Enzootic Activity of Chlamydia in Farms Located in a Hotspot Area for Zoonosis Emergence

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Mosmann, Adrián Díaz, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1014196/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Chlamydias are obligated intracellular Gram-negative bacteria, considered important zoonotic pathogens, broadly present in several bird species and responsible for economic losses in animal production. We analyzed the presence of Chlamydial species with zoonotic risk in farm animals in a highly biodiverse area and with great human circulation, the Argentine, Brazil and Paraguay tri-border area. We surveyed nine farms in an area and nasally swabbed a total of 62 animals. DNA was extracted and specific PCR was performed to identify chlamydial species. We detected Chlamydia spp . in 6.5% (4/62) of the animals tested, positive samples belonged to cattle and none of them showed symptoms of respiratory disease nor had been diagnose with reproductive diseases. Specific nested PCR confirmed two samples belonged to C. pecorum and two to C. psittaci . We report for the first time Chlamydia circulation with zoonotic risk in the region. Surveys in birds and wild mammals could give a better understanding to know what Chlamydial species are circulating in the wild interface. The zoonotic potential should be taking into account as farm workers and the surrounding population could be silent carriers or have respiratory diseases being underdiagnosed, and therefore should be considered in the differential diagnoses. Veterinary Epidemiology Chlamydia pecorum Chlamydia psittaci Zoonotic Diseases Livestock Argentine-Brazilian- Paraguay border. Figures Figure 1 Figure 2 Introduction Chlamydias are obligated intracellular Gram-negative bacteria, which can cause serious infections both in humans and animals, such as birds and cattle. In the latter, it causes abortions, infertility, conjunctivitis, enteritis, respiratory diseases among others, thus leading to economic losses in productivity. In humans, it is associated with sexually transmitted infections (STIs) and respiratory diseases (Whittum-Hudson & Hudson AP. 2005; Mackern-Oberti et al., 2013) They belong to the family Chlamydiaceae that includes only the genus Chlamydia; containing 15 known species ( C. trachomatis , C. avium , C. buteonis , C. caviae , C. felis , C. gallinacean , C. ibidis , C. muridarum , C. poikilothermis , C. serpentis , C. suis , C. abortus , C. psittaci , C. pecorum and C. pneumoniae ). The last three species have proven to be zoonotically relevant (Longbottom & Coulter 2003; Rohde et al., 2010; Staub et al., 2018) . Chlamydia psittaci is the most widely studied in avian and human populations and was first detected in 1879, in Switzerland, in a family that traded parrots from South America (Harris & Williams 1985). It causes human severe respiratory conditions that can lead to death. Infections in humans initiates by the inhalation of products from fluids of infected birds or by handling products derived from poultry (Vanrompay et al., 2007; Balsamo et al., 2017). In Argentina, reports of this disease dates from the beginning of the last century and new outbreaks are regularly recorded (Frutos et al., 2012a; Cadario et al., 2017). Unlike C. psittaci , the description of C. pneumoniae and C. pecorum dates from the early 1990s. At first it was thought that C. pneumoniae only affected humans, but over time studies proved its zoonotic potential (Mitchell et al., 2010; Roulis et al., 2013) . In humans, it causes respiratory infections in adults and children, and at least 70% of the population may eventually become expose to this pathogen (Kuo et al., 1995). Although its zoonotic potential is still under research, C. pecorum is an important pathogen of domestic livestock, mainly sheep and cows and is relevant to the conservation of koala ( Phascolarctos cinereus ) populations in Australia (Polkinghorne et al., 2013). Often asymptomatic, C. pecorum can cause polyarthritis, conjunctivitis, pneumonia, miscarriages, encephalomyelitis and gastrointestinal problems, thus, leading to economic losses (Jelocnik et al., 2015). Though studies are scarce in Argentina, it was detected in birds seized from illegal trafficking in the province of Córdoba (Frutos et al., 2012b). Nonetheless, it is necessary to expand the geographical scope of research and the range of hosts that can act as reservoirs. In the last decade several studies were carried out on the circulation of pathogens with zoonotic relevance in the triple border between Argentina, Brazil and Paraguay (Rivero et al., 2017; Thomaz-Soccol et al., 2018; Valente et al., 2019). This region is a potential hotspot for zoonotic diseases emergence due to the massive interchange of people driven by trade and tourism activities around the Iguazu Falls (Scarpaci 2012). Thus, the area host several protected areas of remnants of Atlantic Forest with a high biodiversity of fauna and flora (Di Bitetti et al., 2003; Galindo-Leal & Câmara 2003 ). Moreover, in all three countries, land-use change has increased due to the expansion of agricultural activities, generating tensions over land tenure and increasing socio-economic inequalities among local residents (Galeano, 2012). Under this scenario, the aim of this work was to study the circulation of Chlamydias of zoonotic relevance in farm animals on the Argentine side of the triple border. Materials And Methods Study site and sample collection. Field sampling was carried out in nine farms located in the province of Misiones, Argentina, in the border of Brazil and Paraguay (25°40′13″S 54°02′34″W) (Figure 1). This rural area limits with two major protected areas, Iguazu National Park in Argentina, and the Iguaçu National Park in Brazil. We involved local stakeholders by identification a first key stakeholder that then, allowed contact with the other owners in the area. We informed the farm owners involved about the project and that all information provided was anonymous and confidential (e.g., the exact location of their properties). Participation was voluntary and they could withdraw with no explanation. Finally, taking into account the guidelines of the International Society of Ethnobiology-ISE (2006), we informed the results and conclusions of the project (ISE 2006). Field work was carried out in 9 farms from September to November 2018. Nasal swabs were obtained with brush-tipped swabs, making circular movements during 10 seconds, and placed them in a 1ml tube containing SPG (sucrose–phosphate–glutamic acid buffer). Figure 1. Map illustrating the study area, the different land uses and farms sampled and those that tested positive for Chlamydia. DNA extraction We subjected 200 µl of sample swabs to DNA extraction using the DNeasy Blood & Tissue KitQIAGEN® (Cat No. /ID: 69506) (QIAGEN) commercial kit, following the manufacturer's specifications. DNA extracted from the L2/434Bu strain of Chlamydia trachomatis was used as a positive control. The extracted DNA was stored at 4°C. Generic polymerase chain reaction for Chlamydia spp. The DNA obtained was first subjected for the detection of a 576 bp fragment of the variable domains II, III and IV of the ompA gene of Chlamydia. The process was performed according to Frutos et al., (2015). Oligonucleotides were selected according to Sachse and Hotzel (2003). The selected external oligonucleotides primers were 191CHOMP (GCI YTI TGG GAR TGY GGI TGY GCI AC) and CHOMP 371 (TTA GAA ICK GAA TTG IGC RTT IAY GTG IGC IGC). DNA fragments were amplified (5 µl) by adding 0.2 mM of the respective primers, 0.8 mM of dNTPs and 1 unit of enzyme Gotaq polymerase (Invitrogen, Life Technologies, Carisbad, CA) in final volume 50 µl. We used a C. trachomatis strain L2c (CP002024) from the Chlamydia laboratory of the "Dr. J. M. Vanella" Institute of Virology as positive control. Nested-PCR for the determination of C. psittaci, C. pneumoniae and C. pecorum Based on the positive results of the generic PCR we performed this procedure, as described by Frutos et al., (2015). We used 2 µl of PCR I product and 0.2 mM of the respective internal primers, 0.8 mM of dNTPs and 1 unit of enzyme Gotaq polymerase (Invitrogen, Life Technologies, Carisbad, CA) were added in final volume 50 µl. Chlamydia psittaci strain VS225 (CP003793), C. pecorum strain 2047 (GQ228191) and C. pneumoniae strain TWAR 183 from the Chlamydia laboratory of the Institute of Virology "Dr. J. M. Vanella" were used as positive controls. Visualization of the DNA fragments obtained by nested-PCR Amplified DNA fragment were separate by electrophoresis in 1.5% agarose gel containing 0.5 ul/ml of ECO-Gel 20.000X Highway dye and visualized through an ultra-violet transilluminator (Sigma). Sequencing and Phylogenetic analysis PCR products were purified by using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, US) and subjected to direct nucleotide sequencing Sanger reaction in both directions in Macrogen, Inc. (Seoul, Korea). Alignment of nucleotide sequences was performed with MEGA 6.0 software package (Kumar et al., 2018). Phylogenetic trees were generated using the Maximum Likelihood method with MEGA 6.0 software package. A bootstrap re‐sampling analysis was performed (1,000 replicates) to test tree robustness. The reference strains used for the phylogenetic trees were obtained from the NCBI GenBank Database. Nine reference sequences of the ompA region of the C. pecorum genome (403 bp) were included in this analysis. The corresponding accession numbers, country of origin, host and clinical condition of these isolates are shown in Table 1. Results A total of 62 nasal swabs were obtained from two horses, nine pigs and 51 cows. Chlamydia spp. infection were obtained in 6.5% (4/62) of the animals sampled. All positive samples belonged to cattle from two of the nine sampled farms (Figure 1)). Specific nested PCR confirmed the occurrence of C. pecorum (n = 2) and C. psittaci (n = 2). In farm "A" 1 out of 10 animals tested positive for C. psittaci and in farm "J" 3 out of 7 animals tested positive, two for C. pecorum and one for C. psittaci . None of the samples were positive for Chlamydia pneumoniae. Genetic diversity and relationships of isolated C. pecorum and C. psittaci strains were analyzed by sequencing ompA and rpoB genes. Obtained sequences were deposited in GenBank under the following accession numbers: MW888425 and MW888425 for the ompA region of the C. psittaci, MW888427 and MW888428 for the ompA region of the C. pecorum. Chlamydia psittaci isolated strains (J006 and A003) grouped with strains previously described in both birds and mammals from different regions of the world as well as with strains identified in the central region of Argentina (Figure 2). Chlamydia pecorum strains (J005 and J008) grouped together with strains isolated from birds in Argentina and different mammals such as livestock and koalas (Figure 2). Discussion In cattle, C. pecorum and C. psittaci can generate subclinical infections or respiratory symptoms, encephalomyelitis or genital sexual-borne infections, affecting fertility, as well as miscarriages with great losses in production (Li et al., 2016; Barati et al., 2017). Although there are reports of Chlamydia pecorum in humans, its zoonotic potential is yet to be understood (Frutos et al., 2015). Some authors suggest that C. pecorum infection is endemic in livestock worldwide, but prevalence is yet unclear, since veterinary chlamydial diagnostics are limited to study genus only (Sachse et al., 2009; Walker et al., 2015). Li et al., (2016) reported prevalences of several chlamydia species from both dairy and beef cattle production in China. Although in their research they did not test nasal swabs, they tested whole blood, vaginal swabs, feces and milk. In their study they reported high prevalence of C. pecorum in fecal samples, indicating C. pecorum as an endemic species optimally adapted to cattle, so it easily spreads throughout the intestinal tract. Our findings in nasal mucosa could indicate that C. pecorum it is not only present in intestinal tract; suggesting the aerial transmission, and support the idea of a prevalent pathogen in cattle worldwide. Moreover, several authors refer to the fact that C. pecorum may be asymptomatic in cattle, as our findings show (Reinhold et al., 2008; Poudel et al., 2012; Li et al., 2016). In cattle, C. psittaci causes low milk productivity, respiratory disease, abortions, and its pathogenicity has been demonstrated experimentally (Borel et al., 2006; Ostermann et al., 2013; Van Loo et al., 2014). However, it is likely that C. psittaci infections in cattle occur in sporadic events, due to possible interactions with birds (Li et al., 2016). Li et al., (2016) reports a much lower prevalence (10%), comparing to C. pecorum (75%). In their study, sequenced strains had a genetic similarity close to pigeons. In that sense the circulation of C. psittaci is documented for several bird species in Argentina (Frutos et al., 2016). Although C. psittaci infection is mainly associated with birds, the findings of this work indicate that mammals are also a source of C. psittaci and may be carriers of strains associated with birds, as proposed by Frutos et al., (2014). While our study was restricted to farm animals, it is important to highlight that the study site is located in the vicinity to an area of high wildlife conservation value, the Iguazu National Park in Argentina and the Iguaçu National Park in Brazil. To date we do not know which species of Chlamydia could affect the local fauna and what their relevance could be at the sanitary and conservation level. In the farms surveyed, many wild species frequent the area, and may have contact with domestic animals. This interaction (domestic/wild hosts) can result in the transmission of pathogens in both ways, by spillover from wildlife to domestic animals or vice versa. As an example, in Australia it has become a huge conservation issue for the koala ( Phascolarctos cinereus ) populations since the introduction of C. pecorum from infected livestock, causing in koalas ocular and reproductive disorders and thus, increasing mortality rates (Polkinghorne et al., 2013; Bachmann et al., 2014). Regarding C. psittaci , birds living in protected areas frequently visit farms, such as parrots and toucans, due to food availability (fruit trees, seeds and palms) and the scarce presence of predators. Cattle can be infected either by ingesting contaminated pastures by bird feces or seeds that they discard during the flight, making it difficult to control and eradicate the disease in the herds. In farming areas close to natural reserves, monitoring chlamydial species that affect wildlife can be an important tool to have a better understanding of the occurrence and potential of emergence of this pathogen in the area. Regarding its zoonotic potential, it is necessary to analyze samples from farmers that may or may not have clinical manifestations of respiratory disease, being the infections under-diagnosed, and that are in close contact with the potential cattle reservoir of Chlamydia spp. In this study, the animals that tested positive and its owners did not show symptoms of respiratory disease, nor have they mentioned cases of recent abortions by the cattle. This is the first study that detected the presence of two species of Chlamydia in the same study area in the triple border area of Argentina, Brazil and Paraguay: Chlamydia pecorum and C. psittaci , and the first report of C. pecorum in bovines for Argentina. The phylogenetic analysis of the strains detected in our study shows a genetic closeness to strains previously detected in central Argentina, which also indicates a regional clustering. Our study provides the basis to deepen the circulation of chlamydia in the area and to understand which animal species could be its hosts and amplifiers. The chlamydial species found are of zoonotic risk and therefore should be included as differential diagnoses in cases of respiratory symptoms in humans. Monitoring birds and poultry in the proximity of the farms is strongly suggested to assess Chlamydial circulation. As in cattle, these diseases should be considered as differential diagnosis in spontaneous abortions or reproductive diseases. Therefore, it is necessary to extend surveillance and deepen studies on chlamydial species involved in animal pathology and their zoonotic potential, since information in the region is scarce. Declarations ACKNOWLEDGMENTS We are grateful to all the farmers of the Andresito and Maria Soledad community who willingly participated and volunteered their time to support the study. We thank Dr. Julia Martinez Pardo for her contribution to generate the maps. This work was supported by the National Scientific and Technical Research Council (CONICET). Author contributions Conceptualization and project design: EV, AD and CC. Field work: EV. Laboratory analysis EV and CC. Bioinformatics analysis CC and JM. Manuscript draft and review EV, JM, CC and AD. CC and AD were the general advisors of the project and contributed equally as the corresponding authors. Availability of data and material: The corresponding author declares that data are available upon request and will be place in an open public repository of CONICET Code availability : Not applicable. Funding Secretariat of Science and Technology/National University of Cordoba Proyecto SeCyT –UNC Cat A. 2018/22 Mincyt Res N°019/17. Ethical approval : Guidelines for the care and use of animals were followed by the ICLAS Ethical Guideline for Researchers. 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Dis., 13(7), 1108. doi: 10.3201/eid1307.070074 Walker E, Lee EJ, Timms P, Polkinghorne A (2015) Chlamydia pecorum infections in sheep and cattle: a common and under-recognised infectious disease with significant impact on animal health.Vet. J., 206(3), 252-260. https://doi.org/10.1016/j.tvjl.2015.09.022 Whittum-Hudson JA, Hudson AP (2005) Human chlamydial infections: persistence, prevalence, and prospects for the future. Nat. Sci. Soc.;13:371–82. https://doi.org/10.1051/nss:2005057 Tables Table 1. Chlamydial strains used in partial ompA DNA sequencing in our study. Chlamydia strain Host Clinical Signs Country origin GenBank accesion Reference C.psittaci Equus caballus Aborted foetus Australia KY287781 Jelocnik et al., 2017 Melopsittacus undulatus Systemic infection Not reported M73035 Kaltenboeck et al., 1993 Amazona aestiva Hepatic disease Brazil MH138293 Vilela et al., 2019 Meleagris gallopavo Nasal discharge Belgium AY762609 Geens et al., 2005 Bos taurus No clinical signs Argentina MW888425 This study Bos taurus No clinical signs Argentina MW888425 This study Diuca diuca No clinical signs Argentina JX399853 Jelocnik et al., 2017 Paroaria coronata No clinical signs Argentina JX399854 Kaltenboeck et al., 1993 Bos taurus Enteritis USA AF269269 Vilela et al., 2019 epizootic Not reported USA AF269268 Geens et al., 2005 Bos taurus Pneumonia Germany EU350138 Jelocnik et al., 2017 C.pecorum Bos Taurus Diarrhea Japan LC021422 Kaltenboeck et al., 1993 Paroaria coronata No clinical signs Argentina JN016882 Vilela et al., 2019 Bos taurus No clinical signs Argentina MW888427 This study Bos taurus No clinical signs Argentina MW888428 This study Gubernatrix cristata No clinical signs Argentina JN016884 Frutos et al., 2015 Capra aegagrus hircus No clinical signs France EU684933 Mohamad et al., 2008 Bos taurus Diarrhea Japan LC021419 Ohtani et al., 2015 Capra aegagrus hircus No clinical signs France EU684932 Mohamad et al., 2008 Phascolarctos cinereus Urogenital infection Australia KU214244 Legione et al., 2016 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Mosmann","email":"","orcid":"","institution":"CONICET: Consejo Nacional de Investigaciones Cientificas y Tecnicas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"P.","lastName":"Mosmann","suffix":""},{"id":68868215,"identity":"0a933010-8482-4bbb-826e-4106727ed771","order_by":2,"name":"Adrián Díaz","email":"","orcid":"","institution":"CONICET: Consejo Nacional de Investigaciones Cientificas y Tecnicas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Adrián","middleName":"","lastName":"Díaz","suffix":""},{"id":68868216,"identity":"0a1b4c48-91de-4371-81b1-1ed6d6c3c1f6","order_by":3,"name":"Cecilia G. Cuffini","email":"","orcid":"","institution":"CONICET: Consejo Nacional de Investigaciones Cientificas y Tecnicas","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Cecilia","middleName":"G.","lastName":"Cuffini","suffix":""}],"badges":[],"createdAt":"2021-10-25 02:08:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1014196/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1014196/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":16359442,"identity":"724dfcec-7b9a-4702-9eec-5fad0fa526db","added_by":"auto","created_at":"2021-12-10 20:56:33","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5663513,"visible":true,"origin":"","legend":"Map illustrating the study area, the different land uses and farms sampled and those that tested positive for Chlamydia.","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-1014196/v1/81607e2a45cf696055f05616.png"},{"id":16359441,"identity":"af8f441c-9b11-462b-85a4-522f0fd30662","added_by":"auto","created_at":"2021-12-10 20:56:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":165429,"visible":true,"origin":"","legend":"Numbers above branches are bootstrap values as a percentage of 1000 pseudo replicates with Maximum Likelihood method. C. pecorum C18-98 was used as a root group. Scale bar shows the percentage sequence diversity. ","description":"","filename":"Figure2.Vanderhoevenetal2021.png","url":"https://assets-eu.researchsquare.com/files/rs-1014196/v1/403b9da2c46a7159e925ff9b.png"},{"id":17050268,"identity":"f40d2c25-5619-4a39-8397-46dace3dcac0","added_by":"auto","created_at":"2022-01-06 10:46:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1683682,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1014196/v1/52794048-0e07-4dc8-bcff-79e892baaece.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eEnzootic Activity of Chlamydia in Farms Located in a Hotspot Area for Zoonosis Emergence\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChlamydias are obligated intracellular Gram-negative bacteria, which can cause serious infections both in humans and animals, such as birds and cattle. In the latter, it causes abortions, infertility, conjunctivitis, enteritis, respiratory diseases among others, thus leading to economic losses in productivity. In humans, it is associated with sexually transmitted infections (STIs) and respiratory diseases (Whittum-Hudson \u0026amp; Hudson AP. 2005; Mackern-Oberti et al., 2013)\u003c/p\u003e\n\u003cp\u003eThey belong to the family Chlamydiaceae that includes only the genus Chlamydia; containing 15 known species (\u003cem\u003eC. trachomatis\u003c/em\u003e, \u003cem\u003eC. avium\u003c/em\u003e, \u003cem\u003eC. buteonis\u003c/em\u003e, \u003cem\u003eC. caviae\u003c/em\u003e, \u003cem\u003eC. felis\u003c/em\u003e, \u003cem\u003eC. gallinacean\u003c/em\u003e, \u003cem\u003eC. ibidis\u003c/em\u003e, \u003cem\u003eC. muridarum\u003c/em\u003e, \u003cem\u003eC. poikilothermis\u003c/em\u003e, \u003cem\u003eC. serpentis\u003c/em\u003e, \u003cem\u003eC. suis\u003c/em\u003e, \u003cem\u003eC. abortus\u003c/em\u003e, \u003cem\u003eC. psittaci\u003c/em\u003e, \u003cem\u003eC. pecorum\u003c/em\u003e and \u003cem\u003eC. pneumoniae\u003c/em\u003e). The last three species have proven to be zoonotically relevant (Longbottom \u0026amp; Coulter 2003; Rohde et al., 2010; Staub et al., 2018) . \u003cem\u003eChlamydia psittaci\u003c/em\u003e is the most widely studied in avian and human populations and was first detected in 1879, in Switzerland, in a family that traded parrots from South America (Harris \u0026amp; Williams 1985). It causes human severe respiratory conditions that can lead to death. Infections in humans initiates by the inhalation of products from fluids of infected birds or by handling products derived from poultry (Vanrompay et al., 2007; \u0026nbsp; Balsamo et al., 2017). In Argentina, reports of this disease dates from the beginning of the last century and new outbreaks are regularly recorded (Frutos et al., 2012a; Cadario et al., 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUnlike \u003cem\u003eC. psittaci\u003c/em\u003e, the description of \u003cem\u003eC. pneumoniae\u003c/em\u003e and \u003cem\u003eC. pecorum\u003c/em\u003e dates from the early 1990s. At first it was thought that \u003cem\u003eC. pneumoniae\u003c/em\u003e only affected humans, but over time studies proved its zoonotic potential (Mitchell et al., 2010; Roulis et al., 2013) . In humans, it causes respiratory infections in adults and children, and at least 70% of the population may eventually become expose to this pathogen (Kuo et al., 1995).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlthough its zoonotic potential is still under research, \u003cem\u003eC. pecorum\u003c/em\u003e is an important pathogen of domestic livestock, mainly sheep and cows and is relevant to the conservation of koala (\u003cem\u003ePhascolarctos cinereus\u003c/em\u003e) populations in Australia (Polkinghorne et al., 2013). Often asymptomatic, \u003cem\u003eC. pecorum\u003c/em\u003e can cause polyarthritis, conjunctivitis, pneumonia, miscarriages, encephalomyelitis and gastrointestinal problems, thus, leading to economic losses (Jelocnik et al., 2015). Though studies are scarce in Argentina, it was detected in birds seized from illegal trafficking in the province of C\u0026oacute;rdoba (Frutos et al., 2012b). Nonetheless, it is necessary to expand the geographical scope of research and the range of hosts that can act as reservoirs.\u003c/p\u003e\n\u003cp\u003eIn the last decade several studies were carried out on the circulation of pathogens with zoonotic relevance in the triple border between Argentina, Brazil and Paraguay (Rivero et al., 2017; Thomaz-Soccol et al., 2018; Valente et al., 2019). This region is a potential hotspot for zoonotic diseases emergence due to the massive interchange of people driven by trade and tourism activities around the Iguazu Falls (Scarpaci 2012). Thus, the area host several protected areas of remnants of Atlantic Forest with a high biodiversity of fauna and flora (Di Bitetti et al., 2003; Galindo-Leal \u0026amp; C\u0026acirc;mara 2003 ). Moreover, in all three countries, land-use change has increased due to the expansion of agricultural activities, generating tensions over land tenure and increasing socio-economic inequalities among local residents (Galeano, 2012). Under this scenario, the aim of this work was to study the circulation of Chlamydias of zoonotic relevance in farm animals on the Argentine side of the triple border.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003ch3\u003eStudy site and sample collection.\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eField sampling was carried out in nine farms located in the province of Misiones, Argentina, in the border of Brazil and Paraguay (25\u0026deg;40\u0026prime;13\u0026Prime;S 54\u0026deg;02\u0026prime;34\u0026Prime;W) (Figure 1). This rural area limits with two major protected areas, Iguazu National Park in Argentina, and the Igua\u0026ccedil;u National Park in Brazil. We involved local stakeholders by identification a first key stakeholder that then, allowed contact with the other owners in the area. We informed the farm owners involved about the project and that all information provided was anonymous and confidential (e.g., the exact location of their properties). Participation was voluntary and they could withdraw with no explanation. \u0026nbsp;Finally, taking into account the guidelines of the International Society of Ethnobiology-ISE (2006), we informed the results and conclusions of the project (ISE 2006).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eField work was carried out in 9 farms from September to November 2018. Nasal swabs were obtained with brush-tipped swabs, making circular movements during 10 seconds, and placed them in a 1ml tube containing SPG (sucrose\u0026ndash;phosphate\u0026ndash;glutamic acid buffer).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 1. Map illustrating the study area, the different land uses and farms sampled and those that tested positive for Chlamydia.\u003c/p\u003e\n\u003ch3\u003eDNA extraction\u003c/h3\u003e\n\u003cp\u003eWe subjected 200 \u0026micro;l of sample swabs to DNA extraction using the DNeasy Blood \u0026amp; Tissue KitQIAGEN\u0026reg; (Cat No. /ID: 69506) (QIAGEN) commercial kit, following the manufacturer\u0026apos;s specifications. DNA extracted from the L2/434Bu strain of \u003cem\u003eChlamydia trachomatis\u003c/em\u003e was used as a positive control. The extracted DNA was stored at 4\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneric polymerase chain reaction for\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eChlamydia\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;spp.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DNA obtained was first subjected for the detection of a 576 bp fragment of the variable domains II, III and IV of the ompA gene of Chlamydia. The process was performed according to Frutos et al., (2015). Oligonucleotides were selected according to Sachse and Hotzel (2003). The selected external oligonucleotides primers were 191CHOMP (GCI YTI TGG GAR TGY GGI TGY GCI AC) and CHOMP 371 (TTA GAA ICK GAA TTG IGC RTT IAY GTG IGC IGC). DNA fragments were amplified (5 \u0026micro;l) by adding 0.2 mM of the respective primers, 0.8 mM of dNTPs and 1 unit of enzyme Gotaq polymerase (Invitrogen, Life Technologies, Carisbad, CA) in final volume 50 \u0026micro;l. We used a \u003cem\u003eC. trachomatis\u0026nbsp;\u003c/em\u003estrain L2c (CP002024) from the Chlamydia laboratory of the \u0026quot;Dr. J. M. Vanella\u0026quot; Institute of Virology as positive control.\u003c/p\u003e\n\u003ch4\u003eNested-PCR for the determination of\u0026nbsp;\u003cem\u003eC. psittaci, C. pneumoniae\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;C. pecorum\u003c/em\u003e\u003c/h4\u003e\n\u003cp\u003eBased on the positive results of the generic PCR we performed this procedure, as described by Frutos et al., (2015). We used 2 \u0026micro;l of PCR I product and 0.2 mM of the respective internal primers, 0.8 mM of dNTPs and 1 unit of enzyme Gotaq polymerase (Invitrogen, Life Technologies, Carisbad, CA) were added in final volume 50 \u0026micro;l.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChlamydia psittaci\u003c/em\u003e strain VS225 (CP003793), \u003cem\u003eC. pecorum\u003c/em\u003e strain 2047 (GQ228191) and \u003cem\u003eC. pneumoniae\u003c/em\u003e strain TWAR 183 from the Chlamydia laboratory of the Institute of Virology \u0026quot;Dr. J. M. Vanella\u0026quot; were used as positive controls.\u003c/p\u003e\n\u003ch4\u003eVisualization of the DNA fragments obtained by nested-PCR\u003c/h4\u003e\n\u003cp\u003eAmplified DNA fragment were separate by electrophoresis in 1.5% agarose gel containing 0.5 ul/ml of ECO-Gel 20.000X Highway dye and visualized through an ultra-violet transilluminator (Sigma).\u003c/p\u003e\n\u003ch4\u003eSequencing and Phylogenetic analysis\u003c/h4\u003e\n\u003cp\u003ePCR products were purified by using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, US) and subjected to direct nucleotide sequencing Sanger reaction in both directions in Macrogen, Inc. (Seoul, Korea).\u003c/p\u003e\n\u003cp\u003eAlignment of nucleotide sequences was performed with MEGA 6.0 software package (Kumar et al., 2018). Phylogenetic trees were generated using the Maximum Likelihood method with MEGA 6.0 software package. A bootstrap re‐sampling analysis was performed (1,000 replicates) to test tree robustness. The reference strains used for the phylogenetic trees were obtained from the NCBI GenBank Database.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNine reference sequences of the ompA region of the \u003cem\u003eC. pecorum\u003c/em\u003e genome (403 bp) were included in this analysis. The corresponding accession numbers, country of origin, host and clinical condition of these isolates are shown in \u0026nbsp;Table 1.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 62 nasal swabs were obtained from two horses, nine pigs and 51 cows.\u0026nbsp;\u003cem\u003eChlamydia\u003c/em\u003e spp. infection were obtained in 6.5% (4/62) of the animals sampled. All positive samples belonged to cattle from two of the nine sampled farms (Figure 1)). Specific nested PCR confirmed the occurrence of \u003cem\u003eC. pecorum\u003c/em\u003e (n = 2) and \u003cem\u003eC. psittaci\u003c/em\u003e (n = 2). In farm \u0026quot;A\u0026quot; 1 out of 10 animals tested positive for \u003cem\u003eC. psittaci\u003c/em\u003e and in farm \u0026quot;J\u0026quot; 3 out of 7 animals tested positive, two for \u003cem\u003eC. pecorum\u0026nbsp;\u003c/em\u003eand one for \u003cem\u003eC. psittaci\u003c/em\u003e. None of the samples were positive for\u0026nbsp;\u003cem\u003eChlamydia pneumoniae.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGenetic diversity and relationships of isolated\u0026nbsp;\u003cem\u003eC. pecorum\u003c/em\u003e and\u0026nbsp;\u003cem\u003eC. psittaci\u003c/em\u003e strains were analyzed by sequencing ompA and rpoB genes. Obtained sequences were deposited in GenBank under the following accession numbers: MW888425 and MW888425 for the ompA region of the\u0026nbsp;\u003cem\u003eC. psittaci,\u003c/em\u003e MW888427 and MW888428 for the ompA region of the\u0026nbsp;\u003cem\u003eC. pecorum.\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChlamydia psittaci\u003c/em\u003e isolated strains (J006 and A003) grouped with strains previously described in both birds and mammals from different regions of the world as well as with strains identified in the central region of Argentina (Figure 2).\u0026nbsp;\u003cem\u003eChlamydia pecorum\u003c/em\u003e strains (J005 and J008) grouped together with strains isolated from birds in Argentina and different mammals such as livestock and koalas (Figure 2).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn cattle, \u003cem\u003eC. pecorum\u003c/em\u003e and \u003cem\u003eC. psittaci\u003c/em\u003e can generate subclinical infections or respiratory symptoms, encephalomyelitis or genital sexual-borne infections, affecting fertility, as well as miscarriages with great losses in production (Li et al., 2016; Barati et al., 2017). Although there are reports of \u003cem\u003eChlamydia pecorum\u003c/em\u003e in humans, its zoonotic potential is yet to be understood (Frutos et al., 2015). Some authors suggest that \u003cem\u003eC. pecorum\u003c/em\u003e infection is endemic in livestock worldwide, but prevalence is yet unclear, since veterinary chlamydial diagnostics are limited to study genus only (Sachse et al., 2009; Walker et al., 2015).\u003c/p\u003e\n\u003cp\u003eLi et al., (2016) reported prevalences of several chlamydia species from both dairy and beef cattle production in China. Although in their research they did not test nasal swabs, they tested whole blood, vaginal swabs, feces and milk. In their study they reported high prevalence of \u003cem\u003eC. pecorum\u003c/em\u003e in fecal samples, indicating \u003cem\u003eC. pecorum\u003c/em\u003e as an endemic species optimally adapted to cattle, so it easily spreads throughout the intestinal tract. \u0026nbsp;Our findings in nasal mucosa could indicate that \u003cem\u003eC. pecorum\u003c/em\u003e it is not only present in intestinal tract; suggesting the aerial transmission, and support the idea of a prevalent pathogen in cattle worldwide. Moreover, several authors refer to the fact that \u003cem\u003eC. pecorum\u003c/em\u003e may be asymptomatic in cattle, as our findings show (Reinhold et al., 2008; Poudel et al., 2012; Li et al., 2016).\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eIn cattle, \u003cem\u003eC. psittaci\u003c/em\u003e causes low milk productivity, respiratory disease, abortions, and its pathogenicity has been demonstrated experimentally (Borel et al., 2006; Ostermann et al., 2013; Van Loo et al., 2014). However, it is likely that \u003cem\u003eC. psittaci\u003c/em\u003e infections in cattle occur in sporadic events, due to possible interactions with birds (Li et al., 2016). Li et al., (2016) reports a much lower prevalence (10%), comparing\u0026nbsp;to \u003cem\u003eC. pecorum\u0026nbsp;\u003c/em\u003e(75%). In their study, sequenced strains had a genetic similarity close to pigeons. In that sense the circulation of \u003cem\u003eC. psittaci\u003c/em\u003e is documented for several bird species in Argentina (Frutos et al., 2016). Although \u003cem\u003eC. psittaci\u003c/em\u003e infection is mainly associated with birds, the findings of this work indicate that mammals are also a source of \u003cem\u003eC. psittaci\u003c/em\u003e and may be carriers of strains associated with birds, as proposed by Frutos et al., (2014).\u003c/p\u003e\n\u003cp\u003eWhile our study was restricted to farm animals, it is important to highlight that the study site is located in the vicinity to an area of high wildlife conservation value, the Iguazu National Park in Argentina and the Igua\u0026ccedil;u National Park in Brazil. To date we do not know which species of Chlamydia could affect the local fauna and what their relevance could be at the sanitary and conservation level. \u0026nbsp;In the farms surveyed, many wild species frequent the area, and may have contact with domestic animals. This interaction (domestic/wild hosts) can result in the transmission of pathogens in both ways, by spillover from wildlife to domestic animals or vice versa. As an example, in Australia it has become a huge conservation issue for the koala (\u003cem\u003ePhascolarctos cinereus\u003c/em\u003e) populations since the introduction of \u0026nbsp;\u003cem\u003eC. pecorum\u003c/em\u003e from infected livestock, causing in koalas ocular and reproductive disorders and thus, increasing mortality rates (Polkinghorne et al., 2013; Bachmann et al., 2014). Regarding \u003cem\u003eC. psittaci\u003c/em\u003e, birds living in protected areas frequently visit farms, such as parrots and toucans, due to food availability (fruit trees, seeds and palms) and the scarce presence of predators. Cattle can be infected either by ingesting contaminated pastures by bird feces or seeds that they discard during the flight, making it difficult to control and eradicate the disease in the herds. In farming areas close to natural reserves, monitoring chlamydial species that affect wildlife can be an important tool to have a better understanding of the occurrence and potential of emergence of this pathogen in the area.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding its zoonotic potential, it is necessary to analyze samples from farmers that may or may not have clinical manifestations of respiratory disease, being the infections under-diagnosed, and that are in close contact with the potential cattle reservoir of \u003cem\u003eChlamydia\u003c/em\u003e spp. In this study, the animals that tested positive and its owners did not show symptoms of respiratory disease, nor have they mentioned cases of recent abortions by the cattle.\u003c/p\u003e\n\u003cp\u003eThis is the first study that detected the presence of two species of Chlamydia in the same study area in the triple border area of Argentina, Brazil and Paraguay: \u003cem\u003eChlamydia pecorum\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;C. psittaci\u003c/em\u003e, and the first report of \u003cem\u003eC. pecorum\u003c/em\u003e in bovines for Argentina. The phylogenetic analysis of the strains detected in our study shows a genetic closeness to strains previously detected in central Argentina, which also indicates a regional clustering. Our study provides the basis to deepen the circulation of chlamydia in the area and to understand which animal species could be its hosts and amplifiers. The chlamydial species found are of zoonotic risk and therefore should be included as differential diagnoses in cases of respiratory symptoms in humans.\u003c/p\u003e\n\u003cp\u003eMonitoring birds and poultry in the proximity of the farms is strongly suggested to assess Chlamydial circulation. As in cattle, these diseases should be considered as differential diagnosis in spontaneous abortions or reproductive diseases. Therefore, it is necessary to extend surveillance and deepen studies on chlamydial species involved in animal pathology and their zoonotic potential, since information in the region is scarce.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eACKNOWLEDGMENTS\u003c/p\u003e\n\u003cp\u003eWe are grateful to all the farmers of the Andresito and Maria Soledad community who willingly participated and volunteered their time to support the study. We thank Dr. Julia Martinez Pardo for her contribution to generate the maps. This work was supported by the National Scientific and Technical Research Council (CONICET).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e Conceptualization and project design: EV, AD and CC. Field work: EV. Laboratory analysis EV and CC. Bioinformatics analysis CC and JM. Manuscript draft and review EV, JM, CC and AD. CC and AD were the general advisors of the project and contributed equally as the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u0026nbsp;\u003c/strong\u003eThe corresponding author declares that data are available upon request and will be place in an open public repository \u0026nbsp;of CONICET\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e: Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e Secretariat of Science and Technology/National University of Cordoba Proyecto SeCyT \u0026ndash;UNC Cat A. 2018/22 Mincyt Res N\u0026deg;019/17.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e: Guidelines for the care and use of animals were followed by the ICLAS Ethical Guideline for Researchers. The Argentine legislation did not require an ethics protocol committee to approve our work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u0026nbsp;\u003c/strong\u003eAll the authors consented to participate in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eAll the authors consent to publication of this article\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBachmann NL, Fraser TA, Bertelli C, Jelocnik M, Gillett A, Funnell O, Flanagan C, Myers G, Timms P, \u0026amp; Polkinghorne A (2014) Comparative genomics of koala, cattle and sheep strains of \u003cem\u003eChlamydia pecorum\u003c/em\u003e. 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Parasitology, 273, 71-79.\u0026nbsp;https://doi.org/10.1016/j.vetpar.2019.08.008\u003c/li\u003e\n \u003cli\u003eVan Loo H, Pardon B, De Schutter P, De Bleecker K, Vanrompay D, Deprez P, Maris J (2014). Detection of \u003cem\u003eChlamydia psittaci\u003c/em\u003e in Belgian cattle with signs of respiratory disease and milk drop syndrome. Vet. Rec., vetrec-2014.\u0026nbsp;DOI:10.1136/vr.102527\u003c/li\u003e\n \u003cli\u003eVanrompay D, Harkinezhad T, Van de Walle M, Beeckman D, Van Droogenbroeck C, Verminnen K, Leten R, Martel A, Cauwerts K. (2007) \u003cem\u003eChlamydophila psittaci\u003c/em\u003e transmission from pet birds to humans. Emerg. Infect. Dis., 13(7), 1108.\u0026nbsp;doi: 10.3201/eid1307.070074\u003c/li\u003e\n \u003cli\u003eWalker E, Lee EJ, Timms P, Polkinghorne A (2015) \u003cem\u003eChlamydia pecorum\u003c/em\u003e infections in sheep and cattle: a common and under-recognised infectious disease with significant impact on animal health.Vet. J., 206(3), 252-260.\u0026nbsp;https://doi.org/10.1016/j.tvjl.2015.09.022\u003c/li\u003e\n \u003cli\u003eWhittum-Hudson JA, Hudson AP (2005) Human chlamydial infections: persistence, prevalence, and prospects for the future. Nat. Sci. Soc.;13:371\u0026ndash;82. https://doi.org/10.1051/nss:2005057\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Chlamydial strains used in partial ompA DNA sequencing in our study.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.175616835994195%\"\u003e\n \u003cp\u003eChlamydia strain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.657474600870827%\"\u003e\n \u003cp\u003eHost\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.1422351233672%\"\u003e\n \u003cp\u003eClinical Signs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.481857764876633%\"\u003e\n \u003cp\u003eCountry origin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.481857764876633%\"\u003e\n \u003cp\u003eGenBank accesion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.593613933236576%\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.467343976777939%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"10\" width=\"11.175616835994195%\"\u003e\n \u003cp\u003e\u003cem\u003eC.psittaci\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.657474600870827%\"\u003e\n \u003cp\u003e\u003cem\u003eEquus caballus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.1422351233672%\"\u003e\n \u003cp\u003eAborted foetus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.481857764876633%\"\u003e\n \u003cp\u003eAustralia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.481857764876633%\"\u003e\n \u003cp\u003eKY287781\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"22.060957910014515%\"\u003e\n \u003cp\u003eJelocnik et al., 2017\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eMelopsittacus undulatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eSystemic infection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eNot reported\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eM73035\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eKaltenboeck et al., 1993\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eAmazona aestiva\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eHepatic disease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eBrazil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eMH138293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eVilela et al., 2019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eMeleagris gallopavo\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNasal discharge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eBelgium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eAY762609\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eGeens et al., 2005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eBos taurus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNo clinical signs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eArgentina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eMW888425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eBos taurus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNo clinical signs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eArgentina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eMW888425\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eDiuca diuca\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNo clinical signs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eArgentina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eJX399853\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eJelocnik et al., 2017\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eParoaria coronata\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNo clinical signs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eArgentina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eJX399854\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eKaltenboeck et al., 1993\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eBos taurus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eEnteritis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eUSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eAF269269\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eVilela et al., 2019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003eepizootic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNot reported\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eUSA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eAF269268\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eGeens et al., 2005\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"11.175616835994195%\"\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.657474600870827%\"\u003e\n \u003cp\u003e\u003cem\u003eBos taurus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.1422351233672%\"\u003e\n \u003cp\u003ePneumonia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.481857764876633%\"\u003e\n \u003cp\u003eGermany\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.481857764876633%\"\u003e\n \u003cp\u003eEU350138\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"22.060957910014515%\"\u003e\n \u003cp\u003eJelocnik et al., 2017\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"9\" width=\"11.175616835994195%\"\u003e\n \u003cp\u003e\u003cem\u003eC.pecorum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"23.657474600870827%\"\u003e\n \u003cp\u003e\u003cem\u003eBos Taurus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.1422351233672%\"\u003e\n \u003cp\u003eDiarrhea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.481857764876633%\"\u003e\n \u003cp\u003eJapan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.481857764876633%\"\u003e\n \u003cp\u003eLC021422\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"22.060957910014515%\"\u003e\n \u003cp\u003eKaltenboeck et al., 1993\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eParoaria coronata\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNo clinical signs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eArgentina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eJN016882\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eVilela et al., 2019\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eBos taurus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNo clinical signs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eArgentina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eMW888427\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eBos taurus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNo clinical signs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eArgentina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eMW888428\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eThis study\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eGubernatrix cristata\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNo clinical signs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eArgentina\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eJN016884\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eFrutos et al., 2015\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eCapra aegagrus hircus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNo clinical signs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eFrance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eEU684933\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eMohamad et al., 2008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eBos taurus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eDiarrhea\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eJapan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eLC021419\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eOhtani et al., 2015\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003eCapra aegagrus hircus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eNo clinical signs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eFrance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eEU684932\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eMohamad et al., 2008\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"26.633986928104576%\"\u003e\n \u003cp\u003e\u003cem\u003ePhascolarctos cinereus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.424836601307188%\"\u003e\n \u003cp\u003eUrogenital infection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eAustralia\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.052287581699346%\"\u003e\n \u003cp\u003eKU214244\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"24.836601307189543%\"\u003e\n \u003cp\u003eLegione et al., 2016\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Chlamydia pecorum, Chlamydia psittaci, Zoonotic Diseases, Livestock, Argentine-Brazilian- Paraguay border.","lastPublishedDoi":"10.21203/rs.3.rs-1014196/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1014196/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Chlamydias are obligated intracellular Gram-negative bacteria, considered important zoonotic pathogens, broadly present in several bird species and responsible for economic losses in animal production. We analyzed the presence of Chlamydial species with zoonotic risk in farm animals in a highly biodiverse area and with great human circulation, the Argentine, Brazil and Paraguay tri-border area. We surveyed nine farms in an area and nasally swabbed a total of 62 animals. DNA was extracted and specific PCR was performed to identify chlamydial species. We detected Chlamydia spp . in 6.5% (4/62) of the animals tested, positive samples belonged to cattle and none of them showed symptoms of respiratory disease nor had been diagnose with reproductive diseases. Specific nested PCR confirmed two samples belonged to C. pecorum and two to C. psittaci . We report for the first time Chlamydia circulation with zoonotic risk in the region. Surveys in birds and wild mammals could give a better understanding to know what Chlamydial species are circulating in the wild interface. The zoonotic potential should be taking into account as farm workers and the surrounding population could be silent carriers or have respiratory diseases being underdiagnosed, and therefore should be considered in the differential diagnoses.","manuscriptTitle":"Enzootic Activity of Chlamydia in Farms Located in a Hotspot Area for Zoonosis Emergence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-12-10 20:56:31","doi":"10.21203/rs.3.rs-1014196/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"66a49af9-b6a1-461e-baf3-450e8cd12c86","owner":[],"postedDate":"December 10th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":9082506,"name":"Veterinary Epidemiology"}],"tags":[],"updatedAt":"2022-03-14T12:46:33+00:00","versionOfRecord":[],"versionCreatedAt":"2021-12-10 20:56:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1014196","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1014196","identity":"rs-1014196","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}