Contagious bovine pleuro-pneumonia (CBPP) is one of the most important trans-boundary livestock diseases in Africa causing severe pneumonia and encapsulated abscesses in the lungs of cattle. Mortality can be high but mostly this disease enters into a latent form reducing productivity and creating the risk of re-emergence form these carriers. Due to the prolonged incubation phase (up to several months) CBPP is often diagnosed very late and confirmed only after days of bacterial culture. As the immune response to this Mycoplasma, the causing agent is still not well understood, the existing serological methods detect 80% of all infected animals, latent carriers not at all and protection of vaccinated animals can not be assessed. The existing vaccines only protect around 60% of the animals not sufficient to keep the disease out of a population. New diagnostic tools comprising of ELISA utilizing highly specific synthetic antigens, molecular detection methods (PCR) and intra-dermal tests could improve the diagnostic and epidemiological capacities in the most affected countries dramatically. Isotopes play a major role in the development and evaluation of these tests. This CRP is aimed at improving the diagnostic capacities of the counterpart laboratories by evaluating the new and existing assays and defining the optimal use of each for a given purpose in accordance with the OIE guidelines. This will support sero-surveillance identifying areas harbouring CBPP and disease free zones for optimal intervention programmes. Molecular epidemiology will help identifying potential genetic risk factors to the aetiology, distribution and prevention of disease.
CBPP is a highly contagious and at the same time chronic disease of cattle, which leads to mortality or dramatic loss of productivity. Despite being highly infectious, symptoms are rarely observed immediately after infection but mostly only months later. This limits the understanding and investigations on the localisation of an outbreak and makes it difficult to identify the contaminating individuals. Therefore the main task was to evaluate the existing diagnostic tests for their fitness for purpose and to search for the most appropriate tools for disease section. As vaccination does deliver only a short lived immunity in not even half the vaccinated animal population, an improved understanding of the protective immune response to design the respective tools and to allow for a rational development of new vaccines was envisaged. Finally molecular techniques to allow for the monitoring of disease spread through genetic markers were intended
.Evaluation of serological tests for CBPP:
The evaluation of 2 new diagnostic tests for fitness for purpose and acceptance by OIE as a prescribed test was performed namely for the CBPP completion ELISA and the LPPQ indirect ELISA. These tests were introduced to all institutes participating and their results in comparison to each other and against the complement fixation test (reference test) were analysed for randomly taken samples, targeted sampling of infected herds, vaccinated animals and post mortem findings. In conclusion the c-ELISA performed best in all settings but recent infections and the statement that vaccinated animals were not giving positive results could not be verified absolutely. Never the less this test is fit for disease control up to a very low prevalence rate. The LPPQ ELISA performed well for sero-surveillance, but is not produced anymore. A newly designed card agglutination tests was evaluated in the field, but had too many (false) positive responders to be useful for an ad hoc disease confirmation.
The c-ELISA is today the recommended test by OIE.
Evaluation of the immune response towards infection:
A major problem in controlling CBPP is the lack of understanding the immune response of cattle to the pathogen. It is speculated, that the pathogen can only be controlled by the cellular immune response. In order to test this, and to develop a test indicative for immunity needed for the development of new vaccines, an experimental setting was developed. Analog to the skin testing of tuberculosis specific unique antigens were produced by the Swiss counterparts and were meant to be intra-dermally injected together with an adjuvant specifically stimulating dendritic cells. Unfortunately the counterpart did not manage to perform the tests in the way foreseen and his results did not give any evidence. Experiments performed at ILRI never the less showed that the presence of specific T-cells (CD4+) presumed to be protective, did not have predictive value for the outcome of infection experiments. The gamma interferon test applied for these studies as an alternative to the skin test did as well not correlate to the lesions found in 15 experimentally infected cows and is therefore as well not suitable. More recent work of ILRI together with IZS Teramo could show an elevated level of TNF alpha in fatally infected animals, but this was to be expected in such severe inflammations. Finally IZS could demonstrate that macrophages were remarkably altering their cellular structures after infection with Mycoplasma mmSC, but only in the presence of pathogen specific antisera, which can explain for instance the delayed onset of symptoms (normally only 3-4 weeks after infection) but does as well not help elucidating a protection mechanism.
Molecular diagnostics:
As molecular diagnostics (PCR) in developing countries are hampered by the need of sophisticated procedures and equipment the CRP aimed at developing a new thermostable test for pathogen detection which can be carried out in the field. Through a technical contract an isothermal PCR (Loop mediated amplification, LAMP) was developed, pretested for specificity in Switzerland and then field evaluated. This test can be carried out in around 1 hour from blood samples taken directly in the field and displays the results including a quality control. This nuclear related test proved to be more sensitive than other molecular tests and has an absolute specificity. A scientific report including evaluation results from different counterpart laboratories is currently in the review phase to be published. This test being cheap ( 2 Euros), rapid and easy to perform ( 3 pipetting steps only) even in the field will enable veterinary services to counteract CBPP infections much more rapidly and thus more efficient. This test can now be used by MS laboratories as the required reagent sets can be commercially obtained. In parallel a published conventional PCR was tested in several laboratories and the best sample preparation technique evaluated. The conclusion for this was to simply boil and spin a sample in a centrifuge and take as assay template the supernatant.
Most of the counterpart laboratories are today capable of performing molecular diagnostic for CBPP.
Molecular epidemiology of CBPP:
As CBPP is a chronic disease, symptoms are readily confused with tuberculosis, severe parasite infections or simply malnutrition. This is the reason for its success to spread as it’s not diagnosed. A very elegant way to show the spread and distribution patterns of an infectious agent is the analysis of its genetic changes in the course of time and thus as well in spatial dimensions as performed for instance for Influenza. At the start of this CRP no work on genetic markers had been carried out and so the mycoplasma reference laboratory at the VUW was asked to analyse 10 Mycoplasma mm. SC strains and other mycoplasma strains for genetic variations. Extensive analysis of all strains on different genetic locations applying genomic fingerprinting employing Randomly Amplified Polymorphic DNA (RAPD) PCR for identification of MmmSC-specific and strain specific PCR products only demonstrated a very high degree of homogeneity among MmmSC strains hampering differentiation among strains of Mycoplasma mycoides subsp. mycoides SC. The 2 SNP differences found in the strains tested were later on utilized by the world reference laboratory for CBPP to describe the evolutionary History of CBPP, which most likely only appeared around 300 years ago. A scientific report by ILRI could demonstrate that bovine mycoplasmas most likely emerged only 10.000 years ago from an ancestor form and its genetic diversity is less than 0.05%. So there is no possibility to perform molecular epidemiology with Mycoplasma mycoides mycoides SC
This CRP had impact on a number of issues, starting from advancing the capacities of veterinary laboratories to quicker and more reliable diagnose CBPP, advancing the LAMP technology to a thermostable platform allowing the shipment and performance of the diagnostics without cooling and finally having added to the scientific understanding of the pathology of CBPP. This gives the IAEA a technological lead in the development of new thermostable diagnostics like 25 years ago with ELISA and adds to the arsenal of molecular diagnostic methods a very simple and cheap alternative to the lab based PCR technologies.
CBPP is currently present in at least 22 MS in Africa and is listed as the cattle disease with the highest impact on productivity and subsequently for the farmer. So addressing this disease is already justified by its socio-economic impact. Additionally there are only a few institutions worldwide working on this disease, as it is categorized “exotic” and requires high security laboratories for research. This hampers progress, specifically as the disease cannot be mimicked by rodent models. So the results as well as the tools and reference material produced during the CRP are of great help to the research institutions. And finally in the context of this CRP the LAMP technology was advanced to a robust diagnostic kit which allows for the first time to perform a molecular test in the field.