Novel strains of Campylobacter cause diarrheal outbreak in Rhesus macaques (Macaca mulatta) of Kathmandu Valley

Campylobacter spp. is often underreported and underrated bacteria that present real health risks to both humans and animals, including non-human primates. It is a commensal microorganism of gastrointestinal tract known to cause gastroenteritis in humans. Commonly found in many wild animals including non-human primates (monkeys- Rhesus macaques) these pathogens are known to be a common cause of diarrhea in humans in many parts of developing and under developed countries. Rhesus macaques from the two holy sites in Kathmandu (Pashupati and Swoyambhu) were included in this cross-sectional study. Diarrheal samples of monkeys were analyzed to detect and characterize the pathogen using 16S rRNA-based PCR screening, followed by DNA sequencing and phylogenetic analysis. Out of a total 67 collected diarrheal samples, Campylobacter spp. were detected in the majority of the samples (n = 64; 96%). DNA sequences of the amplified PCR products were successfully obtained from 13 samples. Phylogenetic analysis identified Candidatus Campylobacter infans (n = 10, Kimura-2 parameter (K2P) pairwise distance values of 0.002287). Remaining three sequences might potentially belong to a novel Campylobacter species/sub-species- closely relating to known species of C. helviticus (K2P pairwise distance of 0.0267). Both Candidatus Campylobacter infans and C. helvitucus are known to infect humans and animals. Additionally, we also detected the bacteria in water and soil samples from the sites. Campylobacter spp. caused the 2018 diarrhea outbreak in Rhesus macaques in the Kathmandu valley. Campylobacter might be one of the important contributing pathogens in diarrheal outbreaks-both in humans and animals (monkeys) in Nepal. Due to close interactions of these animals with humans and other animals, One Health approach might be the most effective way to prevent and mitigate the threat posed by this pathogen.

Campylobacteria is one of the most underrated and under-researched pathogens that is suspected to cause most of diarrhea in Nepal. Zoonosis and reverse zoonosis capacity of campylobacteria has been reported in many literatures. Considering high interactions between monkeys and humans in two of the most visited holy sites in a highly dense metropolitan city of Kathmandu, this is the first study of its kind looking into resevoir, spillover and transmission of a pathogen like Campylobacter from One Health perspective.
We believe by investigating the Campylobacteria outbreak in monkeys, we provide valuable insight into zoonosis/ reverse-zoonosis of One Health concern in humans and wildlife (monkeys). Your estmeed journal with its open source access would be a right portal to dessiminate our findings. And we look forward to your review with positive response. Campylobacter spp. is often underreported and underrated bacteria that present real health risks to both 19 humans and animals, including non-human primates. It is a commensal microorganism of gastrointestinal 20 tract known to cause gastroenteritis in humans. Commonly found in many wild animals including non-21 human primates (monkeys-Rhesus macaques) these pathogens are known to be a common cause of diarrhea 22 in humans in many parts of developing and under developed countries. 23 Rhesus macaques from the two holy sites in Kathmandu (Pashupati and Swoyambhu) were included in this 24 cross-sectional study. Opportunistic diarrheal samples of monkeys were analyzed to detect and characterize 25 the pathogen using 16S rRNA-based PCR screening, followed by DNA sequencing and phylogenetic 26

analysis. 27
Out of a total 67 collected diarrheal samples, Campylobacter spp. were detected in the majority of the 28 samples (n=64; 96%). DNA sequences of the amplified PCR products were successfully obtained from 13 29 samples. Phylogenetic analysis identified Candidatus Campylobacter infans (n=10, Kimura-2 parameter 30 (K2P) pairwise distance values of 0.002287). Remaining three sequences might potentially belong to a 31 novel Campylobacter species/sub-species-closely relating to known species of C. helviticus (K2P pairwise 32 distance of 0.0267). Both Candidatus Campylobacter infans and C. helvitucus are known to infect humans 33 and animals. Additionally, we also detected the bacteria in water and soil samples from the sites. Campylobacter spp. is a zoonotic pathogen that is found in the gut flora of many species ranging from 43 domesticated to wild animals-both free roaming and in captivity. It is capable of infecting humans as well 44 as non-human primates (NHP) causing mild to severe gastrointestinal problems [1][2][3]. Emergence of 45 antibiotic-resistant strains of Campylobacter is a major public health concern as animals carrying the 46 bacteria pose a significant risk to humans via contamination of water sources, food, or through repeated Kathmandu Metropolitan city, Nepal (Image was created using QGIS).

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Both sites were divided into 5 transects, and 5 field teams were mobilized to systematically comb through 95 each transect and collect only diarrheal (loose) fecal samples from monkeys. Feces were collected using 96 sterile swabs in a tube containing phosphate-buffered saline (PBS). A portion of the feces were also 97 collected in silica gel tubes as replicate. Additionally, adjacent soil and water samples from a drinking water 98 source (pond) were also collected to check for any possible environmental contamination. The samples 99 were immediately transported in cold-chain to our lab in Kathmandu and stored at -20 o C freezer for further 100 processing. A total of 67 opportunistic diarrheal fecal samples and surrounding soil samples (n=11) were 101 collected from both the sites. Some water samples (n=5, river and drinking water sources) were also 102 collected. 103

Sequencing and Phylogenetic Analysis 150
Out of the 64 samples, only 13 provided acceptable quality of 16S DNA sequences, which was used to 151 conduct phylogenetic analysis to resolve taxonomy of the Campylobacter isolates. 152 The topology of the phylogenetic tree constructed showed that different species of Campylobacter spp. 153 clustering together in distinct clades. The isolates having same host species grouped together in a same 154 clade with some exceptions. Our isolates clustered into distinct two clades identified-Clade-1 and Clade-2 155 The detection and diagnosis of Campylobacter infection has been challenging due to inefficiency in widely 184 used culture-based detection [41]. However, availability of molecular based diagnostic techniques have 185 proved effective in detecting bacterial species that are normally difficult to culture [42,43]. This might be 186 the first study in Nepal where Campylobacter is directly detected in macaque feces. The Campylobacter 187 was detected in water and soil samples as well, which could increase pathogen spillover possibility amongst 188 various species intermingling at the sites [44]. 189 The phylogenetic analysis of 16S rRNA gene sequences of Campylobacter showed presence of two clades 190 of the bacteria (Fig. 3). The phylogenetic tree in our study also revealed more or less identical 191 ( Supplementary Fig. 4) structure of bacteria clades as found in the study published by Wilkinson D. A., 192 et.al. (2018) [45]. That study used more elaborate whole genome sequence data, and the fact that our study 193 also produced similar Campylobacter clade structure using only 16S rRNA fragment sequence data, 194 validates the utility and accuracy of our technique. 195 The clade-1 isolates clustered together with the newly reported species of Campylobacter (Candidatus C. 196 infans), though neighboring clades consisted of other species as well (Fig. 3). However, these isolates of 197 other species in clade-1 could have been misidentified by the authors, as the K2P distances of isolates found 198 in clade-1 have close distances to Candidatus C. infans, (Supplementary Figure 2) inferring that all isolates 199 in clade-1 could possibly be identified as this newly discovered species. Previous cases of Candidatus C. 200 infans have been isolated from infants from sub-Saharan Africa and South Asia, and from a male (human) 201 in the Netherlands [46]. The Campylobacter spp. isolated in this study all originated from monkey fecal 202 samples, which raises a serious concern over zoonotic capability of these particular isolates [47,48]. 203 Furthermore, an isolate from India found in clade-1 was isolated from sheep alluding to strong evidence to 204 support zoonotic plasticity. 205 Clade-2 contained the three remaining sequences from this study. The clustering of C. helveticus, C. 206 upsaliensis and C. troglodytes in a nearby clades could also be observed as sister clades to clade-2 isolates. 207 Our three isolates formed separate monophyletic clades (Fig. 3) within clade-2 indicating the findings of a 208 probable new species or sub-species of the Campylobacter, which was further supported by the K2P 209 pairwise distance with the closest distance of 0.0267 being with C. helveticus, which is similar to the values 210 of clade-1 isolates against C. hyointestinalis subsp. lawsonii (HQ628645). However, further investigation 211 with a longer 16S rRNA gene fragment or whole genome sequences may be required to properly verify the 212 inferred result. 213 Samples were collected from two different sites separated by dense human settlements-almost making 214 them wooded islands within the urban landscape. The Campylobacter isolates from both the sites clustered 215 together in both clade-1 and clade-2 (Fig. 3). This result suggests there might be some complex interactions 216 taking place between these two animal populations. Since monkeys from these two sites rarely intermingle, 217 the disease spread might be limited as well. However, humans might help spread Campylobacter between 218 these two populations of monkeys. 219 C. hyointestinalis is a commensal organism of pigs whereas C. helveticus is commensal in dogs and cats 220 [45]. Campylobacter can potentially infect monkeys from these animals, and/or through humans as 221 intermediate hosts. Considering other species such as birds, cats and dogs are also interacting closely with 222 the monkeys at these two sites, the Campylobacter reservoir, spillover and transmission are truly playing 223 out in One Health dynamics. Hence, this study highlights the importance of One Health approach to 224 understand and prevent emerging, re-emerging and prevalent infectious diseases. 225 Diarrheal diseases are one of the most devastating public health concerns in Nepal, especially in a densely 226 populated metropolitan cities like Kathmandu. Campylobacter might be one of the important contributing 227 pathogens in diarrheal outbreaks-both in humans and animals (monkeys). We hope that our study will draw 228 attention to this problem and help public health experts in formulating a plan to cure and prevent this kind 229 of outbreak in macaques, thereby, preventing spillover to humans. 230

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DNA sequences are available in the NCBI Genbank with accession number MZ06810 to MZ068112. All 232 the data are included in manuscript including supplementary information. 233