Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray

Abstract

In this preliminary study, a novel DNA microarray system was tested for the diagnosis of bovine piroplasmosis and anaplasmosis in comparison with microscopy and PCR assay results. In the Dakahlia Governorate, Egypt, 164 cattle were investigated for the presence of piroplasms and Anaplasma species. All investigated cattle were clinically examined. Blood samples were screened for the presence of blood parasites using microscopy and PCR assays. Seventy-one animals were acutely ill, whereas 93 were apparently healthy. In acutely ill cattle, Babesia/Theileria species (n=11) and Anaplasma marginale (n=10) were detected. Mixed infections with Babesia/Theileria spp. and A. marginale were present in two further cases. A. marginale infections were also detected in apparently healthy subjects (n=23). The results of PCR assays were confirmed by DNA sequencing. All samples that were positive by PCR for Babesia/Theileria spp. gave also positive results in the microarray analysis. The microarray chips identified Babesia bovis (n=12) and Babesia bigemina (n=2). Cattle with babesiosis were likely to have hemoglobinuria and nervous signs when compared to those with anaplasmosis that frequently had bloody feces. We conclude that clinical examination in combination with microscopy are still very useful in diagnosing acute cases of babesiosis and anaplasmosis, but a combination of molecular biological diagnostic assays will detect even asymptomatic carriers. In perspective, parallel detection of Babesia/Theileria spp. and A. marginale infections using a single microarray system will be a valuable improvement.

Significance of clinical variables and selected biochemical markers in predicting the outcome of bovine anaplasmosis

“…Nonetheless, the efficiency of traditional microscopic examination of Giemsa-stained blood films in detecting carrier animals with low rates of parasitaemia is very limited (Younis et al 2009). The clinical findings presented in this study were in part similar to those previously described (De et al 2012;OIE 2012;El-Ashker et al 2015).…”

“…Ear vein blood samples were also collected and used for blood smear preparation as previously described by El-Ashker et al (2015). After examining more than 50 microscopic fields of blood film, the parasitaemia is quantified and expressed as the percentage of infected erythrocytes.…”

“…A recent report has shown that A. marginale infection in cattle is associated with an acute phase response with elevated serum sialic acid levels (Nazifi et al 2012). Yet, little is known about the molecular characterisation of A. marginale infection in cattle in Egypt, and currently, relatively few reports are available which describe this clinical condition (Younis et al 2009;Salm et al 2011;Abdel Hamid et al 2014;El-Ashker et al 2015). Therefore, the present study was designed to evaluate the acute phase response in cattle naturally infected with A. marginale and to determine the significance of clinical variables as well as selected biochemical markers in predicting the outcome of this disease.…”

First detection on prevalence of Anaplasma marginale in sheep and goat in Karak District, Pakistan

“…marginale prevalence is different, for example, 99.4% reported in Hungry [19], 81% in China [20], 28% in Egypt [3,21], 27.5% in Iran and 16.17% in Ibimirim County, Brazil [22]. It is worthful to mention that a study conducted in Pakistan reported prevalence of 24.47% for anaplasmosis in sheep using indirect ELISA [23], but in the present study higher prevalence 36.8% was observed.…”

“…Although microscopy is gold standard test, prevalence recorded was low as compared to molecular techniques. PCR is more advantageous due to high sensitivity and effectiveness in diagnosing active and carrier state infection [6,17,21]. Similar findings were reported by Salih et al [24] who investigsted the incidence rate of different protozoan species in various livestock through indirect ELISA in Sudan.…”