Pasteurella Multocida Research Articles

Characterization and Potential Application of Phage vB_PmuM_CFP3 for Phage Therapy Against Avian Pasteurella multocida

The rise of antibiotic-resistant bacterial infections necessitates alternative therapeutic strategies, such as phage therapy. This study investigates the potential of phage vB_PmuM_CFP3 (CFP3) as a therapeutic agent against avian cholera caused by Pasteurella multocida (P. multocida). Phage CFP3 was isolated from the feces and wastewater of a laying hen farm and underwent comprehensive biological characterization, including host range, lytic activity, and environmental stability. Transmission electron microscopy revealed CFP3′s typical myovirus morphology, with a head diameter of approximately 60 nm and a tail length of about 120 nm. CFP3 demonstrated high stability across a pH range of 4–10 and temperatures of 30–40 °C, making it suitable for oral administration in poultry. The phage exhibited a latent period of about 90 min and an optimal multiplicity of infection (MOI) of 1. Despite its narrow host range, with a lysis rate of 28.2% against avian-derived type A P. multocida, CFP3′s specificity minimizes impact on non-target bacteria. Whole-genome sequencing revealed a 32,696 bp linear double-stranded DNA genome with 46 predicted open reading frames (ORFs) and no tRNA or antibiotic resistance genes, enhancing its safety profile. Phylogenetic analysis indicated a close evolutionary relationship with Haemophilus phages HP1, HP2, and Pasteurella phage F108. While CFP3 shows promise as a precision therapeutic tool, further in vivo studies are required to evaluate its efficacy and safety. Future research should focus on expanding the phage library, optimizing phage mixtures, and exploring synergistic effects with other antimicrobial strategies. This study provides foundational data supporting the development of CFP3 as a viable alternative to antibiotics for controlling avian cholera.

Non‐functional metastatic thyroid carcinoma with oesophageal invasion and ulceration in a cat

AbstractAn adult, male, neutered, domestic shorthair cat was presented with a 2‐month history of dysphagia and a movable cervical mass. Cytology of the cervical mass was consistent with carcinoma. Thoracic radiographs revealed pleural effusion, cardiomegaly, diffuse patchy interstitial pattern and pulmonary nodules. Fluid analysis of the pleural effusion was consistent with marked septic neutrophilic inflammation, and Pasteurella multocida was isolated. Contrast‐enhanced cervical and thoracic computed tomography scan revealed a cervical mass in the region of the right thyroid lobe, with circumferential invasion of the oesophageal wall and well‐defined pulmonary nodules consistent with distant metastases. Oesophago‐gastroscopy was performed and revealed a proximal, oesophageal, mural mass causing severe intraluminal obstruction. Humane euthanasia was elected. Postmortem examination with histology and immunohistochemistry confirmed a thyroid carcinoma with oesophageal invasion and pulmonary metastases. The oesophageal mucosa was extensively disrupted and ulcerated by the thyroid carcinoma. Additionally, echocardiogram and postmortem examination confirmed cardiomyopathy and congestive heart failure.

Genomic and transcriptomics analysis reveal putative secreted proteins expressed of Pasteurella multocida during 18β-glycyrrhetinic acid treatment

Genomic and transcriptomics analysis reveal putative secreted proteins expressed of Pasteurella multocida during 18β-glycyrrhetinic acid treatment

Infantile Pasteurella multocida Meningitis: Case Report and Review of the Literature

Background: Pasteurella multocida is a rare cause of deep-seated pediatric infections including osteomyelitis and meningitis. We report a case of P. multocida meningitis from Queensland, with a comprehensive review of literature. Methods: Three databases were searched for infants < 12 months of age with confirmed P. multocida meningitis. Mode of transmission, clinical and laboratory features, imaging, treatment and complications were reviewed. Logistic regression analysis was performed to evaluate predictors for outcomes including short-term neurologic complications, any long-term complications and mortality. Results: A total of 74 infants were included, with most cases occurring from indirect household animal contact (42/74; 61%). Bacteremia (38/74; 51%) and seizures (15/74, 20%) were common complications with mortality in 6 children (8%). Young infant age appeared to be the single most important risk factor for bacteremia. Conclusions: Infantile P. multocida meningitis although rare has potentially devastating complications. Younger infants are more likely to develop concomitant bacteremia. Household hand hygiene is imperative after trivial interactions with pets.

Pasteurella multocida from deep nasal swabs and tracheobronchial lavage in bovine calves from Sweden

BackgroundBovine respiratory disease (BRD) is common in intensively raised cattle and is often treated with antibiotics. For practitioners, knowledge of the bacteria involved in an outbreak and their antibiotic susceptibility is warranted. To this end, samples from the upper or lower respiratory tract of calves can be submitted for bacteriological culture and susceptibility testing of relevant isolates. However, it is debated whether isolates from the upper respiratory tract are representative of bacteria causing infections in the lower respiratory tract. In this study, we used MALDI-TOF MS, multilocus sequence typing (MLST) and core-genome multilocus sequence typing (cgMLST) to compare culture results of 219 paired samples (sample pairs) of deep nasal swabs (DNS) and tracheobronchial lavage (TBL). The sample pairs came from 171 calves in 30 calf groups across 25 farms with 48 calves sampled twice.ResultsThe predominant bacterial pathogen was Pasteurella multocida, which was isolated from 37.4% of DNS and 22.4% of TBL. There was no statistically significant difference in isolation frequency of P. multocida between calves considered healthy and those suspected for BRD for DNS (P = 0.778) or TBL (P = 0.410). Among the 49 sample pairs where P. multocida was isolated from TBL, the same species was isolated from DNS in 29 sample pairs (59.2%). Isolates from 28 of these sample pairs were evaluated by MLST, and in 24 pairs (86.0%) P. multocida from DNS and TBL were of the same sequence type (ST). Moreover, cgMLST showed that the genetic distance between isolates within 21 of the 28 sample pairs (75.0%), was less than two alleles, and DNS and TBL isolates were considered identical. In seven sample pairs (25%), the genetic distance was greater, and DNS and TBL isolates were considered nonidentical.ConclusionsPasteurella multocida was readily isolated from DNS and in calves where this species was isolated also from TBL, DNS and TBL isolates were identical in 75% of the sample pairs. This suggests that during an outbreak of BRD, submission of DNS samples from 4 to 6 calves could be a convenient approach for practitioners seeking guidance on P. multocida present in the lower respiratory tract and their antibiotic susceptibility.

Development of a MALDI-TOF MS model for differentiating haemorrhagic septicaemia-causing strains of Pasteurella multocida from other capsular groups

Pasteurella multocida capsular types A, D, and F cause disease in many animal hosts, including bovine respiratory disease in cattle, which is one of the most globally significant animal diseases. Additionally, P. multocida capsular types B and E cause haemorrhagic septicaemia, a devastating disease primarily of cattle, water buffalo, and bison that develops rapidly with high mortality. Haemorrhagic septicaemia mostly occurs in developing countries and has potential to emerge elsewhere in the world. The diagnosis of haemorrhagic septicaemia currently requires recognition of compatible gross or histologic lesions and serotyping or molecular characterization of strains. In this study, we performed genomic characterization of 84 P. multocida strains, which were then used to develop and validate a matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) biomarker-based method for differentiating non-haemorrhagic septicaemia strains of P. multocida from haemorrhagic septicaemia-causing strains. Haemorrhagic septicaemia strain types B:2,5, E:2,5, and B:3,4 were used to maximize diversity. Three automated classification models were generated and then used to develop an assisted model, which utilized two peaks (6419 and 7729 m/z) to accurately differentiate non-haemorrhagic septicaemia-causing strains from haemorrhagic septicaemia-causing strains of P. multocida. The assisted model performed with 98.2 % accuracy for non-haemorrhagic septicaemia strains, 100 % accuracy for classic B:2,5 and E:2,5 strains, and 84.4 % accuracy for combined haemorrhagic septicaemia-causing strains (B:2,5, E:2,5, and B:3,4) with an overall accuracy of 96.9 %. Our results suggest that MALDI-TOF MS may be used to routinely screen P. multocida isolated from diagnostic cases for initial identification of haemorrhagic septicaemia-causing strains, and to determine whether additional characterizations are warranted.

Enhanced ePAD-DNA biosensor incorporating ZnO-NRs: Rapid and reliable electrochemical detection of field-isolated Pasteurella multocida

An electrochemical sensor has received much attention due to its importance for early infection identification, hinting at its critical relevance in diagnostic applications. For the detection of field-isolated strains of Pasteurella multocida, this paper reports the development and fabrication of a DNA-based electrochemical biosensor by integrating zinc oxide (ZnO) nanorods (NRs) into an electrochemical paper-based analytical device (ePAD). One significant improvement over the state-of-the-art features of the sensor is the using paper, an economically viable substrate that can be manufactured in large numbers. These sensors, being categorized under precision instruments with an ecologically friendly design, are ideal for mass production of eco-sensitive substrates. The biosensor is more sensitive and specific as compared to the conventional PCR-based sensing techniques. Moreover, the biosensor exploits the inherent properties of ZnO-NRs to amplify the signal output, whereas ePAD limits detection cost. In addition, a probe targeting the KMT gene of P. multocida was first characterized using conventional PCR and then immobilized onto the ZnO nanorods-modified ePAD surface, which improved the biosensor's ability for the rapid and selective genetic material of the pathogen detection. In addition, the sensor was validated using the methods of Cyclic Voltammetry (CV) and Linear Sweep Voltammetry (LSV). The reported biosensor it provides Linear Response of 0.001-10 μg/ml with LOD 0.001 μg/ml within a response time of 30 s. Subsequently, the biosensor reveals fast kinetics of the reaction as a powerful tool for timely and accurate diagnostics, pointing to its contribution to further development in biosensing technologies in the diagnostic area of infectious diseases at hospitals and clinics.

Peritonitis-related bacterial infections: a large-scale case-series retrospective study in 160 domestic animals (2009-2022).

Bacterial peritonitis infections comprise a life-threatening clinical condition in domestic animals that commonly lead to sepsis and high mortality. A set of bacterial pathogens have been identified in septic peritonitis in livestock and companion animals. Nonetheless, most descriptions are restricted to case reports or limited to only one domestic species, and a restrict number of comprehensive studies involving this infection has focused on a great number of domestic animals. Here, we retrospectively investigated selected epidemiological data (with an emphasis in outcome), clinical signs, bacteriological culturing, and in vitro antimicrobial susceptibility patterns of microorganisms isolated of peritoneal fluid from 160 domestic animals (2009-2023) compatible with septic peritonitis. Bacteria were isolated from 71.9% (115/160) of the peritoneal fluid from 75 dogs (75/115 = 65.2%), 22 cats (22/115 = 19.1%), 14 horses (14/115 = 12.2%), and 4 cattle (4/115 = 3.5%). Among animals with bacterial isolation, Escherichia coli (34/115 = 29.6%), alfa-hemolytic Streptococcus (12/115 = 10.4%), Staphylococcus aureus (8/115 = 6.9%), beta-hemolytic Streptococcus (7/115 = 6.1%), and Pasteurella multocida (6/115 = 5.2%) were predominant in pure culture, in addition to a miscellaneous of other bacteria isolated in minor frequency, e.g., Pseudomonas sp., Trueperella pyogenes, Klebsiella pneumoniae, and Salmonella sp. In general, in vitro susceptibility tests of isolates revealed that florfenicol, chloramphenicol, and amoxicillin/clavulanic acid showed moderate effectivity (≥ 60%). Conversely, most of isolates exhibited resistance mainly to trimethoprim/sulfamethoxazole, enrofloxacin, and penicillin (> 60%). Additionally, multidrug resistance was found in 42.6% (49/115) of the isolates. Data related to the outcome were available in 37.4% (43/115) of animals that had bacterial isolation and, from these, the mortality rate was 79.1% (34/43), with a significant association (p < 0.036) between mortality and septic peritonitis by gram-negative bacteria. Neoplasia (7/43 = 16.3%), pneumonia/pulmonary abscess (5/43 = 11.6%), hepatitis (5/43 = 11.6%), metritis/pyometra (4/43 = 9.3%), and gall bladder rupture (3/43 = 7%) represented the probable main sources of septic peritonitis. Anorexia (34/115 = 29.6%), emesis (29/115 = 25.2%), lethargy (26/115 = 22.6%), respiratory distress (25/115 = 21.7%), ascites (20/115 = 17.4%), and fever (19/115 = 16.5%) were the most frequent clinical signs among animals with bacterial isolation. A variety of bacteria were isolated in the peritoneal fluid of animals, with a predominance of Enterobacteriaceae, streptococci, and staphylococci, highlighting the opportunistic nature of the pathogens in septic peritonitis. High in vitro multidrug resistance of isolates and high mortality of animals reinforce the need for early diagnosis and therapy based on the in vitro antimicrobial profile of the pathogens involved in septic peritonitis. Our results contribute to the etiological characterization, clinical-epidemiological findings, and vigilance of multidrug-resistant bacteria in septic peritonitis among livestock and companion animals.

Peritoneal dialysis-related peritonitis due to Pasteurella multocida in Australia.

Domestic animals are common in Australian households; however, there is little research into the potential risks these animals pose to patients undergoing in-home peritoneal dialysis (PD). Cats and dogs are known to carry many potential pathogens, including Pasteurella multocida. We reviewed the ANZDATA Peritoneal Dialysis Peritonitis Registry for cases of peritonitis due to Pasteurella multocida between 2011 and 2023. Cases identified were younger and more likely to be female compared with the Australian PD population who developed peritonitis due to other organisms. Of the total 32 episodes, 75% were using automated PD with glucose-based solutions. Two cases requiring removal of the PD catheter and transfer to haemodialysis and no deaths were reported. Whilst outcomes were largely favourable, it is likely that many of these cases could have been prevented. Education for people undergoing PD should include information about the potential infectious hazards of domestic animals.

Protection of Rabbits Against Colonization and Morbidity Associated With Toxigenic Pasteurella multocida by Immunization With Inactivated Heat-labile Toxin.

Pasteurella multocida is a significant cause of morbidity and mortality in rabbits, as well as other species. Some isolates elaborate a heat-labile toxin (PMT) that has been shown to be an important virulence factor. Though previous studies have demonstrated protective immunity can be conferred via immunization of rabbits with heat-inactivated PMT (IPMT), we investigated the ability of immunization to impact colonization of P. multocida. Rabbits were immunized at days 0, 7 and 14 with either phosphate buffered saline (PBS), the mucosal adjuvant cholera toxin (CT), IMPT or IPMT + CT. Male New Zealand white rabbits were used and confirmed to be free of P. multocida prior to experimentation. Serum IgG and nasal lavage fluid IgA responses directed against PMT were found in rabbits immunized with IPMT, with or without CT, but not in those immunized with only PBS or CT; and the addition of CT to IPMT enhanced the response. Significantly more P. multocida CFUs (p≤0.05) were cultured from the lungs of rabbits immunized with IPMT, with or without CT, compared to those administered only PBS or CT, although no differences were observed in nasal lavage fluid samples. Further, immunization IPMT, with or without CT, conferred protection against pleuritis and pneumonia. PMT, in addition to its role as a virulence factor, may serve as a colonization factor for P. multocida in the lungs of rabbits.

Porcine Nose Atrophy Assessed by Automatic Imaging and Detection of Bordetella bronchiseptica and Other Respiratory Pathogens in Lung and Nose.

The nasal mucosa is a crucial filtering organ to prevent attachment and invasion of pathogens. To assess nasal health in relation to lung health, transverse cross sections of the nasal turbinates of 121 pigs suffering from respiratory disease and sent for diagnostic necropsy were scored visually and by an artificial intelligence (AI) medical diagnostic application (AI DIAGNOS), resulting in a high correlation of both scores (p < 0.001). Nasal samples of the diseased pigs were examined only for Bordetella (B.) bronchiseptica (PCR and bacteriological culture) and Pasteurella (P.) multocida (bacteriological culture). All pigs showed various degrees of inflammatory lung tissue alterations, and 35.5% of the pigs had atrophy of the nasal turbinates with no relation to detection rates of B. bronchiseptica (54.5%) and P. multocida (29.0%) in the nose. All P. multocida strains from nose samples were negative for the toxA gene so non-progressive atrophic rhinitis was diagnosed. Pigs positive for B. bronchiseptica in the nose were more often positive for B. bronchiseptica in the lung (p < 0.001) and for other bacterial species in the lower respiratory tract (p = 0.005). The new diagnostic application for scoring cross sections of nasal turbinates is a valuable tool for a fast and reproducible diagnostic.

Anti-quorum sensing and anti-biofilm activities of Pasteurella multocida strains

A total of 52 Pasteurella multocida strains of capsular serogroups (A, B and D) were screened for anti-quorum sensing activity against Chromobacterium violaceum. Of which, 12 strains of serogroups A were found to possess anti-quorum sensing activity. Inhibition activity was highest for strain NIVEDIPm9 and lowest for strain NIVEDIPm30 based on zone of pigment inhibition. Further, cell free extract of NIVEDIPm9 strain showed highest anti-biofilm activity in reference E. coli strain and concentration dependent degradation activity of C6-AHL molecule. In whole genome sequence annotation of NIVEDIPm9 strain predicted the presence of four metallo-β-lactamases (MBL) fold metallo-hydrolase proteins. In docking studies, MBL1 and MBL3 proteins showed high binding affinity with autoinduce signalling molecules AHL compound of OH-C10, binding energy value were −6.3 and −6.2 kcal/mol. Interaction study of VAF and quorum sensing molecules showed that OmpA and HgbA proteins were stimulated by all the ten molecules (C4-AHLs, C6-AHLs, C10-AHLs, C14-AHLs, 3-oxo-C10-AHLs, 3OH-C10-HSL, C8-HSL, C10-HSL, C12-HSL, C14-HSL), while toxA gene was stimulated by OH-C10-AHL molecule, sodC gene was stimulated by none. In conclusion, we described the anti-quorum sensing activities of diverse P. multocida strains causing Pasteurellosis in livestock.

The first report of concurrent infection of hemorrhagic septicemia with foot and mouth disease in cattle in Bangladesh

This study aimed to investigate the concurrent infection of Pasteurella multocida (P. multocida) type B:2, which causes Hemorrhagic Septicemia (HS), with cases of Foot and Mouth Disease (FMD) outbreaks in cattle in Bangladesh between March and December 2023. Samples were collected from 11 distinct outbreak areas, totaling 102 samples. These included 54 FMD samples (saliva, tissue epithelium, and morbid tissues such as lung, spleen, and heart) and 54 HS samples (nasal swabs and morbid tissues) from 50 cattle of various ages and sexes, all showing clinical signs of suspected concurrent HS and FMD infection. After sample processing, molecular detection of FMDV and its serotypes was performed using Reverse Transcriptase PCR (RT-PCR) with universal and serotype-specific primers. The HS-causing agent, P. multocida type B:2, was initially identified through cultural and morphological characteristics on various media, followed by Gram’s and methylene blue staining, biochemical tests, and pathogenicity tests through inoculation of isolates into mice. Finally, molecular detection of P. multocida type B:2 was confirmed using PCR with specific primers. Forty-five (83 %) of the 54 FMD suspected samples tested positive for FMDV, with 53 % of these positive for serotype ‘O,’ 17 % for serotype ‘A,’ and 6 % for mixed serotypes ‘O’ and ‘A.’ Among the FMDV-positive samples, 17 (38 %) of the HS-suspected samples tested positive for concurrent infection with P. multocida type B:2. The study reveals that FMDV-induced acute immunosuppression in cattle can lead to complications from concurrent infections, particularly those caused by P. multocida type B:2, resulting in HS alongside FMD.

Comparative evaluation of PCR and loop-mediated isothermal amplification (LAMP) assays for detecting Pasteurella multocida in poultry

ABSTRACT Aims To develop a colourimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Pasteurella multocida in clinical poultry samples and compare the performance of this assay with PCR. A secondary aim was to evaluate a simple DNA extraction method that could enable LAMP-based testing in the field without the need for specialised laboratory equipment. Methods Primer sets for both LAMP and PCR were designed to amplify the KMT1 gene of P. multocida. DNA was extracted from 12 P. multocida isolates using a commercial extraction kit, and subjected to analysis using both LAMP and PCR. The analytical specificity of the LAMP assay was evaluated by testing it against a panel of 12 unrelated bacterial species, and the analytical sensitivity (limit of detection) was determined through testing of serial dilutions of the target DNA and compared to that of PCR. Subsequently, cloacal swabs (n = 40) from a commercial turkey flock were subjected to analysis using both LAMP and PCR assays, using a rapid DNA extraction method and a commercial extraction kit. Clinical sensitivity and specificity of the LAMP assay were calculated in comparison to PCR. Results A single DNA fragment of the expected size (∼ 200 base pairs), was amplified by PCR from 12 P. multocida isolates, which were also all positive by the LAMP assay. The identity of all PCR amplicons was confirmed by sequencing. Both PCR and LAMP showed similar analytical sensitivity, with a LOD of 20 pg of target DNA. As neither PCR nor LAMP assays produced positive results with 12 non-related bacterial species, the analytical specificity was assessed as 100%. However, LAMP demonstrated lower clinical specificity (94.74%) compared to PCR (100%) when 40 clinical samples were tested. None of the DNA samples extracted using the simplified DNA extraction method were amplified by either LAMP or PCR. Conclusion The LAMP assay developed in this study exhibits comparable performance to PCR in detecting P. multocida. Clinical relevance The use of a rapid and portable DNA extraction method, in conjunction with LAMP assays, could create opportunities for point-of-care testing for fowl cholera in field settings.

A surface lipoprotein on Pasteurella multocida binds complement factor I to promote immune evasion.

Pasteurella multocida is the leading cause of wound infections in humans following animals' bites or scratches. This bacterium is also commonly found in the respiratory tract of many mammals and can cause serious diseases resulting in the brutal rapid death of infected animals, especially cattle. To prevent these infections in cattle, a subunit-based vaccine utilizing the surface lipoprotein PmSLP was developed and showed remarkable protection with a single dose administration. Here, we report that PmSLP binds host complement factor I (FI) and facilitates cleavage of complement components C3b and C4b independently of any cofactors (e.g FH, C4BP), thereby allowing the pathogen to evade host defence. Cryo-EM structure of PmSLP bound to FI reveals that PmSLP stimulates FI enzymatic activity by stabilizing the catalytic domain. This is the first time that a bacterial protein has been shown to directly activate FI independent of complement cofactors and target all arms of the complement cascade.

PMCNA_RS00975 activates NF-κB and ERK1/2 through TLR2 and contributes to the virulence of Pasteurella multocida.

Pasteurella multocida is a pathogenic bacterium known to cause hemorrhagic septicemia and pneumonia in poultry. Reports have indicated that certain proteins, either directly involved in or regulating iron metabolism, are important virulence factors of P. multocida. Therefore, understanding virulent factors and analyzing the role of pro-inflammatory cytokines can help us elucidate the underlying pathogenesis. In this study, the PMCNA_RS00975 protein, a putative encapsuling protein encoded by a gene from a specific prophage island of the pathogenic strain C48-1 of P. multocida, was investigated. To further explore the impact of the PMCNA_RS00975 protein on pathogenicity, a PMCNA_RS00975 gene mutant of P. multocida strain C48-1 was constructed using positive selection technology. Subcellular localization was performed to determine the location of the PMCNA_RS00975 protein within P. multocida. The recombinant protein PMCNA_RS00975 of P. multocida was soluble expressed, purified, and its role in pro-inflammatory cytokines was investigated. The mutant exhibited significantly reduced pathogenicity in the mice model. Furthermore, subcellular localization indicated that the PMCNA_RS00975 protein was located at the outer membrane and expressed during infection of P. multocida. Additionally, our experiments revealed that recombinant PMCNA_RS00975 protein promotes the secretion of the IL-6 pro-inflammatory cytokines triggered by the TLR2 receptor via NF-κB and ERK1/2 signaling pathways in the macrophages. This study identified a novel virulence factor in the C48-1 strain, providing a basis for understanding the pathogenesis and directions for the development of attenuated vaccines against P. multocida.

Microbiological Profile of the Upper and Lower Respiratory Tract of Suckling and Weaned Dairy Calves with Acute Respiratory Disease

Bovine respiratory disease (BRD) is a significant global health issue in cattle farming, leading to substantial economic losses. This study analyzed the microbiological profiles of BRD outbreaks in nine dairy cattle herds in southern Brazil. We examined 36 biological samples, including 24 deep nasopharyngeal swabs (NS) and 12 lung tissue, from 29 suckling and 7 weaned heifer calves with acute BRD. PCR and RT-PCR techniques were used to partially amplify the genes of five viruses and four respiratory bacteria. A total of 8 different microorganisms, 4 viruses (bovine viral diarrhea virus, n = 5; bovine coronavirus, n = 3; bovine alphaherpesvirus 1, n = 3; and bovine parainfluenza virus 3, n = 2), and 4 bacteria (Pasteurella multocida, n = 16; Mycoplasma bovis, n = 8; Histophilus somni, n = 7; and Mannheimia haemolytica, n = 4) were identified in 29 (80.5%) samples. Seven samples (four lung tissue and three NS) were negative for all the microorganisms. Mixed infections were more common (62.1%) than single infections (37.9%). Bacterial nucleic acids were more commonly co-detected in NS than in lung tissue. Nucleic acids from a single pathogen were more frequently detected in lung tissues than in NS. M. bovis was the only bacterium detected in the lower respiratory tract. Understanding the microbiological profiles of the respiratory tracts of dairy calves with clinical signs of BRD is crucial for implementing effective biosecurity measures to prevent BRD in suckling and weaned dairy heifer calves.

Development of a Triplex qPCR Assay Based on the TaqMan Probe for the Detection of Haemophilus parasuis, Streptococcus suis Serotype 2 and Pasteurella multocida

Porcine respiratory disease is a significant economic problem for the global swine industry. Haemophilus parasuis (H. parasuis), Streptococcus suis (S. suis), and Pasteurella multocida (P. multocida) are three important pathogenic bacteria of the swine respiratory tract. Notably, the three pathogens not only frequently manifest as mixed infections, but their striking clinical similarities also present difficulties for pig populations in terms of disease prevention and treatment. Thus, we developed a triplex real-time quantitative polymerase chain reaction (qPCR) assay based on a TaqMan probe for the detection of H. parasuis, S. suis serotype 2, and P. multocida. Primers and probes were designed to target the conserved regions of the H. parasuis OmpP2 gene, the S. suis serotype 2 gdh gene, and the P. multocida Kmt1 gene. By optimizing the reaction system and conditions, a triplex qPCR method for simultaneous detection of H. parasuis, S. suis serotype 2, and P. multocida was successfully established. The amplification efficiencies of the standard curves for all three pathogens were found to be highly similar, with values of 102.105% for H. parasuis, 105.297% for S. suis serotype 2, and 104.829% for P. multocida, and all R2 values achieving 0.999. The specificity analysis results showed that the triplex qPCR method had a strong specificity. The sensitivity test results indicated that the limit of detection can reach 50 copies/μL for all three pathogens. Both intra- and inter-assay coefficients of variation for repeatability were below 1%. This triplex qPCR method was shown to have good specificity, sensitivity, and reproducibility. Finally, the triplex qPCR method established in this study was compared with the nested PCR as recommended by the Chinese national standard (GB/T34750-2017) for H. parasuis, the PCR as recommended by the Chinese national standard (GB/T 19915.9-2005) for S. suis serotype 2, and the PCR as recommended by the Chinese agricultural industry standard (NY/T 564-2016) for P. multocida by detecting the same clinical samples. Both methods are reasonably consistent, while the triplex qPCR assay was more sensitive. In summary, triplex qPCR serves not only as a rapid and accurate detection and early prevention method for these pathogens but also constitutes a robust tool for microbial quality control in specific pathogen-free pigs.

Complete genome sequences of eight Pasteurella multocida isolates representing all lipopolysaccharide outer core loci.

Pasteurella multocida (PM) is a major bacterial pathogen that causes fowl cholera disease in both domestic poultry and wild birds. Here, we report the complete genome sequences of eight PM isolates representing all known lipopolysaccharide outer core loci, which are phenotypically expressed as 16 known PM serotypes.

Herd-level occurrence and risk factors associated with respiratory and enteric pathogens from dairy calves in Ontario: A cross-sectional study

This cross-sectional herd-level study aimed to determine the occurrence of and risk factors for pathogens associated with neonatal calf diarrhea and bovine respiratory disease on Ontario dairy farms. From April to August 2022, a convenience sample of 100 dairy farms were visited once. A questionnaire covering farm biosecurity, calving and colostrum management, preweaning nutrition, and housing was administered on-farm. At each farm visit, approximately 5 calves between 2 and 35 d old were randomly selected for fecal sampling. Furthermore, approximately 5 calves between 21 to 122 d old were randomly selected for nasopharyngeal sampling. In total, 363 fecal samples (from 83 dairy farms) and 390 nasopharyngeal swab samples (from 80 dairy farms) were collected. Fecal samples were analyzed individually using a multiplex PCR to identify bacterial and parasitic enteric pathogens. Nasopharyngeal swabs were analyzed as one pooled sample per farm using bacterial culture and real-time PCR. The most common enteric pathogens detected at herd-level were Cryptosporidium parvum (67.4%) and Escherichia coli K99+ (13.2%). The most common respiratory pathogens detected at herd-level were Pasteurella multocida (62.5%), bovine coronavirus (42.5%), and Mycoplasma bovis (21.2%). Multivariable logistic models were built to explore associations between the most common pathogens and herd-level predictors selected from the questionnaire. Herd positivity for C. parvum was positively associated with having more than 61 preweaned calves per year and feeding mainly whole milk to calves. The presence of M. bovis was positively associated with herds that combined manual and automatic milk-feeding systems, and the presence of bovine coronavirus was positively associated with having more than 98 preweaned calves during the year. Univariable Poisson regression models were built to explore the association between the most common pathogens and preweaning calf mortality. Herds that were positive for C. parvum, M. bovis, or bovine coronavirus had a greater risk of preweaning calf mortality. These results provide insights for future research on pathogens associated with NCD and BRD and offer guidance for veterinarians and dairy farmers in implementing disease control measures in dairy calf herds.