Microbiological and Histological Examinations of Endangered Neurergus Kaiseri Tissues Displaying Red-Leg Syndrome

Asian Herpetological Research 2014, 5(3): 204–208 DOI: 10.3724/SP.J.1245.2014.00204

Microbiological and Histological Examinations of Endangered Neurergus kaiseri Tissues Displaying Red-leg Syndrome

Paria PARTO1, Zahra M. S. HAGHIGHI2, Somaye VAISSI1 and Mozafar SHARIFI1*

1 Department of Biology, Faculty of Science, Razi University, Baghabrisham 6714967346, Kermanshah, Iran 2 Faculty of Veterinary Science, Department of basic Science, Razi University, Baghabrisham 6714967346, Kermanshah, Iran

Abstract Presence of the red leg syndrome (RLS) was documented through bacterial and histological examinations in the endangered Kaiser’s mountain newt Neurergus kaiseri obtained from a pet shop. The individuals which were severely infected showed lethargy, appetite loss, weight loss, abdominal skin redness and skin ulcers on hind legs. This study reveals the presence of two bacteria causing RLS on the skin of captive N. kaiseri including Proteus vulgaris and Bacillus cereus. Sections of skin in affected areas and internal organs were examined through standard histological procedures. Histologically, epidermal necrosis and ulcers, epidermal gland depletion, myositis and subcutaneous edema, gastric submucosal edema and hepatomegaly were seen. There were also correlations between the microbial infection and structural changes in tissues of Kaiser’s mountain newt. The severity of the structural changes are related to the level of microbial infection in the target organs and could be sustained by the isolation of P. vulgaris and other pathogens. The presence of the infective bacterial population and their interaction on the skin of the newt may have changed the normal skin flora and facilitate the prevalence of other disease.

Keywords Neurergus kaiseri, red-leg syndrome, lethargy, abdominal skin redness, skin ulcers

1. Introduction (IUCN, 2011). One important aspect of global decline in amphibian populations is that these declines also occurs Amphibians are among the planet’s most threatened in ecologically pristine areas, such as the mountain taxa. Nearly one-third of the world’s 1 856 species are tropical rain forests of Australia and Central America, threatened with extinction (Stuart et al., 2004). Population where human impact from agriculture, deforestation, or declines in recent decades have been especially severe, pollution is thought to be negligible (Cushman, 2006). with up to five species going extinct each year (Stuart Pathogens of different kinds may infect amphibians et al., 2004). Global declines in amphibian population at various life stages and can be the proximate causes have been reported for numerous species. While some of mortality or can lead to sub-lethal damage such as declines are clearly due to habitat loss and habitat severe developmental and physiological abnormalities destruction, others are not associated with obvious (Voyles et al., 2011). Among diseases that affect environmental factors (Beebee and Griffiths, 2005). captive and wild amphibians, the most over diagnosed Other causal hypotheses include the introduction of and misdiagnosed disease is red leg syndrome predators or competitors, increased ultraviolet irradiation, (Densmore and Green, 2007; Hird et al., 1981). Red- acid precipitation, climate change, environmental leg syndrome (RLS) is a widespread infection seen pollution, infectious disease, or a combination of these in frogs, toads, and salamanders. It is recognized by * Corresponding author: Prof. Mozafar SHARIFI, from Razi University, the redness on the underside of the amphibian’s legs Kermanshah, Iran, with his research focusing on amphibian conservation and abdomen (Pasteris et al., 2006). The etiological biology. E-mail: [email protected] agents of RLS are members of the Enterobacteriaceae, Received: 30 November 2013 Accepted: 27 August 2014 such as Proteus vulgaris, P. mirabilis, Pseudomonas No. 3 Paria PARTO et al. Red-leg Syndrome in Neurergus kaiseri 205 aeruginosa, Aeromonashydrophila, and by some was performed manually by viewing a smear under a strains of Staphylococcus epidermidis (Glorioso et al., light microscope. Sections of individual tissue (liver, 1974). Recently, the incidence of Streptococcus iniae, skin and stomach) and skin swabs from the lesion were Chryseobacterium meningosepticum, Chr. indolgenes, aseptically collected at necropsy and also from live newts Citrobacterfreundii and Edwardsiellatarda were also and inoculated onto nutrient and Mac Conkey agar plates. reported (Mauel et al., 2002). Plates are incubated at 30°C in the presence and absence

RLS is an epidemic disease attacking most species of of CO2. Bacteria selected from the pure cultures were frogs and toads in captivity. This disease has rarely been stained with Gram stain and identification of the bacteria reported from Caudata (Densmore and Green, 2007). isolates was carried out with a biochemical profile The purpose of this work was to perform microbiological according to API Identification System (Bromage et al., and histological investigations in organs of infected 1999). Neurergus kaiseri in captivity to evaluate susceptibility of Hepatic lobes (middle fractions), stomach, muscle and any attempt for an efficient captive breeding facility for skin from both healthy and non-healthy animals were this species. aseptically removed, fixed with 10% neutral buffered formalin for 24–48h at room temperature, dehydrated and 2. Materials and Methods embedded in paraffin according to standardized methods for light microscopy (Martoja and Martoja-Pierson, Relatively few species of amphibians occur in Iran, 1970). Six micrometer serial sections of skin, livers, of which only seven species belong to Caudata. In spleens and stomachs were stained with haematoxylin first order streams located on the southern edges of and eosin. Sections were observed with an Olympus the Iranian Plateau, one endemic species, N. kaiseri microscope (Leica Galen III) and were photographed with occurs. Distribution and abundance of N. kaiseri have a digital camera (Leica with Dinocapture 2). been described in southern Zagros Mountains, Iran, and show that this newt is seen in very few numbers 3. Results of streams (Sharifi and Assadian, 2005; Sharifi et al., 2013). Concerns over occurrence and survival of the The microbial examination of the micro-organisms endemic N. kaiseri in very few fragmented streams isolated from the skin of non-healthy N. kaiseri in south-western Iran originated from global decline demonstrating red leg syndrome evidenced the presence of amphibian populations which resulted from a wide of the following bacterial species: Proteus vulgaris and range of threats (Sharifi et al., 2013). The principal and Bacillus cereus isolates. Bacterial isolation revealed immediate threat to the species was illegal collecting that these two species could be obtained, in almost pure for national and international trade (Sharifi et al., 2009). culture, from the skin of all infected newts. These bacteria Following amendment of N. kaiseri to the appendix I could also be isolated to varying degrees from other of the Convention of International Trade of Endangered organs sampled. Species in 2008 international trade of this species Gross lesions were completely uniform in all dead became legalized. Unlike legal control over international newts. Cutaneous blisters and hyperemic abdomen skin trade of this species illegal harvest of this species for were observed (Figure 1). Legs were often swollen national trade as a pet is still continuing. N. kaiseri used with marked subcutaneous edema and focal areas in this study were obtained from a pet shop in Tehran. of hemorrhage within the skeletal muscle. The skin These include 7 adult N. kaiseri which were kept in one contained multifocal areas of epidermal degeneration with aquarium (75 × 45 × 35 cm3). The water temperature abundant dermal edema and a mixed dermal inflammatory in the aquarium ranged from 16–26°C. The aquarium infiltrate that varied from mild to severe. In a few cases, was equipped with an electrical ventilator and a waste the epidermis was focally ulcerated and dermis was pumps. In the aquarium there was a choice of terrestrial exposed with congested and hyperemic blood vessels. habitat provided by small pebbles. The newts were fed by Based on the microbiological results obtained from mealworms (Tenebrio molitor) and blood worms (Glycera skin and different tissues, histological studies were dibranchiata). carried out in N. kaiseri skin, stomach, subcutaneous Blood samples were obtained for hematological tests muscle fibres, skin glands, spleen and liver (Figures from the hearts of 7 sick newts. The blood was drawn 1–5). Clinically, the newts with RLS demonstrated into test tubes containing EDTA. White blood cell count torticolis, stupor and indifference to stimuli. In some 206 Asian Herpetological Research Vol. 5

cases, gross lesions were present. Cutaneous hyperemia was noted especially on the extremities and the dorsal and ventral skin. The legs were often swollen with marked subcutaneous edema and focal areas of haemorrhage within the skeletal muscle. Skin lesions were mostly located on legs and abdomen. The liver and spleen were enlarged. Both organs were discolored with multiple pale foci. Multifocal hemorrhagic myositis with fragmentation of muscle fibers and loss of striation associated with perimysial edema in skeletal muscle of hind limb, degeneration and depletion of subcutaneous seromucous Figure 1 Neurergus kaiseri with red-leg syndrome. Skin ulcerations glands and secondary fungal infection due to presence with inflammatory signs are seen in leg and fingers. of fungal empty thalli, sporangium and round basophilic spores were identified in all dead newts (Figure 4) Hepatomegally and discoloration of liver with multiple pale foci was seen (Figure 2). The stomachs were enlarged with severe submucosal edema (Figure 5).

4. Discussion

In the present study two known pathogens of RLS Proteus vulgaris and Bacillus cereus were isolated from the newt skin. Aquatic medium could be considered as the main cause of dissemination of infectious agents among newts. These pathogens can invade the skin via injury during catching, exposure to high temperatures during shipping, crowded conditions and/or dirty water in tanks. There are other potential vectors normally found Figure 2 Light microscopy photograph of histological section of in the normal skin flora that can cause RLS. There are Neurergus kaiseri liver with RLS showing focal hepatitis, infiltration of mixed inflammatory cells (black arrow) within hepatic par- reports from other groups of bacteria that have caused enchyma. Melanomacrophage cells (white arrow) are also abundant RLS. These include some distinct bacteria belonging to in the section. the genera: Streptococcus, Lactobacillus, Vagococcus and Carnobacterium (Ringo and Gatesoupe, 1998). Moreover, the natural bacterial population and interaction on the skin of amphibians is essential for preventing the bacterial invasion on the skin. Skin flora and microbiota can potentially prevent pathogenic bacterial and fungal invasion through competitive exclusion or antimicrobial secretions. Regarding the cutaneous rout of entry, the skin glands produce and release peptides, that play various roles, in the regulation of physiological functions of the skin or in defence mechanisms against predators’ micro- organisms (Rollins-Smith et al., 2002). Neurergus kaiseri with red leg syndrome demonstrated muscular spasm and were indifferent to external stimuli. Cutaneous lesions appeared to be limited to the extremities and mild hyperemia to deep ulcers were Figure 3 Light microscopy photograph of histological section of Neurergus kaiseri with RLS showing edema and infiltration of also observed in the skin of abdomen. The legs were inflammatory cells (arrows) within subcutaneous muscle fibres. often swollen with marked subcutaneous edema and No. 3 Paria PARTO et al. Red-leg Syndrome in Neurergus kaiseri 207

ranaviruses (Cunningham et al., 1996) and infection by Batrachochytrium dendrobatidis (chytridiomycosis). Because postmortem bacterial invasion of organs is a common phenomenon among aquatic vertebrates, including amphibians and it occurs much more rapidly than in terrestrial, endothermic vertebrates, diagnosis of red leg syndrome is unreliable when it is based solely on bacterial isolation from tissues of an amphibian that died 1 to 3 hours before the necropsy. It is likely that this practice has historically led to considerable over diagnosis of red leg syndrome epizootics among diseased amphibian populations (Densmore and Green, 2007). As it has been reported in frogs the most likely route of the infection may be an infection through the superficial Figure 4 Light microscopy photograph of histological section of skin lesions or gastro-intestinal tract as stated by Glorioso Neurergus kaiseri with RLS showing typical necrosis and depletion of skin glands (G) and edema (E) between muscle bundles. et al. (1974). Skin gland distortion and depletion which was observed in the affected skin areas of N. kaiseri correspond with the similar finding in bullfrogs, studied by Pastries et al. (2006) and could contribute to the pathogen infection. Bad husbandry and inadequate food can also contribute to the spread of the infection. Figure 5 shows the enlarged stomach of a dead N. kaiseri with severes submucosal edema which may have developed as the result of starvation. Captive husbandry in pet shops in Iran is far from satisfactory. In the absence of any governmental control code of practice over well being of aquatic organisms, outbreak of infective disease is likely. Moreover, N. kaiseri and other wild amphibians brought to these pet shops are not captive bred and in most cases the site of collection is not known. These wild caught newts Figure 5 Neurergus kaiseri with red-leg syndrome. Stomach with are collected from the wild without any approval and severe submucosal edema (arrow). documentation from the proper authorities. Moreover, focal areas of hemorrhage within skeletal muscle. Our while in the pet shops there is no measure to prevent the spread of Batrachochytrium dendrobatidis (the amphibian observation showed that the gross lesions are completely chytrid fungus) and other potential pathogens such as uniform in all dead newts, including: cutaneous blisters ranavirus there is a good possibility for this pathogen to and hyperemic abdomen, swollen legs with marked spread from one area to the next or from one individual subcutaneous edema and focal areas of hemorrhage within to another. Bad husbandry in the pet shop could have the skeletal muscle, degeneration of the skin epidermal increased the risk of infective disease and an increase in layers, RBC laden vessels, multifocal hemorrhagic mortality due to crowding, bad nutrition and contact with myositis with perimysial edema in skeletal muscle of pathogenic agents. These factors could weaken natural hind limb, degeneration and depletion of subcutaneous defence mechanisms and in such conditions bacterial seromucous glands and secondary fungal infection, opportunists can invade the animal and cause diseases hepatomegally and the edema of gastric submucosal area (Mauel et al., 2002). which might be the result of inappetance. Multiple and diverse pathogens of amphibians Acknowledgements We thank Razi University and may be present clinically or after death with signs of the Iran National Science Foundation (Contract No. classical red leg syndrome. Other nonbacterial pathogens 91057377) that financially supported this study as a part that may present with similar clinical signs include of a PhD research project. 208 Asian Herpetological Research Vol. 5

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