Molecular fluorescent approach to assessing intraerythrocytic hemoprotozoan Babesia canis infection in dogs

Bicalho, Kelly Alves; Ribeiro, Múcio Flávio Barbosa; Martins‐Filho, Olindo Assis

doi:10.1016/j.vetpar.2004.08.009

Cited by 15 publications

(13 citation statements)

References 11 publications

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“…Flow cytometric assays avoid these limitations. Hydroethidine has been used in studies of B. bovis-and B. canis-infected red blood cells (2,32). This assay relies on the uptake and metabolic conversion of hydroethidine into ethidium by live parasites.…”

Section: Discussionmentioning

confidence: 99%

“…This assay relies on the uptake and metabolic conversion of hydroethidine into ethidium by live parasites. Because conversion does not occur in reticulocytes (2), this assay fails to detect those Babesia species that prefer reticulocytes, such as B. gibsoni (7). In the present study, we adapted the flow cytometric assay that Barkan et al (1) developed to assess parasitemia in mouse models of malaria.…”

Section: Discussionmentioning

confidence: 99%

“…Although considered the gold standard of detection, this test is not ideally suited to quantitatively distinguish reticulocytes from erythrocytes. Inspired by major advances in the diagnosis of malaria using fluorescent nucleic acid stains, flow cytometric assays were developed to assess the viability and growth of B. bovis in red blood cells in vitro (32) and to quantify the percentage of red blood cells infected with B. canis or B. gibsoni in naturally (2) or experimentally infected dogs (7). Although flow cytometry is amenable to multiple and simultaneous detection of surface and intracellular molecules, these studies did not attempt to distinguish reticulocytes from erythrocytes.…”

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Babesia microti Primarily Invades Mature Erythrocytes in Mice

et al. 2006

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Babesia microti is a tick-borne red blood cell parasite that causes babesiosis in people. Its most common vertebrate reservoir is the white-footed mouse. To determine whether B. microti invades reticulocytes, as does the canine pathogen B. gibsoni, we infected the susceptible inbred mouse strains C.B-17.scid and DBA/2 with a clinical isolate of B. microti. Staining of fixed permeabilized red blood cells with 4,6-diamidino-2-phenylindole or YOYO-1, a sensitive nucleic acid stain, revealed parasite nuclei as large bright dots. Flow cytometric analysis indicated that parasite DNA is primarily found in mature erythrocytes that expressed Babesia antigens but not the transferrin receptor CD71. In contrast, CD71-positive reticulocytes rarely contained Babesia nuclei and failed to express Babesia antigens. Accordingly, the frequency of YOYO-1-positive, CD71-negative cells strongly correlated with parasitemia, defined as the frequency of infected red blood cells assessed on Giemsastained blood smears. Importantly, the absolute numbers generated by the two techniques were similar. Parasitemia was modest and transient in DBA/2 mice but intense and sustained in C.B-17.scid mice. In both strains, parasitemia preceded reticulocytosis, but reticulocytes remained refractory to B. microti. In immunocompetent C.B-17 mice, reticulocytosis developed early, despite a marginal and short-lived parasitemia. Likewise, an early reticulocytosis developed in resistant BALB/cBy and B10.D2 mice. These studies establish that B. microti has a tropism for mature erythrocytes. Although reticulocytes are rarely infected, the delayed reticulocytosis in susceptible strains may result from parasite or host activities to limit renewal of the mature erythrocyte pool, thereby preventing an overwhelming parasitemia.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Babesia microti Primarily Invades Mature Erythrocytes in Mice

et al. 2006

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“…Serum samples were submitted to indirect immunofluorescence assay (IFA) with antigens of B. canis vogeli (blood smears from splenectomized dogs that were experimentally infected in our lab) according to Bicalho et al (2004), using a screening dilution of 1:64. To detect antibodies against E. canis, the bacteria were cultivated in DH82 cells, as described by Aguiar et al (2007a), and serum samples were analyzed following the protocol by Silva et al (2010), but with a screening dilution of 1:80.…”

Section: Methodsmentioning

confidence: 99%

Serosurvey for tick-borne diseases in dogs from the Eastern Amazon, Brazil

Spolidorio

Minervino

Valadas

et al. 2013

Rev. Bras. Parasitol. Vet.

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Canine ehrlichiosis and babesiosis are the most prevalent tick-borne diseases in Brazilian dogs. Few studies have focused attention in surveying tick-borne diseases in the Brazilian Amazon region. A total of 129 blood samples were collected from dogs living in the Brazilian eastern Amazon. Seventy-two samples from dogs from rural areas of 19 municipalities and 57 samples from urban stray dogs from Santarém municipality were collected. Serum samples were submitted to Indirect Immunofluorescence Assay (IFA) with antigens of Babesia canis vogeli, Ehrlichia canis, and six Rickettsia species. The frequency of dogs containing anti-B. canis vogeli, anti-E. canis, and anti-Rickettsia spp. antibodies was 42.6%, 16.2%, and 31.7%, respectively. Anti-B. canis vogeli antibodies were detected in 59.6% of the urban dogs, and in 29.1% of the rural dogs (P < 0.05). For E. canis, seroprevalence was similar among urban (15.7%) and rural (16.6%) dogs. For Rickettsia spp., rural dogs presented significantly higher (P < 0.05) prevalence (40.3%) than urban animals (21.1%). This first study on tick-borne pathogens in dogs from the Brazilian eastern Amazon indicates that dogs are exposed to several agents, such as Babesia organisms, mostly in the urban area; Spotted Fever group Rickettsia organisms, mostly in the rural area; and Ehrlichia organisms, in dogs from both areas studied.Keywords: Ehrlichia, Babesia, Rickettsia, dogs, Amazon, Pará state. ResumoEhrliquiose canina e babesiose canina são as doenças parasitárias transmitidas por carrapatos de maior prevalência em cães do Brasil. Poucos estudos pesquisaram doenças transmitidas por carrapatos na região da Amazônia brasileira. Um total de 129 amostras de sangue foram colhidas de cães da Amazônia oriental brasileira. Setenta e dois cães eram de áreas rurais de 19 municípios do Estado do Pará, e 57 amostras foram colhidas de cães errantes vadios da área urbana do município de Santarém-PA. As amostras de soro foram submetidas ao ensaio de imunofluorescência indireta, com antígenos de Babesia canis vogeli, Ehrlichia canis, e seis espécies de Rickettsia. A frequência de cães com anticorpos anti-B. canis vogeli, anti-E. canis, e anti-Rickettsia spp. foi de 42,6%, 16,2% e 31,7%, respectivamente. Anticorpos anti-B. canis vogeli foram detectados em 59,6% dos cães urbanos, e em 29,1% dos cães rurais (P < 0.05). Para E. canis, a soroprevalência foi parecida entre os cães urbanos (15,7%) e rurais (16,6%). Para Rickettsia spp., cães rurais apresentaram prevalência (P < 0.05) significativamente maior (40,3%) do que os cães urbanos (21,1%). Esse primeiro estudo sobre agentes transmitidos por carrapatos entre cães da Amazônia oriental brasileira indica que estes animais estão expostos a vários agentes. Estes incluem Babesia principalmente na área urbana, Riquétsias do grupo da Febre Maculosa principalmente nas áreas rurais, e Erliquia em cães de ambas as áreas, rural e urbana.

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“…To detect anti-B. canis vogeli antibodies, sera were tested by IFAT in house using B. canis vogeli strain Belo Horizonte antigen (15) with a cut-off titer of 1:40 (16) .…”

Section: Serological Methodsmentioning

confidence: 99%

Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

Krawczak

Reis

Silveira

et al. 2015

Rev. Soc. Bras. Med. Trop.

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Introduction:The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites. Methods: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofl uorescence antibody test (IFAT) and by antiLeishmania immunoglobulin G (IgG) antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP) (Bio Manguinhos®/FIOCRUZ/MS) and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios). Results: Of 96 serum samples submitted to serological assays, 4 (4.2%) were positive for Leishmania as determined by ELISA; 12 (12.5%), by IFAT; 14 (14.6%) by rK39 RDT; and 20 (20.8%), by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9%) and 30/96 (31.2%) samples, respectively. No signifi cant association was identifi ed between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05). Conclusions: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil); we also found that the serological tests that were used did not cross-react.

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Molecular fluorescent approach to assessing intraerythrocytic hemoprotozoan Babesia canis infection in dogs

Cited by 15 publications

References 11 publications

Babesia microti Primarily Invades Mature Erythrocytes in Mice

Babesia microti Primarily Invades Mature Erythrocytes in Mice

Serosurvey for tick-borne diseases in dogs from the Eastern Amazon, Brazil

Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

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