The Preliminary Epidemiological Studies Of Common Diseases Ingolden Snub-nosed Monkeys At Shennongjia

The golden snub-nosed monkey(Rhinopithecus roxellanae, R. roxellanae) is one of the rare and precious wild animals of China, and it has been classified as class A protected(a top conservation priority) and endangered(EN) animals in the IUCN Red List. is the most east edge where the golden snub-nosed monkeys were found. There are approximately 1200 golden snub-nosed monkeys in Shennongjia. Among them, 80 free-ranging monkey in Da Longtan; while 9 captive monkey in Xiao Longtan. Epidemic diseases represent one of the major threats to the survival of these animals. Therefore, it is significant to determine epidemiology of the pathogens that potentially affect R. roxellanae so than the corresponding preventative measures could be taken in advance against these pathogen related diseases to actively protect this precious monkey species.In this study, we did the preliminary epidemiological studies of the common diseases based on the current knowledge on other monkey species, including tuberculosis, diarrhea related pathogens and infection of 9 common viruses in golden snub-nosed monkeys at Shennongjia National Nature Reserve. 1. Establishment and preliminary application of an IFN-γ in vitro release assay to detect monkey tuberculosisThis study was aimed to establish an IFN-γ in vitro release assay to detect monkey tuberculosis. By using hybridoma technology, 12 monoclonal antibodies with high titers against Macaca mulatta IFN-γ(Mm IFN-γ) were generated. A sandwich ELISA was established by using one of monoclonal antibodies 3H3 and the rabbit polyclonal antibodies against Mm IFN-γ. The sensitivity of this ELISA was 62.5pg/m L. The results were compared with those detected by commercial Mm IFN-γ detection ELISA kit and the agreement of these two methods was 93.84% with k value of 0.7800.Then 146 rhesus macaques were tested for tuberculosis by using eyelid tuberculin intradermal skin test(TST). By using this TST as the reference method, the commercial Mm IFN-γ detection ELISA kit was used to detect IFN-g in vitro release in the peripheral blood after the whole blood was stimulated with tuberculin of Mycobacterium bovis(b PPD) and M. avium(a PPD). The ROC curve was used to determine the cutoff value which was 114.5 pg/m L with sensitivity of 68.18% and specificity of 91.94% in diagnosis of macaques tuberculosis.By using the Mm IFN-γ in vitro release assay, we also detected 10 golden snub-nosed monkey whole blood samples. The mitogen Con A stimulation was used as positive control. As a result, the whole blood of both Macaca mulatta and golden snub-nosed monkey produced similar levels of IFN-γ which Macaca mulatta was 1050±278.0 pg/m L and golden snub-nosed monkey was 1512±166 pg/m L after stimulation of Con A. It indicated that the Mm IFN-γ in vitro release assay could be used to detect IFN-γ of golden snub-nosed monkeys. However, the IFN-γ levels for PPD and nil stimulation were less than 100 pg/ml, below the cutoff value of 114.5 pg/m L and there is no statistically significant difference between PPD and nil stimulation. Therefore, all the 10 golden snub-nosed monkeys were negative to TB.In conclusion, this study established an IFN-γ in vitro release assay to detect tuberculosis of macaque and golden snub-nosed monkey. 2. Isolation and identification of the diarrheagenic pathogens from golden snub-nosed monkeys with diarrhea at Shennongjia National Nature ReserveThe aim of the current study was to isolate and identify pathogens from the feces of wild snub-nosed monkeys(Rhinopithecus roxellanae). The 76 fecal samples, including 8 diarrheic feces, from 38 Rhinopithecus roxellanae at Shennongjia national nature reserve(Hubei, China) were collected. The detected pathogens included diarrheagenic Escherichia coli, Salmonella and Shigella. As a result, only 2 enteropathogenic Escherichia coli(EPEC) strains were isolated from the diarrheic feces. The analysis with standard antisera identified 2 EPEC strains to be serotype O98. The detection of toxic genes of bfp B, stx1, and stx2 determined these two EPEC strains to be atypical EPEC(a EPEC)(bfp B-, stx1-, and stx2-) and belonged to the phylogenetic group D(Tsp E4C2+, chu A+). In addition, these strains had virulence marker genes eae+, esc V+, intimin b+. The drug susceptibility was tested and and they were highly sensitive to aminoglycosides, fluoroquinolones(ciprofloxacin), and sulfonamides(cotrimoxazole), but surprisingly resistant to the third generation cephalosporin(cefotaxime, ceftazidime) antibiotics which were supposed to be sensitive to E.coli. In addition, between the 2 EPEC strains, strain 1 showed resistance to Tobramycin, Meropenem, Imipenem,while strain 2 were susceptible to them. The LD50 of strain 1 and 2 was 1.31×108(CFU), and 5.26×107(CFU) respectively, indicating a low virulence in mice.In conclusion, this study firstly identified 2 a EPEC strains from 2 wild R. Roxellanae with diarrhea. These strains had a low virulence and highly resistant to the third generation cephalosporin antibiotics. The current study could be significant for further development of preventative measures against potential diseases both in R. Roxellanae and humans. 3. Prevalence of common viral infections in golden snub-nosed monkeys at Shennongjia National Nature ReserveThis survey was conducted to discover the prevalence of common diseases in the wild and captive golden snub-nosed monkeys at Shennongjia National Nature Reserve, China. The blood samples were collected from 10 golden snub-nosed monkeys and the sera were used to detect the antibodies to nine viruses including herpesvirus type 1(B virus, BV), rhesus cytomegalovirus(Rh CMV), simian type D retroviruses(SRV), simian T-cell lymphotropic virus type 1(STLV-1), simian immunodeficiency virus(SIV), simian foamy virus(SFV), simian varicella virus(SVV), hepatitis A virus(HAV) and coxsackievirus(CV) with the commercial kits. As a result, the seroprevalence for BV was 60%(6/10), the highest in all samples; followed by Rh CMV whose seroprevalence was 20%(2/10). No samples were positive for the other 7 viruses. Thus the infection of BV and Rh CMV would be the main threat to the health of golden snub-nosed monkey population at this area and possible the human health.