Transmission of 'Candidatus Anaplasma camelii' to mice and rabbits by camel-specific keds, Hippobosca camelina

Abstract

Anaplasmosis, caused by infection with bacteria of the genus Anaplasma, is an important veterinary and zoonotic disease. Transmission by ticks has been characterized but little is known about non-tick vectors of livestock anaplasmosis. This study investigated the presence of Anaplasma spp. in camels in northern Kenya and whether the hematophagous camel ked, Hippobosca camelina, acts as a vector. Camels (n = 976) and > 10,000 keds were sampled over a three-year study period and the presence of Anaplasma species was determined by PCR-based assays targeting the Anaplasmataceae 16S rRNA gene. Camels were infected by a single species of Anaplasma, 'Candidatus Anaplasma camelii', with infection rates ranging from 63-78% during the dry (September 2017), wet (June-July 2018), and late wet seasons (July-August 2019). 10-29% of camel keds harbored 'Ca. Anaplasma camelii' acquired from infected camels during blood feeding. We determined that Anaplasma-positive camel keds could transmit 'Ca. Anaplasma camelii' to mice and rabbits via blood-feeding. We show competence in pathogen transmission and subsequent infection in mice and rabbits by microscopic observation in blood smears and by PCR. Transmission of 'Ca. Anaplasma camelii' to mice (8-47%) and rabbits (25%) occurred readily after ked bites. Hence, we demonstrate, for the first time, the potential of H. camelina as a vector of anaplasmosis. This key finding provides the rationale for establishing ked control programmes for improvement of livestock and human health.

Fig 1. The map of Kenya showing the sampling sites in Laisamis in Marsabit County.

The following shapefiles were utilised on the map together with the websites where the data was sourced: Kenya administrative boundaries– https://africaopendata.org/dataset/kenya-counties-shapefile; License: https://www.opendefinition.org/licenses/cc-by. Rivers–(ke_major-rivers.zip) https://www.wri.org/resources/data-sets/kenya-gis-data; License:https://www.wri.org/publications/permissions-licensing. All the websites used were under the Creative commons BY 4.0. No base map was used to create the map.

Fig 2. Hippobosca camelina.

Hippobosca camelina was present: (i) in all of the camel herds surveyed, (ii) throughout the year, and (iii) nearly all camels in the herds were infested by keds.

Fig 3. Baseline survey data of livestock keds in Laisamis, northern Kenya.

(A) Ked infestation in domestic animals.Hippobosca camelina, that spends its adult life attached on the host, was predominantly found on camels, indicating its camel-specific preference. However, in rare instances, about 1 to 3 H. camelina flies were collected from sheep and goat herds that were co-herded with camels (but not found on other livestock species studied here). Highest mean ked infestation was recorded on camels, followed by dogs, and then donkeys. (B) The influence of host age and sex on the preference of keds to infest camels. Mature camels had higher average numbers of keds as compared to the young camels. Camel sex did not influence ked infestation (p = 0.5508). (C) Seasonal variations of ked infestations on camels. The highest numbers of keds were recorded on camels during dry season followed by late wet season. The lowest ked numbers were recorded on the camels during the wet season. (D)Proportions of male and female keds on camels. The proportion of female keds was higher than the male flies sampled across the three seasons.

Fig 4. Field’s staining showing Anaplasma sp. infections in representative thin film blood smears.

(A) Naturally infected dromedary camel, (B) Experimental mouse exposed to ked bites, (C) Experimental rabbit post-ked bites. Green arrows point to Anaplasma sp., magnification x100.

Fig 5. PCR—HRM melt curves for detection of Anaplasma sp. in mice, camels, and keds.

DNA sequencing identified Anaplasma sp. in camels (A & C), keds (B & D), and mice (both healthy- E and immunosuppressed mice- F) as ‘Ca. Anaplasma camelii’.

Fig 6. Maximum-likelihood phylogenetic tree of 1000-bp Anaplasma sp. 16S rRNA sequences.

The sequences obtained from this study are shown in pink and blue colours and bootstrap values at the major nodes are the percentage agreement with 1000 bootstrap replicates. Wolbachia endosymbiont KJ814215 (family Anaplasmataceae) was included in the tree as outgroup.