Pasteurella multocida activates apoptosis via the FAK-AKT-FOXO1 axis to cause pulmonary integrity loss, bacteremia, and eventually a cytokine storm.

Pasteurella multocida is an important zoonotic respiratory pathogen capable of infecting a diverse range of hosts, including humans, farm animals, and wild animals. However, the precise mechanisms by which P. multocida compromises the pulmonary integrity of mammals and subsequently induces systemic infection remain largely unexplored. In this study, based on mouse and rabbit models, we found that P. multocida causes not only lung damage but also bacteremia due to the loss of lung integrity. Furthermore, we demonstrated that bacteremia is an important aspect of P. multocida pathogenesis, as evidenced by the observed multiorgan damage and systemic inflammation, and ultimately found that this systemic infection leads to a cytokine storm that can be mitigated by IL-6-neutralizing antibodies. As a result, we divided the pathogenesis of P. multocida into two phases: the pulmonary infection phase and the systemic infection phase. Based on unbiased RNA-seq data, we discovered that P. multocida-induced apoptosis leads to the loss of pulmonary epithelial integrity. These findings have been validated in both TC-1 murine lung epithelial cells and the lungs of model mice. Conversely, the administration of Ac-DEVD-CHO, an apoptosis inhibitor, effectively restored pulmonary epithelial integrity, significantly mitigated lung damage, inhibited bacteremia, attenuated the cytokine storm, and reduced mortality in mouse models. At the molecular level, we demonstrated that the FAK-AKT-FOXO1 axis is involved in P. multocida-induced lung epithelial cell apoptosis in both cells and animals. Thus, our research provides crucial information with regard to the pathogenesis of P. multocida as well as potential treatment options for this and other respiratory bacterial diseases.

Dendritic cell targeting peptide plus Salmonella FliCd flagellin fused outer membrane protein H (OmpH) demonstrated increased efficacy against infections caused by different Pasteurella multocida serogroups in mouse models.

As the major outer membrane protein (OMP) presents in the Pasteurella multocida envelope, OmpH was frequently expressed for laboratory assessments of its immunogenicity against P. multocida infections, but the results are not good. In this study, we modified OmpH with dendritic cell targeting peptide (Depeps) and/or Salmonella FliCd flagellin, and expressed three types of recombinant proteins with the MBP tag (rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, rFliC-OmpH-MBP). Assessments in mouse models revealed that vaccination with rDepeps-FliC-OmpH-MBP, rDepeps-OmpH-MBP, or rFliC-OmpH-MBP induced significant higher level of antibodies as well as IFN-γ and IL-4 in murine sera than vaccination with rOmpH-MBP (P < 0.5). Vaccination with the three modified proteins also provided increased protection (rDepeps-FliC-OmpH-MBP, 70 %; rDepeps-OmpH-MBP, 50 %; rFliC-OmpH-MBP, 60 %) against P. multocida serotype D compared to vaccination with rOmpH-MBP (30 %). In mice vaccinated with different types of modified OmpHs, a significantly decreased bacterial strains were recovered from bloods, lungs, and spleens compared to rOmpH-MBP-vaccinated mice (P < 0.5). Notably, our assessments also demonstrated that vaccination with rDepeps-FliC-OmpH-MBP provided good protection against infections caused by a heterogeneous group of P. multocida serotypes (A, B, D). Our above findings indicate that modification with DCpep and Salmonella flagellin could be used as a promising strategy to improve vaccine effectiveness.

Pharmacokinetic/pharmacodynamic evaluation of gamithromycin against rabbit pasteurellosis.

BACKGROUND: Gamithromycin is an effective therapy for bovine and swine respiratory diseases but not utilized for rabbits. Given its potent activity against respiratory pathogens, we sought to determine the pharmacokinetic profiles, antimicrobial activity and target pharmacokinetic/pharmacodynamic (PK/PD) exposures associated with therapeutic effect of gamithromycin against Pasteurella multocida in rabbits. RESULTS: Gamithromycin showed favorable PK properties in rabbits, including high subcutaneous bioavailability (86.7 ± 10.7%) and low plasma protein binding (18.5-31.9%). PK analysis identified a mean plasma peak concentration (Cmax) of 1.64 ± 0.86 mg/L and terminal half-life (T1/2) of 31.5 ± 5.74 h after subcutaneous injection. For P. multocida, short post-antibiotic effects (PAE) (1.1-5.3 h) and post-antibiotic sub-inhibitory concentration effects (PA-SME) (6.6-9.1 h) were observed after exposure to gamithromycin at 1 to 4× minimal inhibitory concentration (MIC). Gamithromycin demonstrated concentration-dependent bactericidal activity and the PK/PD index area under the concentration-time curve over 24 h (AUC24h)/MIC correlated well with efficacy (R2 > 0.99). The plasma AUC24h/MIC ratios of gamithromycin associated with the bacteriostatic, bactericidal and bacterial eradication against P. multocida were 15.4, 24.9 and 27.8 h in rabbits, respectively. CONCLUSIONS: Subcutaneous administration of 6 mg/kg gamithromycin reached therapeutic concentrations in rabbit plasma against P. multocida. The PK/PD ratios determined herein in combination with ex vivo activity and favorable rabbit PK indicate that gamithromycin may be used for the treatment of rabbit pasteurellosis.

Isolation and Identification of Pasteurella multocida and Mannheimia haemolytica from Pneumonic Small Ruminants and Their Antibiotic Susceptibility in Haramaya District, Eastern Ethiopia.

Background: Pasteurella species are frequently encountered as serious diseases in small ruminants. It is the main cause of respiratory pasteurellosis in sheep and goats of all age groups. Methods: The cross-sectional study was conducted from December 2022 to April 2023 in Haramaya district, eastern Ethiopia, to isolate and identify Pasteurella multocida and Mannheimia haemolytica and estimate their prevalence, associated risk factors, and antimicrobial sensitivity of isolates in small ruminants using a purposive sampling method. A total of 384 samples (156 nasal swabs from clinic cases and 228 lung swabs from abattoir cases) were collected. STATA 14 software was used to analyze the data. In addition, multivariable logistic regression analysis was performed to assess an association of risk factors. Results: Out of the 384 samples examined, 164 were positive for pasteurellosis, resulting in a 42.70% prevalence. Similarly, 63 (38.4%) of the 164 positive results were from nasal swabs, while 101 (61.6%) came from lung samples. M. haemolytica accounted for 126 (76.82%) of the isolates, while P. multocida accounted for 38 (23.17%). Of the 63 nasal swab isolates, 33 (37%) were from goats and 30 (42.8%) were from sheep. And 17 (10.89%) and 46 (29.58%), respectively, were P. multocida and M. haemolytica. Of the 46 (40%) of the 101 (44.3%) isolates of the pneumonic lung, samples were from goats, while 55 (48.47%) were from sheep. In this study, the risk factors (species, age, and body condition score) were found to be significant (p < 0.05). Pasteurella isolates evaluated for antibiotic susceptibility were highly resistant to oxacillin (90.90%), followed by gentamycin (72.72%), and penicillin (63.63%). However, the isolates were highly sensitive to chloramphenicol (90.90%), followed by tetracycline (63.63%), and ampicillin (54.54%). Conclusion: This study showed that M. haemolytica and P. multocida are the common causes of mannheimiosis and pasteurellosis in small ruminants, respectively, and isolates were resistant to commonly used antibiotics in the study area. Thus, an integrated vaccination strategy, antimicrobial resistance monitoring, and avoidance of stress-inducing factors are recommended.

Pasteurella multocida activates Rassf1-Hippo-Yap pathway to induce pulmonary epithelial apoptosis.

Pasteurella multocida is an opportunistic zoonotic pathogen that primarily causes fatal respiratory diseases, such as pneumonia and respiratory syndromes. However, the precise mechanistic understanding of how P. multocida disrupts the epithelial barrier in mammalian lung remains largely unknown. In this study, using unbiased RNA-seq analysis, we found that the evolutionarily conserved Hippo-Yap pathway was dysregulated after P. multocida infection. Given the complexity of P. multocida infection associated with lung injury and systemic inflammatory processes, we employed a combination of cell culture models, mouse models, and rabbit models to investigate the dynamics of the Hippo-Yap pathway during P. multocida infection. Our findings reveal that P. multocida infection activates the Hippo-Yap pathway both in vitro and in vivo, by upregulating the upstream factors p-Mst1/2, p-Lats1, and p-Yap, and downregulating the downstream effectors Birc5, Cyr61, and Slug. Conversely, pharmacological inhibition of the Hippo pathway by XMU-MP-1 significantly rescued pulmonary epithelial cell apoptosis in vitro and reduced lung injury, systemic inflammation, and mouse mortality in vivo. Mechanistic studies revealed that P. multocida induced up-regulation of Rassf1 expression, and Rassf1 enhanced Hippo-Yap pathway through phosphorylation. Accordingly, in vitro knockdown of Rassf1 significantly enhanced Yap activity and expression of Yap downstream factors and reduced apoptosis during P. multocida infection. P. multocida-infected rabbit samples also showed overexpression of Rassf1, p-Lats1, and p-Yap, suggesting that P. multocida activates the Rassf1-Hippo-Yap pathway. These results elucidate the pathogenic role of the Rassf1-Hippo-Yap pathway in P. multocida infection and suggest that this pathway has the potential to be a drug target for the treatment of pasteurellosis.

Discovery of the tigecycline resistance gene cluster tmexCD3-toprJ1 in Pasteurella multocida strains isolated from pigs in China.

Pasteurella multocida is a leading cause of respiratory disorders in pigs. However, the genotypes and antimicrobial resistance characteristics of P. multocida from pigs in China have not been reported frequently. In this study, we investigated 381 porcine strains of P. multocida collected in China between 2013 and 2022. These strains were assigned to capsular genotypes A (69.55%, n = 265), D (27.82%, n =106), and F (2.62%, n = 10); or lipopolysaccharide genotypes L1 (1.31%, n = 5), L3 (24.41%, n = 93), and L6 (74.28%, n = 283). Overall, P. multocida genotype A:L6 (46.46%) was the most-commonly identified type, followed by D:L6 (27.82%), A:L3 (21.78%), F:L3 (2.62%), and A:L1 (1.31%). Antimicrobial susceptibility testing showed that a relatively high proportion of strains were resistant to tetracycline (66.67%, n = 254), and florfenicol (35.17%, n = 134), while a small proportion of strains showed resistance phenotypes to enrofloxacin (10.76%, n = 41), ampicillin (8.40%, n = 32), tilmicosin (7.09%, n = 27), and ceftiofur (2.89%, n = 11). Notably, Illumina short-read and Nanopore long-read sequencing identified a chromosome-borne tigecycline-resistance gene cluster tmexCD3-toprJ1 in P. multocida. The structure of this cluster was highly similar to the respective structures found in several members of Proteus or Pseudomonas. It is assumed that the current study identified the tmexCD3-toprJ1 cluster for the first time in P. multocida.

Pathological analysis and etiological assessment of pulmonary lesions and its association with pleurisy in slaughtered pigs.

The intensification of pig farming has posed significant challenges in managing and preventing sanitary problems, particularly diseases of the respiratory complex. Monitoring at slaughter is an important control tool and cannot be overstated. Hence, this study aimed at characterizing both macroscopical and microscopical lesions and identifying the Actinobacillus pleuropneumoniae (APP), Mycoplasma hyopneumoniae (Mhyo), and Pasteurella multocida (PM) associated with pleurisy in swine. For this, a selected slaughterhouse in São Paulo State underwent a thorough examination of carcasses on the slaughter line, followed by lung sampling. The carcasses and lungs underwent macroscopical examination and were classified according to the score of pleurisy and lung samples were allocated into five groups, being: G0: score 0 - no lesions; G1: score 1; G2: score 2; G3: score 3; and G4: score 4. In total, 217 lung fragments were collected, for the histopathological evaluation and detection of the following respiratory pathogens: APP, Mhyo, and PM by qPCR. The results demonstrated that Mhyo and APP were the most prevalent etiological agents (single and co-identification) in lung samples, in different scores of pleurisies, while bronchopneumonia and bronchus-associated lymphoid tissue (BALT) hyperplasia lesions were the most frequent histopathological findings. Positive correlations were found between the quantification of APP DNA with 1) the score of pleurisy (R=0.254); 2) with the score of lung consolidation in all lung lobes (R=0.181 to R=0.329); and 3) with the score of lung consolidation in the entire lung (R=0.389). The study brings relevant information regarding the main bacterial pathogens associated with pleurisy in pigs and helps with understanding the relationship between the abovementioned pathogens and their impact on the respiratory health of pigs.

Pathogenomic analysis and characterization of Pasteurella multocida strains recovered from human infections.

Pasteurella multocida is an upper respiratory tract commensal in several mammal and bird species but can also cause severe disease in humans and in production animals such as poultry, cattle, and pigs. In this study, we performed whole-genome sequencing of P. multocida isolates recovered from a range of human infections, from the mouths of cats, and from wounds on dogs. Together with publicly available P. multocida genome sequences, we performed phylogenetic and comparative genomic analyses. While isolates from cats and dogs were spread across the phylogenetic tree, human infections were caused almost exclusively by subsp. septica strains. Most of the human isolates were capsule type A and LPS type L1 and L3; however, some strains lacked a capsule biosynthesis locus, and some strains contained a novel LPS outer-core locus, distinct from the eight LPS loci that can currently be identified using an LPS multiplex PCR. In addition, the P. multocida strains isolated from human infections contained novel mobile genetic elements. We compiled a curated database of known P. multocida virulence factor and antibiotic resistance genes (PastyVRDB) allowing for detailed characterization of isolates. The majority of human P. multocida isolates encoded a reduced range of iron receptors and contained only one filamentous hemagglutinin gene. Finally, gene-trait analysis identified a putative L-fucose uptake and utilization pathway that was over-represented in subsp. septica strains and may represent a novel host predilection mechanism in this subspecies. Together, these analyses have identified pathogenic mechanisms likely important for P. multocida zoonotic infections.IMPORTANCEPasteurella multocida can cause serious infections in humans, including skin and wound infections, pneumonia, peritonitis, meningitis, and bacteraemia. Cats and dogs are known vectors of human pasteurellosis, transmitting P. multocida via bite wounds or contact with animal saliva. The mechanisms that underpin P. multocida human predilection and pathogenesis are poorly understood. With increasing identification of antibiotic-resistant P. multocida strains, understanding these mechanisms is vital for developing novel treatments and control strategies to combat P. multocida human infection. Here, we show that a narrow range of P. multocida strains cause disease in humans, while cats and dogs, common vectors for zoonotic infections, can harbor a wide range of P. multocida strains. We also present a curated P. multocida-specific database, allowing quick and detailed characterization of newly sequenced P. multocida isolates.

Spatial and temporal analysis of haemorrhagic septicaemia outbreaks in India over three decades (1987-2016).

Haemorrhagic septicaemia (HS) is an economically important disease affecting cattle and buffaloes and the livelihoods of small-holder farmers that depend upon them. The disease is caused by Gram-negative bacterium, Pasteurella multocida, and is considered to be endemic in many states of India with more than 25,000 outbreaks in the past three decades. Currently, there is no national policy for control of HS in India. In this study, we analysed thirty year (1987-2016) monthly data on HS outbreaks using different statistical and mathematical methods to identify spatial variability and temporal patterns (seasonality, periodicity). There was zonal variation in the trend and seasonality of HS outbreaks. Overall, South zone reported maximum proportion of the outbreaks (70.2%), followed by East zone (7.2%), Central zone (6.4%), North zone (5.6%), West zone (5.5%) and North-East zone (4.9%). Annual state level analysis indicated that the reporting of HS outbreaks started at different years independently and there was no apparent transmission between the states. The results of the current study are useful for the policy makers to design national control programme on HS in India and implement state specific strategies. Further, our study and strategies could aid in implementation of similar approaches in HS endemic tropical countries around the world.

Molecular characterization of Pasteurella multocida from cats and antibiotic sensitivity of the isolates.

BACKGROUND: Companion animals, including dogs and cats, are frequently identified as sources of Pasteurella multocida, a bacterium that can be transmitted to humans and cause infections. OBJECTIVES: This survey defines the prevalence, antibiotic sensitivity, capsular types, lipopolysaccharide (LPS) types and virulence factors of P. multocida isolated from cats. METHODS: A total of 100 specimens from various cat breeds were collected. P. multocida was characterized using both biochemical tests and PCR. Genotypes of isolates were determined using capsular and LPS typing methods. Additionally, virulotyping was performed by detecting the presence of 12 virulence-associated genes. Disk diffusion was used to determine the antibiotic sensitivity of the isolates. RESULTS: The prevalence of P. multocida in cats was 29%. Among the isolates, the majority were capsular type A (96.5%) and type D (3.4%), with a predominant presence of type A. Twenty-six of the isolates (89.66%) belonged to LPS genotype L6, whereas three isolates (10.3%) belonged to genotype L3. Among the 12 virulence genes examined, sodC, oma87, ptfA, nanB and ompH showed remarkable prevalence (100%). The toxA gene was detected in four isolates (13.8%). Variations were observed in other virulence genes. The nanH gene was present in 93.1% of the isolates, whereas the pfhA gene was detected in 58.6% of the isolates. The exbD-tonB, hgbB, sodA and hgbA genes showed prevalence rates of 96.5%, 96.5%, 96.5% and 82.8%, respectively. Additionally, particular capsule and LPS types were associated with specific virulence genes. Specifically, the toxA and pfhA genes were found to be more prevalent in isolates with capsular type A and LPS genotype L6. Most isolates were resistant to ampicillin, clindamycin, lincomycin, streptomycin and penicillin. CONCLUSIONS: According to this epidemiological and molecular data, P. multocida from cats possess several virulence-associated genes and are resistant to antimicrobial medicines commonly used in humans and animals. Thus, it is crucial to consider the public health concerns of P. multocida in humans.

Chronic Pasteurella Multocida Bronchitis Diagnosed 3 Years After Onset of Symptoms.

We report a case of chronic infection with Pasteurella multocida in the lower respiratory tract in a man with a cat. A 77-year-old man presented with recurrent hemoptysis accompanied by bronchiectasis and an opacity in the left lung on chest computed tomography. Although the patient was seropositive for Mycobacterium avium complex, repeated sputum cultures were negative for any specific pathogen. Three years later, he was referred to our hospital for hemoptysis with enhanced opacity in the lower lobe of the left lung. Culture of bronchial lavage fluid obtained via bronchoscopy was positive for P. multocida. The patient was treated with amoxicillin-clavulanic acid for 14 days and was instructed to avoid close contact with his cat. His symptoms and chest imaging findings improved and have not recurred during more than 1 1/2 years of follow up. P. multocida can cause chronic lower respiratory infections.

Rare case of septic shock combined with meningitis caused by Pasteurella multocida without a history of cat and dog bites.

BACKGROUND: Pasteurella multocida is a zoonotic pathogen that mainly causes local skin and soft tissue infections in the human body through cat and dog bites. It rarely causes bacteraemia (or sepsis) and meningitis. We reported a case of septic shock and meningitis caused by P. multocida in a patient without a history of cat and dog bites. CASE PRESENTATION: An 84-year-old male patient was urgently sent to the emergency department after he was found with unclear consciousness for 8 h, accompanied by limb tremors and urinary incontinence. In the subsequent examination, P. multocida was detected in the blood culture and wound secretion samples of the patient. However, it was not detected in the cerebrospinal fluid culture, but its DNA sequence was detected. Therefore, the patient was clearly diagnosed with septic shock and meningitis caused by P. multocida. The patient had no history of cat or dog contact or bite. The patient was subsequently treated with a combination of penicillin G, doxycycline, and ceftriaxone, and he was discharged after 35 days of hospitalisation. CONCLUSION: This report presented a rare case of septic shock and meningitis caused by P. multocida, which was not related to a cat or dog bite. Clinical doctors should consider P. multocida as a possible cause of sepsis or meningitis and should be aware of its potential seriousness even in the absence of animal bites.

Efficient markerless genetic manipulation of Pasteurella multocida using lacZ and pheSm as selection markers.

Pasteurella multocida is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of P. multocida gene function and pathogenesis of P. multocida. Herein, a temperature-sensitive shuttle vector was constructed using lacZ as a selection marker, and markerless glgB, opa, and hyaE mutants of P. multocida were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the lacZ system, and the method could be used for multiple gene deletions. However, the fur mutant was unavailable via this method. Therefore, we constructed a pheSm screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve fur deletion mutant. The transformed strain was sensitive to 20 mM p-chloro-phenylalanine, demonstrating the feasibility of pheSm as a counter-selective marker. The pheSm system was used for markerless deletions of glgB, opa, and hyaE as well as fur that could not be screened by the lacZ system. A comparison of screening efficiencies of the system showed that the pheSm counterselection system was more efficient than the lacZ system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of P. multocida.IMPORTANCEPasteurella multocida is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for P. multocida mutants using lacZ or pheSm screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of lacZ or pheSm as counterselection markers to improve counterselection efficiency in P. multocida. This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of P. multocida.

Epididymitis with Pasteurella multocida isolated from two male Japanese Black beef calves.

Two male Japanese Black calves developed an enlarged scrotum and testis. Orchiectomy was performed and pus was collected during surgery. After removal of the testis, bacteriological and histopathological examinations were conducted to investigate the cause and confirm the diagnosis. Based on the results obtained, both cases were diagnosed with epididymitis caused by an infection with Pasteurella multocida. This is the first study to show that P. multocida causes epididymitis in male calves. Further studies are required to clarify the details underlying the infection of calves with P. multocida.

Attenuated vaccine PmCQ2Δ4555-4580 effectively protects mice against Pasteurella multocida infection.

Pasteurella multocida type A (PmA) mainly causes respiratory diseases such as pneumonia in bovines, leading to great economic losses to the breeding industry. At present, there is still no effective commercial vaccine against PmA infection. In this study, a mutant strain (PmCQ2Δ4555-4580) with brand-new phenotypes was obtained after serially passaging at 42 °C. Whole genome resequencing and PCR analysis showed that PmCQ2Δ4555-4580 missed six genes, including PmCQ2_004555, PmCQ2_004560, PmCQ2_004565, PmCQ2_004570, PmCQ2_004575, and PmCQ2_004580. Importantly, the virulence of PmCQ2Δ4555-4580 was reduced by approximately 2.8 × 109 times in mice. Notably, live PmCQ2Δ4555-4580 could provide 100%, 100% and 40% protection against PmA, PmB and PmF, respectively; and inactivated PmCQ2Δ4555-4580 could provide 100% and 87.5% protection against PmA and PmB. Interestingly, immune protection-related proteins were significantly upregulated in PmCQ2Δ4555-4580 based on RNA-seq and bioinformatics analysis. Meaningfully, by in vitro expression, purification and in vivo immunization, 12 proteins had different degrees of immune protective effects. Among them, PmCQ2_008205, PmCQ2_010435, PmCQ2_008190, and PmCQ2_004170 had the best protective effect, the protection rates against PmA were 50%, 40%, 30%, and 30%, respectively, and the protective rates against PmB were 62.5%, 42.9%, 37.5%, and 28.6%, respectively. Collectively, PmCQ2Δ4555-4580 is a potential vaccine candidate for the prevention of Pasteurellosis involving in high expression of immune protective related proteins.

Characterization and immunological effect of outer membrane vesicles from Pasteurella multocida on macrophages.

Pasteurella multocida is an important bacterial pathogen that can cause diseases in both animals and humans. Its elevated morbidity and mortality rates in animals result in substantial economic repercussions within the livestock industry. The prevention of diseases caused by P. multocida through immunization is impeded by the absence of a safe and effective vaccine. Outer membrane vesicles (OMVs) secreted from the outer membrane of Gram-negative bacteria are spherical vesicular structures that encompass an array of periplasmic components in conjunction with a diverse assortment of lipids and proteins. These vesicles can induce antibacterial immune responses within the host. P. multocida has been shown to produce OMVs. Nonetheless, the precise characteristics and immunomodulatory functions of P. multocida OMVs have not been fully elucidated. In this study, OMVs were isolated from P. multocida using an ultrafiltration concentration technique, and their morphology, protein constitution, and immunomodulatory properties in RAW264.7 cells were studied. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) revealed that the OMVs exhibited typical spherical and bilayered lipid vesicular architecture, exhibiting an average diameter of approximately 147.5 nm. The yield of OMVs was 2.6 × 1011 particles/mL. Proteomic analysis revealed a high abundance of membrane-associated proteins within P. multocida OMVs, with the capability to instigate the host's immune response. Furthermore, OMVs stimulated the proliferation and cellular uptake of macrophages and triggered the secretion of cytokines, such as TNF-ɑ, IL-1ß, IL-6, IL-10, and TGF-ß1. Consequently, our results indicated that OMVs from P. multocida could directly interact with macrophages and regulate their immune function in vitro. These results supported the prospective applicability of P. multocida OMVs as a platform in the context of vaccine development. KEY POINTS: • Preparation and characterization of P. multocida OMVs. • P. multocida OMVs possess a range of antigens and lipoproteins associated with the activation of the immune system. • P. multocida OMVs can activate the proliferation, internalization, and cytokine secretion of macrophages in vitro.

Identification and evolution of ICE-PmuST394: a novel integrative conjugative element in Pasteurella multocida ST394.

BACKGROUND: The emergence of macrolide and tetracycline resistance within Pasteurella multocida isolated from feedlot cattle and the dominance of ST394 in Australia was reported recently. OBJECTIVES: To establish the genetic context of the resistance genes in P. multocida 17BRD-035, the ST394 reference genome, and conduct a molecular risk assessment of their ability to disperse laterally. METHODS: A bioinformatic analysis of the P. multocida 17BRD-035 genome was conducted to determine if integrative conjugative elements (ICEs) carrying resistance genes, which hamper antibiotic treatment options locally, are in circulation in Australian feedlots. RESULTS: A novel element, ICE-PmuST394, was characterized in P. multocida 17BRD-035. It was also identified in three other isolates (two ST394s and a ST125) in Australia and is likely present in a genome representing P. multocida ST79 from the USA. ICE-PmuST394 houses a resistance module carrying two variants of the blaROB gene, blaROB-1 and blaROB-13, and the macrolide esterase gene, estT. The resistance gene combination on ICE-PmuST394 confers resistance to ampicillin and tilmicosin, but not to tulathromycin and tildipirosin. Our analysis suggests that ICE-PmuST394 is circulating both by clonal expansion and horizontal transfer but is currently restricted to a single feedlot in Australia. CONCLUSIONS: ICE-PmuST394 carries a limited number of unusual antimicrobial resistance genes but has hotspots that facilitate genomic recombination. The element is therefore amenable to hosting more resistance genes, and therefore its presence (or dispersal) should be regularly monitored. The element has a unique molecular marker, which could be exploited for genomic surveillance purposes locally and globally.

Pathogenic and genomic characterization of rabbit-sourced Pasteurella multocida serogroup F isolates recovered from dead rabbits with respiratory disease.

Pasteurella multocida serogroup F can infect a number of animals. However, the pathogenicity and genomic features of this serogroup are still largely unknown. In the present study, the pathogenicity and genomic sequences of 19 rabbit-sourced P. multocida serogroup F isolates were determined. The 19 isolates were highly pathogenic for rabbits causing severe pathologic lesions and high mortality in inoculated rabbits. Nevertheless, the pathologic lesions in rabbits caused by the 19 isolates were distinct from those caused by the previously reported high-virulent serogroup F strains J-4103 (rabbit), P-4218 (turkey), and C21724H3km7 (chicken). Moreover, the 19 isolates were avirulent to white feather broilers. The genomes of the 19 isolates were determined to understand the pathogenicity of these isolates. The finding of a number of functional genes in the 19 isolates by comparison with the low-virulent rabbit-sourced serogroup F strain s4 might contribute to the high virulence of these isolates. Notably, polymorphisms were determined in the lipopolysaccharide outer core biosynthetic genes natC and gatF among the serogroup F strains of different hosts. However, the sequences of natC and gatF from rabbit-sourced strains (except for SD11) were identical, which might be responsible for the host specific of the 19 isolates. The observations and findings in this study would be helpful for the understanding of the pathogenicity variation and host predilection of P. multocida. IMPORTANCE: The 19 rabbit-sourced Pasteurella multocida serogroup F isolates showing high virulence to rabbits were avirulent to the broilers. Notably, polymorphisms were determined in the lipopolysaccharide outer core biosynthetic genes natC and gatF among all serogroup F strains of different hosts. However, the sequences of natC and gatF from rabbit-sourced strains (except for SD11) were identical, which might be responsible for the host specific of the 19 isolates.

Evaluation of the effect of therapeutic durations on small ruminant bacterial pneumonia.

BACKGROUND: Sheep and goat production in Ethiopia is hindered by numerous substandard production systems and various diseases. Respiratory disease complexes (RDC) pose a significant threat to the productivity of these animals. Pneumonia is a common manifestation of respiratory disease complexes and often necessitates a prolonged course of antibiotic treatment. This study aimed to optimize and propose the ideal duration of therapy for pneumonia in sheep and goats. METHODS: The study was conducted from February to June 2021 at the Veterinary Teaching Hospital of the College of Veterinary Medicine and Agriculture, Addis Ababa University. The study recruited 54 sheep and goats presented to the hospital for treatment with a confirmed RDC as determined based on clinical signs and bacteriological methods. The animals were randomly allocated to 5 groups each group receiving 10% oxytetracycline (Phenxyl, Phenix, Belgum) intramuscularly for a duration of 3, 4, 5, 6 and 7 consecutive days. The treatment outcomes were assessed by recording vital signs (body temperature, respiratory rate, heart rate, coughing, and nasal discharges), performing lung ultrasonography (L-USG) as well as collection of nasal swabs for bacterial isolation and molecular identification before and after completion of the treatment. An ordered logistic regression model with random effects was employed to determine the optimal therapeutic duration, taking into account the cumulative scores of the outcome variables across the different groups. RESULTS: Among the 54 sheep and goats treated with 10% oxytetracycline, a total of 74.07% (95% CI, 60.35-85.04) achieved complete recovery, as confirmed through clinical, ultrasound, and bacteriological methods. In Group 1 (G1), out of 12 sheep and goats, 8 (83.0%) recovered completely; in Group 2 (G2), out of 11 animals, 9 (82.0%) recovered completely; in Group 3 (G3), out of 11 animals, 10 (93.0%) recovered completely; in Group 4 (G4), out of 9 animals, 9 (100.0%) recovered completely; and in Group 5 (G5), out of 11 animals, 10 (91.0%) recovered completely. Bacteriological examination of nasal swabs indicated involvement of M. hemolytica in 27 (50.00%) and P. multocida in 13 (24.07%) of pneumonic animals. Detection of specific marker genes confirmed only five of the presumptive M. hemolytica isolates, whilst no isolates tested positive for P. multocida. Post-treatment samples collected from recovered animals did not yield any M. hemolytica nor P. multocida. Based on results from clinical signs, L-USG, and bacterial infection variables, the group of sheep and goats treated for seven consecutive days (G5) showed the highest recovery score compared to the other groups, and there was a statistically significant difference (coefficient (ß) = - 2.296, p = 0.021) in variable score between G5 and G1. These findings suggest that the administration of 10% oxytetracycline for a full course of seven consecutive days resulted in symptomatic and clinical recovery rates from respiratory disease in sheep and goats.