Pesq. Vet. Bras. 34(11):1069-1072, novembro 2014
Evaluation of a MPB70-ELISA to differentiate Mycobacterium bovis- from M. avium-sensitized swine1
Carla D. Marassi2, Flávia C.S. de Oliveira3, Sonia R. Pinheiro3, Sergio S. Azevedo3, Francisco R.M. Soto3, Walter Oelemann4, Walter Lilenbaum2* and Silvio A. Vasconcellos3
ABSTRACT.- Marassi C.D., Oliveira F.C.S., Pinheiro S.R., Azevedo S.S., Soto F.R.M., Oelemann W., Lilenbaum W. & Vasconcellos S.A. 2014. Evaluation of a MPB70-ELISA to differenti- ate Mycobacterium bovis- from M. avium-sensitized swine. Pesquisa Veterinária Brasi- leira 34(11):1069-1072. Departamento de Microbiologia e Parasitologia, Universidade Fe- deral Fluminense, Rua Professor Hernani Mello 101, Lab 309, Niterói, RJ 24210-130, Brazil. E-mail: [email protected] Swine are susceptible to different mycobacteria species, being Mycobacterium bovis an M. avium determines a
agent of tuberculosis, with most significant zoonotic risks, while - malsgranulomatous or to differentiate lymphadenitis among mycobacterial with low zoonotic species. risk. In order Currently to improve performed the TB intradermal diagnosis, serologicaltests present assays some have important been developed, limitations, with such encouraging as the lack results. of ability The to purpose detect ofanergic this study ani was to evaluate the performance of a MPB70-ELISA in 82 piglets divided into four groups: sensitized by inactivated M. bovis, M. avium, inoculated with oil adjuvant, or with saline solution. The test was able to discriminate between an animal sensitized by M. bovis and animals of the three other groups, including M. avium-sensitized animals; for this reason, we suggest that MPB70-ELISA could be used as a complementary tool for discriminating the agent of the mycobacteriosis, and therefore to diagnose tuberculosis in a swine herd. INDEX TERMS: Diagnosis, ELISA MPB70, swine, Mycobacterium bovis, Mycobacterium avium.
RESUMO.- [Avaliação de um MPB70-ELISA para diferen- ou de diferenciar entre as espécies micobacterianas. Com o ciar entre suínos sensibilizados com Mycobacterium intuito de melhorar o diagnóstico de TB, testes sorológicos bovis ou M. avium.] Suínos são suscetíveis a diferentes es- têm sido desenvolvidos, com resultados encorajadores. O pécies de micobactérias, sendo Mycobacterium bovis agen- objetivo do presente estudo foi avaliar um MPB70-ELISA te de tuberculose (TB), com claro risco zoonótico, enquan- em 82 leitões divididos em quatro grupos: sensibilizados to M. avium determina uma linfadenite granulomatosa (LG) por M. bovis, por M. avium, inoculados com óleo adjuvante de baixo risco zoonótico. Os testes intradérmicos atualmen- ou com solução salina. O teste foi capaz de discriminar en- te realizados apresentam algumas limitações importantes, tre os animais sensibilizados com M. bovis dos demais três como a falta de habilidade em detectar animais anérgicos grupos, incluindo aqueles que foram sensibilizados com M. avium; desta forma, sugere-se que o MPB70-ELISA poderia 1 Received on July 12, 2014. ser utilizado como ferramenta complementar para discri- Accepted for publication on October 2, 2014. minar o agente da micobacteriose, e portanto diagnosticar 2 Laboratório de Bacteriologia Veterinária, Departamento de Microbio- TB em um plantel de suínos. logia e Parasitologia, Instituto Biomédico, Universidade Federal Flumi- nense (UFF), Lab 309, Rua Prof. Hernani Mello 101, Niterói, RJ 24210-130, TERMOS DE INDEXAÇÃO: Diagnóstico, ELISA MPB70, suínos, Brazil. *Corresponding author: [email protected] Mycobacterium bovis, Mycobacterium avium. 3 Laboratório de Zoonoses Bacterianas, - Departamento de Medicina Ve- terinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária, Universidade de São Paulo (USP), Av. Prof. Dr. Orlando Marques de Paiva INTRODUCTION 87, São Paulo, SP 05508-270, Brazil. Swine can be infected by several mycobacteria, causing 4 Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro (UFRJ), Cidade Universitária, Rio de Janeiro, RJ variable degrees of disease. Among them, there are some 21941-902, Brazil.
agents with clear and significant zoonotic risk, such as 1069 1070 Carla D. Marassi et al. mycobacteria from the tuberculosis complex, as Mycobac- avium D4 strain (Group A); 21 piglets were inoculated with an oil terium bovis suspension of inactivated Mycobacterium bovis AN5 strain (Group et al. 2010). Conversely, other mycobacteria, such as mem- B); 20 piglets were inoculated with oil adjuvant (Group C), and bers of the Mycobacterium, agent of bovine avium tuberculosis complex (MAC),(TB) (Wisselink lead to a 20 piglets were inoculated with saline solution (Group D). After granulomatous lymphadenitis (GL)in head and mesenteric a 30-day interval (Day 90), all animals were tested by CIT. All the - lymph nodes that, despite its economic importance due to cial recommendations (Brasil 2011). the condemnation of carcasses (Faldyna et al. 2012), pres- inoculationsSera samples. and tests Blood were samples performed for ELISA according were to collected Brazilian from offi - - compromised people. ted before sensitization (Day 0); at the moment of sensitization ent Diagnosisa lower public of swine health TB risk, relies often on restrictedintradermal to tests,immuno but (Dayall 82 60) animals and 30in threedays afterdifferent sensitization moments: (Day at the 90). first Samples CIT conduc were always collected a few moments before injection of antigens for for the single intradermal test (SIT) that uses M. bovis pu- CIT. there is a lack of definite patterns of interpretation, either MPB70-ELISA. ELISAs were conducted according to the intradermal test (CIT), that uses bovPPD and also M. avium protocol previously validated for bovines (Marassi et al. 2011). PPDrified (avPPD). protein Althoughderivative theoretically (bovPPD) or those for the tests comparative should be a 0.05M carbonate buffer, (pH 9.8) and used to coat a 96 well able to distinguish TB (M. bovis) from GL (M. avium), those plateBriefly, (Nunc MPB70 Maxisorb was diluted – Sigma-Aldrich) to a concentration overnight of 0.5µg/mLat 8°C. After in - - tests.agents Besides share a that, number intradermal of antigens tests that are makes based thaton the differ de- NaClthree 0.2M, washes, pH the7.2) plateand incubated was blocked for 2hs/37ºC. with a solution the plate compo was tectionentiation of difficultthe cell-mediated and often immune not reliable response by current (CMI) trig skin- washedsed of 5% three skim times milk and diluted swine in sera PBST were buffer diluted (0.05% 1/100 Tween20, in PBST gered by the mycobacterial infection. However, as the infec- tion progresses, antibodies became more easily detectable dilutions were incubated at 37°C under continuous agitation for (Walravens et al. 2002), and, depending on the employed buffer with 1% of skim milk and distributed in duplicate. Sera - by a peroxidase conjugated anti-swine IgG (Sigma-Aldrich) was used60 minutes. at 1/40,000 After fiveand washes,incubated a secondary for one hour antibody at room composed tempe- MPB70capture isantigen one of (as the MPB70), most studied specific antigens antibodies used can in ELISAs be de rature under continuous agitation. The reaction was revealed fortected TB evendiagnosis at the in early ruminants stages of(Marassi the disease et al. (Wiker 2011). 2011). It has with TMB (Sigma-Aldrich) (1mg/mL) and stopped with 1N HCl been employed as a complementary tool, mainly providing the detection of anergic animals that fail to be detected by wavelength(Merck) after of 30 450 min. nm. in the dark. Optical density (OD) readings intradermal tests (Marassi et al. 2009, Waters et al. 2011, wereStatistics. taken using Comparison a spectrophotometer between tests (model and differences 680 BioRad) among at a Zarden et al. 2013). samples were analyzed by t-Test and Friedman analysis perfor- Few studies addressed the diagnosis of mycobacterial med by MedCalc® statistics program (version 12.4). infections in swine. Although serological assays have been developed with promising results (Boadella et al. 2011, RESULTS - - lease assay (Faldyna et al. 2012), they are mainly focused onWisselink the diagnosis et al. 2010), of lymphadenitis, as well as an Interferon-gammawhich is determined re valuesSerum samplesof the four taken experimental at day 0 presented groups werelow ODs 0.05, in thewith ELI no - differenceSA, confirming among that the all four animals tested were groups negative. (P>0.05). The averageSimilar ing the diagnosis of M. bovis infections in swine and/or de- results were obtained for samples collected at the moment signedby MAC. to To discriminate our knowledge, between there infections are no studies determined address by of the sensitization of the animals (Day 60). M. bovis or MAC. Therefore, the purpose of this study was to evaluate the - performance of a MPB70-ELISA in swine experimentally fferences could be observed among the four groups. Avera- ge ODsNevertheless, values were for 0.05 samples for group taken A,at 0.12Day 90, for important group B, and di sensitized by M. bovis-or M. avium-oil suspensions and its discriminatory capability. 0.02 for groups C and D. In Group A, 18/21 presented ODs
MATERIALS AND METHODS values ≤0.10 while for Group B the contrary wasP<0.05), observed as Animals. This research was protocoled as number 1696/2009, andwell 11/21as between swine group presented B and ODs the controls≥0.10. Difference (Groups Cbetween and D), thosewhich twowere groups not different was statistically among themselves significant ( (>0.05). Mo- Medicine and Animal Science of University of São Paulo. A com- P certified by the Bioethical Commission of the School of Veterinary reover, average ODs of groups A, C and D at Day 90 did not - mercial herd certified as free of tuberculosis located in São Paulo, Brazil, for at least five years was studied. Until 21 days-old, ani- (P<0.05)significantly of that differ on from Day 0those and ofDay Day 60. 0 Results or Day 60,are whiledepicted for gativemals were CIT, 82kept animals in individual were randomly boxes with chosen controlled to be studied. temperature All of inGroup Figure B average1. OD at Day 90 was significantly different themand milking. presented When physiological piglets were parameters 30 days-old of normality. (Day 0), after a ne Experimental groups. After a 60-day interval period, the 82 DISCUSSION animals (90 days old/Day 60) were divided into four groups for sensitization. Subsequently, 21 piglets were intramuscularly ino- As expected, since we have studied a TB-free herd, average culated (IM) with an oil suspension of inactivated Mycobacterium OD values were low for all tested animals, with no signi-
Pesq. Vet. Bras. 34(11):1069-1072, novembro 2014 Evaluation of a MPB70-ELISA to differentiate Mycobacterium bovis- from M. avium-sensitized swine 1071
Importantly, our results could clearly demonstrate the testing moment (Day 0) and at the sensitization moment capability of MPB70-ELISA for detecting M. bovis-sensitized (Dayficant 60). difference Those dataamong reduce the four the groups,possibility both of ata biasthe firstand animals with no cross-reaction with Mycobacterium avium- indicate that the alterations that were later observed were -sensitized animals. This can be useful for epidemiological most probably due to the injection of the mycobacterial oil understanding and, consequently, for control programs. suspensions. Although humoral response in swine is not often investiga- In contrast, at Day 90, i.e. 30 days after sensitization, ted, recently a rapid serological test using bovine PPD and a dual path platform for bovine TB diagnosis was reported for animals of Group B (inoculated with inactivated Myco- for M. bovis-infected wild boars (Boadella et al. 2011), with bacteriuma significant bovis increase), and 11/21on average of that ODs group was presentedobserved highonly encouraging results. This study demonstrated that swine - also present a detectable humoral response after mycobac- lues were unaltered for the animals of Groups A, C and D terial infection. Therefore, ELISA can be employed for that (Fig.1).ODs (≥0.10). Importantly, at that moment average ODs va species, with similar advantages that have been observed When compared to studies conducted in ruminants na- in cattle and other species, such as the possibility of testing turally infected with M. bovis (Waters et al. 2011), all ODs samples as a batch, possibility of retesting inconclusive re- sults and no need of a second visit to the herd (Marassi et not totally unexpected. ELISA was performed on non-in- al. 2011). fectedwere relatively animals from low. a Although free herd, unusual, which had our most findings probably were Swine may be infected by either M. bovis or M. avium, no previous contact with either virulent or environmental determining different diseases (TB or GL, respectively) with different zoonotic and Public Health impacts. Thus, cross-reactions may confuse the serological diagnosis and (Marassimycobacteria. et al. Additionally,2011) and animals it is well-known were sensitized that ELISAs only consequently reduce the reliability of serological tests in once,performed which with reduces a single the purified intensity protein of the lead reaction to lower (Wisse ODs-
forthat M. species bovis when non-specific antigens and ELISAs are employing employed. etlink al. et 2012) al. 2010). and Additionally,in the present it hasstudy been we reported have only that tested age thatMPB70 protein is a as secreted antigens protein have been known used as with species-specific encouraging youngcan influence animals. ELISA results in swine (Garcia-Bocanegra results for detecting (Wiker etTB al. in 2011), different species, as bovines (Waters et al. 2011), goats (Marassi et al. 2009) and bu- ffalos (Zarden et al. 2013). Therefore, the discrimination between those two agents is mandatory, and we are not aware of any other study that has addressed that issue before.
CONCLUSION The present study demonstrated that MPB70-ELISA
differentiate between the Mycobacterium bovis- and M. notavium- onlysensitized detected animals. sensitized animals but could specifically We propose that MPB70-ELISA could be used as a com- plementary tool for identifying the agent of the mycobacte- riosis in a swine herd.
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