Objective. The aim of this study was to identify the prevalence of endoparasites and ectoparasites in ex situ snakes at the Barranquilla Zoo. Materials and Methods. Stool samples and ectoparasites of 2 colubrids (Leptophis ahaetulla and Spilotes pullatus), 2 diapsids (Oxybelys aeneus and Thamnodynastes paraguanae) and 13 boids (9 specimens of Boa constrictor, 2 of Epicrates maurus and 2 Eunectes murinus) were analyzed using flotation method in saturated sugar solution and direct observation. Results. The results showed that 65% (11/17) of the animals were positive to the presence of parasites. In relation to the positive specimens, 91% (10/11) belong to the Boidae family, presenting mostly nematodes, in the dipsadid’s samples mainly protozoans were observed and the colubrid’s samples were negative. Regarding ectoparasites, 2 species of ticks (Amblyomma dissimile and Ornothodoros turicata) were found in specimens of Boa constrictor, Epicrates maurus and Spilotes pullatus. Conclusions. The identification of parasites in this research represents a contribution to the study of parasitology in snakes ex situ in Colombia. It provides information to develop preventive measures against pathological agents and allows comparing results with other studies with animals in the wild and under human care.

Zoos are institutions that have several animal species under their care. They also incorporate plans of education for conservation within their mission and offer an experience to visitors in order to make them aware of subjects related to the loss of biodiversity (1). Additionally, the husbandry of animals ex situ presents several challenges such as animal welfare, space requirements and veterinary attention (2), since living in a controlled environment does not exempt animals from presenting parasites or any other health disorder.

Snakes can have ectoparasites and endoparasites both in situ and ex situ, however, those are rarely studied and therefore not well known. Apart from the above, only one individual or very small populations are usually examined. However, certain studies describe nematodes such as Dracunculus brasiliensis, found in Eunectes murinus (3), oxyurids in Boa constrictor and Python regius (4), Cruzia sp., Kalicephalus subulatus, Styphlodora horridum (5) Eimeria spp., Capillaria spp., Sarcocystis spp., Kapsulotaenia spp. (6) Rhabdias spp., Porocephalus spp. in Epicrates cenchria (7), and ticks like Rhipicephalus sp., Aponomma sp. and Amblyomma sp. (8).

Reports of the prevalence of gastrointestinal endoparasites and ectoparasites in snakes in Colombia are scarce. A literature review resulted in only two studies (9,10), which describe the finding of Amblyomma dissimile in two specimens of Boa constrictor and one specimen of Spilotes pullatus. Considering the importance of expanding the knowledge about parasites in snakes, the present study aimed to identify the prevalence of gastrointestinal endoparasites and ectoparasites in snakes under human care at the Barranquilla Zoo.

Area of Study. The study was performed at the Barranquilla Zoo’s herpetarium, located in the city of Barranquilla - Colombia. Geoclimatic conditions: Barranquilla has an average minimum temperature of 25°C and a maximum of 33.5°C, rains that range between 70-178 mm/month and a relative humidity of 84%. Coordinates: 11°00´38″N 74°47´49″O (IDEAM Colombia).

Animals for study. The study was performed during 12 weeks with the snake species listed on Table 1. Two of the 9 specimens of B. constrictor were in the same terrarium, and the remaining specimens were located in individual terrariums.

All the snakes listed in Table 1 were under human care for over two years, with the exception of two B. constrictor. Both boas entered the institution one year prior to the collection of samples for this study, after undergoing the quarantine process described below in the preventive medicine section.

The specimens were fed with mice (Mus musculus), rats (Rattus rattus) or chickens (Gallus gallus), bred for this purpose in the bioterium of the Barranquilla Zoo. The feeding frequency (weekly or twice a month) and the type of prey varied depending on the size of each specimen.

The animals bred at the bioterium were fed with commercial pellets with sawdust as substrate which was changed weekly. Additionally, stool analysis were performed (direct fecal smear and Sheather technique) in pool samples every 3 months with de specimens from the bioterium. During the first week of sample collection for this study, the rodents were tested positive to Hymenolepis diminuta and Aspiculuris tetraptera.

Description of the terrarium. The snakes were located at the Herpetarium area which included 18 terrariums. The enclosures had concrete walls on three sides (laterals and back), one metallic mesh roof (for ventilation), and the front on template glass to allow the observation of the animals. The terrariums had an approximate dimension of 1.1 x 1.1 m x 1.5 m. They also contained live plants, dry leaves, and different substrates (sand, rocks or gravel). The terrariums do not have any heating systems due to Barranquilla’s climate conditions. The design of the enclosures does not allow the snakes to move between terrariums, neither permits free ranging animals to enter, except insects such as cockroaches or beetles. However, animals such as iguanas, birds, squirrels and other free rodents can walk on the roof of the terrariums and even be in contact with the mesh located on the top.

Cleaning and disinfection of the terrariums. Cleaning and disinfection of the water bowls consisted in washing them with soap and water daily, as well as the disinfection with sodium hypochlorite at 1% once a week. The complete disinfection of each terrarium was performed when an animal was transferred to a new enclosure or due to specific sanitary conditions. The disinfection consisted in changing the substrate and spray glutaraldehyde at 0.05% and quaternary ammonium at 0.01% at walls, hangers and substrate.

Preventive Medicine. Annually, a clinical exam was performed to the snakes located in the Herpetarium. Said medical exam consisted of: a complete clinical exam, collection of blood samples (complete hemogram and biochemical exams) and stool samples. The animals received treatment against parasites when tested positive. New individuals in the collection went through a minimum period of 3 months in quarantine, isolated from the main collection. Each individual required three negative samples (direct fecal smear, Sheather technique and modified Ziehl-Neelsen technique) and one clinical evaluation before being transferred to the Herpetarium. Additionally, biochemical tests (uric acid, albumin, aspartate aminotransferase and creatine phosphokinase) were performed depending on the size of the snake.

Sample analysis. Approximately, 2 to 5 g of feces were collected from the area each animal was located. These samples were stored in labelled plastic containers under refrigeration at 4°C until analysis (refrigeration time that did not surpass 24 hours). The recollection of a total of three samples were initially planned for each animal. The ectoparasite samples were taken directly from the animal’s skin and deposited in eppendorf tubes with 70% alcohol. The stool samples were analyzed through a direct fecal smear and floating techniques. The later was implemented through sugar saturated solution described by Kassa et al (11). On the other hand, a stereoscope was used to identify the ectoparasites through anatomical characteristics that allowed the classification according to Quiroz´s description (12). The specific prevalence reported were obtained by dividing the number of individuals with parasites, by the number of individuals evaluated, and multiplying it by one hundred.

Finally, the ethical, technical, scientific and administrative norms for animal research contained in Law 84 by the Colombian Congress were taken into account to sample collection, handling, and preservation procedures. Also, this study was performed during preventive medicine procedures approved by the Barranquilla Zoo’s Veterinary Department.

During the present study, 43 stool samples were collected from a total of 17 specimens (Table 2).

65% (11/17) of the snakes tested positive for parasites. Among the positive, 91% (10/11) were boids. The nematodes were found more significantly in boids, while in dipsadinae’s snakes protozoa were detected. The presence of protozoa was identified as flagellates and endoparasites belonging to the Eimeriidae family, and nematodes like Capillaria sp., larvated and non-larvated eggs belonging to Strongylida and Rhabditiformes larvae (Figure 1).

Within the positive boids, nematodes presented greater prevalence than protozoa (Eimeriidae family and Flagellates), being identified in 100% of the boids (10/10), while protozoa was found only in 20% (2/10). In the E. murinus specimens, Capillaria sp., was identified, as well as endoparasites belonging to the Eimeriidae family, being the only boids with multiple parasites. In the Colubridae and Dipsadinae families (Leptophis ahaetulla, Oxybelis aeneus, Spilotes pullatus, and Thamnodynastes paraguanae), only one sample tested positive. (T. paraguanae), identifying Rhabdifitormes larvae and flagellates. Flagellates were identified in 3 of 4 samples analyzed of the specimen T. paraguanae (Table 2).

A prevalence of 24% (4/17) was determined for non-larvated eggs of nematodes from Strongylida, 18% (3/17) for larvated eggs of nematodes from Strongylida, 12% (2/17) for parasites of the Eimeriidae family and Capillaria sp., 24% (4/17) for Rhabditiform larvae, and 6% (1/17) for flagellates.

The presence of parasites in the positive samples (Table 2) varied as following: 36% (4/11) of B. constrictor were positive for non-larvated eggs of Strongylida, 27% (3/11) had larvated eggs from Strongylida, and 9% (1/11) had Rhabditiform larva. Regarding E. murinus, the Eimeriidae family and Capillariasp. were found in the two specimens evaluated. Likewise, Rhabditiform larvae was observed in the two individuals of E. maurus evaluated. Finally, three of four samples of the only T. paraguanae specimen evaluated tested positive for flagellates and one for Rhabditiform larva.

Apart from the above, seven samples of ectoparasites were found in the terrariums of the following species: 5 Boa constrictor, one Spilotes pullatus and one Epicrates maurus (Table 3). The tick Ornithodoros turicata of the Argasidae family (Figure 2) was identified in six samples. Additionally, the tick Amblyomma dissimile of the Ixodidae family (Figure 3) was identified in one sample belonging to B. constrictor.

The previously named ectoparasites presented greater prevalence in boids than in Colubrid’s snakes, since only one colubrid (S. pullatus) tested positive for the presence of ectoparasites. However, it must be taken into account that the number of boids is much higher than that of colubrids in the present study. The tick species with the largest prevalence was Ornithodoros turicata, being found in six specimens of B. constrictor and in one individual of S. pullatus. Meanwhile, Amblyomma dissimile was only identified in one specimen of B. constrictor, as previously mentioned.

In the present study, 65% (11/17) of the animals tested positive for gastrointestinal parasites, as described in table 2. Two species of protozoa were identified (Flagellates and parasites of the Eimeriidae family) and 4 types of nematodes (non-larvated eggs and larvated eggs of nematodes of the Strongylidae order, Capillaria sp. and Rhabditiform larvae). 27% (3/11) of the individuals had multiple parasites and 73% (8/11) only had nematodes (Table 2).

The high prevalence of nematodes with direct life cycles is attributed to their characteristic of presenting free life parasitic forms. That allows them to survive in the environment for long periods of time, awaiting the precise moment to infect the host, which favors reinfection (10). On the other hand, boids (Boa constrictor, E. maurus and E. murinus) represented 91% (10/11) of the positive specimens, in which both protozoa and nematodes were identified. However, it must be considered that boids are the taxonomic group with the largest number of individuals in the present study.

The specimens were asymptomatic to the clinical signs typical of infections by nematodes and protozoa. For example: anorexia, lethargy, postprandial regurgitation, obstruction, vascular lesions, dispnea, weight loss, acute enteritis, and diarrhea (5, 13, 14). The exception was the only specimen of T. paraguanae presented aqueous and mucous feces during the entire study, one of the symptoms attributed to parasites from flagellates (15).

The results of this study are in accordance with the literature regarding the parasites identified (14). However, parasites from the Eimeriidae family were reported to be the Coccidia with greater incidence in ex situ reptiles, while in this study the prevalence was low (12%). Other authors (11,13,15,16,17), reported the identification of nematodes (Strongylus sp. and Strongyloides sp.), Capillaria sp., flagellates and Eimeriasp., in snakes. These results match with those in the present study. Flagellate protozoa were reported as a parasite largely found in ex situ snakes (18), however, this differs from the results obtained in this work, as only one specimen tested positive for parasitosis due to flagellates. On the other hand, the presence of multiple parasites was more common than simple parasitosis agreeing with a study performed in Iran (19).

Some authors found the parasite Hymenolepis spp. in snake samples and have classified them as pseudo-parasites, as they come from infected rodents, without being pathogenic for snakes (3,20). However, this relation was not present in this investigation, since these parasites were not identified in the coprological exams, although the food sources (rodents) were contaminated (Hymenolepis diminuta and Aspiculuris tetraptera).

Regarding the identification of ectoparasites, the tick Amblyomma dissimile was identified in a specimen of B. constrictor, as reported in literature (9,10). It is important to highlight this species as a transmitter of Hepatozoon sp. and Rickettsia sp., and that it affects species like Epicrates spp. (7,8). Although A. dissimile only affected one of the 17 specimens in this study, we cannot dismiss its presence in the future since it is possible for it to be contaminating the substrate or other parts of the exhibition. Apart from this, the parasite has been reported as the tick that most affects ex situ reptiles in Central and South America (8). Like the above, some authors report A. dissimile as the tick that most affects snakes of the B. constrictor species (21). Likewise, others report findings of species of Ammblyoma sp. in E. murinus (22). However, different from these reports, E. murinus did not present ectoparasites in the present study. Finally, in contrast with A. dissimile reported in colubrids (23), this ectoparasite was not present in colubrids assessed in this investigation.

Apart from the prior tick species, we identified Ornithodoros turicata, which has already been reported in other countries as affecting reptiles (24). As far as we know, this is the first report of said tick species as ectoparasite in snakes in Colombia. Likewise, we observed that this tick originally from the United States and Mexico is also recognized as a vector of the spirochaete (Borrelia turicatae). A pathogen that causes recurrent fever and is a possible transmitter of the African swine fever (24). It is worth highlighting that O. turicata was found in samples of six boidae (B. constrictor andE. maurus) and only one colubrid (S. pullatus). In relation to the possibility of virus transmission, we should note that a specimen of B. constrictor died in the eighth week of the study with neurological symptoms. The death could possibly be associated with inclusion body disease (IBD), after an infestation of this species of tick. Although, this diagnosis was not confirmed due to the fact that the specimen presented advanced autolysis that did not allow taking samples for histopathology. It is possible that there is a relation between this tick species and the retrovirus that causes IBD.

In conclusion, the variety of endoparasites and ectoparasites found in this study is in accordance with literature reports. However, the prevalence in this study differed from other authors considering that it was developed with animals under human care. The food and water sources of each species studied have cleaning and disinfection plans that reduce the probability of occurrence of internal parasites contracted through feeding. In addition, the terrariums have a cleaning and disinfection plan that notably reduces the presence of ectoparasites. On the other hand, there are free-ranging iguanas (Iguana iguana) within the zoo’s installations, which could be a source of parasite transmission. Considering that only direct cycle parasites were found and that all the snakes passed through the quarantine period testing negative for parasites, it is likely that these specimens were contaminated from contact with iguana feces that were circulating in the area or by insects serving as paratenic hosts. Finally, the identification of the parasites of ophidians in this work is an important contribution for the parasitology of this group of reptiles in Colombia. Since it provides information to develop preventive measures against pathological agents and allows comparing results with other studies with animals in the wild and under human care.

The authors of the present article state that they do not have any type of conflict of interest related to the financial, intellectual, family or proprietary part that would constitute a conflict, whether effective potential or apparent.