8fontanilla.pmd Philippine Survey of Nematode Parasite Infection 72 SCIENCE DILIMAN (JULY-DECEMBER 2014) 26:2, 72-84 Phil ippine Survey of Nematode Parasite Infection and Load in the Giant African Snail Achatina f ul ica indicate Angiostrongyl us cantonensis infection in Mindanao Daisy May A. Constantino-Santos* Brian S. Santos Johanne Myrrh E. Soriano Jon Stewart H. Dy Ian Kendrich C. Fontanilla Institute of Biology University of the Philippines Diliman _______________ *Corresponding Author ISSN 0115-7809 Print / ISSN 2012-0818 Online ABSTRACT Achatina fulica is a ubiquitous land snail commonly found throughout the Philippines. As a generalist feeder and being able to survive in a wide range of habitat types and conditions, the snail can easily establish itself in a new area after introduction. It also acts as host to a variety of parasites, including nematodes, which may accidentally infect humans. In this study, A. fulica individuals from 13 areas in the Philippines were sampled and analyzed for nematode infection rate and load. Of the 393 i n d i v i d u a l s s a m p l e d , 8 0 ( 2 0 % ) w e r e f o u n d t o b e i n f e c t e d , w i t h 5 0 4 9 nematodes isolated. The infection rates and parasite load were highly v a r i a b l e . O v e r a l l , t h e p a r a s i t e l o a d r a n g e s f r o m 1 t o 8 6 7 p e r s n a i l . Representative nematodes from A . fulica from Plaridel (n=8) and Davao C i t y ( n = 2 6 ) i n M i n d a n a o w e r e s u b j e c t e d t o D N A e x t r a c t i o n , P C R a m p l i f i c a t i o n , a n d s e q u e n c i n g o f t h e S S U r R N A g e n e , w h i c h i s t h e universal barcode for nematodes. Sequences successfully matched with the dog lungworm Oslerus osleri for the Plaridel nematodes and the rat l u n g w o r m A n g i o s t r o n g y l u s c a n t o n e n s i s f o r t h e D a v a o C i t y n e m a t o d e s , r e s p e c t i v e l y. T h e l a t t e r i s k n o w n t o i n f e c t h u m a n s a n d c a n c a u s e eo s i n o p h i l i c m e n i n g o e n ce p h a l i t i s . T h i s s t u d y p r e s e n t s t h e f i r s t r e p o r t of A. cantonensis in A. fulica from Mindanao and raises a public health concern. Keywords: Achatina fulica, nematode, Philippines, SSU rRNA , Oslerus osleri, Angiostrongylus cantonensis D.M.A. Constantino-Santos and others 73 INTRODUCTION The Giant African Land Snail, Achatina fulica (Family Achatinidae), is a ubiquitous snail with characteristic reddish brown markings on the shell (Jarrett 1931, Raut and Barker 2002) and has the ability to adapt to a wide range of environmental conditions (Cobbinah and others 2008). Its habitat and diet include a wide variety of plant species (Jarrett 1931, Raut and Barker 2002). A. fulica originated from eastern coastal Africa but has spread throughout Asia and the Pacif ic, including the Philippines, during World War II. A . fulica was introduced to these regions by the Japanese to be used as an alternative food source (Alicata 1966, Latonio 1971). A. fulica is capable of carrying large numbers of parasitic nematodes that may infect humans (Kliks and Palumbo 1992). The snail is a known intermediate host of the rat lungworm Angiostrongylus cantonensis (Tsai and others 2004, Cobbinah and others 2008, Zhang and others 2009, Fontanilla and Wade 2012, Constantino- Santos and others 2014). A. cantonensis infection causes eosinophilic meningitis in humans, and outbreaks of the disease have been associated with exposure to infected A . f u l i ca (Tsai and others 2004). Humans may become infected by A. cantonensis via consumption or contact with an infected snail. Consumption of A. fulica is quite common due to its high protein, iron, and calcium content ( Ts a i a n d others 2003, Cobbinah and others 2008). Infection by contact with A . fulica was documented in Taiwan, especially among children playing with the snail (Tsai and others 2004). Apart from A. cantonensis, A. fulica has also been shown to harbor Rhabditis sp. (Seehabutr 2005), the cat lungworm Aelurostrongylus abstrusus (de Andrade-Porto and others 2012), and the dog lungworm Ancylostoma caninum (Constantino-Santos and others 2014). Numerous studies in the Philippines have targeted gastropods, including A. fulica, to determine the presence of a specif ic nematode, A . cantonensis, but these were limited to Luzon (Salazar and Cabrera 1969, Westerlund and Chamberlain 1969, Latonio 1971, Fontanilla and Wade 2008). A recent study by Constantino-Santos and others (2014) demonstrated the presence of two medically important nematodes, A . cantonensis and A . caninum, and 12 other unidentif ied nematodes in A. fulica populations found in Metropolitan Manila. They used the SSU rRNA gene to identify the nematodes or determine their closest match. In this study, a survey of the nematode parasite load and infection rate in A. fulica populations was conducted in 13 different parts of the Philippines. Furthermore, nematodes in the Mindanao populations, which were surveyed for the f irst time, were identif ied through DNA sequencing of the SSU rRNA gene. Philippine Survey of Nematode Parasite Infection 76 ·  RESULTS AND DISCUSSION All areas sampled were found to have infected snails except for Batac, Ilocos Norte. Infection rates for infected populations, however, varied, ranging from 3% to 39%. Even the parasite load of infected snails exhibited high variability, ranging from 1 to 867 nematodes per snail. Davao City, in particular, had the highest infection rate and total number of parasites (Table 1). The variation in parasite load could be a function of the relative age of the snail. Sithithaworn and others (1991) found a positive correlation between the age of the snail based on its shell size and the parasite load; they noted a mean parasite load of 5478 per infected snail in the oldest snail group (>6.60 cm shell length). On the other hand, the variation in rate of infection across the different A. fulica populations may be due to the patchy distribution of both the parasite and snail host. For instance, Bisseru (1971) observed high variation of infection rates in A. fulica even within a small geographic area in West Malaysia, with two populations exhibiting no infection. The prevalence of the parasite is subject to the availability of the hosts, both def initive and intermediate, for the parasite to complete its life cycle. Eight nematode samples from Plaridel were successfully subjected to direct sequencing of the 5’ end of the SSU rRNA gene and gave higher than 99.5% identity with Nematoda sp. Fontanilla (GenBank EF514918) and Oslerus osleri (GenBank AY295812) based on BLAST results. The BLAST results for the Plaridel nematodes Bacoor, Cavite 30 3 (10%) 53 (2-49) Baguio City, Benguet 30 4 (13%) 82 (1-68) Batac, Ilocos Norte 30 0 (0%) 0 (0) Biasong, Cebu 30 4 (13%) 10 (1-5) Legazpi, Albay 30 8 (27%) 792 (1-743) Boac, Marinduque 30 11 (37%) 184 (2-160) Butuan City, Agusan del Norte 30 5 (17%) 1478 (5-867) Davao City, Davao 30 11 (37%) 2300 (1-791) Matanao, Davao del Sur 30 2 (7%) 8 (2-6) Plaridel, Misamis Occidental 33 13 (39%) 74 (1-27) Tagbilaran City, Bohol 30 10 (33%) 43 (1-18) Taytay, Rizal 30 8 (24%) 24 (1-7) Tres de Avril, Cebu 30 1 (3%) 1 (1) Total 393 80 (20%) 5049 (0-867) Table 1. Infection rates and parasite load of A. ful ica specimens from 13 sites in the Philippines Site Sample size Number of infected snails (Percentage) Total number of nematodes (Parasite Load Range) D.M.A. Constantino-Santos and others 77 are summarized in Table 2. Following the 99.5% threshold value proposed by Floyd and others (2002) in identifying nematodes using the SSU rRNA gene, these nematodes are therefore in the same molecular operational taxonomic unit (MOTU) as Oslerus osleri. O. osleri is previously classif ied as Filaroides osleri. It is a metastrongyle parasite with worldwide distribution and is a widely occurring tracheal parasite capable of causing respiratory disease in domestic and wild canids such as dogs. It is transmitted by direct contact and oral ingestion of the f irst-stage larva (Foreyt and Foreyt 1981, Outerbridge and Taylor 1998). The life cycle of this nematode does not involve an intermediate host (Outerbridge and Taylor 1998). Mucociliary apparatus carries the eggs and the infective f irst- stage larva from the tracheal bifurcation to the oropharynx, where they are either swallowed and shed in the feces or shed in the saliva. The primary sources of the parasite are the asymptomatic dogs (Outerbridge and Taylor 1998). Following infection, the f irst-stage larvae penetrate the mucosa of the gastrointestinal tract. It then travels to the right side of the heart via lymphatics or the hepatic venous circulation where the larva migrates to the lungs via pulmonary arteries (Yao and others 2011). Development from the f irst-stage larva to adult occurs in the respiratory tract where tracheobronchial nodules are formed, thus completing the life cycle (Clayton and Lindsay 1979). Maternal grooming is assumed to be the major transmission route in domestic dogs; for free-ranging canids, the regurgitative feeding of the young by parents appeared to be the major means of infection (Clayton and Lindsay 1979, Bowman 2009). The presence of O. osleri in A. fulica in Plaridel, Misamis Occidental could be a result of accidental infection as the snail is not the def initive or f inal host of the parasite and may have only been shed via the feces by an infected dog. A previous study by Constantino-Santos and others (2014) also detected the presence of worms most similar to O. osleri (99.1% identity) and O. rostratus (99.5%–99.7% identity) in A. fulica populations in Metro Manila, Philippines. Nevertheless, detection of O. osleri in Plaridel indicated that A. fulica could serve as another route of infection for humans, especially to those who come in contact with these snails. Twenty six nematode samples from Davao City had the 5’ end of their SSU rRNA gene successfully sequenced and identif ied as Angiostrongylus cantonensis (GenBank GQ181114) based on BLAST results and Floyd’s 99.5% threshold value, with f ive of them having 100% identity with A. cantonensis, as shown in Table 2. Philippine Survey of Nematode Parasite Infection 78 Plaridel, Misamis Occidental 15-2A EF514918 Nematoda sp. Fontanilla D17-D1 446/446(100%) 0/446(0%) AY295812 Oslerus osleri 444/446(99.6%) 0/446(0%) 15-2B EF514918 Nematoda sp. Fontanilla D17-D1 406/406(100%) 0/406(0%) AY295812 Oslerus osleri 405/406(99.8%) 0/406(0%) 15-3A EF514918 Nematoda sp. Fontanilla D17-D1 423/423(100%) 0/423(0%) AY295812 Oslerus osleri 421/423(99.5%) 0/423(0%) 15-3B EF514918 Nematoda sp. Fontanilla D17-D1 416/416(100%) 0/416(0%) AY295812 Oslerus osleri 414/416(99.5%) 0/416(0%) 15-5A EF514918 Nematoda sp. Fontanilla D17-D1 387/388(99.7%) 0/388(0%) AY295812 Oslerus osleri 386/388(99.5%) 0/388(0%) 15-5B EF514918 Nematoda sp. Fontanilla D17-D1 420/420(100%) 0/420(0%) AY295812 Oslerus osleri 418/420(99.5%) 0/420(0%) 15-13 EF514918 Nematoda sp. Fontanilla D17-D1 422/422(100%) 0/422(0%) AY295812 Oslerus osleri 420/422(99.5%) 0/422(0%) 22-4 EF514918 Nematoda sp. Fontanilla D17-D1 420/421(99.8%) 0/421(0%) AY295812 Oslerus osleri 418/421(99.3%) 0/421(0%) Davao City, Davao DV1 GQ181114 Angiostrongylus cantonensis 428/429(99.8%) 0/429(0%) DV3 GQ181114 Angiostrongylus cantonensis 439/439(100%) 0/439(0%) DV4 GQ181114 Angiostrongylus cantonensis 439/439(100%) 0/439(0%) DV5 GQ181114 Angiostrongylus cantonensis 428/429(99.8%) 0/429(0%) DV6 GQ181114 Angiostrongylus cantonensis 438/439(99.8%) 0/439(0%) DV7 GQ181114 Angiostrongylus cantonensis 434/436(99.5%) 0/436(0%) DV3A JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3B JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3C JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3D JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3E JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3F JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3G JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3H JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3I JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3J JN663725 Angiostrongylus cantonensis 460/460(100%) 0/460(0%) DV3K JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3L JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3N JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3O JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3Q JN663725 Angiostrongylus cantonensis 460/460(100%) 0/460(0%) DV3S JN663725 Angiostrongylus cantonensis 460/460(100%) 0/460(0%) DV3T JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3X JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3Y JN663725 Angiostrongylus cantonensis 466/467(99.8%) 1/467(0.2%) DV3AA JN663725 Angiostrongylus cantonensis 468/469(99.8%) 1/469(0.2%) Table 2. Identity of nematodes from Achatina ful ica in Plaridel, Misamis Occidental and Davao City, Davao based on BLAST Specimen Code Accession Number Best Match % Similarity Gaps D.M.A. Constantino-Santos and others 79 This study demonstrates that A. fulica in Davao facilitates the spread of A. cantonensis. The absence of A. cantonensis in Plaridel, on the other hand, is not unexpected. In a study by Bisseru (1971), 27 sites in West Malaya were surveyed for A. cantonensis from A. fulica, and two of these sites did not even yield any parasite. For instance, Padang Besar in Perlis had 0% infection rate from 100 snails sampled, whereas Alor Star, Kedah, which was 65 km away, yielded 28.5% infection rate from 70 snails. The snail intermediate hosts, the rat def initive hosts, or even the parasites, have a patchy distribution themselves; most likely, only those snails found in areas with rats that harbored the parasite were the ones infected (Fontanilla and Wade 2012). Cases of A. cantonensis infection in rodents and molluscs have been documented in the Philippines; however, most of these were found in several provinces in Luzon (Salazar and Cabrera 1969, Westerlund and Chamberlain 1969, Garcia 1979, Antolin and others 2006) and one area in the Visayas (Guerrero and Guerrero 1972). This is the f irst reported case of A. cantonensis in Mindanao in either the snail intermediate host or the rat def initive host. The presence of A . cantonensis in Mindanao is not surprising considering the distribution of rats and A. fulica all over the Philippines. This information, however, is signif icant from a public health perspective. These nematodes cause eosinophilic meningitis or meningoencephalitis (Wan and Weng 2004). They are now generally recognized as the causative agent of human eosinophilic meningitis, also called angiostrongyliasis (Panha 1988), which is characterized by inflammation of the meninges in the human brain and the presence of higher levels of eosinophils in the cerebral spinal fluid (Senanayake and others 2 0 0 3 ) . A three-year study done by Latonio (1971) focused on four cases of eosinophilic meningoencephalitis and two cases of myeloencephalitis symptom-complex from patients in the Philippines. He reasoned that infection might not be due to direct consumption, since A. fulica is not normally consumed by Filipinos. Rather, it is possibly due to the prevalence of A. fulica among edible plants, which could have been contaminated by the snail with A. cantonensis, either through their feces or mucus, before they were consumed by humans (Wallace and Rosen 1969, Marquardt and others 2000). Alternatively, handling infected snails, particularly by children who play with them, could have been another possible route of infection (Wan and Weng, 2004). Occurrence of A. cantonensis in Canton, China was f irst described by Chen (1935) in rats. Rodents are considered its def initive host (e.g. , Rattus rattus, R. norvegicus) whereas intermediate hosts include snails (e.g. , A c h a t i n a f u l i ca , P o m a ce a Philippine Survey of Nematode Parasite Infection 80 canaliculata) and slugs (e.g. , Imerinia plebeia, Leavicaulis alte) (Bartschi and others 2003). The basic life cyle of A . cantonensis involves a def initive mammalian host and an intermediate molluscan host. The adult worms live in the pulmonary arteries of their def initive hosts, where the females also lay their eggs. These hatch into f irst- stage larvae, which are then transmitted to the rats’ feces via the trachea and gastrointestinal tract. These larvae enter their intermediate hosts, such as molluscs, through ingestion of the excrement, wherein they turn into third-stage larvae after two successive molts (Lee and Yen 2005). An infected mollusc can carry a highly variable number of second– and third–stage juveniles depending on the degree of infection (Caldeira and others 2007). These third-stage larvae infect the rat host through consumption of the intermediate host. The larvae migrate to the central nervous system where further development occurs until they reach adulthood. From there, they then return to the pulmonary arteries where they undergo sexual maturation (Qvarnstrom 2007). Humans, upon ingestion of these molluscs in their raw forms, become accidental hosts, manifesting the infection as eosinophilic meningitis (Senanayake and others 2003). The third-stage larvae, similar to what occurs in rodents, migrate to the central nervous system, which consists of the brain and spinal cord tissues. However, these larvae often remain in the central nervous system of human hosts; nevertheless, rare cases exist wherein they continue migration to the lungs. Their presence in the brain and spinal cord causes tissue damage and subsequent inflammation (Qvarnstrom 2007). To prevent human infection, the most effective method is to educate people to maintain good sanitation in food preparation areas, not to eat raw or undercooked snails, and to avoid eating raw vegetables that may harbor inconspicuous or juvenile snails or slugs in regions where A . cantonensis is present (Yang and others 2013). As Davao A. fulica snails carry the said parasite, it is important to heighten the awareness of people in the area of the possibility of infection through consuming raw vegetable crops associated with the snails or playing or handling these snails. 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Veterninary Parasitology 179(1-3): 123-9. Zhang L.H. , Chen J.L. , Dong W.R. 2009. Analysis of a survey of the infection of Achatina f u l i c a w i t h A n g i o s t r o n g y l u s c a n t o n e n s i s i n F o s h a n , G u a n g d o n g P r o v i n c e . A c t a Parasitologica et Medica Entomologica Sinica 16: 244-246. _____________ Daisy May A. Constantino-Santos is a PhD student at the Institute of Biology, University of the Philippines (UP), Diliman and a University Research Associate at the Natural Sciences Research Institute, UP DIliman. She obtained her BSc Biology at UP Baguio and her MSc Biology (Genetics) at UP Diliman. She specializes in Molecular Genetics. Brian S. Santos is an Instructor and a PhD student at the Institute of Biology, UP Diliman. He obtained his BSc Biology and MSc Biology (Genetics) at the Institute of Biology, UP Diliman. He specializes in Molecular Population Genetics and Morphometrics. Johanne Myrrh E. Soriano is currently a medical student at the Far Eastern University Nicanor Reyes Medical Foundation. She obtained her BSc Biology at UP Diliman. Jon Stewar t H. Dy is currently a medical student at the St. Luke’s College of Medicine. He obtained his BSc Biology at UP Diliman. Ian Kendrich C. Fontanilla is an Assistant Professor and head of the DNA Barcoding Laboratory, Institute of Biology, University of the Philippines Diliman. He received his PhD in Genetics from the University of Nottingham, United Kingdom. He specializes in Molecular Genetics and Molecular Phylogenetics.