RADIOSENSITIVITY OF SPORES OF Paenibacillus larvae ssp. larvae IN HONEY

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1 2007 International Nuclear Atlantic Conference - INAC 2007 Santos, SP, Brazil, September 30 to October 5, 2007 ASSOCIAÇÃO BRASILEIRA DE ENERGIA NUCLEAR - ABEN ISBN: RADIOSENSITIVITY OF SPORES OF Paenibacillus larvae ssp. larvae IN HONEY Wanderley Mendes de Almeida 1, Helio de Carvalho Vital 2 and Dulce Maria Tocchetto Schuch 3 1 Serviço de Inspeção de Produtos de Origem Animal Ministério da Agricultura, Pecuária e Abastecimento Avenida Rodrigues Alves, 129, Centro Rio de Janeiro, RJ sipa-rj@agricultura.gov.br 2 Divisão de Defesa Química, Biológica e Nuclear Centro Tecnológico do Exército (CTEx) Avenida das Américas, 28705, Guaratiba Rio de Janeiro, RJ vital@ctex.eb.br 3 Ministério da Agricultura, Pecuária e Abastecimento. Estrada da Ponta Grossa, 3036, Belém Novo, Porto Alegre, RS micro-lara-rs@agricultura.gov.br ABSTRACT Irradiation, usually used in combination with other conventional methods of conservation, has been proven to be an efficient tool to ensure the safety of many types of foods by destroying pathogenic microorganisms and extending their shelf-lives. This work has investigated the efficacy of gamma irradiation to inactivate spores of the bacterium Paenibacillus larvae that causes the American foulbrood, a highly contagious disease still exotic in Brazil that kills bees and contaminates honey, preventing its commercialization and causing great economical losses. In this study, 60g samples of two types of honey inoculated with 3.5x10 3 spores/ml of that bacterium were irradiated with doses of 0, 5, 7.5, 10, 12.5 and 15 kgy and counted. The analyses indicated a mean reduction of 97.5±0.7% in the number of viable spores exposed to 5 kgy. The application of doses of 7.5 kgy or higher yielded no viable spores above the detection threshold (10/mL). In addition the value of D 10 (3.1±0.3 kgy) was estimated and the logarithm of the population of viable spores of Paenibacillus larvae subsp. larvae was determined as linear and quadratic polynomial functions of the radiation dose. The results indicated that the dose of 10 kgy could be insufficient to assure complete sterilization of honey in some cases while suggesting that 25 kgy would perform such task adequately. 1. INTRODUCTION Being a very important food worldwide, honey can be contaminated by very resistant spores of pathogenic bacteria and molds. The American foulbrood (AFB) is the most serious bacterial disease of honey bee (Apis Mellifera L.) brood and is caused by the spore stage of the gram-positive bacterium Paenibacillus larvae subsp. larvae. The fact that the spores can remain viable and last indefinitely on beekeeping equipment causes great concern because the disease is extremely contagious and spreads easily via contaminated equipment, hive tools, and beekeepers` hands, usually destroying entire colonies and causing great economical losses [1].

2 Each infected brood can be used to produce billions of long-living spores that are highly resistant to heat and chemical agents. That is a very impressive figure considering that as few as 35 spores may trigger the disease. As more and more broods become infected and die, the colony dwindles and eventually collapses [2]. Fortunately the AFB is still exotic in Brazil, although it has already been detected in several neighboring countries. Therefore safety procedures must be adopted in order to prevent it from entering the country through the import of contaminated honey [3]. Irradiation is a safe and efficient method for sterilizing products, including foods [4-6]. This work investigated the effects of gamma radiation on the inactivation of spores of P. larvae in honey. 2. EXPERIMENTS The experiments included the following steps, listed in chronological order: (1) preparation of suspensions with spores of P. larvae, (2) Preparation of samples of honey inoculated with the suspensions, (3) Irradiation and (4) bacteriological counting of the samples. Excepting the third step, all others were performed at Lanagro/RS, a reference laboratory for analysis and detection of P. larvae [7]. Two mixtures of several types of honey commercialized in Brazil were investigated, one for each set of experiments. Having different consistencies but the same initial concentration of spores of P. larvae (3.5 x 10 3 /ml), the material was initially stored at room temperature in 600 ml and 450 ml bottles and later transferred to 10 and 12 smaller closed plastic recipients during the first and the second series of experiments, respectively for gamma irradiation with different doses [3]. The exposure to a 49 kci 137 Cs source was performed in the experimental irradiation facility of CTEx (RJ) [8] at a dose rate of 1.8 kgy and yielded 5, 7.5 and 10 kgy with an estimated error of ±10%. All samples were returned to Lanagro/RS soon after irradiation for microbiological analyses. Several higher doses up to 50 kgy were also tested and resulted in no detectable growth of microorganisms in the samples. 3. RESULTS AND DISCUSSION The analyses included the identification and counting of viable colonies of P. larvae among spores of other bacteria in the irradiated and non-irradiated samples. The major results from the two series of experiments (I and II) for 0, 5, 7.5 kgy (or higher) are listed in Table 1. Considering the non-irradiated samples to correspond to no reduction in the population (100% survivors), those irradiated with 5 kgy were found to undergo some 97.0% and 98.7% reductions in the number of viable spores, as determined during the first and second series of experiments, respectively. In addition apparent inactivation of all colonies of bacteria and moulds in the samples was observed for doses equal or higher than 7.5 kgy.

3 Table 1. Number of viable spores of P. larvae in honey for doses of 0, 5 and 7.5 kgy Dose (kgy) 0 5 Series of Experiment N o. of Viable Spores Surviving Fraction Percent Inactivation I 164±25 164/ II 77±8 77/ D 10 I 5±3 5/ ± II 1 1/ Mean ± ± I, II By assuming a linear relationship between the logarithm of the surviving fraction of spores of P. larvae and the dose, the value (D 10 ) needed to secure one log cycle reduction of the initial population (D o ) can be calculated. The last column of Table 1 lists such results, including the mean value for D 10 (3.1 kgy) obtained by least-squares fitting. Table 2 provides information on the population of viable spores of P. larvae by assuming a logarithmic relationship between the population of survivors and the dose. Table 2. Parameters associated to the inactivation of spores of Paenibacillus larvae by irradiation calculated according to the Survival Curve Method using D 10 =3.1 kgy Dose (D) (kgy) D/D 10 Number of Spores/mL Relative Population of Viable Spores x x x x x x x x x x10-8 However it is also known that as the dose increases, so does the severity of attacks of radiolytic products on the DNA of microorganisms, adding to the direct action of the ionizing radiation. In addition mechanisms of repair of damages to DNA eventually collapse at very high doses, disrupting the initial linearity and causing the survival curve to bend down. Such

4 trend may also be observed in the survival curves for irradiated spores of Clostridium botulinum [9]. Thus a further approach may be used to obtain a more realistic survival curve by considering the limit for detection of P. larvae, estimated to be as low as 5.5 spores/ml (50% chance of detection) [10] and lower than 1.0x10 1 spores/ml (100% chance of detection). Moreover inspection of Table 2 reveals that the surviving population of P. larvae irradiated with 7.5 kgy (corresponding to 13/mL) would have probably been detected. The fact that it escaped detection indicates that the concentration of viable spores was in fact lower than that threshold. Thus in order to be conservative, it was assumed as equal to the upper limit of that threshold. That assumption provides a third data point, so that a secondorder polynomial of the decimal logarithm of the relative population of survivors (N/No) as a function of the radiation dose (D) can be fitted to the data, as Eq. 1 describes, leading to the values listed in Table 3. Log 10 N/N o = D D (1) Table 3. Population of viable spores of Paenibacillus larvae as function of gamma dose estimated according to a second-order polynomial fitting Dose (D) (kgy) Concentration of Viable Spores/mL Relative Population of Viable Spores 0 3.5x x x x x x x x x x x10-12 Comparison of Tables 2 and 3 leads to the conclusion that compared to the quadratic approximation, the linear one predicts a larger number of surviving spores especially at higher doses. However, both approximations indicate that irradiation with 10 kgy is insufficient to ensure a complete sterilization of P. larvae in honey, since the treatment is able to reduce the population of viable spores by some 4 log cycles only. The results for 25 kgy are 8 and 12 log cycles drops, according to the first- and second-order approximations, respectively. The latter figure being consistent with the 12-log-cycles drop usually needed to ensure a safe sterilization. However both doses, 10 kgy and 25 kgy, have been used in commercial sterilization of honey, combs and beekeeping equipment [11-12].

5 4. CONCLUSIONS The highly contagious, widespread and destructive AFD is still exotic in Brazil and serious efforts must be undertaken to prevent if from entering the country. Among them irradiation of imported honey and combs and second-hand beekeeping materials and hive equipment is recommended. In this work the mean value of D 10 for spores Paenibacillus larvae subsp. larvae in honey has been determined as 3.1±0.3 kgy across the first two-log-cycles reduction in the population. The results also indicate that 10 kgy causes a 4-log-cycles drop in the population of surviving spores of P. larvae, thus that dose is not sufficient to promote complete sterilization in cases of severe contamination. In contrast, extrapolation of the data suggests that irradiation with 25 kgy may be appropriate to ensure a safe sterilization of imported honey and second-hand beekeeping equipment that may bear residues of contaminated honey. REFERENCES 1. A General overview on AFB and EFB Pathogen, Way of Infection, Multiplication, Clinical symptoms and Outbreak, (2003). 2. American Foulbrood, (2007). 3. Radiossensibilidade de Esporos de Paenibacillus larvae subsp. larvae em Mel, (2006). 4. J. F. Diehl, Safety of Irradiated Foods, Marcel Dekker, Inc., New York, USA (1990). 5. Facts about Food Irradiation, (1999). 6. N. K. Hernandes; H. C. Vital; A. U. O. Sabaa-Srur, Irradiação de Alimentos: Vantagens e Limitações, Boletim do SBCTA, Vol. 37, n.2, pp (2003). 7. Isolamento de esporos de Paenibacillus larvae subsp. larvae no Brasil, (2003). 8. H. C. Vital, L. F. G. Pires, R. Q. Lima, S. O. Vellozo, Experimentos Dosimétricos no Irradiador Gama do IPE, Anais do V Encontro Nacional de Aplicações Nucleares (V ENAN), Rio de Janeiro, Brazil, Oct 15-20, Vol. 1, p. 48 (2000). 9. W. M. Urbain, Food Irradiation, Academic Press, Inc., Orlando, USA (1986). 10. D. M. T Schuch, R. H. Madden, A. Sattler, An Improved Method for the Detection and Presumptive Identification of Paenibacillus larvae subsp. larvae Spores in Honey, Journal of Apicultural Research, Vol. 40, n.2, pp (2001). 11. W. Migdal, H. B. Owczarczyk., B. Kedzia, E. Holderna-Kedzia, D. Madajczyk, Microbiological decontamination of natural honey by irradiation, Radiation Physics and Chemistry, Vol. 57, n.3, pp (2000). 12. Foul brood disease of honey bees: recognition and control, (2007).