Co-migrating and shared antigens of selectedPasteurella haemolytica untypable strains

Bacterial cell envelope preparations from eight untypable strains ofPasteurella haemolytica were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting with rabbit antisera prepared against the eight untypable strains (one untypable strain per rabbit) and with cattle antisera prepared againstP. haemolytica serotypes 1, 2, 5, 6, 9 and against one heterologous untypable strain. Numerous comigrating and shared antigens were recognized by the eight rabbit antisera and theP. haemolytica serotype cattle antisera. Comigrating antigens at 43 and 30 kilodaltons (kDa) were recognized by all eight rabbit antisera. Shared antigens, detected by all eight rabbit antisera when reacting againstP. haemolytica serotype 1, were recognized at 43, 32, 30, 20 and 15 kDa.     

Serological and molecular comparison of Mannheimia haemolytica and Pasteurella trehalosi strains isolated from wild and domestic ruminants in the French Alps

Over a period of 17 years, 84 bacterial isolates identified as Mannheimia haemolytica or M. glucosida, and 52 isolates identified as Pasteurella trehalosi were detected in the lungs of domestic and wild ruminants in the French Alps. The isolates were serotyped according to their surface capsular antigens, and those sharing common antigens were further characterized by pulsed field gel electrophoresis. The results showed that the bacterial isolates included in the study clustered according to the host species from which they were isolated. These findings indicate that the transmission of serotypes of M. haemolytica, M. glucosida or P. trehalosi from an animal host in which they are common to another species sharing the same geographical space may be a rare epidemiological event.     

Effects of Mannheimia haemolytica leukotoxin on apoptosis and oncosis of bovine neutrophils

OBJECTIVE: To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosis and oncosis in bovine neutrophils and to examine the role of calcium ions (Ca2+) in LKT-induced apoptosis. SAMPLE POPULATION: Neutrophils isolated from blood samples obtained from healthy calves. PROCEDURE: Neutrophil suspensions were exposed to lytic or sublytic dilutions of LKT and then examined by use of transmission electron microscopy (TEM) or gel electrophoresis. Contribution of extracellular Ca2+ to LKT-induced apoptosis was investigated by incubating neutrophils with LKT or control solutions in buffer containing 1 mM CaCl2 or in Ca2+-free buffer containing 1 mM ethylene glycol-bis (b-aminoethyl ether)-N,N-tetraacetic acid (EGTA) prior to diphenyl amine analysis. RESULTS: Examination by TEM revealed that bovine neutrophils exposed to lytic dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apoptosis, had changes consistent with apoptosis. Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of extracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributable to the calcium ionophore A23187, but it did not have significant effects on apoptosis induced by LKT or staurosporin. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of LKT to cause apoptosis instead of oncosis is concentration-dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, depending on the LKT concentration in pneumonic lesions. Furthermore, although Ca2+ promotes A23187-induced apoptosis, it is apparently not an essential second messenger for LKT-induced apoptosis.     

Diversity of Mannheimia haemolytica and Pasteurella trehalosi Serotypes from Apparently Healthy Sheep and Abattoir Specimens in the Highlands of Wollo,North East Ethiopia

The prevalence and serotypic diversity of Mannheimia [Pasteurella] haemolytica and Pasteurella trehalosi from nasal swabs, sera and abattoir specimens from sheep in the highlands of Wollo, North East Ethiopia was investigated. Prevalence rates of 83% and 75% of these microorganisms were found in the serum samples and nasal swabs, respectively, from apparently healthy sheep. In a local abattoir, 205 lungs were investigated, 34% of which showed pneumonia, from which samples were collected from 51 lungs and the same number of corresponding tonsils. Mannheimia and Pasteurella species were isolated from 59% of these pneumonic lungs and 69% of the respective tonsils. M. haemolytica serotypes accounted for 41 (59%) and P. trehalosi for 11 (32%) of the isolates from the abattoir specimens. The majority (67%) of isolates from nasal swabs were P. trehalosi, M. haemolytica being isolated f rom 4 (13%) of the swabs. M. glucosida was isolated only from the tonsils. The predominant serotypes of the isolates from both the nasal swabs and the abattoir specimens were M. haemolytica A1 (17%) and P. trehalosi T4 (16%) and T3 (13%). P. trehalosi T15 was less commonly encountered, while M. haemolytica A9 and A13 were not isolated. Studies on sera from 100 sheep indicated that antibodies against M. haemolytica serotype A1 (14%) were most common, followed by A5 and A8 (each 10%) and A9 and P. trehalosi T3 (each 9%) and T4 (8%). Antibodies against M. glucosida or serotype A11 occurred in 2% of the sera. Multiple serotypes were common in all types of samples. The importance of including in vaccines the most prevalent serotypes involved in the pneumonia of sheep in the area is discussed.     

Novel hemotropic Mycoplasma species in white-tailed deer (Odocoileus virginianus)

Globally, hemotropic Mycoplasma spp. are emerging or re-emerging zoonotic pathogens that affect livestock, wildlife, companion animals, and humans, potentially causing serious and economically important disease problems. Little is known about hemotropic Mycoplasma spp. prevalence, host-specificity, or route of transmission in most species, including wildlife. DNA amplification by PCR targeting the 16SrRNA and the RNaseP genes was used to establish the presence and prevalence of hemotropic Mycoplasma spp. in a white-tailed deer (O. virginianus) population in eastern North Carolina. Sixty-five deer (89%) tested positive for hemotropic Mycoplasma spp. where sequence analysis of the 16SsRNA and the RNaseP genes indicated the presence of at least three distinct species. This study represents the first detection of three distinct hemotropic Mycoplasma species in white-tailed deer and the first report of two novel hemotropic Mycoplasma species.     

Mannheimia haemolytica serotype A1 exhibits differential pathogenicity in two related species, Ovis canadensis and Ovis aries

Mannheimia haemolytica causes pneumonia in both bighorn sheep (BHS, Ovis canadensis) and domestic sheep (DS, Ovis aries). Under experimental conditions, co-pasturing of BHS and DS results in fatal pneumonia in BHS. It is conceivable that certain serotypes of M. haemolytica carried by DS are non-pathogenic to them, but lethal for BHS. M. haemolytica serotypes A1 and A2 are carried by DS in the nasopharynx. However, it is the serotype A2 that predominantly causes pneumonia in DS. The objectives of this study were to determine whether serotype A1 exhibits differential pathogenicity to BHS and DS, and to determine whether leukotoxin (Lkt) secreted by this organism is its primary virulence factor. Three groups each of BHS and DS were intra-tracheally administered either 1 x 10(9)cfu of serotype A1 wild-type (lktA-Wt group), Lkt-deletion mutant of serotype A1-(lktA-Mt group), or saline (control group), respectively. In the lktA-Wt groups, all four BHS died within 48h while none of the DS died during the 2-week study period. In the lktA-Mt groups, none of the BHS or DS died. In the control groups, one DS died due to an unrelated cause. Necropsy and histopathological findings revealed that death of BHS in the lktA-Wt group was due to bilateral, fibrinohemorrhagic pneumonia. Although the A1-Mt-inoculated BHS were clinically normal, on necropsy, lungs of two BHS showed varying degrees of mild chronic pneumonia. These results indicate that M. haemolytica serotype A1 is non-pathogenic to DS, but highly lethal to BHS, and that Lkt is the primary virulence factor of M. haemolytica.     

Identification of Pasteurella multocida isolates of ruminant origin using polymerase chain reaction and their antibiogram study

A total of 100 isolates of Pasteurella multocida from various ruminant species (cattle, buffalo and sheep) belonging to different parts of country were identified using Pasteurella multocida-PCR (PM-PCR) and capsular PCR assays. PM-PCR revealed an amplicon of approximately 460 bp in all the isolates tested. As regards capsular PCR, 36 of 38 cattle isolates and 30 of 34 buffalo isolates were found to belong to capsular serogroup B whereas rest of the cattle and buffalo isolates belonged to serogroup A of P. multocida. In case of sheep, a total of 26 out of 28 isolates were positive for serogroup A specific PCR while remaining 2 amplified a PCR product specific for serogroup F of P. multocida. All the isolates were subjected to antibiotic sensitivity testing using 17 different antibiotics. Enrofloxacin was found to be most potent antibiotic as it was effective against 94% of the isolates followed by ofloxacin (93%), chloramphenicol (93%), doxycycline (89%), tetracycline (86%) and ciprofloxacin (84%). Vancomycin, bacitracin and sulfadiazine were ineffective against P. multocida isolates showing 84%, 75% and 82% resistance, respectively. Further, the antibiogram also revealed the development of resistance against multiple drugs among various isolates of the organism.     

Pasteurella haemolytica leukotoxin: chemiluminescent responses of peripheral blood leukocytes from several different mammalian species to leukotoxin- and opsonin-treated living and killed Pasteurella haemolytica and Staphylococcus aureus

A luminol-dependent chemiluminescence (LDCL) assay was used to assess the response of polymorphonuclear leukocyte (PMN) preparations from 4 species of ruminants (ie, cattle, sheep, goats, and antelopes) and 6 species of nonruminants (ie, swine, dogs, cats, rabbits, horses, and persons) to both opsonized and nonopsonized preparations of living and heat-killed Pasteurella haemolytica and Staphylococcus aureus and to opsonized and nonopsonized heat-killed strains of each bacterium in the presence of sterile culture supernatant (leukotoxin) from P haemolytica. The LDCL responses of PMN preparations from each of the species studied were greater for living than for heat-killed S aureus. The most efficient LDCL emission was observed with reaction mixtures containing opsonized living S aureus. Regardless whether they contained killed or living bacteria, the opsonized S aureus preparations elicited LDCL emissions more efficiently than did the corresponding nonopsonized preparations. Living P haemolytica cells and their sterile culture supernatant inhibited the LDCL emissions of phagocytically stimulated PMN preparations from ruminants, but not those from nonruminants. The LDCL response of ruminant PMN to nonopsonized living P haemolytica was characterized by the development of a peak response at 10 minutes of incubation followed by a precipitous decrease and a subsequent complete cessation of chemiluminescence. The peak LDCL response was higher for opsonized living P haemolytica than for nonopsonized living bacteria, and the increased response lasted longer. However, opsonization of living P haemolytica with the serum samples tested only temporarily spared the ruminant PMN preparations from the detrimental effects of leukotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Pasteurella haemolytica A1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals.

To further define the role of Pasteurella haemolytica A1 leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis, its in vitro effects on bovine neutrophils were investigated. Leukotoxin-containing culture supernatant, from P. haemolytica, stimulated a neutrophil respiratory burst as measured by the generation of oxygen-derived free radicals O2- and H2O2. This effect was immediate because preincubation of neutrophils with the culture supernatant for 5 min or longer substantially suppressed this respiratory burst. This suppression was due to cytolysis of the neutrophils. Prolonged incubation of neutrophils with the same culture supernatant caused further cytolysis and degranulation. Heat-inactivated P. haemolytica culture supernatant that had lost its cytotoxic properties failed to stimulate respiratory burst by neutrophils. Furthermore, the respiratory burst, cytolysis and degranulation were abrogated only by leukotoxin-neutralizing monoclonal and polyclonal antibodies, but not by antibodies against the lipopolysaccharide. These studies show that the leukotoxin component in the culture supernatant was responsible for the generation of oxygen-derived free radicals and proteolytic enzymes from neutrophils which may participate in direct lung injury.     

Preliminary evaluation of exotic tick species and exotic pathogens imported on migratory birds into the British Isles

Field studies were carried out to determine whether ticks are being imported into the British Isles on migratory birds. During spring and autumn migration 2004, ticks were collected from ringed birds at 11 bird observatories and 3 inland Riparia riparia colonies. A total of 38 ticks of 4 species (Ixodes ricinus, I. frontalis, I. lividus, I. arboricola) were collected from 12 species of bird. Ticks were tested for viruses in the Flavivirus and Nairovirus genera, with no positives found. This data demonstrates that ticks are being imported into the British Isles on migratory birds with future work recommended to determine the quantity of ticks imported and to detect low prevalence pathogens.     

The effects of stressful exercise on leukocytes in cattle with experimental pneumonic pasteurellosis

Seven yearling bulls were treated with stressful exercise and intrabronchial Pasteurella haemolytica A1. Group 1 bulls (nos. 1–4) underwent treadmill exercise and, 24 days later, intrabronchial instillation of P. haemolytica A1. Group 2 bulls (nos. 5–7) underwent treadmill exercise, followed 30 min later by intrabronchial P. haemolytica A1. Blood lactic acid values were raised (p<0.05) by treadmill exercise only, but plasma cortisol was raised (p<0.05) by treadmill exercise and by P. haemolytica A1 infection. Neutrophils in bronchoalveolar lavage (BAL) differed from control values 24 h after treadmill exercise, and 1 h and 4 h after P. haemolytica A1 infection.Respiratory disease was more severe and the gross lung lesions were larger in group 2 bulls than in group 1 bulls. P. haemolytica A1 was recovered from the livers, spleens and mesenteric lymph nodes of group 2 but not group 1 bulls, suggesting that group 2 bulls had experienced bacteraemia. Decreased neutrophils in BAL fluid from group 2 bulls at 1 h and 4 h after infection suggests that exercise transiently inhibited neutrophil egress from the blood to the alveoli; BAL neutrophils peaked at 1 h and 4 h after infection in group 1 bulls but declined at 24 h. We conclude that group 2 bulls were made more susceptible to experimental pneumonic pasteurellosis by stressful exercise.Abbreviations ADCC antibody dependent, cell-mediated cytotoxicity - AM alveolar macrophages - BAL bronchoalveolar lavage - CFU conlony-forming units     

Salmonella in free-living exotic and native turtles and in pet exotic turtles from SW Spain

We screened 78 native and 94 exotic turtles from natural ponds and 39 exotic pet turtles for presence of Salmonella, resulting with infection rates of 6.61%, 6.4%, and 5.1%, respectively. Concurrent shedding of multiple serotypes of the bacteria was only detected in one pet turtle. Eleven isolates were obtained in free-living turtles, including serotypes commonly found in reptiles and also the serotype Typhimurium, which is commonly related to human infections. In pet turtles, the five serotypes isolated were different to those isolated in free-living turtles and had been reported to cause reptile-associated salmonellosis in humans. These results confirm the risk of transmission of Salmonella from free-living and pet turtles to humans, demanding the necessity of regulation of pet turtle trade in Europe.     

Enhancement of the increase in intracellular calcium concentration in stimulated neutrophils byMycoplasma hyopneumoniae

Neutrophils isolated from the peripheral blood of pigs free of infection withMycoplasma hyopneumoniae were loaded with a fluorescent indicator (Fura-2) for detection of cytosolic free calcium concentration. The kinetics of the intracellular calcium flux were examined after incubation with or without a pathogenic or a non-pathogenic strain ofM. hyopneumoniae. The basal intracellular calcium concentration was not altered by incubation withM. hyopneumoniae. However, the relative increase in cytoplasmic calcium concentration caused by the addition of opsonized zymosan was significantly (p<0.05) higher in neutrophils incubated withM. hyopneumoniae as compared to neutrophils not incubated withM. hyopneumoniae. Additionally, after zymosan stimulation, the intracellular calcium concentration was greater in neutrophils incubated with a pathogenic strain ofM. hyopneumoniae than in those incubated with a non-pathogenic strain. This suggests thatM. hyopneumoniae alters the signal transduction mechanisms in neutrophils and that this alteration may be related to virulence.Abbreviations [Ca]_i intracellular concentration of calcium - CCU colour changing units/ml - Fura-2/AM pentaacetoxymethyl ester - PBS phosphate-buffered saline, pH 7.2 - TNF tumour necrosis factor     

猪源多杀性巴氏杆菌荚膜分型及外膜蛋白H基因序列分析

为了解国内猪源多杀性巴氏杆菌外膜蛋白H基因的变异情况及与荚膜型之间的相关性,本试验采用PCR方法对44株猪源多杀性巴氏杆菌进行荚膜分型和ompH基因的扩增测序。结果显示,44株菌株中22株为荚膜A型,17株为荚膜B型,5株为荚膜D型;44株菌株的ompH基因开放阅读框在1 002~1 056bp之间;SignaIP 4.1预测结果显示,信号肽为N端20个氨基酸残基;ProtParam分析结果显示,成熟蛋白氨基酸残基数量在313~331aa之间,推测的分子质量在33.83~36.46ku之间。序列分析结果显示,44株菌株核苷酸同源性为86.2%~100.0%,氨基酸同源性为86.0%~100.0%;ompH基因核苷酸序列遗传进化树结果显示,荚膜A型、B型和D型菌株分别在不同的分支。试验结果表明,猪源多杀性巴氏杆菌ompH基因在不同血清型之间具有较高的同源性,与荚膜型之间存在相关性。     

REP-PCR Analysis of Pasteurella multocida Isolates from Wild and Domestic Animals in India

Repetitive extragenic palindromic sequence-based PCR (REP-PCR) was used to characterize 67 field isolates of Pasteurella multocida originating from different animal species and geographical regions of India. REP-PCR was found to be rapid and reproducible (three repeats were done). These isolates yielded different 23 profiles which were clustered into eight groups. The discrimination index was moderate (D value 0.83). Somatic and antigenic typing of the isolates did not reveal any correlation with REP-PCR profiles. There was no host-specific, type-specific, region-specific or pathenogenicity-specific pattern. The REP profiles of isolates obtained from wild animals were similar to those obtained from domestic animals. Two common bands were present in all the isolates irrespective of somatic or antigenic types. The results were not comparable with earlier findings, which had shown high discrimination index and correlation with disease presentation. Saxena, M.K., Singh, V.P., Kumar, A.A., Chaudhuri, P., Singh, V.P., Shivachandra, S.B., Biswas, A. and Sharma, B., 2006. REP–PCR analysis of Pasteurella multocida isolates from wild and domestic animals in India. Veterinary Research Communications, 30(8), 851–861     

Molecular Variability among Strains of Pasteurella multocida Isolated from an Outbreak of Haemorrhagic Septicaemia in India

The applicability of conventional and molecular methods for rapid detection and differentiation of Pasteurella multocida serogroup B isolates involved in an outbreak of haemorrhagic septicaemia affecting Indian buffaloes, was studied. Five isolates were obtained and were subjected to phenotypic and genotypic characterization. None of the five isolates could be differentiated on the basis of cultural, biochemical, pathogenicity and antimicrobial sensitivity patterns. Polymerase chain reaction (PCR)-based techniques were found to be specific and sensitive for rapid detection and differentiation of isolates. Repetitive extragenic palindromic (REP-) PCR, enterobacterial repetitive intergenic consensus (ERIC-) PCR and single-primer PCR differentiated all the five isolates into different profiles. All the isolates involved in the outbreak were found to have a genetic profile different from standard P. multocida strain (P52). However, three isolates had similar profiles, whereas each of the remaining two had a different profile. The study indicates the involvement of multiple strains of P. multocida in a single outbreak of haemorrhagic septicaemia in buffaloes. The results also indicate that molecular methods of detection and typing are superior to conventional methods for rapid epidemiological investigations of haemorrhagic septicaemia.     

Identification of avian strains of Pasteurella multocida in India by conventional and PCR assays

The prevalence of capsular and somatic serotypes were studied among 123 Pasteurella multocida strains isolated from chickens (n = 94), ducks (22), quails (4), turkeys (2) and geese (1) from different geographical regions of India. All strains exhibited similar cultural and morphological characteristics. Ninety-two of the isolates belonged to serotype A:1, the most prevalent serotype, with serotypes A:3, A:1,3, D:3 and F:3 having two isolates each. Only one isolate was positive for serotypes A:4 and D:1. Twenty isolates were untyped. A multiplex capsular PCR assay generated amplicons of sizes 460, 1044, 657 and 854 bp in 106 isolates identified as capsular serotype-A, 15 in serotype D and two in serotype F. Capsular types B and E were not detected in any of the avian isolates studied. The present findings suggest that a multiplex capsular PCR assay may be suitable for the rapid initial identification serotypes P. multocida during epidemiological studies of fowl cholera.     

Ribotyping of Indian Isolates of <Emphasis Type="Italic">Pasteurella multocida</Emphasis> Based on 16S and 23S rRNA Genes

The applicability of ribotyping based on 16S and 23S rRNA was evaluated for molecular epidemiological studies. Forty-eight isolates of Pasteurella multocida isolated from different hosts and geographical locations and one reference isolate were ribotyped. Only four ribotypes were found. All the isolates including reference isolate from wild carnivores had the same ribotype, though they had different serotypes. The isolate from a tiger had one band in addition to the bands present in the major ribotype. The isolates from lions represented two ribotypes; of these ribotypes, one (r2) had an additional band of 3.6 kbp, which was absent in all other ribotypes. The second ribotype (r4) from a lion had one band missing (6 kbp) that was present in the other ribotypes. These isolates were further typed using ERIC-PCR and REP-PCR. With ERIC-PCR and REP-PCR, higher D values of 0.83 and 0.89 were obtained. The current study revealed that ribotyping is not a very efficient typing tool for use in molecular epidemiology for differentiation of isolates.