A description of parasites from Iranian snakes

ARTICLE IN PRESS Experimental Parasitology ■■ (2014) ■■–■■

Contents lists available at ScienceDirect

Experimental Parasitology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r

1 2 3 4 5 6 7

Full length article

A description of parasites from Iranian snakes Vahid Nasiri a,b,*, Iraj Mobedi c, Abdolhossein Dalimi b, Abbas Zare Mirakabadi d, Fatemeh Ghaffarifar b, Shohreh Teymurzadeh d, Gholamreza Karimi a, Amir Abdoli b, Q2 Habibollah Paykari a a

8 9 10 11 12 13 14 15

Department of Parasitology, Razi Vaccine and Serum Research Institute, Karaj, Alborz, Iran Departments of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran c Department of Parasitology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran d Department of Venomous Animals and Antivenom Production, Razi Vaccine and Serum Research Institute, Karaj, Alborz, Iran b

H I G H L I G H T S

•

16 17 18 19 20 21 22 23 24

• •

G R A P H I C A L

A B S T R A C T

These are the first data on the parasitic fauna of Iranian terrestrial snakes. Little is known of the parasitic fauna of terrestrial snakes in Iran. This study demonstrates the high prevalence of parasites among Iranian snakes.

25

26 27

Q1

* Corresponding author. E-mail address: [email protected] (V. Nasiri). http://dx.doi.org/10.1016/j.exppara.2014.09.007 0014-4894/© 2014 Elsevier Inc. All rights reserved.

Please cite this article in press as: Vahid Nasiri, et al., A description of parasites from Iranian snakes, Experimental Parasitology (2014), doi: 10.1016/j.exppara.2014.09.007

ARTICLE IN PRESS V. Nasiri et al./Experimental Parasitology ■■ (2014) ■■–■■

2

A R T I C L E

I N F O

Article history: Received 23 March 2014 Received in revised form 19 September 2014 Accepted 24 September 2014 Available online

Q3

Keywords: Iranian snakes Parasitic fauna Intestinal parasites Hemoparasites

A B S T R A C T

Little is known of the parasitic fauna of terrestrial snakes in Iran. This study aimed to evaluate the parasitic infection rates of snakes in Iran. A total of 87 snakes belonging to eight different species, that were collected between May 2012 and September 2012 and died after the hold in captivity, under which they were kept for taking poisons, were examined for the presence of gastrointestinal and blood parasites. According to our study 12 different genera of endoparasites in 64 (73.56%) of 87 examined snakes were determined. Forty one snakes (47.12%) had gastrointestinal parasites. In prepared blood smears, it was found that in 23 (26.43%) of 87 examined snakes there are at least one hemoparasite. To our knowledge, these are the first data on the internal parasitic fauna of Iranian terrestrial snakes and our findings show a higher prevalence of these organisms among them. © 2014 Elsevier Inc. All rights reserved.

1. Introduction More than 3500 species of snakes have been found around the

Q4 world, less than 10% of which are venomous (Bawaskar, 2004; BlackQ5 Q6 man and Dillon, 1992; Meenatchisundaram and Michael, 2009; Q7 Warrel, 2005). In Iran, 69 species of snakes assigned to 37 genera in six families have been identified, of which 36 species are nonvenomous, 25 species are venomous and 8 species are semivenomous (Dehghani, 2010; Latifi, 2000; Zare Mirakabadi and Teymurzadeh, 2008). The class Reptilia, consisting of more than 6000 species, is host to a wide variety of protozoan and metazoan parasites and virtually 100% of free-ranging reptiles harbor some kind of parasites and however, the presence of these organisms is not necessarily associated with a disease state (Barnard and Upton, 1994; McFarlen, 1991). Breeding snakes in captivity, particularly in a semi-extensive system, is an activity that exposes the animals to the action of several parasites (Mader, 1996). Intestinal helminths, pentastomids, and intestinal and blood protozoa are common endoparasites of captive or wild reptiles that these animals may serve as definitive, intermediate, accidental or paratenic hosts (Frye, 1991; Greiner and Mader, 2006; Hernandez-divers, 2006; Jackson and Cooper, 1981; Macarthur et al., 2004). The prevalence of parasites with a monoxenic cycle is higher than that of heteroxenic-cycle parasites, since the latter require an intermediate host to complete their cycle (Reyl, 2001). The transmission of monoxenic-cycle parasites is favored by the physical and organic conditions of the captivity, thus a snake can contaminate other animals or even reinfect itself with its own feces. In addition, the captivity conditions or the related stress may be responsible for the acquisition or increase of the parasitic infection (Klingenberg, 1993). The result of this parasitism is competition with the host animal for food, removal of tissue and fluid, blocking of lymph and blood vessels, edema, ulcerations, necrosis, and anemia (Barnard, 1983; Frank, 1981; Frye, 1991; Marcus, 1981).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Although a considerable species of reptiles are present in Iran’s ecosystems, there is not enough information about the parasitic fauna of reptiles and their role in transmission of veterinary and zoonotic disease (Youssefi et al., 2013). In a research in Iran, a Natrix natrix snake was found to be infected with the worm that belong to the genus Ophiotaenia (Youssefi et al., 2010). In a case report study, myiasis due to Musca domestica was described in a Pseudocerastes persicus snake and a lesion was found on its body where 14 live larvae of M. domestica was removed (Dehghani et al., 2012). An investigation Q8 showed that 1 turtle (Mauremys caspica caspica), 11 grass snakes (Natrix natrix) and 5 dice snakes (Natrix tessellata) have been in- Q9 fected with Telorchis assula in Mazandaran, north of Iran (Youssefi et al., 2013). In another research, one European glass lizard, Pseudopus apodus, and three European grass snakes, Natrix natrix, were examined for helminths and found that Pseudopus apodus harbored one species of Nematoda, Entomelas entomelas and N. natrix harbored 1 species of Digenea, Telorchis assula, 1 species of Cestoda, Ophiotaenia europaea, and 1 species of Nematoda, Rhabdias fuscovenosa (Halajian et al., 2013). Recently it have been reported that 18 snakes, including 9 N. natrix and 9 N. tessellata from Mazandaran Province, north of Iran were infected with parasitic helminths including 1 Nematode: Rhabdias fuscovenosa (larva), 1 Digenea: Telorchis assula and 1 Cestoda: Ophiotaenia europaea (Yossefi et al., 2014). In this study the parasitic fauna of native Iranian snakes that were Q10 taken from the wild and kept in captivity were investigated. 2. Materials and methods A total of 87 snakes representing eight species that were collected between May 2012 and September 2012 from various provinces of Iran sent to the department of Venomous Animals and Antivenom Production, Razi Vaccine and Serum Research Institute. These parasites were kept under captivity and after ding Q11 transferred immediately to the Parasitology laboratory of Razi

Table 1 The taxonomic characterization of examined snakes and number of their parasites. Scientific name of snakes

Common name

Number of examined snakes

Number of positive snakes for intestinal parasitic

Number of positive snakes for hemoparasites

Number of positive snakes for any parasites

Pseudocerastes persicus fieldi Naja oxiana Vipera albicornuta Vipera lebetina obtusa Vipera ursinii eriwanensis Agkistrodon intermedius caucasicus Natrix natrix Coluber caspius gmelin Total

Persian horned viper Central Asian cobra Zigzag mountain viper West-Asian blunt-nosed viper Transcaucasian meadow viper Caucasian pit viper European grass snake Caspian whip snake

23 10 5 20 2 23 1 3 87

10 7 5 10 2 5 1 1 41

13 3 0 6 0 0 0 1 23

23 10 5 16 2 5 1 2 64

Please cite this article in press as: Vahid Nasiri, et al., A description of parasites from Iranian snakes, Experimental Parasitology (2014), doi: 10.1016/j.exppara.2014.09.007

ARTICLE IN PRESS V. Nasiri et al./Experimental Parasitology ■■ (2014) ■■–■■

1

Fig. 1. Acanthocephalan larval detected on the intestinal wall surface.

2

3

Fig. 2. The detected Telorchis assula from Natrix natrix.

Please cite this article in press as: Vahid Nasiri, et al., A description of parasites from Iranian snakes, Experimental Parasitology (2014), doi: 10.1016/j.exppara.2014.09.007

3

ARTICLE IN PRESS 4

1

V. Nasiri et al./Experimental Parasitology ■■ (2014) ■■–■■

Fig. 3. The detected (A) Ophiotaenia sp. and (B) one unknown species from Natrix natrix.

2

3

Fig. 4. The detected nematodes belong to superfamily Rhabditoidea, Strongyloides sp. from one Vipera ursinii eriwanensis and four Naja oxiana.

Please cite this article in press as: Vahid Nasiri, et al., A description of parasites from Iranian snakes, Experimental Parasitology (2014), doi: 10.1016/j.exppara.2014.09.007

ARTICLE IN PRESS V. Nasiri et al./Experimental Parasitology ■■ (2014) ■■–■■

Fig. 7. The detected Blastocystis sp. from one Vipera lebetina obtuse.

1

5

5 6

Fig. 5. The detected Nematode: Ascarididae from intestine of one Naja oxiana.

Vaccine and Serum Research Institute. The snake’s body wall was 7 opened by a longitudinal incision and organ surfaces, mesenteries 8 of the body cavity, the lumen of the digestive tract and the lungs 9 were examined visually for parasites. Subsequently, the viscera were 10 placed in a Petri dish with normal physiological saline and exam11 ined for helminths under a dissecting microscope. The recovered 12 helminths were cleaned carefully and, fixed in 70% alcohol and 13 stained with acetocarmine. The Protozoan parasites were identi14 fied by prepared direct wet mount smears and modified Ziehl– 15 Neelsen staining of fecal samples and examination of them under 16 light microscope. The fecal samples were placed in vials with 2.5% 17 K2Cr2O7 and 10% formalin for examination of the fecal contents for 18 any parasite eggs or oocysts and sporulation of any oocysts found 19 in the samples. 20 For identification of hemoparasites, thin blood smears were pre21 pared from cardiac blood of each snake and then air dried, fixed 22 in absolute methanol, stained with Giemsa stain and examined under 23 light microscope. 24 Collected parasites were deposited in the Museum of Parasitol- Q12 25 ogy Department, Razi Vaccine and Serum Research Institute, Karaj, 26 Alborz, Iran. 27

2

28

3 4

Fig. 6. The detected Eimeria sp. from one Vipera ursinii eriwanensis and one Vipera albicornuta.

Fig. 8. The detected Nyctotheroides sp. from one Pseudocerastes persicus fieldi.

Please cite this article in press as: Vahid Nasiri, et al., A description of parasites from Iranian snakes, Experimental Parasitology (2014), doi: 10.1016/j.exppara.2014.09.007

29

ARTICLE IN PRESS V. Nasiri et al./Experimental Parasitology ■■ (2014) ■■–■■

6

1

Fig. 9. The detected Hexamita batrachorum (Giemsa stain).

2 3 4 5 6 7 8 9

3. Results 3.1. Intestinal parasites 3.1.1. Intestinal worm infection Twelve different genus of endoparasites were determined in 64 (73.56%) out of 87 examined snakes. In the majority of snakes, two

or more species of parasites were found (see details in Table 1). At necropsy, an Acanthocephalan larval stage (Centrorhynchus corvi) was found on the outer surface of the intestinal wall of seven snakes (2 Vipera lebetina obtuse; 2 Vipera albicornuta; 2 Naja oxiana and 1 Coluber caspius gmelin). Local necroses were ob- Q13 served on the intestinal wall surface of snakes with Centrorhynchus corvi (Fig. 1).

10

11

Fig. 10. Intraerythrocytic gametocytes in Naja oxiana.

Please cite this article in press as: Vahid Nasiri, et al., A description of parasites from Iranian snakes, Experimental Parasitology (2014), doi: 10.1016/j.exppara.2014.09.007

12 13 14 15 16 17 18

ARTICLE IN PRESS V. Nasiri et al./Experimental Parasitology ■■ (2014) ■■–■■

1 2

7

Fig. 11. Intraerythrocytic hemoparasites in Pseudocerastes persicus field. (A–D) Intraerythrocytic gametocytes with different pattern of erythrocyte shape changes. (E) Developing schizonts in the liver of infected snake. (F) Unknown organism from liver.

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

An adult stage of one species of trematode (Digenea: Telorchiidae: Telorchis assula) (Fig. 2), two species of cestoda (A: Ophiotaenia sp. and B: one unknown species) (Fig. 3) were detected from the intestine of a Natrix natrix (European Grass Snake). Adult stages of two genus of nematode were detected, including: one belonging to the superfamily Rhabditoidea (Strongyloides sp.) from the intestine of five snakes (one Vipera ursinii eriwanensis and four Naja oxiana) (Fig. 4) and one that belongs to family Ascarididae from the intestine of one Naja oxiana (Fig. 5). 3.1.2. Intestinal protozoa infection Eimeria sp. was detected in two snakes (one Vipera ursinii eriwanensis and one Vipera albicornuta) and in both snakes with Eimeria sp. infection, petechia was observed in the mucous layer of the intestinal tract (Fig. 6). A large number of Blastocystis sp. was

Fig. 12. Intraerythrocytic gametocytes in Coluber caspius gmelin.

Please cite this article in press as: Vahid Nasiri, et al., A description of parasites from Iranian snakes, Experimental Parasitology (2014), doi: 10.1016/j.exppara.2014.09.007

19

ARTICLE IN PRESS 8

1

V. Nasiri et al./Experimental Parasitology ■■ (2014) ■■–■■

Fig. 13. Intraerythrocytic gametocytes in Vipera lebetina obtusa.

2 detected in one snake (Vipera lebetina obtuse) (Fig. 7). Moreover, Cili3 ated protozoa (Nyctotheroides sp.) was detected in a snake 4 (Pseudocerastes persicus fieldi) (Fig. 8). 5 A large numbers of Hexamita batrachorum (a flagellate proto6 zoa belonging to Phylum Sarcomastigophora, Class Mastigophora) was 7 detected in intestinal mucosa and stool of 27 snakes (9 Pseudocerastes 8 persicus fieldi, 8 Vipera lebetina obtuse, 3 Agkistrodon intermedius 9 caucasicus, 1 Vipera ursinii eriwanensis, 2 Vipera albicornuta and 4 10 Naja oxiana) (Fig. 9). This parasite was the most frequent organ11 ism found in snakes (31.03%). 12 13 3.2. Hemoparasites 14 15 Hemoparasites were observed in 23 (26.43%) out of 87 exam16 ined snakes. Many of the infected red blood cells were distorted in 17 size and shape; however, some infected cells were similar to 18 uninfected erythrocytes. We could not to see sporogonic stages, 19 thus we could not determine the parasite’s genus. Microscopic ex20 amination of Giemsa stained slides showed infection of red blood 21 cells of 13 Pseudocerastes persicus fieldi, 6 Vipera lebetina obtusa, 3 22 Naja oxiana and 1 Coluber caspius gmelin with Hemoparasites 23 (Figs. 10–13). 24 25 4. Discussion 26 27 The reptiles have important impacts on different aspects of their 28 ecology and thus demands deep research regarding the influence 29 of these animals and their flora on people and autochthonous animal 30 species. The varieties of different pathogens in these species are very 31 large. The presence of several pathogens in one host and stressful 32 situations can have a negative influence on the health status. Fur33 thermore, there is a possibility of pathogen transmission to human. 34 A healthy reptile has a number of pathogens, all kept in check by 35 a healthy immune system and the beneficial gut flora. When a reptile 36 is highly stressed or under prolonged moderate to severe stress, the 37 immune system falters. In cases of improper environmental tem38 peratures, starvation, or prolonged dehydration, the beneficial gut 39 flora die off and organisms benign in small numbers gain ascen40 41 Q14 dancy and start causing problems (Rataja et al., 2011). It is widely known that under captivity the stress to which 42 animals are submitted to can induce the settlement of several pa43 thologies, mainly those related to parasites (Leinz et al., 1989; Santos 44 et al., 2008; Siqueira et al., 2009) and because parasites can reach 45

high infection loads in hosts in captivity, can be very problematic and may cause the death of hosts (Klingenberg, 1993). So for conservation efforts involving captive breeding, an understanding of the parasites of a particular species is crucial. With this view, it is necessary to provide data on the parasitic fauna of Iranian snakes, in order to permit the establishment of prophylactic procedures to properly reduce the mortality of captive animals. The present study was carried out to evaluate the parasitic infections in wild snakes. The study was proposed because we observed that, in captivity, snakes presented a high prevalence (above 70%) of infection by different parasite species. The present investigation showed that 64 (73.56%) of 87 analyzed snakes arrived for captivity already infected by at least one parasite species. The results from this study are important for the sanitary handling of these important animals during captivity. Other different species were also Q15 detected that similar parasite were described in the previous literatures (Klingenberg, 2000; Mader, 1996; Telford, 2009). In two snakes a wide dilatation and destruction of gastrointestinal tract was seen and Modified Ziehl–Neelsen staining was positive for Eimeria sp. The Hemogregarines (Apicomplexa: Adeleorina) are, intraerythrocytic protozoan parasites that infect a wide variety of vertebrates (Davies and Johnston, 2000; Telford, 2009) and are considered common Hemoparasites of reptiles and this was also true in our study. In conclusion, to our knowledge, these are the first data on the parasitic fauna of Iranian terrestrial snakes and our findings show a higher prevalence of these organisms among them. Control and prevention of many parasitic diseases is associated with breaking the cycle of transmission, and there is no shadow of doubt that precise information about diseases and their causative agents is the major key to control of them and thus, further precise parasitological investigations are required due to the noticeable unexplored area of our country in order to ascend our knowledge concerning parasites of snakes and probable zoonotic and veterinary importance of them. References Barnard, S.M., 1983. A review of some fecal pseudoparasites of reptiles. J. Zoo. Anim. Med. 14, 79–88. Barnard, S.M., Upton, S.J., 1994. A Veterinary Guide to the Parasites of Reptiles: Protozoa. Krieger Publishing Co., Malabar, FL. 154 pp. Davies, A.J., Johnston, M.R.L., 2000. The biology of some intraerythrocytic parasites of fishes, amphibia and reptiles. Adv. Parasitol. 45, 1–107.

Please cite this article in press as: Vahid Nasiri, et al., A description of parasites from Iranian snakes, Experimental Parasitology (2014), doi: 10.1016/j.exppara.2014.09.007

46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89

ARTICLE IN PRESS V. Nasiri et al./Experimental Parasitology ■■ (2014) ■■–■■

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Q16

Dehghani, R., 2010. Animal Natural Toxins. Environmental Toxicology, first ed. Tehran Tak Derakhat and Kashan University of Medical Sciences, p. 389-45. Dehghani, R., Sedaghat, M.M., Sabahi Bidgoli, M., 2012. Wound myiasis due to Musca domestica (Diptera: Muscidae) in Persian horned viper, Pseudocerastes persicus (Squamata: Viperidae). J. Arthropod Borne Dis. 6 (1), 86–89. Frank, W., 1981. Endoparasites. In: Cooper, J.E., Jackson, O.F. (Eds.), Diseases of the Reptilia, vol. 1. Academic Press, London, pp. 291–358. Frye, F.L., 1991. Biomedical and Surgical Aspects of Captive Reptile Husbandry, second ed. Krieger Publishing, Melbourne, FL. Greiner, E.C., Mader, D.R., 2006. Parasitology. In: Mader, D.R. (Ed.), Reptile Medicine and Surgery, second ed. Saunders Elsevier, St. Louis, MI, pp. 43–364. Halajian, A., Bursey, C.R., Goldberg, S.R., Gol, S.M.A., 2013. Helminth parasites of the European glass lizard, Pseudopus apodus (Squamata: Anguidae), and European grass snake, Natrix natrix (Serpentes: Colubridae), from Iran. Comp. Parasitol. 80, 151–156. Hernandez-divers, S.J., 2006. Reptile parasites – summary table. In: Mader, D.R. (Ed.), Reptile Medicine and Surgery, second ed. Saunders Elsevier, St. Louis, MI, pp. 1159–1170. Jackson, O.F., Cooper, J.E., 1981. Cooper, J.E., Jackson, O.F. (Eds.), Diseases of the Reptilia, vol. 1 & 2. Academic Press, New York, NY. Klingenberg, R.J., 1993. Understanding Reptile Parasites: A Basic Manual for Herpetoculturists & Veterinarians. Advanced Vivarium Systems, Lakeside, CA. Klingenberg, R.J., 2000. Diagnosing parasites in old world Chameleons. Exotic DVM 1, 17–21. Latifi, M., 2000. The Snakes of Iran, third ed. Environment Protection Organization, Tehran. Leinz, F.F., Janeiro-Ciquini, T.R.F., Ishizuka, M.M., 1989. Sobrevivência de Bothrops jararacussu (Serpentes, Viperidae, Crotalinae) mantidas em cativeiro. Mem. Inst. Butantan 51, 33–38. Macarthur, S., Wilkinson, R., Meyer, J., 2004. Medicine and Surgery of Tortoises and Turtles. Blackwell Publishing, Ames, IA.

9

Mader, D.R., 1996. Reptile Medicine and Surgery. WB Saunders, Philadelphia, PA. Marcus, L.C., 1981. Veterinary Biology and Medicine of Captive Amphibians and Reptiles. Lea & Febiget, Philadelphia, PA. McFarlen, J., 1991. Commonly occurring reptilian intestinal parasites. In: Proceedings of the American Association of Zoo Veterinarians. Calgary, Canada, pp. 120–127. Rataja, V., Lindtner-Knific, R., Vlahovic´, K., Mavri, U., Dovcˇ, A., 2011. Parasites in pet reptiles. Acta Vet. Scand. 53, 33. Reyl, 2001. Parasitologia, third ed. Guanabara Koogan, Rio de Janeiro. Santos, K.R., Takahira, R.K., Rall, V.L.M., Calderón, C., Sequeira, J.L., Silva, R.J., 2008. Pulmonary, microbiological and hematological changes in Crotalus durissus terrificus (Serpentes, Viperidae) parasitized by nematodes of the genus Rhabdias (Nematoda, Rhabdiasidae). Arq. Bras. Med. Vet. Zootec. 60, 667–674. Siqueira, L.R., Panizzutti, M.H.M., Muniz-Pereira, L.C., Pinto, R.M., 2009. Gross lesions induced by nematodes of Bothrops jararaca and Bothrops alternatus in Brazil with two records. Neotrop. Helminthol. 3, 29–33. Telford, S.R., 2009. Hemoparasites of the Reptilia: Color Atlas and Text. CRC Press, Boca Raton, London, and New York. Yossefi, M.R., Nikzad, R., Nikzad, M., Mousapour, A., Ramazanpour, S.H., Rahimi, M., 2014. High helmintic infection of the European grass snake, Natrix natrix and the dice snake, Natrix tessellate (Serpentes: Colubridae) from Iran. Asian Pac. J. Trop. Dis. 4 (Suppl. 1), S263–S267. Youssefi, M.R., Hoseini, S.H., Mobedi, I., Ahoo, M.B., Rajabloo, M., 2010. Ophiotaenia europaea, from Natrix natrix of north of Iran. World J. Zool. 5, 272–274. Youssefi, M.R., Mousapour, A.A., Nikzad, R., Mobedi, I., Rahimi, M.T., 2013. First report of Telorchis assula (Digenea: Telorchiidae) in three reptile species from North of Iran. World J. Zool. 8 (3), 243–244. Zare Mirakabadi, A., Teymurzadeh, S., 2008. Venomous Snakes of Iran, Prevention, First Aids and Treatment. Teymurzadeh and Tabib Press, Tehran.

Please cite this article in press as: Vahid Nasiri, et al., A description of parasites from Iranian snakes, Experimental Parasitology (2014), doi: 10.1016/j.exppara.2014.09.007

32 33 34 35 36 37 38 Q17 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59