Anaplasma marginale and A. phagocytophilum in cattle in Tunisia

M’ghirbi et al. Parasites & Vectors (2016) 9:556 DOI 10.1186/s13071-016-1840-7 RESEARCH Open Access Anaplasma marginale and A. phagocytophilum in cattle in Tunisia Youmna M’ghirbi1, Marwa Bèji1, Beatriz Oporto2, Fatma Khrouf1, Ana Hurtado2 and Ali Bouattour1* Abstract Background: Tick-borne diseases caused by Anaplasma species put serious constraints on the health and production of domestic cattle in tropical and sub-tropical regions. After recovering from a primary infection, cattle typically become persistent carriers of pathogens and play a critical role in the epidemiology of the disease, acting as reservoirs of the Anaplasma spp. Methods: In this study a duplex PCR assay was used for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle using two primer pairs targeting msp4 and msp2 genes, respectively. We used this method to analyze DNA preparations derived from 328 blood cattle samples that were collected from 80 farms distributed among Tunisia’s four bioclimatic zones. Results: The prevalence of the A. marginale infection (24.7 %) was significantly higher and more widespread (in all bioclimatic areas) than that of A. phagocytophilum (0.6 %), which was found in a mixed infection with A. marginale. Conclusions: The duplex PCR assay used proved to be a rapid, specific and inexpensive mean for the simultaneous detection of Anaplasma marginale and Anaplasma phagocytophilum in cattle blood. It allowed us to report the identification of A. phagocytophilum for the first time in cattle in Tunisia and confirm the presence of A. marginale in cattle from several geographical areas of the country. Further epidemiological studies undertaken using this assay will help improve the surveillance of the associated diseases in the regions where they are endemic. Keywords: Anaplasma marginale, Anaplasma phagocytophilum, Cattle, Duplex PCR assay, Tunisia Background Among tick-borne diseases, bovine anaplasmosis is considered to be one of the most important in ruminants worldwide, causing significant economic losses in tropical and subtropical areas [1]. The socioeconomic impact of the disease and the restrictions on trading infected animals internationally led the Office International des Epizooties (OIE) Animal Health Code to categorize anaplasmosis as a disease that required a notification of its presence [2]. Because outbreaks are seasonal and infection rates are stable, the significance of anaplasmosis is underestimated in endemic areas [3]. Cattle can be infected by several Anaplasma species, like A. marginale, A. phagocytophilum, A. centrale and A. bovis [4–6]. Anaplasma marginale is one of the most prevalent tick* Correspondence: 1 Université Tunis El Manar, Institut Pasteur de Tunis, Laboratoire d’Epidémiologie et de Microbiologie Vétérinaire, Service d’Entomologie Médicale, 1002 Tunis-Belvédère, Tunisia Full list of author information is available at the end of the article transmitted rickettsial diseases of cattle in the world [7]. Highly pathogenic, especially in cattle up to two years old, it causes a disease that produces progressive anemia and icterus [8]. Several decades ago A. phagocytophilum (formerly known as Ehrlichia phagocytophila, E. equi and human granulocytic ehrlichiosis agent), was identified in cattle; it may also infect humans [9]. Known to cause tickborne fever in cattle, it causes not only high fever, but also coughs, miscarriages, decreased milk production and loss of appetite [10]. In areas infested by several tick vector species and where animal husbandry practices include vaccination with live A. centrale bacteria (Israel, Africa, Australia and parts of South America), cattle can be coinfected with two or more Anaplasma species [11, 12]. Disease treatment and prevention strategies focus on using reliable diagnostic tests to accurately and precisely identify infected cattle. While inoculating splenectomized cattle with whole blood has been the gold standard for determining persistent A. marginale infections in cattle, it © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. M’ghirbi et al. Parasites & Vectors (2016) 9:556 Page 2 of 8 is not required for routine testing [13]. Bovine anaplasmosis is diagnosed by identifying Anaplasma in Giemsa-stained blood smears from clinically suspect animals during the acute phase of the disease. However, this method is not useful for detecting presymptomatic and carrier animals. Currently, the competitive enzyme-linked immunosorbent assay (cELISA) is one of the most common diagnostic techniques used to identify the bovine anti-major surface protein 5 (antiMSP5) of Anaplasma marginale [14]. It is considered to be a reliable screening test for cattle infected with A. marginale and to establish their carrier state. However, cross-reactivity has been reported when the cELISA is used to classify cattle infected with A. marginale and/or A. phagocytophilum [15, 16]. Several other serological tests have been used extensively in epidemiological studies of anaplasmosis despite the fact that they do not discriminate between different, antigenically similar Anaplasma species [16, 17]. Yet highly sensitive and specific, molecular methods have been developed to identify A. marginale and A. phagocytophilum DNA [18–22]. To develop a robust diagnostic method, an appropriate target needs to be selected in order to accurately and precisely determine an infection. In Tunisia, Rickettsiales species including A. phagocytophilum, A. bovis, A. marginale, A. centrale, Ehrlichia canis, Ehrlichia sp. and A. platys have recently been detected in horses, cattle, small ruminants, camels, dogs and ticks [23–29]. A molecular assay based on a singlestep duplex PCR, was used to simultaneously detect and differentiate A. marginale and A. phagocytophilum and determine their distribution in cattle from Tunisia. Methods Design of primers A. marginale msp4 gene sequences and A. phagocytophilum msp2 gene sequences were aligned with those of other related species of the genera Anaplasma and Ehrlichia using Vector NTI 8.0 software (Informax Inc., North Bethesda, MD, US). Primers (Table 1) were designed to specifically amplify a 420 bp fragment of the msp4 gene of A. marginale and used in combination with the previously designed primer pair to amplify a 334 bp fragment of the msp2 gene of A. phagocytophilum [30]. Cloning and sequencing the msp4 A. marginale gen (...truncated)