| Acute hemorrhagic syndrome of Chinese soft-shelled turtles, Pelodiscus sinensis, occurred in pond cultivated turtles farms in Zhejiang province since2003, was a new out-breaking disease, with the symptoms of red plaque/spot on plastron and forelimbs, swelled neck and limbs, melanotic lungs, hemorrhagic swelled mosaic liver, serious hyperaemia and haemorrhagia or necrosis on mucosa of throat, intestine and stomach, and hyperaemia erosion on ovary of female turtles. This disease, though not so common like other turtle diseases, can bring high mortality to diseased farms, causing very serious lost.The current studies are on etiological agent of acute hemorrhagic syndrome of Chinese soft-shelled turtles, with isolation, identification, pathogenicity tests and PCR detection of bacteria, virus and mycoplasma. Twelve Chinese soft-shell turtle samples with typical acute hemorrhagic syndrome were collected during2006-2012. The dominant bacteria colonies were picked up after reclones for2-3times and used for16s rRNA gene PCR with the universal primers. The bacteria isolates were identified as Aeromonas sp, Edwardsiella sp. Proteus sp, Lactococcus sp and Salmonella sp. All isolates were used for injection challenging with healthy turtles after cultured for16-24in trypticase soy broth (TSB) Aeromonas and Salmonella isolates show more strong virulence with the LD501.47-5.18×107CFU/ml for Aeromonas, with the similar symptoms like native diseased turtles. Turtles injected with Edwardsiella sp, Proteus sp, Lactococcus sp showed no virulence ti turtles.The virus suspension was prepared by throughout homogenizing viscera from moribound turtles, treated with penicillin and strepotocmycin at4℃overnight, followed by filtration with0.2μm filter membrance. The bacteria-free suspension was injected intramuscularly to healthy turtle at the dilution of1:10、1:20、1:100. Three of twelve samples can develop typical symptoms similar with the native diseased turtles. The viral virulence can be passaged for5generations with the continuous injections. The primers of iridovirus, herpesvirus were used for virus detection for Chinese soft-shelled turtles with typical symptoms. Iridovirus was detected in three samples, no correlation was found with the viral challenging and PCR results. EM observations reveal.the irido-like virions in sample YH06-1and WL12-1. The bacteria-free virus suspension was used for virus culture in three fish cell lines, EPC, CIK, CO. No cell cytopathic effects(CPE) were observed after five generations of viral passages. The universal primers of mycoplasma were used for mycoplasma PCR detection, with two positive turtle samples, YH06-1and SX12-1, but mycoplasma cannot be isolated from the positive samples because failuar of bacteria inhibition by thallium acetate.From the results of different pathogens research, it could be concluded that the etiological agent for acute hemorrhagic syndrome of Chinese soft-shell turtles may not be a single pathogen but caused by multi-pathogens and stress, similar to rhinitis-stomatitis complex, a common serious disease reported in tortoise and turtles.A Salmonella spp SX12-1was isolated and confirmed as the possible causative pathogen of which caused serious disease in Shaoxing in Sep.,2012. Challenged with injection of health turtles by cultivated Salmonella spp.at the dose of1×107CFU/ml、1×108CFU/ml、1×1109CFU/ml, the sick turtles were found4days after injection,with the symptoms swelled neck, hemorrhagic swelled mosaic liver, serious hyperaemia and haemorrhagia on mucosa of intestine and stomach. LD50of SX12-1strain was3.16×106CFU/ml. Rabbit antiserum against Salmonella spp SX12-1was prepared and the double-antibody Sandwich ELISA was developed with high sensitivity and good specificity, with the minimum detection dosage of1×104CFU/ml. It could be considered as a potentiall method for Salmonella spp. |
PDF Full Text Request