{"id":6739,"date":"2023-08-22T18:51:31","date_gmt":"2023-08-22T16:51:31","guid":{"rendered":"https:\/\/veterinarska-stanica-journal.hr\/?post_type=article&#038;p=6739"},"modified":"2023-08-22T18:51:31","modified_gmt":"2023-08-22T16:51:31","slug":"molecular-screening-of-nocardia-spp-in-wild-mammals-a-cross-sectional-study","status":"publish","type":"article","link":"https:\/\/journal.h3s.org\/?article=molecular-screening-of-nocardia-spp-in-wild-mammals-a-cross-sectional-study","title":{"rendered":"Molecular screening of <em>Nocardia<\/em> spp. in wild mammals: a cross-sectional study"},"content":{"rendered":"<p><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2023\/07\/ManuelaMATOS.jpg\" alt=\"\" width=\"168\" height=\"208\" class=\"alignright size-full wp-image-6740\" \/><\/p>\n<p style=\"text-align: center;\">M. <strong>Matos<\/strong>*, S. <strong>Jo\u00e3o<\/strong>, A. C. <strong>Matos<\/strong>, L. <strong>Figueira<\/strong>, L. <strong>Cardoso<\/strong>, M. <strong>de Lurdes Pinto<\/strong> and A. C. <strong>Coelho<\/strong><\/p>\n<hr \/>\n<div class=\"autorinfo\"><strong>Manuela MATOS<\/strong>*, PhD, (Corresponding author, e-mail: mmatos@utad.pt), Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Tr\u00e1s-os-Montes e Alto Douro (UTAD); <strong>S\u00e9rgio JO\u00c3O<\/strong>, MSc, Department of Veterinary Sciences, UTAD, Vila Real, Portugal; <strong>Ana Cristina MATOS<\/strong>, MSc, PhD, Research Centre for Natural Resources, Environment and Society, Polytechnic Institute of Castelo Branco, Q-RURAL \u2013 Quality of Life in the Rural World, Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal; <strong>Lu\u00eds FIGUEIRA<\/strong>, DVM, Centre Research for Natural Resources, Environment and Society, Polytechnic Institute of Castelo Branco, Q-RURAL &#8211; Quality of Life in the Rural World, Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal; <strong>Lu\u00eds CARDOSO<\/strong>, DVM, MSc, PhD, Department of Veterinary Sciences, School of Agrarian and Veterinary Sciences (ECAV), Animal and Veterinary Research Centre (CECAV), UTAD, Vila Real, Portugal; <strong>Maria de Lurdes PINTO<\/strong>, DVM, MSc, PhD, Department of Veterinary Sciences, ECAV, CECAV, UTAD, Vila Real, Portugal; <strong>Ana Cl\u00e1udia COELHO<\/strong>, DVM, MSc, PhD, Department of Veterinary Sciences, ECAV), CECAV, UTAD, Vila Real, Portugal<\/div>\n<div class=\"doi\"><a href=\"https:\/\/veterinarska-stanica-journal.hr\/pdf\/55\/55-2\/molecular-screening-of-nocardia-spp-in-wild-mammals-a-cross-sectional-study.pdf\" target=\"_blank\" rel=\"noopener\"><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2021\/03\/pdf.png\" alt=\"\" width=\"32\" height=\"18\" class=\"alignleft size-full wp-image-1504\" \/><\/a><a href=\"https:\/\/doi.org\/10.46419\/vs.55.2.5\" rel=\"noopener\" target=\"_blank\">https:\/\/doi.org\/10.46419\/vs.55.2.5<\/a><\/div>\n<\/p>\n<p><a name=\"menu\"><\/a><\/p>\n<div id=\"menu\">\n<div class=\"block grey mid\"><span class=\"small\"><a class=\"btn\" href=\"#Abstract\">Abstract<\/a><a class=\"btn\" href=\"#Introduction\">Introduction<\/a><a class=\"btn\" href=\"#Material\">Material and Methods<\/a><a class=\"btn\" href=\"#Results\">Results<\/a><a class=\"btn\" href=\"#Conclusions\">Conclusions<\/a><a class=\"btn\" href=\"#Acknowledgments\">Acknowledgments<\/a><a class=\"btn\" href=\"#Literatura1\" onclick=\"toggle_visibility('Literatura');\">References<\/a><a class=\"btn\" href=\"#Sazetak\">Sa\u017eetak<\/a><\/span><\/div>\n<\/div>\n<p><a name=\"Abstract\"><\/a><a class=\"alignright\" href=\"#\" onclick=\"scrollToTop();return false\"> &#9650;<\/a><\/p>\n<blockquote>\n<h2>Abstract<\/h2>\n<hr \/>\n<p>Nocardiosis is an emerging zoonosis caused by <em>Nocardia<\/em> spp. bacteria. The disease is linked to infections in immunocompromised individuals, and is potentially lethal when it turns systemic and is left untreated. The development of reliable and valid diagnostic methods is crucial to the identification of these pathogenic agents, particularly in wild mammals, which are potential reservoirs. The aim of this study was to determine the molecular prevalence of <em>Nocardia<\/em> spp. in wild mammals in Portugal. A sample of 114 wild mammals belonging to the orders <em>Canidae<\/em>, <em>Cervidae<\/em>, <em>Erinaceidae<\/em>, <em>Herpestidae<\/em>, <em>Leporidae<\/em>, <em>Mustelidae<\/em>, <em>Viverridae<\/em>, and <em>Suidae<\/em> were studied. Tissue samples (<em>n<\/em>=206) were collected from different organs. The molecular prevalence of <em>Nocardia<\/em> in wild mammals was 55.3% (95% CI: 45.7-64.6%). <em>Nocardia<\/em> spp. were detected in nine of ten species studied: 83.3% (95% CI: 62.2-100%) in Egyptian mongoose (<em>Herpestes ichneumon<\/em>), 63.2% (95% CI: 41.5-89.9%) in red fox (<em>Vulpes vulpes<\/em>), 45.9% (95% CI: 29.8-62.0%) in red deer (<em>Cervus elaphus<\/em>), 44.4% (95% CI: 28.2-60.6%) in wild boar (<em>Sus scrofa<\/em>), 3\/3 beech martens (<em>Martes foina<\/em>), 2\/2 Eurasian otters (<em>Lutra lutra<\/em>), 1\/1 European badger (<em>Meles meles<\/em>), 1\/1 genet (<em>Genetta genetta<\/em>), and 1\/2 hedgehog (<em>Erinaceus europeaeus<\/em>). Of the 206 tissue samples studied by molecular techniques, <em>Nocardia<\/em> spp. were detected in 37.4% (95% CI: 36.7-38.1%), with a predominance in mesenteric lymph nodes (13.1%; 95% CI: 12.4-13.9%) and kidneys (9.2%; 95% CI: 8.5-9.9%). These results provide new insight into the prevalence of <em>Nocardia<\/em> in wild mammals and highlight the need for surveillance of wildlife as a potential reservoir of these emergent pathogens.<\/p>\n<p><strong>Key words:<\/strong> <em>Nocardia spp., PCR, Portugal, wild mammals<\/em><\/p><\/blockquote>\n<p><a name=\"Introduction\"><\/a><a class=\"alignright\" href=\"#menu\"> &#9650;<\/a><\/p>\n<h2>Introduction<\/h2>\n<hr \/>\n<p>Nocardiosis is an emergent granulomatous zoonosis that until the early 21<sup>st<\/sup> century was considered a rare opportunistic disease and only a clinical curiosity (Corti and Villafane-Fioti, 2003; Rodrigues <em>et al<\/em>., 2009; Li <em>et al<\/em>., 2020). In fact, nocardiosis typically manifests as an opportunistic disease, with 60% of affected patients having immunosuppressive conditions, though infection may also develop in immunocompetent patients.<br \/>\nThe most common clinical presentation in immunocompetent patients includes superficial cutaneous disease, lymphocutaneous disease, mycetomas, and eye infections that may occur after traumatic inoculation (Ambrosioni <em>et al<\/em>., 2010; Baio <em>et al<\/em>., 2013).<\/p>\n<p>Nocardia spp. are Gram-positive filamentous rods, and members of the family <em>Nocardiaceae<\/em> (together with the genus <em>Rhodococcus<\/em>). They are difficult to isolate in culture due to their fastidious growth (3\u20135 days). In humans, they can cause pulmonary infection, most commonly, primary cutaneous infection and dissemination to other sites as the brain or other internal organs, thus becoming systemic and potentially fatal (Abreu <em>et al<\/em>., 2015; Conville <em>et al<\/em>., 2017).<\/p>\n<p>The increase of cases of infection by <em>Nocardia<\/em> spp. in domestic or wild animals included in consumption, and in humans themselves, has contributed to the status of this emerging zoonosis (Condas <em>et al<\/em>., 2015; Matos <em>et al<\/em>., 2015; Eroksuz <em>et al<\/em>., 2016; D\u00edaz-Santana <em>et al<\/em>., 2022). Currently, there are more than 100 described species of <em>Nocardia<\/em>, with 30 of these being important to humans (Brown-Elliott <em>et al<\/em>., 2006; Betr\u00e1n <em>et al<\/em>., 2016; Lebeaux <em>et al<\/em>., 2019). The use of molecular techniques, such as amplification by polymerase chain reaction (PCR) of the gene encoding RNA of ribosome subunit 16 (Torres <em>et al<\/em>., 1996; Kandi, 2015) has allowed new species to be identified or existing species to be differentiated (Conville <em>et al<\/em>., 2000).<br \/>\nThese different species have already been found in domestic animals (Biberstein <em>et al<\/em>., 1985; Emeruwa, 1986; Maldonado <em>et al<\/em>., 2004; Ribeiro <em>et al<\/em>., 2008; Mund <em>et al<\/em>., 2021; Yoon <em>et al<\/em>., 2022) and in wildlife (Gezuele, 1972; Kinde <em>et al<\/em>., 1992; Vemireddi <em>et al<\/em>., 2007; Domenis <em>et al<\/em>., 2009; Matos <em>et al<\/em>., 2015; Ito <em>et al<\/em>., 2021).<\/p>\n<p>The number of reported cases of nocardiosis has gradually increased worldwide. In North America, according to the Center for Disease Control and Prevention (CDC), between 500 and 1000 new cases of <em>Nocardia<\/em> spp. infection occur annually (Bafghi <em>et al<\/em>., 2014).<\/p>\n<p>To the best of the authors\u2019 knowledge, in the Iberian Peninsula, <em>Nocardia<\/em> spp. have only been found in wild boar (<em>Sus scrofa<\/em>) (Matos <em>et al<\/em>., 2015), with no cases of nocardiosis reported from red fox (<em>Vulpes vulpes<\/em>), Egyptian mongoose (<em>Herpestes ichneumon<\/em>), European badger (<em>Meles meles<\/em>), Eurasian otter (<em>Lutra lutra<\/em>), red deer (<em>Cervus elaphus<\/em>), genet (<em>Genetta genetta<\/em>) or hedgehog (<em>Erinaceus europaeus<\/em>).<\/p>\n<p>Determination of <em>Nocardia<\/em> prevalence in wild populations can provide new insight into the likelihood of pathogen transmission. The aim of this study was to determine the molecular prevalence of <em>Nocardia<\/em> spp. in wild mammals in Portugal.<\/p>\n<p><a name=\"Material\"><\/a><a class=\"alignright\" href=\"#menu\"> &#9650;<\/a><\/p>\n<h2>Material and Methods<\/h2>\n<hr \/>\n<h3>Population and samples<\/h3>\n<p>A cross-sectional study was conducted to estimate the molecular prevalence of <em>Nocardia<\/em> spp. in samples from wild mammals in Portugal.<\/p>\n<p>Between 2010 and 2015, during the <em>hunting season, a molecular study of Nocardia<\/em> spp. was performed on 206 tissue samples from 114 randomly obtained free-ranging wild mammals found dead (on roads or otherwise) or hunted in the municipalities of Idanhaa-Nova (39\u00b055\u203211\u2033North, 7\u00b014\u203212\u2033West) and Penamacor (40\u00b010\u20328\u2033North, 7\u00b010\u203214\u2033West), district of Castelo Branco, east-central Portugal.<\/p>\n<p>The examined 114 wild mammals represented 10 different species belonging to eight taxonomic families (Table 1).<\/p>\n<figure id=\"attachment_6744\" aria-describedby=\"caption-attachment-6744\" style=\"width: 660px\" class=\"wp-caption aligncenter\"><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2023\/07\/table01-molecular-screening.png\" alt=\"\" width=\"660\" height=\"560\" class=\"size-full wp-image-6744\" srcset=\"https:\/\/journal.h3s.org\/wp-content\/uploads\/2023\/07\/table01-molecular-screening.png 660w, https:\/\/journal.h3s.org\/wp-content\/uploads\/2023\/07\/table01-molecular-screening-300x255.png 300w\" sizes=\"auto, (max-width: 660px) 100vw, 660px\" \/><figcaption id=\"caption-attachment-6744\" class=\"wp-caption-text\"><strong>Table 1<\/strong>. Distribution of Nocardia spp. by taxonomic families and species in wild animals collected and examined between 2010 and 2015.<\/figcaption><\/figure>\n<p>Samples from ungulates were obtained immediately after hunting, while the carcasses of the other wild mammals were transported to the Laboratory of Histopathology of the University of Tr\u00e1s-os-Montes and Alto Douro, Portugal, where they were subject to a standard necropsy procedure prior to sampling. Collection site, date, and state of carcass preservation according to gross inspection were recorded.<\/p>\n<h3>Histopathology and culture<\/h3>\n<p>At necropsy, samples of the mesenteric lymph nodes, mediastinal lymph nodes, retropharyngeal lymph nodes, kidneys, lungs, bladder, tonsils, ileocecal valves, brain, and liver were collected for molecular and histopathological analyses. Samples for PCR were frozen at -20\u00b0C before processing. Samples for histopathology were fixed in 10% neutral buffered formalin, routinely processed for paraffin embedding and stained with haematoxylin &#038; eosin and by the Ziehl\u2013Neelsen method for acid-fast bacteria (AFB). Cultures were performed with tissue samples according to established microbiological protocols (Saubolle and Sussland 2003). Briefly, material was inoculated onto Columbia blood agar plates, and incubated at 35\u00b0C for 48 h.<\/p>\n<h3>DNA extraction and PCR amplification<\/h3>\n<p>Genomic DNA was extracted from all samples using the Dneasy Blood and Tissue kit (Qiagen<sup>\u00ae<\/sup>, Hilden, Germany).<br \/>\nNG1 (5\u2019ACCGACCACAAGGGG3\u2019) and NG2 (GGTTGTAACCTCTTCGA) primers were used for PCR amplification of a 16S ribosomal RNA (rRNA) gene segment specific for the genus <em>Nocardia<\/em> (Laurent <em>et al<\/em>., 1999). PCR was performed in a 20 \u00b5L volume using the Taq Master Mix Bioron<sup>\u00ae<\/sup> (2x), 1 \u00b5L each primer (10 \u00b5M) and 30 ng DNA. Positive and negative controls were tested in each PCR cycle.<br \/>\nNocardia DNA was used as the positive control and sterile water as the negative control. Amplification was performed in a thermocycler programmed as follows: 10 min for the initial denaturation step at 94\u00baC, 30 cycles (94\u00b0C for 60 s, 52\u00b0C for 20 s, 72\u00b0C for 60 s) and a 10 min final extension step at 72\u00b0C. Amplification was evaluated by means of 1% agarose gel electrophoresis and the presence of a 596 bp fragment (Laurent <em>et al<\/em>., 1999) was regarded as a positive result. Gel visualisation and image capture were performed with the Molecular Imager Gel DocTM XR equipment associated with the Image LabTM software (Bio-Rad, Hercules, CA, USA).<\/p>\n<h3>Data analysis<\/h3>\n<p>For this study, an animal was regarded as infected if <em>Nocardia<\/em> was detected by PCR in at least one tissue. Molecular prevalence of <em>Nocardia<\/em> spp. was described as absolute frequencies, percentages and 95% confidence intervals. The chi-square test compared molecular positivity values between demographic variables. A <em>p<\/em> &lt; 0.05 was considered as statistically significant. Data analysis was performed using SPSS<sup>\u00ae<\/sup> 25.<\/p>\n<p><a name=\"Results\"><\/a><a class=\"alignright\" href=\"#menu\"> &#9650;<\/a><\/p>\n<h2>Results<\/h2>\n<hr \/>\n<p>Based on PCR, 63 out of the 114 wild mammals were found infected with <em>Nocardia<\/em> spp. The molecular prevalence of <em>Nocardia<\/em> spp. in animals was 55.3% (95% CI: 45.7-64.6%). <em>Nocardia<\/em> spp. was detected in nine of the ten species examined (Table 1): 12\/19 red foxes (<em>V. vulpes<\/em>) (63.2%; 95% CI: 41.5-84.9%), 3\/3 beech martens (<em>Martes foina<\/em>) (100%), 2\/2 Eurasian otters (<em>L. lutra<\/em>) (100%), 1\/1 European badger (<em>M. meles<\/em>) (100%), 1\/1 genet (<em>G. genetta<\/em>) (100%), 10\/12 Egyptian mongooses (<em>H. ichneumon<\/em>) (83.3%; 95% CI: 62.2-100%), 1\/2 hedgehogs (<em>E. europaeus<\/em>) (50.0%; 95% CI: 1.3-98.7%), 16\/36 wild boar (<em>S. scrofa<\/em>) (44.4%; 95% CI: 28.2-60.6%), 17\/37 red deer (<em>C. elaphus<\/em>) (45.9%; 29.8-62.0%). Infection was found in seven taxonomic families: <em>Canidae<\/em> (63.2% positive), <em>Cervidae<\/em> (55.3%), <em>Erinaceidae<\/em> (50.0%), <em>Herpestidae<\/em> (83.3%), <em>Mustelidae<\/em> (100%), <em>Suidae<\/em> (44.4%), and <em>Viverridae<\/em> (100%). No positive tissues were found from wild rabbits (<em>Oryctolagus cuniculus<\/em>). <em>Nocardia<\/em> spp. was not confirmed in culture.<br \/>\nOf the 206 tissue samples studied by PCR, <em>Nocardia<\/em> spp. were detected in 77 tissues (37.4%; 95% CI: 36.7-38.1%), mainly in mesenteric lymph nodes (13.1%; 95% CI: 12.4-13.9%) and kidneys (9.2%; 95% CI: 8.5\u20139.9%). In addition, the microorganism was detected in other tissues such as lungs (5.3%; 95% CI: 4.6-5.9%), bladder (2.4%; 95% CI: 1.7-3.1%), mediastinal lymph nodes (2.4%; 95% CI: 1.7-3.1%), tonsils (1.9%; 95% CI: 1.2-2.6%), retropharyngeal lymph nodes (0.97%; 95% CI: 0.3-1.7%), ileocecal valves (0.97%; 95% CI: 0.3-1.7%), brain (0.49%; 95% CI: 0.2-1.2%) and liver (0.49%; 95% CI: 0.2-1.2%). The differences were found to be statistically significant (<em>P<\/em>=0.019) (Table 2).<\/p>\n<figure id=\"attachment_6745\" aria-describedby=\"caption-attachment-6745\" style=\"width: 660px\" class=\"wp-caption aligncenter\"><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2023\/07\/table02-molecular-screening.png\" alt=\"\" width=\"660\" height=\"358\" class=\"size-full wp-image-6745\" srcset=\"https:\/\/journal.h3s.org\/wp-content\/uploads\/2023\/07\/table02-molecular-screening.png 660w, https:\/\/journal.h3s.org\/wp-content\/uploads\/2023\/07\/table02-molecular-screening-300x163.png 300w\" sizes=\"auto, (max-width: 660px) 100vw, 660px\" \/><figcaption id=\"caption-attachment-6745\" class=\"wp-caption-text\"><strong>Table 2<\/strong>. Distribution of positive PCR results in 206 tissue samples from 114 wild mammals.<\/figcaption><\/figure>\n<p>Histological lesions consistent with a diagnosis of <em>Norcadia<\/em> spp. infection were detected in 65 animals (57.0%; 95% CI: 56.1-57.9%). These results are presented in Table 3.<\/p>\n<figure id=\"attachment_6746\" aria-describedby=\"caption-attachment-6746\" style=\"width: 660px\" class=\"wp-caption aligncenter\"><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2023\/07\/table03-molecular-screening.png\" alt=\"\" width=\"660\" height=\"113\" class=\"size-full wp-image-6746\" srcset=\"https:\/\/journal.h3s.org\/wp-content\/uploads\/2023\/07\/table03-molecular-screening.png 660w, https:\/\/journal.h3s.org\/wp-content\/uploads\/2023\/07\/table03-molecular-screening-300x51.png 300w\" sizes=\"auto, (max-width: 660px) 100vw, 660px\" \/><figcaption id=\"caption-attachment-6746\" class=\"wp-caption-text\"><strong>Table 3<\/strong>. Comparison of PCR results with gross findings in 114 wild mammals.<\/figcaption><\/figure>\n<p>These lesions consisted of abscesses (1\/114; 0.9%; 95% CI: 0.0-1.8%), granulomas with caseo-calcareous features (21\/114; 18.4%; 95% CI: 17.5-19.3%), dermatitis (1\/114; 0.9%; 95% CI: 0.0-1.8%), lymphadenopathy (2\/114; 1.8%; 95% CI: 0.21-6.19%), purulent lymphadenitis (39\/114; 34.2%; 95% CI: 25.6-43.7%) and orchitis (1\/114; 0.9%; 95% CI: 0.0-1.8. From the animals that also rendered positive results in PCR, abscesses were observed in one animal, granulomas with caseous calcification in seven animals, dermatitis in one animal, lymphadenopathy in one animal, orchitis in one animal, and purulent lymphadenitis in 16 animals. Granulomatous lymphadenitis was consistent with tuberculosis-like lesions, with multifocal lesions centred by caseous and\/or liquefied material or showing calcification at the centre of the lesion, followed by inflammatory infiltrate and a thin capsule of surrounding fibrous tissue.<br \/>\nGram-stained smears from those lesions revealed Gram-positive short filaments, coccoid forms, and branching rods in all samples. In the Ziehl-Neelsen stain, samples were partially acid fast.<\/p>\n<p>Regarding healthy animals, 32.5% of animals without visible lesions had a positive PCR result. Regarding animals with gross lesions, 22.8% had positive results (Table 3).<\/p>\n<p>The occurrence of <em>Nocardia<\/em> spp. was more frequent in adult males (26.3%) and less frequent in juvenile females (6.1%).<br \/>\nMost cases occurred in Idanha-a-Nova (47; 72.3%), Penamacor (17; 26.2%) and Castelo Branco (1; 1.5%).<\/p>\n<p>As shown in Table 4, PCR was positive in 33.3% of animals with caseo-calcareous granulomas and in 41% of the purulent lymphadenitis cases (Table 4).<\/p>\n<figure id=\"attachment_6747\" aria-describedby=\"caption-attachment-6747\" style=\"width: 660px\" class=\"wp-caption aligncenter\"><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2023\/07\/table04-molecular-screening.png\" alt=\"\" width=\"660\" height=\"198\" class=\"size-full wp-image-6747\" srcset=\"https:\/\/journal.h3s.org\/wp-content\/uploads\/2023\/07\/table04-molecular-screening.png 660w, https:\/\/journal.h3s.org\/wp-content\/uploads\/2023\/07\/table04-molecular-screening-300x90.png 300w\" sizes=\"auto, (max-width: 660px) 100vw, 660px\" \/><figcaption id=\"caption-attachment-6747\" class=\"wp-caption-text\"><strong>Table 4<\/strong>. Distribution of PCR results by gross findings in 65 wild mammals.<\/figcaption><\/figure>\n<p>A comparison of the frequency of pathological findings according to their PCR result in species is given in Table 5.<\/p>\n<figure id=\"attachment_6748\" aria-describedby=\"caption-attachment-6748\" style=\"width: 660px\" class=\"wp-caption aligncenter\"><img loading=\"lazy\" decoding=\"async\" src=\"https:\/\/veterinarska-stanica-journal.hr\/wp-content\/uploads\/2023\/07\/table05-molecular-screening.png\" alt=\"\" width=\"660\" height=\"439\" class=\"size-full wp-image-6748\" srcset=\"https:\/\/journal.h3s.org\/wp-content\/uploads\/2023\/07\/table05-molecular-screening.png 660w, https:\/\/journal.h3s.org\/wp-content\/uploads\/2023\/07\/table05-molecular-screening-300x200.png 300w\" sizes=\"auto, (max-width: 660px) 100vw, 660px\" \/><figcaption id=\"caption-attachment-6748\" class=\"wp-caption-text\"><strong>Table 5<\/strong>. Frequency of pathological findings falling into different PCR test result categories.<\/figcaption><\/figure>\n<p>The results show that regarding the animal species affected, the most frequent findings were in wild boar and red deer.<br \/>\nRed foxes and Egyptian mongooses had the most frequent PCR positive without lesions.<\/p>\n<p><a name=\"Discussion\"><\/a><a class=\"alignright\" href=\"#menu\"> &#9650;<\/a><\/p>\n<h2>Discussion<\/h2>\n<hr \/>\n<p>The number of cases of <em>Nocardia<\/em> spp. recorded in animals and humans, and its range of hosts, has increased due to an increased number of immunocompromised individuals worldwide (Barry <em>et al<\/em>., 2022; Palomba <em>et al<\/em>., 2022), the greater attention by the scientific community to this microorganism, and improvements in diagnostic techniques.<\/p>\n<p>The present study provides information on the <em>Nocardia<\/em> spp. status of wild mammals in Portugal, confirming the presence of the pathogen(s) in these animal populations. Some previous studies revealed the presence of this genus in wild boar (Matos <em>et al<\/em>., 2015). Nevertheless, this is the first report of <em>Nocardia<\/em> spp. molecular detection in red fox, Egyptian mongoose, European badger, Eurasian otter, red deer, hedgehog, or genet, suggesting a large proportion of these are infected with or exposed to zoonotic <em>Nocardia<\/em> spp.<\/p>\n<p>In this study, small sample sizes resulted in the inability to calculate prevalence estimates for some species. However, the results indicate evidence of infection in these species. Given the high proportion of positive samples, it seems that <em>Nocardia<\/em> infection is relatively common among wild mammals in Portugal.<br \/>\nThe population sample size of different wildlife species should also be taken into account. For some species, the small sizes obtained could reflect their restricted range and relatively low abundance, e.g., for the Eurasian otter (Ruiz-Olmo <em>et al<\/em>., 2011). No evidence of infection was detected in wild rabbits, although only two animals were examined. Therefore, it is not possible to clearly ascertain whether infection occurs in this species.<\/p>\n<p>The fact that a high prevalence was found in species such as the Egyptian mongoose, red fox, wild boar and red deer suggests a possible association between transmission and the trophic hierarchy within the analysed territory.<br \/>\nThis hypothesis is presented as a result of the level these species occupy in the food chain, as carnivores, suggesting a potential infection by ingestion of infected animals or contact with infected carcasses. This hypothesis is reinforced by analysis of the affected organs. The most affected organs were lymph nodes, with special reference to mesenteric nodes (Ramiro-Puig <em>et al<\/em>., 2008). Positive results were obtained from organs such as kidneys, lungs, bladder, tonsils, ileocecal valve, brain, and liver. The presence of Nocardia in different organs is consistent with disseminated nocardiosis (Saubolle and Sussland, 2003; Li <em>et al<\/em>., 2022). Cerebral nocardiosis is a rare entity in humans, but has been reported (Sayer <em>et al<\/em>., 2022; Sher <em>et al<\/em>., 2022).<\/p>\n<p>Histopathological analysis showed that most of the infected animals did not present any relevant <em>post mortem<\/em> lesions. Even in those with lesions, there was a predominance of purulent lymphadenitis, an unspecific response in an attempt to restore immunity. Reports of <em>Nocardia<\/em> infection in free-living terrestrial mammal species are rare (Matos v., 2015). The majority of reports come from free-ranging aquatic organisms and captive teleost and shellfish, as well as from marine mammals (St. Leger <em>et al<\/em>., 2018; D\u00edaz-Santana <em>et al<\/em>., 2022).<\/p>\n<p>Nocardiosis due to nonspecific clinical manifestations can be misdiagnosed as tuberculosis or fungal lung disease (Pan <em>et al<\/em>., 2021). Given the limitations of conventional diagnostic techniques, PCR is an effective tool to diagnose <em>Nocardia<\/em> (Rouzaud <em>et al<\/em>., 2018).<\/p>\n<p>Results suggest that <em>Nocardia<\/em> spp. circulate widely in the study area, which is a serious concern for wildlife. Our findings have also revealed a public health concern because professionals with frequent animal exposure, like veterinarians, biologists or hunters and other professional workers, who come into contact with these species can have an occupational risk of infection when handling infected tissues or samples (Garland-Lewis <em>et al<\/em>., 2017; Bournez <em>et al<\/em>., 2019). Consequently, our results indirectly suggest that the local human population may be exposed to infections with <em>Nocardia<\/em>, since hunted animals were killed for consumption, and the results of the present study need to be analysed in the scope of the One Health approach. Therefore, populations in contact with wild mammals should be instructed to prevent direct and indirect contact.<\/p>\n<p><a name=\"Conclusions\"><\/a><a class=\"alignright\" href=\"#menu\"> &#9650;<\/a><\/p>\n<h2>Conclusions<\/h2>\n<hr \/>\n<p>Despite some limitations, molecular diagnostic methods, such as PCR, are an accurate and rapid way of identifying pathogens, without the recurrent need to culture the bacteria. With this study, it was possible to successfully detect the presence of <em>Nocardia<\/em> spp. in different animal organs and samples. The results highlight the need for constant surveillance of this infectious agent, not only to prevent infections to humans or animals, but also to predict the dissemination and evolution of this agent in a poorly controlled and often neglected environment, as is the case of nature reserves and wild animals in these habitats.<\/p>\n<p><a name=\"Acknowledgments\"><\/a><a class=\"alignright\" href=\"#menu\"> &#9650;<\/a><\/p>\n<h2>Acknowledgments<\/h2>\n<hr \/>\n<p>The authors would like to thank to Laboratory of Genetics and Biotechnology and the Laboratory of Histology and Veterinary Pathology of the Department of Veterinary Sciences of the University of Tr\u00e1s-os-Montes-e-Alto-Douro. This work was supported by strategic research projects UIDP\/CVT\/00772\/2020, UIDB\/04033\/2020 and LA\/P\/0059\/2020 financed by the Portuguese Foundation for Science and Technology (FCT).<\/p>\n<p><a name=\"Literatura1\"><\/a><br \/>\n<strong>References<\/strong><span style=\"color: #808080;\"><a onclick=\"toggle_visibility('Literatura');\" ><span style=\"color: #808080; cursor:pointer;\"> [&#8230; show]<\/span><\/a><\/span><\/p>\n<div id=\"Literatura\" style=\"display: none;\">&nbsp;<a class=\"alignright\" href=\"#menu\" onclick=\"toggle_visibility('Literatura');\"> &#9650;<\/a><\/p>\n<p style=\"font-size: small;\"><em>1.\tABREU, C., N. ROCHA-PEREIRA, A. SARMENTO and F. MAGRO (2015): Nocardia infections among immunomodulated inflammatory bowel disease patients: A review. World J. Gastroenterol. 21, 6491- 6498. 10.3748\/wjg.v21.i21.6491<br \/>\n2.\tAMBROSIONI, J., D. LEW and J. GARBINO (2010): Nocardiosis: updated clinical review and experience at a tertiary center. 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SAYER (2022): Brain abscesses caused by Nocardia farcinica in a 44-year old woman with multiple myeloma: a rare case and review of the literature. Am. J. Case Rep. 23, e937952. 10.12659\/AJCR.937952<br \/>\n39.\tSHER, M., T. HEARD, W. MUNCKHOF and C. SCHWINDACK (2022): First case report of cerebral abscess from Nocardia terpenica and literature review. Clin. Neurol. Neurosurg. 220, 107341. 10.1016\/j.clineuro.2022.107341<br \/>\n40.\tST. LEGER, J., S. RAVERTY and A. MENA (2018): In: Pathology of Wildlife and Zoo Animals. Terio, K. A., McAloose, D., St. Leger, J., eds. Academic Press; Cambridge, MA, USA, pp. 533-568 10.1016\/B978-0-12-805306-5.00022-5<br \/>\n41.\tTORRES, R. D., C. A. OLETTA and H. ZLOTNIK (1996): A rapid and gentle method for isolation of genomic DNA from pathogenic Nocardia spp. Clin. Diagn. Lab. Immunol. 3, 601-604. 10.1128\/ cdli.3.5.601-604.1996<br \/>\n42.\tVEMIREDDI, V., A. SHARMA, C. C. WU and T. L. LIN (2007): Systemic nocardiosis in a reindeer (Rangifer tarandus tarandus). J. Vet. Diagn. Invest. 19(3), 326-329. 10.1177\/104063870701900320<br \/>\n43.\tYOON, J. S., H. SO, B. JOO, J. PARK, I. S. JEONG, G. J. LEE and J. PARK (2022): A Canine case of Nocardia africana infection detected by matrix-assisted laser desorption ionization-time-of- flight mass spectrometry. Vet. Sci. 9, 265. 10.3390\/vetsci9060265<br \/>\n<\/em><\/p>\n<\/div>\n<p><a name=\"Sazetak\"><\/a><a class=\"alignright\" href=\"#\" onclick=\"scrollToTop();return false\"> &#9650;<\/a><\/p>\n<blockquote>\n<h2>Molekularni probir <em>Nocardia<\/em> spp. u divljih sisavaca &#8211; presje\u010dna studija<\/h2>\n<hr \/>\n<div class=\"info\"><strong>Manuela MATOS<\/strong>, PhD, Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Tr\u00e1s-os-Montes e Alto Douro (UTAD); <strong>S\u00e9rgio JO\u00c3O<\/strong>, MSc, Department of Veterinary Sciences, UTAD, Vila Real, Portugal; <strong>Ana Cristina MATOS<\/strong>, MSc, PhD, Research Centre for Natural Resources, Environment and Society, Polytechnic Institute of Castelo Branco, Q-RURAL \u2013 Quality of Life in the Rural World, Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal; <strong>Lu\u00eds FIGUEIRA<\/strong>, DVM, Centre Research for Natural Resources, Environment and Society, Polytechnic Institute of Castelo Branco, Q-RURAL &#8211; Quality of Life in the Rural World, Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal; <strong>Lu\u00eds CARDOSO<\/strong>, DVM, MSc, PhD, Department of Veterinary Sciences, School of Agrarian and Veterinary Sciences (ECAV), Animal and Veterinary Research Centre (CECAV), UTAD, Vila Real, Portugal; <strong>Maria de Lurdes PINTO<\/strong>, DVM, MSc, PhD, Department of Veterinary Sciences, ECAV, CECAV, UTAD, Vila Real, Portugal; <strong>Ana Cl\u00e1udia COELHO<\/strong>, DVM, MSc, PhD, Department of Veterinary Sciences, ECAV), CECAV, UTAD, Vila Real, Portugal<\/div>\n<hr \/>\n<p>Nokardioza je emergentna zoonoza koju prouzro\u010di bakterija <em>Nocardia<\/em> spp. Bolest je povezana s infekcijama u imunokompromiranih pojedinaca te je potencijalno smrtonosna ukoliko postane sistemska i ne lije\u010di se. Razvoj pouzdane i valjane dijagnosti\u010dke metode je klju\u010dan za identifikaciju ovih patogena, posebice u divljih sisavaca koji su potencijalni rezervoari me\u0111u divljim \u017eivotinjama. Cilj je ove studije bio odrediti molekularnu prevalenciju <em>Nocardia<\/em> spp. u divljih sisavaca u Portugalu. Istra\u017eili smo prakti\u010dni uzorak od 114 divljih sisavaca iz porodice: <em>Canidae<\/em>, <em>Cervidae<\/em>, <em>Erinaceidae<\/em>, <em>Herpestidae<\/em>, <em>Leporidae<\/em>, <em>Mustelidae<\/em>, <em>Viverridae<\/em> i <em>Suidae<\/em>. Prikupljeni su uzorci tkiva (<em>n<\/em>=206) iz razli\u010ditih organa. Molekularna prevalencija bakterije <em>Nocardia<\/em> u divljih sisavaca bila je 55,3 % (95% CI: 45,7-64,6 %). <em>Nocardia<\/em> spp. otkrivena je u devet od deset ispitanih vrsta: 83,3 % (95% CI: 62,2-100 %) u egipatskih mungosa (<em>Herpestes ichneumon<\/em>), 63,2 % (95% CI: 41,5-89,9 %) u crvenih lisica (<em>Vulpes vulpes<\/em>), 45,9 % (95% CI: 29,8-62,0 %) u obi\u010dnih jelena (<em>Cervus elaphus<\/em>), 44,4 % (95% CI: 28,2-60,6 %) u divljih svinja (<em>Sus scrofa<\/em>), 3\/3 kune bjelice (<em>Martes foina<\/em>), 2\/2 europske vidre (<em>Lutra lutra<\/em>), 1\/1 euroazijskog jazavca (<em>Meles meles<\/em>), 1\/1 europske cibetke (<em>Genetta genetta<\/em>) te 1\/2 je\u017ea (<em>Erinaceus europeaeus<\/em>). Od 206 uzoraka tkiva ispitanih molekularnim tehnikama, bakterija <em>Nocardia<\/em> spp. otkrivena je u 37,4 % (95% CI: 36,7-38,1 %), s tim da je prevladavala u mezenteri\u010dnim limfnim \u010dvorovima (13,1 %; 95% CI: 12,4-13,9 %) i bubrezima (9,2 %; 95% CI: 8,5-9,9 %). Ovi rezultati daju nove uvide u prevalenciju bakterije <em>Nocardia<\/em> u divljih sisavaca i nagla\u0161avaju potrebu za nadziranjem divljih \u017eivotinja kao potencijalnih rezervoara ovih emergentnih patogena.<\/p>\n<p><strong>Klju\u010dne rije\u010di:<\/strong> <em>Nocardia spp., PCR, Portugal, divlje \u017eivotinje<\/em><\/p><\/blockquote>\n","protected":false},"excerpt":{"rendered":"<p>M. Matos*, S. Jo\u00e3o, A. C. Matos, L. Figueira, L. Cardoso, M. de Lurdes Pinto and A. C. Coelho Manuela<\/p>\n","protected":false},"author":8,"featured_media":0,"menu_order":6,"comment_status":"closed","ping_status":"open","template":"","format":"standard","meta":{"footnotes":""},"categories":[21],"tags":[2041,25,1663,2042],"issuem_issue":[1992],"ppma_author":[1631,2038,1627,2039,1630,2040,1632],"class_list":["post-6739","article","type-article","status-publish","format-standard","hentry","category-original-scientific-articles","tag-nocardia-spp","tag-pcr","tag-portugal","tag-wild-mammals","issuem_issue-veterinarska-stanica-55-2"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v26.6 - https:\/\/yoast.com\/wordpress\/plugins\/seo\/ -->\n<title>Molecular screening of Nocardia spp. in wild mammals: a cross-sectional study - CROATIAN VETERINARY JOURNAL<\/title>\n<meta name=\"description\" content=\"The development of reliable and valid diagnostic methods is crucial to the identification of these pathogenic agents, particularly in wild mammals, which are potential reservoirs. The aim of this study was to determine the molecular prevalence of Nocardia spp. in wild mammals in Portugal.\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/journal.h3s.org\/?article=molecular-screening-of-nocardia-spp-in-wild-mammals-a-cross-sectional-study\" \/>\n<meta property=\"og:locale\" content=\"en_GB\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Molecular screening of Nocardia spp. in wild mammals: a cross-sectional study - CROATIAN VETERINARY JOURNAL\" \/>\n<meta property=\"og:description\" content=\"The development of reliable and valid diagnostic methods is crucial to the identification of these pathogenic agents, particularly in wild mammals, which are potential reservoirs. 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