Competitive Exclusion between Piroplasmosis and Anaplasmosis Agents within Cattle

There are about 869 species of ticks described so far [1], most of which are responsible for the transmission of a huge diversity of microorganisms belonging to almost all the trees of life (viruses, bacteria, protozoa, fungi, and nematodes) [2–4]. Despite the strong opportunity for interaction between these pathogenic species, little is known about the competitive interactions between tick-borne diseases within the vector and the vertebrate host (see [3] for review). Severity of symptoms is often found associated with coinfection by different pathogens (e.g., [3,5–7]). Moreover, dual infections may affect the therapeutic strategies [5]. Exploring the incidence of co-infections and testing for possible interactions (positive or negative) thus represent important objectives. Theileria annulata, Babesia bovis, and Anaplasma marginale are among the most economically important haemoparasitic tickborne diseases of ruminants worldwide [8] and represent a serious economic challenge, particularly in developing countries [9–11]. In this paper we analyse the co-occurrence of these pathogens within individual bovine hosts in northeastern Algeria (North Africa) from one original dataset and one published dataset from the same region [12] that we have reanalysed, and test for the existence of positive or negative associations between these pathogens. We then discuss the implications of our findings in terms of therapeutic strategies and further studies. All samples were collected around Boutheldja, a small town located on the northeast border of Algeria (close to Tunisia) and east of Annaba in Algeria (GPS coordinates 368 459 7.0 N; 88 109 0 E) corresponding to an area of 113.53 km (Figure S1). The sampling period extended from July to December 2004. Ill cattle were diagnosed by private veterinarians, who were contacted directly by their owners. The main criteria used for diagnosis were the presence of the ticks, a hyperthermic condition, presence of icterus, ganglionic hypertrophy, feebleness, lack of appetite, anorexia, anaemia, and dehydration. In case of positive diagnostic a blood smear was carried out, as described in Table 1. Parasites were then identified according to morphology, localisation in the erythrocytes, and using the key proposed by Morel in 1981 [13]. We also reanalysed the data from a published descriptive work [12] undertaken in 2002 in the same part of Algeria on 54 cattle with an identical approach as described above. Four species were identified in the samples: T. annulata, B. bovis, A. marginale, and Theileria orientalis. Note that Anaplasma are rickettsial bacteriae while the others are apicomplexan sporozoan (piroplasms), and that transovarial transmission is only known for Babesia sp. and not for the three other species. In the area investigated, Theileria sp. are transmitted by different Hyaloma ticks, B. bovis is transmitted by the tick Boophilus annulatus, and A. marginale by B. annulatus and Rhipicephalus bursa [4]. All are intracellular parasites [14]. In 2004, the most frequently encountered pathogenic agent was T. annulata (47.6% of infected cattle), followed by A. marginale (40.5%), B. bovis (33.3%), and T. orientalis (2.4%). In 2002, as presented by [12], some differences were observed with 74%, 24%, 8%, and 16% for T. annulata, A. marginale, B. bovis, and T. orientalis, respectively. The comparison of microbe prevalences between 2002 and 2004 samples was undertaken with an approximation of the Fisher exact test with 1,000,000 randomisations using the module Struc of Genepop version 3.1.c [15]. Randomisation tests gave significant differences (p-values 0.036) for all species but A. marginale (p-value1⁄4 0.117). In 2004 (original sample), 12 hosts appeared negative, 15 were infected by T. annulata only, ten by B. bovis only, seven by A. marginale, and four were infected both by B. bovis and A. marginale, five by T. annulata and A. marginale, and one by T. orientalis and A. marginale. In 2002 [12], four hosts appeared negative, 29 were infected by T. annulata only, three by B. bovis only, four by A. marginale, three by T. orientalis, and one was infected both by B. bovis and A. marginale, five by T. annulata and A. marginale, two by T. orientalis and A. marginale, and three by T. annulata and T. orientalis. Presence or absence of a particular micropathogen was treated as a phenotypic character. Thus there were four such phenotypic characters each taking the value 1 (absence) or 2 (presence). To study the association between these four characters a correlation coefficient, initially designed for genetic data, was adapted to our data. This coefficient of correlation is noted Rij, where i and j stands for the pair of