First PCR Confirmed anthrax outbreaks in Ethiopia-Amhara region, 2018-2019.

BACKGROUND: Anthrax is a disease that affects humans and animals. In Ethiopia, anthrax is a reportable disease and assumed to be endemic, although laboratory confirmation has not been routinely performed until recently. We describe the findings from the investigation of two outbreaks in Amhara region. METHODS: Following reports of suspected outbreaks in Wag Hamra zone (Outbreak 1) and South Gondar zone (Outbreak 2), multi-sectoral teams involving both animal and public health officials were deployed to investigate and establish control programs. A suspect case was defined as: sudden death with rapid bloating or bleeding from orifice(s) with unclotted blood (animals); and signs compatible with cutaneous, ingestion, or inhalation anthrax ≤7 days after exposure to a suspect animal (humans). Suspect human cases were interviewed using a standard questionnaire. Samples were collected from humans with suspected anthrax (Outbreak 1 and Outbreak 2) as well as dried meat of suspect animal cases (Outbreak 2). A case was confirmed if a positive test was returned using real-time polymerase chain reaction (qPCR). RESULTS: In Outbreak 1, a total of 49 cows died due to suspected anthrax and 22 humans developed symptoms consistent with cutaneous anthrax (40% attack rate), two of whom died due to suspected ingestion anthrax. Three people were confirmed to have anthrax by qPCR. In Outbreak 2, anthrax was suspected to have caused the deaths of two livestock animals and one human. Subsequent investigation revealed 18 suspected cases of cutaneous anthrax in humans (27% attack rate). None of the 12 human samples collected tested positive, however, a swab taken from the dried meat of one animal case (goat) was positive by qPCR. CONCLUSION: We report the first qPCR-confirmed outbreaks of anthrax in Ethiopia. Both outbreaks were controlled through active case finding, carcass management, ring vaccination of livestock, training of health professionals and outreach with livestock owners. Human and animal health authorities should work together using a One Health approach to improve case reporting and vaccine coverage.

Molecular phylogeny of Amblyomma exornatum and Amblyomma transversale, with reinstatement of the genus Africaniella (Acari: Ixodidae) for the latter.

The phylogeny of the hard tick genus Amblyomma Koch, 1844 deserves special attention, because several poorly studied tick species associated with reptiles still bear the name of this genus, although they may not belong to it. This study focuses on the phylogeny of two such species with uncertain taxonomic status, i.e., Amblyomma transversale (Lucas, 1845) and Amblyomma exornatum Koch, 1844, analyzing two mitochondrial (cytochrome c oxidase subunit 1 and 16S rRNA) and two nuclear (18S and 28S rRNA) genes. In the cox1 phylogenetic analysis, both Am. transversale and Am. exornatum were part of a sister group to all other Metastriata, whereas in the 16S rRNA gene analysis, Am. transversale belonged to a sister group to three subfamilies (Amblyomminae Neumann, 1911; Haemaphysalinae Hoogstraal and Aeschlimann, 1982; Bothriocrotoninae Klompen, Dobson and Baker, 2002), and Am. exornatum formed a sister group to other Amblyomminae. However, based on the 18S and 28S rRNA genes, Am. transversale belonged to a sister group of either Bothriocrotoninae alone or of both Bothriocrotoninae and Haemaphysalinae, respectively. In the latter two phylogenetic analyses Am. exornatum always clustered within Amblyomminae. Morphological comparisons revealed that Am. transversale has at least four unique characters and shares a high number of traits with the genera Robertsicus Barker and Burger, 2018 and Archaeocroton Barker and Burger, 2018, as well as with the subgenus Alloceraea Schulze, 1918 (represented by Haemaphysalis inermis Birula, 1895). These results justify that the genus Africaniella Travassos Dias, 1974 should be reinstated, and the species name of Am. transversale should be used as Africaniella transversale (Lucas, 1845).

Molecular screening for Anaplasmataceae in ticks and tsetse flies from Ethiopia.

Hard ticks and tsetse flies are regarded as the most important vectors of disease agents in Sub-Saharan Africa. With the aim of screening these blood-sucking arthropods for vector-borne pathogens belonging to the family Anaplasmataceae in South-Western Ethiopia, four species of tsetse flies (collected by traps) and seven species of ixodid ticks (removed from cattle) were molecularly analysed. DNA was extracted from 296 individual ticks and from 162 individuals or pools of tsetse flies. Besides known vector-pathogen associations, in Amblyomma cohaerens ticks sequences of Anaplasma marginale and A. phagocytophilum were detected, the latter for the first time in any ticks from cattle in Africa. In addition, part of the gltA gene of Ehrlichia ruminantium was successfully amplified from tsetse flies (Glossina pallidipes). First-time identification of sequences of the above pathogens in certain tick or tsetse fly species may serve as the basis of further epidemiological and transmission studies.

Identification of novel Coxiella burnetii genotypes from Ethiopian ticks.

BACKGROUND: Coxiella burnetii, the etiologic agent of Q fever, is a highly infectious zoonotic bacterium. Genetic information about the strains of this worldwide distributed agent circulating on the African continent is limited. The aim of the present study was the genetic characterization of C. burnetii DNA samples detected in ticks collected from Ethiopian cattle and their comparison with other genotypes found previously in other parts of the world. METHODOLOGY/PRINCIPAL FINDINGS: A total of 296 tick samples were screened by real-time PCR targeting the IS1111 region of C. burnetii genome and from the 32 positive samples, 8 cases with sufficient C. burnetii DNA load (Amblyomma cohaerens, n = 6; A. variegatum, n = 2) were characterized by multispacer sequence typing (MST) and multiple-locus variable-number tandem repeat analysis (MLVA). One novel sequence type (ST), the proposed ST52, was identified by MST. The MLVA-6 discriminated the proposed ST52 into two newly identified MLVA genotypes: type 24 or AH was detected in both Amblyomma species while type 26 or AI was found only in A. cohaerens. CONCLUSIONS/SIGNIFICANCE: Both the MST and MLVA genotypes of the present work are closely related to previously described genotypes found primarily in cattle samples from different parts of the globe. This finding is congruent with the source hosts of the analyzed Ethiopian ticks, as these were also collected from cattle. The present study provides genotype information of C. burnetii from this seldom studied East-African region as well as further evidence for the presumed host-specific adaptation of this agent.

Influence of the biotope on the tick infestation of cattle and on the tick-borne pathogen repertoire of cattle ticks in Ethiopia.

BACKGROUND: The majority of vector-borne infections occur in the tropics, including Africa, but molecular eco-epidemiological studies are seldom reported from these regions. In particular, most previously published data on ticks in Ethiopia focus on species distribution, and only a few molecular studies on the occurrence of tick-borne pathogens or on ecological factors influencing these. The present study was undertaken to evaluate, if ticks collected from cattle in different Ethiopian biotopes harbour (had access to) different pathogens. METHODS: In South-Western Ethiopia 1032 hard ticks were removed from cattle grazing in three kinds of tick biotopes. DNA was individually extracted from one specimen of both sexes of each tick species per cattle. These samples were molecularly analysed for the presence of tick-borne pathogens. RESULTS: Amblyomma variegatum was significantly more abundant on mid highland, than on moist highland. Rhipicephalus decoloratus was absent from savannah lowland, where virtually only A. cohaerens was found. In the ticks Coxiella burnetii had the highest prevalence on savannah lowland. PCR positivity to Theileria spp. did not appear to depend on the biotope, but some genotypes were unique to certain tick species. Significantly more A. variegatum specimens were rickettsia-positive, than those of other tick species. The presence of rickettsiae (R. africae) appeared to be associated with mid highland in case of A. variegatum and A. cohaerens. The low level of haemoplasma positivity seemed to be equally distributed among the tick species, but was restricted to one biotope type. CONCLUSIONS: The tick biotope, in which cattle are grazed, will influence not only the tick burden of these hosts, but also the spectrum of pathogens in their ticks. Thus, the presence of pathogens with alternative (non-tick-borne) transmission routes, with transstadial or with transovarial transmission by ticks appeared to be associated with the biotope type, with the tick species, or both, respectively.

Detection of Francisella-like endosymbiont in Hyalomma rufipes from Ethiopia.

The expanding family of Francisellaceae includes the genus Francisella, where several pathogen bacteria, e.g. the zoonotic F. tularensis, and different Francisella-like agents belong to. Francisella-like endosymbionts (FLEs) are widespread in hard and soft ticks and their pathogenicity is unknown. The examination of 296 ticks collected in Ethiopia was performed for the detection of F. tularensis and FLEs using polymerase chain reaction (PCR) assays based on the amplification of 16S rRNA, sdhA and tul4 gene fragments. FLE was described in one Hyalomma rufipes tick based on the 16S rRNA and sdhA gene sequences. The 16S rRNA gene fragment was identical with the ones detected previously in Rhipicephalus sanguineus and Hyalomma marginatum marginatum in Bulgaria. The presence of endosymbionts with identical 16S rRNA gene sequence in both Rhipicephalus and Hyalomma species further supports the hypotheses, that certain FLEs had independent evolution from their tick hosts.