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.     

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.     

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.     

Variable number tandem repeat (VNTR) typing of strains of Mycoplasma mycoides subspecies mycoides small colony isolated from the north-eastern regions of Italy between 1990 and 1993

The origin of the outbreaks of contagious bovine pleuropneumonia (CBPP) in Italy between 1990 and 1993 were never successfully traced mainly due to the close similarity of the strains of the causative mycoplasma, Mycoplasma mycoides subspecies mycoides small colony (MmmSC) and the limitations of the typing tools available at the time. In this report we examined a selection of strains isolated in the Veneto and Friuli Venezia Giulia regions of Italy by the highly discriminatory variable number tandem repeat (VNTR) procedure. Results were analysed for the first time by a capillary sequencer-based method. It was shown that all the MmmSC strains were genetically very similar and all belonged to the same profiles for both VNTR 4 and 5. This suggests that the outbreaks in Northern Eastern Italy, which eventually spread to other parts of the country, originated from a single source.     

Characterization of the in vitro core surface proteome of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host–pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host–pathogen interactions.     

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.     

Mycoplasma synoviae invades non-phagocytic chicken cells in vitro

Mycoplasma synoviae and Mycoplasma gallisepticum are major poultry pathogens, but their strains differ significantly in invasiveness and pathogenicity. Recent studies have demonstrated that M. gallisepticum invades chicken erythrocytes (CER) and chicken embryonic fibroblasts. The aim of this study was to determine whether M. synoviae also invades chicken cells. Using the gentamicin invasion assay, relative invasion frequency (RIF) of four M. synoviae strains was determined for CER, chicken embryonic cell line (CEC-32) and/or primary chicken chondrocytes (CCH). All tested strains of M. synoviae were capable of invading chicken cells within 24 h after infection. The type strain WVU 1853 showed significantly higher invasiveness in CER (RIF 7.5 ± 1.5%) and CEC-32 (RIF 7.0 ± 0.3%) than field strain ULB 02/T6 and M. gallisepticum strain R_low. Surprisingly, WVU 1853, which is capable of causing synovitis and arthritis in chickens, was less invasive for CCH with a RIF (1.2 ± 0.3%) similar to that of R_low (1.1 ± 0.1%). This is the first study documenting the invasiveness of M. synoviae strains for non-phagocytic chicken cells.     

Seroprevalence of antibodies to Encephalitozoon cuniculi and Encephalitozoon intestinalis in humans and animals

The presence of antibodies against Encephalitozoon cuniculi (E. cuniculi) and Encephalitozoon intestinalis (E. intestinalis) was examined in 215 samples from humans and in 488 samples from five different species of domestic and companion animals in Slovakia. The 215 human samples and samples from 90 swine, 123 non-infected cattle (cattle), 24 cattle infected with bovine leukosis virus (BLV-positive cattle), 140 sheep and 111 dogs were examined by the enzyme-linked immunosorbent assay (ELISA). Samples with serum titres 1:200 or higher were considered as positive. Specific anti-E. cuniculi antibodies were found in humans (0.9%), swine (52%), cattle (2%), sheep (9%) and dogs (15%) except for the BLV-positive cattle at the titre of 1:200. The titre of 1:400 was detected only in humans (0.5%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:200 was confirmed in humans (6%), swine (51%), cattle (11%), BLV-positive cattle (13%) and dogs (6%) but not in sheep. The anti-E. intestinalis antibodies reached the 1:400 in humans (1%), swine (4%) and BLV-positive cattle (17%). The presence of specific anti-E. intestinalis antibodies at the titre of 1:600 was observed only in one swine (1%). Significant differences were observed in animals at titres 1:200 and 1:400 (chi-squared test: p < 0.0001) for both pathogens and in humans only for E. cuniculi at the titre of 1:400 (chi-squared test: p < 0.0075).     

A recombinant chimera composed of repeat region RR1 of Mycoplasma hyopneumoniae adhesin with Pseudomonas exotoxin: in vivo evaluation of specific IgG response in mice and pigs

Using the binding and translocation domain of Pseudomonas exotoxin A [domain III deleted PE termed PE(ΔIII)] as a vehicle, this study characterized and evaluated a novel application of PE toxin in Mycoplasma hyopneumoniae adhesin used as an immunogen. PCR and sequence analysis revealed that 16 copies of AAKPV(E) in tandem repeat region 1 (RR1) of M. hyopneumoniae 97 kDa adhesion were successfully fused to the downstream of PE(ΔIII) to create a subunit vaccine, i.e. PE(ΔIII)-RR1. This chimeric protein, over-expressed in inclusion bodies of E. coli BL21(DE3)pLysS, was characterized by a monoclonal antibody (MAb) F2G5 prepared against RR1 of the 97 kDa adhesin and was readily purified. The data indicated that the epitope recognized by MAb F2G5 was located in the structure of PE(ΔIII)-RR1. Using ELISA and Western blot analyses, the specific IgG immune response against RR1 and whole adhesin in mice immunized with PE(ΔIII)-RR1 was found more marked than that in mice immunized with the M. hyopneumoniae whole cells. Similarly, PE(ΔIII)-RR1 also stimulated a remarkable IgG response against RR1 in pigs compared to that in pigs immunized with the conventional M. hyopneumoniae vaccine. The PE(ΔIII)-RR1 would be potentially useful for the future development of a M. hyopneumoniae adhesin vaccine.     

Natural infection by Cryptosporidium sp., Giardia sp. and Eimeria leuckarti in three groups of equines with different handlings in Rio de Janeiro, Brazil

To detect Cryptosporidium sp., Giardia sp. and Eimeria leuckarti in horses, fecal samples were collected from three different handling horse groups from the state of Rio de Janeiro, Brazil. Group A was composed of “Mangalarga Marchador” pure breed horses, Group B was formed by horses of a Military Corporation and Group C by stray horses captured by the Center of Zoonosis Control Paulo Dacorso Filho. A total of 396 fecal samples were collected, 212 samples from Group A, 154 samples from Group B and 30 from Group C. The material was submitted to the centrifugation - flotation technique and staining by the safranin-methylene blue technique and analyzed. Oocysts of Cryptosporidium sp. were identified in 0.75% of the samples (n = 3); cysts of Giardia sp. in 0.5% (n = 2) and oocysts of E. leuckarti in 0.5% (n = 2). One case of E. leuckarti in group A and one of Cryptosporidium sp. in group B were observed. In group C were observed two cases of Cryptosporidium, two of Giardia and one of E. leuckarti,. Horses of group C were more parasitized by the three protozoans than animals from the other groups (p < 0.01). It was possible to verify that factors related to the animals, like host individual susceptibility and sanitary factors may influence the occurrence of natural infections by gastrointestinal protozoans, although the age did not have influence. This study reports, for the first time, the occurrence of Cryptosporidium sp., Giardia sp. and E. leuckarti in equines of the State of Rio de Janeiro.     

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.     

Echinococcosis in pigs and intestinal infection with Echinococcus spp. in dogs in southwestern Lithuania

Cystic echinococcosis is a major emerging zoonosis in many Eastern European and Asian countries. Post slaughter examinations of 684 pig livers in Lithuania revealed significantly higher numbers of Echinococcus granulosus infections in animals from family farms (13.2%; 95% CI 10.7–16.2) as compared with those from industrial farms (4.1%; 95% CI 0.8–11.5). The prevalence was also significantly higher in pigs older than 1 year than in younger ones. In addition, in 0.5% of the pigs from the family farms, infertile and calcified E. multilocularis lesions were identified by PCR. Faecal samples from rural dogs (n = 240) originating from 177 family farms in 12 villages were investigated for taeniid eggs with two methods. Significantly more dogs excreting taeniid eggs were diagnosed with the flotation/sieving method (n = 34) as compared to the modified McMaster method (n = 12). Multiplex PCR performed with DNA from taeniid eggs isolated from faeces of 34 dogs revealed 26 infections with Taenia spp., 9 with E. granulosus and 2 with E. multilocularis (4 cases with concurrent Taenia spp. and E. granulosus or E. multilocularis infections). Genotyping of E. granulosus cyst tissues from 7 pigs, 1 head of cattle and from E. granulosus eggs from 8 dog faeces revealed the genotype G6/7 (‘pig/camel strain’) in all cases. The high infection pressure with Echinococcus spp. in family farms necessitates initiating control programs.     

Contagious bovine pleuropneumonia in northern tanzania,culture confirmation and serological studies

Summary After an absence of about 25 years contagious bovine pleuropneumonia (CBPP) appeared again in 1990 in Tanzania. It was preceded by a spread in Kenya to an area bordering Tanzania. Due to the frequent cattle movements across the border it was soon introduced into Loliondo in northern Tanzania. One month after the first cases, CBPP was suspected in a total of 9 herds comprising 1,500 cattle. However, few animals showed clear clinical signs and frequent antibiotic treatment at an early stage further obscured the clinical picture. In one herd with acute cases, the diagnosis was confirmed by autopsy andMycoplasma mycoides subsp.mycoides, SC type, was isolated. From this herd several serum samples were positive in the complement fixation test and gave high absorbance values in an ELISA withM. mycoides subsp.mycoides antigen. From 5 other herds with suspected cases blood samples were negative by the complement fixation test but in the enzyme-linked immunosorbent assay at least one in each herd was positive.

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Pleuroneumonia Contagiosa Bovina En Tanzania Septentrional, Confirmacion Mediante Cultivo Y Estudios Serologicos

Resumen Después de un período de 25 años sin ningún brote, la pleuroneumonía contagiosa bovina apareció de nuevo en Tanzania en 1990, después de que la enfermedad se extendiera a una zona de Kenya fronteriza con Tanzania. Debido a los frecuentes movimientos de ganado vacuno a través de la frontera, la enfermedad se declaró pronto en Loliondo, en el norte de Tanzania. Un mes después de los primeros casos, 9 rebaños, que contabilizaban en total 1500 animales, eran sospechosos de estar infectados. Sin embargo, pocos animales mostraron síntomas clínicos claros y et tratamiento precoz con antibióticos contribuyó a disminuir la intensidad de las manifestaciones clinicas. En un rebaño en el que hubo casos agudos el diagnóstico se confirmó mediante necropsia y se aislóMycoplasma mycoides subsp.mycodies del tipo SC. Varias muestras de sangre de este rebaño dieron resultado positivo en el test de fijación del complemento y dieron valores de absorbancia altos en un test de ELISA conM. mycoides subsp.mycoides. Las muestras de sangre provenientes de otros 5 rebaños en los que se sospechó la existencia de la enfermedad dieron resultado negativo en el test de fijación de complemento mientras que al menos un animal de cada rebaño dio resultado positivo en el test ELISA.

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Peripneumonie Contagieuse Bovine Dans Le Nord De La Tanzanie, Confirmation Par Culture Et Etudes Serologiques

Résumé Après une absence d'environ 25 ans la péripneumonie contagieuse bovine est de nouveau apparue en 1990 en Tanzanie. Elle a été prècedée d'une progression au Kenya vers une zone bordant la Tanzanie. Par suite de mouvements frontaliers fréquents du bétail, elle a été beintôt introduite dans la région de Loliondo, dans le nord de la Tanzanie. Un mois après les premiers cas, 9 troupeaux totalisant 1500 têtes ont été supectés. Cependant, peu d'animaux ont présenté des signes cliniques nets et, de surcroît, les traitements antibiotiques entrepris en début de maladie obscurissent le tableau clinique. Le diagnostic a été confirmé à l'autopsie dans un troupeau présentant des cas cliniques etMycoplasma mycoides subsp.mycoides, type SC, a été isolé. Plusieurs échantillons de sérum de ce troupeau ont été positifs en fixation du complément et ont donné de grandes valeurs d'absorption dans un test ELISA avec un antigèneM. mycoides subsp.mycoides. Pour 5 autres troupeax avec des cas suspects, les échantillons de sang ont été négatifs en fixation du complément mais un au moins pour chaque troupeaux a été positif dans un test ELISA.

     

In vitro antimycoplasmal activity of six Jordanian medicinal plants against three Mycoplasma species

The in vitro effect of six Jordanian traditional medicine plant methanolic extracts were tested against 32 isolates of Mycoplasma species; Mycoplasma mycoides subsp. mycoides LC (6), Mycoplasma capricolum subsp. capricolum (8) and M. putrefaciens (18), all isolated from either nasal swabs or milk, from sheep and goats in different regions in Jordan. All Mycoplasma species showed susceptibility to Artemisia herba-alba and Artemisia arborescens with MIC ranges from 3.125–12.5 mg/ml. Allium sativum and Punica grantum showed limited activity against some Mycoplasma isolates. Olea europea and Citrullus colocynthis showed no in vitro activity against any of the Mycoplasma species tested. Artemisia herba-alba and Artemisia arborescens may therefore be useful for the treatment of mycoplasma infections. The authors are British Civil Servants and as such their work is subject to British Crown Copyright. This means the exclusive copyright for the article cannot be transferred.     

Functional and antigenic properties of GlpO from Mycoplasma mycoides subsp. mycoides SC: characterization of a flavin adenine dinucleotide-binding site deletion mutant

L-α-glycerophosphate oxidase (GlpO) plays a central role in virulence of Mycoplasma mycoides subsp. mycoides SC, a severe bacterial pathogen causing contagious bovine pleuropneumonia (CBPP). It is involved in production and translocation of toxic H₂O₂ into the host cell, causing inflammation and cell death. The binding site on GlpO for the cofactor flavin adenine dinucleotide (FAD) has been identified as Gly ₁₂−Gly₁₃−Gly ₁₄−Ile₁₅−Ile₁₆−Gly ₁₇. Recombinant GlpO lacking these six amino acids (GlpOΔFAD) was unable to bind FAD and was also devoid of glycerophosphate oxidase activity, in contrast to non-modified recombinant GlpO that binds FAD and is enzymatically active. Polyclonal monospecific antibodies directed against GlpOΔFAD, similarly to anti-GlpO antibodies, neutralised H₂O₂ production of M. mycoides subsp. mycoides SC grown in the presence of glycerol, as well as cytotoxicity towards embryonic calf nasal epithelial (ECaNEp) cells. The FAD-binding site of GlpO is therefore suggested as a valuable target site for the future construction of deletion mutants to yield attenuated live vaccines of M. mycoides subsp. mycoides SC necessary to efficiently combat CBPP.     

Local and systemic cytokine responses during larval penetration in cattle experimentally infested with Hypoderma lineatum (Diptera: Oestridae)

Local and systemic cytokine responses were studied in 3 groups of cattle, with 4 animals each, experimentally infested with Hypoderma lineatum (De Villers) first instars (L1). The first group was undergoing a primary infestation (G-1), the second group was undergoing a secondary infestation (G-2) and the third group was infested for their third consecutive year (G-3). Cattle were infested with 25 L1 deposited on the skin. Blood and skin samples were taken at 0, 6, 12, 48, 96 and 144 h post-infestation (h.p.i.). Interleukin 10 (IL-10), IL-4 and interferon gamma (IFN-γ) production was studied by immunohistochemistry and sandwich ELISAs. IL-4⁺ cells showed a significant increase at 6 h.p.i. in both reinfested groups (G-2 and G-3) when compared with G-1. In all groups the number of IL-4⁺ cells decreased significantly at 48 h.p.i. IL-10⁺ cells increased in G-1 at 6 and 48 h.p.i., whereas in both reinfested groups increased at 12 h.p.i. with a peak at 48 h.p.i. IFN-γ⁺ cells showed a significant increment at 6 h.p.i. in all groups, followed by a rapid descent at 12 (G-1 and G-2) and 48 h.p.i. (G-3). Penetration of the skin by H. lineatum did not have any significant effect on IFN-γ serum concentrations and, except for IL-10 there were no correlation between local production and serum concentrations of cytokines. The increase of both Th1 (IFN-γ) and Th2-type cytokines (IL-4 and IL-10) indicates that bovine T-cell response during the first phases of the infestation by H. lineatum is apparently a Th0 response.     

Development of an improved vaccine for contagious bovine pleuropneumonia: an African perspective on challenges and proposed actions

Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides (Mmm) is an economically very important cattle disease in sub-Saharan Africa. CBPP impacts animal health and poverty of livestock-dependent people through decreased animal productivity, reduced food supply, and the cost of control measures. CBPP is a barrier to trade in many African countries and this reduces the value of livestock and the income of many value chain stakeholders. The presence of CBPP also poses a constant threat to CBPP-free countries and creates costs in terms of the measures necessary to ensure the exclusion of disease. This opinion focuses on the biomedical research needed to foster the development of better control measures for CBPP. We suggest that different vaccine development approaches are followed in parallel. Basic immunology studies and systematic OMICs studies will be necessary in order to identify the protective arms of immunity and to shed more light on the pathogenicity mechanisms in CBPP. Moreover a robust challenge model and a close collaboration with African research units will be crucial to foster and implement a new vaccine for the progressive control of this cattle plague.     

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.     

Effect of Staphylococcus aureus and Streptococcus uberis on apoptosis of bovine mammary gland lymphocytes

The aim of this study was to determine whether lymphocyte apoptosis is modulated by infections caused by Staphylococcus aureus and Streptococcus uberis. Samples of cell populations were obtained by lavage of the mammary glands at 4 intervals (24, 48, 72 and 168 h) following infection. The percentage of apoptotic lymphocytes peaked at 168 h after challenge with S. aureus or S. uberis. Subsequent experiments focused on in vitro cultivation of mammary gland lymphocytes with S. aureus and S. uberis. These experiments showed a lower percentage of apoptotic lymphocytes following 3 h of cultivating cells with bacteria than after cultivation without bacteria. The results demonstrate that during both experimental infection of bovine mammary glands with S. aureus or S. uberis and during in vitro cultivation of lymphocytes with S. aureus or S. uberis, apoptosis of lymphocytes is delayed.