| Contagious caprine pleuropneumonia (CCPP) is a highly contagious respiratory infectious disease, originated from Mycoplasma capricolum subsp. capripneumoniae (Mccp). Typical cases of CCPP are characterized with extreme fever, high morbidity and mortality, with lesions of pleuritis, croupous pneumonia and interstitial pneumonia. CCPP has caused great damage to goat farming of many African and Asian countries, been listed as a notifiable infectious disease by World Organisation for Animal Health. Epidemics and dead cases of CCPP in wild goats are very rare up to now.Feline viral rhinotracheitis (FVR) is a contagious upper respiratory infectious desease caused by feline herpesvirus type1(FHV-1), occurring in many countries besides China. Dead cases may be seen particularly in neonatal kittens. But none dead case, isolation and identification of FHV-1in South China tigers has been reported previously.From2012to2013, an incident happened to Tibetan antelope with abundant dead cases in Naqu district of Tibet, which was confirmed as CCPP for the first time worldwidely, by a number of methods including transmission electron microscopy, histopathological examination, special PCR/RT-PCR, phylogenetic analysis, isolation and identification of the pathogens, and et al. An epidemiological survey and risk analysis was conducted subsequently, whose results indicate that CCPP had emerged probably as early as in2006, and that the risk for CCPP to be endemoepidemic either in Tibetan Antelopes or in goats, to transmit between them, is very high.In addition, a fatal case of South China tiger was diagnosed as FVR by similar assays. The research contents and results are as follows.1. Field vetrinary survey and preliminary diagnosis of the suspected epidemics in Tibetan antelope.1.1. By questionnaire and field veterinary survey, unexplained pneumonia cases in domestic goats and in wild Tibetan antelopes were recorded, which had very similar symptoms with CCPP. Furthermore, the number of Tibetan antelope in Shenzha, Shuanghu, Nima and Bange county, Naqu district, Tibet, was statisticed.1.2. In the clinical examination for the dead Chirus, the symptoms with effluence of white fluid spumescence were observed, and necropsy was performed on13of the antelope at sites within the Shenzha, Shuanghu, and Nima localities of the Naqu area. Gross pathologic lesions were localized exclusively to the lung with severe pleuropneumonia and partial hepatization in lung. The lungs of affected antelopes displayed a thickening of the interlobular septa, pleuritis, and an accumulation of straw-colored pleural fluid. The pleural exudate solidified to form a gelatinous covering on the lung. Smaple of lung of total13antelopes were collected. 1.3. Samples of lung tissue from5of the antelope were selected for histologic examination. Four of the samples showed fibrinous pneumonia with serofibrinous fluid and an inflammatory cell infiltrate consisting mainly of lymphocytes in the alveoli and bronchioles. One sample showed pulmonary edema with a protein-rich fluid effusion in alveoli. Electron micrograph of lung tissue of the SH3dead Chiru, indicated plemorphic mycoplasma-like particles appearing as short rods and spirals with diameters ranging from100nm to300nm.1.4. The lung samples from each of the13Tibetan antelope were screened by PCR for evidence of M. mycoides cluster, with specific pimers of16s rRNA gene, and the results of agarose gel electrophoresis indicated that a785bp target fragment was amplified from11samples. The11clones were sequenced and blasted. The partial16s rRNA sequence had higher sequence identity (99.8%) between the11clones, had sequence identity (more than99.2%) with Mm cluster, and had the highest identity with Mccp isolates. Then a316bp target fragment was amplified from each of11samples by PCR with specific pimers targeted for arcD gene of Mccp.The author concluded preliminarily here, the incident with abundant dead antelopes was diagnosed as CCPP caused by Mccp, on the basis of above results.2. Isolation and identification of Mccp and confirmation of the high lethality of Mccp to Tibetan antelope.2.1. Lung tissue from each of the13antelopes was minced and inoculated into modified Hayflick broth, and11of13samples showed growth of mycoplasma after2-3passages in culture.2.2The presence of mycoplasma-like particles in the11growth-positive cultures was confirmed by electron microscopy indicating plemorphic mycoplasma-like particles appearing as short rods, and by PCR for evidence of M. mycoides cluster and M. bovis, with results that316bp and562bp fragment were amplified seperately targeted for arcD gene and H2gene of Mccp. H2PCR products of SH3culture was extracted, cloned, sequenced and Phylogeneticly analyzed. The partial H2sequence had higher sequence identity with Mccp isolates (99.3%～99.7%) than with Mcc isolates (90.2%～91.2%), demonstrating that the Mccp isolated from infected Tibetan antelope belongs to the same clade with Mccp strains isolated in Africa and Asia.2.3. By PCR or reverse transcription PCR, lung samples from the13dead Tibetan antelope were also tested for an additional16potential pathogens besides BTV, MVV, CAEV, FMDV, RPV, BPIV, Cb, Cw, Ps, Mb, Ml, MmmLC/Mmc, Mcc, MmmSC and Movi. No pathogens other than Mccp were detected.Thereby, the author had enough evidences to give the conclusion that11Mccp isolates were successfully obtained, and that Mccp was the sole pathogen being highly fatal to Tibetan antelopes, causing the outbreak of CCPP in Naqu area.3. The epidemic characteristics and outbreak risk of CCPP in Tibetan antelope.3.1. CCPP outbreaks were confirmed in Tibetan antelopes(200dead Tibetan antelopes in Naqu district and dometic goats (500dead dometic goats in Ali district) in2013. Sequencing results indicated that the partial arcD gene sequence had high identity between them, and had identity of99.4%～99.8%with other Mccp isolates.3.2. The statistic results show, that CCPP had continued for505days with some interval, that CCPP had caused2869wild ruminants dead belonging to2subfamilies and4genera, and that the dead cases were found in26sites of10towns of4counties.3.3. The analysis of epidemic characteristics indicates, that mortality in Tibetan antelope population is up to18.92%, its dead case number being2648with91.54%in the total, and that almost all epidemic sites dispersed aroud Selincuo lake with ratio of84.04%in the total cases found within the irregular-cycled area. The epidemic CCPP demonstrated a attenuating trend but could be divided into two periods with obvious differences between them. The female-male ratio of all identifyed cases is68:32.3.4. Our results suggested that CCPP had transmitted to domestic goats in Ali, and to Chirus in Naqu since2006, based on the epidemic characteristics, the historic distribution, the information gained by questionare and sample detecting. Argali is most possible host of Mccp. The outbreak of CCPP may be result of synergistic effect originating from intense weather change in September and epidemic risk factors, including goat introduction and transferring, overloading grazing and migration of Chirus. For CCPP, pandemic risk is low, but suffused risk into the neighbouring areas even the neighbouring provinces, and endemic risk in dometic and wild goats, is higher.3.5. About preventive and control measures, the author’s suggestion was conducting researches on inactivated vaccine of CCPP for the dometic goats, deep-going Epidemiological survey and oral vaccine with drinking for the wild ruminants. Futhermore, strengthened quarantine in the habitat, strengthened monitoring of wildlife population, and strengthened management of CCPP as a notifiable disease either for MO A or for SFA should be performed as soon as possible.4. Isolation and identification of FHV-1from South China tiger and epidemic risk assessment of FVR.4.1. Trachea specimen of a dead South China tiger from Shenzhen Wildlife Zoo, was screened for three potential pathogens including FHV-1, CDV/FeDV and FCV, by specific PCR/RT-PCR. The result of agarose gel electrophoresis indicated that only a292bp targeted for TK gene fragment of FHV-1was amplified, whose positive clone was sequenced and blasted. A partial253bp sequence was obtained, and alignment analysis determined that the TK gene shared a high identity with that of other FHV-1isolates. A566bp fragment of the gB (glycoprotein B) gene was found to share100%identity with that of other FHV-1isolates.4.2. FK81cell cultures showed a distinct cytopathic effect, at72h post-inoculation with trachea homogenates. The electron micrograph of cell cultures revealed circular or oval herpesvirus-like particles. Furthermore, PCR/RT-PCR showed that the supernatants of cell cultures were positive only for FHV-1.4.3. Challenged cats exhibited sneezing and increasing palpebral secretion after6d post-inoculation, obvious signs in upper respiratory, changes in the cat’s body temperatures generating a curve with a single peak, and virus shedding in the infected cats from the6th to20th after inoculation.4.4. A retrospective investigation as well as epidemic risk assessment was conducted, and negative PCR results were found for the swab samples collected from stray cats and captive leopards and tigers. That indicated widespread infection was unlikely to occur in the zoo, but sporadic’cases in felids are of high risk.4.5. To better prevent the endangered South China tiger suffering from FVR, strengthened management on breeding, normalizing disease control measures, drug storage for emergence, precisely survey in more zoos and risk management are suggested. Vaccination in tiger needs deep consideration, to the author’s knowledge.In this part, a FHV-1strain fatal to South China tiger was isolated and identified, the risk of sporadic cases even dead cases in endangered population by FVR was pointed out, and preventive&control measures were also suggested.In conclusion, the laboratory confirmative diagnosis, epidemiological survey and risk analysis, on twice CCPP outbreaks in freely ranging Tibetan antelope populations and on FVR case in a breeding South China Tiger, were conducted respectively in this study. The results and findings are important for State Forstry Administration to formulate preventive and control measures, and to protect the endangered wildlife. |
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