Investigation Of A Wild Juvenile Giant Panda’s Death In Wolong Nature Reserve

Wolong Nature Reserve is located in Sichuan Province,China,covering an area of2,000 square kilometers.It is hometown to many wild animals,including giant pandas.There are ～149 wild giant pandas in Wolong Nature Reserve,accounting for ～8% of the total number of wild giant pandas in China.In addition,Shenshuping Base of China Giant Panda Protection and Research Center located here,which has dozens of captive giant pandas.There are also a large number of other wild animals in Wolong Nature Reserve,including tufted deer,sichuan golden monkey,samba deer,long tailed impala,andantelope.Studies have shown that Canine Parvovirus(CPV)can infect a variety of wild animals,including giant pandas,leading to hemorrhagic enteritis.As an important species resource in Wolong Nature Reserve,the survival and reproduction of wild animals are closely related to the development of Wolong Nature Reserve.Canine Parvovirus infection can cause serious acute infectious diseases in wild animals,thereby affecting the ecological stability of Wolong area.Inthisstudy,we performed autopsy,pathological analysis,pathogen isolation and death cause analysis on a dead juvenile wild giant panda carcass came from Wolong,Sichuan province,and carried out epidemiological investigation and phylogenetic analysis of canine parvovirus in Wolong Nature Reserve and surrounding cities,which provides some basic data for the prevention and control of canine parvovirus disease in Wolong Nature Reserve.Experiment 1.Anatomical and histological examination of a dead juvenile wild giant panda in Wolong Nature ReserveIn November 2020,a dead juvenile wild giant panda was found during the routine field inspection in the reserve.We conducted the appearance examination,anatomical observation and histopathological examination on the dead juvenile giant panda.The results showed that the cub died at Niutoushan,Wolong Nature Reserve,Sichuan,China,with an altitude of 2817 m and latitude 103°12’ N,longitude 31°0’ E.The panda cub was male,weighing 1.215 kg,with a body length of 26 cm,a tail length of 5.5 cm,a head length of 11 cm,a forelimb length of 11 cm,and a hind limb length of 10 cm.The mouth and nose were clean and free of foreign bodies.It had not opened its eyes and was about 1 month old.The body had messy fur,without trauma.However,diarrheal stool was present perianal.With gross anatomy detection,there were congestion in the right abdomen,bleeding in the right lung,partial indentation in the liver,multiple congestion andexpansion in the intestinal tract.After dissection,the parenchymal tissues and organs were collected for pathological examination.The cardiac muscle bundle was broken and dissolved,the intermuscular was widened with a small number of macrophages and neutrophils.Less white pulp compared with normal were found in spleen.Pulmonary capillary congestion was significant,and alveolar wall thickened without obvious inflammatory infiltration.A large number of neutrophils were infiltrated in the intestinal lymph nodes,while the whole intestinal mucosa was necrotic and dissolved.Protein cast,with obvious expansion of renal capsule were found in kidney.There were a few macrophages and neutrophils in thymus.The variant epithelium of bladder was intact,without obvious histological lesions.The pathological results indicated myocarditis,compensatory emphysema,acute enteritis and immune insufficiency.Experiment 2.Etiological examination of intestinal infectious diseases in the juvenile wild giant panda death case.Since the pathology illustrated that the panda had intestinal inflammation,we further used colloidal gold and PCR methods to detect common infectious intestinal pathogens of giant panda,including Canine Parvovirus(CPV),Rotavirus(RV),Canine Distemper virus(CDV),Canine Adenovirus(CAV),and Canine Coronavirus(CCV).The results were positive for CPV and negative for CDV,CCV,RV,and CAV.Combined with the results of gross anatomy and histopathology,the main cause of death of the wild juvenile giant panda was systemic sepsis caused by CPV infection.CPV isolated from the juvenile panda had 426 Glu in the VP2 protein,so it was identified as CPV-2c.it also had three amino acid mutations(F267Y,Y324 I and Q370R).This is the first study that investigated CPV-2c parvovirus infection in giant pandas.Genetic evolution analysis found that thepanda isolated CPV-2c was clustered with CPV-2c isolated from China and other Asian countries,far away from the European strain,suggesting that the cause of infection of the dead giant panda may be caused by the transmission of native animals.Experiment 3.Investigation of canine parvovirus in the feces of wild animals in Wolong Nature ReserveCPV infection is the main cause of death in this juvenile panda,illustrating that a serious CPV epidemic may have occurred in Wolong Nature Reserve.The pathogen tracing and transmission risk assessment are the key points for further research.We immediately screened 72 fecal samples of wild animals in Wolong Nature Reserve collected and preserved by the laboratory from January to November 2020,and 62 fecal samples of wild animals newly collected in December 2020 by PCR.All the wild animal fecal samples preserved in the laboratory were negative for CPV,but 9 of the newly collected wild animal feces were CPV positive(the positive rate was 14.06%,the positive animals were giant panda,leopard cat and badger),indicating that the CPV epidemic in Wolong Nature Reserve developed rapidly and the rate of CPV pathogens carrying in wild animals was high.According to the sampling location analysis,it was found that the positive samples were close to the human gathering areas,especially the G350 National Highway,and there was a possibility of mutual transmission with domestic and stray dogs.Experiment 4.Epidemic investigation and evolutionary analysis of canine parvovirus in Wolong Nature ReserveWith beautiful environment,high altitude and cool climate,Wolong has always been one of the best places for Chengdu people to spend summer holidays.In Experiment 3,it was found that the fecal samples of wild animals carrying CPV were distributed close to the Chengdu Wolong section of G350 National Highway.Therefore,we further collected fecal samples of dogs suspected of CPV infection in Wolong Reserve and surrounding cities(Chengdu and Ya’an)for PCR identification,as well as amino acid and genetic evolution analysis to explore the transmission route and epidemic risk of CPV in wild animals in Wolong.It turned out that most CPV strains in Chengdu and Ya’an were also CPV-2c type,which had a great possibility of mutual transmission.Therefore,the source of infection of this CPV epidemic in Wolong Nature Reserve was likely to be domestic dogs in the surrounding cities.Effective measures should be taken to control the prevalence of CPV in domestic dogs and stray dogs and reduce the impact of CPV on wild animals.Conclusion: The main cause of death of the juvenile wild giant panda in Wolong Nature Reserve was systemic multiple organ damage caused by CPV-2c infection.The CPV-2c variant existed in a variety of wild animals in the reserve,which might be related to the transmission of domestic dogs in the surrounding area.