Risk factors associated with contagious caprine pleuro-pneumonia in goats in pastoral areas in the Rift Valley region of Kenya.

A cross-sectional study to determine risk factors associated with sero-prevalence of contagious caprine pleuro-pneumonia (CCPP) in goats was carried out between the months of March, 2014 and March, 2015 in Pokot East, Turkana West and Kajiado Central Sub-counties. A semi-structured questionnaire focusing on risk factors for CCPP was completed for each flock whose serum samples were collected. A logistic regression model was developed to assess the association between the risk factors and CCPP sero-positivity. Of the 54 flocks, 49 (90.7%) presented at least one sero-positive animal. Two hundred and four of the 432 goats tested sero-positive at monoclonal antibody based competitive Enzyme-linked immuno-sorbent assay (c-ELISA), hence a sero-prevalence of 47.2% (95% CI=42.5- 51.9). Previous exposure of flocks to CCPP (p<0.001, OR=52.8; CI=6.45, 432), distant sources of veterinary drugs (p<0.001, OR=6.17; CI=3.41, 11.1), movement of goats to dry season feeding areas (p<0.001, OR=4.31; CI=2.39, 7.75) and markets as a source of new introductions to the flock (p=0.033, OR=1.86; CI=1.05, 3.27) were identified as risk factors significantly associated with CCPP sero-prevalence. The findings provide further evidence supporting the high prevalence and endemic state of the disease in pastoral flocks and hence there is need for adequate measures to be put in place to control the disease effectively.

Contagious bovine pleuropneumonia. A reassessment of the efficacy of vaccines used in Africa.

Contagious bovine pleuropneumonia is a major threat for cattle in Africa. Since 1956 the T1/44 strain has been used as a vaccine, and later on, T1sr, a streptomycin-resistant variant that gives fewer post-vaccinal reactions had been developed. These vaccines are known not to be very efficient but they normally should provide protection for about eight months. However, recent emergency vaccinations, performed in various countries in the southern part of the continent apparently met with failure, casting doubts on the identity as well as the protection afforded by the T1sr strain. A vaccine trial has been designed to reassess the real protection afforded by these vaccines in face of recently isolated pathogenic strains. Great care has been taken to test the original vaccinal strains at a dose corresponding to the minimum requirement by international standards. The test was performed in Cameroon, Kenya, and Namibia as to take into account the genetic diversity that exists among the pathogenic strains. In those conditions, the protection rate at three months varied from 33 to 67%, whatever the strain used, T1/44 or T1sr. These results call for additional research for vaccine development and careful planning of strategies in the fight against CBPP.

Genetic diversity and evolution of Mycoplasma capricolum subsp. capripneumoniae strains from eastern Africa assessed by 16S rDNA sequence analysis.

Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae), the causal agent of contagious caprine pleuropneumonia (CCPP), is a member of the so-called Mycoplasma mycoides cluster. These mycoplasmas have two rRNA operons in which intraspecific variations have been demonstrated. The sequences of the 16S rRNA genes of both operons from 13 field strains of M. capripneumoniae from three neighbouring African countries (Kenya, Ethiopia, and Tanzania) were determined. Four new and unique polymorphism patterns reflecting the intraspecific variations were found. Two of these patterns included length differences between the rrnA and rrnB operons. The length difference in one of the patterns was caused by a two-nucleotide insert (TG) in the rrnB operon and the length difference in the other pattern was due to a three-nucleotide deletion, also in the rrnB operon. Another pattern was characterised by a polymorphic position caused by a mutation that is known to cause streptomycin resistance in other bacterial species. The strain with this pattern was also found to be resistant to streptomycin. Streptomycin resistant clones were selected from four M. capripneumoniae strains to further investigate the correlation of this mutation to streptomycin resistance. Mutations in the 16S rRNA genes had occurred in two of these strains. The fourth pattern included a new polymorphism in position 1059. The results show that polymorphisms in M. capripneumoniae strains can be used as epidemiological markers for CCPP in smaller geographical areas and to study the molecular evolution of this species.

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.

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.

Diagnosis and control of contagious caprine pleuropneumonia.

The diagnosis of contagious caprine pleuropneumonia (CCPP) has often been considered difficult. This is because of the confusion that can arise with other mycoplasmoses of small ruminants. Symptoms and lesions can be similar and the isolation of M. capricolum subsp. capripneumoniae (MccF38) requires skilled technicians. Once MccF38 strains are isolated, their identification should not be difficult. New techniques, such as polymerase chain reaction, now offer the possibility of identifying MccF38 directly from dried samples. However, the isolation of MccF38 strains is always required for an official declaration of infection. Until now, the official serological test has been the complement fixation test; the main drawbacks being lack of sensitivity and specificity and also the short persistence of antibodies detected by this technique. The specific competition enzyme-linked immunosorbent assay has now been developed and should enable wide serological enquiries to determine the real prevalence of the disease. Antibiotic treatments are effective but may not prevent persistence in latent carriers. An inactivated vaccine with saponin as an adjuvant has been produced in Kenya, which protects goats for approximately one year.

Rabbit encephalitozoonosis in Kenya.

Encephalitozoon cuniculi infection was diagnosed in a laboratory rabbit breeding colony at Muguga, Kenya. This is the first report of the disease in rabbits in Kenya. Post-mortem examination showed gross renal lesions and the presence of the parasite in histological sections of the cerebrum and cerebellum. On Gram stain, spores were observed in the kidney sections.

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.

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.

Late lesions of experimental contagious caprine pleuropneumonia caused by Mycoplasma capricolum ssp. capripneumoniae.

A clinical, bacteriological, serological and patho-anatomical study was carried out on 12 goats surviving the acute stage of contagious caprine pleuropneumonia (CCPP), experimentally produced with Mycoplasma capricolum ssp. capripneumoniae (M. capripneumoniae), with the major aims of investigating the chronic stage of the disease and elucidating the possibility of a carrier state beyond the acute fulminant phase. The goats were killed 9, 16, 82 or 126 days after the onset of acute clinical signs. On day 9, clinical signs included low grade fever and persistent coughing. Thereafter, only intermittent coughing was recorded. Serum titres of complement-fixing antibodies to M. capripneumoniae were high at the period of fever but dropped thereafter. Post-mortem examination showed acute fibrinous pleuropneumonia on days 9 and 16, and chronic pleuropneumonia on days 82 and 126, including sequester formations in goats killed on day 126. Mycoplasma capripneumoniae was isolated on days 9 and 16 but not on later occasions. The study showed that goats recovered from acute CCPP may have lesions for a long time thereafter but provide no evidence of a carrier state among long-term survivors.

Diagnosis of contagious caprine pleuropneumonia by detection and identification of Mycoplasma capricolum subsp. capripneumoniae by PCR and restriction enzyme analysis.

Contagious caprine pleuropneumonia (CCPP), one of the most serious and dramatic diseases of goats, is caused by Mycoplasma capricolum subsp. capripneumoniae (M. capripneumoniae). This organism is very difficult to isolate and to correctly identify. In a previous report we described a method for the rapid detection and identification of M. capripneumoniae. This method is based on a PCR system by which a segment of the 16S rRNA gene from all mycoplasmas of the M. mycoides cluster can be amplified. The PCR product is then analyzed by restriction enzyme cleavage for the identification of M. capripneumoniae DNA. This system has now been further evaluated with respect to specificity and diagnostic efficacy for the identification and direct detection of the organism in clinical material. Identification by restriction enzyme analysis of amplified DNA from mycoplasmas of the M. mycoides cluster was verified for 55 strains, among which were 15 strains of M. capripneumoniae. The PCR was applied to clinical samples from the nose, ear, pharynx, pleural fluid, and lung tissue containing M. capripneumoniae or other mycoplasmas. As expected, mycoplasmas belonging to the M. mycoides cluster could be detected by the PCR. Restriction enzyme analysis of the PCR products could then be applied for the identification of M. capripneumoniae. Clinical samples and cultures containing M. capripneumoniae were dried on filter paper, to try an easier sample transport method, and were tested by PCR. M. capripneumoniae DNA could be detected in the dried specimens, but the sensitivity of the PCR test was reduced.