[Present Situation of Contagious Bovine Pleuropneumonia in Central African Republic]. / Situation Actuelle de la Péripneumonie Contagieuse Bovine en République Centrafricaine.

Aims: The aim of this study was to analyse the current situation of Contagious Bovine Pleuropneumonia (CBPP) in the Central African Republic (CAR) by seroprevalence analysis, as well as isolation and characterization of strains of the etiologic agent, Mycoplasma mycoides mycoides (Mmm), circulating in livestock breeding regions. Material and methods: The strains obtained were subjected to whole genome sequencing by Illumina technology and genotyped using the eMLST technique based on 62 genes of the Mmm core genome. Their sensitivity to tetracycline was assessed by determination of the minimum inhibitory concentration (MIC) on agar. A seroprevalence analysis by competitive ELISA was conducted in livestock breeding regions (West, Centre and East CAR), including both zebu and taurine cattle breeds, and both males and females. Results: The three strains isolated in the three regions of the CAR shared exactly the same genomic sequence. Phylogenetic analysis showed that they were closely related to a strain isolated in the CAR in 1991, also sequenced in this study, and clustered with Mmm strains originating from East and Central Africa. The recent isolates presented increased MIC values, though they were still sensitive to tetracycline. The global CBPP prevalence in the CAR was estimated at 12.5% with no significant differences observed between cattle breeding regions, nor between males and females. However, a significantly higher prevalence was observed in zebu compared to taurine cattle, most likely in relation to their herding system based on cattle transhumance and nomadic pastoralism. Conclusion: CBPP is enzootic in the CAR in spite of control campaigns based on use of the live T1 vaccines, which have shown little efficacy due to poor implementation in the field. New strategies combining controlled use of antibiotics and inactivated vaccines, with increased thermostability, should be well received by livestock keepers and allow a better control of CBPP in the region. The fact that the recent Mmm isolates are still resistant to tetracycles is encouraging.

Fatal transmission of contagious caprine pleuropneumonia to an Arabian oryx (Oryx leucoryx).

Contagious caprine pleuropneumonia (CCPP) is an infectious respiratory disease mainly affecting domestic goats. As CCPP has never been documented in grazing antelopes (subfamily hippotraginae), they were not considered susceptible. Mycoplasma capricolum subspecies capripneumoniae (Mccp) was isolated from pleural liquid collected during the necropsy of a severely emaciated Arabian oryx with mild nasal discharge. The Mccp isolate was then genotyped using a multilocus sequence scheme; the sequence type was identical to the Mccp strain previously identified in a sand gazelle from a nearby enclosure. This case shows for the first time that members of the hippotraginae subfamily, here the Arabian oryx, can be affected by CCPP. In addition, genotyping shows that the oryx was most probably infected, at a distance, by sand gazelles.

Variable Number Tandem Repeat (VNTR) analysis reveals genetic diversity within Mycoplasma mycoides mycoides small colony isolates from Nigeria.

A Variable Number Tandem Repeat (VNTR) analysis was conducted on thirteen (13) M. mycoides mycoides Small Colony isolates from Nigeria using Tandem Repeat (TR) 34 which is a predicted lipoprotein located within the hypothetical protein MAG6170. The analysis revealed diversity within the M. mycoides mycoides Small Colony isolates with five different VNTR types indicated. Some correlation was determined between the VNTR types and their geographical origin. VNTR analysis may represent a useful, rapid first-line test for use in molecular epidemiological analysis of M. mycoides mycoides Small Colony for possible outbreak tracing and disease control.

Mycoplasma leachii sp. nov. as a new species designation for Mycoplasma sp. bovine group 7 of Leach, and reclassification of Mycoplasma mycoides subsp. mycoides LC as a serovar of Mycoplasma mycoides subsp. capri.

The Mycoplasma mycoides cluster consists of six pathogenic mycoplasmas causing disease in ruminants, which share many genotypic and phenotypic traits. The M. mycoides cluster comprises five recognized taxa: Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), M. mycoides subsp. mycoides Large Colony (MmmLC), M. mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capricolum (Mcc) and M. capricolum subsp. capripneumoniae (Mccp). The group of strains known as Mycoplasma sp. bovine group 7 of Leach (MBG7) has remained unassigned, due to conflicting data obtained by different classification methods. In the present paper, all available data, including recent phylogenetic analyses, have been reviewed, resulting in a proposal for an emended taxonomy of this cluster: (i) the MBG7 strains, although related phylogenetically to M. capricolum, hold sufficient characteristic traits to be assigned as a separate species, i.e. Mycoplasma leachii sp. nov. (type strain, PG50(T) = N29(T) = NCTC 10133(T) = DSM 21131(T)); (ii) MmmLC and Mmc, which can only be distinguished by serological methods and are related more distantly to MmmSC, should be combined into a single subspecies, i.e. Mycoplasma mycoides subsp. capri, leaving M. mycoides subsp. mycoides (MmmSC) as the exclusive designation for the agent of contagious bovine pleuropneumonia. A taxonomic description of M. leachii sp. nov. and emended descriptions of M. mycoides subsp. mycoides and M. mycoides subsp. capri are presented. As a result of these emendments, the M. mycoides cluster will hereafter be composed of five taxa comprising three subclusters, which correspond to the M. mycoides subspecies, the M. capricolum subspecies and the novel species M. leachii.

Detection of contagious caprine pleuropneumonia in East Turkey.

A study was implemented to investigate the presence of contagious caprine pleuropneumonia (CCPP) in East Turkey. This study was based on clinical surveillance in the field, surveillance at regional slaughterhouses and regular submission of suspected lesions to regional laboratories. The results showed that the agent of CCPP, Mycoplasma capricolum subspecies capripneumoniae (Mccp), could be detected by culture and specific polymerase chain reaction from 37.5% (12/32) of lung samples taken from goats of ten different herds. This agent was also isolated from two of 13 sheep samples (one from the lung and the other from a nasal swab). Mycoplasma capricolum subsp. capripneumoniae was isolated in pure culture and characterised at a finer molecular level. The East Turkish isolate was found to be closely related to another strain of Turkish origin, as well as to Mccp strains isolated in Tunisia. The isolation of Mccp from sheep lung lesions brings the strict host-specificity of this pathogen into question. It may also indicate that Mccp presents a risk for wildlife in the region. Such results, the authors believe, demonstrate that adequate risk assessments should be undertaken in Turkey and neighbouring countries.

Real-time polymerase chain reaction assays for the detection of members of the Mycoplasma mycoides cluster.

AIM: To develop real-time PCR assays for the detection and differentiation of members of the Mycoplasma mycoides cluster. METHODS: Five real-time PCR assays were designed to allow differentiation of members of the M. mycoides cluster: an assay for detection of the M. mycoides subspecies, viz M. mycoides subsp mycoides large colony (MmmLC), M. mycoides subsp capri (Mmc), and M. mycoides subsp mycoides small colony (MmmSC); one for the detection of the M. capricolum subspecies, viz M. capricolum subsp capricolum (Mcc), M. capricolum subsp capripneumoniae (Mccp), and Mycoplasma sp bovine group 7 (BG7); and three for the specific detection of MmmSC, Mccp, and BG7. A panel of 74 Mycoplasma isolates from various geographical origins and a panel of 21 other bacterial isolates were used to evaluate the sensitivity and specificity of the assays. RESULTS: The assays displayed 100% analytical sensitivity in detecting all target Mycoplasma isolates. The analytical detection limit for the assays to detect the M. mycoides subspecies, M. capricolum subspecies, and MmmSC was determined to be 100 fg of genomic DNA, while the Mccp and BG7 assays had a detection limit of 100 fg and 10 fg of genomic DNA, respectively. The M. mycoides subspecies assay had a detection limit of 10(3) (SD 10(2)) cfu/ml milk, 10(4) (SD 10(4)) cfu per swab, and 10(3) (SD 10(3)) cfu/g lung in inoculated samples. The assays displayed 100% specificity when applied to non-target bacterial isolates and to 110 culture-negative milk samples. CONCLUSIONS: The assays were highly sensitive and specific, and provide accurate detection and differentiation of the members of the M. mycoides cluster.

A PCR for the detection of mycoplasmas belonging to the Mycoplasma mycoides cluster: application to the diagnosis of contagious agalactia.

Contagious agalactia is a mycoplasmal infection caused by Mycoplasma agalactiae, Mycoplasma mycoides subsp. mycoides LC, M. mycoides subsp. capri, Mycoplasma capricolum subsp. capricolum and Mycoplasma putrefaciens. Identification of the causative organisms is usually performed by isolation and classical biochemical and serological tests, though this is a lengthy and cumbersome process for mycoplasmas. Specific PCR assays have been developed for the identification of Mycoplasma agalactiae and M. putrefaciens. For members of the M. mycoides cluster existing PCR tests are based on the amplification of highly conserved genes coding for ribosomal proteins, hence a possibility of cross-reactions. The gene glk, coding for a glucokinase, that is found in this cluster is very distantly related to any other bacterial glucokinase described so far. It was therefore chosen as target to design a new PCR test. The validation was performed independently in three laboratories in France and India using over 100 mycoplasma strains of various geographical origins. All strains belonging to the M. mycoides cluster were detected by amplification of the expected PCR product (428 bp) while no amplification was obtained from M. agalactiae strains. Our results demonstrate the universality of this PCR in spite of the great heterogeneity found within this cluster. This new tool may be of great help for the implementation of control measures directed towards contagious agalactia.

A serological investigation into contagious caprine pleuropneumonia (CCPP) in Ethiopia.

For a comparison of serological tests for CCPP, sera from 767 goats were examined. They were subjected to three tests: complement fixation test (CFT) with Mycoplasma capricolum subspecies capripneumoniae antigen; blocking ELISA (B-ELISA) with Mycoplasma capricolum subspecies capripneumoniae antigen; and CFT with Mycoplasma mycoides subspecies mycoides small colony type antigen. Antibodies were detected by these three tests in 23%, 2% and 12%, respectively, of sera from districts in which CCPP had not been reported, and in 60%, 83% and 87%, respectively, in sera from areas in which CCPP had been reported. The specificity of the tests is discussed. The use of the B-ELISA test for the diagnosis and for epidemiological studies of CCPP is strongly recommended.

A specific PCR for the identification of Mycoplasma capricolum subsp. capripneumoniae, the causative agent of contagious caprine pleuropneumonia (CCPP).

Contagious caprine pleuropneumonia is a severe infectious disease of goats in Africa and the Middle East. It is caused by a fastidious mycoplasma, Mycoplasma capricolum subsp. capripneumoniae, a member of the "M. mycoides cluster". Members of this cluster share genomic and antigenic features, which result in common biochemical and serological properties, complicating species identification. Two species of this cluster, M. mycoides subsp. capri and M. mycoides subsp. mycoides large colony biotype, are very often isolated from clinical cases resembling contagious caprine pleuropneumonia. Furthermore, in the laboratory, M. capricolum subsp. capripneumoniae can be easily confused with the closely related capricolum subspecies. Considering these constraints and the scarcity of available methods for identification, a specific polymerase chain reaction was developed. A DNA fragment of 7109 bp containing genes coding for the arginine deiminase pathway (ADI) was chosen as target sequence for the selection of a specific primer pair. The full ADI operon from M. capricolum subsp. capripneumoniae strain GL100 was sequenced. Polymorphism within this locus was analyzed by comparison with the sequence from the closely related IPX strain (M. capricolum subsp. capricolum). It varied from 0.6% to 3.5%. The highest divergence was found in a region coding for arcD. Therefore, this gene was chosen as target for the specific amplification of a 316 bp-long DNA fragment. The specificity of this PCR was validated on 14 M. capricolum subsp. capripneumoniae strains and 27 heterologous strains belonging to the "M. mycoides cluster" and M. putrefaciens. This new PCR will be a valuable tool for the surveillance of contagious caprine pleuropneumonia.

Contagious bovine pleuropneumonia vaccines and control strategies: recent data.

Contagious bovine pleuropneumonia is one of the most threatening transboundary cattle disease in Africa. However, with the exception of Botswana, very few African countries were able to implement eradication strategies for this disease, after it had recently re-infected a number of countries. Previous experimental studies have shown that emergency vaccination campaigns, based on a single injection, were not inducing a sufficient protection level to prevent further spread of the disease. In addition, post-vaccinal reactions were sometimes reported in the field when using vaccine strain T1/44, leading cattle owners to refuse the vaccination. On the contrary, antibiotics are used quite often in the field but there are insufficient data to assess their efficacy properly. Therefore experimental studies were implemented: (i) to check if higher dosages of the vaccine would be able to induce higher protection rates and (ii) to elucidate the origin of the post-vaccinal reactions observed with T1/44 and (iii) to gain preliminary results on the efficacy of long-acting tetracycline. The first experiment included the use of three doses of vaccine strains T1/44 and T1sr: 10(7), 10(8) and 10(9) mycoplasmas per dose. T1/44 seemed to induce a higher protection (70%) than T1sr (60%). However, there was no observable dose effect for these vaccine strains. The second experiment was performed by injecting various MmmSC strains subcutaneously into susceptible cattle. One of these strains was an isolate obtained from a "Willems" reaction following a vaccination with T1/44. This isolate, called T1B, induced typical invading oedema at the injection site in a similar way to the pathogenic strain, whereas the original T1/44 vaccine strain did not. These findings indicate that the strain has reverted to virulence. Finally the antibiotic trials showed that long-acting tetracycline was able to reduce the losses due to the disease but could not prevent the persistence of viable MmmSC in treated animals. The consequences of these findings are discussed. They reinforce the need for additional research on new vaccines able to elicit longer lasting protection. However, once continuing additional field research is obtained, it should allow better defined strategies to be put in place. Meanwhile, immediate action should be taken to prevent the further spread of CBPP in the Southern part of Africa.

Experimental contagious caprine pleuropneumonia: a long term study on the course of infection and pathology in a flock of goats infected with Mycoplasma capricolum subsp. capripneumoniae.

Contagious caprine pleuropneumonia (CCPP) is a major threat to goat farming in parts of Africa and Asia. It classically causes acute high morbidity and mortality early in infection, but little is known of its long term epizootiology and course. In this study, 10 goats were inoculated with Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae) and then mixed with 15 goats for contact transmission. The disease course was monitored in each goat for 56-105 days, whereafter the goats were killed and necropsied. Varying features signifying infection occurred in altogether 17 goats (7 inoculated, 10 in-contact). Clinical signs were severe in 8 goats but no fatalities occurred. Only 6 goats had serum antibody titres against M. capripneumoniae in ELISA. Fourteen goats (5 inoculated, 9 in-contact) had chronic pleuropulmonary lesions compatible with CCPP at necropsy and 7 of those showed M. capripneumoniae antigen in the lung by immunohistochemistry. Neither cultivation nor PCR tests were positive for the agent in any goat. The results indicate that the clinical course of CCPP in a flock may be comparatively mild, M. capripneumoniae-associated lung lesions may be present at a late stage of infection, and chronic infection may occur without a significant serological response.

Contagious bovine pleuropneumonia vaccines, historic highlights, present situation and hopes.

Contagious bovine pleuropneumonia (CBPP) is a contagious infection of cattle caused by a mycoplasma, M. mycoides subsp. mycoides SC (MmmSC). It induces lesions of pleuropneumonia in acute cases and the formation of pulmonary "sequestra" in chronic cases. The disease is prevalent mostly in Africa, where it is responsible for high losses, but it has also been sporadically present in Southern Europe until 1999. Vaccination is now prohibited in most countries except in Africa. An empirical "inoculation" procedure was developed as early as 1852 in Europe but it may have been used even earlier in Africa. The inoculation of pleural fluid was performed at the tip of the tail in Europe and on the bridge of the nose in Africa. It conferred good protection but induced a high number of fatal cases. Various inactivated preparations have been tested in the past with inconclusive results leading sometime to some protection and some other time to a sensitisation of the immunised animals. Such preparations have never been used in the field. Attenuated MmmSC strains have been developed in the 1950s and used extensively in the field both in Africa and Australia. The best known vaccine strains are KH3J, T1/44 and T1sr. Vaccination campaigns have succeeded in reducing considerably the CBPP prevalence in these two continents but eradication was achieved in Australia only by switching to strict measures of animal movement control and a stamping-out policy. The search for new CBPP vaccines has become a major issue for African countries that are facing an increase in outbreaks. The rationale for this search is based on a better understanding of the mycoplasma virulence mechanisms that could lead to a targeted attenuation of MmmSC strains. It is also based on a better understanding of the bovine immune response that may be driven to a pathogenic inflammatory response or conversely to a better balanced response leading to protection.

A specific PCR for the detection of Mycoplasma putrefaciens, one of the agents of the contagious agalactia syndrome of goats.

Mycoplasma putrefaciens is listed as one of the etiologic agents of the contagious agalactia syndrome by the world organisation for animal health. This species has been characterized only recently, 1974, and the number of outbreaks caused by this microorganism so far is very scarce. It induces mastitis in infected goats although other symptoms such as arthritis in adults and septicaemia in kids are also frequently described. Up to now, the identification of M. putrefaciens relied on classical isolation and identification techniques which present a number of limitations. Specific primers for PCR have been designed based on sequence comparisons of the ArcB gene among the 'Mycoplasma mycoides cluster' and related species such as Mycoplasma cottewii and Mycoplasma yeatsii. Sequence alignments confirmed the taxonomic position of M. putrefaciens, which is related to the 'M. mycoides cluster' but also very close to M. yeatsii. The polymorphism observed amongst the different ArcB sequences allowed the determination of a primer pair yielding a specific amplification of a 316 bp-long DNA fragment by PCR. This PCR was validated in two different laboratories with a variety of mycoplasma strains isolated from goats. This new PCR technique will be very useful for a quicker determination of M. putrefaciens strains as well as a better understanding of the prevalence of M. putrefaciens infections.

Molecular epidemiology of contagious bovine pleuropneumonia by multilocus sequence analysis of Mycoplasma mycoides subspecies mycoides biotype SC strains.

Contagious bovine pleuropneumonia is a bacterial disease caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC), and included in list A of the Office International des Epizooties. It is one of the major constraints to cattle raising in sub-Saharan and south-western Africa and also a threat to all countries currently free of the disease. MmmSC strains were considered very homogeneous until 1995, when various techniques such as enzymatic restriction of whole DNA or Southern blotting showed that this was not the case. These techniques are unfortunately difficult to standardize and require the extraction of DNA from an MmmSC culture. We therefore decided to investigate the possibility of constructing a molecular epidemiology tool based on multilocus sequence analysis (MLSA) with PCR amplification of various loci followed by sequencing. Six loci were found suitable for this purpose and an additional PCR was designed to detect the presence of an 8.8kb deletion described by others in some strains. Fifteen different MLSA profiles were evidenced in our study. They allowed a clear distinction between European, south-western African and sub-Saharan strains. In addition, the results obtained on strain PO1967 confirmed its European origin, even though it does not exhibit the 8.8kb deletion. This new tool for contagious bovine pleuropneumonia may prove particularly useful for identifying MmmSC strains in countries at risk from contamination. It can also easily be refined by adding more strains or other loci of interest.

Abattoir-based survey of contagious bovine pleuropneumonia in cattle in Turkey.

Blood samples collected from 945 cattle at four local abattoirs in Turkey were examined for contagious bovine pleuropneumonia (CBPP) by the complement fixation test (CFT) and competitive ELISA (cELISA). In addition, the carcases of the animals were examined macroscopically at the abattoirs and 62 lung samples which had lesions suggestive of CBPP were collected for bacteriological culture. To identify suspicious isolates the PCR was used in addition to the routine biochemical tests. By the CFT, two of the 945 serum samples were seropositive, and by the cELISA, four of them were seropositive. In the bacteriological culture of the lungs, growth was observed in 18 (29 per cent) of the samples by the observation of turbidity in the broths. However, when these broths were inoculated into an agar base, growth was observed in only three (4.8 per cent) samples. These isolates were identified as Mycoplasma species on the basis of biochemical tests. In the PCR analysis of DNA extracted from the broths, none of the isolates was identified as Mycoplasma mycoides subspecies mycoides small colony or one of the members of the M mycoides cluster, but amplification was obtained in only eight (44.4 per cent) of 18 samples, using Mycoplasma-genus specific primers. These DNA samples were examined further with primers specific to 16S rRNA and were then sequenced and compared with the databanks; DNA homologies at different levels were observed in five samples, with Mycoplasma alkalescens, Mycoplasma canadense, Mycoplasma bovis and Mycoplasma bovigenitalium.

Genetic evolution of Mycoplasma capricolum subsp. capripneumoniae strains and molecular epidemiology of contagious caprine pleuropneumonia by sequencing of locus H2.

Contagious caprine pleuropneumonia (CCPP) is a major threat to goat farming in developing countries. Its exact distribution is not well known, despite the fact that new diagnostic tools such as PCR and competitive ELISA are now available. The authors developed a study of the molecular epidemiology of the disease, based on the amplification of a 2400 bp long fragment containing two duplicated gene coding for a putative membrane protein. The sequence of this fragment, obtained on 19 Mycoplasma capricolum subsp. capripneumoniae (Mccp) strains from various geographical locations, gave 11 polymorphic positions. The three mutations found on gene H2prim were silent and did not appear to induce any amino acid modifications in the putative translated protein. The second gene may be a pseudogene not translated in vivo, as it bore a deletion of the ATG codon found in the other members of the "Mycoplasma mycoides cluster" and as the six mutations evidenced in the Mccp strains would induce modifications in the translated amino acids. In addition, an Mccp strain isolated in the United Arab Emirates showed a deletion of the whole pseudogene, a further indication that this gene is not compulsory for mycoplasma growth. Four lineages were defined, based on the nucleotide sequence. These correlated relatively well with the geographical origin of the strains: North, Central or East Africa. The strain of Turkish origin had a sequence similar to that found in North African strains, while strains isolated in Oman had sequences similar to those of North or East African strains. The latter is possibly due to the regular import of goats of various origins. Similar molecular epidemiology tools have been developed by sequencing the two operons of the 16S rRNA gene or by AFLP. All these various techniques give complementary results. One (16S rRNA) offers the likelihood of a finer identification of strains circulating in a region, another (H2) of determining the geographical origin of the strains. These tools can make a very useful contribution to understanding the epidemiology of CCPP.

Phage displayed peptides and anti-idiotype antibodies recognised by a monoclonal antibody directed against a diagnostic antigen of Mycoplasma capricolum subsp. capripneumoniae.

A monoclonal antibody (Mab 4.52) raised against Mycoplasma capricolum subsp. capripneumoniae (Mccp) cell lysate was used as a template to obtain substitute antigens recognised by its paratope. Two approaches were investigated: a 17-mer random peptide library displayed on the surface of a filamentous phage was screened by panning on the immobilised Mab 4.52 and anti-idiotype antibodies were generated by immunising a chicken with the F(ab')(2) fragments of the antibody. Analysis of the peptide sequences displayed by the isolated phages identified two peptides. Both contained two cysteine residues and had identical or similar amino acids in positions 5 (P), 8 (I/L) and 13 (L). The fusion phages were also recognised by Mab 4.52 in enzyme-linked immunosorbent assay (ELISA) and binding was shown by surface plasmon resonance. One of the peptides was a markedly better inhibitor (67%) of the binding of Mab 4.52 to its original antigen than the other (20%) at 1mg/ml. After absorption, to remove isotypic and allotypic reactivities, the anti-idiotype IgY was specifically recognised by Mab 4.52 in ELISA and was able to inhibit its binding to the original antigen, whereas anti-idiotype antibodies raised against a bluetongue virus-specific antibody had no effect. In spite of unequivocal binding of the anti-idiotype antibodies and the fusion phages to the paratope of Mab 4.52, goat antisera appeared not to react with either of the surrogate antigens. In contrast, the test sera bound to the original antigen suggesting that Mab 4.52 does not recognise exactly the same antigenic site as antibodies in the goat antisera.

Specific PCR identification of the T1 vaccine strains for contagious bovine pleuropneumonia.

A specific PCR test for the identification of the vaccine strains T1, T1/44 and T1sr, was developed. This PCR reaction is based on variations of DNA sequences in a region flanking one IS1296 copy. The specific primer pair MmmSCP1-T1M2 amplifies a 700-bp long DNA fragment in the T1 vaccine strains and gives no amplification with the 60 other Mycoplasma mycoides subsp. mycoides SC strains tested. This PCR will permit to distinguish the T1 strain from all other vaccine strains and therefore avoid possible confusions. In addition, it should enable better investigations of post-vaccinal reactions.