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.

Naked-eye on-site detection platform for Pasteurella multocida based on the CRISPR-Cas12a system coupled with recombinase polymerase amplification.

Pasteurella multocida (P. multocida) is an important pathogenic bacterium that poses a serious threat to the development of the livestock economy and human health. Currently, the existing methods for P. multocida detection are time-consuming and require complex professional operations, limiting the application of field detection. In the study, we presented a single-pot naked-eye CRISPR-Cas12a platform (Cas12a-NEye) for the detection of P. multocida. The round tube cover allowed more Cas12a detection solution to be temporarily stored than the flat cap, enabling single-pot assays and avoiding aerosol contamination. The positive samples generated obvious red using naked eye using no excitation light and the negative samples generated blue. The limit of detection (LOD) was a single copy, without cross-reactivity with other closely related bacteria. Furthermore, we validated this platform using 16 P. multocida clinical lung samples and obtained consistent results with the real-time quantitative polymerase chain reaction (qPCR) method. The entire experimental process included rapid DNA extraction (<1 h) and Cas12a-NEye assay (25 min), which was accomplished within 1.5 h. Thus, this "sample-to-answer" platform has significant potential for P. multocida detection.

Limitations of bacterial culture, viral PCR, and tulathromycin susceptibility from upper respiratory tract samples in predicting clinical outcome of tulathromycin control or treatment of bovine respiratory disease in high-risk feeder heifers.

A cross-sectional prospective cohort study including 1026 heifers administered tulathromycin due to high risk of clinical signs of bovine respiratory disease (BRD), measured poor association between BRD clinical outcomes and results of bacterial culture and tulathromycin susceptibility from BRD isolates of deep nasopharyngeal swabs (DNS) and adequate association with viral polymerase chain reaction (PCR) results from nasal swabs. Isolation rates from DNS collected on day-0 and at 1st BRD-treatment respectively were: Mannheimia haemolytica (10.9% & 34.1%); Pasteurella multocida (10.4% & 7.4%); Mycoplasma bovis (1.0% & 36.6%); and Histophilus somni (0.7% & 6.3%). Prevalence of BRD viral nucleic acid on nasal swabs collected exclusively at 1st BRD-treatment were: bovine parainfluenza virus type-3 (bPIV-3) 34.1%; bovine viral diarrhea virus (BVDV) 26.3%; bovine herpes virus type-1 (BHV-1) 10.8%; and bovine respiratory syncytial virus (BRSV) 54.1%. Increased relative risk, at 95% confidence intervals, of 1st BRD-treatment failure was associated with positive viral PCR results: BVDV 1.39 (1.17-1.66), bPIV-3 1.26 (1.06-1.51), BHV-1 1.52 (1.25-1.83), and BRSV 1.35 (1.11-1.63) from nasal swabs collected at 1st BRD-treatment and culture of M. haemolytica 1.23 (1.00-1.51) from DNS collected at day-0. However, in this population of high-risk feeder heifers, the predictive values of susceptible and resistant isolates had inadequate association with BRD clinical outcome. These results indicate, that using tulathromycin susceptibility testing of isolates of M. haemolytica or P. multocida from DNS collected on arrival or at 1st BRD-treatment to evaluate tulathromycin clinical efficacy, is unreliable.

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.

Microbiología de las infecciones causadas por mordeduras de perros y gatos en personas: una revisión / Microbiology of infections caused by dog and cat bites: a review

INTRODUCCIÓN: Las mordeduras causadas por animales de compañía constituyen el 5% de las heridas traumáticas registradas en los servicios de urgencias. OBJETIVO: Conocer los principales agentes infecciosos presentes en las mordeduras provocadas por perros y gatos, tanto de forma individual como conjunta; así como los factores que favorecen la infección. METODOLOGÍA: Se realizó una búsqueda bibliográfica en Pub-Med con la siguiente estrategia de búsqueda: (("Bites, Human"[Mesh]) OR "Bites and Stings"[Mesh]) AND "Infection"[Mesh]. Se incluyeron 24 trabajos en la síntesis cualitativa escritos en lengua inglesa o española, casos clínicos o descriptivos y publicados entre los años 2000 y 2019. RESULTADOS: Las especies más frecuentemente aisladas fueron Capnocytophaga canimorsus en perros y Pasteurella multocida en gatos. La existencia de comorbilidades en el individuo, la mordedura en la mano, o la presencia de signos de alteración del estado general del individuo mordido fueron considerados como factores de riesgo para el desarrollo de la infección tras la mordedura. CONCLUSIONES: Todos los afectados por mordeduras animales deben recibir asistencia médica y considerar la administración de una pauta de profilaxis antimicrobiana con el fin de reducir el riesgo de shock séptico. Por otro lado, es importante advertir al laboratorio de microbiología de la naturaleza de las muestras clínicas obtenidas para alcanzar el mejor diagnóstico etiológico.

BACKGROUND: Bites caused by pets constitute 5% of the traumatic injuries registered in the emergency services. AIM: To know the main infectious agents present in dog and cat bites, both individually and jointly, in humans, as well as the predisposing factors that favor infection and its spread. METHODS: A bibliographic search was carried out in PubMed with the following search strategy: (("Bites, Human" [Mesh]) OR "Bites and Stings" [Mesh]) AND "Infection" [Mesh]. Twenty-four papers were included in the qualitative synthesis written in English or Spanish, clinical or descriptive cases and published between 2000 and 2019. Results: Most frequently isolated species were Capnocytophaga canimorsus in dogs and Pasteurella multocida in cats. The existence of comorbidities in the individual, the bite on the hand, or the presence of signs of alteration of the general state of the bitten individual were considered as risk factors for the development of infection after the bite. CONCLUSIONS: All patients with animal bites should receive medical assistance, and the administration of an antibiotic prophylaxis regimen should be considered to reduce the risk of septic shock. Besides, it is important to advise the microbiology laboratory of the nature of the clinical samples obtained in order to reach the best etiological diagnosis.

Genomic diversity and molecular epidemiology of Pasteurella multocida.

Pasteurella multocida is a bacterial pathogen with the ability to infect a multitude of hosts including humans, companion animals, livestock, and wildlife. This study used bioinformatic approaches to explore the genomic diversity of 656 P. multocida isolates and epidemiological associations between host factors and specific genotypes. Isolates included in this study originated from a variety of hosts, including poultry, cattle, swine, rabbits, rodents, and humans, from five different continents. Multi-locus sequence typing identified 69 different sequence types. In-silico methodology for determining capsular serogroup was developed, validated, and applied to all genome sequences, whereby capsular serogroups A, B, D, and F were found. Whole genome phylogeny was constructed from 237,670 core single nucleotide variants (SNVs) and demonstrated an overall lack of host or capsular serogroup specificity, with the exception of isolates from bovine sources. Specific SNVs within the srlB gene were identified in P. multocida subsp. septica genomes, representing specific mutations that may be useful for differentiating one of the three known subspecies. Significant associations were identified between capsular serogroup and virulence factors, including capsular serogroup A and OmpH1, OmpH3, PlpE, and PfhB1; capsular serogroup B and HgbA and PtfA; and capsular serogroup F and PtfA and PlpP. Various mobile genetic elements were identified including those similar to ICEPmu1, ICEhin1056, and IncQ1 plasmids, all of which harbored multiple antimicrobial resistance-encoding genes. Additional analyses were performed on a subset of 99 isolates obtained from turkeys during fowl cholera outbreaks from a single company which revealed that multiple strains of P. multocida were circulating during the outbreak, instead of a single, highly virulent clone. This study further demonstrates the extensive genomic diversity of P. multocida, provides epidemiological context to the various genotyping schemes that have traditionally been used for differentiating isolates, and introduces additional tools for P. multocida molecular typing.

Fulminant Septic Shock with Pasteurella multocida in a Young Infant: No Bite, Scratch, or Lick!

This is a case of Pasteurella multocida septic shock encountered in a 7-week-old infant without any bites, scratch marks, or history of licks by pet animals (dog and cats in household). The infant required 3 days of vasopressor support and 4 days of mechanical ventilation to achieve normal hemodynamics. This is an unidentified route of transmission and our literature search for this topic discovered reported cases of life-threatening presentation with Pasteurella infections in the absence of a bite or any form of invasive contact with animals. We believe that this is an important public safety message to restrict animal contact of young infants to prevent severe infection.

Occurrence of Pasteurella multocida in Dogs Being Trained for Animal-Assisted Therapy.

Animal-assisted therapy (AAT) is a non-pharmacological therapy aimed at people with physical and/or mental disabilities. Therefore, it is necessary to carry out interventions that guarantee its benefits for patients while also avoiding the risk of zoonoses due to contact with the animals or their mucous membranes. The present study aimed to detect the occurrence of Pasteurella multocida in the oral cavity of dogs attending a "dog educational centre" and training for AAT interventions. In addition, some of the potential predictable factors of infection (i.e., age, sex, breed, and living conditions) were analyzed. In total, 25/200 dogs examined (12.5%; 95% confidence interval = 8.4-18.1%) were positive for P. multocida, as confirmed by PCR. Sex, breed, and living conditions were risk factors associated with P. multocida as revealed by the logistic regression analysis. Specifically, cross-bred female dogs living prevalently outdoors were significantly associated with the presence of P. multocida (p < 0.05). This study represents the first epidemiological survey of the prevalence of P. multocida in the oral cavity of dogs involved subsequently in AAT interventions, highlighting the potential risk of P. multocida infection in patients, often belonging to risk categories (e.g., children, the elderly, and immunocompromised individuals). Therefore, healthcare guidelines could be suggested to integrate the current literature related to the health check of dogs involved in AAT. In this way, it could be ensured that, even with bodily contact during AAT, the risk of pathogen transmission by the co-therapist dog can be avoided.

Strength of association between isolation of Pasteurella multocida and consolidation lesions in ovine pneumonic pasteurellosis.

This study investigated the association of Pasteurella multocida isolation and the molecular characteristics of the isolates with the presence of pneumonic lesions in lambs at slaughter to assess its importance as a causative agent of pneumonic pasteurellosis compared with Mannheimia haemolytica. P. multocida was isolated from the 13.9% and 2.7%, and M. haemolytica from the 36.4% and 26.8%, of lungs with and without lesions, respectively (P < 0.05). Both microorganisms were frequently coisolated (23.2% and 12.5% from lungs with and without lesions, respectively). Isolation of P. multocida alone exhibited greater strength of association with pneumonic lesions (OR 11.4; 95% CI 3.2-40.6) than that exhibited by M. haemolytica alone (OR 3.0; 95% CI 1.6-5.4). Cluster analysis grouped the lungs into four clusters characterized by the isolation of M. haemolytica or P. multocida alone (clusters 1 and 4), coisolation of both microorganisms (cluster 3), and isolation of neither (cluster 2). Cluster 4 lungs exhibited higher frequencies of pneumonic lesions (87.5%) and severe (20.8%) and moderate (25.0%) lesions. Lungs coinfected with both pathogens (cluster 3) did not exhibit a higher frequency of severe and moderate consolidation lesions (6.1% and 14.3%, respectively), suggesting that P. multocida and M. haemolytica do not act synergically to cause more severe pneumonic infections. The greater strength of association of P. multocida isolation with pneumonic lesions together with the higher severity of the lesions caused could indicate a greater role played by this pathogen in the aetiopathogenesis of pneumonic pasteurellosis in sheep than is commonly assumed.

Antimicrobial resistance of Pasteurella multocida type B isolates associated with acute septicemia in pigs and cattle in Spain.

BACKGROUND: Pasteurella multocida is the etiological agent responsible for several diseases in a wide range of hosts around the world and thus, causes serious economic losses. Acute septicemia associated with capsular type B P. multocida has recently emerged in Europe and continuous outbreaks of these acute processes have been described in Spain since they were first detected in pigs in 2009 and cattle in 2015. The scarcity of studies on the antimicrobial susceptibility of this capsular type of P. multocida and growing concern about the general increase of antimicrobial resistance mean that studies related to the performance of type B P. multocida against antibiotics are necessary to establish accurate treatments and to monitor antimicrobial resistances. RESULTS: Seventy-six isolates of P. multocida type B from pigs and cattle with acute septicemia were tested for susceptibility to 10 different antimicrobials. Bovine isolates were susceptible to all the antibiotics we tested except for lincomycin (94.4% of isolates were resistant). However, the antimicrobials we tested were less effective against swine isolates, of which none were susceptible to lincomycin. Furthermore, 29.3% swine isolates were resistant to tetracycline, 27.6% to penicillin, 20.7% to oxytetracycline, 17.3% to chloramphenicol, 15.5% to gentamicin, and 3.4% to enrofloxacin; no resistance to ceftiofur was detected. No multidrug resistant isolates were detected from cattle, while 25.86% of swine isolates were resistant to three or more antibiotic classes. CONCLUSIONS: In this study, the lower resistance rates and multidrug resistant isolates reported for P. multocida type B derived from cattle compared to those isolated from pigs may be related to the increased use of antibiotics in the porcine industry in Spain. Lincomycin is not recommended for the treatment of acute septicemia in pigs or cattle, rather, the use of ceftiofur, enrofloxacin, or gentamicin is indicated as an emergency treatment in the early stages of disease; once the susceptibility results are known, the use of tetracyclines, penicillin, or chloramphenicol should be prioritized. The increase in multidrug resistant isolates and antimicrobial resistance rates indicates that more attention should be paid to prevention as well as the responsible use of antibiotics.

Storage time and temperature affect the isolation rate of Mannheimia haemolytica and Pasteurella multocida from bovine bronchoalveolar lavage samples.

BACKGROUND: A microbiological diagnosis is essential to better target antimicrobial treatment, control and prevention of respiratory tract infections in cattle. Under field conditions, non-endoscopic broncho-alveolar lavage (nBAL) samples are increasingly collected. To what extent the highly variable turnaround time and storage temperatures between sampling and cultivation affect the isolation rate of bacterial pathogens is unknown. Therefore, the objective of this experimental study was to determine the effect of different storage temperatures (0 °C, 8 °C, 23 °C and 36 °C) and times (0,2,4,6,8,24,48 h) on the isolation rate and concentration of Pasteurellaceae in nBAL samples from clinically affected animals. RESULTS: At a storage temperature temperature of 36 °C isolation rates of Mannheimia haemolytica and Pasteurella multocida were significantly reduced 6 h and 48 h after sampling, respectively. At room temperature (23 °C), a decrease in M. haemolytica and P. multocida isolation rate was noticed, starting at 24 and 48 h after sampling, respectively, but only significant for P. multocida at 48 h. The presence of microbial contamination negatively affected the isolation of P. multocida in clinical nBAL samples, but not of M. haemolytica. CONCLUSION: Optimal M. haemolytica and P. multocida isolation rates from clinical nBAL samples are obtained after storage at 0 °C or 8 °C, provided that the sample is cultivated within 24 h after sampling. The maximum period a sample can be stored without an effect on the M. haemolytica and P. multocida isolation success varies and is dependent on the storage temperature and the degree of microbial contamination.

Pasteurella multocida Infections with Unusual Modes of Transmission from Animals to Humans: A Study of 79 Cases with 34 Nonbite Transmissions.

Pasteur discovered the causative agent of fowl cholera (Pasteurella multocida) in 1880. Since then, multiple zoonotic infections affecting humans have been reported. P. multocida infections usually result from bites of cats or dogs. The earliest reports of nonbite transmissions (NBTs) were attributed to cat scratches and lung colonization. More recently, multiple modes of unusual NBTs have been reported, including animal exposures with no direct contact. Here, we report 79 cases of pet-associated infections, with 34 NBTs. Previously unreported and unsuspected, novel modes of NBTs presented include stepping on dog drool infecting a submetatarsal ulcer, contamination of a wound by socks covered with cat hair and dander resulting in P. multocida bacteremia, stumbling over a dog and falling while drunk and abrasions contaminated with dog saliva resulting in wound infection, and severe epiglottitis and supraglottitis after eating peanut butter and crackers half eaten by a dog. Cat bites were more common than dog bites. Both bite and nonbite infections were more common in the elderly, with more older patients in the nonbite group. Upper extremity bites were more than lower extremity bites for both cats and dogs. NBTs were associated with more co-morbidities and resulted in more life-threatening infections than bites, confirming the findings of a prior smaller series. Open wounds were the most common point of entry for nonbite infections, with majority in the lower extremity. Based on this study and prior reports, pet owners must protect open wounds and individuals with certain underlying conditions and infants should avoid pet exposure completely. Our findings and animal transmission of bite and nonbite P. multocida infections reported in literature are summarized.

Microbial diversity involved in the etiology of a bovine respiratory disease outbreak in a dairy calf rearing unit.

The etiological agents involved in a bovine respiratory disease (BRD) outbreak were investigated in a dairy heifer calf rearing unit from southern Brazil. A battery of PCR assays was performed to detect the most common viruses and bacteria associated with BRD, such as bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine alphaherpesvirus 1 (BoHV-1), bovine coronavirus (BCoV), bovine parainfluenza virus 3 (BPIV-3), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Bronchoalveolar lavage fluid (BALF) samples were taken from 21 heifer calves (symptomatic n = 15; asymptomatic n = 6) that, during the occurrence of the BDR outbreak, were aged between 6 and 90 days. At least one microorganism was detected in 85.7 % (18/21) of the BALF samples. Mixed infections were more frequent (72.2 %) than single infections (27.7 %). The interactions between viruses and bacteria were the most common in coinfections (55.5 %). The frequencies of BRD agents were 38.1 % for BRSV, 28.6 % for BVDV, 33.3 % for BCoV, 42.85 % for P. multocida, 33.3 % for M. bovis, and 19 % for H. somni. BoHV-1, BPIV-3, and M. haemolytica were not identified in any of the 21 BALF samples. Considering that BALF and not nasal swabs were analyzed, these results demonstrate the etiological multiplicity that may be involved in BRD outbreaks in dairy calves.

Characterization of Pasteurella multocida isolates from rabbits in China.

Pasteurella multocida is the causative agent of a wide range of diseases (pasteurellosis) and is a zoonotic pathogen in humans. The molecular epidemiology of P. multocida from rabbits in some southern European countries has been characterized, and the associations of some populations with the respiratory niche or virulence factors have been suggested. However, the population structure of P. multocida from rabbits in China has not been well characterized. In this study, 30 P. multocida isolates from rabbits without epidemiological relations in China were clustered using mutilocus sequence typing (MLST). Then, the genotypes of virulence factors (capsule, lipopolysaccharides, HgbB, and PfhA) of these isolates were determined via multiplex PCR methods. Next, the virulence of the isolates in a mice model was established by determining the 50 % lethal dose. Finally, the associations between MLST types and the prevalence of genotypes, virulent strains, or clinical origins were characterized. The P. multocida isolates identified in this work included 3 major clonal complexes: CC9, CC74, and ST129. CC9 exhibited cpsA(F)L3, and was associated with a higher prevalence of rhinitis; CC74 exhibited cpsAL6, and was associated with higher prevalences of hgbB+pfhA- and pneumonia; ST129 exhibited cpsAL1, and was associated with higher prevalences of high-virulence strains and septicemia. The results provided insights into P. multocida from rabbits in China and suggested the use of strains from different populations in future P. multocida pathogenesis and vaccine studies.

First outbreak of bovine haemorrhagic septicaemia caused by Pasteurella multocida type B in Spain - Short communication.

This paper describes the first documented outbreak of haemorrhagic septicaemia (HS) caused by Pasteurella multocida type B in cattle in Spain. This acute, highly fatal septicaemia causes major economic losses in cattle and buffaloes in many areas of Asia and Africa. In other species and in European countries it is an infrequently reported disease. Acute septicaemic pasteurellosis occurred in a free-range farm of 150 cattle and 70 beef calves in Southern Spain. Twenty-one calves and one cow were affected, of which three calves and the adult cow died. Postmortem examination revealed characteristic oedema in the ventral area of the neck and the brisket region, and widespread haemorrhages in all organs. Pure cultures of P. multocida were obtained from all tissues and organs studied. The aetiological agent was further confirmed by molecular and biochemical analysis as P. multocida capsular type B, biovar 3. Although the source of infection could not be determined, wildlife may play an important role. The use of tulathromycin in the initial stage of the disease might be related to the low morbidity and mortality of this outbreak. After using an autogenous vaccine no more cases of HS were observed.

Using genomics to understand inter- and intra- outbreak diversity of Pasteurella multocida isolates associated with fowl cholera in meat chickens.

Fowl cholera, caused by Pasteurella multocida, continues to be a challenge in meat-chicken-breeder operations and has emerged as a problem for free-range meat chickens. Here, using whole-genome sequencing (WGS) and phylogenomic analysis, we investigate isolate relatedness during outbreaks of fowl cholera on a free-range meat chicken farm over a 5-year period. Our genomic analysis revealed that while all outbreak isolates were sequence type (ST) 20, they could be separated into two distinct clades (clade 1 and clade 2) consistent with difference in their lipopolysaccharide (LPS) type. The isolates from the earlier outbreaks (clade 1) were carrying LPS type L3 while those from the more recent outbreaks (clade 2) were LPS type L1. Additionally, WGS data indicated high inter- and intra-chicken genetic diversity during a single outbreak. Furthermore, we demonstrate that while a killed autogenous vaccine carrying LPS type L3 had been successful in protecting against challenge from L3 isolates it might have driven the emergence of the closely related clade 2, against which the vaccine was ineffective. The genomic results also revealed a 14 bp deletion in the galactosyltransferase gene gatG in LPS type L3 isolates, which would result in producing a semi-truncated LPS in those isolates. In conclusion, our study clearly demonstrates the advantages of genomic analysis over the conventional PCR-based approaches in providing clear insights in terms of linkage of isolate within and between outbreaks. More importantly, it provides more detailed information than the multiplex PCR on the possible structure of outer LPS, which is very important in the case of strain selection for killed autogenous vaccines.

Pathogen-specific risk factors in acute outbreaks of respiratory disease in calves.

Respiratory tract infections (bovine respiratory disease) are a major concern in calf rearing. The objective of this study was to identify pathogen-specific risk factors associated with epidemic respiratory disease in calves. A cross-sectional study was conducted, involving 128 outbreaks (29 dairy, 58 dairy-mixed, and 41 beef) in Belgium (2016-2018). A semiquantitative PCR for 7 respiratory pathogens was done on a pooled nonendoscopic bronchoalveolar lavage sample for each herd. Potential risk factors were collected by questionnaire and derived from the national cattle registration databank. Most outbreaks occurred between October and March, and single and multiple viral infections were detected in 58.6% (75/128) and 13.3% (17/128), respectively. Bovine coronavirus (BCV) was the most frequently isolated virus (38.4%), followed by bovine respiratory syncytial virus (bRSV; 29.4%) and parainfluenzavirus type 3 (PI-3; 8.1%). Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni were detected in 33.3, 41.2, 89.1, and 36.4% of the herds, respectively. Specific risk factors for BCV detection were detection of M. haemolytica [odds ratio (OR) = 2.8 (95% confidence interval = 1.1-7.5)], increasing herd size [OR = 1.3 (1.0-1.8) for each increase with 100 animals] and detection of BCV by antigen ELISA on feces in calves in the last year [OR = 3.6 (1.2-11.1)]. A seasonal effect was shown for bRSV only {more in winter compared with autumn [OR = 10.3 (2.8-37.5)]}. Other factors associated with bRSV were PI-3 detection [OR = 13.4 (2.1-86.0)], prevalence of calves with respiratory disease [OR = 1.02 (1.00-1.04) per 1% increase], and number of days with respiratory signs before sampling [OR = 0.99 (0.98-0.99) per day increase]. Next to its association with BCV, M. haemolytica was more frequently detected in herds with 5 to 10 animals per pen [OR = 8.0 (1.4-46.9)] compared with <5 animals, and in herds with sawdust as bedding [OR = 18.3 (1.8-191.6)]. Also, for H. somni, housing on sawdust was a risk factor [OR = 5.2 (1.2-23.0)]. Purchase of cattle [OR = 2.9 (1.0-8.0)] and housing of recently purchased animals in the same airspace [OR = 5.0 (1.5-16.5)] were risk factors for M. bovis. This study identified pathogen-specific risk factors that might be useful for the development of customized control and prevention and for the design of decision support tools to justify antimicrobial use by predicting the most likely pathogen before sampling results are available.