Evaluating the transmission dynamics and host competency of aoudad (Ammotragus lervia) experimentally infected with Mycoplasma ovipneumoniae and leukotoxigenic Pasteurellaceae.

Feral populations of aoudad (Ammotragus lervia) occur in Texas bighorn sheep (Ovis canadensis) habitat and pose several conceptual ecological threats to bighorn sheep re-establishment efforts. The potential threat of disease transmission from aoudad to bighorn sheep may exacerbate these issues, but the host competency of aoudad and subsequent pathophysiology and transmissibility of pneumonic pathogens involved in the bighorn sheep respiratory disease complex is largely unknown. Because the largest population-limiting diseases of bighorn sheep involve pathogens causing bronchopneumonia, we evaluated the host competency of aoudad for Mycoplasma ovipneumoniae and leukotoxigenic Pasteurellaceae. Specifically, we described the shedding dynamics, pathogen carriage, seroconversion, clinical patterns, and pathological effects of experimental infection among wild aoudad held in captivity. We found that aoudad are competent hosts capable of maintaining and intraspecifically transmitting Mycoplasma ovipneumoniae and Pasteurellaceae and can shed the bacteria for 53 days after exposure. Aoudad developed limited clinical signs and pathological findings ranged from mild chronic lymphohistiocytic bronchointerstitial pneumonia to severe and acute suppurative pneumonia, similarly, observed in bighorn sheep infected with Mycoplasma spp. and Pasteurellaceae bacteria, respectively. Furthermore, as expected, clinical signs and lesions were often more severe in aoudad inoculated with a combination of Mycoplasma ovipneumoniae and Pasteurellaceae as compared to aoudad inoculated with only Mycoplasma ovipneumoniae. There may be evidence of interindividual susceptibility, pathogenicity, and/or transmissibility, indicated by individual aoudad maintaining varying severities of chronic infection who may be carriers continuously shedding pathogens. This is the first study to date to demonstrate that aoudad are a conceptual disease transmission threat to sympatric bighorn sheep populations due to their host competency and intraspecific transmission capabilities.

Polymeric nanocarrier-based adjuvants to enhance a locally produced mucosal coryza vaccine in chicken.

Infectious coryza (IC) is an acute upper respiratory disease of chicken caused by Avibacterium (A.) paragallinarum. This disease results in an increased culling rate in meat chicken and a marked decrease in egg production (10% to more than 40%) in laying and breeding hens. Vaccines were first used against IC and effectively controlled the disease. Nanotechnology provides an excellent way to develop a new generation of vaccines. NPs have been widely used in vaccine design as adjuvants and antigen delivery vehicles and as antibacterial agents; thus, they can be used as inactivators for bacterial culture. In this research, the antibacterial effects of several nanoparticles (NPs), such as silicon dioxide with chitosan (SiO2-CS), oleoyl-chitosan (O.CS), silicon dioxide (SiO2), and iron oxide (Fe3O4), on A. paragallinarum were studied. Additionally, different A. paragallinarum vaccines were made using the same nanomaterials at a concentration of 400 µg/ml to help control infectious coryza disease in chicken. A concentration of 400 µg/ml of all the NPs tested was the best concentration for the inactivation of A. paragallinarum. Additionally, this study showed that the infectious coryza vaccine adjuvanted with SiO2 NPs had the highest immune response, followed by the infectious coryza vaccine adjuvanted with Fe3O4 NPs, the infectious coryza vaccine adjuvanted with SiO2-CS NPs, and the infectious coryza vaccine adjuvanted with O.CS NPs in comparison with the infectious coryza vaccine adjuvanted with liquid paraffin (a commercial vaccine).

Vaginitis with purulent vaginal discharge caused by artificial insemination using frozen Histophilus somni-contaminated semen.

In April 2020, two cows in Japan, developed reproductive disorders accompanied by vaginitis with purulent discharge within 3 days of artificial insemination (AI) with the same lot of frozen semen. Histophilus somni was isolated from the vaginal swabs of both cows as well as from the same lot of frozen semen used for the AI. This incident marks the first reported case of H. somni infection in cattle through AI. The major outer membrane protein gene sequences and pulsed-field gel electrophoresis profiles of the isolates were identical. Moreover, we investigated the antimicrobial activity of 12 frozen semen straws against an H. somni isolate using a disk diffusion test. These straws were sourced from five AI centers and included the same lot of semen used for the AI. Although the composition of semen diluents from individual AI centers is not publicly available, both the same lot of frozen semen used in the AI and other lots produced by the same manufacturer showed lower antimicrobial activity than semen from other manufacturers. These results strongly suggest that the two vaginitis were caused by AI using H. somni-contaminated frozen semen because of insufficient antimicrobial activity to inhibit bacterial growth. The minimum inhibitory concentrations of the six antimicrobials recommended for addition to frozen semen in isolates were below the recommended concentrations, suggesting that proper addition could have prevented this incident. This highlights the importance of conducting periodical checks on the antibacterial activity of frozen semen to prevent the transmission of pathogens via AI.

Improving the detection of integrative conjugative elements in bovine nasopharyngeal swabs using multiplex recombinase polymerase amplification.

Bovine respiratory disease (BRD) is an important health and economic burden to the cattle industry worldwide. Three bacterial pathogens frequently associated with BRD (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) can possess integrative and conjugative elements (ICEs), a diverse group of mobile genetic elements that acquire antimicrobial resistance (AMR) genes (ARGs) and decrease the therapeutic efficacy of antimicrobial drugs. We developed a duplex recombinase polymerase amplification (RPA) assay to detect up to two variants of ICEs in these Pasteurellaceae. Whole genome sequence analysis of M. haemolytica, P. multocida, and H. somni isolates harbouring ICEs revealed the presence of tnpA or ebrB next to tet(H), a conserved ARG that is frequently detected in ICEs within BRD-associated bacteria. This real-time multiplex RPA assay targeted both ICE variants simultaneously, denoted as tetH_tnpA and tetH_ebrB, with a limit of detection (LOD) of 29 (95% CI [23, 46]) and 38 genome copies (95% CI [30, 59]), respectively. DNA was extracted from 100 deep nasopharyngeal swabs collected from feedlot cattle on arrival. Samples were tested for ICEs using a real-time multiplex RPA assay, and for M. haemolytica, P. multocida, H. somni, and Mycoplasma bovis using both culture methods and RPA. The assay provided sensitive and accurate identification of ICEs in extracted DNA, providing a useful molecular tool for timely detection of potential risk factors associated with the development of antimicrobial-resistant BRD in feedlot cattle.

Prevalence and genomic-based antimicrobial resistance analysis of Avibacterium paragallinarum isolates in Guangdong Province, China.

Infectious coryza (IC) is an acute infectious respiratory disease in chickens that is caused by Avibacterium paragallinarum (A. paragallinarum). A. paragallinarum poses a significant threat to poultry health due to its virulence and multidrug resistance. This study isolated and identified 21 A. paragallinarum isolates from Guangdong between 2022 and 2023. Biochemical tests showed that 100% of A. paragallinarum isolates fermented glucose but did not ferment alginate and galactose, and only YZ18 was nicotinamide adenine dinucleotide independent. To determine the genetic relatedness between these isolates and NCBI reference strains, whole-genome-based phylogenetic analysis was employed. In addition, analysis of the 2,000 bp-length hmtp210 gene showed that the hmtp210 gene was strongly associated with A. paragallinarum serotypes. Meanwhile, a PCR assay for serotyping A. paragallinarum was developed based on the hmtp210 gene, this assay has high sensitivity and specificity. The antimicrobial susceptibility of isolates was assessed using the disk diffusion method. The antibiotic resistance genes of isolates were analyzed using the genomic method. Phenotypic resistance to ampicillin (95.2%), streptomycin (95.2%), methotrexate-sulfamethoxazole (90.5%), and tetracycline (85.7%) was most frequent among the isolates. All of the isolates exhibited resistance to multiple drugs, and furthermore, the isolates possessed a collective total of 14 genes associated with antibiotic resistance. This study will contribute to advancing our knowledge of A. paragallinarum antibiotic resistance and provide a scientific basis for the prophylaxis and treatment of IC, and the subsequent rational design of potential clinical therapeutics.

Subcutaneous application of hyperimmune serum against Histophilus somni recombinant proteins affects serum antibody reactivity in beef calves.

BACKGROUND: Respiratory tract diseases cause significant economic loss in beef cattle. This study aimed to determine whether the application of hyperimmune serum (HS) containing antibodies against selected antigens of Gram-negative bacteria would improve the health and growth of different breeds of beef calves kept on three farms. Two recombinant protein antigens (Histophilus somni rHsp60 and rOMP40) were used to immunize four cows to produce HS. Eighty seven beef calves (Charolaise n = 36, Limousine n = 34, and crossbreed n = 17) were included into study. One hundred milliliters of serum were administered subcutaneously to 43 beef calves (Charolaise n = 18, Limousine n = 17, and crossbreed n = 8) twice, between 1 and 5 and 21-28 days of life. Calves were examined three times, and blood samples were taken to evaluate immunoglobulin M, G1, and G2, fibrinogen, serum amyloid A, and haptoglobin concentrations and reactivity of these Ig classes of antibodies against H. somni rHsp60 and rOMP40. Average daily weight gain during the first month and until weaning was calculated. RESULTS: HS showed higher (p ≤ 0.05) reactivity in calf sera against H. somni rHsp60 and OMP40 in IgG1 and IgG2. In experimental calves, compared to control calves, the reactivity of IgG1 against rOMP40 in the second sampling was higher in Limousine calves (p ≤ 0.001) and in the other two herds (p ≤ 0.05). Serum IgG2 antibody activity against H. somni rHsp60 in the second sampling was higher in experimental calves than in control calves in charolaise (p ≤ 0.05) and limousine (p ≤ 0.001) herds. The reactivity of IgG2 against rOMP40 in the second sampling of experimental calves was higher in herds with Charolaise and Limousine calves (p ≤ 0.001) and in crossbred calves (p ≤ 0.05). In the third sampling, serum IgG1 antibody reactivity against rOMP40 in Limousine calves was higher (p ≤ 0.05) in the experimental group. Among the other evaluated parameters, only SAA in the second sampling in the herd with Charolaise calves and heart rate in the herd with Limousine calves were significantly higher in the control calves (p ≤ 0.05). CONCLUSION: The application of HS to calves in all herds had an impact on specific reactivity in IgG1 and IgG2 classes against H. somni rOMP40 and rHsp60, antigens which were used for serum production.

Contribution of Hfq to gene regulation and virulence in Histophilus somni.

Histophilus somni is one of the predominant bacterial pathogens responsible for bovine respiratory and systemic diseases in cattle. Despite the identification of numerous H. somni virulence factors, little is known about the regulation of such factors. The post-transcriptional regulatory protein Hfq may play a crucial role in regulation of components that affect bacterial virulence. The contribution of Hfq to H. somni phenotype and virulence was investigated following creation of an hfq deletion mutant of H. somni strain 2336 (designated H. somni 2336Δhfq). A comparative analysis of the mutant to the wild-type strain was carried out by examining protein and carbohydrate phenotype, RNA sequence, intracellular survival in bovine monocytes, serum susceptibility, and virulence studies in mouse and calf models. H. somni 2336Δhfq exhibited a truncated lipooligosaccharide (LOS) structure, with loss of sialylation. The mutant demonstrated increased susceptibility to intracellular and serum-mediated killing compared to the wild-type strain. Transcriptomic analysis displayed significant differential expression of 832 upregulated genes and 809 downregulated genes in H. somni 2336Δhfq compared to H. somni strain 2336, including significant downregulation of lsgB and licA, which contribute to LOS oligosaccharide synthesis and sialylation. A substantial number of differentially expressed genes were associated with polysaccharide synthesis and other proteins that could influence virulence. The H. somni 2336Δhfq mutant strain was attenuated in a mouse septicemia model and somewhat attenuated in a calf intrabronchial challenge model. H. somni was recovered less frequently from nasopharyngeal swabs, endotracheal aspirates, and lung tissues of calves challenged with H. somni 2336Δhfq compared to the wild-type strain, and the percentage of abnormal lung tissue in calves challenged with H. somni 2336Δhfq was lower than in calves challenged with the wild-type strain. In conclusion, our results support that Hfq accounts for the regulation of H. somni virulence factors.

Rodentibacter caecimuris comb. nov., arising from the heterotypic synonymy between Pasteurella caecimuris Lagkouvardos et al. 2016 and Rodentibacter heylii Adhikary et al. 2017.

The misclassification of the species Pasteurella caecimuris Lagkouvardos et al. 2016 along with the heterotypic synonymy between P. caecimuris and Rodentibacter heylii Adhikary et al. 2017 has long been recognized. However, no formal assignment of P. caecimuris to its correct taxonomic position has been made accordingly and therefore the nomenclatural consequences have not been implemented. In the present study, the author first re-evaluates the taxonomic relationships of P. caecimuris using genome-based approaches, confirming the need of reclassification to the genus Rodentibacter and presenting evidence of the synonymy between R. heylii and P. caecimuris. Next, the author proposes a new name Rodentibacter caecimuris comb. nov. and, based on the priority of their specific epithets, treats Rodentibacter heylii as a later heterotypic synonym of Rodentibacter caecimuris.

Case reports involving coinfection with Avibacterium paragallinarum and Ornithobacterium rhinotracheale in broiler chickens and Avibacterium endocarditis in broiler breeding hens in Poland.

ABSTRACTThe study describes three clinical cases of infection with Avibacterium spp.. In case no. 1, respiratory clinical signs and high mortality (0.7-4.2% daily; total 21.2%) in Ross 308 broiler chickens were shown to be caused by coinfection with sequence type 9 of O. rhinotracheale presumptive serotype A and A. paragallinarum presumptive serotype B. The identical (pulsed-field gel electrophoresis) restriction pattern (pulsotype) of seven A. paragallinarum isolates indicated that infectious coryza in broilers was caused by the same clone. In cases 2 and 3, sudden increased deaths in Ross 308 broiler breeders (especially males) with lesions in the endocardium (valvular or mural endocarditis) were shown to be caused by A. endocarditis. Among nine antibiotics tested, florfenicol was the only antibiotic to which all A. paragallinarum and O. rhinotracheale isolates were susceptible. Out of the eight antibiotics tested, 11 A. endocarditis isolates from both clinical cases of infective endocarditis were susceptible to penicillin, amoxicillin, doxycycline and florfenicol. The A. endocarditis isolates tested in both clinical cases had different PFGE patterns (pulsotypes), but identical within a case. The causes of infectious coryza and infective endocarditis in the cases presented have not been determined. In the prevention of infectious diseases in large-scale livestock farming, it is very important to follow the rules of biosecurity.

GWAS and comparative genomics reveal candidate antibiotic resistance genes in the avian pathogen Gallibacterium anatis for six widespread antibiotics.

Gallibacterium anatis is a Gram-negative bacterium found in the respiratory and genital tracts of various animals, primarily poultry. Its association with septicemia and high mortality in poultry, along with the rise in multidrug-resistant strains, has amplified concerns. Recent research uncovered significant variability in antibiotic resistance profiles among G. anatis isolates from different Austrian flocks, and even between different organs within the same bird. In response, in the present study 60 of these isolates were sequenced and a combination of comparative genomics and genome-wide association study (GWAS) analysis was applied to understand the genetic variability of G. anatis across flocks and organs and to identify genes related to antibiotic resistance. The results showed that each flock harbored one or two strains of G. anatis with only a few strains shared between flocks, demonstrating a great variability among flocks. We identified genes associated with resistance to nalidixic acid, trimethoprim, cefoxitin, tetracycline, ampicillin and sulfamethoxazole. Our findings revealed that G. anatis may develop antibiotic resistance through two mechanisms: single-nucleotide mutations and the presence of specific genes that confer resistance. Unexpectedly, some tetracycline-resistant isolates lacked all known tetracycline-associated genes, suggesting the involvement of novel mechanisms of tetracycline resistance that require additional exploration. Furthermore, our functional annotation of resistance genes highlighted the citric acid cycle pathway as a potential key modulator of antibiotic resistance in G. anatis. In summary, this study describes the first application of GWAS analysis to G. anatis and provides new insights into the acquisition of multidrug resistance in this important avian pathogen.

Gallibacterium anatis infection in poultry: a comprehensive review.

Gallibacterium anatis (G. anatis), a member of the Pasteurellaceae family, normally inhabits the upper respiratory and lower genital tracts of poultry. However, under certain circumstances of immunosuppression, co-infection (especially with Escherichia coli or Mycoplasma), or various stressors, G. anatis caused respiratory, reproductive, and systemic diseases. Infection with G. anatis has emerged in different countries worldwide. The bacterium affects mainly chickens; however, other species of domestic and wild birds may get infected. Horizontal, vertical, and venereal routes of G. anatis infection have been reported. The pathogenicity of G. anatis is principally related to the presence of some essential virulence factors such as Gallibacterium toxin A, fimbriae, haemagglutinin, outer membrane vesicles, capsule, biofilms, and protease. The clinical picture of G. anatis infection is mainly represented as tracheitis, oophoritis, salpingitis, and peritonitis, while other lesions may be noted in cases of concomitant infection. Control of such infection depends mainly on applying biosecurity measures and vaccination. The antimicrobial sensitivity test is necessary for the correct treatment of G. anatis. However, the development of multiple drug resistance is common. This review article sheds light on G. anatis regarding history, susceptibility, dissemination, virulence factors, pathogenesis, clinical picture, diagnosis, and control measures.

Classification of Bisgaard taxon 6 and taxon 10 as Exercitatus varius gen. nov., sp. nov.

Forty-one isolates of Bisgaard taxon 6 obtained from guinea pigs, pandas, pigs and muskrat and isolates of taxon 10 from horses and horse bites in humans were subjected phenotypic characterization. Production of acid from (-)-d-mannitol, (-)-d-sorbitol and (+)-d-glycogen separated taxon 10 (positive) from taxon 6 (negative), while from two to 11 phenotypic characteristics separated taxa 6 and 10 from the 32 genera of Pasteurellaceae reported so far. Forty-four strains were genetically characterized. Sequencing of 16S rRNA genes documented a monophyletic relationship at the species level and the highest 16S rRNA gene sequence similarity of 95.6 % to other species was found between strain CCUG 15568T and the type strain of Mannheimia glucosida (CCUG 38457T). Digital DNA-DNA hybridization (dDDH) values predicted from whole genomic sequences between CCUG 15568T and other characterized strains of taxa 6 and 10 were 69.3-99.9 %. The average nucleotide identity values were higher than 95 % for all strains. The highest dDDH value of 29 % outside the taxa 6 and 10 group was obtained with the genome of the type strain of [Actinobacillus] succinogenes, indicating a separate taxonomic status at species level to taxa 6 and 10. The phylogenetic comparison of concatenated conserved protein sequences showed the unique position of the taxa investigated in the current study which qualified for the status of a new genus since the highest identity was found with Basfia with 79 %, well below the upper threshold between genera of 85 %. Based upon the low genetic similarity to other genera of the family Pasteurellaceae and a unique phenotype, we suggest that Bisgaard taxa 6 and 10 should be classified as Exercitatus varius gen. nov., sp. nov. The G+C of the type strain of Exercitatus varius, 8.5T (=CCUG 15568T=DSM 115565T), is 46.2 mol%, calculated from the whole genome.

Assessing Methods for Replacement of Soiled Bedding Sentinels in Cage-level Exhaust IVC Racks.

Soiled bedding sentinel programs have long been the cornerstone of rodent health monitoring surveillance. Many recent studies have evaluated methods to replace live animals in these programs; however, the type of ventilated rack being used greatly influences the detection rate of adventitious pathogens. This study evaluated 4 alternative sampling techniques across 5 distinct vivaria and assessed their accuracy in detecting 5 pathogens. Testing was done in an operational (real-world) setting using IVC racks that vent air at the cage level. The 5 agents surveyed were mouse norovirus, Helicobacter spp., Rodentibacter spp. Entamoeba muris, and Spironucleus muris. Samples were collected for subsequent PCR assays as follows: 1) cages with live sentinels exposed to soiled bedding; 2) filter paper placed on the lid of an unoccupied cage containing soiled bedding; 3) filter paper placed in the bedding of an unoccupied cage that contained soiled bedding; 4) swabs from an unoccupied sentinel cage that contained soiled bedding; and 5) pooled swabs from colony cages admixed with swabs from soiled bedding sentinel mice. Cumulative accuracy for all pathogens of interest was highest with the existing soiled bedding sentinel program, followed by pooled swabs of colony cages mixed with swabs from occupied soiled bedding sentinel cages. Soiled bedding sentinel cages detected mouse norovirus, Helicobacter spp., and S. muris with the highest accuracy; the pooled swabs were best in detecting Rodentibacter spp. and E. muris. The findings suggest that with the type of rack and caging used in our facilities, the soiled bedding sentinel method has highest concurrence with the expected health status of an animal room, and the results from this method can be enhanced with the addition of pooled swabs of colony animals.

Development of a new multiplex quantitative PCR for the detection of Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae.

Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae are important porcine pathogens responsible for polyserositis, polyarthritis, meningitis, pneumonia, and septicemia causing significant economic losses in the swine industry. A new multiplex quantitative polymerase chain reaction (qPCR) was designed on one hand for the detection of G. parasuis and the virulence marker vtaA to distinguish between highly virulent and non-virulent strains. On the other hand, fluorescent probes were established for the detection and identification of both M. hyorhinis and M. hyosynoviae targeting 16S ribosomal RNA genes. The development of the qPCR was based on reference strains of 15 known serovars of G. parasuis, as well as on the type strains M. hyorhinis ATCC 17981T and M. hyosynoviae NCTC 10167T . The new qPCR was further evaluated using 21 G. parasuis, 26 M. hyorhinis, and 3 M. hyosynoviae field isolates. Moreover, a pilot study including different clinical specimens of 42 diseased pigs was performed. The specificity of the assay was 100% without cross-reactivity or detection of other bacterial swine pathogens. The sensitivity of the new qPCR was demonstrated to be between 11-180 genome equivalents (GE) of DNA for M. hyosynoviae and M. hyorhinis, and 140-1200 GE for G. parasuis and vtaA. The cut-off threshold cycle was found to be at 35. The developed sensitive and specific qPCR assay has the potential to become a useful molecular tool, which could be implemented in veterinary diagnostic laboratories for the detection and identification of G. parasuis, its virulence marker vtaA, M. hyorhinis, and M. hyosynoviae.

Pathogen Prevalence Estimates and Diagnostic Methodology Trends in Laboratory Mice and Rats from 2003 to 2020.

Rodents used in biomedical research are maintained as specific pathogen-free (SPF) by employing biosecurity measures that eliminate and exclude adventitious infectious agents known to confound research. The efficacy of these practices is assessed by routine laboratory testing referred to as health monitoring (HM). This study summarizes the results of HM performed at Charles River Research Animal Diagnostic Services (CR-RADS) on samples submitted by external (non-Charles River) clients between 2003 and 2020. Summarizing this vast amount of data has been made practicable by the recent introduction of end-user business intelligence tools to Excel. HM summaries include the number of samples tested and the percent positive by diagnostic methodology, including direct examination for parasites, cultural isolation and identification for bacteria, serology for antibodies to viruses and fastidious microorganisms, and polymerase chain reaction (PCR) assays for pathogen-specific genomic sequences. Consistent with comparable studies, the percentages of pathogen-positive samples by diagnostic methodology and year interval are referred to as period prevalence estimates (%PE). These %PE substantiate the elimination of once common respiratory pathogens, such as Sendai virus, and reductions in the prevalence of other agents considered common, such as the rodent coronaviruses and parvoviruses. Conversely, the %PE of certain pathogens, for example, murine norovirus (MNV), Helicobacter, Rodentibacter, and parasites remain high, perhaps due to the increasing exchange of genetically engineered mutant (GEM) rodents among researchers and the challenges and high cost of eliminating these agents from rodent housing facilities. Study results also document the growing role of PCR in HM because of its applicability to all pathogen types and its high specificity and sensitivity; moreover, PCR can detect pathogens in samples collected antemortem directly from colony animals and from the environment, thereby improving the detection of host-adapted, environmentally unstable pathogens that are not efficiently transmitted to sentinels by soiled bedding.

Identification and initial characterization of Hfq-associated sRNAs in Histophilus somni strain 2336.

Small RNAs (sRNA), in association with the global chaperone regulator Hfq, positively or negatively regulate gene expression in bacteria. For this study, Histophilus somni sRNAs that bind to Hfq were identified and then partially characterized. The Hfq-associated sRNAs in H. somni were isolated and identified by co-immunoprecipitation using anti-Hfq antibody, followed by sRNA sequencing. Sequence analysis of the sRNA samples identified 100 putative sRNAs, out of which 16 were present in pathogenic strain 2336, but not in non-pathogenic strain 129Pt. Bioinformatic analyses suggested that the sRNAs HS9, HS79, and HS97 could bind to many genes putatively involved in virulence/biofilm formation. Furthermore, multi-sequence alignment of the sRNA regions in the genome revealed that HS9 and HS97 could interact with sigma 54, which is a transcription factor linked to important bacterial traits, including motility, virulence, and biofilm formation. Northern blotting was used to determine the approximate size, abundance and any processing events attributed to the sRNAs. Selected sRNA candidates were confirmed to bind Hfq, as determined by electrophoretic mobility shift assays using sRNAs synthesized by in vitro transcription and recombinant Hfq. The exact transcriptional start site of the sRNA candidates was determined by RNA ligase-mediated rapid amplification of cDNA ends, followed by cloning and sequencing. This is the first investigation of H. somni sRNAs that show they may have important regulatory roles in virulence and biofilm formation.

Basic Characterization of Natural Transformation in Avibacterium paragallinarum.

Avibacterium paragallinarum is the pathogen involved in infectious coryza (IC), an acute infectious upper respiratory disease in chickens. The prevalence of IC has increased in China in recent years. There is a lack of reliable and effective procedures for gene manipulation, which has limited the research on the bacterial genetics and pathogenesis of A. paragallinarum. Natural transformation has been developed as a method of gene manipulation in Pasteurellaceae by the introduction of foreign genes or DNA fragments into bacterial cells, but there has been no report on natural transformation in A. paragallinarum. In this study, we analyzed the existence of homologous genetic factors and competence proteins underlying natural transformation in A. paragallinarum and established a method for transformation in it. Through bioinformatics analysis, we identified 16 homologs of Haemophilus influenzae competence proteins in A. paragallinarum. We found that the uptake signal sequence (USS) was overrepresented in the genome of A. paragallinarum (1,537 to 1,641 copies of the core sequence ACCGCACTT). We then constructed a plasmid, pEA-KU, that carries the USS and a plasmid, pEA-K, without the USS. These plasmids can be transferred via natural transformation into naturally competent strains of A. paragallinarum. Significantly, the plasmid that carries USS showed a higher transformation efficiency. In summary, our results demonstrate that A. paragallinarum has the ability to undergo natural transformation. These findings should prove to be a valuable tool for gene manipulation in A. paragallinarum. IMPORTANCE Natural transformation is an important mechanism for bacteria to acquire exogenous DNA molecules during the process of evolution. Additionally, it can also be used as a method to introduce foreign genes into bacteria under laboratory conditions. Natural transformation does not require equipment such as an electroporation apparatus. It is easy to perform and is similar to gene transfer under natural conditions. However, there have been no reports on natural transformation in Avibacterium paragallinarum. In this study, we analyzed the presence of homologous genetic factors and competence proteins underlying natural transformation in A. paragallinarum. Our results indicate that natural competence could be induced in A. paragallinarum serovars A, B, and C. Furthermore, the method that we established to transform plasmids into naturally competent A. paragallinarum strains was stable and efficient.

Foremost report of the whole genome of Spirabiliibacterium mucosae from India and comparative genomics of the novel genus Spirabiliibacterium.

Several Pasteurella like organisms isolated from various avian species were recently reclassified into new genus based on whole genome sequence analysis. One such Pasteurella like organism, Bisgaard taxon 14 was classified as Spirabiliibacterium mucosae. In the present study, a Gram-negative organism was isolated from ailing pigeons with respiratory infection from a farm in Tamil Nadu, India and the organism was misidentified as Burkholderia mallei by Vitek 2 compact system based on biochemical characterization. Since, B. mallei is highly pathogenic and zoonotic, to further confirm, 16S rDNA sequencing and analysis was carried out which revealed that the strain belonged to Bisgaard taxon 14 (Spirabiliibacterium mucosae). To further confirm the findings, whole genome sequencing of the isolate was performed. Whole genome phylogeny and average nucleotide identity (ANI) analysis showed that the genome was closely matching with Spirabiliibacterium mucosae type strain 20,609 /3. Hence, the strain from pigeon was named as Spirabiliibacterium mucosae TN_CUL_2021 and the genome was submitted in NCBI SRA database. The genome of S. mucosase TN_CUL_2021 is only the second genome available worldwide in the NCBI database. Comparative genome analysis of 26 Pasteurellaceae family strains revealed 1101 genes specific for Spirabiliibacterium mucosae. Similarly, luxS virulence gene was found only in S. mucosae and Bisgaardia hudsonensis strains. Since there are only 2 genomes available in the NCBI genome database, further studies on isolation of S. mucosae needs to be carried out to identify its epidemiology and pathogenesis so as to develop better diagnostic assays and vaccines.

Identification, 16S rRNA-based characterization, and antimicrobial profile of Gallibacterium isolates from broiler and layer chickens.

Gallibacterium spp., particularly G. anatis, have received much attention as poultry pathogens in recent years. We report here the presence and antimicrobial resistance profile of 69 Gallibacterium isolates obtained from 2,204 diagnostic submissions of broiler and layer chickens in 2019-2021. Gallibacterium-positive chickens had lesions primarily in the respiratory tract, reproductive tract, and related serosal surfaces. Gallibacterium spp. were initially identified based on their typical cultural characteristics on blood agar. The isolates were confirmed by a genus-specific PCR spanning 16S-23S rRNA and MALDI-TOF mass spectrometry. Phylogenetic analysis based on 16S rRNA gene sequence revealed distinct clades. Of the 69 isolates, 68 clustered with the reference strains of G. anatis and 1 with Gallibacterium genomospecies 1 and 2. Antimicrobial susceptibility testing of 58 of the 69 isolates by a MIC method showed variable responses to antimicrobials. The isolates were all susceptible to enrofloxacin, ceftiofur, florfenicol, and gentamicin. There was a high level of susceptibility to trimethoprim-sulfamethoxazole (98.0%), streptomycin (98.0%), amoxicillin (84.0%), sulfadimethoxine (71.0%), and neomycin (71.0%). All of the isolates were resistant to tylosin. There was resistance to penicillin (98.0%), erythromycin (95.0%), clindamycin (94.0%), novobiocin (90.0%), tetracycline (88.0%), oxytetracycline (76.0%), and sulfathiazole (53.0%). A high rate of intermediate susceptibility was observed for spectinomycin (67.0%) and sulfathiazole (40.0%). Our findings indicate a potential role of G. anatis as an important poultry pathogen and cause of subsequent disease, alone or in combination with other pathogens. Continuous monitoring and an antimicrobial susceptibility assay are recommended for effective treatment and disease control.

Examination of the horizontal gene transfer dynamics of an integrative and conjugative element encoding multidrug resistance in Histophilus somni.

Integrative and conjugative elements (ICEs) are self-transferable mobile genetic elements that play a significant role in disseminating antimicrobial resistance between bacteria via horizontal gene transfer. A recently identified ICE in a clinical isolate of Histophilus somni (ICEHs02) is 72 914 base pairs in length and harbours seven predicted antimicrobial resistance genes conferring resistance to tetracycline (tetR-tet(H)), florfenicol (floR), sulfonamide (Sul2), aminoglycosides (APH(3″)-Ib, APH(6)-Id, APH(3')-Ia), and copper (mco). This study investigated ICEHs02 host range, assessed effects of antimicrobial stressors on transfer frequency, and examined effects of ICEHs02 acquisition on hosts. Conjugation assays examined transfer frequency of ICEHs02 to H. somni and Pasteurella multocida strains. Polymerase chain reaction assays confirmed the presence of a circular intermediate, ICE-associated core genes, and cargo genes in recipient strains. Susceptibility testing examined ICEHs02-associated resistance phenotypes in recipient strains. Tetracycline and ciprofloxacin induction significantly increased the transfer rates of ICEHs02 in vitro. The copy numbers of the circular intermediate of ICEHs02 per chromosome exhibited significant increases of ∼37-fold after tetracycline exposure and ∼4-fold after ciprofloxacin treatment. The acquisition of ICEHs02 reduced the relative fitness of H. somni transconjugants (TG) by 28% (w = 0.72 ± 0.04) and the relative fitness of P. multocida TG was decreased by 15% (w = 0.85 ± 0.01).