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

Paria PARTO, Zahra M. S. HAGHIGHI, Somaye VAISSIand Mozafar SHARIFI*

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

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

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

Paria PARTO1, Zahra M. S. HAGHIGHI2, Somaye VAISSI1and Mozafar SHARIFI1*

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

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

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.

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

1. Introduction

Amphibians are among the planet’s most threatened taxa. Nearly one-third of the world’s 1 856 species are threatened with extinction (Stuart et al., 2004). Population declines in recent decades have been especially severe, with up to five species going extinct each year (Stuart et al., 2004). Global declines in amphibian population have been reported for numerous species. While some declines are clearly due to habitat loss and habitat destruction, others are not associated with obvious environmental factors (Beebee and Griffiths, 2005). Other causal hypotheses include the introduction of predators or competitors, increased ultraviolet irradiation, acid precipitation, climate change, environmental pollution, infectious disease, or a combination of these(IUcN, 2011). One important aspect of global decline in amphibian populations is that these declines also occurs in ecologically pristine areas, such as the mountain tropical rain forests of Australia and central America, where human impact from agriculture, deforestation, or pollution is thought to be negligible (cushman, 2006).

Pathogens of different kinds may infect amphibians at various life stages and can be the proximate causes of mortality or can lead to sub-lethal damage such as severe developmental and physiological abnormalities (Voyles et al., 2011). Among diseases that affect captive and wild amphibians, the most over diagnosed and misdiagnosed disease is red leg syndrome (Densmore and Green, 2007; Hird et al., 1981). Redleg syndrome (RLS) is a widespread infection seen in frogs, toads, and salamanders. It is recognized by the redness on the underside of the amphibian’s legs and abdomen (Pasteris et al., 2006). The etiological agents of RLS are members of the Enterobacteriaceae, such as Proteus vulgaris, P. mirabilis, Pseudomonas

aeruginosa, Aeromonashydrophila, and by some strains of Staphylococcus epidermidis (Glorioso et al., 1974). Recently, the incidence of Streptococcus iniae, chryseobacterium meningosepticum, chr. indolgenes, citrobacterfreundii and Edwardsiellatarda were also reported (Mauel et al., 2002).

RLS is an epidemic disease attacking most species of frogs and toads in captivity. This disease has rarely been reported from caudata (Densmore and Green, 2007). The purpose of this work was to perform microbiological and histological investigations in organs of infected Neurergus kaiseri in captivity to evaluate susceptibility of any attempt for an efficient captive breeding facility for this species.

2. Materials and Methods

Relatively few species of amphibians occur in Iran, of which only seven species belong to caudata. In first order streams located on the southern edges of the Iranian Plateau, one endemic species, N. kaiseri occurs. Distribution and abundance of N. kaiseri have been described in southern Zagros Mountains, Iran, and show that this newt is seen in very few numbers of streams (Sharifi and Assadian, 2005; Sharifi et al., 2013). concerns over occurrence and survival of the endemic N. kaiseri in very few fragmented streams in south-western Iran originated from global decline of amphibian populations which resulted from a wide range of threats (Sharifi et al., 2013). The principal and immediate threat to the species was illegal collecting for national and international trade (Sharifi et al., 2009). Following amendment of N. kaiseri to the appendix I of the convention of International Trade of Endangered Species in 2008 international trade of this species became legalized. Unlike legal control over international trade of this species illegal harvest of this species for national trade as a pet is still continuing. N. kaiseri used in this study were obtained from a pet shop in Tehran. These include 7 adult N. kaiseri which were kept in one aquarium (75 × 45 × 35 cm3). The water temperature in the aquarium ranged from 16–26°c. The aquarium was equipped with an electrical ventilator and a waste pumps. In the aquarium there was a choice of terrestrial habitat provided by small pebbles. The newts were fed by mealworms (Tenebrio molitor) and blood worms (Glycera dibranchiata).

Blood samples were obtained for hematological tests from the hearts of 7 sick newts. The blood was drawn into test tubes containing EDTA. White blood cell count was performed manually by viewing a smear under a light microscope. Sections of individual tissue (liver, skin and stomach) and skin swabs from the lesion were aseptically collected at necropsy and also from live newts and inoculated onto nutrient and Mac conkey agar plates. Plates are incubated at 30°c in the presence and absence of cO2. Bacteria selected from the pure cultures were stained with Gram stain and identification of the bacteria isolates was carried out with a biochemical profile according to API Identification System (Bromage et al., 1999).

Hepatic lobes (middle fractions), stomach, muscle and skin from both healthy and non-healthy animals were aseptically removed, fixed with 10% neutral buffered formalin for 24–48h at room temperature, dehydrated and embedded in paraffin according to standardized methods for light microscopy (Martoja and Martoja-Pierson, 1970). Six micrometer serial sections of skin, livers, spleens and stomachs were stained with haematoxylin and eosin. Sections were observed with an Olympus microscope (Leica Galen III) and were photographed with a digital camera (Leica with Dinocapture 2).

3. Results

The microbial examination of the micro-organisms isolated from the skin of non-healthy N. kaiseri demonstrating red leg syndrome evidenced the presence of the following bacterial species: Proteus vulgaris and Bacillus cereus isolates. Bacterial isolation revealed that these two species could be obtained, in almost pure culture, from the skin of all infected newts. These bacteria could also be isolated to varying degrees from other organs sampled.

Gross lesions were completely uniform in all dead newts. cutaneous blisters and hyperemic abdomen skin were observed (Figure 1). Legs were often swollen with marked subcutaneous edema and focal areas of hemorrhage within the skeletal muscle. The skin contained multifocal areas of epidermal degeneration with abundant dermal edema and a mixed dermal inflammatory infiltrate that varied from mild to severe. In a few cases, the epidermis was focally ulcerated and dermis was exposed with congested and hyperemic blood vessels.

Figure 1 Neurergus kaiseri with red-leg syndrome. Skin ulcerations with inflammatory signs are seen in leg and fingers.

Figure 3 Light microscopy photograph of histological section of Neurergus kaiseri with RLS showing edema and infiltration of inflammatory cells (arrows) within subcutaneous muscle fibres.

Based on the microbiological results obtained from skin and different tissues, histological studies were carried out in N. kaiseri skin, stomach, subcutaneous muscle fibres, skin glands, spleen and liver (Figures 1–5). clinically, the newts with RLS demonstrated torticolis, stupor and indifference to stimuli. In some 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 glands and secondary fungal infection due to presence 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 in the normal skin flora that can cause RLS. There are reports from other groups of bacteria that have caused RLS. These include some distinct bacteria belonging to 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’ microorganisms (Rollins-Smith et al., 2002).

Figure 4 Light microscopy photograph of histological section of Neurergus kaiseri with RLS showing typical necrosis and depletion of skin glands (G) and edema (E) between muscle bundles.

Figure 5 Neurergus kaiseri with red-leg syndrome. Stomach with severe submucosal edema (arrow).

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 also observed in the skin of abdomen. The legs were often swollen with marked subcutaneous edema and focal areas of hemorrhage within skeletal muscle. Our observation showed that the gross lesions are completely uniform in all dead newts, including: cutaneous blisters and hyperemic abdomen, swollen legs with marked subcutaneous edema and focal areas of hemorrhage within the skeletal muscle, degeneration of the skin epidermal layers, RBc laden vessels, multifocal hemorrhagic myositis with perimysial edema in skeletal muscle of hind limb, degeneration and depletion of subcutaneous seromucous glands and secondary fungal infection, hepatomegally and the edema of gastric submucosal area which might be the result of inappetance.

Multiple and diverse pathogens of amphibians may be present clinically or after death with signs of classical red leg syndrome. Other nonbacterial pathogens that may present with similar clinical signs include 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 skin lesions or gastro-intestinal tract as stated by Glorioso 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 are collected from the wild without any approval and documentation from the proper authorities. Moreover, while in the pet shops there is no measure to prevent the spread of Batrachochytrium dendrobatidis (the amphibian chytrid fungus) and other potential pathogens such as ranavirus there is a good possibility for this pathogen to spread from one area to the next or from one individual to another. Bad husbandry in the pet shop could have increased the risk of infective disease and an increase in mortality due to crowding, bad nutrition and contact with pathogenic agents. These factors could weaken natural defence mechanisms and in such conditions bacterial opportunists can invade the animal and cause diseases (Mauel et al., 2002).

AcknowledgementsWe thank Razi University and the Iran National Science Foundation (contract No. 91057377) that financially supported this study as a part of a PhD research project.

Beebee T. J. c., Griffiths R. A.2005. The amphibian decline crisis: A watershed for conservation biology? Biol conserve, 125: 271–285

Bromage E. S., Thomas A., Owens L.1999. Streptococcus iniae, a bacterial infection in barramundi Lates calcarifer. Dis Aquat Org, 36: 177–181

cunningham S. D., Anderson T. A., Schwab A. P., Hsu F. c.1996. Phytoremediation of soil contaminated with organic pollutants. Adv Agron, 56: 55–114

cushman S. A.2006. Effects of habitat loss and fragmentation on amphibians: A review and prospectus. Biol conserv, 128: 231–240

Densmore c. L., Green D. E.2007. Diseases of amphibians. ILAR J, 48: 235–254

Glorioso J. c., Amborski R. L., Amborski G. F., culley D. c.1974. Microbiological studies on septicemic bull- frogs (Rana catesbeiana). Am J Vet Res, 35: 1241–1245

Hird D. W., Diesch S. L., McKinnel R. G.1981. Aeromonas hydrophila in wildcaught frogs and tadpoles (Rana pipiens) in Minnesolta. Lab Anim Sci, 31: 166–169

IUcN2011. The IUcN Red List of Threatened Species 2011. 2

Martoja R., Martoja-Pierson M.1970. TB cnicas de histologيa animal. S. A. Barcelona: Toray Masson, 176–208

Mauel M. J., Miller D. L., Frazier K. S., Hines M. E.2002. Bacterial pathogens isolated from cultured bullfrog (Rana catesbeiana). Microbes Infect, 13: 25–32

Pasterisa S. E., Buhlera M. I., Nader-Macı´asb M. E.2006. Microbiological and histological studies of farmed-bullfrog (Rana catesbeiana) tissues displaying red-leg syndrome. Aquaculture, 251: 11–18

Ringo E., Gatesoupe F .J.1998. Lactic acid bacteria in fish: a review. Aquaculture, 160 (3-4): 177–203

Rollins-Smith L. A., carey c., Longcore J., Doersam J. K., Boutte A., Bruzgal J. E., conlon J. M.2002. Activity of antimicrobial skin peptides from ranid frogs against Batrachochytrium dendrobatidis, the chytrid fungus associated with global amphibian declines. Dev comp Immunol, 26: 471–479

Sharifi M., Assadian S.2005. Reproductive cycle of the Yellow Spotted Newt Neurergus microspilotus (caudata: Salamandridae) in Western Iran. Russian J Herpetol, 12: 63–68

Sharifi M., Papenfuss T., Rastegar-Pouyani N., Anderson S., Kuzmin S.2008. Neurergus kaiseri. In IUcN (Ed.), IUcN Red List of Threatened Species Version 2009. Gland, Switzerland: IUcN (http://www.iucnredlist.org, accessed 22 Octo ber 2009).

Sharifi M., Farasat H., Barani-Beiranvand H., Vaissi S., Foroozanfar E.2013. Notes on distribution and abundance of the endangered Kaiser’s mountain newt, Neurergus kaiseri (caudata: Salamandridae), in southwestern Iran. Herpetol conserv Biol, 8(3): 724−731

Stuart S. N., chanson J. S., cox N. A., Young B. E., Rodrigues A. S. L., Fischmann D. L., Waller R. W.2004. Status and trends of amphibian declines and extinctions worldwide. Science, 306: 1783–1786

Voyles J., Rosenblum E. B., Berger L.2011. Interactions between Batrachochytrium dendrobatidis and its amphibian hosts: a review of pathogenesis and immunity. Microbes Infect, 13: 25–32

*corresponding author: Prof. Mozafar SHARIFI, from Razi University, Kermanshah, Iran, with his research focusing on amphibian conservation biology.

E-mail: sharifimozafar2012@gmail.com

Received: 30 November 2013 Accepted: 27 August 2014