Analysis of the immunoproteome of Mycoplasma mycoides subsp. mycoides small colony type reveals immunogenic homologues to other known virulence traits in related Mycoplasma species

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small colony type (MmmSC) has been eradicated in the developed world, but it is still present in many countries of sub-Saharan Africa. After initially successful control measures in the 1960s it has been spreading due to a lack of money, fragmentation of veterinary services, uncontrolled cattle movement, insufficient vaccine efficacy and sensitivity of current diagnostic tests.In this study we used two-dimensional polyacrylamide gel electrophoresis followed by immunoblot with sera from MmmSC-infected animals and MALDI-ToF mass spectrometry to identify novel immunogenic proteins as candidate molecules for improved diagnostics and vaccines. We identified 24 immunogens recognized by pooled sera from experimentally infected cattle. Furthermore, a serum from an animal with acute clinical disease as well as severe pathomorphological lesions recognized 13 additional immunogens indicating variation in the antibody responses to CBPP amongst cattle. Most immunogens showed compelling similarity to protein/gene sequences in the two ruminant pathogens M. capricolum subsp. capricolum and M. mycoides subsp. mycoides large colony type both belonging to the mycoides cluster. Three of these proteins, namely glycerol-3-phosphate oxidase, adenylosuccinate synthase, and glyceraldehyde-3-phosphate dehydrogenase, had no compelling homologue in the other distantly related bovine pathogen M. agalactiae. In addition, translation elongation factor Tu, heat shock protein 70, pyruvate dehydrogenase, and FKBP-type peptidyl-prolyl isomerase, which have been found to mediate adhesion to host tissue in other mycoplasmas were shown to be expressed and recognized by sera. These proteins have potential for the development of improved diagnostic tests and possibly vaccines.     

Investigations of Outbreaks of Contagious Caprine Pleuropneumonia in Eritrea

Mycoplasmas were isolated from freeze-dried lung samples from goats from the western lowlands of Eritrea suspected of being affected by contagious caprine pleuropneumonia. The goats belonged to two herds in which mortality and morbidity rates were high. Mycoplasma capricolum subsp. capripneumoniae was identified in most samples by the polymerase chain reaction and by conventional serological tests. The latex agglutination test detected more positive serum samples in both herds than did the complement fixation test. Following cloning, the isolates of M. capricolum subsp. capripneumoniae were analysed biochemically and shown to be metabolically similar. They oxidized glucose, N-acetylglucosamine, pyruvate and L-lactate with high affinity and mannose, glucosamine and 2-oxobutyrate with low affinity; they were unable to utilize maltose, trehalose, fructose or ethanol. Major improvements were seen in the growth yield of the Eritrean strains with the addition of pyruvate to the medium. Thus, it may be that organic acids are important energy sources for these strains and may be used in addition to or in place of glucose. In contrast to most other strains of the M. mycoides cluster, the Eritrean strains produced large amounts of hydrogen peroxide during the oxidation of NADH by lysed cells. This characteristic had previously been reported for strain M. F38, the type strain of M. capricolum subsp. capripneumoniae, although strain F38 did not metabolize sugars. Hydrogen peroxide has long been considered a pathogenicity factor in mycoplasma infections. This is the first isolation of M. capricolum subsp. capripneumoniae from Eritrea.     

Characterisation of protein and antigen variability among Mycoplasma mycoides subsp. mycoides (LC) and Mycoplasma agalactiae field strains by SDS-PAGE and immunoblotting

Mycoplasma mycoides subsp. mycoides (LC) (Mmm LC) and Mycoplasma agalactiae are the most important mycoplasma species involved in the contagious agalactia syndrome. A total of 25 field strains from Spain and the two type strains were analysed by SDS-PAGE and immunoblotting. Two polyclonal antisera (PAbs) raised against a pool of strains of each mycoplasma species were used. The results revealed a high degree of protein variability among the field strains. The type strain of Mmm LC appeared to be representative of the field strains of this species, whereas this was not the case with the M. agalactiae type strain. Whereas M. agalactiae is known to possess a gene family regulating surface antigen diversity, there is a need to study the mechanisms used byMmm LC to generate antigenic variability in more detail.     

The efficacy of some wild medicinal plant extracts on the survival and development of third instar larvae of <Emphasis Type="Italic">Chrysomyia albiceps</Emphasis> (Wied) (Diptera: Calliphoridae)

Four crude wild plants extract of Artemisia herba-alba Asso, Artemisia monosperma Del., Euphorbia aegyptiaca Boiss. and Francoeuria crispa (Forsk.) extracted with four successive solvents; hexane, diethyl ether, ethyl acetate and ethanol were evaluated against the third instar larvae of Chrysomyia albiceps using dipping and thin film techniques. In dipping technique, larvae were immersed in the concentrations of plant extracts for 30 seconds. However, in thin film technique, larvae were exposed to thin layer from each plant extract in the Petri-dishes. Results showed that all extracts had toxic effects on larvae in both two treatments. Hexane and diethyl ether extracts of A. herba-alba and ethyl acetate extract of A. monosperma recorded the highest effect in both two treatments. Ethanol extracts of E. aegyptiaca and A. monosperma were the highest in dipping and thin film treatments, respectively. In dipping treatment, most plant extracts revealed extending effect on pupae especially while in thin film treatment most larvae which succeeded to develop to pupae produced normal flies. Deformed pupae were only recorded with the high concentration of diethyl extracts of A. herba-alba. Hexane, diethyl ether and ethyl acetate extracts of F. crespa only produced low percentages of deformed flies. Histological examination conducted on larvae confirmed that extracts in thin film treatment penetrated to the gut and destroyed its epithelial cells and wall. It concluded that the crude extracts of the four tested plants can be used in controlling of C. albiceps larvae while hexane extracts of E. aegyptiaca, A. herba-alba and A. monosperma are considered the most promising plant preparations against the larvae by using thin film technique.     

Mycoplasma detection and isolation from one-humped camels (Camelus dromedarius)

In scientific literature, a small amount of information is found concerning mycoplasmosis in camel species. Mycoplasma (M.) arginini, Acholeplasma (A.) laidlawii, and Acholeplasma oculi have been reported to be isolated from these host species. Serologically positive results have been reported for Mycoplasma mycoides subsp. mycoides SC type, Mycoplasma capricolum subsp. capripneumoniae, and M. mycoides subsp. capri. The aims of this study were to detect, isolate, and identify mycoplasmas from camels (Camelus dromedarius). Initially, saliva and ear smears plus conjunctival and vaginal secretions were taken from five female animals, but only conjunctival secretions in three male animals, all belonging to the same farm. An unknown mycoplasma was isolated from one of the vagina samples. Additionally, another unknown and uncultured mycoplasma was detected with molecular biology in the same sample. In the second stage, 23 vaginal secretions were taken from the same farm plus another secretion from a different one. Ten isolates of the same unknown and previously isolated mycoplasma were detected, nine of them recovered from the vagina of female camels. Some mycoplasmas have been related to reproductive disorders; however, there is no evidence that the isolated mycoplasmas are related to such disorders.     

Kinetics of substrate oxidation and hydrogen peroxide production by Mycoplasma mycoides subsp. mycoides Large Colony (LC) type and Mycoplasma mycoides subsp. capri

Mycoplasma mycoides subsp. mycoides Large Colony (LC) type is a pathogen of goats causing contagious agalactia and respiratory disease, found on all continents where small ruminants are kept. It shares close genetic characteristics with M. mycoides subsp. capri. Substrate oxidation by 22 strains of M. mycoides subsp. mycoides LC from nine countries was compared with that of eight strains of M. mycoides subsp. capri from five countries. There was considerable similarity in the substrates used, but substrate saturation coefficients (K_s) varied for different substrates. Substrate utilization patterns and K_s values did not (1) significantly differentiate the LC strains from each other, (2) show any correlation with geographical origin, or (3) distinguish the LC strains from the capri strains. These results support previous studies justifying the reclassification of these subspecies as a single species.     

Detection of Mycoplasma mycoides sub-species mycoides small colony by a specific capture/enrichment monoclonal antibody-based sandwich ELISA

The present study describes the development of a specific Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC) monoclonal antibody (MAb), 6E3, and its application in a sandwich ELISA (sELISA) format. Mab 6E3 reacted only to the 12 MmmSC within the 32 M. mycoides cluster strains and 12 representative strains of other bovine, ovine and caprine associated mycoplasmas examined. A capture/enrichment format of the sELISA that combined MAb 6E3 with a previously developed MAb 3H12 that cross reacted with Mmm Large Colony [Rodriguez, F., Ball, H.J., Finlay, D., Campbell, D., Mackie, D.P., 1996. Detection of Mycoplasma mycoides sub-species mycoides by monoclonal antibody-based sandwich ELISA. Veterinary Microbiology 51, 69–76], retained MmmSC specificity and improved the sensitivity from the 1.2 × 10⁷ cfu/ml for a standard 2 h capture stage sELISA down to as low as 2 cfu/ml for a 72 h capture. A low level of false positives (1%) was observed when this assay was applied to 200 bovine respiratory and milk samples submitted for diagnostic investigation. This simple and specific sELISA provides a suitable assay for screening large numbers of samples for CBPP.     

Changes in Lymphocyte Subsets in the Bronchus‐associated Lymphoid Tissue of Goats Naturally Infected with Different Mycoplasma Species

The distribution of cells containing lysozyme, S‐100 protein, CD3, CD4, CD8, major histocompatibility complex class II antigen and immunoglobulin G (IgG) was analysed in the bronchus‐associated lymphoid tissue (BALT) of goats naturally infected with three Mycoplasma species. This study included the immunohistochemical characterization of the pneumonic lesions of 18 goats (3–5 months old) infected with one of the following Mycoplasma species: M. mycoides ssp. mycoides, Large Colony type (goats no. 1–6), M. mycoides ssp. capri (goats no. 7–12) and M. capricolum ssp. capricolum (goats no. 13–18). Microscopically, infected animals showed a moderate bronchointerstitial pneumonia, characterized by lymphoid hyperplasia of the BALT and infiltration of mononuclear cells in the alveolar walls and airways. The main cellular type in the BALT was represented by CD3⁺ T lymphocytes, and the ratio of CD4⁺:CD8⁺ cells was >2. The BALT showed large germinal centres mainly composed of IgG⁺ B lymphocytes, with numerous S‐100⁺ follicular dendritic cells. The presence of follicular dendritic cells confirmed the high degree of organization of this lymphoid tissue. The immunohistochemical results showed that activated T lymphocytes, particularly in the CD4 subset, and IgG⁺ B cells, play a major role in the immune response of the caprine lung infected with these species of mycoplasmas.     

Field trial of a combined vaccine against caprine contagious agalactia: humoral immune response in lactating goats

Two vaccines against Mycoplasma agalactiae and Mycoplasma mycoides subsp. mycoides (large colony type) were developed using inactivated strains selected in previous characterization studies. Formaldehyde and phenol were used as the inactivating agents for vaccines A and B, respectively. Aluminium hydroxide plus purified saponin (Quil-A) were added to both vaccines as adjuvant. The field trial was designed to evaluate the specific humoral immune response to the two mycoplasma species in lactating goats over a period of 7 months. The vaccines were tested on 120 goats randomly assigned to three groups of 40 animals each. Two groups received two injections of vaccine A or B respectively, and a third group remained in the herd as control. Antibody titres determined by ELISA indicated a significant difference between both vaccines and the control group over a 6-month period. Immunoblotting assays also revealed the production of antibodies against the two mycoplasma species. Further field trials are underway to evaluate the efficacy and protection conferred to the animals by these specific antibodies.     

A mycoplasma from chronic caprine pleuropneumonia in Kenya

Summary A new mycoplasma was isolated from cases of chronic caprine pleuropneumonia in Kenya. It belonged to the speciesMycoplasma mycoides being a member of Al-Aubaidi's Group 8. When inoculated into goats and sheep the organism caused pleuropneumonia and local subcutaneous lesions. The pleuropneumonia was not contagious. In contrast the organism was non-pathogenic in cattle.

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Resumen Se aisló un nuevo micoplasma de casos clínicos de pleuroneumonía caprina crónica en Kenia. El agente pertenece a la especieMycoplasma mycoides, siendo un miembro del grupo 8 Al-Aubaidi. El organismo produjo pleuroneumonía lesiones subcutáneas cuando se inoculó en cabras y ovejas. La enfermedad producida no fue contagiosa y el organismo no fue patógeno para los bovinos.

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Résumé Un nouveau mycoplasme a été isolé à partir de cas de pleuropneumonie caprine chronique au Kenya. Il appartient à l'espèceM. mycoides, membre du Groupe 8 d'Al Aubaidi. Inoculé à des chèvres et des moutons, ce germe cause une pleuropneumonie et des lésions sous cutanées locales. La pleuropneumonie n'est pas contagieuse. Par contre, le germe n'est pas pathogène pour les bovins.

     

Molecular Epidemiology of Contagious Bovine Pleuropneumonia in Tanzania Based on Amplified Fragment Length Polymorphism and Pulsed‐field Gel Electrophoresis Analysis

The genetic diversity of 60 field strains of Mycoplasma mycoides ssp. mycoides, small colony type (M. mycoides SC), comprising 56 isolates from cattle in Tanzania, one from Kenya, two from Botswana and one from Portugal, as well as the type (PG1^T) and vaccine (T₁‐SR49) strains, was investigated. The strains were analysed for variations in the EcoRI and Csp6I restriction sites in the genomic DNA using the amplified fragment length polymorphism (AFLP) technique, and variations in the BamHI restriction sites using pulsed‐field gel electrophoresis (PFGE). Six AFLP types were detected among the analysed strains. The AFLP profiles of the type and vaccine strains were indistinguishable from each other. Indistinguishable AFLP profiles were found for 55 Tanzanian field strains, one of them isolated in 1990 and the other 54 isolated in 1998/1999, although one strain isolated in 1999 showed a different profile. Strains from different countries revealed different AFLP profiles. Six PFGE types were detected among the analysed strains, with all the 56 Tanzanian field strains displaying indistinguishable PFGE profiles. Strains from different countries revealed different PFGE profiles, and so did the type and vaccine strains. The strong genomic homogeneity among M. mycoides SC strains associated with outbreaks of contagious bovine pleuropneumonia in different regions of Tanzania suggests that the outbreaks of the disease in the 1990–99 period might have been caused by a single epidemic clone. Moreover, this study has demonstrated that AFLP and PFGE are potential tools for molecular epidemiological studies of M. mycoides SC infections.     

Antigen heterogeneity among Mycoplasma mycoides subsp. mycoides SC isolates: discrimination of major surface proteins

The protein and antigen profiles of 60 isolates, strains and the type strain PG1 of Mycoplasma mycoides subsp. mycoides SC were compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblot analysis. Analysis using contagious bovine pleuropneumonia antisera and hyperimmune rabbit sera against several representative strains revealed some differences in protein profiles and variability in antigens among strains from different geographic regions. The most common antigenic bands had the molecular masses of 110, 95, 80, 69, 62, 60, 48, 44, 39 and 38 kDa. There were differences among European strains, where a larger group coming from Italy lacked the p98 antigen, thus, with one exception, distinguishing the Italian strains from Portuguese, French and Spanish strains. African, Australian and PG1 strains showed heterogenic profiles, with quantitative differences and in a few strains some antigenic bands were absent. The group constituting African, Australian and PG1 strains was characterised by the presence of 71.5/70 kDa antigens, which were not detected in European strains. Mycoplasma mycoides subsp. mycoides SC membrane proteins were characterised by Triton X-114 partitioning and p110, p98, p95, p62/60 and p48 were identified as immunogenic antigens. The simultaneous presence of these five antigens was common to all the sera examined and, therefore, indicates the diagnostic potential of immunoblotting. Most immunodominant antigens are surface-exposed proteins as determined by the trypsin treatment.     

Seroprevalence of and risk factors for Mycoplasma mycoides subspecies capri infection in small ruminants in Northern Jordan

During the period from January 2002 to December 2003, serum samples were collected from 104 small ruminant flocks consisting of 18 sheep flocks, 27 goat flocks, and 59 mixed flocks containing both sheep and goats in northern Jordan. Only female sheep and goats were sampled. At least five females aged over 2 years per flock per species were sampled and examined for Mycoplasma mycoides subspecies capri using the latex agglutination test. To increase the chances of detecting positive flocks, sick or older ewes were sampled. Specific information was obtained using a questionnaire to identify potential risk factors for M. mycoides subsp. capri seropositivity in small ruminants. The true flock-level seroprevalences of M. mycoides subsp. capri were 34%, 32%, and 38% in small ruminants (sheep and goats), sheep, and goats, respectively. Differences between flock-level seroprevalences in sheep and goats were not significant (p = 0.7). Multivariable logistic regression analysis of 21 production and health management practices showed four to be associated with M. mycoides subsp. capri seropositivity including flocks which were grazed and fed concentrate supplement (OR = 4.6), improper cleaning of milking utensils (OR = 4.7), buying new animals to replace culled ones (OR = 0.3), and treating against helminths when clinical signs of helminth infections appear (OR = 0.4).     

Serological response to T1 strainMycoplasma mycoides in calves born of previously vaccinated dams

Summary Calves from dams vaccinated two years before calving may have an irregular complement-fixing (CF) antibody response following vaccination with the T₁ strain ofMycoplasma mycoides var.mycoides (M. mycoides). These calves, including those with no detectable CF response, were resistant to subcutaneous challenge with virulentM. mycoides. The CF antibody to primary vaccination was 2-mercaptoethanol (2-ME) sensitive. After re-vaccination and challenge the CF antibody was 2-ME resistant indicating a secondary response.

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Resumen Los terneros de madres vacunadas 2 a?os antes del parto pueden tener una respuesta de anticuerpos fijadores de complemento de forma irregular después de la vacunación con la cepa T1 deMycoplasma mycoides. Estos terneros, incluyendo aquellos que no tuvieron una respuesta de anticuerpos fijadores de complemento detectables, resistieron al desafío por la vía subcutànea cuando se les inoculó conMycoplasma mycoides virulento. El primer anticuerpo en estos terneros era sensible al 2-mercapto etanol (2-ME). Sin embargo, después de la revacunación y exposición por desafío con 3 meses de intervalo, el anticuerpo era resistente a la acción del 2-Mercapto etanol, indicando una respuesta secundaria. En los terneros controles desafiados, se observaron tanto la respuesta primaria comol a respuesta de anticuerpos secundaria. El anticuerpo dirigido contra elMycoplasma mycoides galactan usado en la prueba indirecta de Hemaglutinación, era sensible a la acción del 2-Mercapto etanol.

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Résumé Les veaux issus de mères vaccinées 2 ans avant la parturition peuvent avoir de fa?on irrégulière des anticorps fixant le complément après la vaccination par la souche T₁ deMycoplasma mycoides. Ces veaux, y compris ceux qui n'ont pas d'anticorps fixant de complément décelables, ont résisté à l'épreuve par voie sous-cutanée à l'aide d'une souche virulente. Les anticorps précoces observés chez ces veaux sont des anticorps sensibles au mercaptoethanol. Cependant après re-vaccination et après infection d'épreuve au bout de 3 mois, les anticorps sont devenus résistants au mercaptoethanol, ce qui indique une réponse secondaire. Chez les veaux qui sont les témoins de l'épreuve, on a décelé des réspones en anticorps primaires et secondaires. Les anticorps vis-à-vis du galactan deMycoplasma mycoides, utilisé dans le test d'hémagglutination indirecte, sont sensibles au mercaptoethanol.

     

Depleting proteins from the growth medium of Mycoplasma capricolum unmasks bacterium-derived enzymatic activities

Mycoplasma constitutes a unique group of bacteria best characterized as lacking peptidoglycan and having one of the smallest genomes of all free-living prokaryotes. Members of this group also represent important pathogens of humans, animals, and plants. Our understanding of the interaction between these pathogens and their hosts is limited, partly due to our inadequate knowledge of the secreted enzymes and virulence factors of these pathogens. Analysis of secreted proteins of mycoplasma has been hampered by their fastidious growth requirements where protein-rich growth supplements are required. Simple ultrafiltration of the complete medium through a 10 kDa cut-off membrane successfully removed virtually all of the polypeptides in the medium and supported the growth of Mycoplasma capricolum (type California kid). This modification (AM medium) exposed the activities of a number of enzymes produced by this bacterium during growth including; acid and alkaline phosphatase, gelatinase, and β-lactamase activities. We also show that the spent culture medium contained hemolysin activity.     

A study on Contagious Caprine Pleuropneumonia (CCPP) in goats at an export oriented abattoir, Debrezeit, Ethiopia

300 goat serum samples from an export-oriented abattoir were tested for contagious caprine pleuropneumonia antibodies by the complement fixation test. The disease prevalence was 31% with no significant differences (P > 0.05) between the regions “Borena”, “Bale”, “Afar” and “Jinka” or the age of the goats (P > 0.05). Gross pathology and histopathology of the lung primary lesions were indicative of pleuropneumonia caused by Mycoplasma capricolum subsp. capripneumoniae.     

A passive haemagglutination test for detection of antibodies against mycoplasma mycoides subsp. mycoides using glutaraldehyde-fixed sheep erythrocytes

A simple passive haemagglutination test using the microtitre technique was developed for the detection of antibodies against Mycoplasma mycoides subsp. mycoides infection in cattle. Of the four different concentrations (0.1, 0.2, 0.25 and 0.5%) of glutaraldehyde used for the fixation of sheep erythrocytes, antigens prepared with erythrocytes fixed with 0.2 and 0.25% concentrations of glutaraldehyde gave the best results.The test was found to be very practical, sensitive, specific and reproducible. It also compares favourably with the complement fixation test.     

In vitro antiviral activity of chestnut and quebracho woods extracts against avian reovirus and metapneumovirus

Field evidences have suggested that a natural extract, containing tannins, could be effective against poultry enteric viral infections. Moreover previous studies have shown that vegetable tannins can have antiviral activity against human viruses. Based on this knowledge three different Chestnut (Castanea spp.) wood extracts and one Quebracho (Schinopsis spp.) wood extract, all containing tannins and currently used in the animal feed industry, were tested for in vitro antiviral activity against avian reovirus (ARV) and avian metapneumovirus (AMPV). The MTT assay was used to evaluate the 50% cytotoxic compounds concentration (CC₅₀) on Vero cells. The antiviral properties were tested before and after the adsorption of the viruses to Vero cells. Antiviral activities were expressed as IC₅₀ (concentration required to inhibit 50% of viral cytopathic effect). CC₅₀s of tested compounds were >200 μg/ml. All compounds had an extracellular antiviral effect against both ARV and AMPV with IC₅₀ values ranging from 25 to 66 μg/ml. Quebracho extract had also evident intracellular anti-ARV activity (IC₅₀ 24 μg/ml). These preliminary results suggest that the examined vegetable extracts might be good candidates in the control of some avian virus infections. Nevertheless further in vivo experiments are required to confirm these findings.     

Assessing the effectiveness of intubation as a challenge model in contagious bovine pleuropneumonia vaccine experiments

A study was carried out to assess the effectiveness of a bronchoscope in administering a pathogenic field strain of Mycoplasma mycoides subsp. mycoides (MmmSC) in cattle challenge experiments. Out of 16 animals inoculated using the bronchoscope, 10 (62.2%) showed clinical disease as evidenced by fever and 15 (93.8%) displayed typical lesions of CBPP from which MmmSC was isolated. Serum samples collected weekly were tested by Complement Fixation Test (CFT) and competitive enzyme-linked immunosorbent assay (c-ELISA). Antibodies to MmmSC were detected in 10 out of the 16 animals by the CFT and 11 out of the 16 animals by c-ELISA. The onset of clinical disease was as early as 2 days post-inoculation, and most of the animals developed clinical disease 2 to 3 weeks post-infection. These results clearly demonstrate that nasotracheal inoculation of pathogenic strain of MmmSC with the aid of a bronchoscope can lead to early onset of clinical disease; similar to previous studies but with higher numbers of animals showing clinical disease. This is in contrast with previous studies where early clinical disease was observed in as little as 15% of inoculated animals. This nasotracheal inoculation method using a bronchoscope can, therefore, be adopted for use in experimental challenge infections of cattle. This method is found to be a better replacement to the contact transmission method whose drawback includes extra cost of donor animals and unpredictable rate and timing of transmission from intubated to challenge animals.     

共表达K88/987p菌毛产肠毒素性大肠杆菌的分离鉴定及其卵黄抗体的制备

为制备针对一些大型养猪场产肠毒素性大肠杆菌(enterotrxigenic Escherichia coli,ETEC)分离株的特异性卵黄抗体(egg yolk immunoglobulin,IgY),试验对从这些养殖场分离的ETEC分离株菌毛基因类型进行了PCR鉴定,纯化该分离株的菌毛蛋白免疫蛋鸡制备IgY,对该IgY的效价、特异性和体外抑菌效果进行了检测。结果表明,该分离株具有K88和987p 两种菌毛基因,纯化后的分离株菌毛具有较强的免疫原性,经3次免疫后产生的IgY对K88和987p全菌和菌毛的效价可达到1:64 000,分离株菌毛IgY能特异地与K88和987p反应,与K99、F41无交叉反应,5 mg/mL分离株菌毛IgY在体外具有很好的抑菌效果。