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Institute of Molecular Pathogenesis (IMP)

National Reference Laboratory for Contagious Bovine Pleuropneumonia

Disease and infectious agent

Regarding to the Epizootic Disease Act in the latest version Contagious Bovine Pleuropneumonia (CBPP) is a notifiable, highly contagious bacterial disease of the respiratory tract of cattle. High morbidity and mortality are typical for this disease, causing immense economic losses within the farm industry.

Susceptive is cattle of all ages and of all types (such as buffalo, bison, yak, and water buffalo). The disease is characterized by an extensive and marked inflammation of the lung (“marbled” pattern of lung tissue) and fibrinous pleuritis. The course of the disease often sharpens from subacute to chronic.

Animals surviving Contagious Bovine Pleuropneumonia and which are chronically ill serve as important sources of infection. Due to the hidden danger harbored in chronically infected animals, many countries, when trading animals, insist on written proof of negative antibody results given by the complement fixation test (CFT). Despite the fact that nowadays the disease occurs only in some parts of Africa, South America, and Asia (last detection in Europe: Italy, 1993; Portugal, 1999) a permanent danger of reintroducing Contagious Bovine Pleuropneumonia into Europe because of intense animal trading and open borders within Europe can not be underestimated. Right now though there is no concrete danger of the disease being reintroduced into Germany, where the last case was reported in 1926.

Mycoplasma mycoides subsp. mycoides Small Colony Type (MmmSC) is the infectious agent causing Contagious Bovine Pleuropneumonia. This agent belongs to the genus Mycoplasma within the family of Mycoplasmataceae (class Mollicutes). They are known as the smallest microorganisms living in cell free media (0,3-0,8 µm). Mycoplasma species have no cell wall and appear on special solid medium typically as “fried egg”-like colonies.

There are no plans for extensive research projects at our institute right now, since at the time CBPP is not a concrete danger for Germany. Scientific activities therefore concentrate on the investigation of doubtful samples and on the optimization of diagnostic methods available as well as the development and validation of new diagnostic methods for CBPP.

Tasks:

  1. Support of examination offices by the investigation of difficult diagnostic cases, especially helping with
    - Cultivation of the agent and detection of M. mycoides by polymerase chain reaction (PCR)
    - Differentiation of Mycoplasma isolates regarding the valid taxonomy by PCR and indirect immunfluorescence technique
    - Investigation of doubtful sera by using the complement fixation test (CFT)
    - Characterization of suspicious sera by immunoblotting
  2. Improvement of already used methods as well as the development of new diagnostic techniques and their intern validation
  3. Pursuit of the current epidemic situation in the world

Due to media requirements and the slow growth of Mycoplasma, the cultivation of the agent is sometimes difficult and always time consuming. Often cross-reactions appear by using the immunofluorescence test as well as serological detection techniques, which can influence specificity negative.

These cross-reactions are likely to appear because of the close relationship of MmmSC to a number of other Mycoplasma species. Mycoplasma species listed below belong to the so called Mycoplasma-mycoides-cluster, that shares a number of common antigens and therefore makes it sometimes impossible to distinguish them with traditional detection methods.

Important Mycoplasma species for differential diagnostics are

  • M. mycoides subsp. capri
  • M. capricolum subsp. capricolum
  • M. capricolum subsp. capripneumoniae
  • M. leachii

The diagnosis of CBPP often relies only on the complement fixation test (CFT), which is recommended by the Office International des Epizooties (OIE). Yet this serological method is known for its low sensitivity and problems with specificity. In the past there have been cases reported where cattle were tested positive by CFT in Germany. That these positive reactions are caused by cross-reactions could be shown only by immunoblotting, another serological method recommended by OIE, which is characterized by a much higher specificity and sensitivity.

Immunoblotting uses separation by electrophoresis of the MmmSC antigen followed by immobilization of the separated proteins on a membrane. This antigen-covered membrane will then be incubated with the sera to be investigated including control sera. Reactive bands are made visible by a change of color due to an enzymatic reaction. A sample is proven positive, if certain bands with a defined molecular weight (110, 98, 95, 62/60, and 48 kDa) can be detected. With this method a more precise result regarding the presence of antibodies against MmmLC is possible and so immunoblotting is recommended in cases of unclear CFT results.

Another alternative method recommended by the OIE, besides CFT, is a commercial antibody ELISA (CIRAD, France), but this test is not licensed in Germany and can therefore only be used with special permission.

PCR is known to be extremely faster than then the cultivation method, which has to be following by an indirect immunofluorescence. PCR results are available after 2 days in comparison to results from the culture method, which may take up to 20 days.

This is even more important when getting serological suspicious results since until MmmSC free status is proven, precaution procedures have to take place (e.g. live stock protection).

Other disadvantages of indirect immunofluorescence versus PCR are the following:

  • Subjective examination of the results (depending on the experience of the investigator)
  • False positive results can occur after using vaccines
  • Complement binding antibodies were produced more then 10 days after an onset of clinical signs so that an early recognition of infectious herds is often impossible

For these reasons the reference laboratory developed a PCR method based on two primer pairs: one pair is detecting all members of the Mollicutes, while the other is species specific, which allows clear specification of the infectious agent.

Lung tissue, lung lymphnodes, nasal swabs, pleural liquid, and blood, as well as sperm samples and synovial liquid can be used in this PCR. Besides a high specificity a high sensitivity of the PCR has been proven, so that this method is in accordance with requirements for a fast diagnostic procedure and can be used now as a routine procedure. Besides culturing, PCR is recommended by the OIE for the direct identification of the Agent.

  • Culture of tissue samples (first bouillon, followed by culturing on solid media): in case of growth (microscopic evaluation of the agar plate): performance of indirect immunofluorescence technique on the cultivated bacteria
  • In addition: parallel PCR with tissue samples and/or nasal swabs
  • Application of CFT on sera (prescribed test for international trade)
  • In case of positive or uncertain CFT results, we recommend to do immunoblotting as well