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,

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 (Rana rugulosa or 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, , East , report the etiological investigation of the unexpected , , Burma, and 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 , 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 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 Chinese edible 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. Issue , () beef extract peptone and gauze’s medium no.1 with or The further observation showed that all the groups without blood. But none of the colonies developed in martin were mouthful mucus, swelling abdomen, bleed spots in the medium. The result of gram stain assay showed that seven skin by inoculation with E. tarda isolates. After autopsy, it strains were rod and gram negative, and one is globe and was exhibited that the liver, lungs and kidney were distinctly gram positive. congestive, and gastrointestinal tract was inflated greatly. Species identification of isolates: The Eight strains were These data suggested that the E. tarda was the most potential classified in four genus groups, six species by BLAST pathogen in this survey. In addition, though few frogs were analysis of 16S rRNA gene sequences. The dominant and dead in the groups treated with C. freundii, M. luteus and P. communal species is E.tarda, which can be isolated from hauseri, it was not considered in statistical significance. both of the heart bloods and visceral tissues from the checked Drug sensitivity analysis of E.tarda: The three E. tarda dead baby crocodiles, and three strains were obtained. Others strains were trailed for sensitivity and resistance against 20 five strains, Citrobacter freundii, Enterobacter asburiae, selected antibiotics. The results (Table 2) showed that the Proteus hauseri, Proteus penneri and Micrococcus luteus, sensitivities of the three strains were divergent. Strain CSet03 were isolated either from the liver or from the spleen. M. luteus is resistant to eight antibiotics. CSet08 is resistant against was minimally isolated and the only gram positive strain. 10 antibiotics, and CSet04 has resistance against over 14 Virulence of isolates antibiotics. So the resistance is CSet04> CSet08 All the three E.tarda isolated strains: CSet03, CSet04 and >CSet03.The order is same as the virulence of the strains. CSet08 were obviously pathogenic after intraperitoneal Seven antibiotics were concurrently no suppressive to all injection of the frogs compared with the others. Median lethal the three strains. Only three antibiotics out of the 20 tested drugs, that are ceftazidime pentahydrate, norfloxacin dose (LD50), a dose at which half the frogs are killed, was determined to evaluate the in vivo toxicity. The values of baccidal and amikacin, were high effective to the three E. tarda isolated strains. LD50 were determined by the graphical calculation method 5 of Bliss (Table 1). For CSet03, the level of LD50 is 2.0×10 Crocodiles are ancient, large, vital and rare wild 4 CFU; for CSet04, the level of LD50 is 5.7×10 CFU; for animals, whose ancestors have roamed the earth since the 4 CSet08, the dose of LD50 is 7.5×10 CFU. So the virulence time of the dinosaurs. In order to produce meat, leather, and is CSet04> CSet08 >CSet03. other goods from crocodiles and alligators, many species of

Table 1: Artificial infection tests of the isolated strains strains Dose(CFU) Number of death (four frogs per group) Mortality rate% LD50 1d 2d 3d 4d 5d 6d CSet03 2×106 0 2 1 0 0 0 75 2.0×105 2×105 0 0 1 1 0 0 50 2×104 0 0 0 1 0 0 25 CSet04 2×106 0 2 1 1 0 0 100 5.7×104 2×105 0 1 1 1 0 0 75 2×104 0 0 1 0 0 0 25 CSet08 2×106 0 1 2 1 0 0 100 7.5×104 2×105 0 0 1 1 0 0 50 2×104 0 0 1 0 0 0 25 CScf05 2×106 0 0 0 1 0 0 25 - 2×105 0 0 0 1 0 0 25 2×104 0 0 0 0 0 0 0 CSea02 2×106 0 0 0 0 0 0 0 - 2×105 0 0 0 0 0 0 0 2×104 0 0 0 0 0 0 0 CSph01 2×106 0 0 0 1 0 0 25 - 2×105 0 0 0 0 0 0 0 2×104 0 0 0 0 0 0 0 CSpp06 2×106 0 0 0 0 0 0 0 - 2×105 0 0 0 0 0 0 0 2×104 0 0 0 0 0 0 0 CSml07 2×106 0 0 1 0 0 0 25 - 2×105 0 0 0 0 0 0 0 2×104 0 0 0 0 0 0 0 Note: CS:C. siamensis; et: E. tarda; cf: C. freundii; ea: E. asburiae; ph: P. hauseri; pp: P. penneri; ml: M. luteus. 4 INDIAN JOURNAL OF ANIMAL RESEARCH Table 2: The antibiotic sensitivity test of the E. tarda isolates Antibiotics Conc (g/piece) Inhibition zone diameter (mm) /Antibiotic sensitivity CSet03 CSet04 CSet08 Ceftazidime Pentahydrate 30 23/S 24/S 25/S Norfloxacin Baccidal 10 28/S 17/S 23/S Amikacin 30 18/S 21/S 20/S Norfloxacin 5 24/S 16/I 17/S doxycycline 30 18/S 0/R 17/S Amoxicillin 20 18/S 0/R 0/R Ofloxacin for Injection 5 25/S 0/R 0/R Enrofloxacin 5 25/S 0/R 0/R spectinomycin 100 17/I 15/I 16/I Cefazolin 30 14/I 0/R 13/I Kanamycin 30 16/I 13/R 16/I streptomycin 10 13/I 0/R 0/R Gentamicin 10 12/R 14/I 14/I neomycin 30 2/R 12/R 15/I Carbenicillin 100 11/R 16/R 0/R Erythromycin 15 13/R 0/R 11/R Tetracycline Tablets 30 0/R 0/R 0/R Penicillin 10 0/R 0/R 0/R Lincolmensin 2 0/R 0/R 0/R Cephalosporin VI 30 0/R 0/R 0/R Note: S: Sensitive; I: Intermediate; R: Resistant. both crocodiles and alligators have been farmed since the characteristics, E. tarda is divided into three groups: early 20th century. But the commercial operations were truly biogroup 1, biogroup 2 and wild type (Ewing et al., 1965; raised up in the 1960s, when wild alligators were hunted Grimont et al., 1980; Walton et al., 1993). Only the bio group nearly to extinction, and many species were placed under 1 is mostly associated with zoo animals, such as reptiles and official protection by that time. birds; the wild type strains are principally found in human Importing and Farming crocodile first grew up in and fish; biogroup 2 strains have only been isolated from China with the demand for skins since 1993 (Erhu et al., humans (Alcaide et al., 2006). 2005), which can fetch hundreds of dollars in international At present, it is still not directly proved that E. tarda market. Currently, the breeding areas have been distributed can cause disease among crocodiles and alligators. The in Guangdong, Hainan, Fujian, Anhui, Hubei, Zhejiang and reason may be associated with the people’s low concern, Shanghai in China. However, few crocodilian businesses are uneasy accomplishment of the artificial infection to crocodile successful, due to the low reproduction rate, high morbidity and alligator, owing to rarity, expensiveness and and mortality with poor raising technique and infectious aggressiveness. For example, one captive crocodile was dead diseases. Some statistical data revealed that the death rate in Beijing Zoo in 2013. The main isolates were E. tarda of Nile crocodile, Estuarine crocodile and Siamese crocodile from the autopsy, according to the investigation of China were 16%, 7.85% and 9.13% in captivity, respectively University of Agriculture, but the results were not published (Haiyan et al., 2011). So, it is important to improve the till now for no crocodile available for experimental study. feeding level and drop the incidence of unnatural death by Similarly, New Zealand Ministry of Agriculture and Forestry strengthening the research on biological habit and disease reported that E. tarda was identified from crocodiles with control of captive and farmed crocodiles. “non-specific” septicemia in personal communication in In this report, E. tarda is the predominate and the 2007 (New Zealand Ministry of Agriculture and Forestry, most virulent species among the isolated strains, which is a 2007), and considered that E. tarda is a potential hazard in worldwide distributional gram-negative enterobacterium, the commodity. But for the same reason, there is no formal predominantly in fresh and marine water, mud, and the and public paper published. intestine of fish and other marine animals. As a zoonosis, E. Mouse is the most common experimental animal tarda can infect mammals and a variety of animals, which in medical and pharmaceutical research. But in this study, inhabit the water environment, including fish, bird, we tried to use easily purchased and cheaper Chinese edible and reptiles, and causes Edwardsiella septicemia, frog as a model for crocodile infection testing, considered which often was associated with septic shock and high of the closer relationship between frog and crocodile than mortality. Based on the physical and biochemical mice. Notably, in pre-experiment, we found that the frogs Vol. Issue , () can bear starving for several weeks. So, fasting was adopted molecular mechanism probably be shared between in the virulence assay to avoid the extra pollution produced antimicrobial resistance and bacterial virulence in E. tarda. by foods and feces. Simultaneously, the high multiple drug resistance of the three In fact, as an enteric opportunistic pathogen, only stains also illustrated that resistance had accelerated by the the adhesion, invasion and even migration occur, E. tarda is overuse of antibiotics in current breeding. In this case, the able to cause disease (Wang et al., 2005). Furthermore, antibiotics: Ceftazidime pentahydrate, norfloxacin and ascites is one of typical symptoms in some infected fishes, amikacin were recommended to be prescribed. And to such as yellow catfish (Zhengfeng et al., 2008). So, in this prevent the recurrence of death, maintaining appropriate case, the hint of massive ascetic fluids and the isolation of raising number of crocodiles in one habitat, and keeping E. tarda in viscera and blood were in good agreement with clean, sanitary aquatic environment also were advised. Since the above infection process and clinical symptoms, so E. then, three years past, no such case has taken place again in tarda was strongly suggested the cause of sudden death this winter and spring. time. ACKNOWLEDGEMENTS Integrating the results of the virulence and drug This work is supported by grants from the National sensitivity test, we found that the virulence of the isolated E. Natural Science Foundation of China (31460699) and tarda strains from strong to weak is CSet04, CSet08 and Scientific Research Foundation for Returned Scholars, CSet03, and the number of resistant antibiotics from most Ministry of Education of China [Department of International to lest is the same order. This consistency indicated that some Cooperation and Exchanges (2014) 1685].

REFERENCES Alcaide, E., Herraiz, S. and Esteve, C. (2006). Occurrence of Edwardsiella tarda in wild European eels Anguilla anguilla from Mediterranean Spain. Diseases of aquatic organisms, 73: 77-81. Bauer, A.W., Kirby, W.M.M., Sherris, J.C. and Tuck, M. (1996). Antibiotics susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol, 45:493–496. Bedell, M.A., Largaespada, D.A., Jenkins, N.A. and Copeland, N.G. (1997). Mouse models of human disease. Part II: recent progress and future directions. Genes Dev, 11: 11-43. Bliss, C.I. (1953). The calculation of the dosage-mortality curve. Ann Appl Biol, 22:134–167. Erhu, G. and Yue, Z. (2005). The status and developing measures of alien crocodile breeding in China. Forest resources management, 4:51-54. Ewing, W.H., McWhorter, A.C., Escobar, M.R., Lubin, A.H. (1965). Edwardsiella, a new genus of Enterobacteriaceae based on a new species, E. tarda. International Journal of Systematic and Evolutionary Microbiology, 15: 33-38. Grimont, P.A., Grimont, F., Richard, C. and Sakazaki, R. (1980). Edwardsiella hoshinae, a new species of Enterobacteriaceae. Current Microbiology, 4: 347-351. Haiyan, C and Denghua, Z. (2011). Discussion on crocodile breeding techniques and prospect analysis. Aquaculture (China), 12:10-14. Lieschke, G.J. and Currie, P.D. (2007). Animal models of human disease: zebrafish swim into view. Nat Rev Genet, 8:353-67. New Zealand Ministry of Agriculture and Forestry (2007). Import risk analysis: Crocodilia and eggs of Crocodilia from Australia. IMAF Biosecurity New Zealand, ISBN: 978-0-478-31101-3 (Print) ISBN: 978-0-478-31102-0 (Online). Simpson, B.K. and Bezuijen, M.R. (2010). Siamese crocodile Crocodylus siamensis. Crocodiles status survey and conservation action plan. 2nd ed. Darwin: Crocodile Specialist Group, pp: 120-126. Walton, D.T., Abbott, S.L. and Janda, J.M. (1993). Sucrose-positive Edwardsiella tarda mimicking a biogroup 1 strain isolated from a patient with cholelithiasis. Journal of clinical microbiology, 31: 155-156. Wang, I.K., Kuo, H.L., Chen, Y.M., Lin, C.L. and Chang, H.Y., Chuang, F. R., and Lee, M. H. (2005). Extra intestinal manifestations of Edwardsiella tarda infection. International Journal of Clinical Practice, 59: 917-921. Warkentin, I.G., Bickford, D. Sodhi, N.S. and Bradshaw, C.J. (2009). Eating frogs to extinction. Conserv Biol, 23:1056-9. Weisburg, W.G., Barns, S.M., Pelletier, D.A., Lane, D.J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol, 173: 697–703. Zhengfeng, D., Hui, X., Wenyi, B., Liping, L. and Guanghong, W. (2008). Pathogenic bacteria causing ascites disease of cultured yellow catfish (pelteobagrus fulvidraco). Journal of Huazhong Agricultural University (China), 05: 639-643.