Gastrointestinal parasitic helminths of bats from a cave in Luzon Island, Philippines

Bat sampling was conducted in Cavinti Underground River and Caves Complex (CURCC) (14°16′50.85″N, 121°38′5.68″E) located in Barangay Paowin, Cavinti at the eastern side of Laguna Province in the Philippines (Fig. 1). Bat sampling was conducted on May 22 – 30, 2014. The cave system was discovered by a local logger in 1980 and was opened to tourists in 2013. Bats were collected from three caves, namely, Cathedral, Minalokan, and Kalaw, as well as the surrounding karst forest areas. Cathedral and Minalokan cave entrances are traversed by a river. Vegetation around the caves is mainly secondary growth forest adjacent to agroforests and coconut plantations. Plants belonging to the families Melastomataceae, Euphorbiaceae, and Moraceae (Ficus spp.) dominate the surrounding vegetation.

Fig. 1.

Location map of Cavinti, Laguna (left) and sampling sites in Cavinti Underground River and Cave Complex (CURCC) (right).

Bats were captured using mist nets (12 × 2.6 m with 36 mm mesh and four shelves) placed 0 – 3 m above ground, in a series of five nets. A total of 20 nets were set in cave entrances as well as in potential flyways outside the cave (i.e. forest clearings, near streams and rivers). These nets were watched over for an hour after dusk (1800h to 1900h) and were regularly checked every 10 minutes especially during emergence to mitigate potential harm to bat captures. The nets were then left overnight for eight nights then checked early morning the next day to retrieve any captures. Individual bats captured were put separately into clean cloth bags. Bat identification was based on Ingle and Heaney (1992). Morphometric data such as weight, sex, and age category (juvenile, sub-adult, adult) of the bats captured were identified and recorded. In particular, age classification of bats was determined by illuminating the dorsal side of the wing and assessing the extent of fusion in the epiphyseal plates of the phalanges; bats exhibiting unfused epiphyseal plates were classified as juveniles, whereas those with fused plates were considered adults (Kunz, 1988). Photographs of the species collected were also taken.

Each bat was sacrificed by placing the cloth bag in a Ziploc with cotton moistened with ethyl acetate. The abdominal cavity was opened, and the digestive tract removed intact and placed in a clean Petri dish. The intestinal tract was gently teased to remove intestinal contents as well as the lining of intestine to remove embedded parasites. Helminths found were picked, counted, and placed in cold saline solution to evert proboscis for identification purposes. The helminths were then placed in tubes filled with 70 % ethanol for further processing in the laboratory.

The helminths were stained and identified using a light microscope. For trematodes and cestodes, isolated samples were directly placed onto depression slides, stained using Acetocarmine stain then de-stained using acid ethanol (1ml HCL: 100 ml 70 % Ethanol) after five minutes to clear some of the red tinge off of the specimen. Total time for clearing of specimen was variable depending on the tinge on the specimen; some might take longer while others have shorter clearing times. Basic ethanol (1ml 10 % NH3: 100ml 70 % Ethanol) was then used to replace the acidic ethanol and add a bluish tinge to some of the parasites’ external and internal structures and add a color gradient to the specimen for easier viewing of its structure and organs. The basic ethanol was then pipetted out and the stained specimen was then dehydrated using a series of serial dilutions of ethanol (i.e. 70 %, 80 %, 90 % and 100 % ethanol). The specimen was placed in each dilution for 15 minutes to extract all water from the specimen. The specimens were then cleared in Xylene, then mounted on a slide using Canada balsam. Specimens that were not immediately mounted were stored in either xylene or 100 % ethanol.

For nematodes, collected specimens were subjected to the same serial dilution of ethanol as mentioned above. After dehydration, the nematodes were placed in two washings of clove oil for at least 30 minutes each. These were fixed in a 3:1 ratio of clove oil and Canada balsam, then mounted on a slide using Canada balsam.

Identification of isolated helminths were done through the aid of published journals and diagnostic keys by Inglis (1968), Prudhoe and Manger (1969), Mészáros (1973), Fischthal and Kuntz (1975), Lotz and Palmieri (1985), Wong and Anderson (1986), Kifune et al. (2001), Okafor et al. (2004), Bray et al. (2008), and Hechinger (2012).

Prevalence (P) and mean intensity (MI) of infection for each helminth taxon were calculated based on Bush et al. (1997), and values were computed using Quantitative Parasitology (QP) version 3.0 (Rozsa et al., 2000). Chi-square test of independence was used to determine differences in the prevalence of helminths among all the samples while Mann-Whitney (U-test) was used to compare mean intensity between host sex, diet, roosting site, and age; both analyses were done using SPSS version 20 (SPSS Inc., Chicago, IL, USA)

Among the three helminth groups, highest prevalence and intensity of infection were observed for trematodes (Table 1). This is in accordance with other studies which reported a higher percentage of bats being infected with trematodes compared with other helminth groups (Nickel & Hansen, 1967; Blankespoor & Ulmer, 1970; Ubelaker, 1970; Coggins, 1988; Hilton & Best, 2000). Further, insectivorous bats were the only positive for trematode infection which is consistent with other studies conducted from other regions (Ubelaker, 1970; Coggins, 1988; García-Vargas et al., 1996; Pérez-Ponce de León, 2001). This can be attributed to the fact that insectivorous bats primarily consume insects, which are commonly recognized as intermediate hosts for trematodes (Ubelaker, 1970; García-Vargas et al., 1996).

Knight and Pratt (1955) described the first life cycle of a trematode with bat as a final host such that bat feces containing parasite eggs reach water sources, in which the miracidia hatch and infect freshwater snails. Cercariae shed by the latter enter a second aquatic intermediate host, usually insect larvae, and develop into metacercariae, which encyst and are infective to bats as the final host. In some cases, bats can also become infected by trematodes when a free swimming cercaria is ingested when drinking from contaminated water source (Noguiera et al., 2004). Thus, considering the typical pattern of digenean life cycles, the more frequently the host comes into contact with water, the higher the likelihood of infection by these helminths (Pérez-Ponce de León, 2001; Niewiadomska & Pojmanska, 2011). As such, the high prevalence of trematodes may also be due to the availability of water sources in the sampling site.

Trematodes isolated from the sampled bats in this study include Acanthatrium sp., Prosthodendrium sp., and Plagiorchis sp. (Fig. 2):

1. Acanthatrium sp.

Fig. 2.

Trematodes isolated from bats collected in Cavinti Underground River and Cave Complexes (CURCC), Laguna: A. Prosthodendrium sp. adult B. Acanthatrium sp. adult. C. Plagiorchis sp. adult. AC—Acetabulum, CP—cirrus pouch, OS—Oral Sucker, OV—Ovary, PH—Pharynx, T—Testes, UT—Uterus, VT—Vitellaria

This genus was first described by Faust (1919) for all lecithodendriid trematodes having spines in the genital atrium and having pretesticular vittelaria. Spines are visible in the external tegument. The oral sucker is positioned at the anterior terminal end and the acetabulum positioned approximately at the middle portion of the fluke (Fig. 2A). Bulbous pharynx is also visible, leading to the intestinal ceca. Testes are entire and ovoid, located at each side of the fluke and positioned slightly posterior the acetabulum. The ovary is located on the right of the fluke and can usually be seen slightly anterior or posterior to the acetabulum.

In Japan, this trematode was also isolated from the gut of bats such as Rhinolophus ferrumequinum and Myotis pruinosus (Kifune et al., 2001). In Texas, a species of this genus, Acanthatrium alicatai, infected cave-dwelling bats such as Myotis velifer and Antrozous pallidus (McAllister et al., 2007). In general, Acanthatrium transmission to bats involves cercariae developing into sporocyst inside a snail, penetrating caddisfly larvae and bats become infected by eating infected adult caddisflies (Knight & Pratt, 1955).

In this study, this lecithodendrid trematode was isolated from three species of bats sampled namely, Miniopterus paululus, Rhinolophus rufus, and Rhinolophus inops (Table 2). All three bat host species are new records for the genus Acanthatrium.

Sedlock et al. (2014) observed species of Rhinolophus having insects of order Trichoptera to be a part of their diet. Since Trichopteran and coleopteran insects are known intermediate hosts of Acanthatrium, rhinolophids have a high chance of being infected as observed in this study (Ubelaker, 1970). Meanwhile, Miniopterus diet consists of a wide range of insects including dipterans, hemipterans, and isopterans and to a lesser extent, lepidopterans and coleopterans which could also have permitted the infection by Acanthatrium (Jacobs, 1999; Ubelaker, 1970).

2. Prosthodendrium sp.

The genus Prosthodendrium was established for all lecithodendriid flukes having pretesticular vitteline glands. Prosthodendrium can be differentiated from Acantharium with the former being slightly bigger and more ovoid to circular in shape, sometimes appearing to be wider than they are long. It lacks cuticular spines and the prostate mass does not overlap with the acetabulum. Ovary is located posterolateral on the right side of the acetabulum and slightly below the line of the testis (Fig. 2B).

There were records of this helminth occurring in bats in other countries. In Japan, they were isolated from gut of vespertillionids (i.e. Myotis ikkonikovi, Myotis yanbarensis, Myotis pruinosus, Myotis natteri and Miniopterus fuscus) and Rhinolophus ferrumequinum (Kifune et al., 2001). Prosthodendrium oscidia and Prosthodendrium chilostomum were also isolated from Taphozous melanopogon and Pipistrellus pulveratus in Cambodia and were previously recorded in neighboring Asian countries such as Japan, China, Taiwan, Vietnam, Arabia, India, Afghanistan, Iraq, Egypt, and Yemen (Kifune et al., 2001).

Hipposiderids such as Hipposideros armiger and H. bicolor were also known hosts of this trematode (Fischthal & Kuntz, 1975; Lotz, 1985). In this study, this trematode was exclusively isolated from one species, H. diadema, making it a new host record of this this trematode species (Table 2). A possible mode of infection of this trematode could have been ingestion of metacercariae encysted in dragon fly larvae larvae (naiads) of the Family Libeluidae (Vajrasthira & Yaemput, 1971).

3. Plagiorchis sp.

Plagiorchis is one of the largest families in the order Plagiorchiida. Its taxonomy is currently problematic because of high morphological similarity between different forms and species of this genus. In general, these trematodes have elongated body and a ventral sucker that is slightly larger or at least equal in size to the oral sucker. They also feature a long cirrus-sac along the longitudinal body axis, and the anterior margin of the vitellarium does not extend to the posterior margin of the ventral sucker (Sharpilo & Tkack, 1992). Specimens collected from this study have a short esophagus leading to bifurcating ceca that extends almost the entire length of the fluke (Fig. 2C). It has an acetabulum positioned well above the midline of the body. The ovary can be located below the acetabulum and above the two testis that are positioned either in tandem or one on top the other. The uterus when gravid fills almost the entirety of the worm with eggs.

This trematode has been reported infecting species of bats of the genus Tadarida, Rhinolophus, Miniopterus, Myotis, and Pipistrellus (Nahhas et al., 2005; Horvat et al., 2016). One species, Plagiorchis vespertilionis, is a common bat trematode but has been reported to infect humans (Guk et al., 2007). This was also recently reported for the first time in the Philippines and was isolated from the gut of mouse-eared bat (Myotis sp.) (Salcedo, 2021). Thus, this is the second account of this zoonotic parasite in the country. In this study, Plagiorchis was isolated from an individual of Hipposideros pygmaeus, providing a new host record for this trematode. Transmission of this trematode to bats could be due to ingestion of cercariae encysted in larvae of Ephemeria, Trichoptera, mosquitoes (Culex), and dragonfly nymphs (Ubelaker, 1975).

Although studies have shown a high degree of specificity of nematode species in bats of Suborder Microchiroptera, very little is known about their transmission dynamics and life cycle (Ubelaker, 1970; Barus & Rysavy, 1971). These parasites can either have an indirect or direct life cycle. For instance, the nematode Litosomoides yutajensis, a vector-transmitted filarial worm from a mormoopid bat was observed to have an indirect life cycle, with filarioids transmitted by haematophagous arthropods when feeding on the host (Anderson, 1992; Guerrero, 2006). Nematode larvae have also been recorded from blood-sucking ticks and insects (Beaver & Burgdorfer, 1984; Bain & Renz, 1993; Spratt & Nicholas, 2002). Meanwhile, Strongylacantha glycirrhiza which mostly infects rhinolophid bats has a direct life cycle: eggs from bat’s feces are passed into surroundings and infects other intermediate hosts when third-stage larvae penetrate the skin or are ingested (Anderson, 1992). Some species of nematode larvae also have the ability to escape from the adult female vulva by perforating the cuticle and bats making contact with each other at roosting site allows the infective larvae to penetrate the next host allowing higher intensity of infection (Ubelaker, 1970). Beetles and cockroaches also often serve as intermediate host of nematodes for other mammals (Cram, 1931; Martin, 1976).

Two nematode taxa were isolated from bats in this study:

1. Nycteridostrongylus sp.

Isolated worms have a dorsal esophageal tooth at the cephalic extremity (Fig. 3A). The cephalic extremity is somewhat enclosed within a bursa. The samples also have a prominent cervical organ of fixation observed at the anterior extremity of the worms. This structure looks like an expanded flap originating from the anterior and enlarging into a bulbous flap with visible striations.

Fig. 3.

Nematode Nycteridostrongylus sp. (A) anterior part showing cephalic head and (B) copulatory bursa.

Nycteridostrongylus was first described by Baylis (1930) to be a common parasite of genus Miniopterus. Only three species of Nycteridostrongylus were described with one species, Nycteridostrongylus uncicollis, restricted to bats of genus Miniopterus (Wong and Anderson 1986). Thomas (1959) isolated this nematode in Miniopterus blepotis captured in Australia while Gibson et al. (2005) and Meszaroz (1973) reported this trematode to infect Miniopterus schreibersii in London and Miniopterus fuligonosus in Vietnam, respectively. However, no definite life cycle and transmission dynamics involving bats was established.

In this study, 44 individuals of Nycteridostrongylus were isolated from Miniopterus paululus. In addition, the occurrence of this nematode in other bat species such as Rhinolophus rufus, Rhinolophus inops, and Hipposideros diadema gives additional host record of this nematode aside from Miniopterus.

2. Toxocara sp.

Isolated Toxocara samples were milky-white in color. Male and female Toxocara were morphologically distinct from each other with males having highly curved tails while the females had none (Fig. 4E). Females were also generally larger and longer with an average length of 106.60 ± 8.68 mm as compared to the males which had average length of 57.00 ± 4.9mm. The worms have three distinct lips which were observed upon close examination of the anterior end (Figs. 4A and 4C). The reproductive organs, copulatory spicule and vulva, are also visible.

Fig. 4.

Male Toxocara sp. (A) anterior and (B) posterior part. Female Toxocara sp. (C) anterior and (D) posterior part. (E) Toxocara sp. adult male (top) and female (bottom).

Currently, 26 species of Toxocara are recognized, most of which are non-transmissible to humans (Ziegler & Macpherson, 2019). Of these, two species are known to infect bats: Toxocara pearsei known only from South and Central America with Natalus tumidirostris and Peropteryx macrotis as known recorded host, while Toxocara pteropodis is a known zoonotic nematode infecting flying foxes (Pteropus sp.) and has been recorded in Oceania, Australia, India, Indonesia, and Papua New Guinea (Ziegler & Macpherson, 2019).

Prociv (1987) has previously isolated worms similar to T. pteropodis from Rousettus amplexicaudatus specimens collected from Luzon Island, Philippines. In this study, this nematode was also exclusively isolated from Rousettus amplexicaudatus. Average total length measurements of male (57.00 + 4.9mm) and female (106.60 + 8.68mm) Toxocara samples isolated in this study were also within the range of sizes obtained by Prociv (1987). A detailed examination of eggs and preserved specimens of Toxocara from this study is pending to compare with T. pteropodis.

Studies on cestodes parasitizing bats are relatively few compared to other helminth classes. A number of cestode genera have been observed infecting chiropterans including Hymenolepis, Taenia, and Oochoristica (Prudhoe and Manger, 1969; Murai, 1976; Sawada & Harada, 1986). Coleopterans (beetles) are often involved as an intermediate host (Morgan & Hawkins, 1951; Yamaguti, 1961; Ubelaker, 1970). Low occurrence of parasitism by cestodes in bats was observed in some studies (Nogueira et al., 2004; Vargas et al., 2009; Angoma et al., 2020). Similarly, cestode also had the lowest infection rate among the three classes of helminths in this study. This group was represented by only one taxon:

Vampirolepis sp.

Vampirolepis is closely related to the tapeworms of genus Hymenolepis and both belong to the family Hymenolepididae. Isolated Vampirolepis samples in this study have scolex with four suckers that are approximately 100 μm in diameter (Fig. 5A). Rostellum was retracted or egested with rows of hooks lining its crown. This cestode was isolated in the small intestine of the following species of bat host: Hipposideros diadaema, Hipposideros pygmaeus, Miniopterus paululus, Rousettus amplexicaudatus, Ptenochirus jagori, and Rhinolophus rufus (Table 2).

Fig. 5.

Cestode Vampirolepis sp. scolex (A), mature segment (B), and gravid segment (C).

Vampirolepis has been reported parasitizing bats in countries like China, Brazil, Chile, Japan, South Africa, and Hungary (Sawada, 1970; Sawada et al., 1998; Noguiera et al., 2004; Junker et al., 2008; Muñoz et al., 2010) with mean intensity ranging from one to two adult worms per bat and prevalence ranging from one to 30 %, depending on the bat species.

Sawada (1976) hypothesized that based on the ecological standpoint of bats in caves, bat tapeworm life cycle would involve insects from bat guano as intermediate hosts. Bats may be infected by ingesting these insects that have cysticercoids. The cysticercoid will then grow in the bat’s small intestine. This may explain the infection of cestodes in insectivorous cave-dwelling bats in this study (i.e. Hipposideros diadaema, Hipposideros pygmaeus, Miniopterus paululus, Rhinolophus rufus). However, frugivorous bats, Ptenochirus jagori and Rousettus amplexicaudatus, were also infected by Vampirolepis. Although fruits and nectars are the main dietary component of these species, some studies suggest that fruit bats may add comparatively higher protein foods, mainly insects, to their diets (Morrison, 1980; Giraldo-Martínez et al., 2023). For instance, Galorio and Nuñeza (2014) found traces of digested insect parts in stomachs of Ptenochirus jagori and Rousettus amplexicaudatus in the Philippines. Thus, it is possible that these frugivorous bats ingested infected insects when foraging resulting to cestode infection.

Data showed a significantly higher number of infected male individuals (49.50 %) than female (33.30 %) and that these values are statistically significant (x² = 4.574, p = 0.032) (Table 3). On the other hand, intensity of infection was higher in female bats (MI = 16±0.1/infected bat) than males (MI = 4±1.3/infected bat), although these values were not significantly different between sexes (U = 3149.5, p = 0.231).

Many studies report the influence of host sex on parasite prevalence, intensity, and aggregated distribution (Zuk & McKean, 1996; Poulin, 1996; Lord et al., 2012; Giraldo-Martínez et al., 2023). Male-biased infection of helminths are frequently observed in mammals and other vertebrate groups (Klein, 2004). This is due to sex hormones, mainly testosterone, which may have beneficial effects on helminth development (Haukisalmi et al., 1988). Testosterone could depress both cell mediated and humoral immune responses resulting in higher susceptibility to parasitism in males (Grossman, 1989). Female hosts meanwhile have sex hormone estrogen which was reported to increase hosts’ resistance to helminth infection by enhancing humoral immunity and inhibiting cell mediated responses (Klein, 2004).

Helminth intensity has no significant difference between the two sexes which is consistent with previous studies on parasite assemblages of small mammals like bats (Esteban et al., 2001; Lord et al., 2012) and rodents (Feliu et al., 2006). However, some studies report higher intensity and prevalence of helminth infection in other female mammalian hosts: Trichostryonglyus retortaeformis in rabbits, Schistosoma mansoni and Taenia crassiceps in mice, and Hymenolepis nana in rats (Klein, 2004).

Our result showed that insectivorous bats have significantly higher helminth prevalence compared to fruit bats (x² = 5.503, p = 0.019) (Table 3). Insectivorous bats also have a significantly higher number of helminths harbored for each infected bat than frugivores (U = 2550.00, p = 0.006).

Since helminths are typically acquired through the food that bats consume (Holmes, 1964; Phillips, 1966), insectivorous bats may experience more prevalent and higher MI of helminth infections due to their insect diet, which serve as intermediate hosts for many helminth species. Likewise, since fruit bats generally eat fruits and nectar, there is a lower possibility of being infected by helminths. Fruit bats observed in this study only harbored cestodes and nematodes. This suggests that fruit bats may consume insects as a part of their diets or get infected through the incidental ingestion of intermediate hosts. Frugivorous bats have long been observed to consume insects as a source of protein to supplement their diet (Gardner, 1977; Thomas, 1984; Giraldo-Martínez et al., 2023), which could explain infection by helminths.

Cave-dwelling bats have a significantly higher number of infected individuals compared to non-cave dwelling bats (x² = 5.467, p = 0.019). Mann-Whitney U-test meanwhile showed significant difference in MI between cave-dwelling and non-cave dwelling bats (U = 1505.5, p = 0.009).

This is the first study to compare helminth assemblages between cave and non-cave dwelling bats. Cave-dwelling bats generally have relatively larger population size, higher degree of clumping, and frequently interact with other cave-dwelling bat species compared to non-cave dwelling bats making them more vulnerable to parasite infection. Host colony or group size and roosting behavior of bats have been shown to influence density of infection of ectoparasites, resulting in increased prevalence and intensity of infection with increase in host group size (Ter Hofstede & Fenton, 2005). Grooming behavior might be a potential transmission method for helminths in cave-dwelling bats. Helminth eggs left on their bodies during defecation could be ingested when the bats groom themselves. In addition, since some helminths were also hypothesized to have cave arthropods as intermediate hosts, cave-dwelling bats have a higher chance of being infected with helminths (Sawada, 1976). Lastly, some species of nematode larvae that are released when eggs from bat roosts hatch, may penetrate the skin of bats when they come into contact, making cave roosting bats more vulnerable to infection (Ubelaker, 1970).

Age class of sampled bats was classified as juvenile, subadult, and adult. However, no juvenile bats were collected in this study. Data showed that helminth prevalence and MI between the two age classes has no significant difference (x² = 0.202, p = 0.653 and U = 2339.00, p = 0.171, respectively) It is possible that similarities in the habits and roosting locations of both age class for each species may account for the similarities in their infection rates since there are similar opportunities for exposure to pathogens.

Although not statistically significant, helminth infection was observed to be slightly more prevalent in adult bats (P = 41.1 %, MI = 9±0.9/infected bat) than sub-adult bats (P = 37.2 %, MI = 4±0.4/infected bat. Previous studies have also shown higher infection rates in adult bats compared to younger bats owing to the increased foraging efficiency of older bats (Hamilton & Barclay, 1998; Adams & Pedersen, 2000; Lord et al., 2012). Further, adult bats have a wider range of diet including hard-bodied prey items, so a higher diversity of helminth infection is expected, compared to younger bats whose diet is limited to smooth-bodied insects or milk (Aguirre et al., 2003). Future studies could increase the sample size for each species to reveal more distinct patterns or variations in infection rates between subadult and adult bats.