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Glaesserella parasuis (GPS) is an important bacterial pathogen of swine. Serotype identification has presented a bottleneck in GPS research since it was first identified as the pathogen causing Glässer's disease in pigs in 1910. This paper presents a systematic review of the history of the development and application of gel immunodiffusion (GID), indirect hemagglutination assay (IHA), and polymerase chain reaction (PCR) typing methods for GPS, and the discovery of their shared antigenic basis. It provides a systematic theoretical overview of the immunology and principles underlying the three typing methods and offers new ideas for research into the prevention and control of Glässer's disease. In 1992, GPS was first classified into serotypes 1-15 using GID based on GPS heat-stable antigens, but about 25% of the strains were found to be non-typeable, and the composition of their antigens for serotyping was unclear. In 2003, the IHA method was established based on saline-extracted antigens of GPS, whose sensitivity and typing rate were higher than for GID, although about 15% of strains were still found to be non-typeable. The results of IHA and GID typing are roughly consistent, since they share the same GPS surface polysaccharide serotyping antigens, although whether these are capsular polysaccharides, lipopolysaccharides, or other polysaccharides, remains to be determined. In 2013, the Capsular polysaccharide (CPS) synthetic gene clusters from GPS serotypes 1-15 were successfully analyzed, confirming that CPS is essential for the formation of antigens for serotyping. In 2015, primers were designed based on the specific target genes of GPS capsules to establish a PCR typing method (H-PCR) for GPS, which, however, could not identify serotypes 5 and 12. In 2017, a new PCR typing method (J-PCR) was established based on the specific target genes of GPS capsules, which could identify serotypes 5 and 12. A combination of the two PCR typing methods enables the typing of almost all GPS strains, and the consistency with GID and IHA was verified using molecular biological methods. The antigenic basis of the three typing methods was shown to involve the GPS capsule. PCR typing methods are characterized by simple operation, fast speed, and low cost, and can successfully solve many problems in GID and IHA serotyping, and so have become widely adopted., (© 2024. The Author(s).)

This study aimed to determine the prevalence and serovar distribution of salmonellae in liver, heart, and spleen (LHS) and gizzard (G) of slaughtered broilers. For this, a total of 60 sample units, comprised of 30 LHS and 30 G collected from 3 slaughterhouses, were analysed by reference methods for detection and serotyping as revised ISO 6579-1:2017 and ISO 6579-3:2014, respectively. Also, Salmonella-specific real-time PCR (Salm-PCR) was used for species confirmation, while Salmonella Enteritidis (S. Enteritidis) and Salmonella Typhimurium (S. Typhimurium) specific real-time PCR (SE/ST-PCR) was evaluated to determine its efficiency for rapid detection of the serovars mandated in current legal regulations compared to standard serotyping. All LHS (100%-30/30) and 90% (27/30) of G samples harbored Salmonella with an overall prevalence of 95% (57/60) in samples examined, where all isolates were confirmed as Salmonella by Salm-PCR. The most prevalent serovar in broiler giblets was S. Virchow (80.70%-46/57) followed by S. Enteritidis (19.30%-11/57). SE/ST-PCR (%17.54-10/57) could not detect one G isolate, which was serotyped as S. Enteritidis by standard serotyping. High relative accuracy (98.25%), sensitivity (100%) and specificity (100%), and agreement between methods (κ: 0.94) verified SE/ST-PCR's potential to be used as an alternative in rapid detection of S. Enteritidis and S. Typhimurium. Data on high Salmonella prevalence in broiler giblets of slaughterhouse origin, and detection of the pathogen by the implementation of all requirements indicated in the revised ISO 6579-1:2017 standard method, enabling the determination of actual prevalence in the samples with high sensitivity and specificity is of significance for public health. Additionally, identification of S. Virchow as the dominant serovar followed by S. Enteritidis with a relatively lower prevalence, and absence of S. Typhimurium in broiler giblets are important findings for Turkiye. This up to date data, obtained by strict application of ISO 6579-3:2014 procedures, indicated a shift in circulating serovars in the broiler industry. The objective findings in this study would bring awareness to national/international literature, and may be of use in future improvements in legal regulations., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Avian cellulitis in broilers, caused by avian pathogenic Escherichia coli, is a major cause for carcass rejections during meat inspection, resulting in significant economic losses. In this study, we analysed E. coli isolates obtained from broiler chickens affected by cellulitis for their genetic relatedness and antimicrobial resistance phenotype and genotype. The objective was to determine whether there is a clonal spread or whether these clinical isolates differ. For this purpose, E. coli was isolated from swab samples collected from diseased broilers across 77 poultry farms in Germany, resulting in 107 isolates. These isolates were subjected to serotyping, PCR-based phylotyping and macrorestriction analysis with subsequent pulsed-field gel-electrophoresis for typing purposes. In addition, the presence of virulence genes associated with avian pathogenic E. coli (APEC) was investigated by PCR. Antimicrobial susceptibility of the isolates was examined by the disk diffusion method according to CLSI guidelines and subsequently, the presence of corresponding resistance genes was investigated by PCR. Typing results revealed that a significant proportion of the isolates belonged to serotype O78:K80, which is one of the major APEC serotypes. Phylogenetic grouping showed that phylogenetic group D was most commonly represented (n = 49). Macrorestriction analysis showed overall heterogenous results, however, some clustering of closely related isolates was observed. The level of antimicrobial resistance was high, with 83.8% of isolates non-susceptible to at least one class of antimicrobial agents and 40% of isolates showing resistance to at least three classes. The most frequently observed resistance was to ampicillin, mediated by bla TEM (n = 56). However, few isolates were non-susceptible to ciprofloxacin (n = 8) and none of the isolates was resistant to 3rd generation cephalosporins or carbapenems. Overall, the results show that genetically diverse APEC associated with avian cellulitis can be found among and within German poultry farms. While most isolates were antimicrobial resistant, resistance levels to high(est) priority critically important antimicrobials were low., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to determine Salmonella contamination levels, presence and serovar distribution in broiler carcasses before and after chilling, as well as to evaluate the effectiveness of chilling process. A total of 96 pooled neck skin samples (PNSS) of 48 prechill (PreC) and 48 postchill (PosC) carcasses, representing 480 broilers collected in 6 mo' period were analyzed using ISO 6579-2:2012 Miniaturized Most Probable Number (ISO-mMPN) technique. Species confirmation and serovar identification was performed by Salmonella-specific real-time PCR (Salm-PCR) and conventional serotyping, respectively. Mean Salmonella count was 1.84 log 10 MPN/g in PreC, and 1.48 log 10 MPN/g in PosC samples, indicating a statistically significant reduction of 0.36 log 10 MPN/g (p < 0.05) in the counts by plant's air chill system. Salmonella positivity reduced from 97.9% (47/48) in PreC to 85.42% (41/48) in PosC samples, confirmed by Salm-PCR with identified serovars as S. Virchow (89.77 %) followed by S. Schwarzengrund (9.09%) and S. Bredeney (1.14%). Persistence of high load and prevalence of Salmonella with serovar Virchow dominance (other than the ones mandated in current guidelines) in the final product contributes significant and up to date data to relevant literature, and provides unbiased epidemiological reference to legal authorities for future relevant revisions., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Actinobacillus pleuropneumoniae (APP) is a bacterium frequently associated with porcine pleuropneumonia. The acute form of the disease is highly contagious and often fatal, resulting in significant economic losses for pig farmers. Serotype diversity and antimicrobial resistance (AMR) of APP strains circulating in north Italian farms from 2015 to 2022 were evaluated retrospectively to investigate APP epidemiology in the area. A total of 572 strains isolated from outbreaks occurring in 337 different swine farms were analysed. The majority of isolates belonged to serotypes 9/11 (39.2%) and 2 (28.1%) and serotype diversity increased during the study period, up to nine different serotypes isolated in 2022. The most common resistances were against tetracycline (53% of isolates) and ampicillin (33%), followed by enrofloxacin, florfenicol and trimethoprim/sulfamethoxazole (23% each). Multidrug resistance (MDR) was common, with a third of isolates showing resistance to more than three antimicrobial classes. Resistance to the different classes and MDR varied significantly depending on the serotype. In particular, the widespread serotype 9/11 was strongly associated with florfenicol and enrofloxacin resistance and showed the highest proportion of MDR isolates. Serotype 5, although less common, showed instead a concerning proportion of trimethoprim/sulfamethoxazole resistance. Our results highlight how the typing of circulating serotypes and the analysis of their antimicrobial susceptibility profile are crucial to effectively manage APP infection and improve antimicrobial stewardship., (© 2024. The Author(s).)

Introduction: Shiga toxin-producing Escherichia (E.) coli (STEC) are zoonotic foodborne pathogens of significant public health importance. While ruminants are considered the main reservoir, wild animals are increasingly acknowledged as carriers and potential reservoirs of STEC. The aim of this study was to determine the occurrence of STEC in a total of 59 faecal samples of hunted wild boars (Sus scrofa) from two different regions in Switzerland (canton Thurgau in northern Switzerland and canton Ticino in southern Switzerland), and to characterise the isolates using a whole genome sequencing approach. After an enrichment step, Shiga-toxin encoding genes (stx) were detected by real-time PCR in 41 % (95 % confidence interval (95 %CI) 0,29 - 0,53) of the samples, and STEC were subsequently recovered from 22 % (95 %CI 0,13 - 0,34) of the same samples. Seven different serotypes and six different sequence types (STs) were found, with O146:H28 ST738 (n = 4) and O100:H20 ST2514 (n = 4) predominating. Subtyping of stx identified isolates with stx1c/stx2b (n = 1), stx2a (n = 1), stx2b (n = 6), and stx2e (n = 6). No isolate contained the eae gene, but all harboured additional virulence genes, most commonly astA (n = 10), hlyE (n = 9), and hra (n = 9). STEC O11:H5, O21:H21, and O146:H28 harboured virulence factors associated with extra-intestinal pathogenic E. coli (ExPEC), and STEC O100:H20 and O155:H26 possessed sta1 and/or stb and were STEC/enterotoxigenic E. coli (ETEC) hybrid pathotypes. Our results show that wild boars are carriers of STEC which may be distributed in the environment, possibly leading to the contamination of agricultural crops and water sources. The serogroups included STEC O146 which belongs to the most common non-O157 serogroups associated with human illness in Europe, with implications for public health. Since Stx2e-producing STEC have frequently been reported in swine and pork, STEC O100:H20 harbouring stx2e in faeces of wild boars may be relevant to free-range systems of pig farming because of the potential risk of transmission events at the wildlife-livestock interface.

Background: Glaesserella parasuis is the causative agent of Glässer's disease in pigs. Serotyping is the most common method used to type G. parasuis isolates. However, the high number of non-typables (NT) and low discriminatory power make serotyping problematic. In this study, 218 field clinical isolates and 15 G. parasuis reference strains were whole-genome sequenced (WGS). Multilocus sequence types (MLST), serotypes, core-genome phylogeny, antimicrobial resistance (AMR) genes, and putative virulence gene information was extracted., Results: In silico WGS serotyping identified 11 of 15 serotypes. The most frequently detected serotypes were 7, 13, 4, and 2. MLST identified 72 sequence types (STs), of which 66 were novel. The most predominant ST was ST454. Core-genome phylogeny depicted 3 primary lineages (LI, LII, and LIII), with LIIIA sublineage isolates lacking all vtaA genes, based on the structure of the phylogenetic tree and the number of virulence genes. At least one group 1 vtaA virulence genes were observed in most isolates (97.2%), except for serotype 8 (ST299 and ST406), 15 (ST408 and ST552) and NT (ST448). A few group 1 vtaA genes were significantly associated with certain serotypes or STs. The putative virulence gene lsgB, was detected in 8.3% of the isolates which were predominantly of serotype 5/12. While most isolates carried the bcr, ksgA, and bacA genes, the following antimicrobial resistant genes were detected in lower frequency;  blaZ (6.9%), tetM (3.7%), spc (3.7%), tetB (2.8%), bla-ROB-1 (1.8%), ermA (1.8%), strA (1.4%), qnrB (0.5%), and aph3''Ia (0.5%).   CONCLUSION: This study showed the use of WGS to type G. parasuis isolates and can be considered an alternative to the more labor-intensive and traditional serotyping and standard MLST. Core-genome phylogeny provided the best strain discrimination. These findings will lead to a better understanding of the molecular epidemiology and virulence in G. parasuis that can be applied to the future development of diagnostic tools, autogenous vaccines, evaluation of antibiotic use, prevention, and disease control., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Analysis of the VP1 gene sequence of the foot and mouth disease virus (FMDV) is critical to understanding viral evolution and disease epidemiology. A standard set of primers have been used for the detection and sequence analysis of the VP1 gene of FMDV directly from suspected clinical samples with limited success. The study validated VP1-specific degenerate primer-based reverse transcription polymerase chain reaction (RT-PCR) for the qualitative detection and sequencing of serotype O FMDV lineages circulating in India. The novel degenerate primer-based RT-PCR amplifying the VP1 gene can circumvent the genetic heterogeneity observed in viruses after cell culture adaptation and facilitate precise viral gene sequence analysis from clinical samples., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Korean Society of Veterinary Science.)

We describe phenotypic and genetic characterization of an atypical Japanese Actinobacillus pleuropneumoniae isolate OT761. Nucleotide sequence analysis revealed that gene clusters involved in capsular polysaccharide and O-polysaccharide (O-PS) biosynthesis of the isolate were nearly identical to those of serovar 2 reference strain. The main difference found between the O-PS loci is the shortening of 31 amino acids from the C terminus of WcaJ in the atypical isolate due to a 93 bp deletion at the 3' end of wcaJ gene. Immunoblot analysis revealed that this isolate could not produce O-PS. Taken together, our results showed that the C-terminal domain of the A. pleuropneumoniae WcaJ plays a critical role in enzyme function of WcaJ involved in the biosynthesis of O-PS.

Non-typhoidal Salmonella (NTS) poses a global threat to public health. Poultry, one of the main reservoirs of NTS, is usually not clinically affected by most NTS, yet the economic losses to the poultry industry due to control and mitigation efforts, and due to negative publicity can be tremendous. NTS strains are routinely characterized into serotypes in a time-consuming, labor-intensive multistep process that requires skilled personnel. Moreover, the discriminatory power of serotyping is limited compared to other subtyping methods. Whole-genome sequence data enable the identification of genetic variation within serotypes. However, sequencing is often limited by available resources, and analyzing and interpreting the genetic data may be time-consuming. Source tracing during epidemiological outbreak investigations requires rapid and efficient characterization of strains to control pathogen spread. Here we designed a multiplex polymerase chain reaction (PCR) assay for the detection of genetic variants of Salmonella Muenchen, a serotype that has emerged in Israel in the last 3 yr in both clinical human cases and different hosts. Test sensitivity of 99.21% and specificity of 94 to 100% were determined using in-silico PCR with a dataset of 18,282 NTS assemblies from 37 NTS serotypes. Similarly, test sensitivity of 100% and specificity of 96.2 to 100% were determined in-vitro with 120 NTS isolates of 52 serotypes. Moreover, the test enabled differentiation between the common sequence types of serotype Muenchen using both approaches. As opposed to traditional serotyping and other subtyping methods, the designed test allows for rapid and cost-efficient detection of the emerging S. Muenchen serotype and its variants in a single step. Future development of similar assays for other dominant serotypes may help reduce the time and cost required for detection and initial characterization of dominant NTS strains. Overall, these tests will be beneficial to both public health and for reducing of the economic losses to the poultry industry due to NTS infections., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Conventional Salmonella surveillance requires a week for isolation, confirmation, and subsequent serotyping. We previously showed that this could be reduced by 24 h by combining the pre-enrichment and enrichment steps into a single selective pre-enrichment step and was tested on directly after picking. The goal of this study was 2-fold: 1) to evaluate the use of selective pre-enrichment through each step of processing, including postintervention when the Salmonella load is reduced, and 2) to assess any changes in serovar populations in Salmonella positive samples. Duplicate carcass drip samples, each representative of 500 broiler carcasses, were collected by catching processing water drip under moving carcass shackle lines in each of three commercial broiler slaughter plants. Samples were collected post-pick, post-inside-outside bird wash (IOBW), and post-chill; duplicate wing rinses were performed pre- and post-antimicrobial parts dip. Each processing plant was sampled 6 times for a total of 180 samples collected. The number of Salmonella positives identified with selective pre-enrichment conditions (48/180) was similar to traditional selective enrichment culture conditions (52/180), showed good concordance in recovery rate between the 2 culture methods (Fisher's exact test, P = 0.72). We also found that the incidence of Salmonella reduced dramatically after antimicrobial intervention (post-pick 66.7% vs. post chill 8.3%). When serovar populations were evaluated in Salmonella positive samples using CRISPR-SeroSeq, we detected four different Salmonella serovars, Kentucky, Infantis, Schwarzengrund, and Typhimurium, and their incidence rose between post-pick and post-IOBW. The relative abundance of Infantis within individual samples increased between post-pick and post-IOBW while the relative abundance of the other 3 serovars decreased. These results suggest that a selective pre-enrichment step reduces the time required for Salmonella isolation without negatively affecting detection and serovar profiles in culture positive samples were not altered between culture conditions used., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Septicemia caused by Riemerella anatipestifer ( R. anatipestifer ) is a serious problem in the duck industry worldwide, and it is currently one of the major concerns for duck farming in Vietnam.., Aim: This study was conducted to identify the causative agent of septicemia in ducks in Vietnam. The antimicrobial susceptibility and serotypes of R. anatipestifer isolates were also determined to provide valuable information for disease treatment and vaccine development., Methods: Riemerella anatipestifer was isolated using blood agar and chocolate agar media. The commercial API 20NE microtest system and the partial nucleotide sequence analysis of the 16s rRNA were used to identify R. anatipestifer strains. Serotypes were determined by slide agglutination test using standard antisera against R. anatipestifer . The disk diffusion method was utilized to investigate the antimicrobial susceptibility of R. anatipestifer isolated strains., Results: A total of 408 samples were collected from ducks with typical symptoms of septicemia for R. anatipestifer isolation. Sixty-nine R. anatipestifer strains were identified. Serotyping results showed that 30 out of 69 bacterial strains were classified as serotypes 1, 6, 8, 10, and 20, with serotype 10 being the most prevalent. The antimicrobial susceptibility test revealed that 100% of the bacterial isolates were susceptible to Amoxicillin/clavulanic acid and Imipenem. On the contrary, the majority of R. anatipestifer strains were resistant to Nalidixic acid (89.9%), Streptomycin (75.4%), and Norfloxacin (72.5%)., Conclusion: This is the first ever report in terms of identification, serotyping, and antimicrobial susceptibility tests of R. anatipestifer causing septicemia in ducks of Vietnam, providing useful scientific information for treatment as well as vaccine development to control the disease.

Streptococcus suis is one of the most important swine bacterial pathogens causing economic losses. This report presents the serotype distribution of S. suis recovered from diseased pigs in Québec from January 2015 to June 2020. Serotypes 1/2 and 2 predominated, followed by serotypes 7, 3, 5, 4, 9, 1, and 14. Compared to previously reported data, very few changes could be observed concerning the serotype distribution, indicating a relative stability. Half of the untypable isolates did not belong to the species S. suis sensu stricto , as determined by recN polymerase chain reaction. Less than 10% of "real S. suis " isolates were untypable. The genetic diversity of S. suis serotypes 1, 2, and 14, as analyzed by multilocus sequence typing, was mainly represented by sequence type (ST)1, ST28, ST25, and ST94. All ST1 isolates (considered highly virulent) belonged to either serotype 1 or 14., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Background: Listeria monocytogenes (L. monocytogenes) is a serious zoonotic and food transmitted human pathogen causing meningitis and abortions. Several outbreaks of listeriosis have been associated with the consumption of ready-to-eat food products; dairy, meat, fish, and contaminated fruits and vegetables worldwide., Aim: This study was designed to detect and characterize L. monocytogenes isolated from local and imported fish in Jordan., Methods: A total of 170 fish (70 local and 100 imported), of which 140 fresh and 30 frozen samples were used in this study. Listeria monocytogenes was cultured and initially identified using conventional microbiological methods. For confirmation and serotyping of the L. monocytogenes isolates, PCR techniques were used. Using oral and intraperitoneal administration, mice were used to determine the pathogenicity and LD 50 of the isolated L. monocytogenes ., Results: A total of 72 Listeria spp. isolates were cultured from fish. Of those, 24 were positively identified as L. monocytogenes. Other strains of Listeria spp. were L. ivanovii (21), L. innocua (11), and L. grayi (16). Serotyping of the L. monocytogenes indicated that 14 isolates belonged to the 1/2b, 3b serotypes whereas 10 isolates belonged to the 4a and 4c serotypes. All isolates were virulent to mice with an LD 50 dose ranging from 3 × 10 10 CFU/ml to 3 × 10 7.5 CFU/ml. All the virulent isolates belonged to the serotype 1/2b. Histopathologically, dead mice showed multiple necrotic lesions in the liver and spleen., Conclusion: Results of this study showed the presence of potentially pathogenic L. monocytogenes in fresh and frozen, local, and imported fish in Jordan. Strict monitoring and quality control regulatory measures must be adopted to prevent future outbreaks of food poisoning associated with fish consumption., Competing Interests: The authors declare that they have no conflicts of interests.

The presence of pathogenic Shiga toxin-producing Escherichia coli (STEC) in dairy products represents a public health concern because of its ability to produce the toxins Stx1 and Stx2, which cause intestinal diseases. Monitoring the stages of milk production and checking dairy products for contamination are crucial steps to ensure dairy safety. This study aimed to report the occurrence of thermotolerant coliforms, E. coli, and STEC strains in pasteurized dairy products and to evaluate the antibiotic resistance profiles, serotypes, and characterizations of the STEC isolates by pulsed-field gel electrophoresis. We obtained a total of 138 pasteurized dairy products from 15 processing plants in Bahia, Brazil, to examine coliforms, E. coli, and STEC strains. We found that 43% of samples (59/138) contained thermotolerant coliforms, and 30% (42/138) did not comply with Brazilian regulations. Overall, 6% (9/138) were positive for E. coli and 4% (5/138) were positive for STEC. We recovered 9 STEC isolates from pasteurized cream (2/9), Minas Padrão cheese (2/9), Minas Frescal cheese (4/9), and ricotta (1/9). All isolates were stx2-positive, and 2 were eae-positive. All isolates were negative for the "big 6" STEC serogroups, belonging instead to serotypes ONT:HNT, ONT:H12, O148:H-, OR:H40, OR:HNT, and O148:HNT. Pulsed-field gel electrophoresis revealed 100% genetic similarity among 3 isolates from 2 different samples produced in the same production facility, which may suggest cross-contamination. As well, we found isolates that were 98% similar but in samples produced in different production facilities, suggesting a mutual source of contamination or a circulating strain. Two STEC strains exhibited resistance to streptomycin. Although the isolates presented a low resistance profile and no strain belonged to the "big 6" pathogenic group, the circulation of stx2-positive STEC strains in ready-to-eat products highlights the importance of epidemiological surveillance inside the Brazilian dairy chain., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Glaesserella parasuis strains were characterized by serotyping PCR, vtaA virulence marker Leader Sequence (LS)-PCR, clinical significance, and geographic region. Overall, the serovars 4, 5/12, 7, 1, and 13 were the most commonly detected. Serovars of greatest clinical relevance were systemic isolates that had a higher probability of being serovar 5/12, 13, or 7. In comparison, pulmonary isolates had a higher likelihood of being serovars 2, 4, 7, or 14. Serovars 5/12 and 13 have previously been considered disease-associated, but this study agrees with other recent studies showing that serovar 7 is indeed associated with systemic G. parasuis disease. Serovar 4 strains illustrated how isolates can have varying degrees of virulence and be obtained from pulmonary, systemic, or nasal sites. Serovars 8, 9, 15, and 10 were predominantly obtained from nasal samples, which indicates a limited clinical significance of these serovars. Additionally, most internal G. parasuis isolates were classified as virulent by LS-PCR and were disease-associated isolates, including serovars 1, 2, 4, 5/12, 7, 13, and 14. Isolates from the nasal cavity, including serovars 6, 9, 10, 11, and 15, were classified as non-virulent by LS-PCR. In conclusion, the distribution of G. parasuis serovars remains constant, with few serovars representing most of the strains isolated from affected pigs. Moreover, it was confirmed that the LS-PCR can be used for G. parasuis virulence prediction of field strains worldwide.

Serotyping is the most common method to characterize field isolates of Actinobacillus (A.) pleuropneumoniae, the etiological agent of porcine pleuropneumonia. Based on serology, many farms seem to be infected and antibodies against a wide variety of serovars are detectable, but, so far it is unknown to what degree respective serovars contribute to outbreaks of clinical manifest disease. In this study, 213 German A. pleuropneumoniae field isolates retrieved for diagnostic purposes from outbreaks of porcine pleuropneumonia between 2010 and 2019 were genetically serotyped and analyzed regarding their apx-toxin gene profile using molecular methods. Serotyping revealed a prominent role of serovar 2 in clinical cases (64% of all isolates) and an increase in the detection of this serovar since 2010 in German isolates. Serovar 9/11 followed as the second most frequent serovar with about 15% of the isolates. Furthermore, very recently described serovars 16 (n = 2) and 18 (n = 8) were detected. Most isolates (93.4%) showed apx-profiles typical for the respective serovar. However, this does not hold true for isolates of serovar 18, as 75% (n = 6) of all isolates of this serovar deviated uniformly from the "typical" apx-gene profile of the reference strain 7311555. Notably, isolates from systemic lesions such as joints or meninges did not harbor the complete apxICABD operon which is considered typical for highly virulent strains. Furthermore, the extremely low occurrence (n = 1) of NAD independent (biovar II) isolates in German A. pleuropneumoniae was evident in our collection of clinical isolates.

Actinobacillus pleuropneumoniae serotype 17, one of the two most recent serotypes described, has been isolated from diseased pigs in North America. Yet, no serological test for surveillance has been developed so far. An enzyme-linked immunosorbent assay (ELISA) using the long-chain lipopolysaccharide antigen (LC-LPS) of this serotype is described. As predicted by previous genetic data on the O-antigen locus, cross reactions were observed between this serotype and serotypes 3, 6, 8, and 15. Although animals infected by serotype 17 would be detected using the current serotype 3 LC-LPS ELISA, better results may be obtained when plates are coated with the antigen purified from the homologous serotype., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Glaesserella parasuis is an important pathogen in swine production. It acts as a primary pathogen in systemic Glässer´s disease and as a secondary pathogen in Porcine Respiratory Disease Complex. In this study, a collection of 308 isolates from carrier animals and individuals with respiratory or Glässer´s disease isolated 2012-2019 in Germany was analysed. Isolates were characterized for serovar implementing two different PCR methods. Additionally, two different PCR methods for pathotyping isolates were applied to the collection and results compared. Serovar 6 (p < 0.0001) and 9 (p = 0.0007) were correlated with carrier isolates and serovar 4 was associated with isolates from animals with respiratory disease (p = 0.015). In systemic isolates, serovar 13 was most frequently detected (18.9%). Various other serovars were isolated from all sites and the ratio of serovar 5 to serovar 12 was approximately 1:2. These two serovars together represented 14.3% of the isolates; only serovar 4 was isolated more frequently (24.7%). The pathotyping method based on the leader sequence (LS = ESPR of vta) was easy to perform and corresponded well to the clinical background information. Of the carrier isolates 72% were identified as non-virulent while 91% of the systemic isolates were classified as virulent (p < 0.0001). Results of the pathotyping PCR based on 10 different marker genes overall were in good agreement with clinical metadata as well as with results of the LS-PCR. However, the pathotyping PCR was more complicated to perform and analyze. In conclusion, a combination of the serotyping multiplex-PCR and the LS-PCR could improve identification of clinically relevant G. parasuis isolates, especially from respiratory samples.

We present the largest and most representative study of the serological diversity of Dichelobacter nodosus in England. D. nodosus causes footrot and is one of the top five globally important diseases of sheep. The commercial vaccine, containing nine serogroups, has low efficacy compared with bivalent vaccines. Our aim was to investigate the prevalence and distribution of serogroups of D. nodosus in England to elucidate whether a bivalent vaccine could protect the national flock. Farmers from 164 flocks submitted eight interdigital swabs from eight, preferably diseased, sheep. All serogroups, A-I, were detected by PCR in 687/1150 D. nodosus positive swabs, with a prevalence of 2.6-69.3% of positive swabs per serogroup. There was a median of two serogroups per flock (range 0-6). Serogroups were randomly distributed between, but clustered within, flocks, with 50 combinations of serogroups across flocks. H and B were the most prevalent serogroups, present in > 60% of flocks separately but in only 27% flocks together. Consequently, a bivalent vaccine targeting these two serogroups would protect 27% of flocks fully (if only H and B present) and partially, if more serogroups were present in the flock. We conclude that one bivalent vaccine would not protect the national flock against footrot and, with 50 combinations of serogroups in flocks, flock-specific vaccines are necessary.

We previously developed a multiplex PCR assay for the differentiation of serovar 1a, 1b, 2 and 5 strains of Erysipelothrix rhusiopathiae. In this study, we analyzed the serovar-defining chromosomal region of a serovar 2 swine isolate, which was PCR-positive for both serovars 1a and 2 by the multiplex PCR assay. Genetic analysis of the chromosomal region revealed that, as in serovar 1a strains, the ERH&#95;1440 gene, which is usually truncated or missing in serovar 2 strains, was intact in this strain. This paper first shows an E. rhusiopathiae serovar 2 strain possessing an intact ERH&#95;1440 gene and suggests that care may be needed when determining the serovar of such rare strains by PCR assay.

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.

Streptococcus suis is one of the most important bacterial swine pathogens worldwide and is an emerging pathogen in humans. There are 29 serotypes, and serotyping, which is based on the antigenicity of the capsular polysaccharide (CPS) or on its coding genes, is often part of routine identification and provides further information regarding S. suis virulence and zoonotic potential. Serotypes 2 and 14 possess high zoonotic potential, and serotype 1/2 is the serotype most frequently isolated from diseased pigs in North America. PCR has replaced antibody-based techniques to perform serotyping. However, traditional PCR is not able to differentiate serotype 2 from 1/2 and serotype 1 from 14, given that the only difference in the cps loci of those serotype pairs is a nonsynonymous single-nucleotide polymorphism. We developed a mismatch amplification mutation assay (MAMA)-PCR that was able to correctly serotype 148 isolates previously known to be serotypes 1, 2, 1/2, or 14. This technique will be highly useful in animal and human health laboratories performing PCR serotyping of S. suis isolates.

The genetic relatedness and antimicrobial susceptibility profiles of Salmonella isolated from poultry and their environment were determined. One broiler breeder flock (BBF1) and 2 broiler flocks (BF1 and BF2) were reared over a 1.75-year period on the same poultry research farm. Hatching eggs were obtained from BBF1 to produce BF1 chicks, while BF2 chicks were progeny of a separate, unsampled broiler breeder flock. BF1 and BF2 were reared in the same housing facilities but 6 mo apart. Salmonella isolates were collected via litter sock sampling (BF1), cecal excision (BF1 and BF2), or cloacal swabs (BBF1). Serotyping identified Salmonella enterica subsp. enterica serovar Altona (SA) in BBF1 and S. enterica subsp. enterica serovar Senftenberg (SS) in BF1 and BF2. Genotypic fingerprinting was achieved with Rep-PCR using the (GTG) 5 primer and revealed sequence homology among Senftenberg isolates from BF1 and BF2. For each isolate, the minimum inhibitory concentration was determined for 27 antimicrobial agents using Sensititre plates with formularies specific to antimicrobials used in poultry production or those used to control gram negative pathogens. Isolates from the 3 flocks were resistant to clindamycin, erythromycin, novobiocin, penicillin, and tylosin tartrate and demonstrated intermediate resistance to azithromycin, florfenicol, and spectinomycin. These data demonstrated that serovar Altona and Senftenberg were harbored by poultry, the latter appeared to persist in broiler flocks, and both serotypes shared similar patterns of antimicrobial susceptibility in an integrated research operation. In the case of multiple Salmonella isolates, combining genotypic fingerprinting methods with serotyping of representative isolates would reduce the number of samples required for serotyping and more clearly identify relatedness of isolates. These methods facilitate effective surveillance in poultry production systems, thus allowing for implementation of precise Salmonella control measures., (Copyright © 2020. Published by Elsevier Inc.)

A 1-month-old rabbit, imported as a pet by a distributor, died suddenly in the quarantine period in Japan due to suppurative pleuropneumonia. A bacterial isolate from its right lung was identified as Pasteurella multocida serotype A: 11. The isolate was classified as ST204 using the RIRDC scheme of multilocus sequence typing, suggesting that the isolate was genetically related to European isolates of the same sequence type listed in the PubMLST database and not to four other isolates that originated from past imported rabbits. In the immunohistochemical assay, an antiserum recognizing the somatic serotype 11 antigen generated from chicken could specifically detect P. multocida, indicating that the antiserum for somatic serotyping was useful for immunohistochemical diagnosis of rabbit pasteurellosis.

This study reports the results of diagnostic and molecular typing methods for 18 Avibacterium paragallinarum isolates obtained from outbreaks of infectious coryza in commercial layer flocks in the Netherlands. Isolation, biochemical identification, species-specific PCR tests and classical serotyping were performed. In addition, molecular typing by Enterobacterial Repetitive Intergenic Consensus-Based Polymerase Chain Reaction (ERIC-PCR) and sequence analysis of the partial HPG2 region of A. paragallinarum were applied and results of both techniques were compared. Moreover, the pathogenicity of an isolate of the most common genotype detected in the Netherlands was determined in an animal experiment. All 18 Avibacterium isolates were nicotinamide adenine dinucleotide-dependent . All isolates were detected by the species-specific conventional PCR while 33% of the isolates were missed by the species-specific real-time PCR. Sequence analysis showed a probe mismatch as a result of a single nucleotide polymorphism (G1516A). Modification of the probe of the real-time PCR was necessary to overcome false negative results. Molecular typing showed that sequence analysis of the partial HPG2 region was in concordance with ERIC-PCR results and indicated the presence of two major genotypes. Serotyping showed the presence of serovars A-1, A-2 and B-1. There was no correlation between genotyping results and serotyping results. Inoculation of an isolate of the most prevalent genotype, and belonging to serovar A-1, into brown layer hens demonstrated the pathogenicity of this isolate.

Rapid identification of the infecting Salmonella serovar from porcine diagnostic samples is vital to allow implementation of appropriate on-farm treatment and management decisions. Although identification at the serogroup level can be rapidly achieved at most veterinary diagnostic laboratories, final Salmonella serovar identification often takes several weeks because of the limited number of reference laboratories performing the complex task of serotyping. Salmonella serogroup B, currently the dominant serogroup identified from swine clinical samples in the United States, contains serovars that vary from highly pathogenic to minimally pathogenic in swine. We determined the frequency of detection of individual group B serovars at the Iowa State Veterinary Diagnostic Laboratory from 2008 to 2017, and validated a multiplex real-time PCR (rtPCR) to distinguish pathogenic serogroup B serovars from those of lesser pathogenicity. Our results indicate that, since 2014, Salmonella enterica ssp. enterica serovar 4,[5],12:i:- has been the dominant serovar identified from swine clinical samples at the ISU-VDL, with S. Typhimurium now the second most common serovar identified. We developed a rtPCR to allow rapid differentiation of samples containing S. 4,[5],12:i:- and S. Typhimurium from samples containing serovars believed to be of less pathogenicity, such as S. Agona and S. Derby. When combined with enrichment culture, this rtPCR has the ability to significantly improve the time to final serovar identification of the 2 most commonly identified pathogenic Salmonella serovars in swine, and allows rapid implementation of serovar-specific intervention strategies.

Salmonella laboratorial detection is usually carried out by bacteriological culture and serological methods. Salmonella isolates are then classified into more than 2,650 serotypes with different somatic (O) and flagellar (H) antigenic combinations. More recently, DNA analysis methods were developed and applied for the identification of Salmonella serotypes, including intergenic spacer regions (ISRs) that separates DNA-encoding ribosomal subunits (rRNA gene) in Salmonella genomes. The present study aimed to evaluate the nucleotide diversity of the ISRs in 2 rRNA operons (rrnB and rrnH) for the assignment of Salmonella serotypes. A total of 63 Salmonella isolates (bacterial cultures) from 21 serotypes were obtained in the period of 2014 to 2017. DNA was extracted, and PCRs were used to detect the genus Salmonella and 4 important serotypes: Enteritidis, Gallinarum, Heidelberg, and Typhimurium. ISRs of the operons rrnB and rrnH were amplified by PCR and then sequenced. All sequence results were submitted to BLASTn search and were aligned in comparison to 72 Salmonella reference nucleotide sequences. The results demonstrated that 60 (95.2%) samples returned a sequence of the same serotype (determined by the traditional serological procedure) after searching in BLASTn and/or in the alignment with the reference sequences for both operons (rrnB and rrnH). These PCR-sequencing procedures had a general agreement index of 0.792 based on the Kappa analysis, 98.7% sensitivity value, 100% specificity, and 98.4% accuracy. Three different phylogenetic trees were generated from the alignments with the sequences of rrnH, rrnB, and rrnH plus rrnB and isolates clustered in specific branches according to the serotypes., (© 2019 Poultry Science Association Inc.)

Enterotoxigenic Escherichia coli (ETEC) is the most common and global cause of neonatal calf diarrhea, but there is a little information regarding calf ETEC strains in Argentina. In this study, five ETEC isolates from diarrheic dairy calves (2-10 d old) from Buenos Aires and Cordoba, Argentina were characterized on the basis of virulence gene (VG) pattern, O:H serotyping, hemolytic phenotype, phylogenetic group affiliation, antimicrobial (AM) resistance profile, and presence of integron class 1 and 2. The five isolates were examined by polymerase chain reaction (PCR) for the presence of 18 bovine VGs and showed the following genotypes: F5 + /F41 + / sta + (D242), F5 + / sta + (D158), F5 + / sta + (D157), F5 + (D151-9), and F5 + / iucD + (D151-5). These VGs confer pathogenic potential and most of them are associated with the ETEC pathotype. The five isolates showed a non-hemolytic phenotype, belonged to five different serotypes: O101:H - , O141:H - , O60:H - , ONT:H10, and ONT:H - , and were assigned to the phylogenetic group A by the quadruplex Clermont PCR method. The AM resistance of the three isolates D242, D157, and D151-5 was determined by agar disk diffusion method for 24 AMs and they exhibited a multi-resistance phenotype (resistance to four different AM classes: Cephalosporins, Penicillins, Macrolides, and Ansamycins). In addition, class 1 integrons were found in the isolate D151-5 containing the dfrA17-aadA5 gene cassette and in the bovine ETEC reference strain FV10191 containing the dfrA1-aadA1 gene cassette. The present study revealed for the first time the occurrence of multi-resistant ETEC associated with neonatal diarrhea in dairy calves in Argentina. This finding may be used for diagnostic and therapeutic purposes., Competing Interests: The authors declare that there is no conflict of interest.

Shiga toxin-producing Escherichia coli (STEC), Enteropathogenic E. coli (EPEC), and Enterotoxigenic E. coli (ETEC) make up an important group of pathogens causing major animal and public health concerns worldwide. The aim of this study was to determine the prevalence of different pathotypes of E. coli in captive wildlife. We analyzed 314 fresh fecal samples from captive wildlife, 30 stool swabs from animal caretakers, and 26 feed and water samples collected from various zoological gardens and enclosures in India for the isolation of E. coli, followed by pathotyping by multiplex PCR. The overall occurrence rate of E. coli was 74.05% (274/370). The 274 E. coli isolates were pathotyped by multiplex PCR targeting 6 genes. Of them, 5.83% were pathotyped as EPEC, 4.74% as STEC, and 1.09% as ETEC. The 16S rRNA genes from the selected isolates were amplified, sequenced, and a phylogenetic tree was constructed. The phylogenetic tree exhibited indiscriminate genetic profiling and some isolates from captive wild animals had 100% genetic identity with isolates from caretakers, suggesting that captive wildlife may serve as a reservoir for infection in humans and vice-versa. The present study demonstrates for the first time the prevalence of these E. coli pathotypes in captive wildlife in India. Our study suggests that atypical EPEC strains are more frequent than typical EPEC strains in captive wildlife. Discovering the implications of the prevalence of these pathotypes in wildlife conservation is a challenging topic to be addressed by further investigations.

Background: Prior to 2010, the lagoviruses that cause rabbit hemorrhagic disease (RHD) in European rabbits (Oryctolagus cuniculus) and European brown hare syndrome (EBHS) in hares (Lepus spp.) were generally genus-specific. However, in 2010, rabbit hemorrhagic disease virus 2 (RHDV2), also known as Lagovirus europaeus GI.2, emerged and had the distinguishing ability to cause disease in both rabbits and certain hare species. The mountain hare (Lepus timidus) is native to Sweden and is susceptible to European brown hare syndrome virus (EBHSV), also called Lagovirus europaeus GII.1. While most mountain hare populations are found on the mainland, isolated populations also exist on islands. Here we investigate a mortality event in mountain hares on the small island of Hallands Väderö where other leporid species, including rabbits, are absent., Results: Post-mortem and microscopic examination of three mountain hare carcasses collected from early November 2016 to mid-March 2017 revealed acute hepatic necrosis consistent with pathogenic lagovirus infection. Using immunohistochemistry, lagoviral capsid antigen was visualized within lesions, both in hepatocytes and macrophages. Genotyping and immunotyping of the virus independently confirmed infection with L. europaeus GI.2, not GII.1. Phylogenetic analyses of the vp60 gene grouped mountain hare strains together with a rabbit strain from an outbreak of GI.2 in July 2016, collected approximately 50 km away on the mainland., Conclusions: This is the first documented infection of GI.2 in mountain hares and further expands the host range of GI.2. Lesions and tissue distribution mimic those of GII.1 in mountain hares. The virus was most likely initially introduced from a concurrent, large-scale GI.2 outbreak in rabbits on the adjacent mainland, providing another example of how readily this virus can spread. The mortality event in mountain hares lasted for at least 4.5 months in the absence of rabbits, which would have required virus circulation among mountain hares, environmental persistence and/or multiple introductions. This marks the fourth Lepus species that can succumb to GI.2 infection, suggesting that susceptibility to GI.2 may be common in Lepus species. Measures to minimize the spread of GI.2 to vulnerable Lepus populations therefore are prudent.

Background: Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the most highly infectious diseases in livestock, and leads to huge economic losses. Early diagnosis and rapid differentiation of FMDV serotype is therefore integral to the prevention and control of FMD. In this study, a series of serotype-specific reverse transcription recombinase polymerase amplification assays combined with lateral flow dipstick (RPA-LFD) were establish to differentiate FMDV serotypes A, O or Asia 1, respectively., Results: The serotype-specific primers and probes of RPA-LFD were designed to target conserved regions of the FMDV VP1 gene sequence, and three primer and probe sets of serotype-specific RPA-LFD were selected for amplification of FMDV serotypes A, O or Asia 1, respectively. Following incubation at 38 °C for 20 min, the RPA amplification products could be visualized by LFD. Analytical sensitivity of the RPA assay was then determined with ten-fold serial dilutions of RNA of VP1 gene and the recombinant vector respectively containing VP1 gene from FMDV serotypes A, O or Asia1, the detection limits of these assays were 3 copies of plasmid DNA or 50 copies of viral RNA per reaction. Moreover, the specificity of the assay was assessed, and there was no cross reactions with other viruses leading to bovine vesicular lesions. Furthermore, 126 clinical samples were respectively detected with RPA-LFD and real-time PCR (rPCR), there was 98.41% concordance between the two assays, and two samples were positive by RPA-LFD but negative in rPCR, these were confirmed as FMDV-positive through viral isolation in BHK-21 cells. It showed that RPA-LFD assay was more sensitive than the rPCR method in this study., Conclusion: The development of serotype-specific RPA-LFD assay provides a rapid, sensitive, and specific method for differentiation of FMDV serotype A, O or Asia1, respectively. It is possible that the serotype-specific RPA-LFD assay may be used as a integral protocol for field detection of FMDV.

Background: All over the world, Shiga toxin-producing Escherichia coli (STEC) are considered as important zoonotic pathogens. Eight serogroups have the greatest role in the outbreaks and diseases caused by STEC which include O26, O45, O103, O111, O113, O121, O145 and O157. Ruminants, especially cattle are the main reservoirs but the role of small ruminants in the epidemiology of human infections has not been thoroughly assessed in many countries. The objective of this research was to investigate the pathogenic potential of the STEC strains isolated from slaughtered goats. In this study, a total of 57 STEC strains were recovered from 450 goats and characterized by subtyping of stx genes, O-serogrouping, phylo-typing and DNA fingerprinting., Results: Amongst 57 STEC strains isolated from goats, the prevalence of stx1 was significantly more than stx2 (98.2% vs. 24.5%; P ≤ 0.05), and 22.8% of strains harbored both stx1 and stx2 genes. Three (5.2%) isolates were characterized as EHEC, which carried both eae and stx genes. A total of five stx-subtypes were recognized namely: stx1c (94.7%), stx1a (53.7%), stx2d (21%), stx2c (17.5%), and stx2a (15.7%). In some parts of the world, these subtypes have been reported in relation with severe human infections. The stx subtypes predominantly occurred in four combinations, including stx1a/stx1c (35%), stx1c (31.5%), stx1c/stx2a/stx2c/stx2d (5.2%) and stx1c/stx2c/stx2d (%5.2%). In serogrouping, the majority of STECs from goats did not belong to the top 8 serogroups but two strains belonged to O113, which has been recognized as an important pathogenic STEC in Australia. Interestingly, none of stx + eae + isolates belonged to the tested serogroups. In phylo-typing the isolates mostly belonged to phylo-group B1 (82.4%), followed by phylo-group A (12.3%). STEC strains showed a substantial diversity in DNA fingerprinting; there were 24 unique ERIC-types (with a ≥95% similarity) among the isolates., Conclusions: Despite the fact that the top 8 STEC serogroups were uncommon in caprine strains, the presence of highly pathogenic stx subtypes indicates that small ruminants and their products can be considered as an overlooked public health risk for humans, especially in developing countries which consume traditional products.

The gel diffusion precipitin test (GDPT) and restriction endonuclease analysis (REA) have commonly been used in the serotyping and genotyping of Pasteurella multocida. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis has become the gold standard for other organisms, offering higher resolution than previously available methods. We compared WGS to REA and GDPT on 163 isolates of P. multocida to determine if WGS produced more precise results. The isolates used represented the 16 reference serovars, isolates with REA profiles matching an attenuated fowl cholera vaccine strain, and isolates from 10 different animal species. Isolates originated from across the United States and from Chile. Identical REA profiles clustered together in the phylogenetic tree. REA profiles that differed by only a few bands had fewer SNP differences than REA profiles with more differences, as expected. The GDPT results were diverse but it was common to see a single serovar show up repeatedly within clusters. Several errors were found when examining the REA profiles. WGS was able to confirm these errors and compensate for the subjectivity in analysis of REA. Also, results of WGS and SNP analysis correlated more closely with the epidemiologic data than GDPT. In silico results were also compared to a lipopolysaccharide rapid multiplex PCR test. From the data produced in our study, WGS and SNP analysis was superior to REA and GDPT and highlighted some of the issues with the older tests.

We investigated the feasibility of an assay based on target-specific primer extension, combined with a suspension array, for the multiplexed detection and typing of a veterinary pathogen in animal samples, using Streptococcus suis as a model pathogen. A procedure was established for simultaneous detection of 6 S. suis targets in pig tonsil samples (i.e., 4 genes associated with serotype 1, 2, 7, or 9, the generic S. suis glutamate dehydrogenase gene [ gdh], and the gene encoding the extracellular protein factor [ epf]). The procedure was set up as a combination of protocols: DNA isolation from porcine tonsils, a multiplex PCR, a multiplex target-specific primer extension, and finally a suspension array as the readout. The resulting assay was compared with a panel of conventional PCR assays. The proposed multiplex assay can correctly identify the serotype of isolates and is capable of simultaneous detection of multiple targets in porcine tonsillar samples. The assay is not as sensitive as the current conventional PCR assays, but with the correct sampling strategy, the assay can be useful for screening pig herds to establish which S. suis serotypes are circulating in a pig population.

Salmonella enterica isolates (n = 122), including 32 serotypes from 113 dogs and 9 cats, were obtained from household dogs (n = 250) and cats (n = 50) during 2012-2015. The isolates were characterized by serotyping, antimicrobial resistance phenotyping and genotyping, and virulence gene screening. Serovars Weltevreden (15.6%) and Typhimurium (13.9%) were the most common. The majority (43%) of the isolates were multidrug resistant. The dog isolates (12.3%) harbored class 1 integrons, of which the dfrA12 - aadA2 cassette was most frequent (66.7%). The only class integron in serovar Albany was located on a conjugative plasmid. Two ESBL-producing isolates ( i.e ., a serovar Krefeld and a serovar Enteritridis) carried bla TEM and bla CTX-M , and the bla TEM gene in both was horizontally transferred. Of the plasmid-mediated quinolone resistance genes tested, only qnrS (4.9%) was detected. Most Salmonella isolates harbored invA (100%), prgH (91.8%), and sipB (91%). Positive associations between resistance and virulence genes were observed for bla PSE-1 / orgA , cmlA / span , tolC , and sul1 / tolC ( p ＜ 0.05). The results suggest that companion dogs and cats are potential sources of S. enterica strains that carry resistance and virulence genes and that antimicrobial use in companion animals may select for the examined Salmonella virulence factors.

Background: Pseudomonas aeruginosa is an important human opportunistic pathogen responsible for fatal nosocomial infections worldwide, and has emerged as a relevant animal pathogen. Treatment options are dramatically decreasing, due to antimicrobial resistance and the microorganism's large versatile genome. Antimicrobial resistance profiles, serotype frequency and genomic profile of unrelated P. aeruginosa isolates of veterinary origin (n = 73), including domesticated, farm, zoo and wild animals mainly from Portugal were studied. The genomic profile, determined by DiversiLab system (Rep-PCR-based technique), was compared with the P. aeruginosa global population structure to evaluate their relatedness., Results: Around 40% of the isolates expressed serotypes O6 (20.5%) and O1 (17.8%). A total of 46.6% of isolates was susceptible to all antimicrobials tested. Isolates obtained from most animals were non-multidrug resistant (86.3%), whereas 11% were multidrug resistant, MDR (non-susceptible to at least one agent in ≥ three antimicrobial categories), and 2.7% extensively drug resistant, XDR (non-susceptible to at least one agent in all but two or fewer antimicrobial categories). Resistance percentages were as follows: amikacin (0.0%), aztreonam (41.1%), cefepime (9.6%), ceftazidime (2.7%), ciprofloxacin (15.1%), colistin (0.0%), gentamicin (12.3%), imipenem (1.4%), meropenem (1.4%), piperacillin + tazobactam (12.3%), ticarcillin (16.4%), ticarcillin + clavulanic acid (17.8%), and tobramycin (1.4%). Animal isolates form a population with a non-clonal epidemic structure indistinguishable from the global P. aeruginosa population structure, where no specific 'animal clonal lineage' was detected., Conclusions: Serotypes O6 and O1 were the most frequent. Serotype frequency and antimicrobial resistance patterns found in P. aeruginosa from animals were as expected for this species. This study confirms earlier results that P. aeruginosa has a non-clonal population structure, and shows that P. aeruginosa population from animals is homogeneously scattered and indistinguishable from the global population structure.

Streptococcus suis (S. suis) is an important swine pathogen and an emerging zoonotic agent. Most clinical S. suis strains express capsular polysaccharides (CPS), which can be typed by antisera using the coagglutination test. In this study, 79 S. suis strains recovered from diseased pigs in Canada and which could not be typed using antisera were further characterized by capsular gene typing and sequencing. Four patterns of cps locus were observed: (1) fifteen strains were grouped into previously reported serotypes but presented several mutations in their cps loci, when compared to available data from reference strains; (2) seven strains presented a complete deletion of the cps locus, which would result in an inability to synthesize capsule; (3) forty-seven strains were classified in recently described novel cps loci (NCLs); and (4) ten strains carried novel NCLs not previously described. Different virulence gene profiles (based on the presence of mrp, epf, and/or sly) were observed in these non-serotypeable strains. This study provides further insight in understanding the genetic characteristics of cps loci in non-serotypeable S. suis strains recovered from diseased animals. When using a combination of the previously described 35 serotypes and the complete NCL system, the number of untypeable strains recovered from diseased animals in Canada would be significantly reduced.

Background: Enterococcus cecorum (EC) infection currently is one of the most important bacterial diseases of modern broiler chickens but can also affect ducks or other avian species. However, little is known concerning pathogenesis of EC and most studies concentrate on examinations of EC strains from broilers only. The objective of this study was to compare pathogenic and commensal EC strains from different animal species concerning different phenotypic and genotypic traits., Results: Pathogenic and commensal EC strains were not clearly separated from each other in a phylogenetic tree based on partial sequences of the 16S-rRNA-gene and also based on the fatty acid profile determined with gas chromatography. C 12:0 , C 14:0 , C 15:0 , C 16:0 , C 17:0 , C 18:0 , C 18:1 w7c, C 18:1 w9c and C 20:4 w6,9,12,15c were detected as the major fatty acids. None of the 21 pathogenic EC strains was able to utilize mannitol, while 9 of 29 commensal strains were mannitol positive. In a dendrogram based on MALDI-TOF MS data, pathogenic strains were not clearly separated from commensal isolates. However, significant differences concerning the prevalence of several mass peaks were confirmed between the two groups. Two different antisera were produced but none of the serotypes was predominantly found in the pathogenic or commensal EC isolates. Enterococcal virulence factors gelE, esp, asa1, ccf, hyl and efaAfs were only detected in single isolates via PCR. No virulence factor was found significantly more often in the pathogenic isolates. The chicken embryo lethality of the examined EC isolates varied from 0 up to 100%. The mean embryo lethality in the pathogenic EC isolates was 39.7%, which was significantly higher than the lethality of the commensal strains, which was 18.9%. Additionally, five of the commensal isolates showed small colony variant growth, which was never reported for EC before., Conclusions: Pathogenic and commensal EC isolates from different animal species varied in chicken embryo lethality, in their ability to metabolize mannitol and probably showed divergent mass peak patterns with MALDI-TOF MS. These differences may be explained by a separate evolution of pathogenic EC isolates. Furthermore, different serotypes of EC were demonstrated for the first time.

Salmonella (S.) enterica and Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens. Here, we report the prevalence of S. enterica and STEC in feces of 316 zoo animals belonging to 61 species from Chile. S. enterica and STEC strains were detected in 7.5% and 4.4% of animals, respectively. All Salmonella isolates corresponded to the serotype Enteritidis. To the best of our knowledge, this is the first report of S. Enteritidis in the culpeo fox ( Lycalopex culpaeus ), black-capped capuchin ( Sapajus apella ) and Peruvian pelican ( Pelecanus thagus ) and the first STEC report in Thomson's gazelle ( Eudorcas thomsonii )., Competing Interests: There is no conflict of interest.

The aim of our study was to reveal the molecular basis of the serologic nontypeability of 2 Actinobacillus pleuropneumoniae field isolates. Nine field strains of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia, were isolated from pigs raised on the same farm and sent to our diagnostic laboratory for serotyping. Seven of the 9 strains were identified as serovar 15 strains by immunodiffusion tests. However, 2 strains, designated FH24-2 and FH24-5, could not be serotyped with antiserum prepared against serovars 1-15. Strain FH24-5 showed positive results in 2 serovar 15-specific PCR tests, whereas strain FH24-2 was only positive in 1 of the 2 PCR tests. The nucleotide sequence analysis of gene clusters involved in capsular polysaccharide biosynthesis of the 2 nontypeable strains revealed that both had been rendered nontypeable by the action of ISApl1, a transposable element of A. pleuropneumoniae belonging to the IS30 family. The results showed that ISApl1 of A. pleuropneumoniae can interfere with both the serologic and molecular typing methods, and that nucleotide sequence analysis across the capsular gene clusters is the best means of determining the cause of serologic nontypeability in A. pleuropneumoniae., (© 2016 The Author(s).)

Background: Actinobacillus pleuropneumoniae (APP) causes porcine pleuropneumonia (PP)., Objective: Serotypes and antimicrobial resistance patterns in APP isolates from pigs in Korea were examined., Methods: Sixty-five APP isolates were genetically serotyped using standard and multiplex PCR (polymerase chain reaction). Antimicrobial susceptibilities were tested using the standardized disk-agar method. PCR was used to detect β-lactam, gentamicin and tetracycline-resistance genes. The random amplified polymorphic DNA (RAPD) patterns were determined by PCR., Results: Korean pigs predominantly carried APP serotypes 1 and 5. Among 65 isolates, one isolate was sensitive to all 12 antimicrobials tested in this study. Sixty-two isolates was resistant to tetracycline and 53 isolates carried one or five genes including tet(B), tet(A), tet(H), tet(M)/tet(O), tet(C), tet(G) and/or tet(L)-1 markers. Among 64 strains, 9% and 26.6% were resistance to 10 and three or more antimicrobials, respectively. Thirteen different antimicrobial resistance patterns were observed and RAPD analysis revealed a separation of the isolates into two clusters: cluster II (6 strains resistant to 10 antimicrobials) and cluster I (the other 59 strains)., Conclusion: Results show that APP serotypes 1 and 5 are the most common in Korea, and multi-drug resistant strains are prevalent. RAPD analysis demonstrated that six isolates resistant to 10 antimicrobials belonged to the same cluster.

The H6N1 avian influenza virus has circulated in Taiwan for more than 40 years. The sporadic activity of low pathogenic H5N2 virus has been noted since 2003, and highly pathogenic H5N2 avian influenza virus has been detected since 2008. Ressortant viruses between H6N1 and H5N2 viruses have become established and enzootic in chickens throughout Taiwan. Outbreaks caused by Novel highly pathogenic H5 avian influenza viruses whose HA genes were closely related to that of the H5N8 virus isolated from ducks in Korea in 2014 were isolated from outbreaks in Taiwan since early 2015. The avian influenza virus infection status is becoming much more complicated in chickens in Taiwan. This necessitates a rapid and simple approach to detect and differentiate the viruses that prevail. H6N1, H5N2 and novel H5 viruses were simultaneously subtyped and pathotyped in this study using reverse transcription loop-mediated isothermal amplification and microarray, with detection limits of 10°, 10(1) and 10° viral copy numbers, respectively. The microarray signals were read by the naked eye with no expensive equipment needed. The method developed in this study could greatly improve avian influenza virus surveillance efficiency.

A multiplex (m)PCR and a PCR followed by restriction fragment length polymorphism (RFLP) analysis of Avibacterium paragallinarum have been proposed as alternatives to conventional serotyping by the Page scheme. We evaluated both methods, and also sequenced the PCR-RFLP target fragment to reexamine the capacity of molecular serotyping. Eleven reference strains and 27 field isolates were used. Many reference strains and isolates were misidentified as Page serogroup B. The sequence analysis revealed 6 profiles based on the matching rates of the target sequence with the 3 reverse primers of the mPCR. The reference strains and field isolates in profiles 1 and 4 were correctly identified as serogroup A or C by the mPCR. The strains and/or isolates in profiles 2, 3, 5, and 6 could be misidentified as serogroup B or as nontypeable by the mPCR. The homology comparison of the sequences showed that the target sequence of the mPCR, called region 2, was not Page serogroup specific, although some Kume serovars, such as A-1 and C-2, were correctly serotyped. In addition, there was a 9 nucleotide deletion in the sequences of profiles 1, 3, and 5, but not of profiles 2, 4, and 6. Overall, we confirmed that the mPCR and PCR-RFLP molecular assays are not suitable for identifying the serogroups of A. paragallinarum isolates. With further study, analysis of region 2 sequences may have potential as a means of recognizing the Kume serovars of A. paragallinarum isolates., (© 2016 The Author(s).)

We observed increasing unserotypable (UT) Actinobacillus pleuropneumoniae isolates using agar gel diffusion (AGD) test. To reanalyze their serovar, we performed rapid slide agglutination (RSA) test and multiplex PCR for 47 UT isolates. Of these, 25 were serovar 1 (UT-serovar 1), 20 were serovar 2 (UT-serovar 2) and 2 were serovar 15 (UT-serovar 15). We examined serotyping antigen extraction temperature to determine heat influence. UT-serovar 1 and 15 were influenced by heat, because their precipitation lines were observed in the case of low antigen extraction temperature. To investigate the relationship between antigenicity and genotype, we performed pulsed-field gel electrophoresis (PFGE) analysis using UT-serovar 2 and 15. The predominant PFGE pattern of UT-serovar 2 was identical to that of serovar 2.

OBJECTIVE To identify important subspecies and serovars of Salmonella enterica in a captive reptile population and clinically relevant risk factors for and signs of illness in Salmonella-positive reptiles. DESIGN Retrospective cross-sectional study. ANIMALS 11 crocodilians (4 samples), 78 snakes (91 samples), 59 lizards (57 samples), and 34 chelonians (23 samples) at the Bronx Zoo from 2000 through 2012. PROCEDURES Data pertaining to various types of biological samples obtained from reptiles with positive Salmonella culture results and the reptiles themselves were analyzed to determine period prevalence of and risk factors for various Salmonella-related outcomes. RESULTS Serovar distribution differences were identified for sample type, reptile phylogenetic family, and reptile origin and health. Salmonella enterica subsp enterica was the most common subspecies in Salmonella cultures (78/175 [45%]), identified across all reptilian taxa. Salmonella enterica subsp diarizonae was also common (42/175 [24%]) and was recovered almost exclusively from snakes (n = 33), many of which had been clinically ill (17). Clinically ill reptiles provided 37% (64) of Salmonella cultures. Factors associated with an increased risk of illness in reptiles with a positive culture result were carnivorous diet and prior confiscation. Snakes had a higher risk of illness than other reptile groups, whereas lizards had a lower risk. Bony changes, dermatitis, and anorexia were the most common clinical signs. CONCLUSIONS AND CLINICAL RELEVANCE This study provided new information on Salmonella infection or carriage and associated clinical disease in reptiles. Associations identified between serovars or subspecies and reptile groups or clinical disease can guide management of Salmonella-positive captive reptiles.

Listeria monocytogenes is capable of infecting almost all animals. However, outbreaks of listeriosis are infrequent in birds. This report describes an outbreak of listeriosis in a small pheasant (Phasianus colchicus) breeder farm with more than 2,000 pheasants from Hubei province of the People's Republic of China. The affected flock consisted of adult and young birds. Approximately 300 young birds and a few adult birds were found dead within a few days of the onset of clinical signs. Twenty-five dead birds were collected for further examination. Histopathological lesions in the visceral organs were characterized by monocyte infiltration and proliferation. Localized encephalitis and meningitis were detected in the brains of dead birds. Gram-positive organisms were observed in heart blood smear, liver, and brain impression smears. The organisms were isolated from fresh liver and were identified as L. monocytogenes serotype 4b based on multiplex polymerase chain reaction (PCR) and hlyA gene sequence analysis. This is the first report describing outbreak of listeriosis in pheasant flock., (© 2015 Poultry Science Association Inc.)

Rapid and accurate diagnosis of foot-and-mouth disease (FMD) and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT) values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples) and specificity (100% each for serotypes O, A and Asia-1 positive samples) for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for the poor recognition of some of the viruses by the earlier assays. These new assays should help in the early detection and typing of serotype O, A and Asia-1 FMDVs circulating in West Eurasia to enable improved disease control.

The present study analyzes the characteristics of Salmonella spp. from broiler chicken farms in Brazil. In total, 82 Salmonella spp. strains were characterized by serotyping, determining susceptibility to antimicrobials, and using pulsed-field gel electrophoresis (PFGE). Fifteen Salmonella serotypes were identified, among which Minnesota (40.24%), Infantis (14.63%), Heidelberg (7.31%), Senftenberg (6.09%), and Mbandaka (6.09%) were the most frequent. Salmonella Minnesota occurred mostly in the state of Mato Grosso do Sul and in one of the broiler companies surveyed. Approximately 60% of the strains were resistant to at least one of the antimicrobials tested. From these isolates, 17.07% were resistant to only one antimicrobial (tetracycline or streptomycin), and 9.75% were resistant to 3 or more antimicrobial classes. Thirteen resistance profiles were characterized, the most frequent of which were the resistance to tetracycline (15.85%); to the combination of trimethroprim with sulfamethoxazole, and tetracycline (10.97%); and to the combination of streptomycin and tetracycline (9.75%). Multiple correspondence analysis revealed that susceptibility or resistance of the analyzed strains and also particular Salmonella serotypes were associated with broiler-producing companies where the samples were collected. Strains presented high intraserotype genetic variability, as shown by the 64 PFGE profiles, suggesting the existence of several contamination sources in the surveyed farms., (© 2015 Poultry Science Association Inc.)

Canine leptospirosis occurs worldwide; however, information on the relationship between Leptospira serotypes/genotypes and virulence in dogs remains limited. We investigated the molecular characteristics of Leptospira interrogans canine isolates belonging to three serogroups using multiple-locus variable-number tandem repeat analysis (MLVA) and the effects of each serotype/genotype on the clinical characteristics of leptospirosis in dogs. MLVA using 11 loci of the three major L. interrogans serogroups in Japan, Australis (32 strains from 21 dogs), Autumnalis (12; 7) and Hebdomadis (66; 39), revealed more divergent genetic heterogeneity within each serogroup than multilocus sequence typing (MLST), and they formed two, three and five clusters (CLs), respectively. Lethal infections were caused by all Leptospira serogroup isolates (70.3 % with Hebdomadis, 83.3 % with Australis and 100 % with Autumnalis) or Leptospira isolates belonging to all the CLs (57.1-100 %) without any significant differences. A significant difference in hyperaemia and haemorrhage of mucus membrane was observed between serogroups Australis and Autumnalis (P = 0.03). Leptospira isolates of Australis CL2 caused no hyperaemia and haemorrhage from mucus membrane, whereas those of Australis CL1, Autumnalis CL3 and Hebdomadis CL1 and CL3 did (P<0.05). Significant differences in creatinine (Cre) levels were observed between serogroups Australis and Hebdomadis (P = 0.02). In addition, significant differences in blood urea nitrogen levels were observed between serogroups Australis and Hebdomadis (P = 0.004) and Australis and Autumnalis (P = 0.02). Based on MLVA types, a significant difference in Cre levels was observed between Hebdomadis CL1 and CL4 (P = 0.0018). Our results indicated that MLVA had a higher discriminatory power and was more concordant with serotyping than MLST. Although all Leptospira serotypes and genotypes caused lethal infections in dogs, the L. interrogans serogroup Australis strains were more likely to cause severe kidney damage than Autumnalis and Hebdomadis, which may be more critical to the outcome of infected dogs than haemorrhage. Our results also suggest that the virulence mechanisms and target organs in dogs may differ by Leptospira genotype., (© 2015 The Authors.)