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.

An improved multiplex PCR for Actinobacillus pleuropneumoniae, Glaesserella australis and Pasteurella multocida.

Glaesserella australis, a newly described bacterial species, has been isolated from pig lungs that displayed lesions very similar to those caused by Actinobacillus pleuropneumoniae, prompting the need for a validated diagnostic tool. In this work, we have altered a multiplex PCR used for the identification of cultures of G. australis, A. pleuropneumoniae and Pasteurella multocida to be more sensitive and then evaluated the use of the altered diagnostic tool on cultures and directly on tissues. The altered multiplex PCR was validated using 47 related species, both type/reference strains and field isolates. The sensitivity was assessed by serial dilutions and used a mixture of target bacteria in different concentrations. Further, 166 lung samples from 54 farms from four Australian States were used to validate the ability of the multiplex PCR to detect bacteria in lung swabs. The multiplex PCR was specific for the three target species. The assay could detect a minimum of 40 colony forming units (CFU) of G. australis, 786 CFU of A. pleuropneumoniae and 238 CFU of P. multocida. The multiplex PCR yielded more positives than coventional bacteriological examination. From a total of 166 lung samples, 51.9%, 51.9% and 5.6% of farms were PCR positive for P. multocida, A. pleuropneumoniae and G. australis, respectively. The results suggested that the new multiplex PCR was specific, sensitive and out performed traditional culture. The prevalence of G. australis was not very high, but it was the dominant pathogen in infected pigs.

On-farm colorimetric detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in crude bovine nasal samples.

This work modifies a loop-mediated isothermal amplification (LAMP) assay to detect the bovine respiratory disease (BRD) bacterial pathogens Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in a colorimetric format on a farm. BRD causes a significant health and economic burden worldwide that partially stems from the challenges involved in determining the pathogens causing the disease. Methods such as polymerase chain reaction (PCR) have the potential to identify the causative pathogens but require lab equipment and extensive sample processing making the process lengthy and expensive. To combat this limitation, LAMP allows accurate pathogen detection in unprocessed samples by the naked eye allowing for potentially faster and more precise diagnostics on the farm. The assay developed here offers 66.7-100% analytical sensitivity, and 100% analytical specificity (using contrived samples) while providing 60-100% concordance with PCR results when tested on five steers in a feedlot. The use of a consumer-grade water bath enabled on-farm execution by collecting a nasal swab from cattle and provided a colorimetric result within 60 min. Such an assay holds the potential to provide rapid pen-side diagnostics to cattle producers and veterinarians.

Identification, HPG2 Sequence Analysis, and Antimicrobial Susceptibility of Avibacterium paragallinarum Isolates Obtained from Outbreaks of Infectious Coryza in Commercial Layers in Sonora State, Mexico.

This is the first extensive report on the identification and characterization of Avibacterium paragallinarum (AVP) isolates obtained from outbreaks of infectious coryza (IC) in IC-vaccinated layer flocks from Sonora State in Mexico. Isolates obtained from IC outbreaks during the years 2007, 2014, 2015, 2017, and 2019 were identified by conventional PCR test and 16S rRNA gene analysis, serotyped by Page serotyping and genotyped by the recently described partial sequence analysis of the HPG2 region. Furthermore, antimicrobial susceptibility profiles were determined by a recently improved minimal inhibitory concentration (MIC) test. The conventional PCR test and the 16S rRNA analyses confirmed the isolates as AVP. Serotyping results showed the involvement of isolates belonging to serotypes A, B, and C in the IC outbreaks. Genotyping of the HPG2 region revealed the presence of sequence type (ST)1, ST4, and ST11, of which the latter has also been identified in Europe. The MIC susceptibility test showed that all tested isolates were susceptible for the majority of tested antimicrobials, including erythromycin and tetracycline, which are important antibiotics for the treatment of IC. The IC situation in Sonora State, Mexico, is complex because of the presence of serotypes A, B, and C. This finding emphasizes the importance of biosecurity in combination with the application of the most optimal vaccination programs in the control of IC in Sonora State, Mexico.

Nota de investigación­Análisis de secuencias de la región HPG2 y susceptibilidad antimicrobiana de aislamientos de Avibacterium paragallinarum obtenidos de brotes de coriza infecciosa en aves de postura comerciales en el estado de Sonora, México. Este es el primer informe extenso sobre la identificación y caracterización de aislamientos de Avibacterium paragallinarum (AVP) obtenidos de brotes de coriza infecciosa (IC) de parvadas de ponedoras vacunadas con coriza infecciosa en el estado de Sonora en México. Los aislamientos obtenidos de los brotes de coriza infecciosa durante los años 2007, 2014, 2015, 2017 y 2019 se identificaron mediante una prueba de PCR convencional y el análisis del gene de ARNr 16S, se serotipificaron mediante el método de Page y se genotipificaron mediante el análisis parcial de secuencias descrito recientemente de la región HPG2. Además, se determinaron los perfiles de susceptibilidad a los antimicrobianos mediante la prueba de concentración mínima inhibitoria (MIC) que ha sido mejorada recientemente. La prueba de PCR convencional y los análisis de secuencias del gene ARNr 16S confirmaron que los aislados eran A. paragallinarum. Los resultados de la serotipificación mostraron la participación de aislamientos pertenecientes a los serotipos A, B y C en los brotes de coriza infecciosa. La genotipificación de la región HPG2 reveló la presencia de secuencias del tipo (ST) 1, ST4 y ST11, de los cuales este último también ha sido identificada en Europa. La prueba de susceptibilidad por concentración mínima inhibitoria mostró que todos los aislados analizados eran susceptibles a la mayoría de los antimicrobianos analizados, incluida la eritromicina y la tetraciclina, que son antibióticos importantes para el tratamiento contra la coriza infecciosa. La situación de coriza infecciosa en el estado de Sonora, México, es compleja por la presencia de los serotipos A, B y C. Este hallazgo enfatiza la importancia de la bioseguridad en combinación con la aplicación de los programas de vacunación óptimos en el control de la coriza infecciosa en el estado de Sonora, México.

Severe pneumonia in a street rat (Rattus norvegicus) caused by Rodentibacter rarus strain RMC2.

Background: Rodents are one of the most dangerous reservoirs and carriers of infectious diseases. Gradually, rats have become predominant in cities, sometimes staying in close vicinity to humans, pets, and other animals. Consequently, they tend to increase the transmission risk of pathogens. Case Description: Here, we report an original case of bacterial pneumonia in a street rat (Rattus norvegicus). The rat was found dead on a street in the chief town of Marseille (France) after being run over by a car. The necropsy of the corpse revealed generalized granulomatous pneumonia in almost all the pulmonary lobes. Lung lesions and predominantly multiple fibro-inflammatory areas are presumably the witness of an infectious etiology. Bacterial isolation was carried out from lung tissues. Colonies were identified by MALDI-TOF MS and confirmed by 16S rRNA sequencing. The following bacteria were identified: Staphylococcus cohnii, Bordetella bronchiseptica, Bordetella parapertussi, Corynebacterium glucuronolyticum, Pelistega suis and Rodentibacter rarus. Based on the histopathological diagnosis and the avoidance approach, the most likely etiological agent of pneumonia is therefore R. rarus, a little-known Pasteurellales bacterium that is closely related to Rodentibacter pneumotropicus. Conclusion: These data emphasize the severity of R. rarus infection in rodents. Thus, pointing out a potential risk for other animals (dogs, cats, and birds), as well as humans. The health monitoring program for rodents and rabbits pasteurellosis should now include R. rarus. Therefore, the pathological effect of the Rodentibacterspecies and/or strains needs to be better explored.

An unusual case of multiple hepatic and pulmonary abscesses caused by Aggregatibacter aphrophilus in a young man: a case report.

BACKGROUND: Aggregatibacter aphrophilus, formerly known as Haemophilus aphrophilus, belongs to the HACEK organisms, a group of pathogens classically associated with infectious endocarditis. A. aphrophilus is a rarely found pathogen, though abscess formation in various organs has been described, typically due to spread from an infected heart valve. Here we describe the unusual case of multiple hepatic abscesses caused by A. aphrophilus. CASE PRESENTATION: A 33-year-old Caucasian man presented at our hospital with fever and malaise, elevated inflammatory markers, and liver enzymes. Imaging was compatible with multiple liver and pulmonary abscesses, without evidence of endocarditis. Cultures of blood and liver abscess material remained without growth. Polymerase chain reaction finally revealed Aggregatibacter aphrophilus in the liver tissue. The patient recovered fully within 6 weeks of doxycycline treatment. CONCLUSIONS: There are only a few case descriptions of liver abscesses caused by A. aphrophilus. As a ubiquitous organism in the gastrointestinal tract, A. aphrophilus may reach the liver via the portal venous system, as well as through hematogenous spread from the oropharynx. HACEK organisms are notoriously difficult to grow on culture, which highlights the diagnostic importance of eubacterial PCR.

Pulmonary Aggregatibacter actinomycetemcomitans infection masquerades as malignancy in a patient with periodontitis.

A 49-year-old man with a 37.5 pack-year smoking history presented with a suspected neoplasm of the right lung following the discovery of a metabolically active mass on positron emission tomography-CT imaging. The patient, who demonstrated poor oral hygiene, had a history of irregular problem-oriented dental visitation. Having excluded malignancy through histologic investigations, Aggregatibacter actinomycetemcomitans-a well-established periodontal pathogen-was subsequently cultured from his pulmonary aspirate. The patient was therefore managed with systemic antimicrobials and adjunctive dental extractions to eliminate the likely source of infection, whereafter the mass resolved without complication. This case corroborates previous reports of extraoral isolation of A. actinomycetemcomitans, which may mimic cancer clinically and radiographically. While a definitive causative link between untreated periodontitis and systemic infection remains to be elucidated, such cases present a compelling argument in favour of promoting oral health to prevent systemic disease.

Differentiation among the most important Rodentibacter species by multiplex PCR assays targeting the ITSile+ala sequences of the rRNA operons.

Screening for the Rodentibacter species is part of the microbiologic quality assurance programs of laboratory rodents all over the world. Nevertheless, currently there are no PCR amplification techniques available for the diagnostic of R. ratti, R. heidelbergensis and of a Rodentibacter related ß-haemolytic taxon. The aim of this study was to utilize the differences in the sequence of the Internal Transcribed Spacer (ITS) regions of R. pneumotropicus, R. heylii, R. ratti, R. heidelbergensis and of the ß-haemolytic Rodentibacter taxon for the design of specific PCR assays for these species. The ITSile+ala sequence variations allowed the design of specific forward and reverse primers for each species included, that could be combined in different multiplex assays. The performance characteristics specificity and sensitivity registered for each primer pair against a diverse collection of Pasteurellaceae isolated from rats and mice and of further non-Pasteurellaceae strains was 100% for all five Rodentibacter species included. In addition, the PCR assays displayed high limits of detection and could be successfully used for detection of Rodentibacter spp. DNA in clinical swabs of laboratory mice and rats. Overall, the assays described here represent the first PCRs able to diagnose R. ratti, R. heidelbergensis and the ß-haemolytic Rodentibacter taxon, whose diagnostic to species level could further facilitate better understanding of their geographic distribution, prevalence, and biology in the future.

Aggregatibacter actinomycetemcomitans pneumonia mimicking lung cancer in a previously healthy 12-year-old child from Saudi Arabia: a case report.

Aggregatibacter actinomycetemcomitans formely known as Actinobacillus actinomycetemcomitans is a known part of the normal oral flora. It can sometimes cause oral or rarely extra-oral infections secondary to hematogenous extension or aspiration. It is a rare cause of invasive pneumonia. It can resemble tuberculosis or lung cancer in its presentation. Making the diagnosis in such case is crucial for better management that usually require tissue biopsy. We report a case of Aggregatibacter actinomycetemcomitans invasive pneumonia in a 12-year-old previously healthy boy from Saudi Arabia. After a prolonged course of antibiotics, clinical and radiological follow up showed complete resolution of the infection.

Etiology, epidemiology, pathology, and advances in diagnosis, vaccine development, and treatment of Gallibacterium anatis infection in poultry: a review.

Gallibacterium anatis is a Gram-negative bacterium of the Pasteurellaceae family that resides normally in the respiratory and reproductive tracts in poultry. It is a major cause of oophoritis, salpingitis, and peritonitis, decreases egg production and mortality in hens thereby severely affecting animal welfare and overall productivity by poultry industries across Europe, Asia, America, and Africa. In addition, it has the ability to infect wider host range including domesticated and free-ranging avian hosts as well as mammalian hosts such as cattle, pigs and human. Evaluating the common virulence factors including outer membrane vesicles, fimbriae, capsule, metalloproteases, biofilm formation, hemagglutinin, and determining novel factors such as the RTX-like toxin GtxA, elongation factor-Tu, and clustered regularly interspaced short palindromic repeats (CRISPR) has pathobiological, diagnostic, prophylactic, and therapeutic significance. Treating this bacterial pathogen with traditional antimicrobial drugs is discouraged owing to the emergence of widespread multidrug resistance, whereas the efficacy of preventing this disease by classical vaccines is limited due to its antigenic diversity. It will be necessary to acquire in-depth knowledge on important virulence factors, pathogenesis and, concerns of rising antibiotic resistance, improvised treatment regimes, and novel vaccine candidates to effectively tackle this pathogen. This review substantially describes the etio-epidemiological aspects of G. anatis infection in poultry, and updates the recent development in understanding the pathogenesis, organism evolution and therapeutic and prophylactic approaches to counter G. anatis infection for safeguarding the welfare and health of poultry.

Rapid identification of respiratory bacterial pathogens from bronchoalveolar lavage fluid in cattle by MALDI-TOF MS.

Respiratory tract infections are a major health problem and indication for antimicrobial use in cattle and in humans. Currently, most antimicrobial treatments are initiated without microbiological results, holding the risk of inappropriate first intention treatment. The main reason for this empirical treatment is the long turnaround time between sampling and availability of identification and susceptibility results. Therefore the objective of the present study was to develop a rapid identification procedure for pathogenic respiratory bacteria in bronchoalveolar lavage fluid (BALf) samples from cattle by MALDI-TOF MS, omitting the cultivation step on agar plates to reduce the turnaround time between sampling and identification of pathogens. The effects of two different liquid growth media and various concentrations of bacitracin were determined to allow optimal growth of Pasteurellaceae and minimise contamination. The best procedure was validated on 100 clinical BALf samples from cattle with conventional bacterial culture as reference test. A correct identification was obtained in 73% of the samples, with 59.1% sensitivity (Se) (47.2-71.0%) and 100% specificity (Sp) (100-100%) after only 6 hours of incubation. For pure and dominant culture samples, the procedure was able to correctly identify 79.2% of the pathogens, with a sensitivity (Se) of 60.5% (45.0-76.1%) and specificity (Sp) of 100% (100-100%). In mixed culture samples, containing ≥2 clinically relevant pathogens, one pathogen could be correctly identified in 57% of the samples with 57.1% Se (38.8-75.5%) and 100% Sp (100-100%). In conclusion, MALDI-TOF MS is a promising tool for rapid pathogen identification in BALf. This new technique drastically reduces turnaround time and may be a valuable decision support tool to rationalize antimicrobial use.

Histophilus somni myocarditis and leptomeningitis in feedlot cattle: case report and occurrence in South America.

We investigated deaths in a group of feedlot steers in Argentina. The main findings in 3 steers autopsied were pulmonary congestion and edema, necrotizing myocarditis, pericarditis, suppurative leptomeningitis, and bronchopneumonia. Histophilus somni was detected by bacterial culture and immunohistochemistry in the hearts of the 3 animals. Partial sequences of the 16S rRNA gene of a H. somni isolate had 99% similarity with other H. somni sequences in GenBank. Most reports of H. somni septicemia in cattle originate from North America and western Europe. There is scant information about cardiac histophilosis in South America. A survey of diagnostic laboratory personnel in 7 South American countries documented various forms of bovine histophilosis in Argentina, Brazil, Uruguay, and Venezuela.

Insights into Pasteurellaceae carriage dynamics in the nasal passages of healthy beef calves.

We investigated three bovine respiratory pathobionts in healthy cattle using qPCR optimised and validated to quantify Histophilus somni, Mannheimia haemolytica and Pasteurella multocida over a wide dynamic range. A longitudinal study was conducted to investigate the carriage and density of these bacteria in the nasal passages of healthy beef calves (N = 60) housed over winter in an experimental farm setting. The three pathobiont species exhibited remarkably different carriage rates and density profiles. At housing, high carriage rates were observed for P. multocida (95%), and H. somni (75%), while fewer calves were positive for M. haemolytica (13%). Carriage rates for all three bacterial species declined over the 75-day study, but not all individuals became colonised despite sharing of environment and airspace. Colonisation patterns ranged from continuous to intermittent and were different among pathobiont species. Interval-censored exponential survival models estimated the median duration of H. somni and P. multocida carriage at 14.8 (CI95%: 10.6-20.9) and 55.5 (CI95%: 43.3-71.3) days respectively, and found higher density P. multocida carriage was associated with slower clearance (p = 0.036). This work offers insights into the dynamics of pathobiont carriage and provides a potential platform for further data collection and modelling studies.

Dermacoccus sp. isolated from a brain abscess in a 4-year-old child.

Dermacoccus spp. have rarely been reported as human pathogens. We describe a case of a 4-year-old boy with congenital heart disease who was diagnosed with a brain abscess. The abscess was drained and the sample grew Streptococcus intermedius, Aggregatibacter aphrophilus and Dermacoccus sp.. Dermacoccus grew after 5 days of incubation and the patient was treated with meropenem.

Sensitive and immunogen-specific serological detection of Rodentibacter pneumotropicus infections in mice.

BACKGROUND: Rodentibacter (R.) pneumotropicus colonizes the respiratory and urogenital tracts of laboratory mice with a reported moderate serological prevalence from 4 to 13%. Thus, regular tests to identify this pathogen in mice are recommended for animal facilities. However, a recent study indicated that current serological assays are partly insensitive, as C57BL/6 and BALB/c mice infected with R. pneumotropicus were incorrectly screened as seronegative. RESULTS: Here, we report a systematic analysis of protein and lipopolysaccharides antigens by immunoblot and ELISA that allowed establishing a sensitive test system able to differentiate between R. pneumotropicus and the closely related species R. heylii. Furthermore, the main immunogen, designated as 'characteristic antigen for Rodentibacter of laboratory origin 1' (CARLO-1), was identified by two-dimensional gel electrophoresis followed by immunoblot and tandem mass spectrometry in a preparation of outer membrane proteins. An indirect ELISA relying on the recombinantly expressed protein provided high sensitivity, specificity, and selectivity. The corresponding carlo1 gene was highly conserved (> 97%) among 21 isolates of R. pneumotropicus and R. heylii. CONCLUSION: The newly identified protein CARLO-1 is well suited for the sensitive and specific serological detection of Rodentibacter infections in mice. Indirect differentiation of R. pneumotropicus and R. heylii infections may be possible using an ELISA based on a whole-cell antigen preparation. All four established ELISA systems using a whole-cell preparation, lipopolysaccharides, outer-membrane proteins and protein CARLO-1 as antigen, respectively, outperformed a commercial ELISA in terms of sensitivity.

Identification of Lonepinella sp. in Koala Bite Wound Infections, Queensland, Australia.

We report 3 cases of koala bite wound infection with Lonepinella koalarum-like bacteria requiring antimicrobial and surgical management. The pathogens could not be identified by standard tests. Phylogenetic analysis of 16S rRNA and housekeeping genes identified the genus. Clinicians should isolate bacteria and determine antimicrobial susceptibilities when managing these infections.

Development of a multiplex real-time PCR assay using two thermocycling platforms for detection of major bacterial pathogens associated with bovine respiratory disease complex from clinical samples.

Bovine respiratory disease complex (BRDC) is one of the most significant diseases of cattle. Bacterial pathogens involved in BRDC include Mannheimia haemolytica, Mycoplasma bovis, Histophilus somni, and Pasteurella multocida. We developed and evaluated a multiplexed real-time hydrolysis probe (rtPCR) assay using block-based Peltier and rotary-based thermocycling on lung tissue, nasal swabs, and deep nasopharyngeal swabs. The rtPCR results were compared to culture or a gel-based M. bovis PCR using statistical analysis to determine optimum quantification cycle (Cq) cutoffs to maximize agreement. The limits of detection were 1.2-12 CFU/reaction for each pathogen. M. haemolytica was the most prevalent organism detected by rtPCR, and was most frequently found with P. multocida. The rtPCR assay enabled enhanced levels of detection over culture for all pathogens on both thermocycling platforms. The rotary-based thermocycler had significantly lower Cq cutoffs (35.2 vs. 39.7), which maximized agreement with gold standard culture or gel-based PCR results following receiver operating characteristic analysis to maximize sensitivity (Se) and specificity (Sp). However, overall assay Se and Sp were similar on both platforms (80.5% Se, 88.8% Sp vs. 80.1% Se, 88.3% Sp). Implementation of these tests could enhance the detection of these pathogens, and with high-throughput workflows could reduce assay time and provide more rapid results. The assays may be especially valuable in identifying coinfections, given that many more antemortem samples tested in our study were positive for 2 or more pathogens by rtPCR ( n = 125) than were detected using culture alone ( n = 25).