An Investigation of Sudden Death in Farmed Infant Siamese Crocodiles During Winter and Spring in Hainan, China

B-591 [1-5] Indian J. Anim. Res., AGRICULTURAL RESEARCH COMMUNICATION CENTRE Print ISSN:0367-6722 / Online ISSN:0976-0555 www.arccjournals.com/www.ijaronline.in An investigation of sudden death in farmed infant Siamese crocodiles during winter and spring in Hainan, China Guiying Guo1, Jiahao Jiang1, Nuo Yang1, Peng Wang3, Liling Zhang4, Ying Wang1, Jifeng Zeng2* and Jiping Zheng1* Department of Biotechnology, Hainan University, Haikou 570228, China. Received: 12-04-2016 Accepted: 17-09-2016 DOI:10.18805/ijar.v0iOF.6999 ABSTRACT Sudden deaths without any specific clinical sign occurred among one year old young ones on Siamese crocodile (Crocodylus siamensis) farms in Hainan province, China, during winter and spring in 2012 - 2013. The autopsy showed that the internal organs seemed normal except full of clear ascites in abdominal cavity. There were total six bacterial species isolated, Edwardsiella tarda, Proteus penneri, Proteus vulgaris, Enterobacter asburiae, Citrobacter freundii and Micrococcus luteus from either the heart bloods or the spleens and livers. The virulence of isolated strains was tested by intraperitoneal inoculation in female Chinese edible frogs (Rana rugulosa or Hoplobatrachus rugulosus). The six-day mortality revealed that E. tarda was the most pathogenic, whereas P. penneri and E. asburiae exhibited no infection at all, and the rest demonstrated similar inconspicuous toxicity. All these findings suggest that E. tarda would be the principal suspect causing death of Siamese crocodile. The sensitivities of the E. tarda isolates were tested against 20 kinds of antimicrobial drugs. The results showed that ceftazidime, norfloxacin and amikacin were highly sensitive to the isolates of E.tarda. Key words: Crocodylus siamensis, Edwardsiella tarda, Hoplobatrachus rugulosus, Sudden death. INTRODUCTION 2005). During 2012-2013, asymptomatic deaths occurred Siamese crocodile (Crocodylus siamensis) is a in succession among infant crocodiles on farms in the cities small freshwater crocodile, native to the most of Southeast of Sanya and Wenchang, Hainan province. In this paper, we Asia, including Indonesia, Brunei, Laos, East Malaysia, report the etiological investigation of the unexpected Cambodia, Thailand, Burma, and Vietnam etc. But currently, mortality on Wenchang Crocodile Farm. the species is already extirpated in the wild or nearly extinct But the matter of exigency is how to recognize and from 99% of its original range except Cambodia by the confirm the real pathogens from the suspected isolates increasing threat from human disturbance, habitat occupation original from the dead cases in this report, because it is too and illegal capture. It was even believed that the species cost to study Siamese crocodile directly as the infection test was almost or completely extinct in the wild in 1992 species, and would also be strongly opposed from the wildlife (Simpson et al., 2010). Although Siamese crocodile looks protection. Moreover, the conventional experimental like unfriendly, it has a huge economic value in terms animals, for example, mice (endotherm) and zebrafish of meat for human consumption and skins for the (ectotherm), are difficult or impossible to replicate such kind luxurious leather industry. Farming crocodiles is not only of diseases from reptiles, which obviously more fit to an important and lucrative activity, but also has a positive understanding of the human disease (Bedell et al., 1997 and effect on the preservation of the wild ones from hunting and Lieschke et al., 2007). In general, an ideal animal model killing. usually meets a determined taxonomic equivalency to Since the mid-1980s, a large number of crocodiles Siamese crocodile, so as to react to disease in a way that were exported from Cambodia to stock commercial farms resembles as crocodiles. In phylogeny and genetic in China, but successful breeding was only in several relatedness, the reptiles would be the best choice, but in terms provinces, such as Hainan, due to the similar wild habitats of the reptiles, such as turtles, crocodilians, snakes, of Siamese crocodile. Nonetheless, the environment amphisbaenians, lizards and tuatara, all of them are difficult conductive to crocodile rearing is also ideal for infection. to use for either high expense and rarity or lack breeding Therefore, deaths always take place every year (Erhu et al., techniques. *Corresponding author’s e-mail: [email protected], [email protected] 1Department of Biotechnology, Hainan University, Haikou 570228, China. 2Department of Animal Science, Hainan University, Haikou 570228, China. 3Beijing Institute of Biotechnology, Beijing 100071, China. 4Hainan Dosho Python technique Co., Ltd, Haikou 57000, China. 2 INDIAN JOURNAL OF ANIMAL RESEARCH Here, we tried to use amphibian Chinese edible frog 1 2 3 4 M 5 6 7 8 (Rana rugulosa or Hoplobatrachus rugulosus) as the testing animal of virulence assay, for the closer relationship with reptiles than mice and zebrafish in evolution and ecological habitation. The Chinese edible frog, commonly known as 1.5kb (16S rDNA) ‘field chicken’ for the leg taste in China, is widely farmed and easy purchased from the market in China, Malaysia, and Thailand (Warkentin et al., 2009). So, the population is large enough for robust experimental designs. The results of frog mortality examination revealed that only Edwardsiella tarda Fig 1: PCR products of 16S rRNA gene from isolates. Lane 1-8: isolates from sudden death infant Siamese crocodiles. Lane M: is the most virulent isolated species from the dead Siamese CL5kb ladder (5000, 3000, 2000, 1500, 750, 500, 300, 100 bp). crocodiles in this time, while others lead to very slight or no infectious symptoms at all. were determined by BLAST (Basic Local Alignment Search MATERIALS AND METHODS Theorem) analysis with the sequencing results. Cases: Two dead one-year-old Siamese crocodiles from Pathogenicity of isolates and LD50 Determination: Female Wenchang Farm, Hainan were brought in ice box to our lab Chinese edible frogs (mean±sd 100±10 g) were purchased of microbiology for necropsy and pathogens isolation during from Frog Feeding Company, Haikou. For keeping the frog’s November 2013. Gross examination revealed that both were semi-aquatic habits, a simple basin breeding device was approximately one kg weight, and the scales are bright designed. In brief, a standard brick (240 mm×115 mm× without any scratch or scar. At necropsy, colorless and 90 mm) was put in a 35 cm-diameter basin, then, freshwater odorless liquid were found filled in abdominal cavity. No was poured in till at the height of 90 mm. After that, 4 frogs other macroscopic lesion was observed in the organs. were placed in one basin as one group, then a net was covered Cellulous pathogen isolation and microscopic observations around to prevent escaping. To confirm the health status, all For isolation and purification of the non-viral frogs were fasted in quarantine with water for one week pathogens, the viscera tissues: liver, lung, spleen, and kidney before infection. For experimental infection, the healthy frogs were pierced and probed by inoculation loop or sterile cotton- were divided randomly into four groups (four frogs per tipped applicator. The heart blood was collected by cardiac group). Three groups were treated by intraperitoneal injection puncture. Then each sample was streaked onto four different with 100 l suspension of each isolated strain in 1×PBS blood agar plates. That were Brian Heart Infusion (BHI) at the doses of 2×105 CFU/ml, 2×106 CFU/ml and 2×107 (bacteria), beef extract peptone (bacteria), martin (fungi) and CFU/ml. The control group was injected with 1×PBS. All gauze’s medium no.1 (antinomyces) plates, separately, then symptoms and death numbers were recorded in six days after 0 cultured at 28 C with or without 5% CO2 for three to five inoculation. Using the SPSS software, the 50% lethal dose days till the colonies were observed. All the colonies were (LD50) with 95% confidence limits was calculated by probit re-streaked onto new plates for repurification. analysis of Bliss (Bliss, 1953). For microscopic observations, Gram stain Drug sensitivity test: Antimicrobial susceptibility was procedure was performed to distinguish the bacterial isolates tested by the Kirby-Bauer disk diffusion method (Bauer et into Gram-positive or Gram-negative bacteria. al., 1996) according to Clinical and Laboratory Standards 16S rRNA sequencing: Single colony was Institute guidelines (WS/T-125-1999). Briefly, 100 l inoculated in nutrient broth and grown overnight. The overnight liquid culture of pathogenic strain was uniformly Genomic DNA of 1.5 ml broth culture was isolated and spread on agar medium, and drug-sensitive discs (OXOID, purified by using bacterial genomic DNA kit (Aidlab, China). UK) were placed with at least 30-mm intervals on medium. DNA fragment (1.5 kb) of 16S rRNA gene was amplified The inhibition zone diameters were examined after from genome with universal primers (Forward primer 27F: incubation for 24 h at 28 °C. Large zones of inhibition 5'-AGAGTTTGATCCTGGCTCAG-3' and reverse primer indicate that the organism is susceptible, while small or no 1429R: 5’-GGTTACCTTGTTACGACTT-3’) (Weisburg et zone of inhibition indicates resistance. Escherichia coli al., 1991). PCR reaction was done by initial denaturation at (ATCC 25922) and Staphylococcus aureus (ATCC25923) 94 °C for 3 min, followed by 25 cycles of denaturation at served as controls. 94 °C for 30 second, annealing at 55 °C for 30 second and RESULTS AND DISCUSSION extension at 72 °C for 1 minute. The final extension was Culture and morphology characteristics of isolates: Eight conducted at 72 °C for 10 minutes. The 1.5 kb PCR product different colonies were obtained from the viscera tissues of (fig 1) was purified and ligased with T-vector for sequencing two dead Siamese crocodiles in three days by using streaking at Sangon Biotech Company (China). The bacterial species plate methods. All the eight strains can be grown and formed typical bacteria-like smooth colonies, on the plates of BHI, Vol.

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