PARASITES & PARASITOLOGISTS

MOST UPDATED INFORMATIONF FROM AN EXPERT 

Domenico Otranto,Dip[artimento di Sanita publica e Zootecnia, Facolta di Medicina Veterinaria, Universita di Bari

The genus Thelazia (Spirurida, Thelaziidae) includes 16 species of nematode parasites commonly refereed as ‘eyeworms’ which affect the eyes and associated tissues of several animals (e.g. canids, felids, ruminants, equids) and humans. The adult parasites live under the nictitating membrane of the eye and the mature females release first-stage larvae (L1s) into the lachrymal secretions, which are subsequently ingested by the arthropod intermediate host (non-biting diptera), where they undergo development until the infective third-stage larvae (L3s). Thelazia callipaeda, also known as the ’oriental eyeworm’, infects a range of definitive hosts (e.g. dogs, cats, foxes, wolves, rabbits, hares and humans).

Department of Veterinary Public Health of the Faculty of Veterinary Medicine, University of Bari, PO Box 7, 70010 Valenzano (Bari), Italy. website: www.baryparasitology.it

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Introduction

Nematodes belonging to the genus Thelazia (Spirurida, Thelaziidae), commonly known as eyeworms, are parasites living in the orbital cavities and associated tissues of several mammals, including humans (Anderson, 2000). Transmission occurs by means of secretophagous non-biting flies which feed on animal lachrymal secretions and become infected with the first stage larvae (L1) released by the adult females in the lachrymal secretions of the hosts. In the arthropod vector larvae undergo development from the L1 to the third infective stage (L3), while remaining encapsulated in different parts of the vectors’ body (depending upon the different species of Thelazia) and migrating through the arthropod coeloma to the labella. Thelazia L3s emerge from the labella of infected flies as soon as they feed on the lachrymal secretions of receptive animals and develop into the adult stage in the ocular cavity. Out of the 16 species of Thelazia described, two i.e. T. callipaeda Railliet and Henry, 1910 and T. californiensis Price, 1930, may infect carnivores and humans (Skrjabin et al., 1967). Indeed, T. callipaeda can also infect cats, foxes, rabbits and wolves (Kozlov, 1961; Skrjabin et al., 1967; Otranto et al., 2007), while T. californiensis causes disease in sheep, deer, coyotes and bears (Anderson, 2000). Both adult and larval stages of eyeworms are responsible for eye disease with symptoms ranging from mild (e.g. lacrimation, ocular discharge, epiphora) to severe (e.g. conjunctivitis, keratitis and, corneal opacity or ulcers) (Figures 1 and 2).

So far, T. californiensis has been reported only in Western USA (Skrjabin et al., 1967; Doezie et al., 1996), whilst T. callipaeda infections have been registered in men and dogs from the Russian Federation and the Far East (i.e. Indonesia, Thailand, China, Korea, Myanmar, India and Japan (Bhaibulaya et al., 1970, Kosin et al., 1989, Hong et al., 1995).

On the tracks of the “oriental eyeworm in Europe”: epidemiological and molecular evidences

Because of its distribution T. callipaeda has been known for a long time as the “oriental eye-worm” (Anderson, 2000). However, over the last 20 years infections by T. callipaeda have been reported in dogs from northern Italy (Rossi and Bertaglia, 1989), and Basilicata region (southern Italy) with prevalence of infection up to 60.14% (Otranto et al., 2003). In southern Italy also cats, foxes and, for the first time, wolves have been found to be affected by T. callipaeda (Otranto et al., 2003; 2007).The existence of a sylvester life cycle of T. callipaeda has been demonstrated in foxes in areas with high prevalence of canine thelaziosis, thus indicating that foxes may act as one of the main reservoir of T. callipaeda in pets (Rossi et al., 2002; Otranto et al., 2003). Following the above reports, and probably due to improved awareness by parasitologists and practitioners, new cases of thelaziosis have also been registered sporadically in France (Chermette et al., 2004) and Germany (Hermosilla et al., 2004) in dogs which had spent some time, mostly during summer, in northern Italy. Very recently, autochthonous cases of thelaziosis affecting dogs and cats have been described in South western France (Dordogne area) (Dorchies et al., 2007) and Switzerland (Schnyder et al., 2007). The increased number of reports of thelaziosis in dogs, cats and foxes in Europe raised a number of questions concerning the provenance of T. callipaeda, as to whether thelaziosis by T. callipaeda is autochthonous or had been imported from the ex-USSR and/or Asian countries (Otranto et al., 2003a). For instance, the latitudes of the European countries where canine thelaziosis is endemic (between 39° and 46° N) are similar to those of the Asian countries where canine and human thelaziosis had been previously reported (between 10° and 45° N for India and Japan, respectively) (Otranto et al., 2003a). In order to exploit the origin of this infection in Europe, a molecular investigation employing a mitochondrial DNA marker (Otranto et al., 2005b) demonstrated that, despite a relatively high degree of genetic variability among T. callipaeda isolates from Asia (i.e. China and Korea), no genetic variation occurs among individual nematodes collected from different host species (i.e. dogs, cats and foxes) and localities within Europe (i.e. Italy, Germany and the Netherlands). Genetic homogeneity within T. callipaeda from dogs, foxes and cats from three countries in Europe indicated a strict affiliation of this nematode to the intermediate host rather than to the definitive hosts and that the distribution of the parasite would be expected to coincide with that of the vector (Otranto et al., 2005b).

As a first step toward a more comprehensive picture of T. callipaeda it was necessary to fill the gaps into the knowledge on its biology on the intermediate hosts.

T. callipaeda in the definitive host: an original endo-ecto parasitic nematode!

Exploring the biology of Thelazia spp., one immediately realizes that this genus of spirurids probably represents one of the most particular taxon among parasitic nematodes for the tight relationship it has established with the definitive- and intermediate-hosts. Indeed, T. callipaeda as a ‘nematode’ should be considered an ‘endoparasite’, but the adult and larval stages live in the anterior chamber of the eye, thus being exposed to the air and to the external environment just like ectoparasites. Moreover, due to the localization of the parasites, thelaziosis can be treated topically, by direct instillation of drugs into the eyes (Otranto and Traversa, 2005).

In a recent study aiming to contribute new insights into the chrono-biology of T. callipaeda in the definitive host under field conditions, eyeworms were collected from 31 naturally infected dogs from Basilicata region (southern Italy), where a high prevalence of infection (up to 60.14%) was recorded (Otranto et al., 2003), over one year from January 2002 to December 2003 (Otranto et al., 2004). Conjunctival secretions were also collected and examined for the presence of larval stages. The presence of blastomerized eggs in the uterus of females throughout the period – except for the period from May to November – indicated a seasonal activity in the reproduction of T. callipaeda, coinciding with the presence/absence of the vector. In fact, L1s were found in the lachrymal secretions of dogs during summertime (June-July), ready to be ingested by flies feeding on the lachrymal secretions, whilst during March-April and October, the increased number of L4s in the conjunctival sac of infected dogs indicated that the flies intermediate hosts of T. callipaeda were active from early spring to early fall (Otranto et al., 2004). Basic knowledge on the chrono-biology of T. callipaeda constituted an important starting point for further studies to identify the intermediate hosts and to investigate risk likelihood for definitive hosts under field conditions.

Thelazia callipaeda’s intermediate host: simply a “zoophilic fruitfly”

Knowledge on the species of vector/s involved in the transmission of T. callipaeda is needed in order to understand the origin of thelaziosis in Europe and, therefore, to plan reliable control strategies.

The number of surveys on Thelazia spp. intermediate hosts available in literature is scant, mainly due to the difficulties in retrieving larvae in the vector body and to the low prevalence and mean intensity of infected flies. Investigations have been carried out mainly on the vectors of Thelazia spp. affecting cows in North America, Slovakia and the ex-USSR, both by dissection of experimentally and naturally infected flies or, more recently, by detecting Thelazia DNA in infected flies (reviewed in Otranto and Traversa). Despite of the number of information available on the intermediate hosts of Thelazia spp. affecting livestock, little information was available on the intermediate hosts of T. callipaeda. One single report described Musca domestica (Diptera, Muscidae) as vector of T. callipaeda in the Asian Pacific regions (Shi et al. 1988). However, in a recent study in which M. domestica flies were allowed to feed directly on the eyes of an infected dog or fed with L1 of T. callipaeda, it has been demonstrated that this species of fly is unlikely to act as a vector of T. callipaeda (Otranto et al., 2005).

Following the first report by Kozlov (1963), Phortica variegata Fallén 1823 (Drosophilidae, Steganinae) and Phortica okadai Okada 1956 have been shown, under experimental conditions, to act as a vector of T. callipaeda in Europe and China, respectively (Otranto et al. 2005a; Wang et al. 2002). This finding is of relevance considering that, unlike the vectors of other Thelazia species (i.e. Musca spp.) Phortica flies are drosophilids (commonly known as ‘fruitflies’) which display an “unusual” behaviour since they feed on lachrymal secretions of humans and carnivores. Both adult and larval stages of these species of drosophilids may be easily confused with other species belonging to the same genus. In addition, very little is known on the biology and ecology of P. variegata. In a recent study the population dynamics of P. variegata flies under natural conditions has been monitored demonstrating that, in the area of Italy where dog thelaziosis is endemic, the highest number of P. variegata is collected in July –August (Otranto et al., 2006). In the same study, distributional data for ~250 sites in which P. variegata has been collected in Europe were analysed using a desktop implementation of the Genetic Algorithm for Rule-Set Prediction (GARP) in order to identify suitable environments for the development of P. variegata across Italy and Europe (Figure 4) (Otranto et al. 2006). The results showed with high degree of confidence that large areas of central Europe are likely to be suitable habitat for this species of drosophilid (Otranto et al. 2006).

The population dynamics of P. variegata flies in their natural environment and their feeding preferences (on vegetables and/or animal lachrymal secretions) were also examined in relationship to their role as vectors of T. callipaeda in Basilicata region (Otranto et al., 2006a). From April to November 2005 P. variegata flies (557 males and 412 females) were collected weekly. After morphological identification (Maca, 1977; Bachli et al., 2005), the flies were dissected or subjected to a PCR assay to detect the presence of T. callipaeda larvae. The zoophilic preferences of P. variegata were assessed by collecting flies around the eyes of a human bait or around a fruit bait. A total of 1.34% of P. variegata flies were found positive for T. callipaeda larvae at dissection (0.83%), while 2.81% were demonstrated to harbour T. callipaeda DNA (Otranto et al., 2006 IJP). Interestingly, only male flies were collected around the eyes, whereas a male/female ratio of 1:4 was registered around the fruit bait. Again, only P. variegata males were found to be infected by T. callipaeda thus raising the hypothesis that only P. variegata males may act as an intermediate host under natural conditions (Otranto et al., 2006).

The above results immediately stimulated new-intriguing questions on the role that the gender of insects plays in the transmission of parasitic diseases…

Phortica variegata males as vectors of Thelazia callipaeda: does co-evolution enforce the parasitism?

It is known that the transmission of pathogens by insect vectors is specifically associated with adult females, as demonstrated for several blood–feeding insects (e.g., mosquitoes, ceratopogonids, sandflies, blackflies and tabanids), due to the need of a blood meal to complete their gonadotrophic cycle (Lehane, 1991; Attardo et al., 2005). In fact, after the blood meal, female insects lay a significantly higher number of eggs compared with the unfed ones (Attardo et al., 2005), thus increasing the number of potential ABD vectors. With this in mind, the detection of T. callipaeda exclusively in a male arthropod vector is of great interest from both parasitological and ecological standpoints and represents a unique case in which a male insect feeding on vertebrate host secretions acts as vector of ABDs under natural conditions (reviewed by Otranto et al., 2008). The retrieval of L2s encysted in the testes of P. variegata males suggests that T. callipaeda and the male drosophilid have undertaken co-evolution (Otranto et al., 2006). Moreover, the fact that females do not feed around the eyes of humans and the absence of T. callipaeda-infected females support the proposal that the zoophilic behaviour of P. variegata is linked specifically to the male gender as well as environmental and biological factors and dietary needs (e.g., high-protein supplementation). This finding, along with the relatively broad range of hosts for T. callipaeda (humans, rabbits, dogs, wolves, cats and foxes), lends credence to the proposal that this nematode and its arthropod vector have co-evolved, as further confirmed by the finding of a unique mitochondrial haplotype existing across T. callipaeda specimens collected from different countries in Europe and from different definitive hosts [Otranto et al., 2005]. It has been proposed that, following an original (occasional) P. variegata meal on “eye secretions”, T. callipaeda might have ‘used’ this fly to complete its larval development. At the meantime, it is possible that the presence of T. callipaeda larvae in the body as well as in the proboscis of the infected fly (Figure 3) might have ”forced” Phortica to feed on lachrymal secretions as a source of food, since it was easier to ‘drink’ from lachrymal secretions than feed on fruit scraps [Otranto et al., 2006]. Thus, it was speculated that the specificity in the development of T. callipaeda in male P. variegata might have biased the zoophilic behaviour of the fly.

Human thelaziosis: a new disease for the Old Continent.

Nematode helmints (e.g. filarioids and spirurids) transmitted by arthropods may cause life-threatening diseases, particularly in developing countries. From this standpoint, human infections by T. callipaeda have received little attention, despite the high prevalence recorded in some Asiatic countries (reviewed in Shen et al., 2007), and human infections by T. californiensis have only been reported occasionally in the USA (Doezie et al., 1996). Since its first report in Beijing (China) (Stuckey, 1917), human thelaziosis (HT) by T. callipaeda has been described in several areas of the former Soviet Union (Miroshnichenko et al., 1988) and Asian continent, including China (Chen et al., 1954), Korea (Min et al., 2002), Japan (Koyama et al., 2000), Indonesia (Kosin et al., 1989), Thailand (Yospaiboon et al., 1989; Bhaibulaya et al., 1970), Taiwan (Cheung et al., 1998) and India (Singh and Singh, 1993). HT occurs predominantly in poor, rural communities with low health and socio-economic standards particularly where domestic dogs and other animals (e.g. cats and foxes) are heavily affected (Wang et al., 2002b; 2003a). Despite its significance, HT is still a neglected disease for the majority of ophtalmologists and physicians, being probably under-estimated due the misleading clinical symptoms (see below) and to the fact that most clinical reports have been published in Chinese, Russian or Korean and thus have not been accessible to the majority of the international scientific community (reviewed in Shen et al., 2006). However, the prevalence and relevance of HT has been increasing in China over the last two decades.

For instance, 370 new cases of HT have been reported mainly in elders and children from 17 provinces in the south-eastern China within the year 2005 (in Shen et al. 2006).

The first four cases of human thelaziosis in Europe have been diagnosed in Cuneo (Piedmont region, Italy) in patients coming from an area of north-western Italy and south-eastern France (Otranto and Dutto, 2008), where the infection had been previously reported in dogs, cats and foxes (Otranto et al., 2003; Dorchies et al., 2007). Patients presented similar clinical symptoms (i.e. exudative conjunctivitis, lachrymation and foreign body sensation) lasting from a few days to weeks before being referred for medical examination and complained for floating “filaments” on the eye surface. The nematodes collected were identified as T. callipaeda based on morphological keys (Otranto et al., 2003) and molecular characterization of a partial sequence of the mitochondrial cytochrome c oxidase subunit 1 gene. All the cases of human thelazioisis were reported during summer (from June to August), when the fly acting as vector of T. callipaeda is most active (late spring to fall seasons in Southern Europe).

Diagnosis, treatment and control

The first reports of human infection by T. callipaeda in Italy and France (Otranto and Dutto, 2008) highlight the importance of including this arthropod borne disease among the differential diagnoses of bacterial or allergic conjunctivitis. Indeed, the seasonality of human thelaziosis may help the correct aetiological diagnosis of the disease, also considering that spring and summer are the seasons in which allergic conjunctivitis (e.g. by pollens) occurs most frequently, thus causing troubles for the differential diagnosis. This is particularly important in the early phases of the infection, when mostly larval stages, which are small and difficult to detect and identify, are present in the conjuctival sac of the definitive host. Similarly, the clinical diagnosis of HT may be difficult when only a small number of nematodes is present and the clinical signs (related to an inflammatory response) mimic allergic conjunctivitis. Untimely or incorrect treatments of the infection may result in delay in the recovery mainly in children and the elders, where the transmission of the nematodes is most likely to result in a clinical disease. The clinical manifestations of HT (reviewed in Shen et al., 2006) are related to the number of nematodes present in the eye, their location, the host immune response and the occurrence of secondary infection with bacteria (e.g. Pasteurella spp., Chlamydia spp. and Staphylococcus spp.). Symptoms of thelaziosis are mainly due to the lateral serration of the T. callipaeda cuticle which is responsible for mechanical damage of the conjunctival and corneal epithelium. HT may result in mild conjunctivitis, follicular hypertrophy of the conjunctiva, foreign body sensation, epiphora, itchiness, congestion, swelling, hypersensitivity to light, and keratitis. Due to the presence of the nematodes, infected subjects often scratch their eyes, inducing bacterial secondary infection and consequently complicating the primary diagnosis.

With a careful clinical and ophthalmological examination the tangled worms may be seen, mostly in the conjunctival sac and medial or lateral canthus of the eye of infected patients. Nevertheless, cases of thelaziosis where the worms were isolated from the anterior chamber have been reported (reviewed in Shen et al., 2006). In the latter cases, the localization of the eyeworms in the anterior or posterior chambers or in the vitreous body and retina are often linked to the presence of fibrous tissue, inducing clinical symptoms, such as decreased vision, black spots in visual field, photophobia, excessive lacrimation, ocular congestion, aqueous humor turbidity, and sometimes, purulent exudation under the anterior chamber (reviewed in Shen et al., 2006).

Diagnosis of canine and human thelaziosis is usually based on the finding of the adult nematode on the conjunctiva at the clinical and ophthalmological examinations. The larvae or adult nematodes appear as a thin, creamy-white thread in the conjunctival sac. The morphological key for the species identification of the nematode are available (Otranto et al., 2003). Briefly, the parasites are characterized by the presence of a buccal capsule and their mouth opening shows a hexagonal profile. The internal margins of the buccal capsule are everted and subdivided by excavations into six festoons. T. callipaeda females are characterized by the vulva located anteriorly to the oesophagus-intestinal junction while males present five pairs of postcloacal papillae.

Nematodes can be collected by flushing saline from the medial canthus. The eye secretions should then be submitted to a microscopical parasitic examination to detect the typical newborn L1. However, the clinical diagnosis of thelaziosis in animals and humans can be difficult if small number of adults are present. In fact, clinical symptoms are mainly caused by third (L3) and/or fourth (L4) immature stages and appear to be similar to those of allergic conjunctivitis.

Adult and larvae may be removed mechanically by rinsing the conjunctival sac with sterilized saline and fluids or picking them out with fine forceps or cotton swab. In the latter case local anesthesia with 1% dicaine is recommended for detection and recovery of T. callipaeda (Shen et al., 2006). The symptoms described above generally resolve quickly. Prevention of HT includes strategies aimed to control the fly populations (Shen et al., 2006). Bed nets to prevent the contact with the fly intermediate host could also be useful, as well as treatment of all infected domestic animals.

For the treatment of dog thelaziosis the topic instillation of organophosphates (Rossi and Perruccio, 1989) or moxidectin 1% (Lia, et al., 2004) is highly efficacious against T. callipaeda. Imidacloprid 10% and moxidectin 2.5% in spot-on formulation showed to be effective for the control of dog thelaziosis within five (90.47%) to nine (95.23%) days after treatment and it has been demonstrated to overcome problems linked to the mechanical removal of parasites or to the restraining of the animals for the local instillation of drugs in the eyes (Bianciardi and Otranto, 2005). Recently, the administration of an injectable sustained-release formulation of moxidectin provided one season protection against T. callipaeda infection in dogs from an endemic area of Northern Italy (Rossi et al., 2007). The above chemoprophylaxis approach would more likely reduce the prevalence of dog thelaziosis, and therefore the risk for human infections in endemic areas.

Domenico  Otranto  scientists  group

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