Is Mycetoma a Vector-Borne Disease: The First Report on the Detection of Madurella mycetomatis in Ticks

Currently, there is a massive gap the mycetoma knowledge in particular in its epidemiological characteristics, the infection route, the predisposing factors and the host susceptibility. With this background, the present cross-sectional descriptive entomological study was conducted to determine the possible role of arthropod vectors in the transmission of eumycetoma as well as the knowledge, attitude and practice (KAP) among the villagers towards that in a mycetoma endemic village at Sennar State, Sudan. The study showed an abundance of indoors and outdoors arthropod vectors, and that included ticks, mosquitoes, sandflies, cockroaches and houseflies in the studied area. Ticks were more frequent, and they belonged to three genera and four species, and the later included Hyalomma (H.) anatolicum (11.03%), Hyalomma (H.) rufipes (0.67%), Rhipicephalus (R.) everts (73.1%) and Amblyoma (A.) lepidium (15.2%). The different types of the collected arthropod vectors were pooled in groups, and each group was screened for the presence of the Madurella (M.) mycetomatis DNA, the most frequent causative agents of eumycetoma in the studied area. The DNA was extracted, and amplification of the genomic rRNA genes was done by using universal pan fungal primers and specific M. mycetomatis primers. One pool containing R. evertsi DNA samples and one sample of H. Rufipes DNA gave positive results following PCR amplification of the universal fungal positive primers while H. rufipes sample gave positive results for M. mycetomatis using a specific primer. An association between the animals’ dungs, ticks and mycetoma transmission can be suggested from this study. However, further in-depth studies are needed to verify that. Author summary Mycetoma is a severely neglected tropical disease characterised by painless subcutaneous tumour-like swellings frequently noted in the extremities. There is a massive knowledge gap in transmission, infection route, and historically, it is believed to be associated with minor trauma caused by thorn pricks. This study was designed to determine the possible role of arthropods in mycetoma transmission in an endemic area in Sudan during the cold dry season. Pools of medically important arthropods were screened for mycetoma causative agents using DNA based method. The villagers’ habits and knowledge on arthropod vectors were examined using a pre-designed questionnaire. The results showed various presences of many arthropod vectors. Ticks were found in high prevalence, and densities in domestic animals found inside houses and the villagers had high contact level with the ticks in comparison to other vectors. The study reports for the first time, the detection of the causative agents of mycetoma in a pool of ticks. More studies on the possible role of ticks in the transmission of mycetoma diseases are badly needed to delineate the possible role of ticks on transmission of mycetoma.


Introduction
a tropical climate with an annual rainfall of 600 mm and with varies relative humidity 122 of 18% to 80% and temperature varies between 20 0 C and 40 0 C. The soil is mainly 123 black cotton one, which cracks during the dry season and expands when the rain   135 Entomological surveillance for indoor resting and outdoor disease vectors was 136 carried out in twenty randomly selected houses at the studied village. Informed 137 written consent was obtained from the head of each house. In each of the selected 138 houses, a combination of different collection methods was conducted, and that 139 included light traps, sticky paper traps, active search and Knockdown methods [16]. 140 Light and sticky paper traps in addition to active search methods were used for the 141 collection of outdoor nocturnal vectors from animal shelters. Two light traps were 142 suspended with the fan 40-50cm above the ground level. Traps were set one hour 143 before the sunset and collected early next morning before the sunrise. The traps were transported to the field laboratory where sandflies and mosquitoes were sorted 145 by sex and genus and were preserved in 70% ethanol for later identification to 146 species level. For sticky trap collection, A4-sized white sticky traps (10 per night for 147 three nights) coated with sesame oil were used to capture sandflies from outdoor 148 habitats. A set of 10 sticky traps were hung vertically in a row of 30 cm above the 149 ground supported by wooden sticks. Sticky traps were removed early in the morning, 150 sandflies were removed using forceps and stored in 80% ethanol in labelled vials for 151 further identification.

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The Knockdown and active search collection (direct pick up) methods were used to 153 collect indoor (inside rooms) diurnal resting mosquitoes and sandflies vectors. For 154 Knockdown method, the rooms were sprayed early morning between 6:00 and 155 8:00am with commercial insecticide (Pif Paf). White sheets (2 x 2 meters) were laid 156 on all flat surfaces over the entire floor and beds in the room, and all doors and 157 windows were closed. Rooms were sprayed in a clockwise direction and care was 158 taken to start spraying from the roof and all open spaces or holes in the walls until 159 the room was filled with a fine mist. Then the room was quickly closed. After about 160 15 minutes, the door was opened, and the sheets were picked one at a time from 161 their corners. The sheets were carried outside, and all knocked down arthropods 162 were collected outside the rooms in daylight using forceps. Adult mosquitoes were 163 stored dry in labelled Petri dishes using silica gel. Other arthropods were preserved 164 in 80% ethanol in labelled falcon tubes.

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Collection of tick samples from domestic animals was carried out by a veterinary 166 doctor in addition to two trained volunteers from the local community. Ticks were directly collected from the invested domestic animals found in animal shelters (cows 168 and goats only) and preserved in labelled Petri dishes using silica gel. Also, an 169 active search for immature stages of ticks was carried out in some animal shelters.

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Search was focused on specific areas that included underneath fresh and dry animal 171 dungs, water tanks and fresh plants offered as animal feed.   metal beats were added in microcentrifuge tube. 500µl from ATL buffer was added, 205 and the tube was put in the tissue lyser machine for 10mins/frequency 30, 206 centrifuged at 10,000x for one minute. The supernatant was placed into a new tube, 207 and 25µl proteinase K was added and incubated in a water bath at 56̊0C for 30mins.

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Samples were centrifuged at 8000g for three mins. The clear supernatant was put 209 into a new tube, and 300µl absolute ethanol was added and shaken briefly, then the 210 tube was centrifuged to remove the drops from inside of the lid. The solution was put 211 in a QIAGEN mini-column and centrifuged (8000g/3mins). 750µl AW1 solution was 212 added into the column and centrifuged (8000g/3mins). The lid of the column was closed and was put in a new collection tube and centrifuged (15000g/1min). The 214 column was placed in a new 1.5ml tube, and 70µl AE buffer was added inside the 215 column and centrifuged (8000g/3mins). Samples were stored at -20 0 C.  Ticks were found in animal shelters present inside houses with very high levels of 251 infestation and density; 100% infestation rate and densities that often range between 252 hundreds and thousands in cows, sheep and goats. It is observed that unclean 253 animal shelters, availability of animal dung, high density and diversity of animals 254 accompanied with no veterinary services provided suitable environment for ticks.

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The contact level between humans, animals and ticks is considerably high inside 256 animal shelters in comparison to grazing areas which led to a shift in the normal life cycle of ticks as breeding can take place inside animal shelters within the houses 258 rather than at outdoor grazing areas.

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Also, domestic animals (especially goats and sheep) were frequently found inside 260 rooms and as villagers allow them to enter rooms during the hot hours in the middle 261 of the day as the inside rooms provide a place with more suitable temperature. Also 262 the same scenario happens during rainy season to protect their animals from rains.  for M. mycetomatis using a specific primer (Figs 1,2). The present study showed high infestation of ticks accompanied with high densities   In conclusion, an association between the animals' dungs, ticks and mycetoma 420 transmission can be suggested from this study.