Parasite infections in free-ranging amphibians seem to appear obligatory worldwide, and thus, very high prevalences of up to 90% have previously been described (Coggins and Sajdak 1982; Al-Sorakhy and Amr 2003; Amin et al. 2012). For instance, Rizvi et al. (2011) sampled free-ranging amphibians in an Indian Wildlife Sanctuary (Haryana) and found that endemic common dicroglossid frogs (Euphlyctis cyanophlyctis) were frequently infected (52.9%) by nematodes. In contrast to this wildlife study, there is very little knowledge on parasitic infections of dicroglossid frogs (E. cyanophlyctis) kept in captivity. While comparing our prevalence data with previous published studies, it should be considered that most of these surveys were conducted in wild animals, and this fact might explain prevalence differences. Most likely to dicroglossid frogs, other free-ranging amphibians are also showing higher parasitic prevalences when compared with those kept in captivity (Coggins and Sajdak 1982; Amin et al. 2012). Moreover, sensitivity and specificity of applied DSFS to detect helminth and protozoan stages might have influenced observed prevalence as different diagnostic methods in former wildlife studies have been used (Rizvi et al. 2011; Amin et al. 2012).
Despite the fact that extrinsic risk factors, such as habitat changes, habitat losses, predatory pressure, and poor water quality can directly affect parasitic burdens and prevalences in free-ranging amphibians (Vaucher 1990; Kehr and Hamann 2003; Marcogliese and Pietrock 2011; Thiemann and Wassersug 2000), very little is still known whether these factors might also influence the outcome of parasitic burdens in pet amphibians kept in households or zoos (Mutschmann 2010).
In this study, helminth infections occurred frequently in investigated animals (Table 4). All nematode species found in this survey have been reported to possess pathogenic significance for amphibians (Mutschmann 2010; Amin et al. 2012; Langford and Janovy 2009; Langford 2010; Yildirimhan et al. 2012). Correspondingly, amphibians are well-known to be parasitized by numerous nematode families, such as Trichinellidae, Rhabditidae, Strongyloididae, Ascarididae, Cosmocercodidae, Oxyuridae, Heterakidae, Camalladae, Gnathostomatidae, Habronematidae, Filaroidae and Physalopteridae. For amphibians, particularly rhabditidean helminths are considered as pathogenic endoparasites (Mutschmann 2010; Amin et al. 2012; Yildirimhan et al. 2012). The genus Strongyloides is known to cause protein-loss enteropathy in various anuran hosts (Patterson-Kane et al. 2001). Cosmopolitan adult female Rhabdias lungworms are capable of parthenogenesis and known to parasitize lung tissues of different amphibian hosts, including various toad and frog species (Langford 2010; Fernández Loras et al. 2011), while males live in earth/ground substrates (geohelminths). Amphibian hosts become infected by oral uptake or percutaneous infection of exogenous infective third-stage larvae (L3) which then migrate via blood/lymph system into the lungs (Langford and Janovy 2009; Langford 2010). In lungs, adult Rhabdias females start producing eggs through parthenogenesis. Thus, amphibian rhabdiosis might result in pulmonary tissue damage and/or eosinophilic pneumonia (Densmore and Green 2007). In free-ranging amphibians, Rhabdias infections seem to occur frequently and sometimes result in pneumonia (Kuzmin et al. 2003; Mohammad et al. 2010; Fernández Loras et al. 2011). Consistently, Rhabdias spp. infection rates for captive German amphibians were rather high in this study (19.3%) and resulted in the most prevalent parasites. Rhabdias/Strongyloides infection rates varied significantly within taxon, i.e., caudates were less frequently infected (3.13%) than anurans (22.83%). Nonetheless, it is well known from literature that Rhabdias is more frequently parasitizing frogs/toads (Langford and Janovy 2009; Langford 2010). In line, Rhabdias ranae seems not capable to infect caudates and to be restricted to frogs/toads as suitable hosts, but in the past two decades, first Rhabdias infections in caudates have been reported (Kuzmin et al. 2001; Kuzmin et al. 2003; Eisenberg and Pantchev 2009). Therefore, it seems assumable that anurans might be more often infected with Rhabdias than caudates, especially because the correlation was rather high (r = 0.44) when comparing these two amphibian groups (Cohen 1988). Clinical relevance of rhabdiosis was also underlined in dissections, since in one adult male Australian green tree frog (L. caerulea), a Rhabdias spp.-infected lung was found and which might have caused severe pneumonia, hepatitis, and nephritis. Nonetheless, other pathogens could not be ruled out as the same animal showed secondary bacterial infections with Chryseobacterium indologenes (+++) and Pseudomonas fluorescens (+) isolated from the frog’s coeloma. Alongside Rhabdias, other nematode genus, i.e., Oswaldocruzia, was frequently diagnosed (2.8%) in domestic kept amphibian pets. Oswaldocruzia nematodes infect amphibian hosts exclusively by the oral uptake of exogenous infective L3 (Hendrikx 1983). Noteworthy, a cutaneous Pseudocapillaroides xenopi infection was diagnosed in an adult African clawed frog (X. laevis). This X. laevis-infected animal suffered not only of a severe verminous dermatitis but also of secondary Gram-negative P. fluorescens (+++), Aeromonas hydrophila (++), and Citrobacter braakii (++) dermal infections. The amphibian nematode P. xenopi infects the epidermis and can cause clinically symptoms, such as erythematous/erosive dermatitis, with characteristic roughness of affected skin, petechiae, and dermal ulcera (Cunningham et al. 1996; Mutschmann 2010). P. xenopi can complete its direct life cycle within epidermis of frogs/toads in which burrowing activities of subdermal nematodes can lead to the damage of parasitized skin. Therefore, P. xenopi-infected animals are more susceptible for bacterial and/or fungal secondary dermal infections (Cunningham et al. 1996), as confirmed in our investigation.
According to protozoan enteric infections, in 14 cases (8.7%), potentially pathogenic, flagellated protozoan genera, such as Proteromonadida, Reteromonadida, Diplomonadida, and Trichomonadida, were additionally diagnosed. Nonetheless, the literature considers many of these enteric flagellates as commensals within intestinal tract of amphibians (Densmore and Green 2007; Mutschmann 2010). Conversely, some genera of diplomonadids (Giardia, Hexamita, Spironucleus) and trichomonadids (Monocercomonas, Hexamastix, Tritrichomonas) can cause weight loss, general edema, and enteritis in severely infected animals.
The clinical relevance of flagellated protozoan infections was demonstrated during conducted dissections: Out of all dissected animals, four (3.7%) died because of severe Tritrichomonas spp.- and/or Spironucleus spp.-derived enteritis. These animals showed severe catarrhalic- to hemorrhagic-necrotic enteritis combined with secondary bacterial infections (e.g., Pseudomonas spp./Sphingobacterium spp.) of liver and gut mucosa.
Only five animals (3.7%) were positive for Blastocystis spp. infections. Conversely to our findings, Yoshikawa et al. (2004) found anurans and newts from distinct locations in Japan to be infected with Blastocystis showing very high prevalences (47.8–100%) by using in vitro culture diagnostic methods. Our observed Blastocystis prevalence might have been higher if this in vitro cultivation method would have been applied, but it cannot be excluded that this parasite is simply less frequently found in German pet amphibians. Unfortunately, there is still very little knowledge on amphibian-related blastocystiosis. The same holds true for its possible impact on animal health kept in captivity (Mutschmann 2010). Nevertheless, Blastocystis should be considered as potentially pathogenetic protozoan species and infections should be considered according to clinical symptoms. Moreover, during dissections we here diagnosed Entamoeba spp. cysts in three (2.8%) animals.
Several studies have focused on gastrointestinal apicomplexan coccidian parasites in amphibians (Duszynski et al. 2007). So far, monoxenous coccidian genera Eimeria, Goussia, Hyaloklossia, and Cystoisospora (former Isospora according to new nomenclature) have been described in diverse amphibian host species (Duszynski et al. 2007), and for further review a disposed online version (http://biology.unm.edu/coccidia/anura.html) is recommended. In accordance with these reports, we also diagnosed un-sporulated coccidian oocysts in one animal (0.6%), but amphibian oocysts were not fully identifiable to species level. Furthermore, non-sporulated Eimeria spp. oocysts were found within gut lumen of one dissected fire salamander, (Salamandra salamandra) but coccidian-derived death was ruled out as this animal was also co-infected with Aplectana spp., Spironucleus spp., and Tritrichomonas sp. and showed a manifested mycotic dermatitis.
Enteropathogenic apicomplexan Cryptosporidium is known to infect also the microvillus border of amphibian gastrointestinal epithelial cells (Jirků et al. 2008). Consistently, we diagnosed Cryptosporidium oocysts in an Australian frog (L. caerulea) via CFS analysis. If here identified Cryptosporidium oocysts were shed during a patent infection or whether they were passed because of Cryptosporidium spp.-infected prey animal consumption (e.g., feeding of baby mice) remains unclear. Since Cryptosporidium can be transmitted by ingestion of infected food animals, poorly treated water as well as direct contact with infective oocysts, it is possible to assume that human infections might occur through ingestion of under-cooked frog (Rana spp.) meat and/or handling and processing of Cryptosporidium-infected frogs as recently demonstrated in Africa (Kia et al. 2017). Former study revealed a high prevalence of Cryptosporidium spp. (35.9%) in the intestine of 117 frogs (Rana spp.) sold at the Hanwa frog market Zaria, Kaduna State, Nigeria, for human consumption (Kia et al. 2017; Kia and Ukuma 2017). Therefore, further public health studies on different transmission routes of this neglected anthropozoonotic parasite should be conducted, including amphibians designated for human consumption (Kia et al. 2017; Kia and Ukuma 2017).
Aside from protozoans, nematodes, cestodes, and trematodes, no acanthocephalan infections were here detected. Nonetheless, during necropsies, also cestode-parasitized animals were found. As such, in three dissected animals (2.8%), various long cestode specimens containing mature proglottids were diagnosed. Noteworthy was a heavily Nematotaenia-infected male Dyeing dart frog (Dendrobates tinctorius), which showed obstipation and congestion of ground substrate in the gut lumen. Amphibians are known to be infected by different cestode genera, i.e., Proteocephalus, Ophiotaenia, Cephalochlamys, Bothriocephalus, Nematotaenia, Distoichometra, Cylindrotaenia, and Baerietta. Clinical symptoms of nematotaeniosis manifest in affected animals during stress and/or in case of heavy infections (Mutschmann 2010). Then, ileus with obstipation, blood loss, necrosis of intestinal mucosa, edema or even death may also occur if untreated (Mutschmann 2010). Interestingly, a digenean trematode infection was found in a deceased yellow-bellied toad (Bombina variegata), showing clinical symptoms, including hydrocoeloma, generalized edema, and pathohistological findings, such as hepatitis, enteritis, nephritis, and a bacterial co-infection (Citrobacter spp. +). Amphibians represent not only intermediate hosts for various digenean trematode orders (e.g., Amphistomida, Echinostomatida, Gasterostomida, Hemiurida, Holostomida, Plagiorchida) but also second or even final hosts. Nevertheless, trematode-driven pathological effects are mostly unknown for amphibians (Mutschmann 2010).
Since many of examined amphibians in this study are considered as threatened endemic species of neotropical regions, e.g., Adelphobates galactonotus, Phyllomedusa bicolor, and Trachycephalus resinifictrix, and thus being kept as zoo animals for conversation reasons, detected parasites in these animals might represent imported parasites from their natural tropical habitats. Therefore, it seems noteworthy to mention that new wild amphibians introduced into zoological gardens should undergo a mandatory quarantine regime in order to avoid further spread of neozoan parasites as suggested elsewhere (Hallinger et al. 2019; 2020).
Interestingly, parasitic infection rates in investigated anurans (51.12%) were significantly higher than the ones observed in caudate species (12.88%). As proposed for zoo animals, it is also recommended that newly purchased frogs, newts, and toads by private owners should be submitted to parasitological examination in order to detect presence of gastrointestinal parasites during quarantine as a routine health screening.