Occurrence of Selected Zoonotic Fecal Pathogens and First Molecular Identification of Hafnia paralvei in Wild Taihangshan Macaques (Macaca mulatta tcheliensis) in China

Rhesus macaques (Macaca mulatta) are hosts to a range of zoonotic and potentially zoonotic pathogens. The present study firstly provides a broader investigation of the presence and prevalence of zoonotic fecal pathogens in wild Taihangshan macaques, a subspecies of rhesus macaque in China. A total of 458 fecal samples were collected between September 2015 and November 2016. Fourteen genera of intestinal parasites (four genera of protozoans and ten genera of helminths) and twelve genera of bacteria were tested for using PCR amplification. The overall samples prevalence of parasitic infection was 98.25%. Entamoeba spp. (89.96%), Balantidium coli (70.09%), and Isospora spp. (28.38%) were the most prevalent protozoa, whereas the predominant prevalent helminths were Trichuris sp. (93.23%), Strongyloides spp. (73.36%), and Oesophagostomum sp. (31.66%). Ten genera of intestinal bacteria were detected in samples of rhesus macaques, including Shigella (31.66%), Escherichia coli (29.91%), Klebsiella pneumoniae (28.38%), Leptospira (26.64%), Campylobacter jejuni (18.34%), Salmonella (13.32%), etc. Eight samples (1.75%) were tested Hafnia-positive based on sequences analysis of 16S rRNA and ampC gene. This is the first molecular characterization of Hafnia infection in NHPs. Our cross-sectional prevalence study provides important information for monitoring the potential transmission of zoonotic infections from wild rhesus macaques.


Introduction
Rhesus macaques (Macaca mulatta) are one of the most widely distributed nonhuman primates (NHPs) in the world. There are more than six kinds of rhesus macaque subspecies in China, and Taihangshan macaque (Macaca mulatta tcheliensis) is a subspecies confined to China southern Taihangshan Mountains area on the Henan-Shanxi border (N 35 ∘ 12 49 , E 112 ∘ 41 25 ) [1]. Rhesus macaques are an increasingly important source of zoonotic diseases [2]. They can share pathogens with livestock and humans and act as reservoirs for several emerging infectious diseases such as malaria [2,3]. Some molecular epidemiological studies have been performed to evaluate the prevalence of bacteria and parasites in NHPs [4][5][6][7][8][9][10]. Enterobacterial pathogens including Shigella spp., Salmonella spp., and Escherichia coli are the most commonly distributed pathogens in NHPs [4][5][6]. A diversity of intestinal parasites including five protozoan genera and six helminths genera was reported to infect NHPs [7,8]. Many of these bacteria and parasites have strong pathogenic potential in both humans and animals. More recently, investigations of parasitic infections have been reported in several subspecies of rhesus macaque samples in China [7,9,10]. However, there are few data regarding the prevalence and distribution of the potential zoonotic fecal pathogens among Taihangshan macaques. Therefore, our objective was to get initial information about the presence and prevalence of selected fecal pathogens in wild Taihangshan macaques. To explore the molecular epidemiology of zoonotic pathogens in Taihangshan macaques, fourteen genera of intestinal parasites (four genera of protozoans and ten genera of helminths) and thirteen genera of bacteria were tested for using PCR amplification. Hafnia was amplified with conventional PCR using the primers specific for the 16S rRNA gene and ampC gene as described previously [11,12]. All primers and conditions used in this study were listed in Supplementary Table 1.

Isolation, Genotyping, and Antibacterial
Susceptibility of the Hafnia. The fecal samples were cultured on nutrient agar plates (Oxoid, UK) in anaerobic conditions at 37 ∘ C for 18 h and typical colonies were transferred at least thrice in the same conditions. The isolates were examined by the Gram stain, 16S rRNA gene and ampC gene sequencing, and biochemical tests using the BD Phoenix Automated Microbiology System (BD). Antibiotic susceptibility testing was performed for Hafnia as previously described [12].

Sequencing and Phylogenetic
Analyses. The PCR products from positive samples were bidirectionally sequenced at BGI Sequencing (Beijing, China). Nucleotide sequences were aligned with reference strains obtained from the GenBank database. The molecular phylogenetic trees were constructed by using the neighbor-joining method executed in MEGA6 [13]. The robustness of the tree topology was assessed with 1000 bootstrap replicates.

Nucleotide Sequence Accession Numbers.
The representative nucleotide sequences of this study have been deposited in the GenBank database under accession number MG923797 for 16S rRNA gene and MK189458 for ampC gene.  (Figure 1).

Discussion
Taihangshan macaque (Macaca mulatta tcheliensis) occupies the northern limit of all rhesus macaque natural populations in the world [1,14]. A national nature reserve in the Taihangshan Mountains area was established by the Chinese government to protect the macaques. In most wildlife populations, infectious diseases are considered as the second leading cause of mortality, right behind predation and malnutrition [15]. Meanwhile, rhesus macaques have the potential to transmit various pathogens to humans and domestic animals. Therefore, monitoring the presence and prevalence of zoonotic infectious agents among this population is important for the general health of humans and animals coming into contact with this population. This study demonstrates a high samples prevalence (98.25%, 450/458) and diversity (three genera of protozoans and eight genera of helminths) of intestinal parasites in Taihangshan macaques in China. Previous studies revealed that the samples prevalence varied significantly with species, geographic region, feeding habitats, and age [7,8]. Similar prevalence was reported in bushmeat monkeys samples (92%) in Cameroon [8], while lower prevalence was found in 34 NHP species samples (54.1%) in China [7] and primates samples (54.5%) at a zoo in Malaysia [16].
Several molecular epidemiological studies have demonstrated Trichuris sp. and Entamoeba spp. were the most frequently detected parasites [8], whereas others reported that Entamoeba spp. [7] or Strongyloides spp. [17] was the most prevalent. Similarly, Trichuris sp., Entamoeba spp., and Strongyloides spp. were the most common parasitic pathogens in our study with a high prevalence of 93.23%, 89.96%, and 73.36%, respectively. Several studies reported that Trichuris sp. and Entamoeba spp. were parasitic with a high potential for transmission to humans and animals because of their simple and direct life cycles [7,18,19].
Balantidium coli detected in our study are multihost parasite capable of infecting animals and humans. Balantidium coli could damage the intestinal mucosal and cause serious diarrhea and dysentery [10]. The prevalence of infection found in the present study (70.09%) with Balantidium coli is consistent with rates previously published in bred rhesus monkeys and baboons [10,20], but higher than cercopithecid monkeys samples [21]. Isospora spp. infections are very common and have a worldwide distribution [22]. The prevalence of Isospora spp. DNA in Taihangshan macaques was quite high (28.38%). On the contrary, the previous study in nonhuman primates samples in China from 2009 to 2015 revealed a relatively lower (1.9%) prevalence rate for this protozoan [7]. Macaques in several regions of China and India have been reported to be infected with Cryptosporidium spp. [7,23,24], while in this study all macaques samples tested by PCR were negative. The negative results suggested that this protozoan is not an important parasite in rhesus macaques in this region.
The results from the present study confirm the local circulation of zoonotic bacterias, including Salmonella, Shigella, Escherichia coli, Campylobacter jejuni, and Yersinia. Infections of these pathogens were spread by the fecal-oral route or contacting with pathogen carriers, often of animal and wildlife origin, as well as consumption of contaminated food and water [27]. Shigella was the most regular pathogen in Enterobacteriaceae and could easily cause human and other animals infection. The samples prevalence of Shigella (31.66%) was higher than that reported by Banish et al. [28]. Nine monkeys with clinical manifestations of diarrhea were all positive for Shigella infection which suggested that Shigella was an important bacterial cause of diarrhea in this region. Different species of NHPs are known to be potential carriers of Salmonella, Escherichia coli, Campylobacter jejuni, Klebsiella pneumoniae, and Leptospira. Published estimates suggest that the samples prevalence of Leptospira was slightly higher in wild-caught vervet monkeys samples [29], while the prevalence of Escherichia coli was lower in wild chimpanzees samples [5]. The similar prevalence of Campylobacter spp. and Klebsiella pneumoniae has been documented in various investigations [30,31]. Even though wild Taihangshan macaques were not known to be exposed to Mycobacterium tuberculosis and Pasteurella multocida, rhesus monkeys are known to be infected with these bacteria. Importantly, wild Taihangshan macaques serving as a reservoir host could facilitate the spread of bacterial infection.
Hafnia, a gram-negative bacterium belonging to the Enterobacteriaceae family presently consists of at least two distinct relatedness groups (Hafnia alvei and Hafnia paralvei) [11,32]. Hafnia has a worldwide distribution including a variety of mammals, birds, reptiles, amphibians, fish, and foods [33]. Hafnia is responsible for infectious diseases in pullets, rats, and horses [34,35] and has been recovered from human clinical specimens, even though it may be opportunistic human pathogens [12,[36][37][38]. Gunthard and Pennekamp [36] performed a study on the clinical significance of Hafnia isolates from 61 patients. The results indicated that Hafnia was isolated from 57 patients (93.4%) with underlying illnesses. More importantly, Hafnia was found to be the sole etiologic agent of invasive disease in 3 patients characterized by septicemia or peritonitis. In our previous study, Hafnia was detected from sample of liver tissue with hemorrhage of a dead rhesus monkey (unpublished data). The present study firstly indicates the existence of Hafnia paralvei in NHPs in China. However, further studies are needed to isolate more strains and characterize the associations between the microbiological findings and clinical data of Hafnia paralvei in Taihangshan macaques.

Conclusion
In conclusion, the present study firstly provides a broader investigation of zoonotic pathogens in wild Taihangshan macaques in China, which detailed the presence and prevalence of bacteria and parasites. Our preliminary results demonstrate high prevalence and diversity of significant zoonotic infections amongst wild Taihangshan macaques. This baseline data provides valuable feedback for monitoring the potential transmission of zoonotic infections from wild rhesus macaques.

Data Availability
The epidemiological data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare no conflicts of interest.