Identification of Corynebacterium ulcerans and Erysipelothrix sp. in Malayan pangolins-a potential threat to public health?

The discovery of severe acute respiratory syndrome-coronavirus-2-like and Middle East respiratory syndrome-coronavirus-like viruses in Malayan pangolins has raised concerns about their potential role in the spread of zoonotic diseases. Herein, we describe the isolation and whole-genome sequencing of potentially zoonotic two bacterial pathogens from diseased Malaysian pangolins (Manis javanica)-Corynebacterium ulcerans and Erysipelothrix sp. The newly identified species were designated as C. ulcerans P69 and Erysipelothrix sp. P66. C. ulcerans P69 exhibited 99.2% whole-genome nucleotide identity to human bacterial isolate 4940, suggesting that it might have zoonotic potential. Notably, C. ulcerans P69 lacked the diphtheria toxin (tox) gene that is widely used in vaccines to protect humans from corynebacterial infection, which suggests that the current vaccine may be of limited efficacy against this pangolin strain. C. ulcerans P69 also contains other known virulence-associated genes such as pld and exhibits resistance to several antibiotics (erythromycin, clindamycin, penicillin G, gentamicin, tetracycline), which may affect its effective control. Erysipelothrix sp. P66 was closely related to Erysipelothrix sp. strain 2-related strains, exhibiting 98.8% whole-genome nucleotide identity. This bacterium is lethal in mice, and two commercial vaccines failed to protect its challenge, such that it could potentially pose a threat to the swine industry. Overall, this study highlights that, in addition to viruses, pangolins harbor bacteria that may pose a potential threat to humans and domestic animals, and which merit attention. IMPORTANCE: This study firstly reports the presence of two potentially zoonotic bacteria, Corynebacterium ulcerans and Erysipelothrix sp., in diseased Malaysian pangolins collected in 2019. The pangolin C. ulcerans is lethal in mice and resists many antibiotics. It clustered with a lethal human strain but lacked the diphtheria toxin gene. Diphtheria toxin is widely used as a vaccine around the world to protect humans from the infection of corynebacteria. The lack of the tox gene suggests that the current vaccine may be of limited efficacy against this pangolin strain. The pangolin Erysipelothrix sp. is the sister clade of Erysipelothrix rhusiopathiae. It is lethal in mice, and two commercial vaccines failed to protect the mice against challenge with the pangolin Erysipelothrix sp., such that this strain could potentially pose a threat to the swine industry. These findings emphasize the potential threat of pangolin bacteria.

Molecular characterization of a novel Spiruromorpha species in wild Chinese pangolin by mitogenome sequence analysis.

Pangolins are susceptible to a variety of gastrointestinal nematodes due to their burrowing lifestyle and feeding habits, and few parasitic nematodes have been reported. Here, a Chinese pangolin with old wounds on its leg and tail was rescued from the Heyuan City, Guangdong Province. The cox1 and SSU rRNA of the worms from the intestine of the Chinese pangolin had the highest sequence identity of 89.58% and 97.95% to the species in the infraorder Spiruromorpha. The complete mitogenome of the worm was further assembled by next-generation sequencing, with a size of 13,708 bp and a GC content of 25.6%. The worm mitogenome had the highest sequence identity of 78.56% to that of Spirocerca lupi, sharing the same gene arrangement with S. lupi and some species in other families under Spiruromorpha. However, the mitogenome between the worm and S. lupi showed differences in codon usage of PCGs, sequences of NCR, and tRNA secondary structures. Phylogenetic analysis showed that the worm mitogenome was clustered with S. lupi in the family Thelaziidae to form a separate branch. However, it is still difficult to identify the worm in the family Thelaziidae because the species in the family Thelaziidae are confused, specifically S. lupi and Thelazia callipaeda in the family Thelaziidae were separated and grouped with species from other families. Thus, the parasitic nematode from the Chinese pangolin may be a novel species in Spiruromorpha and closely related to S. lupi. This study enriches the data on gastrointestinal nematodes in the Chinese pangolin.

First case report of pustules associated with Escherichia fergusonii in the chinese pangolin (Manis pentadactyla aurita).

BACKGROUND: Escherichia fergusonii is a common conditionally pathogenic bacterium that infects humans and animals. E. fergusonii has been reported to cause diarrhea, respiratory disease, and septicemia, but it is rarely reported to cause skin infections in animals. E. fergusonii has been isolated from the skin and muscular tissue of Chinese pangolin (Manis pentadactyla aurita). To date, there have been no reports of Chinese pangolins with clinical signs of skin diseases. CASE PRESENTATION: This case report describes the clinical case of a subadult (bodyweight: 1.1 kg) female Chinese pangolin from wild rescue with pustules and subcutaneous suppurative infection due to E. fergusonii in the abdominal skin. Bacterial culture, Biochemical analysis, PCR and histopathology were utilized to identify the bacteria in the pustule puncture fluid and infected tissue. To the best of our knowledge, this is the first report of E. fergusonii-related pustules on a Chinese pangolin. CONCLUSION: This case report presents the first observed skin infection in a Chinese pangolin. E. fergusonii infection should be considered as a possible differential diagnosis of pustules and subcutaneous suppurative skin conditions in Chinese pangolins, and we also provide several recommendations for the diagnosis and treatment of this disease.

Comparison of Venous Blood Gas and Biochemical Parameters in Sunda Pangolin (Manis javanica) and Chinese Pangolin (Manis pentadactyla) before and after Isoflurane Anesthesia.

Venous blood gas analytes are commonly examined in animals, and the results may be important when evaluating the overall health status of an animal. Pangolins are critically endangered mammals, and there is limited information on their physiological reference values in the literature. The aim of this study was to analyze venous blood gas and biochemical parameters before and during isoflurane anesthesia in wild healthy Sunda and Chinese pangolins. The results obtained showed that the blood gas index trends of the two pangolin species before and after isoflurane anesthesia were the same. After anesthesia, the partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), total carbon dioxide (CO2), mean blood bicarbonate (HCO3-), extracellular fluid compartment (BEecf) base excess and the mean blood glucose (Glu) levels of both pangolin species showed a significant increase compared to the pre-anesthesia period. In contrast, the mean blood potassium (K+), lactate (Lac) and mean blood pH levels were significantly lower. No significant differences in the mean blood sodium (Na+) or blood ionized calcium (iCa) levels were observed during anesthesia. This study is important for future comparisons and understanding the health status of this endangered species.

Virus diversity, wildlife-domestic animal circulation and potential zoonotic viruses of small mammals, pangolins and zoo animals.

Wildlife is reservoir of emerging viruses. Here we identified 27 families of mammalian viruses from 1981 wild animals and 194 zoo animals collected from south China between 2015 and 2022, isolated and characterized the pathogenicity of eight viruses. Bats harbor high diversity of coronaviruses, picornaviruses and astroviruses, and a potentially novel genus of Bornaviridae. In addition to the reported SARSr-CoV-2 and HKU4-CoV-like viruses, picornavirus and respiroviruses also likely circulate between bats and pangolins. Pikas harbor a new clade of Embecovirus and a new genus of arenaviruses. Further, the potential cross-species transmission of RNA viruses (paramyxovirus and astrovirus) and DNA viruses (pseudorabies virus, porcine circovirus 2, porcine circovirus 3 and parvovirus) between wildlife and domestic animals was identified, complicating wildlife protection and the prevention and control of these diseases in domestic animals. This study provides a nuanced view of the frequency of host-jumping events, as well as assessments of zoonotic risk.

Identification of coronaviruses in farmed wild animals reveals their evolutionary origins in Guangdong, southern China.

Coronavirus infections cause diseases that range from mild to severe in mammals and birds. In this study, we detected coronavirus infections in 748 farmed wild animals of 23 species in Guangdong, southern China, by RT-PCR and metagenomic analysis. We identified four coronaviruses in these wild animals and analysed their evolutionary origins. Coronaviruses detected in Rhizomys sinensis were genetically grouped into canine and rodent coronaviruses, which were likely recombinants of canine and rodent coronaviruses. The coronavirus found in Phasianus colchicus was a recombinant pheasant coronavirus of turkey coronavirus and infectious bronchitis virus. The coronavirus in Paguma larvata had a high nucleotide identity (94.6-98.5 per cent) with a coronavirus of bottlenose dolphin (Tursiops truncates). These findings suggested that the wildlife coronaviruses may have experienced homologous recombination and/or crossed the species barrier, likely resulting in the emergence of new coronaviruses. It is necessary to reduce human-animal interactions by prohibiting the eating and raising of wild animals, which may contribute to preventing the emergence of the next coronavirus pandemic.

Correction: Are pangolins the intermediate host of the 2019 novel coronavirus (SARS-CoV-2)?

[This corrects the article DOI: 10.1371/journal.ppat.1008421.].

Identification of Amblyomma javanense and detection of tick-borne Ehrlichia spp. in confiscated Malayan Pangolins.

Due to habitat destruction and illegal hunting and trade, the number of pangolins has been sharply reduced. To protect pangolins from extinction, relevant departments are combined and active action have been taken. A total of 21 confiscated Malayan pangolins were rescued in 2019, but died continuously for unknown reasons. This study aimed to investigate the reasons for the death of these pangolin and rescue them. 19 of the 21 confiscated pangolins had ticks on their body integument. A total of 303 ticks were collected and identified as Amblyomma javanense (A. javanense) according to their morphology and the sequences of 16S rRNA and internal transcribed spacer 2 (ITS2). There were multi-organ damages in the dead pangolins, especially congestion and hemorrhage in lung, heart and kidney and inflammation of which were observed using HE staining. Pathogens' nucleic acid detection showed ticks were only positive for Ehrlichia spp, with 56.7% positive rate of collected ticks (127/224), which was further confirmed in tissues from dead pangolins. Our findings confirm that ehrlichiosis caused by Ehrlichia spp. from A. javanense might accelerate the confiscated pangolin's death. More attention should be payed to tick-elimination work and the diagnoses and treatment of tick-borne diseases in the follow-up rescue operation.

Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins.

The current outbreak of coronavirus disease-2019 (COVID-19) poses unprecedented challenges to global health1. The new coronavirus responsible for this outbreak-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-shares high sequence identity to SARS-CoV and a bat coronavirus, RaTG132. Although bats may be the reservoir host for a variety of coronaviruses3,4, it remains unknown whether SARS-CoV-2 has additional host species. Here we show that a coronavirus, which we name pangolin-CoV, isolated from a Malayan pangolin has 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S proteins, respectively. In particular, the receptor-binding domain of the S protein of pangolin-CoV is almost identical to that of SARS-CoV-2, with one difference in a noncritical amino acid. Our comparative genomic analysis suggests that SARS-CoV-2 may have originated in the recombination of a virus similar to pangolin-CoV with one similar to RaTG13. Pangolin-CoV was detected in 17 out of the 25 Malayan pangolins that we analysed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus from pangolins that is closely related to SARS-CoV-2 suggests that these animals have the potential to act as an intermediate host of SARS-CoV-2. This newly identified coronavirus from pangolins-the most-trafficked mammal in the illegal wildlife trade-could represent a future threat to public health if wildlife trade is not effectively controlled.

Are pangolins the intermediate host of the 2019 novel coronavirus (SARS-CoV-2)?

The outbreak of a novel corona Virus Disease 2019 (COVID-19) in the city of Wuhan, China has resulted in more than 1.7 million laboratory confirmed cases all over the world. Recent studies showed that SARS-CoV-2 was likely originated from bats, but its intermediate hosts are still largely unknown. In this study, we assembled the complete genome of a coronavirus identified in 3 sick Malayan pangolins. The molecular and phylogenetic analyses showed that this pangolin coronavirus (pangolin-CoV-2020) is genetically related to the SARS-CoV-2 as well as a group of bat coronaviruses but do not support the SARS-CoV-2 emerged directly from the pangolin-CoV-2020. Our study suggests that pangolins are natural hosts of Betacoronaviruses. Large surveillance of coronaviruses in pangolins could improve our understanding of the spectrum of coronaviruses in pangolins. In addition to conservation of wildlife, minimizing the exposures of humans to wildlife will be important to reduce the spillover risks of coronaviruses from wild animals to humans.

Severe infection of wild-caught snakes with Spirometra erinaceieuropaei from food markets in Guangzhou, China involves a risk for zoonotic sparganosis.

Wild-caught snakes are a popular and traditional food in China. However, little known to the public, snakes are also intermediate hosts of Spirometra erinaceieuropaei, a food- and water-borne pathogen of sparganosis. Therefore, we investigated the prevalence of S. erinaceieuropaei in 10 popular species of wild-caught snakes in Guangzhou City (Guangdong Province) between July 2009 and July 2010. One hundred and twenty-four specimens of 10 species (including Enhydris plumbea, Zoacys dhumnades, Elaphe radiate, Elaphe taeniura, Elaphe carinata, Ptyas mucosus, Ptyas korros, Naja naja atra, Bungarus fasciatus, and Bungarus multicinctus) were randomly selected from a total of 1,160 wild-caught snakes. They were obtained from food markets in 5 representative districts (Huadou, Panyu, Tianhe, Haizhu, and Conghua). The specimens were killed, necropsied, and examined for parasitic helminths. Of the snakes examined, 29.8% were infected by spargana and the worm burden per infected snake ranged from 1 to 221. Most species were infected except for En. plumbea, B. fasciatus, and B. multicinctus. Prevalence even reached 100% in Zoacys dhumnades. More than half (53.5%) of the spargana were located in muscular tissue, 36.4% in subcutaneous tissue, and 10.1% in the coelomic cavity. The study revealed the potential risk for the zoonotic sparganosis by eating wild-caught snakes and will be helpful in arousing public health concern about the consumption of snake meat.