Teat disorders predispose ewes to clinical mastitis after challenge with Mannheimia haemolytica

In order to study the effects of sheep teat disorders on the protection of the mammary gland, we used a Mannheimia haemolytica isolate, which did not cause clinical mastitis when deposited into intact teats. In the first experiment, this was deposited into the duct of teats with orf (Group A, n=5) or papilloma (Group B, n=3). In the second, teats were chapped and then, the organism was deposited into the duct (Group C, n=7) or on the skin (Group D, n=4). Ewes with healthy teats were controls (Group E, deposition into duct, n=5; Group F, deposition on skin, n=2). The ewes in Groups A, B or C developed clinical mastitis 5 h later, whilst the ewes in Group D developed it 2 d later; no control ewe developed clinical mastitis. In ewes with teat lesions, the organism was isolated from secretion samples and the California Mastitis Test became positive 5 h after challenge; neutrophils and lymphocytes were seen in Giemsa-stained secretion films from Group A or B ewes, whilst macrophages, neutrophils and lymphocytes in films from Group C or D ewes; neutrophils were predominating in films from Group E or F ewes. Inside the teats of Group A, B, C or D ewes, folds, hyperaemia and mucosal thickness were seen; histologically, subepithelial leucocytic infiltration was seen. In Group A or B ewes, no evidence of lymphoid tissue at the teat duct-cistern border was found. In Group C or D ewes, intense erosion and ulceration of the teat skin and conspicuous lymphoid tissue at the teat duct-cistern border, were evident; lesions characteristic of haemorrhagic mastitis were in the mammary parenchyma. In control ewes, subepithelial leucocytic infiltration in the teat duct and lymphoid tissue as above, were evident. We postulate that teat lesions can be predisposing factor to mastitis, by adversely affecting defences and speeding the process of infection and making it more severe.     

溶血性曼氏杆菌致病机制的研究进展

曼氏杆菌病是牛、羊等反刍动物的重要呼吸道传染病,给养牛和养羊业带来较大的经济损失,溶血性曼氏杆菌是该病的病原,作者对其生物学特性、临床症状、毒力因子、致病机制等进行简要概述,为进一步深入研究提供参考。     

溶血性曼氏杆菌PCR检测方法的研究

溶血性曼氏杆菌是导致牛、羊呼吸道传染病的一种病原菌。为快速、准确诊断由本菌导致的疾病 ,从基因库中获得溶血性曼氏杆菌( M.haemolytica )的基因序列 ,再从其基因序列中获得与其它细菌包括 M.annheimiagranulomatis,M.varigena ,M.ruminalis,M.glucosidal ,M.annheimiaspp和多杀性巴氏杆菌等不同的基因片段 ,设计成引物 ,建立了特异性好、敏感性高的 M.haemolytica PCR检测方法。结果表明 ,除溶血性曼氏杆菌为阳性外 ,其余所有细菌均为阴性 ,特异性为 1 0 0 % ,最小检出量为 8× 1 0 2 cfu/ m L 曼氏杆菌或 1 / 1 0个单个菌落。检验人工感染牛 ,检出率为 75%。此PCR检测方法的建立 ,为 M.haemolytica提供了一个快速诊断方法     

Mannheimia haemolytica and the pathogenesis of enzootic bronchopneumonia

Mannheimia (M.) haemolytica (formerly Pasteurella [P.] haemolytica) is the primary aetiological agent of pneumonic pasteurellosis--one of the most important respiratory diseases in cattle and sheep. While bovine pneumonic pasteurellosis is regarded to be mainly caused by M. haemolytica serotype A1, and in Germany during the last years also by serotype A6, sheep can be infected by all serotypes although there is an increased prevalence of serotypes A2 and A5-7. The obligate pathogenicity of M. haemolytica is proven by isolation of pure cultures from pneumonic lungs as well as by infection studies. Knowledge about the virulence mechanisms of M. haemolytica and their molecular basis are fragmentary, most probably due to the complex gene regulation of virulence associated factors in lung tissues. This review summarizes the current literature covering virulence factors to substantiate a model of pathogenesis. After serotype A1 strains have colonized the bovine upper respiratory tract they replace other serotypes by mechanisms unknown to date. After fulminant proliferation in the upper respiratory tract the microorganisms colonize the lower respiratory tract, finally entering alveolar spaces. An inflammatory cascade is initiated by M. haemolytica LPS and Leukotoxin, causing activation of the complement system and release of cytokines. Pathognomonic for bovine pneumonic pasteurellosis is the strong influx of neutrophiles accompanied by accumulation of fibrin, finally causing necrosis of alveolar spaces. Depending on lesion size this fibronecrotizing pneumonia can result in death of the animals. In addition, possible protective antigens are discussed. There is still a great effort in the development of efficacious vaccines against pneumonic pasteurellosis in cattle and sheep caused by various M. haemolytica serotypes worldwide. The scarce knowledge concerning presence and distribution of virulence associated factors in M. haemolytica strains and their role in pathogenesis made it difficult to determine a suitable vaccine candidate in the past. In addition, there is lack of knowledge concerning the variability of virulence factors in individual isolates. Genome sequence analysis of M. haemolytica, enabling proteomics and transciptomics, hopefully will give new insight into the pathogenesis of pneumonic pasteurellosis.     

A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.     

Phenotypic characterisation of Australian sheep and cattle isolates of Mannheimia haemolytica, Mannheimia granulomatis and Mannheimia varigena

Objective To perform a comprehensive phenotypic characterisation of 35 isolates of bacteria previously identified as haemolytic Pasteurella‐Actinobacillus and obtained from cattle and sheep. Design The 35 isolates that had been obtained from Australian animals, 30 from cattle and five from sheep, were compared with reference strains of the five recognised species of the genus Mannheimia ‐ M haemolytica, M glucosida, M granulomatis, M ruminalis and M varigena. Results Thirty‐four of the isolates could be confidently assigned to three species of the genus Mannheimia. Twenty‐nine were M haemolytica, with 25 being isolated from cattle and four from sheep. All but three of the bovine M haemolytica were isolated from pneumonic lungs. Of the three remaining bovine M haemolytica isolates, one was obtained in pure culture from a bovine milk sample and the other two as part of a mixed flora associated with a middle ear infection of a calf suffering mucosal disease. Of the four ovine M haemolytica isolates, two were isolated in pure culture from milk and two, also in pure culture, from pneumonic lungs. Three bovine isolates were identified as M granulomatis ‐ one from a tongue abscess, one from a jaw abscess and one from a lung showing suppurative bronchopneumonia. Two bovine isolates were identified as M varigena‐ one coming from an udder and the other from a spleen. The available diagnostic records provided no information on whether these isolates were associated with a disease process. The remaining isolate was obtained from an ovine tongue abscess and could not be assigned to a recognised species within the genus Mannheimia. Conclusion The study represents the first time that M haemolytica, M granulomatis and M varigena have been recognised as being present in cattle and sheep in Australia. Veterinary laboratories that encounter Pasteurella‐Actinobacillus‐like organisms from cattle and sheep should attempt as complete a characterisation as possible to help improve our knowledge of the disease potential of these organsims.     

Phenotypic characterisation of Australian sheep and cattle isolates of Mannheimia haemolytica,Mannheimia granulomatis and Mannheimia varigena

OBJECTIVE: To perform a comprehensive phenotypic characterisation of 35 isolates of bacteria previously identified as haemolytic Pasteurella-Actinobacillus and obtained from cattle and sheep. DESIGN: The 35 isolates that had been obtained from Australian animals, 30 from cattle and five from sheep, were compared with reference strains of the five recognised species of the genus Mannheimia--M. haemolytica, M. glucosida, M. granulomatis, M. ruminalis and M. varigena. RESULTS: Thirty-four of the isolates could be confidently assigned to three species of the genus Mannheimia. Twenty-nine were M. haemolytica, with 25 being isolated from cattle and four from sheep. All but three of the bovine M. haemolytica were isolated from pneumonic lungs. Of the three remaining bovine M. haemolytica isolates, one was obtained in pure culture from a bovine milk sample and the other two as part of a mixed flora associated with a middle ear infection of a calf suffering mucosal disease. Of the four ovine M. haemolytica isolates, two were isolated in pure culture from milk and two, also in pure culture, from pneumonic lungs. Three bovine isolates were identified as M. granulomatis--one from a tongue abscess, one from a jaw abscess and one from a lung showing suppurative bronchopneumonia. Two bovine isolates were identified as M. varigena--one coming from an udder and the other from a spleen. The available diagnostic records provided no information on whether these isolates were associated with a disease process. The remaining isolate was obtained from an ovine tongue abscess and could not be assigned to a recognised species within the genus Mannheimia. CONCLUSION: The study represents the first time that M. haemolytica, M. granulomatis and M. varigena have been recognised as being present in cattle and sheep in Australia. Veterinary laboratories that encounter Pasteurella-Actinobacillus-like organisms from cattle and sheep should attempt as complete a characterisation as possible to help improve our knowledge of the disease potential of these organsims.     

Characterization of Mannheimia haemolytica biofilm formation in vitro

Mannheimia haemolytica is the primary bacterial agent in the bovine respiratory disease complex. It is thought that M. haemolytica colonizes the tonsillar crypts of cattle as a commensal and subsequently descends into the lungs to cause disease. Many bacterial species persist in the host as biofilms. There is limited information about the ability of M. haemolytica to form biofilms. The aim of this study was to develop an in vitro model for M. haemolytica biofilm formation. We found that M. haemolytica required at least 36 h to form robust biofilms on plastic in vitro when incubated in RPMI-1640 tissue culture medium at 37 °C, with maximal biofilm formation being evident at 48 h. Biofilm formation was inhibited by adding the monosaccharides d(+) galactose and d(+) mannose to the growth medium. Addition of antibodies to the M. haemolytica surface protein OmpA also reduced biofilm formation. Upon evaluating the macromolecules within the biofilm extracellular polymeric substance we found it contained 9.7 μg/cm² of protein, 0.81 μg/cm² of total carbohydrate, and 0.47 μg/cm² of extracellular DNA. Furthermore, proteinase K treatment significantly decreased biofilms (P < 0.05) while α-amylase and micrococcal nuclease decreased biofilms to a lesser extent. M. haemolytica biofilm cells were more resistant than planktonic cells to the antibiotics florfenicol, gentamicin, and tulathromycin. These results provide evidence that M. haemolytica can form biofilms, which could contribute to its ability to persist as a commensal in the bovine upper respiratory tract.     

Quantification of Mannheimia haemolytica leukotoxin by indirect ELISA

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of extracellular leukotoxin (LKT) produced in chemostat culture of Mannheimia haemolytica in a serum-free culture medium. Leukotoxin purified with preparative SDS-PAGE was used for the production of chicken polyclonal antibodies (PAb) that served as the primary detecting antibody. Excising the LKT protein from an analytical SDS-PAGE gel proved an efficient technique for the purification of the toxin. Consequently, the 102 kDa LKT polypeptide purified in this manner served as reference toxin and the resulting calibration curve was modelled using a four parameter logistic fit to relate absorbance to LKT protein concentration. The lower detection limit corresponded to an LKT concentration of 14.5 ng ml(-1). The presence of SDS, serum albumin and the coating pH had a distinct effect on the absorbance values of the indirect ELISA.     

绵羊溶血性曼氏杆菌灭活疫苗免疫效果观察

溶血性曼氏杆菌主要引起牛羊等反刍动物的呼吸系统疾病,2006年,康乐县某养殖场的部分小尾寒羊发生了厌食、腹泻、咳嗽、高烧、流产等症状的传染性疾病.经采取死亡羊只的脾脏、肝脏、胎盘等病料,进行了实验室诊断,确诊为溶血性曼氏杆菌引起的曼氏杆菌病.为有效控制曼氏杆菌病,我们开展了灭活苗免疫效果的观察.     

溶血性曼氏杆菌PCR检测试剂盒的研制及初步应用

在溶血性曼氏杆菌PCR检测方法研究的基础上,进行溶血性曼氏杆菌PCR检测试剂盒的研究及初步应用.结果表明,试剂盒特异性好,批内和批间的试剂盒无差异;试剂盒在-20℃可保存30个月,4℃可保存12个月;应用试剂盒从1000多份临床样品中筛选分离到6株溶血性曼氏杆菌.     

Effect of danofloxacin and tilmicosin on body temperatures of beef calves with pneumonia experimentally induced by inoculation with Mannheimia haemolytica

OBJECTIVE: To examine effects of danofloxacin and tilmicosin on continuously recorded body temperature in beef calves with pneumonia experimentally induced by inoculation of Mannheimia haemolytica. ANIMALS: 41 Angus-cross heifers (body weight, 160 to 220 kg) without a recent history of respiratory tract disease or antimicrobial treatment, all from a single ranch. PROCEDURE: Radiotransmitters were implanted intravaginally in each calf. Pneumonia was induced intrabronchially by use of logarithmic-phase cultures of M. haemolytica. At 21 hours after inoculation, calves were treated with saline (0.9% NaCl) solution, danofloxacin, or tilmicosin. Body temperature was monitored from 66 hours before inoculation until 72 hours after treatment. Area under the curve (AUC) of the temperature-time plot and mean temperature were calculated for 3-hour intervals and compared among treatment groups. RESULTS: The AUCs for 3-hour intervals did not differ significantly among treatment groups for any of the time periods. Analysis of the mean temperature for 3-hour intervals revealed significantly higher temperatures at most time periods for saline-treated calves, compared with temperatures for antimicrobial-treated calves; however, we did not detect significant differences between the danofloxacin- and tilmicosin-treated calves. The circadian rhythm of temperatures before exposure was detected again approximately 48 hours after bacterial inoculation. CONCLUSIONS AND CLINICAL RELEVANCE: Danofloxacin and tilmicosin did not differ in their effect on mean body temperature for 3-hour intervals but significantly decreased body temperature, compared with body temperature in saline-treated calves. Normal daily variation in body temperature must be considered in the face of respiratory tract disease during clinical evaluation of feedlot cattle.     

The association between teat end hyperkeratosis and teat canal microbial load in lactating dairy cattle

Most pathogens that cause bovine mastitis invade the udder lumen through the teat canal. Amino acids and intercellular lipids may support microbial colonisation of the teat canal epithelium by pathogenic microorganisms. The aim of this study was to investigate the association between teat end hyperkeratosis, which is induced by machine milking, and teat canal microbial load. Contralateral teats, which differed in teat end hyperkeratosis scores, were identified in a split-udder experiment. The teat canal's microbial load was evaluated using the wet and dry swab technique. Staphylococcus (S.) aureus, Streptococcus (Sc.) uberis, Escherichia (E.) coli and other coliforms were detected by agar plate cultures. The positive detection of E. coli and the log(10)-transformed E. coli load of a teat canal were significantly associated with the teat end hyperkeratosis score (P<0.05). There were significant differences with respect to positive findings for E. coli, as well as the microbial load of E. coli and Sc. uberis, between the less-calloused and the more-calloused teat of a pair. For S. aureus, no significant associations between hyperkeratosis score and teat canal microbial load were detected. In general, a teat with a highly calloused teat end had an increased teat canal microbial load compared with the contralateral teat, characterised by a lower callosity. The results of the present study indicate that the environmental pathogen load is associated with teat end hyperkeratosis. Further research is needed to identify factors that may affect teat canal microbial load in lactating dairy cattle.     

The N-acetyl-D-glucosamine specific adhesin from Mannheimia haemolytica activates bovine neutrophils oxidative burst

In this work we identified specific bovine leukocytes that were bound by the Mannheimia haemolytica adhesin molecule (MhA) and the biological effect on the leukocytes. Histochemical staining and flow cytometry showed that MhA bind neutrophils (90%) and monocytes (5%). MhA induced an oxidative response in purified neutrophils; this effect was 1.5-fold higher than the effect observed with control cells activated with Zymosan. Cellular binding by MhA was inhibited with GlcNAc and its oligomers, as well as by glycoproteins containing tri- and tetra-antennary N-glycosydically linked glycans. MhA-induced oxidative burst was significantly inhibited by GlcNAc, iodoacetamide, superoxide dismutase, and piroxicam (p<0.05). Our findings suggest that among bovine leukocytes, neutrophils are the main target for MhA, inducing production of oxidative radicals by non-opsonic mechanism that seem to play an important role in tissue damage during mannheimiosis.     

Presence of subepithelial lymphoid nodules in the teat of ewes

A total of 87 clinically healthy ovine teats were examined bacteriologically (by scraping the mucosa) and histologically. Teats examined were those of lactating mammary glands with no bacteria isolated (n = 23); of mammary glands after cessation of lactation with no bacteria isolated (n = 25); of lactating mammary glands with bacteria isolated (n = 22); and of mammary glands after cessation of lactation with bacteria isolated (n = 17). The salient histological feature was subepithelial leucocytic infiltration. In teat cisterns, lymphocytes were the predominant cell type and in teat ducts, lymphocytes and neutrophils were seen in equal proportions. Subepithelial lymphoid nodules, some with germinal centres, were detected in 43 (49%) teats. The majority of lymphoid nodules was observed at the border between teat duct and teat cistern. Presence of bacteria was significantly associated with the presence of leucocytic activity (P < 0.001) and with the presence of lymphoid nodules (P = 0.032). We conclude that the presence of induced subepithelial lymphoid tissue at the border between teat duct and teat cistern appears to be important in protecting the mammary gland during the early stages of bacterial invasion. The findings call for further investigations into the lymphoid structures of the teat; these should elucidate the role and development of mammary mucosa-associated lymphoid tissues and may lead to strategies for enhancing non-specific defence mechanisms of the mammary gland.     

Efficacy of difloxacin in calves experimentally infected with Mannheimia haemolytica

OBJECTIVE: To determine the efficacy of difloxacin, a novel fluoroquinolone antibiotic, in calves experimentally infected with Mannheimia haemolytica (formerly Pasteurella haemolytica). ANIMALS: Seventy-two 3-month-old Holstein calves. PROCEDURES: Calves were inoculated with M haemolytica intratracheally; after they developed clinical signs of pneumonic pasteurellosis, they were randomly assigned to 1 of 6 groups (n = 12/group). Calves in each group were treated with 10% difloxacin (2.5 or 5 mg/kg of body weight), 5% difloxacin (2.5 or 5 mg/kg), enrofloxacin (5 mg/kg), or saline (0.9% NaCl) solution (control group), once daily for 5 days, and clinical signs were scored daily. On day 15, calves were euthanatized, and the percentage of diseased lung tissue was calculated. Swab specimens of the lungs were submitted for bacterial culture. RESULTS: Mortality rate and percentage of diseased lung tissue were significantly higher and cure rate and average daily gain were significantly lower for control calves, compared with calves in the treatment groups; however, no significant differences were found among treatment groups. Mannheimia haemolytica was isolated from the lungs of 10 control calves and from at least 2 calves in each of the treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that difloxacin and enrofloxacin were equally effective for treatment of calves with experimentally induced pneumonic pasteurellosis. However, treatment of infected calves with difloxacin or enrofloxacin may not eliminate the organism.     

Investigations on the species specificity of Mannheimia (Pasteurella) haemolytica serotyping

Eleven serotypes (1, 2, 5-9, 12-14 and 16) have been demonstrated within Mannheimia haemolytica. Subsequent serotyping of 166 Mannheimia haemolytica-like strains, genetically and phenotyphically distinct from Mannheimia haemolytica, and isolated from ruminants, pigs, hares and rabbits showed that 13.2% were typeable, 19 of which were serotype 11 representing strains now being classified as M. glucosida. In addition, three strains belonged to serotypes 6, 9 and 16, respectively. Additionally, the serotyping results of 98 (P.) haemolytica-like isolates from non-ruminant sources collected by the UK Veterinary Investigation Centres during the period 1982-1996 were investigated. None of these isolates have been kept, making further genetic characterization impossible. Among these isolates, 25.5% were typeable representing serotypes 1, 2, 3, 5, 6, 9, 10, 13 and 15. Substantial evidence has been reported indicating that M. haemolytica-like isolates from non-ruminant sources represent species different from M. haemolytica. The present investigation underlines that serotyping does not represent a reliable method for the identification of M. haemolytica or M. glucosida. These observations emphasize that extended phenotypic and genetic characterization is necessary for the proper identification of these organisms.     

Role of Mannheimia haemolytica leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis

Bovine pneumonic pasteurellosis continues to be a major respiratory disease in feedlot cattle despite the recent advances in our understanding of the underlying complexities of causation. The etiological agent, Mannheimia haemolytica, possesses several virulence factors, including capsule, outer membrane proteins, adhesins, neuraminidase, endotoxin and exotoxic leukotoxin. Accumulating scientific evidence implicates leukotoxin as the primary factor contributing to clinical presentation and lung injury associated with this disease. Unlike other virulence factors, leukotoxin shows cell-type- and species-specific effects on bovine leukocytes. Recent investigations have delineated the mechanisms underlying the target-cell-specificity of leukotoxin and how this contributes to the pathogenesis of lung damage. This review summarizes current understanding of the secretion, regulation, mechanisms of action and evolutionary diversity of leukotoxin of M. haemolytica. Understanding the precise molecular mechanisms of leukotoxin is critical for the development of more effective prophylactic and therapeutic strategies to control this complex disease.