Fatal CDV infection in giant pandas
On December 3, 2014, an eight-year old giant panda named Chengcheng presented with jaw trembling and violent convulsions of the limbs. Over the ensuing fourteen weeks, four additional giant pandas housed in the same room or adjacent rooms began to display clinical signs including mucopurulent ocular discharge, nasal and footpad hyperkeratosis, and violent convulsions of the limbs (clinical onset dates are listed in Table 1). Nucleic acids isolated from nasal swabs, urine, feces and blood collected from affected pandas at the time of clinical presentation all tested positive for CDV by RT-PCR. PCR-based tests were negative for other virus previously isolated from giant pandas (canine coronavirus) or viruses regarded as a potential threat to giant pandas (canine adenovirus, canine herpesvirus, and canine parainfluenza virus)41617. CDV-positive giant pandas were monitored and treated with antiserum therapy.
Each of the five CDV-infected giant pandas that displayed clinical signs of infection died 7–34 days following disease onset (Table 1). No CDV serum neutralizing (SN) antibodies were detected in the five infected giant pandas showing clinical signs of CDV infection prior to death (Table 1). However, CDV RNA was detected by RT-PCR in heart, liver, spleen, lung, kidney, intestines and brain of four deceased giant pandas (Table 2). In addition, CDV RNA was detected by RT-PCR from blood and nasal swab samples collected from an asymptomatic giant panda named Zhuzhu, who was previously vaccinated against CDV in 2012 and had high-titer SN antibodies (Table 1). None of the additional sixteen giant pandas in the Shanxi Rare Wild Animal Rescue and Research Center tested positive for CDV by RT-PCR. Uninfected pandas within the Shanxi Rare Wild Animal Rescue and Research Center were placed in isolation on December 26, 2014 and vaccinated with a canarypox-vectored CDV vaccine.
Late in the course of infection, giant pandas exhibited clinical signs of nasal hyperkeratosis (Fig. 1a) and footpad hyperkeratosis (Fig. 1b) which are characteristic of CDV infection in other animals18. Severe pneumonia with dark-red congestion was observed in affected lungs along with small white patches on the surface of the lungs of one CDV-infected giant panda named Fengfeng (Fig. 1c). Lung samples from Fengfeng were examined for histopathological analysis, whereas other samples (e.g ., brain, spleens) were not assessed due to cellular autolysis of tissues collected at necropsies performed >12 hours of after death. Histological analyses of lung tissue from Fengfeng showed interstitial pneumonia with congestion, multinuclear macrophage infiltration in the alveoli, and widening of alveolar septa (Fig. 1d). Histological observations were consistent with those previously reported in the lungs of cynomolgus monkeys and red fox (Vulpes vulpes) infected by CDV1419.
The complete viral genome of the CDV isolate giant panda/SX/2014 was sequenced (Gen Bank accession no. KP793921) and showed highest nucleotide identity to the PS strain of CDV isolated from a dog (98.7% identity, Gen Bank accession no. JN896331) and the CDV-RD-JL strain of CDV isolated from a raccoon dog (Nyctereutes procyonoides, 98.6% identity, Gen Bank accession no. KJ848781) (Fig. 2a). Phylogenetic analysis and multiple sequence alignments based on the H gene sequence revealed that giant panda/SX/2014 belongs to the Asia-1 cluster. The H gene sequence of giant panda/SX/2014 was 99.1% identical to the SD(08)1 and LN(07)1 CDV strains (Gen Bank accession no. FJ810215 and EU325730) isolated from a fox and raccoon dog in Shandong and Liaoning province in China, respectively (Fig. 2b). The giant panda/SX/2014 H gene sequence showed 95.9% identity to a sequence previously recovered from a CDV-infected giant panda during an outbreak at the Chongqing Zoo in 1997 (Gen Bank accession no. AF178038) (Table 3).
Unique features of the H protein of giant panda/SX/2014
The predicted H protein amino acid sequence of giant panda/SX/2014 was identical to H protein amino acid sequences predicted following RT-PCR and sequencing of the H gene of CDV contained in the lung samples of all deceased giant pandas. All H gene sequences were predicted to encode five unique amino acid substitutions in the H protein which have not previously been identified in other CDV isolates belonging to the Asia-1 cluster, including V26M, T213A, K281R, S300N, and P340Q. Additionally, all H gene sequences also encoded a histidine (H) residue at amino acid position 549, which has previously been associated with CDV isolates from non-canid hosts20. The Y549H substitution is involved in H protein binding to the signaling lymphocytic activation molecule (SLAM) receptor and has been hypothesized to contribute to the emergence of highly pathogenic CDV and host range expansion (Table 3).
Here, we have documented CDV infection in six captive endangered giant pandas during an outbreak in the Shanxi Rare Wild Animal Rescue and Research Center in China. Five of six CDV-infected giant pandas died as a result of the infection. Affected giant pandas presented with clinical illness beginning the first week of December, 2014 through March, 2015. All affected giant pandas were housed in the same room or adjacent rooms, suggesting that CDV may have been transmitted between pandas via direct contact and/or respiratory droplets. The surviving panda tested positive for CDV by RT-PCR, but did not develop overt clinical signs of CDV infection and was previously vaccinated against CDV, strongly supporting the utility of CDV vaccination in giant pandas.
CDV transmission to captive animals may potentially occur via direct or indirect contact with infected domestic dogs or wild carnivores. Domestic dogs were considered the likely source of infection for canine distemper in Serengeti lions in 199421. In North America, wild raccoons (Procyon lotor) were thought to be the source of CDV infection in large captive cats in 1991 and 19929. In Japan, raccoon dogs were considered to be the source of outbreak for canine distemper in tigers in 2009 and 201022. In Denmark, it was speculated that wildlife species, such as foxes, raccoon dogs, and ferrets were the sources of CDV infection of farmed mink (Neovison vison)23. In China, CDV infection has been observed in domestic dogs, wild canids (fox, raccoon dogs), and non-canids (mink, monkey) demonstrating the remarkable ability of this pathogen to cross species barriers1524. The reservoir source of CDV leading to the outbreak among giant pandas remains unclear. While there were no carnivores in the Shanxi Rare Wild Animal Rescue and Research Center, it is possible that domestic dogs or other susceptible wild animals carrying CDV in the area were responsible.
The highly variable nature of H gene sequences among viruses belonging to the Morbillivirus genus has been exploited to characterize CDV field strains and investigate relationships among various strains. Sequencing of CDV from giant pandas revealed five unique amino acid changes (V26M, T213A, K281R, S300N, P340Q) encoded by the H gene that have not been observed previously in Asia-1 strains. Amino acid residues at positions 549 in the CDV H protein are implicated in CDV host range restriction and pathogenesis2526. Notably, the H gene of CDV from giant pandas possessed a Y549H substitution which has been associated with the emergence of highly pathogenic CDV and host range expansion. Before this outbreak, the Y549H substitution had only observed in twelve CDV strains isolated from mink, fox and raccoon dogs in Shandong province and an isolate from a mink in Liaoning province in China24. While the Shanxi Rare Wild Animal Rescue and Research Center is located a significant distance away from these provinces (>900 kilometers), these species may represent potential sources of CDV leading to this outbreak among giant pandas. Previous work has shown that serial passage of dog-derived CDV strain 5804 in ferrets led to acquisition of the Y549H substitution27. CDV isolates with a histidine residue at position 549 also showed enhanced virulence in raccoons relative to strains lacking histidine at this position28. We therefore speculate that the high-level of virulence associated with giant panda/SX/2014 infection of giant pandas may be related to the presence of a histidine residue at position 549 of the H protein. The additional unique H protein amino acid substitutions identified may reflect adaptive changes facilitating CDV infection of giant pandas. As an RNA virus, CDV is capable of rapid mutation leading to viral variants that are potentially better equipped for replication in giant pandas, as has been documented for the emergence of the Y549H substitution during serial passage in ferrets27.
Vaccination represents an effective approach to prevent CDV infection of domestic dogs and may have utility in captive giant panda populations. Currently, there are no standard vaccination strategies in place for the prevention of infectious diseases in captive giant pandas in China. The effectiveness of CDV vaccination in giant pandas is supported by the observation that the single panda to survive CDV infection during this outbreak (Zhuzhu) was previously vaccinated against CDV and had high-titer SN antibodies. This animal did not display clinical signs despite recovery of CDV genomic material from blood and nasal swab samples, suggesting that the protective immune responses elicited by CDV vaccination were not sufficient to prevent naturally-acquired CDV infection but may have attenuated disease. Ultimately, universal CDV vaccination of captive giant pandas may be warranted but must be also weighed against potential vaccination risks. Additional studies to better understand the safety and efficacy of CDV vaccines in giant pandas are needed as CDV vaccines are more widely implemented. In a study involving two giant pandas, a commercially available canarypox-vectored CDV vaccine safely elicited SN antibody titers above a level considered to be protective against CDV disease29. Due to the limited supply of the canarypox-vectored CDV vaccine and the potential risks associated with live-attenuated CDV vaccines, most giant pandas in the Shanxi Rare Wild Animal Rescue and Research Center and other organizations involved in giant panda breeding programs are not routinely vaccinated. The documentation of a CDV outbreak among captive giant pandas in China suggests that heightened surveillance and CDV vaccination should be considered in all facilities housing captive giant pandas for successful conservation of this endangered species.
The protocol of the study was carried out in accordance with guidelines of animal welfare of World Organization for Animal Health. All experimental protocols were approved by the Review Board Military Veterinary Research Institute of the Academy of Military Medical Sciences.
Virus detection by PCR and RT-PCR
Nasal swab, urine, fecal and blood samples from each affected panda were collected at the time of clinical disease onset. Viral DNA and RNA were isolated from samples using the AxyPrep Multisource Genomic DNA Miniprep kit (AXYGEN, Union City, USA) and RNeasy Mini kit (QIAGEN, Germantown, MD) according to manufacturer’s protocols. Extracted nucleic acids were tested by RT-PCR for CDV using primers specific for CDV H gene (P1:5′-CGAGTCTTTGAGATAGGGTT-3′ and P2: 5′-CCTCCAAAGGGTTCCCATGA-3′). RT-PCR and PCR testing for other viruses threatening giant pandas (canine adenovirus, canine herpesvirus, canine coronavirus, and canine parainfluenza virus) were performed using previously reported methods1730. RT-PCR testing for CDV was also performed on samples collected from the heart, liver, spleen, lungs, kidneys, intestines, and brain of each deceased giant panda, with the exception of Chengcheng for whom tissue samples were not available. Serum samples were collected from the giant pandas during the outbreak to measure SN antibody titers against CDV.
Histopathological analysis of giant pandas infected with CDV
Necropsies were performed on all deceased giant pandas at which time tissue samples were collected for histologic examination. Lung samples from the giant panda named Fengfeng, who experienced a long illness duration, were selected for histologic review using routine methods. Tissue samples were fixed in 10% phosphate-buffered formalin, embedded in paraffin wax, sectioned, and stained with hematoxylin and eosin prior to analysis.
Virus isolation and sequencing
CDV was isolated from lung and spleen tissue collected from the deceased giant panda Dabao as follows. Tissue samples were inoculated onto monolayers of Vero cells expressing canine SLAM protein, which have been previously used to grow and isolate CDV31. The presence of CDV was confirmed by RT-PCR detection using primers specific for the CDV N gene (P3: 5′-GTGACTGCTCCTGATACTGC-3′ and P4: 5′-ACCAACTCCCATAGCATAAC-3′). The giant panda CDV isolate was named as giant panda/SX/2014. The complete H gene of the giant panda/SX/2014 isolate and virus contained in lung samples from deceased giant pandas was amplified for sequencing by RT-PCR using H gene specific primers (P5: 5′-GTCTTGCCTGATTGTCAGGC-3′ and P6: 5′-GGTTTGATTCAATCGTCGG-3′). The entire genome of the giant panda/SX/2014 CDV isolate was amplified and sequenced using a set of fifteen primer pairs to generate overlapping PCR amplicons (Table S1).
Phylogenetic trees were constructed using molecular evolutionary genetics analysis MEGA 6 software (http://www.megasoftware.net/mega.php) with the neighbor-joining (NJ) method to calculate distance. Bootstrapping with 1,000 replicates was performed to determine the percentage reliability for each internal node.
How to cite this article: Feng, N. et al. Fatal canine distemper virus infection of giant pandas in China. Sci. Rep. 6, 27518; doi: 10.1038/srep27518 (2016).