Parasitism with Acanthocephalus Ranae in Frogs (Pelophylax Ridibundus Pallas 1771), from North-east Romania.

The present study was carried out on 10 frogs, Pelophylax ridibundus (Pallas, 1771), captured near the river Bahlui, Iasi, between April and May, 2019, for the study of digestive helminths and their effect on the digestive tract. Macroscopic examination of the general cavity revealed distension of the small intestine. The sectioning of the intestinal wall revealed a variable number of parasites (1 - 12) with a whitish cylindrical body, anchored in the duodenal mucosa. The morphological study of the parasites confirmed the species as Acantocephalus ranae. The prevalence of the infection was 60 % and the average intensity was 4.83 ± 4.15. Lesions of the small intestine were characterized by catarrhal enteritis, hemorrhagic spots and ulcers on the mucosa, occlusion and obstruction of the intestinal lumen. Histologically, eosinophilic inflammatory infiltrate in the glandular crypts, mucosal and submucosal edema, fibrosis and conjunctival hyperplasia, total mucosal atrophy, were found. The infection with Acanthocephalus ranae in Pelophylax ridibundus caused severe pathological changes, conditioned by the intensity of the infection, aspects revealed for the first time in Romania.

Ranids are an important component of the biodiversity of the aquatic environment in which they also play a host role for many species of parasites. They are of particular interest, as they are used in both biomedical experiments and human nutrition. They are also bioindicators of environmental quality and play an important role in controlling populations of harmful insects, or in the pharmaceutical industry, through the use of biologically active compounds in the skin (Cogalniceanu 2002).

Pelophylax ridibundus Pallas, 1771 sin. Rana ridibunda Pallas, 1771 (the marsh frog) lives in lakes, stagnant waters, and shallow marshes with a lot of vegetation. It is a diurnal species, of medium size (9 – 15 cm) (Cogalniceanu, 2002), which differs according to sex and geographical area (in males: mean 71.84 mm, range: 49.56 – 110.80 mm, and in females: mean 80.10 mm, range: 51.05 – 104.78 mm) (Ayaz et al., 2007), which feeds opportunistically on terrestrial and aquatic invertebrates (insects, spiders, crustaceans, gastropods, beetles) (Bam-e-Zar et al., 2019), and small vertebrates (freshwater fish), and sometimes by cannibalism. It has a wide geographical distribution (Ciudin, 2004).

Ninety-eight percent of ranids are parasitized (Elmajdoub et al., 2018), being definitive, intermediate or paratenic hosts for many species of parasites (trematodes, cestodes, nematodes, acanthocephalans) (Imkongwapang et al., 2014), which affect their state of health (Imasuen et al., 2012). Frog health is important for ecological balance, for the accuracy of the results obtained in experimental laboratories and for human health (Ciudin, 2004). Among the parasitic entities that affect the ranids, acanthocephalosis is differentiated by the magnitude of the conflict between the parasite and the host, ending, in general, by triggering the disease state, the depreciation of the host organism to a lethal end (Aznar et al., 2018). Acanthocephalus ranae (Schrank, 1788) Lühe, 1911 has a wide specificity and a large geographical distribution (Hackman et al., 2011).

It is identified in many species of amphibians, including Triturus karelinii Strauch, 1870, Bombina variegata Linnaeus, 1758, Rana dalmatina Bonaparte, 1940, Rana esculenta Linnaeus, 1758, Rana temporaria Linnaeus, 1758, Rana ridibunda Pallas, 1771, in Bulgaria (Dimitrova & Dimitrova, 2012; Dimitrova & Marinova 2018), Pelophylax esculentus Linnaeus, 1758, in Hungary (Heczeg et al., 2016), Rhinella icterica Spix, 1824, in Brazil (Pilati et al., 2013), Bufo viridis Laurenti, 1768, Rana ridibunda (Karakas, 2015), Hyla orientalis Bedriaga, 1890 (Yakar et al. 2016), Rana dalmatina Bonaparte, 1940 (Dusen, et al., 2009), Pelophylax ridibundus Pallas, 1771 (Koyun et al., 2015), Pelophylax bedriagae Camerano, 1882 (Demir et al., 2015), in Turkey, Pelophylax ridibundus (Leon-Regagnon, 2019), in Iran, and the small intestine, in fish (Sakthivel et al., 2017), in India. The traumatic, irritative, compressive, occlusive, inflammatory, toxic, and spoilage mechanisms used by acanthocephalans during parasitism cause digestive disorders, maldigestion, malabsorption, metabolic disorders, irreversible tissue changes on the gastrointestinal tract, morbidity and mortality (Aznar et al., 2018). Acanthocephalus ranae is transmitted through an intermediate host represented by the isopod crustacean, Caecidotea militaris (Amin, 1982; Pfenning, 2017) and the species Asellus aquaticus (Dimitrova & Dimitrova; 2012, Amin et al., 2019), in which the infectious stage of cystacanth develops. Ingestion of intermediate host crustaceans ensures the development of acanthocephalans in the digestive tract of amphibians, freshwater fish (family Cyprinidae), and other aquatic animals. The continuous decline of amphibian populations is directly or indirectly influenced by human activities. The protection of amphibians is regulated worldwide. According to Law 462/2001 (Regime of natural protected areas, conservation of natural habitats of wild fauna and flora), all species of amphibians are protected. Habitats Directive 92/43 / EEC - consolidated on 01/01/2007 by EU Directive, emphasizes that 10 species are strictly protected and three species (Rana esculenta, R. ridibunda, and R. temporaria) can be economically exploited following impact studies (Cogalniceanu 2002; Herczeg et al., 2016). The purpose of these investigations is to report the species A. ranae in the marsh frog species Pelophylax ridibundus in the area of the river Bahlui-Iasi, and the histopathological effects on the small intestine, aspects not previously reported in Romania.

Material and Methods

Study area and sample collection

The investigations took place on frogs collected near the Bahlui River, Iasi (47° 9’44 “N 27° 35’20” E). The Bahlui River, known as the “muddy river, home of frogs” has a length of 119 km, and an average annual flow of 4.88 m3/s; passes through the city of Iasi and has a basin with an area of 2,007 km2.

Ten frogs of the species Pelophylax ridibundus were captured manually between April and May 2019, for the study of digestive helminths and their effect on the digestive tract. Frogs care and handling were carried out in accordance with institutional guidelines. The frogs were examined individually and euthanized by placing them in an ether container. Through the median incision, the general cavity was opened and the internal organs were examined. In some frogs, the opening of the intestine revealed in variable numbers, cylindrical, whitish formations, fixed in the intestinal wall, blocking the lumen. The parasites were extracted, washed with warm distilled water and deposited in containers with 70 % alcohol, for morphology and prevalence studies. Also, fragments of intestine (duodenum and jejunum) were taken and deposited in containers with 10 % formaldehyde, for histopathological processing.

Sample examination

The extracted parasites were counted, separated by sex and examined morphologically, to identify the species, in accordance with the literature (Kurbanov, 1978; Norton & Esposito, 1994; Heck-mann et al., 2011).

The intestinal fragments were embedded in paraffin, sectioned to 5 μm thick, in serial sections and stained with the Masson trichromic method (Foot, 1933). Tissue examination and microphotography were performed with the Leica 750 DM photon microscope, Leica 750 DM, ICC 750 Camera and Leica Application Suite (LAS), software version 4.20 (Oct. 2012), for image retrieval. (Leica Microsystems GmbH Wetzlar, Germany).

Statistical analysis

The statistical parameters regarding body size in acanthocephalans (Mean, Standard Error, Standard Deviation, Sample Variance, Minimum, Maximum) were analyzed through the descriptive statistics program, Microsoft Office 2016, Microsoft Excel 2016 application.

Ethical Approval and/or Informed Consent

Animal care and handling were carried out in accordance with institutional guidelines.

Results

Necropsy examination

Macroscopic examination revealed in the small intestine of six frogs, a variable (1 – 12) number of parasites, strongly anchored in the duodenal wall, which completely occluded and obstructed the intestinal lumen (Fig. 1). On the mucosa, hemorrhagic spots, edema of the tissues at the place of fixation of the parasites, thickening of the wall and abundant mucus, were observed. Twenty-nine specimens were collected from the small intestine of frogs, of which 11 males and 18 females, with a prevalence of 60 % and an average intensity of 4.83 ± 4.15. The parasites collected have an elongated, cylindrical, whitish-yellow body with relatively small dimensions. Males measured on average 11.16 ± 0.51 mm in length and 1.37 ± 0.55 mm in width. Females measured on average 25.31 ± 3.87 mm in length and 2.06 ± 0.54 mm in width. According to the literature (Kurbanov, 1978; Norton & Esposito, 1994; Heckmann et al., 2011), the collected specimens belonged to the species A. ranae.

Fig. 1

Pelophylax ridibundus: parasites anchored in the intestinal mucosa (digital photo).

Histopathological results

Infected frogs showed variable intestinal lesions, determined by the intensity of parasitism. Histologically, areas of desquamation and detachment of the epithelium from the intestinal villi, epithelial hyperplasia, associated with inflammatory cell infiltrate, arranged at the base of the villi, in the glandular crypts was observed (Fig. 2a). The inflammatory infiltrate was rich in lymphocytes, fibroblasts, and granulocytes in which eosinophils predominated in association with erythrocytes released from hemorrhages induced by parasite fixation (Fig. 2b). In other cases, the histological lesions were more serious, characterized by partial or total destruction of the villi, the disappearance of the mucosal epithelium and discovery of the conjunctival axis (Fig. 2c). The intensity of the infection had severe consequences on the intestinal mucosa, causing fibrosis, and conjunctival hyperplasia of microvilli, complete disappearance of intestinal epithelium, abundant inflammatory infiltrate in glandular crypts (Fig. 2b). Also, catarrhal inflammation of the intestinal mucosa was observed (Fig. 2d), in which, hyperplasia and edematous infiltration of the submucosa and musculosa, ulcerative areas and inflammatory infiltrate, necrobiosis and cell necrosis processes, and areas with total atrophy of the intestinal mucosa were found.

Fig. 2 a, b, c, d.

Pelophylax ridibundus infected with A. ranae. Small intestine. a. Destruction of villi tissue architecture (DVA), epithelial hyperplasia (EH), inflammatory infiltrate (II). b. Around the acanthocephalans (Ac.), the mucosa is injured (IM), with abundant inflammatory cell infiltrate(ICI) in glandular crypts (GC) and villous fibrosis (VF). c. The effect of parasitism consists in partial or total destruction of the villi (VD) the disappearance of the mucosal epithelium (DME) and discovery of the conjunctival axis (DCA), and general atrophy. d. Catarrhal jejunitis: hyperplasia and edematous infiltration of the submucosa and musculosa (HEM), ulcerative areas (UA) and inflammatory infiltrate, necrobiosis and cell necrosis processes, and areas with total atrophy (TA) of the intestinal mucosa (Trichormic Masson stain x 100, 200).

In the intestinal contents, cellular detritus, acanthocephalans eggs, and erythrocytes, and also, a section through the proboscis, and the body of a female with the uterus full of eggs, were observed.

Discussions

The parasitism with Acanthocephalus ranae (Schrank, 1788) Luhe, 1911 is studied by many authors (Kurbanov, 1978; Norton & Esposito, 1994; Heckmann et al. 2011; Dimitrova & Dimitrova, 2012; Koyun et al., 2015; Pfenning, 2017; Aznar et al., 2018), from all over the world. The prevalence of A. ranae infection in Pelophylax ridibundus reported in this paper is in agreement with data from the literature. In general, the prevalence of A. ranae infection in amphibians ranged from 2 % in Bufo viridis (Karakas, 2015), to 39 % – 100 % in Rhinella icterica (Pilati et al., 2013). Yakar et al. (2016), report an infection prevalence of 6.66 % in Hyla orientalis, and Demir et al. (2015), report a 9.25 % prevalence in Pelophylax bedriagae, while Dusen et al. (2009), report prevalence of 45.45 % in Rana dalmatina. The prevalence of A. ranae is similar to the prevalence of another species of acanthocephalans reported in amphibians, in other geographical areas (Corynosoma spp. 2.04 %, in Rana saharica) (Elmajdoub et al., 2018). It is known that A. ranae parasitizes amphibians in the small intestine, but Karakas et al. (2015), report both intestinal and gastric localization of A. ranae species, Rana ridibunda and Bufo viridis. In the present work, A. ranae was found only in the intestine. Pilati et al. (2013), showed that the mean intensity of small intestine parasitism in Rhinella icterica with A. ranae was 25 specimens, and the maximum intensity, 53 specimens, in Pelophylax bedriagae (Demir et al., 2015). In most studies, the mean intensity of acanthocephalans infection ranged from 1.50 in Hyla orientalis (Yakar et al., 2016), to 2.66 in Rana dalmatina (Dusen et al., 2009), or 14.60 in Pelophylax bedriagae (Demir et al., 2015). In the current study, the mean intensity of A. ranae infection in Pelophylax ridibundus was 4.83 (1 – 12). The number of acanthocephalans and long-term parasitism influenced the development of the pathological condition. The irritative, compressive, occlusive, toxic, spoilage mechanisms used by acanthocephalans during parasitism cause inflammation of the intestinal mucosa, irreversible tissue damage to the intestinal mucosa, and mortality, aspects also described by Heckmann et al. (2011). The macroscopic and microscopic lesions described in this paper are in agreement with the study done by Heckmann et al. (2011), which highlights the complete obstruction of the intestinal lumen with acanthocephalans and necrotic tissue, in Rana ridibunda. The same authors described, histologically, extensive hemorrhagic areas and many free erythrocytes in the lumen space and granulocytes visible in the inflammatory infiltrate, in high concentrations. The cells of the intestinal villi are destroyed around the extremity of the acanthocephalan penetrated into the intestinal wall, aspects similar to those described in this paper. Recently, Sakthivel et al. (2017) reported the intestinal parasitism of freshwater fish, Caranx ignobilis species with A. ranae, describing tissue changes similar to those presented in Pelophylax ridibundus. So, in fish, in the intestine, at the site of fixation of the parasite, tissue damage, congestion, edema, erosions, ulcers, and thickening of the intestinal wall were observed. The same authors highlighted histologically, hyperplasia of the intestinal villi and lamina propria, cellular infiltrated areas around the proboscis, aggregation of lymphocytes, and fibroblasts at the site of fixation and inflammation, suggesting that long-term parasitism increases cell infiltration (Sakthivel et al., 2017). The examination of the Pelophylax ridibundus frogs collected near the Bahlui Iasi river revealed, for the first time, the parasitism of the small intestine with Acantocephalus ranae and severe tissue changes induced on the intestinal mucosa, aspects not previously reported in Romania. Future research, conducted on a larger number of amphibians collected from other geographical areas of the country, will contribute to a better knowledge of the helminthic fauna of amphibians in Romania and the impact induced on aquatic populations.