Circulation and Molecular Characterization of Infectious Laryngotracheitis Virus in Poultry Flocks with Respiratory Disorders in Turkey, 2018-2022.

Infectious laryngotracheitis (ILT) is a very serious worldwide respiratory disease of poultry, with many countries reporting ILT infections over the last decade. However, few reports are available regarding ILT disease prevalence in poultry in Turkey. Accordingly, the present study investigated ILT infection in Turkish broiler flocks between 2018 and 2022. Circulating ILT strains were characterized by sequence and phylogenetic analysis of two fragments of the infected-cell protein 4 gene. ILT virus (ILTV) was confirmed by quantitative PCR in 8 of the 21 flocks examined. As in other diseases, co-infections with other respiratory pathogens in confirmed ILT cases may worsen the symptoms and prolong the disease course. The present study confirmed co-infections with infectious bronchitis virus (13/21 tested flocks and 5/8 ILTV-positive flocks), indicating the importance of these pathogens in the occurrence of ILT infections.

Circulación y caracterización molecular del virus de la laringotraqueítis infecciosa en bandadas de aves de corral con trastornos respiratorios en Turquía, 2018­2022. La laringotraqueítis infecciosa (ILT) es una enfermedad respiratoria muy seria de la industria avícola en todo el mundo y muchos países han notificado infecciones por esta enfermedad durante la última década. Sin embargo, hay pocos informes disponibles sobre la prevalencia de laringotraqueítis infecciosa en la avicultura de Turquía. En consecuencia, el presente estudio investigó la infección por laringotraqueítis infecciosa en parvadas de pollos de engorde en Turquía entre los años 2018 y 2022. Las cepas de laringotraqueítis infecciosa circulantes se caracterizaron mediante análisis de secuencias y filogenéticos de dos fragmentos del gene de la proteína 4 de las células infectadas. El virus ILT (ILTV) se confirmó mediante PCR cuantitativa en ocho de las 21 parvadas examinadas. Como ocurre con otras enfermedades, las coinfecciones con otros patógenos respiratorios en casos confirmados de laringotraqueítis infecciosa pueden complicar los signos clínicos y prolongar el curso de la enfermedad. El presente estudio confirmó coinfecciones con el virus de la bronquitis infecciosa (en 13/21 parvadas analizadas y en 5/8 parvadas positivas para laringotraqueítis infecciosa), lo que indica la importancia de estos patógenos en la aparición de infecciones por la laringotraqueítis infecciosa.

Genotyping and antimicrobial resistance profiles of chicken originated Salmonella Enteritidis isolates.

Salmonellosis is a common foodborne zoonosis worldwide. The most common Salmonella serovar in humans is Salmonella enterica subsp. enterica serovar Enteritidis (50.3%) in the world. The main transmission route for S. Enteritidis is consumption of contaminated poultry products. Therefore, it is important to determine the diversity and spread of chicken-originated S. Enteritidis isolates in order to monitor and control salmonellosis. Pulsed-field gel electrophoresis (PFGE) and multiple locus variable number of tandem repeats analysis (MLVA) are frequently used for typing of S. Enteritidis isolates. This study aimed to determine the antimicrobial resistance (AMR) profiles and MLVA and PFGE genotypes of chicken-originated S. Enteritidis isolates. A total of 200 S. Enteritidis isolated from chicken broiler, layer, and breeder flocks from different locations in Turkey were investigated by Kirby-Bauer disk diffusion method, PFGE, and MLVA. The AMR test indicated that 57% of the S. Enteritidis isolates were susceptible to all antimicrobials, while 39% were resistant to at least one antimicrobial. The highest resistance (25%) was against ampicillin. Multi-drug resistance rate was low (21%) and mostly from broiler flocks (93%). All isolates were genotyped into 32 different PFGE genotypes (PT) and 34 different MLVA genotypes (MT). The dominant genotypes were PT6 (12.5%) and MT22 (50%). In specific sample groups, there was a correlation between genotypes, breeding type, geographic location, and isolation years of the isolates. There was no significant difference in the discrimination power of PFGE and MLVA. However, MLVA was more suitable for large sample groups and routine genotyping because it was easier, quicker, and less labor-intensive to use.

Antibiotic Resistance Profile of Rarely Isolated Salmonella Serotypes from Poultry in Turkey.

This study investigated five strains of each serotype of Salmonella Agona, Salmonella Heidelberg, Salmonella Hindmarsh, Salmonella Kouka, Salmonella Muenchen, Salmonella Ottmarchen, Salmonella Saintpaul and Salmonella II, isolated during the 2014-2017 period. Disc diffusion was used to identify the phenotypic profiles of antibiotic resistance to 12 antimicrobials while the presence of antibiotic resistance genes (ARGs) was detected by PCR. The most sensitive serotype was S. Kouka while the most resistant serotypes were S. Agona and S. Heidelberg. MDR was detected most frequently in S. Agona strains, followed by S. Saintpaul, S. Hindmarsch, and S. Ottmarchen. The samples were most susceptible to chloramphenicol and ceftazidime and most resistant to sulfonamide. The resistance genes were detected in phenotypically resistant strains. Among the tetracycline-resistant strains, tet (A) was the most prevalent gene. The results of this study highlight the importance of monitoring antibiotic resistance profiles and related genes, which can spread to form MDR bacteria. Salmonella spp., which significantly contribute to ARG dissemination, should be monitored constantly to protect the closely related health of humans, animals, and the environment. The level of antibiotic resistance observed in this study, even in rarely isolated Salmonella serotypes, also indicates the need for careful and selective use of antibiotics.

Presence of Clostridioides difficile in cattle feces, carcasses, and slaughterhouses: Molecular characterization and antibacterial susceptibility of the recovered isolates.

The aims of this study were to isolate and identify Clostridioides difficile from cattle feces and carcasses, and slaughterhouse samples, and to determine the molecular characteristics and antibacterial susceptibility of the recovered isolates. A total of 220 samples, including 100 cattle fecal samples, 100 cattle carcass surface samples, and 20 slaughterhouse samples were used as the study material. In total, 12 (5.45%) samples, including 11 (11%) cattle fecal samples and 1 (5%) slaughterhouse sample, were found to be positive for C. difficile. On the other hand, all of the carcass samples were negative for C. difficile. A total of 11 (91.66%) isolates, including 10 fecal isolates and 1 slaughterhouse wastewater isolate, were found to be positive for the presence of the toxin genes tcdA and tcdB, whilst 1 fecal isolate was found to be negative for both genes. In addition, 3 different ERIC-PCR profiles were identified in the 11 fecal isolates. The ERIC-PCR profile of the slaughterhouse wastewater isolate was found to be similar to one of the ERIC-PCR profiles obtained from the fecal isolates. All of the isolates were resistant to ciprofloxacin and levofloxacin. Considering that the agent is a spore-forming bacterium shed in feces, the detection of C. difficile isolates of different genotypes, some carrying toxin genes, suggests that feces and slaughterhouse wastewater carrying this bacterium may pose a risk for the contamination of carcasses. The current study revealed that hygiene conditions should be performed to the maximum extent in slaughterhouses.

Campylobacter anatolicus sp. nov., a novel member of the genus Campylobacter isolated from feces of Anatolian Ground Squirrel (Spermophilus xanthoprymnus) in Turkey.

Seventy-four Gram-negative, motile, slightly curved rod-shaped, microaerophilic, oxidase-positive and catalase-negative isolates, recovered from fecal samples of the Anatolian ground squirrel (Spermophilus xanthoprymnus) in Kayseri, Turkey, were subjected to a polyphasic taxonomic study. Results of a genus-specific PCR indicated that all isolates belonged to the genus Campylobacter. 16S rRNA gene sequence analyses revealed the closest match as Campylobacter curvus DSM 6644T with identity levels of 96.41-96.70%. Based on the 16S rRNA gene phylogeny of the 74 isolates, six isolates (faydin-G24, faydin-G52, faydin-G105, faydin-G114, faydin-G129 and faydin-G140T) were chosen as representatives for further characterization. The overall genome relatedness indices for the strain faydin-G140T, compared to the most closely related type strain C. curvus ATCC 35224T, were calculated as 15.2%, 72.5%, and 83.7% for digital DNA-DNA hybridization (dDDH), and average nucleotide identity (ANIb and ANIm), respectively. The G+C content and genome size of the strains ranged between 35.2-35.4 mol% and 1.7-1.8 Mb, respectively. Based on data obtained from the polyphasic taxonomy approach, including phenotypic characterization as well as genomic and chemotaxonomic analyses, these strains are concluded to represent a novel species, for which the name Campylobacter anatolicus sp. nov. is proposed with faydin-G140T as the type strain (=DSM 112311T = LMG 32238T).

The first detection and characterization of goose parvovirus (GPV) in Turkey.

Derzsy's disease, which is seen in goslings (Anser anser domestica) and Muscovy ducks (Cairina moschata), progresses to high mortality and causes significant yield losses. The disease agent is goose parvovirus (GPV), which is common in countries with waterfowl production. It has not previously been reported in Turkey. Using qPCR and sequencing of the VP3 protein-encoding gene, GPV is identified as the causative agent of high mortality among geese between 2018 and 2019. The VP3 sequences were also compared with the similar GenBank sequences phylogenetically. All the sequences were found to be most similar (98.90%) with Polish and Taiwan GPV strains. Phylogenetic analysis of the VP3 gene in strains in Turkey and comparison with strains from other countries demonstrated that the Turkish strains are native to the geography and circulated locally. This study detected the presence of the GPV gene for the first time in Turkey and demonstrated the importance of comparing the vaccine strain and wild type.

Molecular characterisation of hydrogen sulfide negative Salmonella enterica serovar Havana.

Hydrogen sulfide (H2S) detection is a screening method for distinguishing and identifying Salmonella strains from other bacteria in the intestine. Incidences of H2S-negative Salmonella have recently been reported in different countries. Although a high resistance rate against antimicrobial agents has been reported for H2S-positive Salmonella in many regions of the world, there is increasing evidence that high resistance to antibiotics has also increased in many H2S-negative Salmonella isolates. In this study, molecular characterisation of three H2S-negative Salmonella Havana, isolated from cloacal swab samples of broiler chickens, was performed. The phsA, phsB and phsC genes of the phs operon, which is responsible for hydrogen sulfide production, were amplified. Sequence analysis was then performed to identify mutations in the gene cluster. The antimicrobial resistance profiles of the isolates were determined by disc diffusion. Molecular characterisation was performed by multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE). The sequence analysis showed identified five point mutations in the phsA gene and one point mutation in the phsC gene in all isolates. The antibiotic resistance profile showed that the strains were resistant to cefoxitin and ceftazidime. MLST analysis showed that all strains belonged to sequence type (ST) 1621. This study is the first to report the H2S-negative S. Havana serotype.

[Multilocus sequence analysis, biofilm production, antibiotic susceptibility and synergy tests of Burkholderia species in patients with and without cystic fibrosis]. / Kistik fibrozisli ve kistik fibrozisi olmayan hasta grubunda Burkholderia türlerinin multilokus sekans analizi, biyofilm olusturma, antibiyotik duyarlilik ve sinerji testleri.

Burkholderia spp. emerged as important pathogens in the airways of immunocompromised humans, especially those with cystic fibrosis (CF). Failure of identification with conventional techniques, high intrinsic resistance to most antibiotics and biofilm formation can cause difficulties in the treatment of these infections. The aim of this study was to identify Burkholderia spp. strains isolated from CF and non-CF patients with with routine microbiological methods, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and multilocus sequence analysis (MLSA), to determine of the antibiotic susceptibility and synergies, and to evaluate biofilm formation of these isolates. A total of 38 Burkholderia spp. (25 CF, 13 non-CF) from 26 patients were identified by biochemical, phenotypical and matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and sequence types were revealed by multilocus sequence analysis (MLSA). Sequence types of isolates were identified using the PubMLST database. Characteristics of biofilm formation of clinical isolates were evaluated by microplate method. Antibiotic susceptibilities of ceftazidime, meropenem, trimethoprim-sulfamethoxazole (TMP-SXT) and levofloxacin were determined by broth microdilution method according to CLSI (2017) guidelines. Synergy tests were performed by checkerboard method. Clinical isolates were identified as Burkholderia cenocepacia (n= 16), Burkholderia contaminans (n= 11), Burkholderia gladioli (n= 4), Burkholderia dolosa (n= 4), Burkholderia multivorans (n= 2) and Burkholderia seminalis (n= 1). Sequence types of these isolates were determined as ST19, ST72, ST102, ST180, ST482, ST602, ST629, ST740, ST839 and ST1392. The correct identification at the species-level with MALDI-TOF MS was 94-100% for all isolates except B.contaminans. Biofilm formation among the identified species in the study was determined as 53% (n= 20). There was no statistical difference when the biofilm production was evaluated separately among Burkholderia species and biofilm production rates between CF (56%, 14/25) and non-CF (46%, 6/13) Burkholderia isolates (p> 0.05). Overall rates of resistance to ceftazidime, meropenem, TMP-SXT, and levofloxacin of the isolates were 35%, 66%, 50% and 40%, respectively. The antibiotic resistance against Burkholderia spp., isolates obtained from CF patients were more susceptible to ceftazidime, but no significant difference was found for other antibiotics. Synergy was determined between meropenem and TMP-SXT in two isolates. Antagonism was detected in 15 isolates, 12 of them were between meropenem and ceftazidime, three of them were between ceftazidime and TMP-SXT. Numerous resistance mechanisms may lead to higher resistance in this bacteria, whereas the antagonism between meropenem and ceftazidime in this study might be attributed to the expression of beta-lactamases. In this study, the distinctness of sequence types between Burkholderia spp. isolated from CF and non-CF patient, provided a better understanding about the importance of biofilm formation for the infections with these bacteria and emphasized that the management of therapy should be driven by the antibiotic test results.

Pathogenicity, genotyping and antibacterial susceptibility of the Listeria spp. recovered from stray dogs.

The present study aimed to determine the prevalence of Listeria spp. in stray dogs in the Kayseri province of Turkey. In addition, serotyping, genotyping and virulence gene analysis of the isolated Listeria spp. were performed and their pathogenicity and antibacterial susceptibility were investigated. The study included 80 rectal swaps taken from 80 stray dogs of different ages and gender that were sheltered in the Kayseri Municipal Dog Shelter. Listeria selective broth and Listeria selective agar were used for the isolation of Listeria spp. and the isolates were identified using a Microbact 12L (Oxoid, England) identification test kit. 16S rDNA sequencing and species-specific polymerase chain reaction (PCR) were performed for molecular identification of the isolates, multiplex PCR and a serological test were performed for serotyping, and PCR was used for virulence gene analysis. For determining the pathogenicity of L. monocytogenes and L. innocua isolates, a total of 100 mice (50 pregnant and 50 non-pregnant) were used. The mice were infected intraperitoneally; the inoculation dose was 1 × 109 CFU/mL and 0.2 mL was used for each animal. Tissue samples obtained from infected mice were processed for the re-isolation of the Listeria spp. and then stained with hematoxylin eosin and Brown-Brenn Gram stain. The antibiotic susceptibilities of the isolates were determined by the disk diffusion method. Listeria spp. were isolated from 5 (6.25%) of the 80 fecal samples. While 1 of the isolates was identified as L. monocytogenes, 4 of them were identified as L. innocua. Serotyping by serological and molecular methods revealed the isolate of L. monocytogenes to be serotype 1/2a. According to the phylogenetic trees, L. innocua and L. monocytogenes strains were clustered in different groups. The L. monocytogenes isolate was positive for all virulence genes tested. All L. innocua isolates were positive for the plcB gene. While all L. innocua isolates were negative for the lin1068 gene, 3 L. innocua isolates were found to be positive for the lin0558 gene. In mice infected with L. monocytogenes, pathological findings were observed in the uterus, intestines, pancreas, and heart. In mice infected with L. innocua, pathological findings were observed in the stomach, intestines and spleen. L. monocytogenes- or L. innocua-related infections or other inflammatory reactions were not observed in the brains of infected animals. On histopathological examination with Gram stain, an abundance of Listeria spp. was observed in the lesions of the liver, spleen, uterus, and kidney. Moreover, while abortion was observed in all animals infected with L. monocytogenes, it was not observed in any of the animals infected with L. innocua. Antibiotic susceptibility testing revealed that all 5 isolates were sensitive to ampicillin, amoxicillin/clavulanic acid, erythromycin, gentamicin, penicillin G, and trimethoprim-sulfamethoxazole and were resistant to nalidixic acid, streptomycin, and cefuroxime sodium. Considering also the pathogenicity of the isolated microorganisms, it can be suggested that stray dogs as carriers of Listeria spp. are a significant risk to public health. As L. innocua isolates, which are considered apathogenic, led to the occurrence of lesions similar to those caused by L. monocytogenes, detailed studies on the pathogenesis of L. innocua infections caused by L. innocua isolates recovered from various sources are required.

Neonatal calf meningitis associated with Streptococcus gallolyticus subsp. gallolyticus.

Here, we report a case of neonatal calf meningitis due to Streptococcus gallolyticus subsp. gallolyticus (SGG). Clinical, pathological and microbiological findings were evaluated. API Strep, 16S rRNA gene sequencing, rpoB gene sequencing and sodA gene sequencing were used for the complete identification of SGG. This is the first documented report of neonatal calf meningitis due to SGG in veterinary medicine.

[Antimicrobial susceptibilities of clinical Nocardia isolates identified by 16S rRNA gene sequence analysis]. / 16S rRNA gen dizi analizi ile tanimlanan klinik Nocardia izolatlarinin antimikrobiyal duyarliliklari.

Nocardia species are ubiquitous in the environment and responsible for various human infections such as pulmonary, cutaneous, central nervous system and disseminated nocardiosis. Since the clinical pictures and antimicrobial susceptibilities of Nocardia species exhibit variability, susceptibility testing is recommended for every Nocardia isolates. The aims of this study was to determine the antimicrobial susceptibilities of Nocardia clinical isolates and to compare the results of broth microdilution and disc diffusion susceptibility tests. A total of 45 clinical Nocardia isolates (isolated from 17 respiratory tract, 8 brain abscess, 7 pus, 3 skin, 3 conjunctiva, 2 blood, 2 tissue, 2 pleural fluid and 1 cerebrospinal fluid samples) were identified by using conventional methods and 16S rRNA gene sequence analysis. Susceptibility testing was performed for amikacin, ciprofloxacin, ceftriaxone, linezolid and trimethoprim-sulfamethoxazole (TMP-SMX) by broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) criteria recommended in 2011 approved standard (M24-A2) and disk diffusion method used as an alternative comparative susceptibility testing method. Among the 45 Nocardia strains, N.cyriacigeorgica (n: 26, 57.8%) was the most common species, followed by N.farcinica (n: 12, 26.7%), N.otitiscaviarum (n: 4, 8.9%), N.asteroides (n: 1, 2.2%), N.neocaledoniensis (n: 1, 2.2%) and N.abscessus (n: 1, 2.2%). Amikacin and linezolid were the only two antimicrobials to which all isolates were susceptible for both broth microdilution and disk diffusion tests. In broth microdilution test, resistance rates to TMP-SMX, ceftriaxone and ciprofloxacin were found as 15.6%, 37.8% and 84.4% respectively, whereas in the disk diffusion test, the highest resistance rate was observed against ciprofloxacin (n: 33, 73.3%), followed by TMP-SMX (n: 22, 48.9%) and ceftriaxone (n: 15, 33.3%). In both of these tests, N.cyriacigeorgica was the species with the highest resistance to ciprofloxacin (n: 25, 96.2%). When the susceptibility test results were compared, amikacin (κ= 1), linezolid (κ= 1), and ceftriaxone (κ= 0.903) showed very good agreement, whereas ciprofloxacin showed good agreement (κ= 0.672). For TMP-SMX no agreement was found between the two test methods (κ= 0.092). In conclusion, due to the identification of different species with molecular methods and increased frequency of Nocardia infections in recent years, in vitro susceptibility testing for Nocardia species is important to guide the appropriate antimicrobial treatment.

Phylo-typing of clinical Escherichia coli isolates originating from bovine mastitis and canine pyometra and urinary tract infection by means of quadruplex PCR.

BACKGROUND: Escherichia coli is one of the major causative agents of bovine mastitis worldwide, and is typically associated with acute, clinical mastitis. Besides this, E. coli strains which belong to the extra-intestinal pathogenic group are also the major cause of urinary tract infections and pyometra in dogs. OBJECTIVES: In this study, it was aimed to investigate phylo-groups/subgroups in 155 E. coli isolates obtained from acute bovine mastitis, 43 from urinary tract infections of dogs and 20 from canine pyometra by a formerly described triplex PCR and recently described new quadruplex polymerase chain reaction (PCR) method. RESULTS: Group A1 (n = 118; 76%) and B1 (n = 71; 46%) were found to be the most prevalent groups by triplex and quadruplex PCR assays in mastitis isolates, respectively. Phylo-typing of 43 urinary tract isolates also revealed that most of the isolates belonged to A1 (n = 23; 54%) by triplex and B2 (n = 36; 84%) by quadruplex PCR assays. The isolates assigned as group A1 (n = 17; 85%) by triplex PCR could not be classified by quadruplex PCR in pyometra isolates. CONCLUSIONS: The results support the hypothesis that E. coli strains isolated from bovine mastitis cases are environmental. Also, groups C, E and F were identified as new phylo-groups for the first time in acute bovine mastitis cases. The comparison of triplex PCR with quadruplex PCR results revealed that most of the groups assigned in triplex PCR were altered by quadruplex PCR assay.