Ehrlichia canis detection in dogs from Várzea Grande: a comparative analysis of blood and bone marrow samples

ABSTRACT: The objective of this study was to compare the DNA detection of Ehrlichia canis in blood and bone marrow to determine the prevalence of the agent in Várzea Grande, Mato Grosso. Blood samples and bone marrow from 80 dogs of both sexes, different breeds and age, were collected and processed for a cross-sectional study performed using nested PCR. Of the 80 dogs, 61 (76.3%) had E. canis DNA in one of the samples. The buffy coat was positive in 42 dogs (52.5%) and the bone marrow was positive in 33 (41.3%). There was no significant association between the positive biological samples of either the buffy coat or bone marrow and the presence or absence of clinical signs (P=0.49). No risk factor was associated with infection in the studied area. The bone marrow samples were efficient for the molecular diagnosis of canine ehrlichiosis, particularly when there was a negative blood sample, although infection was present.

RESUMO: Este trabalho teve por objetivo comparar a detecção de DNA de Ehrlichia canis em amostras de sangue e medula óssea, além de determinar a ocorrência do agente em Várzea Grande, Mato Grosso. Amostras de sangue e medula óssea de 80 cães, de ambos os sexos, diferentes raças e idade, foram coletados em estudo seccional e processados para realização de nested PCR. Dos 80 cães, 61 (76,3%) apresentaram DNA de E. canis em uma das amostras pesquisadas. A capa leucocitária foi positiva em 42 (52,5%) e a medula óssea em 33 (41,3%). Não foi observada associação significativa com a positividade das amostras biológicas, sangue ou medula óssea, e a presença ou ausência de sinais clínicos (P=0,49). Nenhum fator de risco foi associado à infecção na área pesquisada. A amostra de medula óssea mostrou-se bom sítio para o diagnóstico molecular da erliquiose canina, principalmente quando da infecção com negatividade da amostra sanguínea.

Ehrlichia canis detection in dogs from Várzea Grande: a comparative analysis of blood and bone marrow samples

ABSTRACT: The objective of this study was to compare the DNA detection of Ehrlichia canis in blood and bone marrow to determine the prevalence of the agent in Várzea Grande, Mato Grosso. Blood samples and bone marrow from 80 dogs of both sexes, different breeds and age, were collected and processed for a cross-sectional study performed using nested PCR. Of the 80 dogs, 61 (76.3%) had E. canis DNA in one of the samples. The buffy coat was positive in 42 dogs (52.5%) and the bone marrow was positive in 33 (41.3%). There was no significant association between the positive biological samples of either the buffy coat or bone marrow and the presence or absence of clinical signs (P=0.49). No risk factor was associated with infection in the studied area. The bone marrow samples were efficient for the molecular diagnosis of canine ehrlichiosis, particularly when there was a negative blood sample, although infection was present.

RESUMO: Este trabalho teve por objetivo comparar a detecção de DNA de Ehrlichia canis em amostras de sangue e medula óssea, além de determinar a ocorrência do agente em Várzea Grande, Mato Grosso. Amostras de sangue e medula óssea de 80 cães, de ambos os sexos, diferentes raças e idade, foram coletados em estudo seccional e processados para realização de nested PCR. Dos 80 cães, 61 (76,3%) apresentaram DNA de E. canis em uma das amostras pesquisadas. A capa leucocitária foi positiva em 42 (52,5%) e a medula óssea em 33 (41,3%). Não foi observada associação significativa com a positividade das amostras biológicas, sangue ou medula óssea, e a presença ou ausência de sinais clínicos (P=0,49). Nenhum fator de risco foi associado à infecção na área pesquisada. A amostra de medula óssea mostrou-se bom sítio para o diagnóstico molecular da erliquiose canina, principalmente quando da infecção com negatividade da amostra sanguínea.

Erliquiose canina - relato de caso / Ehrlichiosis canine - case report

Canine ehrlichiosis is a disease caused by species of genus Ehrlichia with great importance in veterinary. The clinical signs as well as haematological changes are variable and it is necessary to use other more specific diagnostic methods. The present work reports the case of a female dog, SRD, presenting hyperthermia, convulsion, uveitis, nystagmus, myoclonus, ataxia, prostration and lymphadenitis. The necropsy report was suggestive of ehrlichiosis.

Erliquiose canina - relato de caso / Ehrlichiosis canine - case report

Canine ehrlichiosis is a disease caused by species of genus Ehrlichia with great importance in veterinary. The clinical signs as well as haematological changes are variable and it is necessary to use other more specific diagnostic methods. The present work reports the case of a female dog, SRD, presenting hyperthermia, convulsion, uveitis, nystagmus, myoclonus, ataxia, prostration and lymphadenitis. The necropsy report was suggestive of ehrlichiosis.(AU)

Ehrlichia canis detection in dogs from Várzea Grande: a comparative analysis of blood and bone marrow samples / Detecção de Ehrlichia canis em cães domiciliados em Várzea Grande: análise comparativa entre amostras de sangue e medula óssea

The objective of this study was to compare the DNA detection of Ehrlichia canis in blood and bone marrow to determine the prevalence of the agent in Várzea Grande, Mato Grosso. Blood samples and bone marrow from 80 dogs of both sexes, different breeds and age, were collected and processed for a cross-sectional study performed using nested PCR. Of the 80 dogs, 61 (76.3%) had E. canis DNA in one of the samples. The buffy coat was positive in 42 dogs (52.5%) and the bone marrow was positive in 33 (41.3%). There was no significant association between the positive biological samples of either the buffy coat or bone marrow and the presence or absence of clinical signs (P=0.49). No risk factor was associated with infection in the studied area. The bone marrow samples were efficient for the molecular diagnosis of canine ehrlichiosis, particularly when there was a negative blood sample, although infection was present.(AU)

Este trabalho teve por objetivo comparar a detecção de DNA de Ehrlichia canis em amostras de sangue e medula óssea, além de determinar a ocorrência do agente em Várzea Grande, Mato Grosso. Amostras de sangue e medula óssea de 80 cães, de ambos os sexos, diferentes raças e idade, foram coletados em estudo seccional e processados para realização de nested PCR. Dos 80 cães, 61 (76,3%) apresentaram DNA de E. canis em uma das amostras pesquisadas. A capa leucocitária foi positiva em 42 (52,5%) e a medula óssea em 33 (41,3%). Não foi observada associação significativa com a positividade das amostras biológicas, sangue ou medula óssea, e a presença ou ausência de sinais clínicos (P=0,49). Nenhum fator de risco foi associado à infecção na área pesquisada. A amostra de medula óssea mostrou-se bom sítio para o diagnóstico molecular da erliquiose canina, principalmente quando da infecção com negatividade da amostra sanguínea.(AU)

LEISHMANIOSE E ERLIQUIOSE CANINA: UMA ABORDAGEM EPIDEMIOLÓGICA E CLÍNICO-LABORATORIAL

O íntimo contato e a domesticação tornou os cães a espécie com maior número de doenças semelhantes aos humanos. A leishmaniose visceral (LV) causada por protozoários Leishmania infantum é uma doença negligenciada com letalidade próximo a 10%. No Brasil, o ciclo da LV é zoonótico e os cães sãoreservatórios urbanos. Casos de leishmaniose visceral canina (LVC) são constantes nos centros urbanos brasileiros e os índices epidemiológicos de LV estão correlacionados com os de LVC. Medidas de prevenção e controle são realizados para prevenir e controlar a LV além de eutanásia de reservatórios positivos. Aerliquiose canina (EC) é uma doença causada por bactérias do gênero Ehrlichia, potencialmente fatal e zoonótica. Essas duas doenças são observadas frequentemente em comorbidade nos cães. Este trabalho visa abordar três situações: a) definir fatores de risco para LVC em área endêmica no Brasil. b) avaliar métodos de diagnóstico para EC na clínica veterinária; e c) relatar um caso de comorbidade LVC, EC e dirofilariose. O estudo transversal analítico foi realizado com 654 cães, destes 521 foram inclusos no modelo de regressão logística múltipla incluindo as seguintes variáveis: sexo, idade, tamanho, positividade para EC, estrato social (ES: 1 a 7), local que dorme, comprimento e cor da pelagem. A análise de regressão logística mostrou que cães de estrato social menos favorecido (OD: 1.39, IC 95%: 1.15 - 1.70, p <0,001),positivos para erliquiose (OD: 2.01, IC 95%: 1.20 - 3.44, p < 0,01), que dormemno quintal (OD: 2.32, IC 95%: 1.44 - 3.79, p < 0,01) e que cães de grande porte (OD: 1.82, IC 95%: 1.03 - 3.15, p < 0,05) tem mais chances de serem positivos para LVC. O segundo trabalho avaliou 52 cães por quatro métodos de diagnóstico para EC comparados pelo modelo de Análise de Classe Latente: citologia de medula óssea (BMC), contagem de plaquetas (PC), teste rápido imunocromatográfico Alere® (Alere) e PCR de sangue periférico (PCR). BMC e PCR apresentaram sensibilidade de 100%, e especificidade de 100% e 64%, respectivamente, enquanto PC teve uma performance baixa, com sensibilidade de 63% e especificidade de 75%. Alere obteve performance moderada com sensibilidade de 81% e especificade de 78%. A concordância dos testes foi calculada pelo coeficiente de kappa, sendo considerada razoável (0,38). Concluiu-se que o diagnóstico para EC exige uma anamnese detalhada somada ao exame físico para determinar a combinação certa de métodos de diagnóstico aplicada. No relato de caso, um cão foi diagnosticado com LVC, EC e dirofilariose em um check-up que permitiu a detecção precoce das comorbidades. Um protocolo terapêutico foi estabelecido, e após 12 meses de tratamento o animal obteve alta clínica com exames negativos. Concluiu-se que EC,estrato social familiar, o local onde o cão dorme e o tamanho do animal são fatores derisco para LVC. Esses fatores podem ser evitados com medidas simples somados à visita veterinária preventiva periódica que permite a detecção dessas comorbidades precocemente promovendo a saúde única, ou seja, a vida dos cães e das famílias multiespécie.

The close contact and antique domesticationoff dogs make them the species with the higher number of diseases like humans. In this context, visceral leishmaniasis (LV), caused by Leishmania infantum protozoans, is a neglected disease with high lethality rate when untreated. In Brazil, LV cycle is zoonotic, and dogs are considered one urban reservoir. Canine visceral leishmaniasis cases (LVC) are constantly found in Brazilian urban regions and epidemiological rates of LV and LVC are correlated. Several studies and measures are done to prevent and control LV, including positive reservoir control by euthanasia. Additionally, canine ehrlichiosis (EC) is an infectious diseases caused by Ehrlichiagender bacteria, potentially fatal and zoonotic. These two diseases are frequently observed in comorbidity, worsening clinical signs, treatment, and prognosis.This work aims three situations: a) describe risk factors for LVC in endemic area of Brazil; b)evaluate diagnostic methods for EC in the veterinary clinic; and c) to report one case of LVC, EC and heartworm comorbidity. In the first study, a cross-sectional study was performed with 654 dogs. From those, 521 were included in the multiple logistic regression model with LVC as the outcome and including the following variables: sex, age, size, EC positivity, families social stratum (ES: 1 to 7), sleeping location, fur length and color. Our results showed that on dog positive for ehrlichiosis (OD: 2.01, IC 95%: 1.20 - 3.44, p < 0,01), from lower social stratum (OD: 1.39, IC 95%: 1.15 - 1.70, p < 0,001), that sleep outside the house (OD: 2.32, IC 95%: 1.44 - 3.79, p < 0,01), and large dogs (OD: 1.82, IC 95%: 1.03 - 3.15, p < 0,05) have more chances to be seropositive for LVC. The second work tested 52 dogs byfour diagnostic methods for EC comparedby Latent Class Analysis model: bone marrow cytology (BMC), platelet count (PC), rapid immunochromatographic Alere® test (Alere) and total blood PCR (PCR). BMC and PCR had 100% sensibility, and 100%, 64% specificity respectively, meanwhilePC had low sensibility of 63% and specificity of 75%. Alere had moderate sensibility of 81% and specificity of 78%. Agreement between tests were calculated fair by kappas coefficient (0,38).The case report consists of one dog diagnosticated with heart worm, LVC and EC back in 2014 during annual check-up by veterinary practitioner. One therapeutic protocol was established and after 12 months of treatment, the animal was discharged from the clinic with negative tests.It was concluded that EC, family social strata, the place where the dog sleeps and the animal's size are risk factors for LVC. These factors can be avoided with simple measures, additionally, the periodic preventive veterinary visit allows the early detection of these comorbidities, promoting One Health, which is, the lives of dogs and multispecies families.

Acute and subclinical Ehrlichia canis infection and cortisol response to ACTH stimulation test

Adrenocortical disturbances are associated with canine ehrlichiosis due to the immunological changes caused by infection and consequent inflammation. Thus, this study aimed to evaluate the occurrence of adrenocortical hormonal changes in dogs naturally infected with Ehrlichia canis (n=21) as confirmed by the presence of anti-E. canis antibodies (Dot-ELISA) and nested PCR (nPCR). Serum cortisol concentrations were assessed by radioimmunoassay before and one hour after ACTH stimulation. Ten healthy dogs were subjected to the same stimulation protocol and used as controls. The results revealed that the dogs with naturally acquired acute and subclinical ehrlichiosis secreted cortisol following ACTH stimulation in similar concentrations to those of healthy dogs.

Acute and subclinical Ehrlichia canis infection and cortisol response to ACTH stimulation test

Adrenocortical disturbances are associated with canine ehrlichiosis due to the immunological changes caused by infection and consequent inflammation. Thus, this study aimed to evaluate the occurrence of adrenocortical hormonal changes in dogs naturally infected with Ehrlichia canis (n=21) as confirmed by the presence of anti-E. canis antibodies (Dot-ELISA) and nested PCR (nPCR). Serum cortisol concentrations were assessed by radioimmunoassay before and one hour after ACTH stimulation. Ten healthy dogs were subjected to the same stimulation protocol and used as controls. The results revealed that the dogs with naturally acquired acute and subclinical ehrlichiosis secreted cortisol following ACTH stimulation in similar concentrations to those of healthy dogs.(AU)

Serum TNF- and IL-10 in Ehrlichia spp. naturally infected dogs

Background: Canine ehrlichiosis (CE) is a disease caused by Ehrlichia canis, transmitted by Rhipicephalus sanguineus and dogs can present variable clinical manifestations, ranging from subclinical, acute to chronic illness. This pathogen preferentially infects leukocytes. In Brazil illness is described nationwide with the seropositivity varying from 4.8% to over 50%. The control of Ehrlichia spp. infection was related to cellular immune response mediated by Th1 while the CE acute phase is related to humoral immune response mediated by Th2, with high production of antibodies. Th1 and Th2 balance can be measured by cytokine profile. TNF-α is associated with inflammatory responses while IL-10 inhibits mechanisms which prevent tissue damage caused by the intense inflammatory response. The aim of this study was to evaluate the TNF-α and IL-10 serum levels in naturally infected dogs by Ehrlichia spp.Material, Methods & Results: Twenty five dogs, both sexes, one to eight years old, weighing 5 to 40 kg, undefined breed were divided in two groups: Control (CT, n = 14) and naturally infected by Ehrlichia spp. dogs (EH, n = 11). Naturally infected dogs were from Zoonosis Control Center. Ehrlichia spp. morulae was visualized in bone marrow aspirates. Blood samples were collected for hematological analysis and serum dosages of TNF-α and IL-10 (commercial kits by sandwich ELISA). The frequency of clinical signs was expressed as percentage. T test, Spearman correlation and linear regression were used when necessary (P ≤ 0.05). Alopecia (45%), generalized lymphadenopathy (36%), onychogryphosis (27%) and conjunctivitis (27%) were the most frequent clinical signs in the EH group. No difference was found between studied groups in hematological and biochemical parameters. TNF-α concentration was 43.3 ± 16.9 pg/mL in CT and 41.1 ± 3.8 pg/mL in EH, while IL-10 concentration was 20.2 ± 8.4 pg/mL in CT and 21.0 ± 5.1 pg/mL in EH.[...](AU)

Serum TNF- and IL-10 in Ehrlichia spp. naturally infected dogs

Background: Canine ehrlichiosis (CE) is a disease caused by Ehrlichia canis, transmitted by Rhipicephalus sanguineus and dogs can present variable clinical manifestations, ranging from subclinical, acute to chronic illness. This pathogen preferentially infects leukocytes. In Brazil illness is described nationwide with the seropositivity varying from 4.8% to over 50%. The control of Ehrlichia spp. infection was related to cellular immune response mediated by Th1 while the CE acute phase is related to humoral immune response mediated by Th2, with high production of antibodies. Th1 and Th2 balance can be measured by cytokine profile. TNF-α is associated with inflammatory responses while IL-10 inhibits mechanisms which prevent tissue damage caused by the intense inflammatory response. The aim of this study was to evaluate the TNF-α and IL-10 serum levels in naturally infected dogs by Ehrlichia spp.Material, Methods & Results: Twenty five dogs, both sexes, one to eight years old, weighing 5 to 40 kg, undefined breed were divided in two groups: Control (CT, n = 14) and naturally infected by Ehrlichia spp. dogs (EH, n = 11). Naturally infected dogs were from Zoonosis Control Center. Ehrlichia spp. morulae was visualized in bone marrow aspirates. Blood samples were collected for hematological analysis and serum dosages of TNF-α and IL-10 (commercial kits by sandwich ELISA). The frequency of clinical signs was expressed as percentage. T test, Spearman correlation and linear regression were used when necessary (P ≤ 0.05). Alopecia (45%), generalized lymphadenopathy (36%), onychogryphosis (27%) and conjunctivitis (27%) were the most frequent clinical signs in the EH group. No difference was found between studied groups in hematological and biochemical parameters. TNF-α concentration was 43.3 ± 16.9 pg/mL in CT and 41.1 ± 3.8 pg/mL in EH, while IL-10 concentration was 20.2 ± 8.4 pg/mL in CT and 21.0 ± 5.1 pg/mL in EH.[...]