Molecular and serological detection of Ehrlichia canis in naturally exposed dogs in Iran: an analysis on associated risk factors

Abstract

The general aim of this study, which was conducted for the first time in Iran, was to evaluate the seroprevalence and geographical distribution of Ehrlichia canis in a dog population in Iran, followed by molecular confirmation using PCR and sequencing. Blood samples were collected from 240 dogs in different areas of Alborz and Tehran Provinces and initially analyzed using the immunofluorescent antibody (IFA) test to detect anti-Ehrlichia canis IgG antibodies. Subsequently, nested PCR was performed based on a fragment of the 16S rRNA gene of E. canis on serologically positive samples. The results showed that 40/240 dogs (16.6%) presented anti-Ehrlichia canis IgG antibodies and that nine of the blood samples from the 40 seropositive dogs (22.5%) contained E. canis DNA, which was confirmed by sequencing. The seroprevalence of E. canis tended to be higher in purebred, one to three-year-old male dogs living in the Plain zone, in rural areas; however, this difference was not statistically significant.Keywords: Ehrlichiosis, Ehrlichia canis, IFA, nested PCR, dogs, Iran. ResumoO objetivo geral deste estudo, que foi feito pela primeira vez no Irã, foi avaliar a soroprevalência e distribuição geográfica de Ehrlichia canis em população de cães no Irã, seguida da confirmação molecular por meio de PCR seguida de sequenciamento. Amostras de sangue de 240 cães de diferentes áreas das Províncias de Alborz e Teerã foram coletadas e, inicialmente, analisadas pelo Reação de Imunofluorescência (IFA) para detecção de anticorpos IgG anti-Ehrlichia canis Subsequentemente, uma reação do tipo nested PCR baseada em um fragmento do gene 16S rRNA de E. canis foi realizada nas amostras sorologicamente positivas. Os resultados mostraram que 40/240 cães (16,6%) apresentaram anticorpos IgG anti-Ehrlichia canis e nove (22,5%) das amostras de sangue dos 40 cães soropositivos continham DNA de E. canis, confirmado por sequenciamento. A soroprevalência de E. canis, embora não estatisticamente significativa, mostrou uma tendência em se apresentar maior em cães machos com 1-3 anos, de raça pura, que vivem em zonas planas e áreas rurais.

Demonstrating the presence of Ehrlichia canis DNA from different tissues of dogs with suspected subclinical ehrlichiosis

“…It could be to the low parasitaemia observed in the natural infection [7,11,[16][17][18][19][20][21]. Besides, other more speci c methods are used as diagnostic, including the immuno uorescence antibody test (IFA) and ELISA (enzyme-linked immunosorbent assay), which are both able of detecting speci c antibodies [21][22][23][24][25][26][27], as well as other molecular techniques such as the polymerase chain reaction (PCR) [1,19,36,[28][29][30][31][32][33][34][35]. Presently, the infectious disease group of the American College of Veterinary Internal Medicine (ACVIM) requires that dogs diagnosed with this disease show suggestive clinical signs and have positive tests, either by serology and/or by PCR [37].…”

Demonstrating the presence of Ehrlichia canis DNA from different tissues of dogs with negative PCR in blood

“…Identi cation of morulae in monocytes in a blood smear is diagnostic of the disease; however, a low frequency of morulae in buffy coat smears has previously been reported, which could be due to the low parasitaemia observed in the natural infection [7,11,[16][17][18][19][20][21]. Besides, other more speci c methods are used as diagnostics, including the immuno uorescence antibody test (IFA) and ELISA (enzyme-linked immunosorbent assay), which are both able to detect speci c antibodies [21][22][23][24][25][26][27], as well as other molecular techniques such as the polymerase chain reaction (PCR) [1,19,[28][29][30][31][32][33][34][35][36]. Presently, the Infectious Disease Group of the American College of Veterinary Internal Medicine (ACVIM) requires that dogs diagnosed with this disease show suggestive clinical signs and have positive tests, either by serology and/or by PCR [37].…”

Demonstrating the presence of Ehrlichia canis DNA from different tissues of dogs with negative PCR in blood

“…It could be to the low parasitaemia observed in the natural infection [7,11,[16][17][18][19][20][21]. Besides, other more specific methods are used as diagnostic, including the immunofluorescence antibody test (IFA) and ELISA (enzymelinked immunosorbent assay), which are both able of detecting specific antibodies [21][22][23][24][25][26][27]. Also, other molecular techniques exist such as the polymerase chain reaction (PCR) [1,19,36,[28][29][30][31][32][33][34][35].…”