Pasteurella Isolates Research Articles

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

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. 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. 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%). 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.

In Vitro Antibacterial Activity of Microbial Natural Products against Bacterial Pathogens of Veterinary and Zoonotic Relevance.

Antimicrobial resistance (AMR) is considered one of the greatest threats to both human and animal health. Efforts to address AMR include implementing antimicrobial stewardship programs and introducing alternative treatment options. Nevertheless, effective treatment of infectious diseases caused by bacteria will still require the identification and development of new antimicrobial agents. Eight different natural products were tested for antimicrobial activity against seven pathogenic bacterial species (Brachyspira sp., Chlamydia sp., Clostridioides sp., Mannheimia sp., Mycobacterium sp., Mycoplasma sp., Pasteurella sp.). In a first pre-screening, most compounds (five out of eight) inhibited bacterial growth only at high concentrations, but three natural products (celastramycin A [CA], closthioamide [CT], maduranic acid [MA]) displayed activity at concentrations <2 µg/mL against Pasteurella sp. and two of them (CA and CT) also against Mannheimia sp. Those results were confirmed by testing a larger collection of isolates encompassing 64 Pasteurella and 56 Mannheimia field isolates originating from pigs or cattle, which yielded MIC90 values of 0.5, 0.5, and 2 µg/mL against Pasteurella and 0.5, 4, and >16 µg/mL against Mannheimia for CA, CT, and MA, respectively. CA, CT, and MA exhibited higher MIC50 and MIC90 values against Pasteurella isolates with a known AMR phenotype against commonly used therapeutic antimicrobial agents than against isolates with unknown AMR profiles. This study demonstrates the importance of whole-cell antibacterial screening of natural products to identify promising scaffolds with broad- or narrow-spectrum antimicrobial activity against important Gram-negative veterinary pathogens with zoonotic potential.

Properties of Pasteurella multocida isolated from animals during the seasonal migration of saigas.

The paper describes data from the study of cultural, morphological, and biochemical properties and the pathogenicity and virulence of epizootic isolates of Pasteurella multocida obtained from cattle and saigas. The study aimed to investigate the properties of P. multocida isolated from saigas and cattle during their seasonal migration, with a focus on its role in the epizootic process and potential transmission to farm animals. The research was conducted in a laboratory setting at the West Kazakhstan Agrarian-Technical University. White mice, saigas, and cattle were examined, and pathological and bacteriological analyses were performed on tissues and secretions. Pathogenicity, virulence, and toxigenicity of the isolated Pasteurella cultures were determined through biological tests on white mice. The morphological, cultural, and biochemical properties of the isolates were studied using standard microbiological methods. The study found that P. multocida isolates from both saigas and cattle exhibited high pathogenicity and virulence when tested on white mice. The isolates from sick and dead animals displayed 65.3 and 83.3% pathogenicity, respectively. The isolates were toxic to white mice, with filtrate dilutions showing 100% toxigenicity. Comparative analysis showed morphological and cultural similarities between Pasteurella isolates from saigas and cattle, confirming their identity. This research demonstrates that P. multocida, isolated from both saigas and cattle, contributes to the epizootic process and poses a threat to farm animals. Saigas, in particular, play a role in disease transmission during seasonal migrations. Understanding the ecological interactions between wild and farm animals is crucial for implementing preventive measures to control the spread of infectious diseases, emphasizing the need for comprehensive monitoring and intervention strategies.

Molecular Detection and Genotyping of P. Multocida, M. Hemolytica, and B. Trehalosi Isolates Targeting the Virulence Associated Genes from Ethiopian Cattle and Sheep

Pasteurellaceae families are usually considered opportunistic pathogens which inhabit normal flora on the mucosal membranes of the upper respiratory and the lower genital tracts of mammals and birds. The majority of P. multocida, M. hemolytica, and B. trehalosi isolates are opportunistic animal pathogens and cause disease only under certain conditions. Some of the target genes are shared by isolates found in the same or different genera.. The alpha / beta hydrolase kmt1 genes part of OMP and SLP are shared by all P.multocida species and FbpA is common gene for both P.multocida and M. hemolytica isolates. Other virulence related genes are used for genotyping of P.multocida, M.hemolytica and B.trehalosi isolates. The objective of the current research was focused on the detection and genotyping of different target genes that can be found in the isolates of P. multocida, M. hemolytica, and B. trehalosi using various PCR primers. The distributions of virulence-associated genes were assessed in the isolates of the three genera. Materials and methods A total of eight isolates from P. multocida, M. hemolytica, and B. trehalosi isolated from clinical cases of cattle and sheep and stored at the Bacteriology laboratory of NVI were used as source of samples for the present study. DNeasy® Blood &amp; Tissue Kit (Qiagen, Germany) was used for the extraction of DNA from bacterial isolates. Different primers were used for genotyping of P. multocida, P.hemolytica and B.trehalosi isolates. Results P.multocida was detected using species-specific kmt1 primer with an amplicon product of 460bp. Four serogroups of P.multocida (A, B, D, &amp;E) were detected using serotype-specific primers with amplification products of 1044bp, 760bp, 657bp, and 511bp respectively. PCR was conducted to HS causing P.multocida type B and detected 620 bp amplicons. SLP genes were detected in all P.multocida serotypes with an amplification product size 1400-1553bp. In addition to that, the four P. multocida serotypes were found to be positive for BRD-PmSLP with an amplification product of 460bp but using recombinant HS-SLP primers resulted in the double band at 460 bp and 541bp for serotypes A and D. However, HS causing type B&amp; E serotypes were detected with an amplified product of 541bp. TbpA2 has been detected with an amplicon size of approximately 750 bp for types (A and D) and 1300bp for type B using TbpA-F2/Rev primers. All of the P.multocida serotypes (A, B, D&amp;E) and M. hemolytica (A: 1) were strongly positive for the iron acquisition FbpA gene with amplification bands at 500bp and 1000bp, respectively. A multiplex PCR as-say was carried out for detection of M. haemolytica A: 1 using PHSSA and Rpt2 primers with amplification products of 327pb and 1022 bp respectively. The three isolates of B. trehalosi were identified using BtsodA primers with the result of 144bp amplicon size. Conclusion From this work, we understood that P.multocida and M. haemolytica shared genetic materials like iron binding proteins (FbpA) and some of the target genes (kmt1 and SLP) were commonly found in all isolates of P. multocida. However, TbpA2 was found to be in most but not in all Pasteurella isolates. We also observed that PHSSA and Rpt2 genes were exclusively found in M. hemolytica isolates whereas Bt-sodA was prominent in all B. trehalosi isolates (T3, T4&amp;T15).

Screening of Antimicrobial Activity of Essential Oils against Bovine Respiratory Pathogens - Focusing on Pasteurella multocida.

Administration of essential oils as natural plant products with antimicrobial activity might be an alternative to antibiotic treatment of bovine respiratory disease. The aim of this study was to analyse the in vitro antimicrobial activity of 11 essential oils against Pasteurella multocida isolated from the respiratory tract of calves using microdilution with determination of minimum inhibitory and bactericidal concentration as well as agar disc diffusion. Additionally, antimicrobial activity against Mannheimia haemolytica and bacteria in the Mannheimia clade was assessed by agar disc diffusion. Seven essential oil mixtures were also tested against all bacterial isolates. P. multocida was strongly inhibited by cinnamon cassia and lemongrass oil followed by coriander, winter savory, thyme, clove, and peppermint oil in the microdilution assays. Eucalyptus, wintergreen, spruce, and star anise oil showed lower activity. Comparison of both methods revealed an underestimation of cinnamon cassia oil activity by agar disc diffusion and conflicting results for wintergreen oil in microdilution, which precipitated in broth. Cinnamon cassia, thyme, wintergreen, lemongrass, and winter savory oil all showed strong antimicrobial activity against M. haemolytica . Bacteria in the Mannheimia clade were mostly inhibited by cinnamon cassia and thyme oil. Pasteurella isolates were more susceptible to inhibition by essential oils than Mannheimia isolates. Essential oil mixtures did not show stronger antibacterial activity than single essential oils. In conclusion, cinnamon cassia and lemongrass as well as coriander, winter savory, and thyme oil are promising candidates for treatment of P. multocida -associated bovine respiratory infections.

Antimicrobial activity of essential oils against Pasteurella spp. isolated from the oral cavity of domestic cats

ABSTRACT: Pasteurella spp. have been identified predominantly in the oral microbiota of domestic cats. However, Pasteurella spp. was significantly more prevalent in cats with inflammatory oral disease; and consequently, it was considered as part of the etiology in this disease. In addition, in animals, Pasteurella spp. have become increasingly resistant to a large number of antimicrobials. Natural products, especially essential oils, could contribute to minimizing this issue. This study determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of six essential oils against Pasteurella spp. isolates from the oral cavity of domestic cats. Our results showed that essential oils with better antimicrobial effectiveness against most of the Pasteurella isolates were lemongrass, tea tree and clove, with inhibition values between 50 to 800 µg mL-1. All essential oils showed bacteriostatic activity against the species of Pasteurella isolated from the domestic cats. These results suggested that lemongrass, tea tree and clove oils have potential to be used in products for oral hygiene and treatment of oral infections in domestic cats.

Antimicrobial Resistance in Bacteria Isolated From Cats and Dogs From the Iberian Peninsula.

Pet animals are assumed to be potential reservoirs in transferring antimicrobial resistance (AMR) to humans due to the extensively applied broad-spectrum antimicrobial agents and their close contact with humans. In this study, microbiological data and antimicrobial susceptibility results of dog (n = 5,086) and cat (n = 789) clinical samples from a private Laboratory of Diagnosis in Barcelona were analyzed. Samples came from different counties of the Iberian Peninsula during 2016–2018. In dogs, clinical samples were most commonly from otitis, and in cats from wounds, respiratory tract infections and conjunctivitis. In both pet groups, Staphylococcus spp. (31% in dogs vs 30% in cats), Streptococcus spp. (19% vs 17%), Pseudomonas spp. (16% vs 10%), Escherichia coli (8% vs 5.6%), and Enterococcus spp. (5.5% vs 6.8%) were shown as the most predominant bacteria. However, higher frequencies of P. aeruginosa, P. canis, and S. pseudintermedius were found in dogs, while S. aureus and P. multocida were more prevalent in cats. The antimicrobial susceptibility testing demonstrated that Enterococcus spp. and Pseudomonas spp. presented the highest levels of AMR in both dogs and cats. Within the Enterobacteriaceae, E. coli showed low levels of AMR compared to Klebsiella, Proteus, or Enterobacter spp. Respiratory tract infections caused by K. pneumoniae presented higher AMR in cats. By contrast, Pasteurella isolates from the respiratory tract were highly sensitive to all the antimicrobials in cats and dogs. Data from this study could be used to guide empirical antimicrobial selection in companion animal veterinary practices in the Iberian Peninsula.

Prevalence and antibiotic susceptibility of Mannheimia haemolytica and Pasteurella multocida isolated from ovine respiratory infection: A study from Karnataka, Southern India.

Background and Aim:Respiratory infection due to Mannheimia haemolytica and Pasteurella multocida are responsible for huge economic losses in livestock sector globally and it is poorly understood in ovine population. The study aimed to investigate and characterize M. haemolytica and P. multocida from infected and healthy sheep to rule out the involvement of these bacteria in the disease.Materials and Methods:A total of 374 healthy and infected sheep samples were processed for isolation, direct detection by multiplex PCR (mPCR), and antibiotic susceptibility testing by phenotypic and genotypic methods.Results:Overall, 55 Pasteurella isolates (27 [7.2%] M. haemolytica and 28 [7.4%] P. multocida) were recovered and identified by bacteriological tests and species-specific PCR assays. Significant correlation between the detection of M. haemolytica (66.6%) with disease condition and P. multocida (19.1%) exclusively from infected sheep was recorded by mPCR. In vitro antibiotic susceptibility testing of 55 isolates revealed higher multidrug resistance in M. haemolytica (25.9%) than P. multocida (7.1%) isolates. Descending resistance towards penicillin (63.6%), oxytetracycline (23.6%), streptomycin (14.5%), and gentamicin (12.7%) and absolute sensitivity towards chloramphenicol were observed in both the pathogens. The antibiotic resistance genes such as strA (32.7%) and sul2 (32.7%) associated with streptomycin and sulfonamide resistance, respectively, were detected in the isolates.Conclusion:The study revealed the significant involvement of M. haemolytica together with P. multocida in ovine respiratory infection and is probably responsible for frequent disease outbreaks even after vaccination against hemorrhagic septicemia in sheep population of Karnataka, southern province of India.

Oral flora of stray dogs and cats in Algeria: Pasteurella and other zoonotic bacteria.

Background and Aim:Knowledge of potentially pathogenic bacteria presents in the oral cavity of dogs and cats may be helpful in determining appropriate treatment for infected bite wounds. About 120.000 people are exposed to dog and cat bites every year in Algeria, but little is known about the dog and cat oral flora causing bite wound complications. The purpose of this study was to identify potential zoonotic bacteria from oral cavity of dogs and cats and to determine their susceptibility to antibiotics to contribute to the treatment of bite wound infection.Materials and Methods:Oral swabs from 100 stray dogs and 100 stray cats were collected and cultured in several media: Chocolate agar, MacConkey agar, and Mannitol Salt Agar. Bacterial isolates were identified using several commercial kits of the analytical profile index and tested for antibiotic susceptibility by disk diffusion method.Results:Overall, 185/200 (92.5%) dogs and cats carried zoonotic bacteria in their mouths, of which 55.13% (102/185) had at least two bacterial pathogens. 374 pathogenic strains belonging to 15 genera were isolated: Eleven were Gram-negative (Proteus, Pasteurella, Escherichia, Moraxella, Klebsiella, Acinetobacter, Enterobacter, Pseudomonas, Aeromonas, and Neisseria Haemophilus) and four were Gram-positive (Staphylococcus, Streptococcus, and Corynebacterium, Bacillus). Fifty-one strains of Pasteurella were isolated from 44 carriers of Pasteurella (21 Pasteurella multocida, 21 Pasteurella pneumotropica, and 9 Pasteurella spp.). Pasteurella strains were tested for antibiotic resistance. Resistance to at least one drug was observed in 8 (15.68%) of Pasteurella isolates and two strains (3.92%) were found to be multidrug-resistant (to two or more drugs). Erythromycin, penicillin, and ampicillin were the antimicrobials to which the isolates showed greater resistance (7.84%, 5.88%, and 3.92%, respectively).Conclusion:To the best of our knowledge, this study is the first in Algeria to detect potential human pathogenic bacteria in the oral cavity of dogs and cats. It reveals that these animals have multiple zoonotic bacteria in their mouths including Pasteurella species, which may be multidrug-resistant.

SOME MICROBIOLOGICAL STUDIES ON PASTEURELLA MULTOCIDA IN RABBITS

To establish this goal, a total of 120 samples of blood and nasal swabs (60 samples from apparently healthy rabbits and 60 samples from diseased ones) were collected from different farms scattered at different localities in Sohag governorate. All samples were submitted for bacteriological examination by conventional culture method. Recovery of pasteurella isolates from apparently healthy and diseased rabbits were (16), (37) isolates respectively, with isolation rate of (26.7%), (61.7%) respectively and total isolation rate of (44.2%). Isolation of pasteurella, colonial and cellular morphological pictures were studied and recorded Biochemical reaction proved that only 15(28.3%) of isolates had typical biochemical properties of pasteurella multocida. Serotyping of (15) pasteurella isolates by using ELISA test revealed that only 13(86.7%) out of 15 pasteurella isolates were positive for ELISA test. The capsular serotyping of the selected (13) Pasteurella multocida isolates were found to be belong to capsular serotype (A). The somatic serotyping proved that (8 isolates) were belonged to serotype 3 with a percentage of (53.3%) followed by (4 isolates) were belonged to serotype 12 with a percentage of (26.7%) and (one isolate) was belonged to serotype 1 with a percentage of (6.7%). The recovered isolates were submitted for molecular identification of KMT1 gene by PCR. Only 14 (93.3%) out of (15) isolates were positive to KMT1 gene and confirmed to be Pasteurella multocida. The occurrence of Pasteurella multocida of examined samples of apparently healthy rabbits and diseased ones were (8.3%) and (15%) respectively.

Identification of clinical Pasteurella isolates by MALDI-TOF — a comparison with VITEK 2 and conventional microbiological methods

The aim of this study was to compare the performance of four methods that are widely used in the clinical microbiology laboratory for identification of Pasteurella species. The 4 methods evaluated were VITEK2, VITEK MS (BioMerieux), and Bruker Biotyper MS (Bruker) as well as traditional biochemical tests. Sequencing of the sodA gene was used as the reference method. Sixty-five isolates of Pasteurella spp. from 65 patients were analyzed. One Pasteurella multocida isolate from American Type Culture Collection (Manassas, VA, USA) was used as a reference. Traditional biochemical tests accurately identified 62/66 (94%) isolates. Both Bruker and Vitek matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) identified 59/66 (89%) strains, but VITEK2 could only identify 32/66 (48.5%) isolates correctly. The mean time to identification using biochemical tests was 20 hours; VITEK2 took 6 hours and MALDI-TOF approximately 10 minutes. In conclusion, MALDI-TOF is a quick method, which accurately identified most isolates of Pasteurella to the species level. Thus, MALDI-TOF constitutes a valuable diagnostic tool in the clinical laboratory.

A study on Ovine pneumonic pasteurellosis: Isolation and Identification of Pasteurellae and their antibiogram susceptibility pattern in Haramaya District, Eastern Hararghe, Ethiopia

BackgroundSheep constitute the second major component of livestock in Ethiopia. However, efficient utilization of this potential resource is hampered by combination of health problems, poor management and feed shortage. Haramaya district is one of the remote settings in Ethiopia where information about the livestock disease is not well documented. Hence this study was conducted to determine the causative agents and their antimicrobial susceptibility pattern of bacterial Pasteurella isolates among pneumonic ovine in Haramaya district, Eastern Hararghe, Ethiopia.ResultsOut of 256 samples examined, Pasterurella was isolated in 64 (25%), of which 38 (59.4%) were from lungs and 26 (40.6%) were from nasal cavities. 87.5% of the isolates were Mannheimia haemolytica and 12.5% were Pasteurella multocida. All of the isolates from the lungs were Mannheimia haemolytica whereas 69% of the isolates from nasals cavities were Mannheimia haemolytica. Age and body temperature were significantly associated with Pasteurella isolates from clinic (P < 0.05). Despite diverse in the site of origins, the isolates exhibited uniformity in sensitivity to a majority of the antibacterial agents. The most effective drug was Cholramphenicol (100%) followed by Sulfamethoxazole (89.1%) and Tetracycline (84.4%). Both species were completely resistant to Gentamycin and Vancomycin.ConclusionMannheimia haemolytica is the most common cause of ovine pneumonic pasteurellosis in the study area. The isolates were susceptible to limited antimicrobial agents. Therefore, the antimicrobial susceptibility test should be conducted before treatment, except for critical cases.

Detection of Pasteurella multocida isolates from local pigs of India by polymerase chain reaction and their antibiogram

Pasteurella multocida has been recognized as an important veterinary pathogen for over a century. Conventional methods for diagnosis of pasteurellosis rely on the detection of the organism by microscopy and its isolation and identification. However, as far as pasteurellosis is concerned, it is not just sufficient to know the identity of the organism. To constitute effective control measures, it is important to know the serotype of the organism. A study was undertaken to characterize the Pasteurella isolates from local pigs in India with clinical respiratory disease by determination of their capsule types and presence or absence of toxin gene. Pasteurella could be isolated from 66.70% of pigs with clinical respiratory disease. All the isolates were confirmed through biochemical characterization and P. multocida-specific polymerase chain reaction. It has also been observed that all the isolates belonged to capsular type D. All the isolates were sensitive to chloramphenicol, chlortetracycline, doxycycline, and enrofloxacin, while the rest of the antibiotics were less effective. It has also been observed that all isolates were resistant to cephalexin, penicillin G, and sulphadiazine. The study revealed the detection of P. multocida serotype D from clinical respiratory diseases of local pigs of India, which could be one of the important respiratory tract pathogens responsible for mortality of local pigs in India.

Isolation, Antimicrobial Resistance, and Virulence Genes of Pasteurella multocida Strains from Swine in China

A total of 233 isolates of Pasteurella multocida were obtained from 2,912 cases of clinical respiratory disease in pigs in China, giving an isolation rate of 8.0%. Serogroup A P. multocida isolates were isolated from 92 cases (39.5%), and serogroup D isolates were isolated from 128 cases (54.9%); 12 isolates (5.2%) were untypeable. P. multocida was the fourth most frequent pathogenic bacterium recovered from the respiratory tract, after Streptococcus suis, Haemophilus parasuis, and Escherichia coli. All isolates were characterized for their susceptibilities to 20 antibiotics and the presence of 19 genes for virulence factors (VFs). The frequency of antimicrobial resistance among P. multocida isolates from swine in China was higher than that reported among P. multocida isolates from swine in from other countries, and 93.1% of the isolates showed multiple-drug resistance. There was a progressive increase in the rate of multiresistance to more than seven antibiotics, from 16.2% in 2003 to 62.8% in 2007. The resistance profiles suggested that cephalosporins, florfenicol, and fluoroquinolones were the drugs most likely to be active against P. multocida. Use of PCR showed that colonization factors (ptfA, fimA, and hsf-2), iron acquisition factors, sialidases (nanH), and outer membrane proteins occurred in most porcine strains. The VFs pfhA, tadD, toxA, and pmHAS were each present in <50% of strains. The various VFs exhibited distinctive associations with serogroups: concentrated in serogroup A, concentrated in serogroup D, or occurring jointly in serogroups A and D. These findings provide novel insights into the epidemiological characteristics of porcine P. multocida isolates and suggest that the potential threat of such multiresistant bacteria in food-producing animals should not be neglected.

Tet(L)-mediated tetracycline resistance in bovine Mannheimia and Pasteurella isolates

Tetracycline-resistant Mannheimia and Pasteurella isolates, which were negative for the tetracycline resistance genes (tet) commonly detected among these bacteria, were investigated for other tet genes present and their location. Mannheimia and Pasteurella isolates were investigated for their MICs of tetracycline and their plasmid content. Identification of tet genes was achieved by PCR. Plasmids mediating tetracycline resistance were identified by transformation and hybridization experiments. Plasmid pCCK3259 from Mannheimia haemolytica was sequenced completely and analysed for its structure and organization. All tetracycline-resistant isolates carried the gene tet(L) either on plasmids or on the chromosome. Two M. haemolytica isolates and one Mannheimia glucosida isolate harboured a common 5.3 kb tet(L) plasmid, designated pCCK3259. This plasmid was similar to the tet(B)-carrying tetracycline resistance plasmid pHS-Tet from Haemophilus parasuis and the streptomycin/spectinomycin resistance plasmid pCCK647 from Pasteurella multocida in the parts coding for mobilization functions. The tet(L) gene was closely related to that of the Geobacillus stearothermophilus plasmid pTB19. However, the translational attenuator responsible for the tetracycline-inducible expression of tet(L) was missing in plasmid pCCK3259. A recombination site was identified downstream of tet(L), which might explain the integration of the tet(L) gene region into a basic pCCK3259 replicon. A tet(L) gene was shown for the first time to be responsible for tetracycline resistance in Mannheimia and Pasteurella isolates. This report demonstrates a lateral transfer of a tetracycline efflux gene in Gram-negative bovine respiratory tract pathogens, probably originating from Gram-positive bacteria.

Identification of a truncated, but functionally active tet(H) tetracycline resistance gene in Pasteurella aerogenes and Pasteurella multocida

Molecular analysis of Pasteurella isolates of animal origin for plasmid-encoded tetracycline resistance genes identified a common tet(H)-carrying plasmid of 5.5 kbp in a single isolate of Pasteurella aerogenes and six isolates of Pasteurella multocida. This plasmid carried a truncated Tn 5706 element in which one of the IS elements, IS 1596, was lost completely and of the other, IS 1597, only a relic of 84 bp was left. Sequencing of the resistance gene region and the flanking areas revealed the presence of a deletion in the 3′ end of the tet(H) gene which shortened the tet(H) reading frame by 24 bp. The amino acid sequence of the respective TetH protein comprised only 392 amino acids. Despite this deletion, the tet(H) gene conferred high level tetracycline resistance not only to the original Pasteurella isolates but also to the respective Escherichia coli JM107 and C600 transformants as confirmed by MIC determination. The deletion was probably the result from recombinational events. Two possible recombination sites involved in the deletion of tet(H) and that of IS 1597 were identified. Macrorestriction analysis of the Pasteurella isolates carrying plasmid pPAT1 confirmed horizontal and vertical transfer of this plasmid.

The immunogenicity and pathogenicity of Pasteurella multocida isolated from poultry in Indonesia

Of 13 field isolates of Pasteurella from chickens and ducks in Indonesia, 10 were confirmed as P. multocida subspecies multocida, one as P. multocida subspecies gallicida and one as P. multocida subspecies septica. Nine were capsular Type A four were Serotype 1, one was Serotype 4, one was Serotype 11, one was Serotypes 4,12, and the remaining six were untypable. Five isolates were pathogenic for mice and two were pathogenic for chickens. Both a trivalent vaccine which included local field isolates and an imported commercial vaccine, were efficacious in layer chickens against challenge with virulent reference and local field strains. Though not statistically significant, the protection provided by the trivalent vaccine against virulent field isolate challenge was slightly better and could provide an improvement over the currently used imported vaccine although further field trials are required. A bacterin vaccine produced from a Serotype 1 field isolate grown in the allantoic sac of embryonated chicken eggs provided chickens with good cross protection against heterologous serotype challenge.

Population structure and diversity of avian isolates of Pasteurella multocida from Australia.

A total of 110 isolates of Pasteurella multocida from Australian poultry and reference strains for the 16 somatic serovars plus the three subspecies (gallicida, multocida, septica) were analysed to examine their population structure and diversity. The 81 field isolates examined by multilocus enzyme electrophoresis (MLEE) were diverse, being divided into 56 electrophoretic types (ETs), with the 19 reference strains in another 15 ETs. The population was clonal and somatic serotyping was not particularly useful in establishing relationships between isolates. The 71 ETs formed three distinct subclusters (A, B and C) at a genetic distance of 0.36. Biovars tended to be associated with these subclusters: A with biovars 1, 3, 4, 5 and 8 and B with biovars 2, 6, 7, 9 and 10. Ribotyping, performed on all 110 isolates using Hpall, recognized 21 ribotypes forming nine clusters (R1-R9). The isolates in ribotype cluster R1 were almost identical to those in MLEE cluster B. Using both MLEE and ribotyping, the 19 non-Australian reference strains were found to be distributed over the full diversity of the Australian isolates of P. multocida. This study has shown that a range of P. multocida clones are associated with fowl cholera in Australia and that many of the Australian isolates are similar to non-Australian reference strains. Both the MLEE results and the ribotyping data identified a previously unrecognized subset of P. multocida strains.

Serotyping and Enzyme Characterization of Pasteurella haemolytica and Pasteurella multocida Isolates Recovered from Pneumonic Lungs of Stressed Feeder Calves

Ninety-one isolates of Pasteurella multocida (Pm)and 124 of Pasteurella haemolytica (Ph) were recovered from the lungs of calves that died of bovine respiratory tract disease (BRTD). Nine Pm enzyme profiles (A through I) and 9 Ph enzyme profiles (J through R) were determined for the Pasteurella isolates. The Pm isolates were relatively evenly divided among the enzyme profiles, with one exception, profile I. The Ph isolates were not evenly distributed among the profiles. Fifty of the 91 Pm isolates were serotyped. Forty-two Pm isolates were positive for capsule type A, and 8 were untypable. Five somatic type antigen profiles (3; 3,4; 3,7; 3,4,7; and 4) were identified among the 50 serotyped Pm isolates; one isolate was untypable. The Ph isolates were further divided through serotyping and grouped as follows: 74 (60%) Pasteurella haemolytica A1 (PhA1), 12 (10%) PhA2, 4 (3%) PhA5, and 34(27%) PhA6. Eighty-one percent of the Ph serotypes were clustered in the M and N enzyme profile. The P enzyme profile was almost unique to PhA2 (8 of 12, 67% of PhA2 isolates). Results of this study indicate a need to collect more data on Ph serotypes at the state veterinary diagnostic laboratories.

Isolation of Pasteurella spp. from free-ranging American bison (Bison bison).

From November 1991 through March 1992, nasal and pharyngeal swab samples were collected from 45 bison (Bison bison) from Yellowstone National Park, Montana (USA) and cultured for Pasteurella spp. Thirteen isolates of Pasteurella spp. were recovered from 10 (22%) of the animals. Ten isolates were from pharyngeal samples in contrast to three isolates from nasal samples. Pasteurella haemolytica (six biotype T, two biotype A, and two biotype 3) was the predominant Pasteurella species. Five biotype T isolates were serotype 4 and the sixth agglutinated in antisera 3, 4, and 10. Both biotype A isolates were untypable with antisera to recognized type strains. Pasteurella multocida was isolated from the pharyngeal samples of one animal. Two isolates could not be identified to species.