Molecular evidence of Babesia infections in Spinose ear tick , Otobius megnini infesting stabled horses in Nuwara Eliya racecourse : A case study

This article is published under the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract: Spinose ear tick, Otobius megnini (Family Argasidae) is a one-host soft tick that parasitizes domesticated animals and occasionally humans. It causes otoacariasis or parasitic otitis in humans and animals and also known to carry infectious agents. Intra aural infestations of O. megnini is a serious health problem in the well-groomed race horses in Nuwara Eliya. Otobius megnini collected from the ear canal of stabled horses in Nuwara Eliya racecourse were tested for three possible infections, Rickettsia, Theileria and Babesia. Genomic DNA was extracted from 22 ticks collected from 11 horses and then pathogen-specific DNA was amplified using PCR. Ticks from two horses (one thoroughbred and one stallion) tested PCR positive for Babesia but not for other infections. None of the horses however, showed any clinical symptoms of babesiosis. This is the first record of Babesia infections in O. megnini. However, the presence of Babesia DNA in O. megnini doesn’t qualify it as the vector of equine babesiosis, horse blood has to be tested for the presence of


INTRODUCTION
The spinose ear tick, Otobius megnini (Dugès 1883) (Acari: Ixodida: Argasidae) is an economically important soft tick, parasitizes livestock mostly cattle, goats, sheep, and horses and infests humans as well (Naudé et al., 2001;Keirans and Pound, 2003;Ariyarante et al., 2016).It is a one-host tick from the New World with a wide geographical distribution and its original centre of distribution is considered to be the southwestern North America, from where it spread to Central and South America and then to the rest of the world (Estrada-Pena et al., 1999;Keirans and Pound, 2003).Since the larva and the nymph of this tick feed inside the ear canal of the host for a long period, it allows the tick to be distributed over a vast geographic region transcontinentally through the distribution of the host animals.Otobius megnini is thought to have reached India in mid-1930s together with cattle or horses brought from Southern Africa.This tick species is recorded in race horses brought from farms in northern India for an auction at the Madras Race Club (Joseph, 1982).There is a speculation that O. megnini was introduced to Sri Lanka from India via horse trading.The first report of O. megnini in Sri Lanka is in 2010 from stable workers and jockeys as an intra-aural infestation (Ariyaratne et al., 2010).In Sri Lanka, O. megnini appears to have a limited distribution with no records of it infesting any other domesticated animals other than horses in the racecourses (Diyes and Rajakaruna, 2016).
Otobius megnini is the causative agent of horse otoacariasis or parasitic otitis.This condition can cause serious injuries and occasionally death in horses (Wall and Shearer, 2008).The common clinical signs include abnormal head carriage, head shaking and head rubbing (Perris, 1995).Early studies report nervous disease (Ramanujachari and Alwar, 1955) and auricular nerve paralysis (FAO, 1958) due to the presence of O. megnini in the ear canal of horses.However, there is no conclusive evidence to support the classification of O. megnini as a paralysis tick.The fact that this tick feeds within the ears of its hosts where inflammatory reactions could affect the balance of the host and lead to symptoms that could be interpreted as being neurological in origin should be considered.Intermittent painful muscle cramps were described in horses that were severely infested with O. megnini (Madigan et al., 1995).Between muscle cramps, horses appear to be normal and once the ticks are removed clinical signs are reduced and recovered within 12 to 36 hrs (Madigan et al., 1995).Recently, from Northern Mexico a two-year-old quarter breed was reported having myotonia and colic associated with the infestation of O. megnini (Zarate-Ramos et al., 2014).
Otobius megnini is known to carry some infectious agents.Coxiella burnetii, the causative agent of Q fever was recovered from O. megnini collected from dairy cattle in Southern California (Jellison et al., 1948).Nymphs of O. megnini were reported infecting two patients with Spotted fever group rickettsioses from Mexico (Bustamante et al., 1946).Studies have shown that O. megnini can be naturally infected with Ehrlichia canis but does not transmit the Ceylon Journal of Science 47(4) 2018: 405-409 406 agent (Ewing et al., 1990).A study carried out in South Africa reported Borrelia burgdorferi seropositive cases of three horse riders and owner of a stable, 71 horses and five dogs but the O. megnini specimens collected from these hosts were negative for the infection (Fivaz et al., 1991).
Equine piroplasmosis and rickettsial infections are common among horses but there are no records of these infections in horses in Sri Lanka.However, human pathogenic Orientia tsutsugamushi, Rickettsia typhi and Rickttsia conorii (Kularatne et al., 2003;Kularatne et al., 2013), Theilaria orientalis, Theilaria annulata (among water buffaloes) (Sivakumar et al., 2012), Babesia bovis, Babesia bigemina (among cattle), Babesia gibsoni and Babesia canis (among dogs) have been reported (Sivakumar et al., 2012) in Sri Lanka.However, little is known about their prevalence, distribution and the vector-tick species.Hence, the present study examined the presence of three common tick-borne pathogens: Rickettsia, Theileria and Babesia in O. megnini infesting stabled horses in Nuwara Eliya.

MATERIALS AND METHODS
Ticks were collected from the ear canals of 11 thoroughbred horses in Nuwara Eliya racecourses during 2014 using small pieces of white open wove cotton bandages (new, cleaned bandage for every removal).The ticks together with earwax were placed in 100 ml plastic vials and brought to the laboratory.From each horse, two fully engorged nymphs were cleaned and preserved in absolute ethanol and were kept at -20 o C. A total of 22 samples were used to analyze from 11 horses for the presence of Rickettsia, Theileria and Babesia infections in the tick.

Molecular analysis
Each nymph was washed in a series of ethanol (70%, 50%, 30%, 10%) keeping 1 hour in each solution and were finally transferred into distilled water for 1 hour.Genomic DNA was extracted using QIAGEN DNeasy Blood and Tissue kit (Cat no: 69504, German) according to manufacturer's instructions.Eluted DNA was kept at -20 o C until the PCR amplification.The PCR reactions for the detection of pathogenic DNA from ticks were performed with 5 μl of extracted DNA, 1.5 μl of each primer (10 mM), 5 μl of 5x PCR buffer, 3 μl of MgCl 2 (25 mM), 2 μl of dNTPs (2 mM), 0.25 µl Taq DNA polymerase and 6.75 µl DEPC water in a final reaction volume of 25 μl.After amplification, the PCR products were visualized by gel electrophoresis (1.5% W/V) with a DNA molecular weight marker (GelPilot 100 bp Plus DNA Ladder, product no.239045, QIAGEN).primers were used to amplify a 530-bp fragment of the Rickettsial ompA gene (Fournier et al., 1998;Santibáñez et al., 2013).A nested PCR was performed in 25 μl volumes as stated above with the primers and 2 μl of the first PCR product as the DNA template.The reaction mixtures were subjected to an initial denaturation step at 95 °C for 5 min, followed by 42 repeated cycles at 95 °C for 40 seconds (denaturation), 57 °C for 40 seconds (annealing), 68 °C for 40 seconds (extension).Amplification was completed by a further 10 min step at 72ºC (final extension).Same thermocycler parameters were used for the nested PCR with 30 cycles.

Detection of Babesia
Detection of Babesia isolates in DNA extracts were performed by amplifying a 284bp fragment of 18S rRNA gene by using Bab 18S forward primer 5′-TAG(AG) GATTGGAGGTCGTCA-3′ and Bab 18S reverse primer 5′-AACGGAATTAACCAGACAAA-3′ (Hildebrandt et al., 2007).Thermal profile used for the amplification was 5 min initial denaturation at 94 °C, followed by repeated 30 cycles of 30 seconds denaturation at 94 °C, 30 seconds annealing at 50 °C, 30 seconds extension at 72 °C.The final extension was 2 min at 72 °C.

RESULTS AND DISCUSSION
Analysis of 22 samples of O. megnini from 11 race horses for the presence of Rickettsia, Babesia and Theilaria infections revealed that only two ticks (18.2%) collected from one thoroughbred and one stallion were PCR positive for Babesia infections.This study provides the first evidence of presence of Babesia in O. megnini.Although the DNA isolated from the tick was sent for sequencing to confirm the Babesia species, the results were not satisfactory.It is important to examine the horse blood for either parasite DNA or antibodies to confirm whether O. megnini acts as a vector for equine babesiosis.None of the horses however showed any symptoms of babesiosis.Recently, microscopic examination of blood samples from some of the horses in this stable has shown no positive results for Babesia or any other infection of blood parasites (Dissanayake et al., 2017).It could be possible that microscopy may have not picked the low parasitemia levels.
It is not clear the way in which O. megini became infected with the pathogen.Many of the race horses in Nuwara Eliya stables including the two horses with Babesia infected ticks were imported from India.Therefore, it can be suspected that infected ticks may have been introduced to Sri Lanka via horse trading.It is important that the horses undergo a proper and thorough quarantine process.Once the larvae attach to the horse ear canal, they feed, moult and come out as a fully engorged nymph.Adults are non-feeding, free living stage.Since, O. megnini is a one host tick and the adult females do not feed, spreading of infectious diseases from one host to another is limited unless the infectious agent shows both transovarial and transstadial transmission like Rickettsia bellii maintained in Ixodes loricatus (Horta et al., 2006).
Equine babesiosis (Equine piroplasmosis) is established in tropical, subtropical regions of the world and it is an important intra-erythrocytic protozoan that causes great economic loss to equine industry (De Waal, 1992).In Asia, equine babesiosis is frequently reported from India, China, Korea and Iran (Gautam and Dwivedi, 1976;Wise et al., 2013).So far, Babesia caballi and B. equi (some reported as Theileria equi but remains controversial) are frequently reported among horses and other equid species including donkeys, mules and zebras (Friedhoff and Soule, 1996).The information on soft ticks as vectors of these two Babesia species is scant.Recently, under laboratory conditions, Ornithodoros moubata has been identified as a suitable candidate to transmit B. equi (Battsetseg et al., 2007).
There was no indication of the presence of Rickettsia and Theilaria infections in O. megnini infesting the racehorses in Nuwara Eliya.The information on the distribution of Rickettsia spp.associating horses is limited.Besides, the relationship between soft ticks and Rickettsia spp. is poorly documented except for Carios capensis, Carios sawaii and Ornithodoros erraticus (Kawabata et al., 2006;Raoult and Parola, 2007).Although spotted fever group rickettsial infections have been recorded in hard ticks collected from some wild and domesticated animals in Sri Lanka, the list doesn't include horses (Liyanarachchi et al., 2015).Coxiella burnetii has been reported in some areas of Sri Lanka and has the capability of causing abortion, stillbirth and neonatal deaths in horses (Angelakis et al., 2012).
In Sri Lanka, O. megnini is reported only from horses in Nuwara Eliya racecourse.Dynamic nature of the life cycle and high population stability has enabled O. megnini to become an invasive tick in Nuwara Eliya racecourses (Diyes and Rajakaruna, 2016;2017).Different strategies have been implemented to control the infestations of O. megnini in the stabled horses but none of them was successful.Recently, the use of flumethrin based acaricides (Diyes et al., 2016), parasitoid scuttle fly, Megaselia scalaris (Diyes et al., 2015) and predator ant species Tapinoma melanocephalum (Diyes et al., 2017) have shown to be effective in controlling the free-living stages of O. megnini.In Sri Lanka O. megnini is reported only from Nuwara Eliya racecourse.Though Babesia was found in O. megnini, it does not confirm its vector capacity.Presence of pathogenic nucleic acid in a tick only indicates the carrier status of the tick but not the interspecies transmission (Estrada-Peña and de la Fuente, 2014).Therefore, further studies are required to elucidate the vector capacity of O. menini for equine babesiosis and to confirm the species status.This tick has a high affinity to infest cattle posing a potential risk of spreading the infestation and associated tick-borne diseases into the nearby dairy farms.