extraction-saliva-haemolymph-salivary-glands-midgut-from-individual

Extraction of Saliva, Haemolymph, Salivary Glands, and Midgut from Individual Ticks (Acari: Ixodidae)

Ticks are blood-feeding ectoparasites recognised for their ability to transmit several infectious pathogens to humans and animals. Tick-borne pathogen ...

International Centre of Insect Physiology and Ecology (icipe)

University of Kwazulu-Natal

Ticks and tick-borne pathogens (TBPs) are major constraints to camel health and production, yet epidemiological data on their diversity and impact on dromedary camels remain limited. We surveyed the diversity of ticks and TBPs associated with camels and co-grazing sheep at 12 sites in Marsabit County, northern Kenya. We screened blood and ticks (858 pools) from 296 camels and 77 sheep for bacterial and protozoan TBPs by high-resolution melting analysis and sequencing of PCR products.  (75.7%),  (17.6%) and  (6.7%) spp. ticks were morphologically identified and confirmed by molecular analyses. We detected TBP DNA in 80.1% of blood samples from 296 healthy camels. " Anaplasma camelii", " Ehrlichia regneryi" and  were detected in both camels and associated ticks, and , ,  and  endosymbionts were detected in camel ticks. We also detected , which is responsible for heartwater disease in ruminants, in  ticks infesting camels and sheep and in sheep blood, indicating its endemicity in Marsabit. Our findings also suggest that camels and/or the ticks infesting them are disease reservoirs of zoonotic Q fever (), ehrlichiosis () and rickettsiosis (), which pose public health threats to pastoralist communities.

Ticks and Tick-Borne Pathogens Associated with Dromedary Camels () in Northern Kenya.

African Trypanosomiases threaten the life of both humans and animals. Trypanosomes are transmitted by tsetse and other biting flies. In Rwanda, the African Animal Trypanosomiasis (AAT) endemic area is mainly around the tsetse-infested Akagera National Park (NP). The study aimed to identify Trypanosoma species circulating in cattle, their genetic diversity and distribution around the Akagera NP.

Occurrence, diversity and distribution of Trypanosoma infections in cattle around the Akagera National Park, Rwanda.

Akagera National Park and its surroundings are home to tsetse flies and a number of their mammalian hosts in Rwanda. A One-health approach is being used in the control and surveillance of both animal and human trypanosomosis in Rwanda. Determination of the infection level in tsetse flies, species of trypanosomes circulating in vectors, the source of tsetse blood meal and endosymbionts is crucial in understanding the epidemiology of the disease in animals and humans in the region. Tsetse flies ( = 1101), comprising  ( = 771) and  ( = 330) were collected from Akagera park and surrounding areas between May 2018 and June 2019. The flies were screened for trypanosomes, vertebrate host DNA to identify sources of blood meal, and endosymbionts by PCR - High Resolution Melting analysis and amplicon sequencing. The feeding frequency and the feeding indices (selection index - W) were calculated to identify the preferred hosts. An overall trypanosome infection rate of 13.9% in the fly's Head and Proboscis (HP) and 24.3% in the Thorax and Abdomen (TA) were found. Eight trypanosome species were identified in the tsetse fly HP and TA, namely: ,  Kilifi,  savannah, , , , ,  and . We found no evidence of human-infective  We also identified eighteen species of vertebrate hosts that tsetse flies fed on, and the most frequent one was the buffalo () (36.5%). The frequently detected host by selection index was the rhinoceros () ( = 16.2). Most trypanosome infections in tsetse flies were associated with the buffalo blood meal. The prevalence of tsetse endosymbionts  and  was 2.8% and 4.8%, respectively. No  and Salivary Gland Hypertrophy Virus were detected. These findings implicate the buffaloes as the important reservoirs of tsetse-transmitted trypanosomes in the area. This contributes to predicting the main cryptic reservoirs and therefore guiding the effective control of the disease. The study findings provide the key scientific information that supports the current One Health collaboration in the control and surveillance of tsetse-transmitted trypanosomosis in Rwanda.

Trypanosomes infection, endosymbionts, and host preferences in tsetse flies ( spp.) collected from Akagera park region, Rwanda: A correlational xenomonitoring study.

 Livestock are key sources of livelihood among pastoral communities. Livestock productivity is chiefly constrained by pests and diseases. Due to inadequate disease surveillance in northern Kenya, little is known about pathogens circulating within livestock and the role of livestock-associated biting keds (genus ) in disease transmission. We aimed to identify the prevalence of selected hemopathogens in livestock and their associated blood-feeding keds.  We randomly collected 389 blood samples from goats (245), sheep (108), and donkeys (36), as well as 235 keds from both goats and sheep (116), donkeys (11), and dogs (108) in Laisamis, Marsabit County, northern Kenya. We screened all samples for selected hemopathogens by high-resolution melting (HRM) analysis and sequencing of PCR products amplified using primers specific to the genera:  and   In goats, we detected  (84.5%), a novel  sp. (11.8%),  (7.3%),  (66.1%), and  (0.8%). We also detected  (93.5%),  (22.2%), and  (38.9%) in sheep. In donkeys, we detected '  Anaplasma camelii' (11.1%),  (22.2%),  (25%), and  (13.9%). In addition, keds carried the following pathogens; goat/sheep keds -  (29.3%)  (0.86%),  (0.86%), and  (51.7%); donkey keds -  (18.2%) and  (63.6%); and dog keds -  (15.7%),  (0.9%),  (0.9%)   (76%),  (46.3%),   (76%), and  (5.6%).  We found that livestock and their associated ectoparasitic biting keds carry a number of infectious hemopathogens, including the zoonotic . Dog keds harbored the most pathogens, suggesting dogs, which closely interact with livestock and humans, as key reservoirs of diseases in Laisamis. These findings can guide policy makers in disease control.

Molecular detection of novel  sp  and zoonotic hemopathogens in livestock and their hematophagous biting keds (genus ) from Laisamis, northern Kenya.

Q fever is a globally distributed, neglected zoonotic disease of conservation and public health importance, caused by the bacterium Coxiella burnetii. Coxiella burnetii normally causes subclinical infections in livestock, but may also cause reproductive pathology and spontaneous abortions in artiodactyl species. One such artiodactyl, the dromedary camel (Camelus dromedarius), is an increasingly important livestock species in semi-arid landscapes. Ticks are naturally infected with C. burnetii worldwide and are frequently found on camels in Kenya. In this study, we assessed the relationship between dromedary camels' C. burnetii serostatus and whether the camels were carrying C. burnetii PCR-positive ticks in Kenya. We hypothesized that there would be a positive association between camel seropositivity and carrying C. burnetii PCR-positive ticks.

Coxiella burnetii serostatus in dromedary camels (Camelus dromedarius) is associated with the presence of C. burnetii DNA in attached ticks in Laikipia County, Kenya.

Tick-borne pathogen (TBP) surveillance studies often use whole-tick homogenates when inferring tick-pathogen associations. However, localized TBP infections within tick tissues (saliva, hemolymph, salivary glands, and midgut) can inform pathogen transmission mechanisms and are key to disentangling pathogen detection from vector competence.

Tissue-specific localization of tick-borne pathogens in ticks collected from camels in Kenya: insights into vector competence.

 ()-infected livestock and wildlife have been epidemiologically linked to human Q fever outbreaks. Despite this growing zoonotic threat, knowledge of coxiellosis in wild animals remains limited, and studies to understand their epidemiologic role are needed. In -endemic areas, ticks have been reported to harbor and spread  and may serve as indicators of risk of infection in wild animal habitats. Therefore, the aim of this study was to compare molecular techniques for detecting  DNA in ticks.

Comparison of test performance of a conventional PCR and two field-friendly tests to detect  DNA in ticks using Bayesian latent class analysis.

Ticks are blood-feeding ectoparasites that harbor diverse pathogens and endosymbionts. Their microbial communities vary based on tick species, stage, sex, geographical location, surrounding environment, and tissue type. Understanding tick microbiota at the tissue level is crucial for unraveling how microbiomes are distributed in tick tissues and influence pathogen transmission. We used V1-V2 16 S rRNA gene sequencing to analyze tissue-specific bacterial compositions (hemolymph, saliva, salivary glands, and midgut) of Amblyomma gemma, Rhipicephalus pulchellus, Hyalomma dromedarii, and Hyalomma rufipes ticks collected from camels in Marsabit County, northern Kenya. The V1-V2 region of the 16 S rRNA gene effectively differentiated 43 Rickettsia africae and 16 Rickettsia aeschlimannii tick samples from other rickettsial species, as well as Coxiella endosymbionts from Coxiella burnetii. In contrast, the V3-V4 region sequences of these species could not be clearly distinguished. Coxiella endosymbionts were most common in Am. gemma and Rh. pulchellus, while Francisella endosymbionts predominated in Hyalomma ticks; both were primarily localized in the salivary glands. High abundances of Coxiella endosymbionts, as well as Pseudomonas, were associated with the absence or low abundance of Rickettsia pathogens in both Am. gemma and Rh. pulchellus, suggesting competitive interactions between these microbes. Additionally, Proteus mirabilis, an opportunistic pathogen of the urinary tract in humans, was found predominantly in Hyalomma ticks, except for the salivary glands, which were most abundant with Francisella endosymbionts. Furthermore, we detected the Acinetobacter, Pseudomonas, and Corynebacterium genera in all the tick tissues, supporting the hypothesis that these bacteria might circulate between camel blood and ticks. Saliva and hemolymph generally harbored more extracellular bacteria than the salivary glands and midgut. This study provides a new approach to unravel tick-endosymbiont-pathogen interactions by examining the tissue localization of tick-borne pathogens and symbionts in Am. gemma, Rh. pulchellus, Hy. dromedarii, and Hy. rufipes from camels in northern Kenya. Our findings establish a baseline for developing an understanding of the functional capacities of symbionts and for designing symbiont-based control strategies.

Exploring the microbiomes of camel ticks to infer vector competence: insights from tissue-level symbiont-pathogen relationships.

Hippobosca camelina (camel ked) is an obligate hematophagous ectoparasite that infests camels. Hematophagy inflicts painful bites leading to myiasis, anemia and pathogen transmission such as Candidatus Anaplasma camelii. A genome assembly for this biting flies is currently unavailable, limiting understanding of its genetics, including the chemosensory system.

De novo genome assembly, annotation, and characterization of chemosensory genes in the camel ked (Hippobosca camelina).

Ticks pose a major threat to livestock and human health in sub-Saharan Africa, with climate change and pastoral movements fueling their spread. Few studies have integrated multiple sample types to characterize tick-borne pathogens (TBPs) in cattle in Kenya. This knowledge gap hinders the development of effective surveillance and control strategies, leaving vulnerable populations and their livestock susceptible to these persistent threats.

Article	Year
Ticks and Tick-Borne Pathogens Associated with Dromedary Camels () in Northern Kenya. Microorganisms\| PubMed ID: 34209060	2021
Occurrence, diversity and distribution of Trypanosoma infections in cattle around the Akagera National Park, Rwanda. PLoS neglected tropical diseases\| PubMed ID: 34910728	2021
Trypanosomes infection, endosymbionts, and host preferences in tsetse flies ( spp.) collected from Akagera park region, Rwanda: A correlational xenomonitoring study. One health (Amsterdam, Netherlands)\| PubMed ID: 37363264	2023
Molecular detection of novel sp and zoonotic hemopathogens in livestock and their hematophagous biting keds (genus ) from Laisamis, northern Kenya. Open research Africa\| PubMed ID: 37396343	2022
Coxiella burnetii serostatus in dromedary camels (Camelus dromedarius) is associated with the presence of C. burnetii DNA in attached ticks in Laikipia County, Kenya. Zoonoses and public health\| PubMed ID: 38627945	2024
Tissue-specific localization of tick-borne pathogens in ticks collected from camels in Kenya: insights into vector competence. Frontiers in cellular and infection microbiology\| PubMed ID: 38698904	2024
Comparison of test performance of a conventional PCR and two field-friendly tests to detect DNA in ticks using Bayesian latent class analysis. Frontiers in veterinary science\| PubMed ID: 38962707	2024
Exploring the microbiomes of camel ticks to infer vector competence: insights from tissue-level symbiont-pathogen relationships. Scientific reports\| PubMed ID: 39955302	2025
De novo genome assembly, annotation, and characterization of chemosensory genes in the camel ked (Hippobosca camelina). BMC genomics\| PubMed ID: 40670921	2025
Molecular characterisation of tick-borne pathogens in cattle in kenya: insights from blood, ticks, and skin swab analyses. BMC veterinary research\| PubMed ID: 41039537	2025

Dennis Getange

Biography

JoVE Journal Publications

Other Publications