Project: Multiplex PCR: A new kit for simultaneous detection of zoonotic pathogens

Pathogens in which, zoonoses are owed, are accountable for hundreds of thousands of infections each year. These pathogenic can infect both human population, leading thousands patients to the hospitals, as well as animal population causing a number of problems such as reduction of animal and milk production, expulsions, etc._x000D_Zoonoses can be caused by a number of pathogens including Brucella, Leptospira, Anaplasma species, Coxiella burnetii and Clamydophila pneumoniae._x000D_Brucellosis is a transmitted disease that is caused by worldwide spread bacteria of the gender of Brucella. Various strains of Brucella can infect lambs, cattle, deer, pigs, dogs, etc. People can be infected via contact with diseased animals or even with waste from these animals. Brucellosis is a frequent disease especially in regions where livestock-farming is highly developed and health control programs are not applied. The degree of mortality is around 2%._x000D_Leptospirosis is an illness caused by the bacterium Leptospira. It has been characterized as a zoonosis that infects humans as well. The bacterium is transmitted by contact with water infected by animal urine, via the eyes, mucus membrane and any possible skin wounds. An alternative way of transmission of the bacterium includes the consumption of infected food._x000D_Coxiella burnetii constitutes the cause of Q fever. Cattle, lambs and sheep constitute the CO reservoirs of the bacterium. Nevertheless, various other animals, including domestic ones, have been found being infected by the bacterium. Transmission of the disease can result via milk, urine, excrements and amniotic fluid. The transmission of bacterium into humans may, also, take place via the respiratory tract when the micro-organism hovers in the air. Few bacteria are enough in order to cause illness. The degree of mortality is around 2,4%._x000D_Bacteria belonging in the group of anaplasmataceae can cause gangliac fever and anaplasmosis (formerly known as human granulocytic ehrichiosis (HGE)). Species of mammals that have been reported as reservoirs are dogs, horses and sheep. Infection from these pathogens can lead to decreased milk production. As much as half of humans infected by Anaplasma phagocytophilum require hospitalization._x000D_Chlamydophila pneumoniae belongs in the group of chlamydiae that may infect humans constituting one of the causative agents of pneumonia (atypical pneumonia). Chlamydiae can be transmitted via droplets of lungs from an infected to a healthy person. Among others, infection may result in meningo-engephalitis, arthritis, myocarditis etc._x000D_One of the methods used for the detection of the above-described pathogens is the polymerase chain reaction (PCR). The advantage of method is the high sensitivity and specificity. The disadvantage of the method is the high cost required. In order to test a sample for the presence of the above pathogens a number of different PCRs are required, each one specific for the detection of a certain microbe. Therefore, the process, for example, for the detection of Brucella, Leptospira, Anaplasma species, Coxiella and Clamydophila pneumoniae in a human sample would require at least 5 different PCRs, 15 hours and the cost would equal with at least 5 times the cost of one single PCR. In case of a positive sample, there would be further need of PCRs in order to identify the exact species, e.g. in the case of Brucella whether the pathogen is Brucella abortus or Brucella melitensis._x000D_For this reason, the project will aim at the production of a technique that will allow the simultaneous detection of the above pathogens with a single PCR (multiplex PCR). _x000D_Such a technique not only will drastically decrease the required time of getting the answer to the veterinarian and/or the doctor, but will, also, decrease dramatically the cost allowing the control of much more samples. Simultaneously, since culturing the above pathogens apart from difficult is, also, exceptionally time-consuming (above one week required) and since antibodies against these pathogens can not be detected during the first few days of the disease, the multiplex PCR can be rendered as first-choice method._x000D_Several attempts have been made in the past to develop similar techniques that will enable the simultaneous detection certain pathogens (eg Brucella and Leptospira or Bartonella and Yersinia pestis). However, such a technique, which will allow the detection, at the same time, of the pathogens described above, has not been developed._x000D_Another technique will, also, be introduced. Western blot can detect the protein of interest from a mixture of proteins. It can provide information about the size of the protein and protein expression. It is an indirect method of detecting the presence of a bacterium, but it is of higher sensitivity than IFA. We intend to apply Western blot for the above pathogens. The absence of any vaccine makes clear that prevention can prove decisive for the correct confrontation of the above pathogens.

Acronym MuPCR (Reference Number: 4352)
Duration 01/06/2008 - 01/06/2011
Project Topic Development and evaluation of new diagnostic kits allowing simultaneous detection of pathogens responsible for zoonotic diseases (Brucella, Leptospira, Anaplasma species, Coxiella and Clamydophila psittaci).
Network Eurostars
Call Eurostars Cut-Off 1

Project partner

Number Name Role Country
3 E.P. & T.A. LABORATORIES AND VETERINARY DIAGNOSTICS LTD Partner Cyprus
3 D.N.ROUSSIS & SIA O.E. Coordinator Greece
3 University of Crete, Medical School, WHO Collaborating Center for Research and Training in Mediterranean Zoonoses Partner Greece