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IJAMBR 5 (2017) 132-139 ISSN 2053-1818

Morphological study on parasitic fauna of four species of in Mizoram, India

Gautam Patra1*, Subhamoy Ghosh1, Madhurendra Bachan2, C. Lalnunpuia1, Ajit Kumar2, Apurba Debbarma2, Basanta Saikia3, Seikh Sahanawaz Alam4 and Jayashre Bhagawati5

1Department of Veterinary Parasitology, College of Veterinary Sciences and Husbandry, Central Agricultural University, Selesih, Aizawl, India. 2Department of Veterinary Parasitology, West Bengal University of Animal and Fishery Sciences, 37 K. B. Sarani, Kolkata-37, West Bengal, India. 3Department of Veterinary Surgery and Radiology, College of Veterinary Sciences and Animal Husbandry, Central Agricultural University, Selesih, Aizawl, India. 4District Microbiologist, Malda Medical College and Hospital, Malda, West Bengal, India. 5FCLA, College of Veterinary Sciences & Animal Husbandry, Central Agricultural University, Selesih, Aizawl, India.

Article History ABSTRACT Received 13 November, 2017 This study revealed the parasitic fauna of different species of lizards from Received in revised form 27 different parts of Mizoram, India. Four different species of lizards, namely House November, 2017 Accepted 07 December, 2017 (Hemidactylus flaviviridis, ), ( indicus, Varanidae), Gecko (Gekko gecko, Gekkonidae) and (Chamaeleo Keywords: zeylanicus, Chameleonidae) were surveyed from different parts of Mizoram, India Lizards, in an attempt to investigate the prevalence of parasites. Faecal samples were Parasites, examined by conventional sedimentation and floatation techniques. Lizards that SEM, were run over by car or sacrificed underwent post-mortem examination. Blood Mizoram, samples were also examined for the presence of any haemoprotozoan infection. India. Light microscopy (LM) and scanning electron microscopy (SEM) were applied for detailed morphological study. The ecto-parasites were identified on the basis of morphology. A total of 69 lizards were examined and found to be infected with three species of nematode (Pharyngodon sp., Rhabdias sp. and Stongyle), one species of trematode (Postorchigenes sp.), three species of tissue protozoa (Eimeria spp., Isospora spp. and Cyclospora spp.) and two species of hamoprotozoa (Trypanosoma sp. and Lankesterella sp.). Only one species of tick belonging to the Amblyomma was found on the body surface of monitor Article Type: lizard. The study clearly indicates that lizards can harbour different parasites Full Length Research Article which can affect the health status of these . ©2017 BluePen Journals Ltd. All rights reserved

INTRODUCTION

In recent times, many reptiles are being collected from evolutionary and reproductive biology, vector borne the wild and kept as home pets. Reptiles are also used diseases and others (Greenberg et al., 1989; Driscoll and as animal models in biomedical research for studies like Henderson, 2008). Reptiles in the wild are naturally cardiovascular physiology, environmental toxicology, infected with a wide range of parasites. Parasitic infections are frequently encountered in captive bred reptiles also. The incidence of parasites in reptiles including lizard has been reported by several studies *Corresponding author. E-mail: [email protected]. (Scullion and Scullion, 2009; Majlathova et al., 2010; Tel: +918582859415. Rataj et al., 2011). Lizards are frequently found in human

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dwellings in tropical countries including India. Several Post mortem examination factors undermine the immune response of reptiles which predispose the opportunity for pathogenic organisms to Twenty dead lizards were brought to the laboratory cause infections and resultant diseases. Reptiles may during the whole period of survey. Post mortem was done also act as carriers for different pathogens which are systematically and thoroughly. Any parasite found was transmitted to other and even to humans (Rataj washed several times with 0.85% normal saline solution et al., 2011; Papini et al., 2011). Mizoram is one of the (NSS) before transferring to 70% alcohol. global hot spots in India for reptilian diversity. The major reptilian families in Mizoram include Gekkonidae, Varanidae and Chameleonidae. Little is known about the Examination of skin for ectoparasites parasitic fauna of lizards in this part of India. The prevalence and the intensity of parasitic infections act as Skins of both live and dead lizards were carefully a source of information regarding the status and the examined for any ecto-parasites. Live ticks were directly possible impact of parasites in the lizard population in dropped into 10% alcohol. For permanent preparation of natural conditions. Therefore, this present study was slides, bodies of ticks were perforated with entomological intended to investigate the prevalence and intensity of pins and boiled with 70% potassium hydroxide solution parasites in three groups of lizards. It also provides for 5-10 min. The treated samples were put in 70% detailed light microscopy (LM) and scanning electron alcohol for 5 min and 90% alcohol 5 min. Finally the microscopy (SEM) study of few parasites of lizards. specimens were kept in carbolic acid for 10 min before mounting in D.P.X. mountant. Amblyomma sp. were identified on the keys provided by Soulsby (1982). MATERIALS AND METHODS

Study area and lizard collection Preparation of sample for SEM

Different areas of Aizawl district of Mizoram were At first, any unwanted materials such as faecal debris, selected as parts of the study area. The lizards were mucus, blood or other body fluids which would hamper captured using fish nets, by hand or by noosing. The microscopical observation were carefully removed by survey was carried out from September 2016 to February washing with nuclease free water (NFW) with the help of 2017. Some lizards killed accidentally were also treated a fine camel brush. For SEM, specimens were washed 4- as sample survey. A total of 69 lizards belonging to the 5 times with 0.2 M Cacodylate buffer (pH - 7.3) and fixed family Gekkonidae (H. flaviviridis and G. gecko), in 2.5% Glutaraldehyde at 4°C for 24 h. The fixed Varanidae (V. indicus) and Chameleonidae (C. samples were washed in phosphate buffer saline (pH - zeylanicus) were examined. 7.2) for three times and then in double distilled water followed by acetone dehydration. After acetone dehydration, the specimens were dried with liquid carbon dioxide at its critical point (that is, 3.5°C at 11 psi). The Collection and examination of faecal samples specimens were then immersed in tetra methyl saline (TMS) for 5-10 min for two changes at 4°C. Then they are Faeces (n=185) were examined fresh. Direct smear, brought to room temperature (25-26°C) to dry. The floatation and sedimentation techniques were employed samples were mounted on aluminium stubs. The parasitic following standard procedure (Souza et al., 2014; Wolf et specimens were then gold coated in a sputter coat and al., 2014). Faecal sample of dead lizards were also finally examined under SEM [(JSM-6360-JEOL)] at the examined. North Eastern Hill University (NEHU), Shillong, Meghalaya, India in Sophisticated Analytical Instrument Facilities (SAIF) Laboratory (Patra et al., 2016). Examination of blood samples

A total of 69 blood samples from each adult lizard RESULTS AND DISCUSSION specimen were collected by puncturing of the caudal vein with disposable sterile syringes. Thin blood smears were A total of 69 lizards from three families (Gekkonidae, prepared and quickly air dried. In case of field samples Varanidae and Chameleonidae) were examined during away from laboratory, slides were stored and brought to the study period and the result of the faecal sample and the laboratory in plastic slide boxes. Slides were stained blood samples is shown in Table 1 in proper percentile with Giemsa stain for 45 min and examined under oil form. immersion at ×1000 magnification. Forty three lizards were found infected with three

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Table 1. Species of parasites infecting lizards from Mizoram, India.

House lizards Chameleon Monitor lizard Type of parasite Gecko (G. gecko) (15) (H. flaviviridis) (37) (C. zeylanicus) (14) (V. indicus) (3) Endo-parasites Nematodes Pharyngodon sp. 20 (54.05%) 05 (35.71%) 04 (26.67%) - Rhabdias sp. 02 (5.40%) 03 (21.43%) - - Strongyle 05 (13.51%) 02 (14.28%) 02 (13.33%) - Trematodes Postorchigenes sp. 01 (2.70%) - - - Intestinal Protozoa Eimeria sp. 04 (10.81%) 02 (14.28%)) 03 (20%) 02 (66.67%) Isospora sp. 01 (2.70%) 01 (7.14%) 01 (6.67%) - Cyclospora sp. - - 03 (20%) - Balantidium sp. - - 01 (6.67%) - Haemoprotozoa Lankesterella sp. 02 (5.40%) - 01 (6.67%) - Trypanosoma sp. 02 (5.40%) 01 (7.14%) - - Ecto-parasites Amblyomma sp. - - - 01 (33.33%)

different species of nematodes (Pharyngodon sp., were found positive for Lankesterella sp. and Rhabdias sp., Strongyle). One lizard was found infected Trypanosoma sp. infection respectively. Only 33.33% of with a species of trematode (Postorchigenes sp.) and the monitor lizards were found positive with ecto-parasite eighteen lizards were found infected with three species of (Amblyomma sp.) infestation. tissue protozoa (Eimeria sp., Isospora sp. and Based on morphometry under LM and SEM, three Cyclospora sp.). Six lizards were also found positive for species of nematodes were identified as Pharyngodon two species of haemoprotozoans namely Trypanosoma sp. (Figures 1a, b, c and d), Strongyloides sp. (Figures sp. and Lankesterella sp. Only one monitor lizard was 2a, b and c) and Strongyle species (Figures 3a, b and c). found infested with Amblyomma sp. One trematode under Postorchigenes genus was found Out of 69 lizards examined, the highest infection rate in one house lizard (Figures 4a and b). Three species of by nematode was found in house lizards by Pharyngodon tissue protozoa were found when faecal floatation sp. (54.05%) followed by chameleon (35.71%) and gecko method was used(Figures 5, 6 and 7). Balantidium sp. (26.67%). The infection rate by Rhabdias sp. was less cysts were found in four faecal samples (Figure 8). While (5.40%) in house and garden lizards and mild in two house lizards were found positive for Lankesterella chameleon (21.43%). The lizards (house lizards, sp. (Figure 9). One garden lizard was found infected with chameleon and gecko) were found infected with Trypanosoma sp. (Figure 10). Light microscopy pictures strongyles in more or less similar rate (13.51, 14.28% of Amblyomma sp. which was recovered from monitor and 13.33% respectively). No infection was found in lizard are shown in Figures 11a and b. monitor lizard by any of the nematode parasites. The present study underscores the prevalence and Only one (2.70%) lizard was found infected with intensity of ecto- and endo-parasitic fauna of four species Postorchigenes sp. Infection with intestinal protozoa was of lizards from different parts of Mizoram, India. Parasites found significantly higher by Eimeria sp in all the groups occasionally cause harmful effects on lizards kept in of lizards. It was found highest in monitor lizard (66.67%) captivity or in the wild (Rataj et al., 2011; Hedley et al., followed by gecko (20%), chameleon (14.28%) and 2013). For systematic identification and classification, all house lizards (10.81%). Mild infection with Isospora sp morphological aspects of parasites must be studied was recorded in different groups of lizards. Cyclospora under light microscopy. The importance of SEM study lies sp was found predominantly in gecko (20%). Balantidium on its remarkable ability to provide three dimensional sp oocyst was detected only in one gecko (6.67%). images with magnification that allow understanding the Infection with haemoprotozoa, Lankesterella sp. and spatial relationship among surface structures Trypanosoma sp. were recorded as 5.4% in house lizards (Hirschmann, 1983; Gibbons, 1986). Faecal floatation in blood smear examination. 6.67% geckos and 7.14% was found superior to other faecal examination

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a b c

d e

Figure 1. a, Pharyngodon sp. (female) (X40); b, Pharyngodon sp. (anterior part) (X100); c, egg of Pharyngodon sp. (X100); d, Larval stage of Pharyngodon sp. (X100); e, Somatic papillae and transverse ridge of Pharyngodon sp (SEM).

a b c

Figure 2. a, Rh abdias sp. (anterior end ) ( X100); b, Rhabdias sp. (anterior end) (mouth part); C, posterior part of Rhabdias sp. (SEM).

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a b

c d

Figure 3. a, Anterior end of Strongyle (SEM) (mouth part); b, Strongyle under SEM; c, Strongyle under SEM; d, Strongyloides sp. egg (X100).

a b

Figure 4. a, Postorchigenes sp. (SEM); b, Body surface of Postorchigenes sp (SEM).

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a b

Figure 5. Sporulated spp. of Isospora sp. (X100).

Figure 7. Unsporulated Oocysts of Cyclospora sp. (X400).

Figure 8. Balantidium cyst (X100).

Patra et al. 138

Figure 9. Giemsa stained blood smear of lizard showing Lankesterella sp. (X1000).

Figure 10. Giemsa stained blood smear of lizard showing Trypanosoma sp. (X1000).

Figure 11. a, Amblyomma sp. in-situ; b, female Amblyomma sp. under LM (X40).

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