Peyraud et al. BMC Veterinary Research 2014, 10:48 http://www.biomedcentral.com/1746-6148/10/48 RESEARCH ARTICLE Open Access An international collaborative study to determine the prevalence of contagious caprine pleuropneumonia by monoclonal antibody-based cELISA Armelle Peyraud1,2, François Poumarat3,4, Florence Tardy3,4, Lucía Manso-Silván1,2, Karomatullo Hamroev5, Tillo Tilloev5, Mullojon Amirbekov5, Karim Tounkara6, Charles Bodjo6, Hezron Wesonga7, Isabel Gacheri Nkando7, Shiferaw Jenberie8, Martha Yami8, Eric Cardinale9, Deodass Meenowa10, Mahmad Reshad Jaumally10, Tahir Yaqub11, Muhammad Zubair Shabbir11, Nadia Mukhtar11, Mohibullah Halimi12, Ghulam Mohammad Ziay12, Willy Schauwers13, Hafizullah Noori14, Ali Madad Rajabi14, Stéphane Ostrowski15 and François Thiaucourt1,2* Abstract Background: Few serological tests are available for detecting antibodies against Mycoplasma capricolum subsp. capripneumoniae, the causal agent of contagious caprine pleuropneumonia (CCPP). The complement fixation test, the test prescribed for international trade purposes, uses a crude antigen that cross-reacts with all the other mycoplasma species of the “mycoides cluster” frequently infecting goat herds. The lack of a more specific test has been a real obstacle to the evaluation of the prevalence and economic impact of CCPP worldwide. A new competitive ELISA kit for CCPP, based on a previous blocking ELISA, was formatted at CIRAD and used to evaluate the prevalence of CCPP in some regions of Kenya, Ethiopia, Mauritius, Tajikistan and Pakistan in an international collaborative study. Results: The strict specificity of the test was confirmed in CCPP-free goat herds exposed to other mycoplasma species of the ‘’mycoides cluster”. Prevalence studies were performed across the enzootic range of the disease in Africa and Asia. Seroprevalence was estimated at 14.6% in the Afar region of Ethiopia, whereas all the herds presented for CCPP vaccination in Kenya tested positive (individual seroprevalence varied from 6 to 90% within each herd). In Mauritius, where CCPP emerged in 2009, nine of 62 herds tested positive. In Central Asia, where the disease was confirmed only recently, no positive animals were detected in the Wakhan District of Afghanistan or across the border in neighboring areas of Tajikistan, whereas seroprevalence varied between 2.7% and 44.2% in the other districts investigated and in northern Pakistan. The test was also used to monitor seroconversion in vaccinated animals. Conclusions: This newly formatted CCPP cELISA kit has retained the high specificity of the original kit. It can therefore be used to evaluate the prevalence of CCPP in countries or regions without vaccination programs. It could also be used to monitor the efficacy of vaccination campaigns as high-quality vaccines induce high rates of seroconversion. Keywords: Contagious caprine pleuropneumonia, Competitive ELISA, Seroprevalence, Kenya, Ethiopia, Mauritius, Tajikistan, Afghanistan, Pakistan, Vaccine quality control * Correspondence: [email protected] 1Centre de coopération internationale en recherche agronomique pour le développement (CIRAD) UMR CMAEE, Montpellier F-34398, France 2INRA, UMR1309 CMAEE, Montpellier F-34398, France Full list of author information is available at the end of the article © 2014 Peyraud et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Peyraud et al. BMC Veterinary Research 2014, 10:48 Page 2 of 9 http://www.biomedcentral.com/1746-6148/10/48 Background the European part of Turkey in 2005 [12] and poses a Contagious caprine pleuropneumonia (CCPP) has been threat to the Balkan countries of the European Union. known as a clinical condition for 140 years [1], but the CCPP has been shown to infect wildlife species held in burden and distribution of this disease remain largely captivity for conservation purposes in Qatar [13] but has unknown. The causal agent of CCPP was first isolated in also been detected in free-ranging wildlife in Tibet [14]. 1976 [2], but it was not given its species name until This is a matter of concern not only for free-ranging en- 1993: M. capricolum subsp. capripneumoniae (Mccp) dangered species in which CCPP may occur [15], but [3]. Conspicuous macroscopic lesions are observed in also for CCPP-free countries importing wild species for animals with acute disease. These lesions are restricted propagation purposes or for zoological collections. to the pleural cavity and consist of unilateral pleuro- CCPP is difficult to control. The vaccines against pneumonia with profuse pleural effusion, but diagnosis CCPP consist of inactivated Mccp antigen with saponin as may still be difficult. Until the development of specific an adjuvant [16]. If effective, these vaccines should induce PCR assays [4,5], it was difficult to obtain laboratory marked seroconversion, and this need-s to be investigated confirmation of CCPP. Autopsies are rarely performed furher. The antibodies induced by vaccination can inter- on goats dying from the disease and, when samples are fere with the results of disease prevalence studies, but sent to the laboratory for isolation of the causal agent, cELISA could be used to assess the efficacy of vaccination other mycoplasmas, such as M. mycoides subsp. capri campaigns. Vaccination efficacy has been assessed in ex- (Mmc), M. capricolum subsp. capricolum (Mcc) and perimental trials, leading to the definition of an optimum M. ovipneumoniae, are often isolated. This is due to the Mccp antigen concentration of 0.15 mg/dose, which yields extremely fastidious growth of Mccp, which is overgrown a protective immune response [17]. by faster growing mycoplasmas. Furthermore, animals The lack of a reliable, commercially available specific are often treated with antibiotics, hampering isolation of ELISA test for detecting Mccp antibodies was recog- the causal agent. Despite great improvements in the for- nized as a key issue during the design of an EU-funded mulation of culture media, the isolation of Mccp remains project aiming to control neglected animal diseases in very difficult [6]. It was thought that the use of PCR for Africa through vaccination (VACNADA). A blocking the molecular detection of Mccp would greatly facilitate ELISA based on the use of a specific monoclonal antibody the diagnosis of CCPP and provide more accurate infor- recognizing Mccp was developed in 1994 [18]. This kit has mation about the prevalence of the disease. recently been modified to obtain a heat-stable kit produced However, there have been very few declarations of to quality assurance standards. We describe here the use of CCPP outbreaks to the OIE in the last 15 years, due to a this new kit to evaluate the prevalence of CCPP in various lack of awareness of this disease and possible confusion countries and to evaluate the seroconversion induced by with other diseases, such as “peste des petits ruminants” the CCPP vaccine. (PPR) or Pasteurella infections. Little is known about the economic impact of CCPP, although participatory Methods epidemiological surveillance, without the need for la- Formatting of the cELISA kit boratory confirmation, may prove a useful approach. For The same components of the kit developed in 1994 [18] example, Turkana pastoralists in Kenya rank CCPP as were used, but the kit was modified in collaboration with one of the principal diseases affecting their goats, to- a private company, IDEXX-Montpellier SAS. The hy- gether with PPR and sarcoptic mange [7]. The impact of bridoma cell line “4.52” developed by CIRAD was revivi- CCPP and the potential threat represented by this dis- fied and subcloned to check its stability. The main ease have been greatly underestimated. objective was then to produce stable precoated plates Until recently, CCPP was believed to be confined to and to design a protocol yielding results similar to those Africa and the Middle East. Its presence in Asia was originally reported, in terms of specificity and sensitivity. confirmed only recently, with the isolation of Mccp Serum samples known to be positive and negative were strains in China [8] and the detection of Mccp by PCR used to achieve this end. in Pakistan [9] and Tajikistan. Molecular epidemiology The final CCPP cELISA kit protocol differed slightly studies have shown that the Mccp strains circulating in from the original published protocol, as it was a strict Asia belong to a specific clade supported by significant competitive assay rather than a blocking assay. We mini- bootstrap values [10]. This indicates that the presence of mized the variability of incubation time by first mixing the disease in Asia is not the result of a recent introduc- diluted sera (1/10) with a fixed quantity of monoclonal tion, which would have resulted in Asian Mccp geno- antibody in a normal non-coated plate and then trans- types closely resembling genotypes found elsewhere. ferring the mixture to a precoated plate and incubating Indeed, it was suggested that CCPP was present in India at 37°C for one hour, with gentle shaking. The plates as early as 1914 [11]. The disease was also recognized in were then incubated for 30 minutes with the conjugate Peyraud et al. BMC Veterinary Research 2014, 10:48 Page 3 of 9 http://www.biomedcentral.com/1746-6148/10/48 and 20 minutes with the tetramethyl-benzidine