205 1Department of Veterinary Medicine, Faculty of Agriculture and Veterinary, University of Prishtina “Hasan Prishtina”, Republic of Kosovo. 2Department of Animal Science, University of Wyoming, Laramie, WY 82071, United States of America. *Corresponding author at: Department of Veterinary Medicine, Faculty of Agriculture and Veterinary, University of Prishtina “Hasan Prishtina”, Republic of Kosovo. E‑mail: driton.sylejmani@uni‑pr.edu. Keywords Antimicrobial susceptibility, Brain, MIC, Ovine listeriosis, Serotyping. Summary In 2018, a case of neural disease suspected of listeriosis was reported in a flock of sheep in Kosovo with the death of ewes and 5 lambs. Samples from the brain of only three dead animals were subjected to histopathological and bacteriological analysis. MALDI-TOF MS was applied to confirm suspected Listeria spp. isolates from culture and multiplex PCR was applied for molecular serotyping. All isolates were tested for antimicrobial susceptibility by microdilution broth method. The histopathological analysis of the brain specimens showed typical changes for Listeria monocytogenes. Listeria spp. was isolated in brain samples from all three animals, and all the isolates were confirmed using MALDI-TOF MS and PCR down to the species level (Listeria monocytogenes). The molecular characterisation using multiplex PCR revealed all isolates as Listeria monocytogenes serotype 4b. All L. monocytogenes isolates were found to be susceptible to penicillin, erythromycin, tetracycline, streptomycin, trimethoprim/ sulfamethosazole, quinupristin/dalfopristin, kanamycin, vancomycin, and gentamicin but resistant to nitrofurantoin and lincomycin. This study shows the emergence of a highly virulent strain in sheep farms in Kosovo and a possible threat to public health. Afrim Hamidi1, Bledar Bisha2, Izedin Goga1, Baolin Wang2, Avni Robaj1 and Driton Shefqet Sylejmani1* A case report of sporadic ovine listerial meningoencephalitis in Kosovo Veterinaria Italiana 2020, 56 (3), 205-211. doi: 10.12834/VetIt.2166.12781.3 Accepted: 29.05.2020 | Available on line: 31.12.2020 the brain along interneuronal connections both in retro- and anteretrograde directions (Oevermann et  al. 2010, Granier et  al. 2011, Henke et  al. 2015). Listeriosis associated with encephalitis is more prevalent among ruminants, especially adult sheep, with an attack rate of 10-20% and a mortality rate of 5-10%, respectively. Lambs as young as 5 weeks old may develop the septicaemic form of the disease, whereas older feedlot lambs (4-8 months old) develop the encephalitic form (Wesley et  al. 2002, Lozano et al. 2011). Ruminants affected by listerial encephalitis generally show marked neurological symptoms, including ataxia, masticatory problems, failure of jaw closure, drooping of ears, upper eye lids and lips, swallowing problems, tongue palsy, circling, head tilt and leaning to one side, drooling of saliva, and facial paralysis (Braun et al. 2002, Kumar et  al. 2007, Dons et  al. 2007, Brugere-Picoux 2008, Rocha et  al. 2013). Once L.  monocytogenes enters the brain in small ruminants, its elimination is Introduction Listeriosis is an infectious disease caused by Listeria monocytogenes, a Gram-positive, intracellular, nonsporulating rod, which affects a wide range of animals as well as humans (George 2002, Nash et al. 1995). In both humans and livestock (sheep, cattle, goats, and less frequently, poultry), listeriosis can manifest as encephalitis, septicaemia, and abortion (Bartt 2000, Kumar et  al. 2007, Brugere-Picoux 2008). The disease usually occurs sporadically, thus outbreaks are only occasionally reported (Walland et  al. 2015). In animals, listeriosis outbreaks are primarily described in small ruminants (goats, sheep) and cattle (Wagner et al. 2005, Budrant et al. 2011, Dreyer et  al. 2015). The neurological form, otherwise known as circling disease, is usually observed in farm animals where the bacterium gains entrance to the body through abrasions of the buccal mucosa. It ascends unilaterally along the trigeminal nerve, resulting in encephalitis. L.  monocytogenes can spread intraaxonally within CASE REPORT 206 Listerial meningoencephalitis in Kosovo Sylejmani et al. Veterinaria Italiana 2020, 56 (3), 205-211. doi: 10.12834/VetIt.2166.12781.3 Case report Case presentation In March 2018, 10 adult ewes as well as 8 lambs from a flock of 277 sheep in central Kosovo displayed symptoms such as depression and anorexia followed by neurological signs. Forty-eight hours from the initial onset of the symptoms, the affected ewes and lambs manifested severe clinical signs such as temperature of 40.5 °C, grinding of teeth (crunch empty), circling and tilting of the head, unilateral facial paralysis, drooping ears and lips and supporting the head against the manger and walls. Within 7 days after the disease onset, 6 adult sheep and 5 lambs died. Samples were obtained from the brainstem of the dead animals. A silage diet had been administered to the flock of sheep. Based on the narrative provided by the sheep flock owner, one of the dead lambs (3 month old), was exclusively fed with milk from an ewe. Histopathological analysis and findings In order to investigate the cause of the disease and the fatalities, samples from three dead animals (ewes and lamb) were obtained from the brainstem to conduct histopathological analysis. The samples were fixed in 10% buffered formalin, dehydrated, and embedded in paraffin, 5  µm sections were stained with hematoxylin and eosin (H&E), and observed with a light microscope with 10x-40x objectives. Examination of the medulla oblongata revealed suppurative encephalitis characterized by microabscesses as well as neuroparenchymal lesions (Figure 2 and 3, black arrow) in all affected animals. Perivascular cuffing of lymphomononuclear cells was observed in the medulla oblongata with enhanced Virchow-Robin spaces (Figure 1, black arrow). Isolation of Listeria monocytogenes from brain samples At necropsy of two dead ewes and one lamb, two brain samples from each animal were collected for bacteriological analysis. Subsequently, samples were cultured in Fraser broth (Merck) and incubated aerobically for 24-48 h at 37 °C. After incubation, an aliquot was streaked onto PALCAM Listeria Selective Agar (Merck) and incubated aerobically for 24-48  h at 37  °C. Presumptive identification of L.  monocytogenes was based on the observation of typical colony morphologies on PALCAM agar, Gram staining, haemolysis patterns, catalase and oxidase reactions, and tumbling motility. The characteristic Gram-positive coccobacilli (short rods) less likely in comparison to cattle (Godreuil et  al. 2003, Rocha et  al. 2013). Sporadic ovine listeriosis cases are mainly attributted to serogroup 1/2a, 1/2b, and 4b (Low et  al. 1993, Kotzamanidis et  al. 2019). L.  monocytogenes is typically found in soil, vegetables, plants, feces, sewage, genital secretions and the nasal mucous membranes of healthy animals. The disease occurs more frequently in the winter and the early spring and is associated with encephalitis after silage intake (Wiedmann et  al. 1994, Bertsch et al. 2013). In these cases, mortality rates of 3.1% and 1.3% have been observed in ewes and lambs, respectively (Nash et al. 1995, Lyon et al. 2008). L.  monocytogenes is commonly present in silage, but it can multiply only in silage which has not been fermented properly (pH 5.0-5.5) (Doumith et  al. 2004, Kumar et  al. 2007). Livestock intended for food production play an important role in the zoonotic transmission of the disease. Humans commonly acquire L.  monocytogenes following consumption of products such as contaminated raw milk and, minimally processed foods of plant origin (contamination likely traced back to animal operations); however, rare cases of direct transmission have been reported (Heger et  al. 1997, Nightingale et  al. 2004). Outbreaks of human listeriosis have been mainly linked to consumption of ready-to-eat (RTE) foods, including soft cheeses. It should be noted that outbreak investigations and source attribution are often complicated by the typically long incubation time of L.  monocytogenes in conjuction with the ubiquity of this pathogen, which often leads to contamination of foods not likely to be associated with listerial presence (Granier et  al. 2011, Amato et  al. 2017, Buchanan et  al. 2017). Further, the occurrence of antibiotic resistance of isolates of L.  monocytogenes from non-human samples has been reported for a number of important antibiotics, including tetracycline, doxycyline, erythromycin and sulfamethoxazole/trimethoprim (Vela et  al. 2001, Granier et  al. 2011, WHO 2016, Amato et al. 2017 ). Well-established breakpoints for use in determination of antimicrobial susceptibility of L.  monocytogenes veterinary clinical isolates are not available, except for select antibiotics such as penicillin G and sulfamethoxazole/trimethoprim, complicating interpretation of antimicrobial susceptibility data and requiring extrapolation based on available information from other closely-related bacteria. The aim of the current study was to provide descriptive analysis of an outbreak in sheep associated with high mortality, determine the cause of fatalities, identify and characterize the etiological agent, and determine the antibiotic susceptibility of the isolates. 207 Sylejmani et al. Listerial meningoencephalitis in Kosovo Veterinaria Italiana 2020, 56 (3), 205-211. doi: 10.12834/VetIt.2166.12781.3 which were catalase positive and oxidase negative, and displayed umbrella shaped growth pattern in motility medium as well as tumbling motility in wet mounts at room temperature were considered as ‘presumptive’ Listeria isolates. Typical Listeria colonies were transferred to cryotubes containing Brain Heart Infusion Broth (BD Difco) with 20% glycerol and maintained frozen at - 20 °C until used for confirmation. Confirmation of isolates of L. monocytogenes Downstream confirmation analyses revealed the presence of L. monocytogenes in six brain samples (three different animals) analyzed (Table I). The confirmation of isolates of L.  monocytogenes was achieved by matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-TOF MS)-based identification. MALDI-TOF MS and biotyping was performed using the BrukerUltraflex  II TOF/TOF in positive ion reflector mode pre-calibrated with bacterial test standard (Bruker) according to the manufacturer’s instructions using an ethanol/ formic acid extraction protocol. Spectral analyses and sample identifications was performed using the BrukerBiotyper software (Ver. 3.1.). Identifications were only accepted if scored ≥  2.0 by the Biotyper algorithm. All isolates were confidently identified by MALDI-TOF MS as L.  monocytogenes (score higher than 2.4). Molecular serotyping of L. monocytogenes L.  monocytogenes strains previously confirmed by MALDI-TOF MS, including five isolates (B1, B2, B3, B4, and B5) from the first sheep, five isolates (B6, B7, B8, B9, and B10) from the second sheep, and eight isolates (B 11, B12, B13, B14, B15, B16, B17, and B18) from the lamb, were subjected to molecular serotyping by targeting six genes of L.  monocytogenes, lmo0737 (691 bp), lmo1118 (906  bp), ORF2819 (471 bp), ORF2110 (597 bp), prfA (274 bp), and prs (370 bp) (Doumith et  al. 2004, Kerouanton et al. 2010). PCR was performed Figure 3. Perivascular infiltration of lympho mononuclear cells in medulla oblongata (Hematoxylin and Eosin stain; 40× Objec.) Figure 2. Microabscess in medulla oblongata (Hematoxylin and Eosin stain; 40× Objec.) Figure 1. Neuroparenchymal lesions from infiltration of lympho mononuclear cells (Hematoxylin and Eosin stain; 40× Objec.). Table I. Number of samples and L. monocytogenes isolates included in the study. Number of samples (brain) Strains* Serotypes 2 (from sheep 1) B1, B2, B3, B4, B5 4b 2 (from sheep 2) B6, B7, B8, B9, B10 4b 2 (from lamb 1) B11, B12, B13, B14, B15, B16, B17, B18 4b *B = Clinical isolates from the brain of sheep and lamb affected of meningoencephalitis. 208 Listerial meningoencephalitis in Kosovo Sylejmani et al. Veterinaria Italiana 2020, 56 (3), 205-211. doi: 10.12834/VetIt.2166.12781.3 quinupristin/dalfopristin, linezolid, nitrofurantoin, kanamycin, ciprofloxacin, vancomycin, lincomycin and gentamicin. The breakpoints used for the determination of the minimum inhibitory concentrations (MIC) using the broth microdilution method, were those recommended by Clinical & Laboratory Standards Institute (CLSI 2014) for closely-related bacteria (Staphylococcus spp., Enterococcus spp., Streptococcus spp.). There was one exception to this pattern, namely for penicillin for which specific Listeria breakpoints are defined. Staphylococcus aureus ATCC 29213 was used as quality control strain, with susceptibility testing results in the expected range. All 18 L.  monocytogenes strains tested were found to be susceptible to penicillin (MIC 0.5-1  µg/ mL), erythromycin (MIC 0.25  µg/mL), tetracycline (MIC 1  µg/mL), ciprofloxacin (MIC 1  µg/mL), tylosin tartrate, sulfamethoxazole/trimethoprim, streptomycin, vancomycin, and gentamicin, but with 2  separate groups, one for genes prfA and prs, and the other four genes lmo0737, lmo1118, ORF2819, and ORF2110. The molecular serotyping using multiplex PCR revealed prfA, prs, ORF2819, and ORF2110 genes from all isolates, indicating that the isolates belonged to L.  monocytogenes serotype 4b. Antimicrobial susceptibility testing of L. monocytogenes isolates All strains were tested for antimicrobial susceptibility by broth microdilution using the Sensititre® GPN3F dehydrated panel (Trek Diagnostic Systems, Cleveland, OH) following the manufacturer’s instructions. The following antimicrobial agents were used for the in vitro microdilution broth testing protocol: penicillin, erythromycin, sulfamethoxazole/ trimethoprim, chloramphenicol, tetracycline, daptomycin, streptomycin, tylosin tartrate, Table II. MICs of 16 antimicrobial agents for L. monocytogenes isolates from ovine meningoencephalitis. Antimicrobials Number of isolates (n = 18) with MIC of (mg/l) 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256 512 1024 Penicillin - - - 2 (11.1) 14 (77.8) 2 (11.1) - - - - - - - - - - Erythromycin - - - 18 (100) - - - - - - - - - - - - Tetracycline - - - - - 18 (100) - - - - - - - - - - Ciprofloxacin - - - - - 18 (100) - - - - - - - - - - Chloramphenicol - - - - - - - - 15 (83.3) 2 (11.1) 1 (5.6) - - - - - Sulfamethoxazole/ Trimethoprim - - - - 18 (100) - - - - - - - - - - - Daptomycin - - - - - - 11 (61.1) 7 (38.9) - - - - - - - - Vancomycin - - - 2 (11.1) - 14 (77.8) 2 (11.1) - - - - - - - - - Streptomycin - - - - - - - - - - - - - - 18 (100) - Nitrofurantoin - - - - - - - - - - - 18 (100) - - - - Tylosin tartrate - - - - - - - 16 (88.9) 2 (11.1) - - - - - - - Gentamicin - - - - - - - - - - - - 18 (100) - - - Quinupristin/ dalfopristin - - - - 12 (66.6) 5 (27.8) 1 (5.6) - - - - - - - - - Lincomycin - - - - - - - - 18 (100) - - - - - - - Linezolid - - - - 1 (5.6) - 2 (11.1) 15 (83.3) - - - - - - - - Kanamycin - - - - - - - - - - - - 17 (94.4) - - 1 (5.6) 209 Sylejmani et al. Listerial meningoencephalitis in Kosovo Veterinaria Italiana 2020, 56 (3), 205-211. doi: 10.12834/VetIt.2166.12781.3 gentamicin, and vancomycin, are quite similar to those reported for strains of L. monocytogenes from ovine meningoencephalitis in a study by Vela and colleagues (Vela et al. 2001). While in other authors' studies L.  monocytogenes isolates have shown resistance to tetracycline (Vela et  al. 2001, Counter et  al. 2007), the isolates in the current study were susceptible to this antimicrobial. Based on the in vitro analysis, it would be expected that penicillin would provide an effective treatment during administration of antibiotic therapy in the current clinical settings. Furthermore, low-level resistance to a fluoroquinolone such as ciprofloxacin has been associated with the presence of efflux pumps and potentially the use of fluoroquinolones in animal production, especially broiler production (Godreuil et  al. 2003). It is not unusual that all isolates displayed resistance to daptomycin, which corroborated findings of other studies which do not recommend the use of daptomycin for treatment of listeriosis cases (Noll et  al. 2018, Spanjaard and Vandenbroucke-Grauls 2008). Multiple mechanisms, including some that are transferable, have been reported to be involved in conferring resistance to lincosamides in Listeria spp. (Bertsch et  al. 2013). Different L.  monocytogenes isolates originating from the same tissues (i.e. brain) were shown to contain different antimicrobial resistance phenotypes. This could also indicate further differences between the different strains of isolates of L.  monocytogenes 4b, including other virulence factors. Furthermore, this study demonstrates the presence of highly pathogenic L.  monocytogenes serotype 4b strains causing a neural form of listeriosis in small ruminants in Kosovo, which is being reported for the first time. Listeriosis-positive sheep flocks may pose a grave public health risk, as a great number of popular food products derived from these animals are often consumed in the region, greatly increasing opportunities for zoonotic transmission. Therefore, in such cases preventive measures have to be undertaken to avoid the transfer of L. monocytogenes to processed milk and other dairy products, although human listeriosis cases are yet to be reported in Kosovo. Lastly, the results of our study indicate that L.  monocytogenes from ovine listeriosis manifests susceptibility to most commonly utilized antimicrobials; however, several instances of intermediate susceptibility and full resistance identified in the tested isolates suggests a future need for close monitoring and surveillance of antimicrobial resistance in L. monocytogenes strains of animal origin in Kosovo. resistant to lincomycin with an observed MIC of 8  µg/mL, daptomycin (MIC 2-4  µ/mL) and intermediate to nitrofurantoin with an observed MIC of 64 µg/mL. Furthermore, a number of isolates were classified as sensitive to some other antimicrobials such as kanamycin (17/18), quinupristin/dalfopristin (17/18), chloramphenicol (15/18), and linezolid (3/18); resistant to chloramphenicol (1/18) and kanamycin (1/18); and intermediate to linezolid (15/18), chloramphenicol (2/18), and quinupristin/ dalfopristin (1/18) (Table II). Discussion The findings of suspected neurological disease presented in this case study were established through histopathology and horizontal isolation of the etiological agent, L.  monocytogenes from brain tissues. Histopathological findings observed within the brainstem of these animals are consistent with those previously described for ruminant encephalitic listeriosis (Maxie and Youssef 2007, Zachary 2012). The finding of a seasonal occurrence of ruminant listeriosis in this study correlates well with the findings reported by Wiedmann and colleagues (Wiedmann et  al. 1994). That specific study reported isolation of Listeria from brains of sheep and goats affected with listerial encephalitis and from silage consumed by the animals in the winter and early spring. We found that only one serotype, serotype 4b, was implicated as the etiological agent in this outbreak. In another similar study conducted in Greece, serotype 4b was reported as the predominant strain (81%) isolated in small animals, in which the neural form of the disease was diagnosed (Giannati-Stefanou et al. 2006). The antimicrobial susceptibility testing results in our study overwhelmingly support the presence of highly  susceptibile L.  monocytogenes isolates (n  =  18). We report the following levels of susceptibility for the isolates tested: penicillin  (100%), erythromycin (100%), tetracycline (100%), ciprofloxacin (100%), tylosin tartrate (100%), sulfamethoxazole/trimethoprim (100%), vancomycin (88.9%), streptomycin (100%), gentamicin (100%), kanamycin (94.4%), quinupristin/dalfopristin (94.4%), and chloramphenicol (83.3%). All isolates (100%) were resistant to lincomycin and daptomycin. Resistance was also observed to chloramphenicol and kanamycin (5.6%). On the other hand, our study indicated that the following proportions of L.  monocytogenes isolates could be included in the intermediate category: nitrofurantoin (100%), linezolid (83.3%), chloramphenicol (11.1%), and quinupristin/dalfopristin (5.6%). The MIC ranges obtained in this study for penicillin, erythromycin, 210 Listerial meningoencephalitis in Kosovo Sylejmani et al. Veterinaria Italiana 2020, 56 (3), 205-211. doi: 10.12834/VetIt.2166.12781.3 Acknowledgements The authors gratefully aknowledge the local veterinarians of the municipality of Ferizaj Kosovo, where the listeriosis outbreak was identified as well as the regional animal health and veterinary inspectorate for the cooperation and the resources made available to acces the farm and investigate the case. Funding This study was supported by own funds of the University of Prishtina and the Department of Animal Science at the University of Wyoming. 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