMHA) (Krauspenhar et al., 2003), increased amounts of bilirubin in the serum, and the presence of R. sanguineu