When confiscated, most Malayan pangolins were in poor health, part of which had severe trauma, and infested with dozens of adults and subadult ticks. Then rescue work measures were carried out. Firstly, ticks on the body surface were gently removed (Figure 1A) and the wounds were cleaned and applied with antibiotics. Afterwards, they were kept in clean rooms specially adapted for pangolins. Finally, pangolins were fed with the artificial diets that consisted of silky ants, mealworms, bee pupa and vitamin by intragastric gavage until they can eat on their own. After 5 ~ 7 days, some pangolins began to eat independently.
Clinical symptoms showed that rescued pangolins were in bad situation, including poor spirit, drowsiness, anorexia, edema of extremities, snotty and coughing. Some pangolins had inexplicable wounds and bleeding on their body surface for about 10 ~ 26 days. A small group of pangolins even excreted tarry stool and hematuria, and erythrocyte and leucocyte could be found in the urine in the later course. There were nervous symptoms such as convulsions before they died.
Then ticks collected from the surface of pangolins were identified using morphological method (Figure 1B) and molecular biotechnology (Figure 2). Based on the presence of eyes, inornate dark brown scutum and stubby round spur in the coxae II, III and IV, which were different from the other species of the Amblyomma, morphologic analysis implied that these ticks collected from the confiscated Malayan pangolins were Amblyomma javanense (A. javanense) (Figure 1B). Subsequently, specific DNA sequences were amplified from whole DNA genomes of 6 individual ticks and sequenced for 16S rRNA (4/6) and ITS2 (6/6) (Supplementary Figure 1A). BLAST results showed that 16S rRNA gene of ticks was close to Amblyomma javanense (A. javanense) with a similarity of more than 99%, and ITS2 region was close to Amblyomma paulopunctatum with a similarity of nearly 92%. Genetic evolution analysis showed that the selected sequences of 16S rRNA gene belong to the same branch as A. javanense (Figure 2A) and ITS2 region sequences formed an individual branch (Figure 2B). Based the results above, the ticks were finally defined as A. javanense. and ITS2 sequences identified in this study were uploaded as A. javanense (Accession number: MK928428.1 and MK928429.1).
In the autopsy of pangolins, ulcerations and blood on the skin surface, edematous body and pale mucous membranes were observed (Figure 3A). Congestion and oedema were shown in most internal organs (Figure 3B-D), including lung, pancreas, spleen, kidney and bladder mucosa. Bleeding points could be seen on the surface of lung, trachea, bronchus and kidney, and the cut surface of the lungs was infiltrated with foamy fluid. In the heart, there were signs of myocardial edema, pericardium effusion and ventricular congestion (Figure 3C). There were congestion and jaundice in the serosa of omasum of the gastrointestinal system, many Angiostrongylus cishonensis adsorbed in the duodenum (data not shown). Part of the interstina parva mucosa was congested, and the mesenteric lymph nodes were highly swollen, congested, edematous and hemorrhagic as well. Infiltrating hemorrhage was seen in the node section. In the kidneys, corticomedullary differentiation was not clear and blood-like fluid accumulated in the renal calyes (Figure 3).
Histopathological analysis revealed marked presence of inflammation in tissues (Figure 4). Microscopic lesions included myocardial failure, sinus hepaticus and lymphoid nodule dilatation with blood stasis, widened splenic cord, multiply lymphocytes, collapsed alveoli, glomerular capillary hyperemia and increased volume, epithelial cells of mucosa necrosis and submucosa congestion in the sialaden, and bladder mucosa folds inward (Figure 4).
The organs of dead pangolins were collected for the detection of viral pathogens using PCR or reverse transcription-PCR (RT-PCR). The results showed the specimens were negative for EMCV, PIV5, AIV, CDV, CHV and CPV (data not shown). Also, pathogens of ticks like Babesia, Hepatozoon, Theileria and Ehrlichia spp. were detected as described above and the results showed that only Ehrlichia spp. was positive in this study (Supplemental Figure 1B). Self-designed primers were used to detect Ehrlichia spp. of 224 individuals of ticks (Male: 140, Female: 84) using PCR and nested PCR in which 56.7% of the specimens were shown to be positive (Table 2), most of which were detected in lung, spleen and blood of dead pangolins. The autopsy and histopathological changes were consistent with the changes in the case of Ehrlichia spp. infection. Ehrlichia spp. infection might be one of the reasons for the death of rescued Malayan pangolins.
According to BLAST result, the 16S rRNA sequences of 3 randomly selected samples were identified as Ehrlichia spp. and were closest to E. chaffeensis and E. ruminantium, with a similarity of above 98%. This observation was consistent to a phylogenetic tree (Figure 5). However, the obtained sequences (about 550 bp) were not long enough to be identified to the species level. When Ehrlichia spp. had been detected, all live pangolins were injected with broad-spectrum tetracycline-class antibiotic doxycycline for medical treatment. Finally, three pangolins were alive before transfer.