Surveillance of wild animals carrying infectious agents based on high-throughput screening platform in the Republic of Korea

Background Infectious diseases transmitted by wild animals are major threats to public health. This study aimed to investigate the potential of rescued wild animals that died of unknown causes as reservoirs of infectious agents. From 2018 to 2019, 121 dead wild animals (55 birds and 66 mammals) were included in this study. All wild animals died during treatment after anthropogenic events. After deaths of animals, necropsies were performed and trachea, lungs, large intestine (including stool), and spleen were collected to determine causes of deaths. A high-throughput screening (HTS) quantitative polymerase chain reaction (qPCR) designed to detect 19 pathogens simultaneously against 48 samples in duplicate was performed using nucleic acids extracted from pooled tissues and peripheral blood samples. If positive, singleplex real-time PCR was performed for individual organs or blood samples. Results The HTS qPCR showed positive results for Campylobacter jejuni (10/121, 8.3%), Campylobacter coli (1/121, 0.8%), Mycoplasma spp. (78/121, 64.5%), and Plasmodium spp. (7/121, 5.7%). Singleplex real-time PCR confirmed that C. jejuni was detected in the large intestine but not in the blood. C. coli was only detected in the large intestine. Mycoplasma spp. were detected in all organs, having the highest proportion in the large intestine and lowest in the blood. Plasmodium spp. was also detected in all organs, with proportions being were similar among organs. Conclusions This study shows that wild animals can become carriers of infectious agents without showing any clinical symptoms. Supplementary Information The online version contains supplementary material available at 10.1186/s12917-023-03714-0.


Background
More than half of known infectious pathogens are zoonotic, 12% of which are causing emerging infectious diseases (EIDs) [1].The number and diversity of diseases are increasing.Many diseases not only threaten human health, but also affect livestock and wild animals [2].Globalization, environmental change, and migrating birds or wild animals living in borders can spread diseases or pathogens more easily and widely.For example, avian influenza (AI) is one of the most well-known viral diseases transmitted from migratory birds to poultry [3].Climate change increases the risk of new inflows of wildlife-derived infectious diseases, especially arthropodborne diseases such as malaria and Japanese encephalitis.It also increases the spread of disease [4].The trade of wild animals and wild animal products can also affect the spread of diseases [5].
Most EIDs are considered to spread from wildlife.The importance of constructing surveillance programs and research is increasing [6].Outbreaks of infectious diseases are recognized mainly by reporting them in clinical cases or by detecting pathogens extracted from the environment or carcasses.Constructing a surveillance program to enable rapid detection and response can help prevent the spread of diseases [7,8].High-throughput quantitative polymerase chain reaction (qPCR) platforms are useful because large-scale detection can be performed rapidly and sensitively [9,10].
The importance of surveilling wildlife-derived infectious diseases is increasing, as is the number of diseases that require surveillance.Monitoring all diseases takes substantial time, finances, and reagent volume.Moreover, research on untamed and living wild animals requires greater effort [11].Although the gold standard varies from disease to disease, molecular diagnosis is commonly considered a useful method [12].Real-time qPCR is often used to detect pathogens because it is capable of performing quantitative analysis with a highsensitivity [13].However, with increasing number of diseases requiring surveillance, it is costly and time-consuming to test all samples for significant results [14].A high-throughput qPCR platform can be used to address this issue to reduce time and cost.Its analytical sensitivity and specificity are not decreased compared to those of singleplex screening [9].
This study was conducted to investigate the potential of wild animals rescued from traumatic distress and died during treatment or were euthanized as carriers of infectious diseases using a high-throughput qPCR platform.

Results of high-throughput qPCR platform
Results of high-throughput qPCR platform showed that samples were positive for Campylobacter jejuni (10/121, 8.3%), Campylobacter coli (1/121, 0.8%), Mycoplasma spp.(78/121, 64.5%), and Plasmodium spp.(7/121, 5.7%) (Table 1).C. coli was detected only in a kestrel.C. jejuni and C. coli were detected in water birds or other wild animals that had been rescued around water bodies such as rivers and seas.Mycoplasma spp.and Plasmodium spp.were detected in various species from rescued areas such as expressways, residential areas, and farmlands.

Results of singleplex qPCR
In birds, singleplex qPCR revealed that C. jejuni was detected in 100% of large intestine samples, 63% of trachea samples, 75% of lung samples, and 50% of spleen samples.C. coli was detected only in the large intestine.Mycoplasma spp. was detected in 76% of large intestine samples, 68% of trachea samples, 63% of lung samples, 29% of spleen samples, and 5% of blood samples.Plasmodium spp. was found in 53% of lung and large intestine samples, 46% of trachea and blood samples, and 38% of spleen samples.In mammals, C. jejuni was detected in 100% of large intestine, trachea, and spleen samples and 50% of lung samples.Mycoplasma spp. was detected in 75% of large intestines, 70% of tracheae, 65% of lungs, 40% of spleens, and 45% of blood samples.Plasmodium spp. was only detected in the trachea with a high Ct value of 37.98.

Histopathological analysis
Pathogens were detected in 82 (67.8%) of 121 animals examined.All patients did not show any clinical signs regarding these pathogens.Morphologically, it was difficult to confirm the effect of infection because most patients showed hemorrhage or tissue damage due to trauma.Twenty-two (22/82, 18.2%) cases, including 4 mammals (4/22, 18.2%) and 18 birds (18/22, 81.8%), showed histopathological lesions such as inflammation or necrosis in internal organs associated with infections.In mammals, only Mycoplasma spp. was detected from three raccoon dogs and one Korean water deer.Among them, in one raccoon dog, Mycoplasma spp. was detected in the lung, large intestine, and trachea.However, histopathological lesions related to pathogens were only found in the lungs (mild interstitial pneumonia) [15].The other raccoon dog with Mycoplasma spp.detected in blood only showed hepatic necrosis.Thus, additional qPCR was performed and Mycoplasma spp. was detected in the liver.The last raccoon dog infected by endoparasites and ectoparasites showed inflammation in kidney, heart, pancreas, large intestine, stomach, lung, and spleen.However, Mycoplasma spp. was only detected in the large intestine, heart, pancreas, and kidney.In one Korean water deer rescued after a car accident, Mycoplasma spp. was detected in the lung and trachea.However, histopathological lesion regarding infection was not confirmed in the lung or trachea.Instead, chronic nephritis showed and additional qPCR detected Mycoplasma spp. in the kidney.In birds, Mycoplasma spp.were detected the most frequently (16/18, 88.9%), followed by Plasmodium spp.(5/18, 27.8%).C. jejuni (2/18, 11.1%), and C. coli (1/18, 5.6%).The association between detected pathogen and histopathological lesion was not distinct except for two cases (1 Chinese Little Bittern and 1 Eurasian Woodcock) of Plasmodium spp.detected in the liver (mild hepatitis) [16].

Molecular analysis ofPlasmodiumspp.
A phylogenetic analysis was conducted based on sequence homologies of Plasmodium spp. in each organ (Fig. 1).Among Plasmodium spp.positive samples through high-throughput qPCR platform and singleplex qPCR, two avian species of Haemoproteus spp.and one  Leucocytozoon spp.were found in three animals through nested and conventional PCR.Therefore, these three animals were excluded.Through phylogenetic analysis, we confirmed that pathogens obtained from different organs of one animal showed homologous sequences except for two cases.For one animal, the sequence from the spleen showed Haemoproteus spp.from nested PCR.However, a conventional PCR revealed Plasmodium spp.For the other animal, sequences from the blood revealed Plasmodium spp. in nested PCR.However, in conventional PCR and sequencing, Haemoproteus spp. was detected.In the analysis of geographical distribution of genotypes (Fig. 2), samples found in a Korean water deer (Hydropotes

Discussion
This study confirmed the presence of major infectious pathogens in the Republic of Korea in wildlife that died or euthanized at wildlife rescued center using a highthroughput platform.In this study, more than one pathogen was detected in 67.8% of examined wild animals.
Pathogen detection was higher in wild birds (40/55, 72.7%) than in wild mammals (42/66, 63.6%).Clinical symptoms corresponding to detected pathogens were not observed in all animals during treatment periods.On the other hand, 18.2% of detected pathogens were confirmed to be associated with histopathological lesions regarding infections.Histopathological lesions related to detected pathogens were only shown in six patients (3 raccoon dogs, 1 Korean water deer, 1 Chinese Little Bittern, and 1 Eurasian Woodcock).Even in these six patients, specific lesions for each pathogen were not shown or no lesion was identified in any organs where pathogens were detected.Therefore, the detection of such pathogens was more likely to be from secondary infections or incidental finding due to the effect of trauma or other infections such as ectoparasites or endoparasites.Considering that all wild animals included in this study were rescued from anthropogenic accidents or injuries, or from serious ectoparasitic infections (severe scabies infection in raccoon dogs), wild animals are carrying pathogens as asymptomatic carriers.
Wild birds are considered as carriers of human camphylobacteriosis [17].The transmission of this pathogen occurs through a fecal-oral route, causing waterborne outbreaks [18].Campylobacter spp. is also a thermophilic pathogen.Birds are considered good reservoir of these pathogens because birds have higher body temperatures than mammals [19].Patients carrying Campylobacter spp.showed common features consistent with those described in previous studies [17][18][19].All patients were waterfowl or the rescued area was near water.In addition, more were detected in birds (14.5%) than in mammals (3%).In addition, patients did not show any symptoms corresponding to these pathogens such as enteritis.Since wild birds living near the water are more likely to asymptomatic reservoirs of Campylobacter spp., monitoring should be conducted for them.
In this study, Mycoplasma spp.had the highest prevalence (64.5%).However, clinical signs and histopathological lesions associated with these pathogens were rarely observed.Only four (4/78, 5.1%) patients showed histopathological lesions in organs from which pathogens were detected.Even in wild animals with histopathological lesions, the direct cause of death was trauma due to accidents, indicating that wild animals might be potential carriers of Mycoplasma spp.A variety of Mycoplasma spp.have been identified in wild animals worldwide.Although the pathogenesis has not been elucidated yet, Mycoplasma spp. is known to cause various diseases [20].Further studies are necessary to identify the correlation between the presence of bacteria and pathological changes in wild animals.
In this study, in all wild animals in which Plasmodium spp. was detected in multiple organs, the same sequence was confirmed as a result of sequencing of pathogens detected for each organ except for two cases.Sequence comparison also revealed that Plasmodium spp.detected in the trachea of Korea water deer (Hydropotes inermis) (18-809) shared 99% sequence similarities with those detected in Chinese little bitterns (Ixobrychus sinensis) .It was unclear why Plasmodium spp., which is highly similar to that isolated from birds, was detected in organs of herbivores.However, cases of pathogenic induction by erythrocyte development of Plasmodium spp.have been reported [21].Although rare, interspecies transmission cases indicate that mammals have contact with birds in the wild [22].A more extensive study on the degree of Plasmodium spp.infection in wild mammals and its genetic similarity with avian isolates if Plasmodium spp. is needed in the future.
In a previous study conducted in the same area [23], Plasmodium spp. was detected in 6 (5.1%) out of 118 birds, whereas it was detected in 6 (10.9%) out of 55 birds in this study, showing a higher prevalence.Plasmodium spp.identified in the previous study were mainly similar to those identified in Asian countries regarding their genetic diversities, while Plasmodium spp.identified in this study became more diverse to include those detected in Europe or the United States.It can also be seen that genotypes between migratory birds or between migratory birds and resident birds are still similar.Sequences of a Plasmodium spp.isolated from a Korean water deer (Hydropotes inermis) (18-809) was most similar to that of a Plasmodium spp.isolated from Japan.They both were in the same clade as Plasmodium spp.detected from Chinese little bitterns (Ixobrychus sinensis) (18-617) and brown hawk owl (Ninox scutulata) .This may indicate that migratory birds' role as mediators of Plasmodium spp. is at risk of expanding to mammals as well as resident birds.However, this study was limited to those with histopathological evaluations among those who died or were euthanized.Since screening criteria of samples were different from those of the previous study, direct comparison of prevalence was limited.A separate study should be conducted to analyze changes in the prevalence of Plasmodum spp. in the study area.

Conclusion
Since pathogens of wild animals can also be pathogens of humans and livestock, various investigations of diseases centered on wild animal carcasses have been conducted.This study confirms that wild animals that appear healthy without having specific pathogen-specific lesions can carry pathogens, demonstrating that wild animals can be asymptomatic carriers of pathogens.Future studies on wildlife-borne diseases should use carcasses and determine the possibility of simple carriers simultaneously.

Sample Collection
A total of 121 wild animals (55 birds and 66 mammals) rescued between January 2018 and December 2019 that died or were euthanized during treatment were included in this study (Table 2).In the case of euthanizing wild animals because they could not be release into the wild, T-61 euthanasia solution (Embutramide, Mebenzonium iodide, Tetracaine hydrochloride infectable solution) was injected at 0.3 mg/kg under unconsciousness after deep inhalation anesthesia with isoflurane.In the course of treatment, animals had no specific clinical symptoms for infectious diseases.No other signs were observed in clinical laboratory tests (complete blood count and clinical chemistry analysis).After death or euthanasia, necropsy and histopathological examination were performed to determine the cause of death.During necropsy, four organ tissues (trachea, lung, large intestine [including stool], and spleen) were collected and stored in ethanol for screening of infectious agents.The peripheral blood collected and used for blood tests during medical treatment and stored was also included in the investigation of infectious agents.

Nucleic acid extraction and reverse transcription
Tissues were washed with distilled water to remove ethanol.Blood and tissue samples were ground separately using TissueLyser II (Qiagen, Hilden, Germany) with nuclease-free water and then mixed together.The total volume was 200 or 250 µL.DNA and RNA were extracted simultaneously using a MagMAX™ Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions.Nucleic acid extraction was also conducted for positive samples using a QIAamp DNA Mini Kit (Qiagen).For RNA viruses, reverse transcriptase PCR was performed using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific).Extracted nucleic acids were stored at -20℃.

Singleplex qPCR
Singleplex qPCR was performed for high-throughput qPCR platform-positive samples to determine which tissues or blood samples contained the pathogen.It was laso performed for additional organ tissues showing histopathologic lesions.For nucleic acid amplification, 2 µL of nucleic acid from each organ and blood was used as a template in a final volume of 20 µL with TaqMan® Fast Advanced Master Mix (Thermo Fisher Scientific), 0.2 µM of each primer, and 0.1 µM of each probe.The thermal cycler protocol consisted of incubation at 50 °C for 2 min, 95 °C for 20 s, and 40 cycles at 95 °C for 1 s and at 60 °C for 20 s.The primer and probe were the same as those designed for the high-throughput qPCR platform panel.
After qPCR, all positive samples were sequenced and compared to BLAST database to determine pathogenic organisms.

Molecular Analysis forPlasmodiumspp.
Two gel-based PCRs and sequencing was performed for phylogenetic analysis of identified Plasmodium spp.One was nested PCR for comparing sequence homologies of identified hemoparasites in a single case and the other one was a conventional PCR for comparing geographical distribution of genotypes.Both steps of nested PCR were performed in a final volume of 50 µL using a HotStarTaq Master Mix Kit (Qiagen).The template consisted of 2 µL of nucleic acid isolated from each organ and blood.The mixture was denatured for 15 min at 95 o C, followed by 40 cycles at 95 o C for 45 s, 50 o C for 45 s, and 72 o C for 1 min, and finally extended at 72 o C for 7 min before maintaining at 4 o C. The first PCR product was used as a template for the second PCR.For nested PCR, HameNF1-HaemNR3 primers were used for the first PCR and HaemF-HaemR2 primers were used for the second PCR.PCR amplicons had an expected size of 617 bp for nested PCR [42].For conventional PCR, the first reaction was performed using 3760 F-4292Rw2 primers.If the fragment was not amplified, a follow-up reaction was performed with smaller fragments using F1-4292Rw2, F3-4292Rw2, and 3760 F-R1 primer pairs.PCR amplicons had an expected size of 400-500 bp [43,44].Phylogenetic analysis was conducted by comparing base pair of cytochrome b sequence.Phylogenetic analysis was performed for two purposes: (1) to compare sequence homology of Plasmodium spp.detected in each organ (Fig. 1); and (2) to estimate geographical distribution of Plasmodium spp.mainly transmitted by wild birds (Fig. 2).For the phylogenetic tree for confirming sequence homology, a sequence of Theileria annulata (GenBank accession number: M63015) was used as outgroup.For comparing geographical diversity, the phylogenetic tree was made using a sequence of Plasmodium spp.(GenBank accession numbers AY099029 and JN164729) as outgroup.Neighbor-joining genetic analysis was performed using MEGA-X.Numbers on branches indicate bootstrap values based on 1,000 replicates.

Fig. 1
Fig.1Sequence homologies of Plasmodium spp.detected in multiple organs in a single case.Plasmodium spp.detected in the same individual showed the same sequence except for two cases.Sequences obtained from this study are written in terms of the year of rescue and detected organs followed by species.Neighbor-joining phylogenetic analysis was performed with MEGA-X.Bootstrap percentage values are shown on branches

Fig. 2
Fig. 2 Phylogenetic analysis of Plasmodium identified in wild birds in this study, showing diverse genotypes.Sequences obtained from this study are written in terms of rescue year and detected organs.Neighbor-joining genetic analysis was performed using MEGA-X.Bootstrap percentage values are shown on branches

Table 1
Prevalence of pathogen and reason for rescue of animals with pathogens detected

Table 2
Information on wildlife species (scientific name) included in this study