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Development and evaluation of a selective medium for Brucella suis
Research in Veterinary Science 93 (2012) 565–567
Contents lists available at SciVerse ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Development and evaluation of a selective medium for Brucella suis Ana Cristina Ferreira a,f,⇑, Cláudia Almendra b,c, Regina Cardoso a, Marta Silva Pereira d, Albano Beja-Pereira b, Gordon Luikart b,e, Maria Inácia Corrêa de Sá a a
Instituto Nacional de Recursos Biológicos (INRB), I.P./LNIV, Laboratório Nacional de Investigação Veterinária, Estrada de Benfica, 701, 1549-011 Lisboa, Portugal CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, 4485-661 Vairão, Portugal c ICBAS, Instituto de Ciências Biomédicas, Universidade do Porto, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal d Fullsense, Zona Industrial de Tondela Zim II, Lote 6, 3460-070 Tondela, Portugal e Flathead Lake Biological Station, University of Montana, Polson, MT 59860, USA f Universidade de Lisboa, Faculdade de Ciências, Centro de Biodiversidade, Genómica Integrativa e Funcional (BioFIG), Edificio ICAT, Campus da FCUL, Campo Grande, 1749-016 Lisboa, Portugal b
a r t i c l e
i n f o
Article history: Received 20 May 2011 Accepted 7 September 2011
Keywords: Brucella suis Wild boar Selective medium Bacteriological diagnosis
a b s t r a c t A new selective medium, named LNIV-M, has been developed for isolation of Brucella suis. In this work, we evaluated the growth of B. suis reference and field strains from domestic pigs in different basal media and the susceptibility to different antibiotics contained in the currently used Farrell’s and modified Thayer-Martin media. We also determined the efficacy of LNIV-M and its diagnostic performance for isolating B. suis from wild boar tissue samples. A total of 1649 samples from 918 hunter-harvested wild boars were cultured in LNIV-M, Farrell’s and modified Thayer-Martin media. One hundred and thirty-nine (8.4%) samples from 63 (6.9%) animals resulted in a positive culture. LNIV-M detected 93.6% and 62.6% of positive animals and samples, respectively, while Farrell’s and modified Thayer-Martin media detected, respectively, 92.1% and 79.4% of positive animals and 58.3% and 59.7% of samples. These results confirm the adequate diagnostic performance of LNIV-M in the isolation of B. suis. Ó 2011 Elsevier Ltd. All rights reserved.
In Europe, Brucella suis biovar 2 is the most commonly isolated biovar in domestic pigs. It is also frequently isolated from wild boar (Sus scrofa) and European hares (Lepus europaeus), which are considered its natural reservoirs (Al Dahouk et al., 2005; Cvetnic et al., 2003; Garin-Bastuji et al., 2000; Godfroid et al., 1994; Godfroid and Käsbohrer, 2002). Wild boar seems to be the main source of infection for domestic pigs in the Iberian Peninsula (EFSA, 2009; Muñoz et al., 2010). Due to the lack of specificity of serological tests, the isolation of brucellae from animals is the unique indisputable test to demonstrate the infection (EFSA, 2009). Primary isolation of the slow growing Brucella species requires selective media to inhibit overgrowing contaminants present in field samples. The most widely used media are Farrell’s medium (FAR; Farrell, 1974) and modified Thayer-Martin’s medium (MTM; Brown et al., 1971; Marín et al., 1996a). FAR is an excellent medium but has inhibitory effects on some Brucella species ⇑ Corresponding author at: Instituto Nacional de Recursos Biológicos (INRB), I.P./ LNIV, Laboratório Nacional de Investigação Veterinária, Estrada de Benfica, 701, 1549-011 Lisboa, Portugal. Tel.: +351 217115284. E-mail addresses: [email protected] (A.C. Ferreira), clau [email protected] (C. Almendra), [email protected] a.pt (R. Cardoso), [email protected] (M.S. Pereira), [email protected] (A. Beja-Pereira), [email protected] (G. Luikart), [email protected] tura.pt (M.I. Corrêa de Sá). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.09.004
including B. suis (De Miguel et al., 2011; Jensen and Halling, 2010; Marín et al., 1996a, 1996b). On the other hand, the MTM is suitable for isolating most brucellae, but lacks inhibitory effects on many overgrowing contaminants, and hinders the assessment of colonial morphology by direct observation of culture plates (De Miguel et al., 2011; Marín et al., 1996a, 1996b). The objective of this work was to develop a new selective medium (named herein LNIV-M) for the isolation of B. suis and evaluate its diagnostic performance in the isolation of B. suis biovar 2. To select basal medium for LNIV-M, we used the reference strains, B. suis 1330 (biovar 1, ATCC 23444), B. suis Thomsen (biovar 2, ATCC 23445) and B. suis 686 (biovar 3, ATCC 23446) and 22 B. suis biovar 2 field strains from domestic pigs, identified and typed according to Alton et al. (1988). Blood Agar Base (BAB), GC medium (GC), Tryptic Soy Agar (TSA) (all from Becton–Dickinson, USA), Brucella Medium Base (BMB; Oxoid, England) and Plommet Medium (PM; Plommet, 1991) were evaluated as basal media by colony counting, alone or supplemented with 5% sterile horse serum (-S; Euro-Lone, Canada) or 1% haemoglobin (-H; Becton–Dickinson, USA). BMB, GC and TSA were also supplemented with 0.1% yeast extract (-YE; Becton–Dickinson, USA). Each strain was grown in TSA slants at 37 °C with 10% CO2 for 48 h, harvested in sterile phosphate-buffered saline (PBS) pH 7.2 and spectrophotometrically (S2100 Diode Array Spectrophotometer, WPA) adjusted in the same
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diluent to about 109 CFU/mL (OD600 = 0.170). Then, six 10-fold dilutions of each suspension were performed in PBS and 0.1 mL of each dilution were plated by triplicate in each medium. After incubation at 37 °C for 5 days in 10% CO2 atmosphere, the mean (n = 3) number of CFU/plate was determined in each medium, and statistically compared by ANOVA followed by LSD multiple comparison test, using the PROC GLM procedure of SAS statistical package (SAS/ STATÒ version 9.2; SAS, 2008; Zar, 1984). Our results proved that B. suis growth was significantly (p < 0.0001) inhibited in GC-S and GC-YE with respect to GC-H, BAB-S or TSA-S as described by others (De Miguel et al., 2011). The later three basal media performed similarly and yielded the highest number of CFU/plate (results not shown). Considering the cost of BAB-S and that GC-H is a no-translucent medium and cumbersome to prepared, TSA-S was finally chosen as LNIV-M basal medium. In a second step, the antimicrobial supplement was formulated according to the results of Minimal Inhibitory Concentration (MIC; Sahm and Washington, 1991) and Concentration Enabling Growth (CEG; Marín et al., 1996a) obtained with the 22 B. suis biovar 2 field strains against bacitracin, vancomycin, polymyxin B, nalidixic acid, cycloheximide, nystatin, colistin, nitrofurantoin and amphotericin B (all from Sigma–Aldrich, Germany), in the range of 512–1 mg/L, using Mueller–Hinton broth and agar (Becton–Dickinson, USA). Results are presented in Table 1. The variations in susceptibility to the antibiotics were notorious, except for amphotericin B and cycloheximide antifungal agents which, as expected, did not affect the growth of B. suis bv. 2. In particular, most of the strains (13/22; 59.1%) were inhibited by low concentrations of polymyxin B (MIC 6 4 mg/L and CEG 6 1 mg/L), but all are resistant to higher concentrations of colistin (MIC P 16 mg/L and CEG P 4 mg/L). These results are in agreement with those obtained by De Miguel and colleagues (2011). Considering the MIC and CEG results (Table 1), LNIV-M was formulated with TSA-S supplemented with 20 mg/L vancomycin (bactericidal for most gram positive bacteria), 4 mg/L colistin and 16 mg/L of nitrofurantoin (active against most gram negative bacteria), and 100 mg/L cycloheximide and 16 mg/L (95 000 IU/L) nystatin (active against yeasts and moulds). The efficacy of LNIV-M for
Table 1 Minimal Inhibitory Concentration (MIC) and Concentration Enabling Growth (CEG) for 22 B. suis field strains. Antibiotics
No. of strains
MIC (mg/L)
CEG (mg/L)
Bacitracin
18 4 2 10 8 2 7 12 3 9 11 2 19 3 8 6 6 2 22 21 1 7 9 5 1
512 256 >512 512 512 256 64 32 16 64 4 1 64 32 512 512 512 512 >512 512 256 512 256 128 64
256 128 512 256 128 64 16 8 4 16 1 <1 32 16 256 128 64 32 512 256 128 256 64 32 16
Vancomycin
Colistin methanosulfonate
Polymixin B sulphate
Nalidixic acid Nitrofurantoin
Amphotericin B Cycloheximide Nystatin
culturing B. suis was compared with that of FAR and MTM media (both prepared as described elsewhere; Marín et al., 1996a; Farrell, 1974), using the B. suis reference and field strains described above. The number of CFU/plate grew in LNIV-M, FAR and MTM selective medium and TSA-S (control) was determined and statistically compared as detail above. The overall results are shown in Table 2. No significant differences were found for LNIV-M and MTM with respect to the TSA-S medium used as control, but FAR significantly (p < 0.05) reduced the number of CFU. These data confirm our preliminary studies and the results obtained for other Brucella species (De Miguel et al., 2011; Marín et al., 1996a, 1996b). Moreover, colonial size on LNIV-M and MTM were smaller than on TSA-S but larger than on FAR. Since MTM contains haemoglobin, the translucent LNIV-M has the advantage to allow a better direct identification of Brucella. Finally, to evaluate the relative diagnostic performance of LNIVM in the primary isolation of B. suis with respect to FAR and MTM, we cultured 1649 samples (spleen; liver; lung; peripheral lymph node; reproductive organs) from 918 hunter-harvested wild boars, sampled during the hunting season (November–March) across Portugal. All tissue samples were processed as described by Alton et al. (1988) and 0.2 mL/plate of each tissue homogenate were cultured in duplicate plates on the three media. Samples were considered positive when at least one CFU was isolated after incubation at 37 °C (10% CO2) for up to 10 days. Brucella species and biovars were identified according to Alton et al. (1988). The number of plates rejected due to overgrowing contaminants in LNIV-M was lower than in MTM but higher than in FAR (data not shown). From the 918 wild boars tested, 63 animals (6.9%) were found to be infected with B. suis biovar 2, showing a total of 139 (8.4%) positive samples (63 spleens; 21 livers; 21 lymph nodes; 21 lungs; 13 reproductive organs). LNIV-M detected 59 out the 63 (93.6%) positive animals and 87 out the 139 (62.6%) positive samples, while FAR and MTM detected, respectively, 58 (92.1%) and 50 (79.4%) of animals, and 81 (58.3%), and 83 (59.7%) of samples. The combined use of LNIV-M and MTM detected 95.2% (60) positive animals, whereas LNIV-M and FAR detected 100% positive animals. The results obtained in the diagnostic performance of FAR and MTM are not in agreement with those from efficacy studies. This can be explained by the high inhibitory effect of FAR for most contaminants present in field samples, allowing the growth of Brucella colonies and showing that this medium is more suitable for diagnostic purpose than MTM. The Cohen’s Kappa coefficient (K) and McNemar’s Chi-square test (v2; Zar, 1984) were used to calculate the level of agreement between media. A p less than 0.05 (p < 0.05) was considered statistically significant. Analysis of samples showed a substantial
Table 2 Susceptibility of B. suis reference and field strains to the new LNIV-M selective culture medium, in comparison to modified Thayer-Martin (MTM) and Farrell (FAR) media. Culture media
TSA-S (control) LNIV-M MTM FAR
CFU/plate (mean ± SD) of B. suisa Reference strains
Field strains
Biovar 1 ATCC 23444
Biovar 2 ATCC 23445
Biovar 3 ATCC 23446
Biovar 2 (n = 22)
84.0 ± 7.81B
75.0 ± 15.10B
78.7 ± 5.51B
72.1 ± 21.57B
77.3 ± 8.08B 68.3 ± 15.04B 22.3 ± 2.08A
83.3 ± 6.66B 73.3 ± 7.57B 25.7 ± 3.06A
81.3 ± 1.53B 46.0 ± 14.00B 21.0 ± 1.00A
67.0 ± 20.77B 64.4 ± 23.12B 48.6 ± 18.74A
a Mean and SD (n = 3 from independent experiments with the corresponding reference strain; or n = 22 field strains) of the number of CFU/plate were determined, after triplicate plating of 0.1 mL of a suspension containing around 103 CFU/ mL in each culture medium. TSA-S was used as control. A, B Values with the same letter indicate that means were statistically equivalent (p > 0.05).
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agreement between media, except for lung tissue, where poor recovery of Brucella was achieved. The best value was observed when comparing LNIV-M and FAR (K = 0.699, 95% CI; v2 = 1.25, p P 0.05, data not shown). In conclusion, for an adequate bacteriological diagnosis of brucellosis and to increase its sensitivity, more than one selective culture medium should be used. Data obtained in this study indicate that LNIV-M is a useful selective medium for isolation of B. suis and the combined use of LNIV-M and FAR has shown to improves the performance of the bacteriological diagnosis of brucellosis in swine. Acknowledgments We acknowledge Professor Rogério Tenreiro from Universidade de Lisboa, Faculdade de Ciências, Centro de Biodiversidade, Genómica Integrativa e Funcional (BioFIG) for his detail suggestions and critical review of the manuscript which helped us to improve it. This work was financed by project Grants from FCT (PTDC/CVT/ 69438/2006 and PTDC/CVT/104050/2008). CA was supported by a National Grant (SFRH/BD30737/2006) Fundação para a Ciência e Tecnologia (FCT) and GL was partially supported by Grants from the US National Science Foundation (Grant DEB 074218), the Walton Family Foundation, CIBIO-UP, and Grants from the Portuguese Science Foundation (PTDC/BIA-BDE/65625/2006 and PTDC/CVT/ 69438/2006). References Al Dahouk, S., Nöckler, K., Tomaso, H., Splettstoesser, W.D., Jungersen, G., Riber, U., Petry, T., Hoffmann, D., Scholz, H.C., Hensel, A., Neubauer, H., 2005. Seroprevalence of Brucellosis, Tularaemia and Yersiniosis in Wild Boars (Sus scrofa) from North-Eastern Germany. Journal of Veterinary Medecine 52, 444– 455. Alton, G.C., Jones, L.M., Angus, R.D., Verger, J.M., 1988. Techniques for the Brucellosis Laboratory. Institute National de la Recherche Agronomique, Paris, France. Brown, G.M., Ranger, C.R., Kelly, D.J., 1971. Selective media for the isolation of Brucella ovis. Cornell Veterinarian 61, 265–280.
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Cvetnic, Z., Mitak, M., Ocepek, M., Lojkic, M., Terzic, S., Jemersic, L., Humski, A., Habrun, B., Sostaric, B., Brstilo, M., Krt, B., Garin-Bastuji, B., 2003. Wild boars (sus scrofa) as reservoirs of Brucella suis biovar 2 in Croatia. Acta Veterinaria Hungaria 51, 465–473. De Miguel, M.J., Marín, C.M., Muñoz, P.M., Dieste, L., Grilló, M.J., Blasco, J.M., 2011. Development of a selective medium for primary isolation of the main Brucella species. Journal of Clinical Microbiology 49, 1458–1463. EFSA, 2009. Scientific Opinion of the Panel on Animal Health and Welfare (AHAW) on a request from the Commission on porcine brucellosis (Brucella suis). The EFSA Journal 1144, 1–112. Farrell, I.D., 1974. The development of a new selective medium for the isolation of Brucella abortus from contaminated sources. Research in Veterinary Science 16, 280–286. Garin-Bastuji, B., Hars, J., Calvez, D., Thiébaud, M., Artois, M., 2000. Brucellose du porc domestique et du sanglier sauvage due à Brucella suis biovar 2 en France. Epidémiologie and Santé Animale 38, 1–5. Godfroid, J., Käsbohrer, A., 2002. Brucellosis in the European Union and Norway at the turn of the twenty-first century. Veterinary Microbiology 90, 135–145. Godfroid, J., Michel, P., Uytterhaiegen, L., De Smedt, C., Rasseneur, F., Boelaert, F., Saegerman, C., Patigny, X., 1994. Brucellose enzootique a Brucella suis biotype 2 chez le sanglier (Sus scrofa) en Belgique. Les Annales de Medicine Vétérinaire 138, 263–268. Jensen, A.E., Halling, S.M., 2010. Effect of polymyxin B and environmental conditions on isolation of Brucella species and the vaccine strain RB51. Comparative Immunology, Microbiology and Infectious Diseases 33, 121–131. Marín, C.M., Alabart, J., Blasco, J.M., 1996a. Effect of antibiotics contained in two Brucella selective media on growth of B. abortus, B. melitensis and B. ovis. Journal of Clinical Microbiology 34, 426–428. Marín, C., Jimenez de Bagüés, M.P., Barberán, M., Blasco, J.M., 1996b. Comparison of two selective media for the isolation of B. melitensis from naturally infected sheep and goats. Veterinary Record 138, 409–411. Muñoz, M.P., Boadella, M., Arnal, M.C., De Miguel, M., Revilla, M., Martinez, D., Vicente, J., Acevedo, P., Oleaga, A., Ruiz-Fons, F., Marin, C., Prieto, J.M., De la Fuente, J., Barral, M., Barberan, M., De Luco, D., Blasco, J.M., Gortazar, C., 2010. Spatial distribution and risk factors of Brucellosis in Iberian wild ungulates. BMC Infectious Diseases 10, 46. Plommet, M., 1991. Minimal requirements for growth of Brucella suis and other Brucella species. Zentralblatt fur Bakteriologie 275, 436–450. Sahm, D.F., Washington, J.A., 1991. Antibacterial susceptibility tests: dilution methods. In: Balows, A., Hausler, W.J., Jr., Hermann, K.L., Isenberg, H.D., Shadomy, H.J. (Eds.), Manual of Clinical Microbiology, fifth ed. American Society for Microbiology, Washington, D.C., pp. 1105–1116. SAS Institute, 2008, . SAS/STAT Version 9.2, User’s Guide, second ed. SAS Institute Inc., Carry, NC, p. 208. Zar, J.H., 1984. Biostatistical Analysis, second ed. Prentice Hall International, London.
Contents lists available at SciVerse ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Development and evaluation of a selective medium for Brucella suis Ana Cristina Ferreira a,f,⇑, Cláudia Almendra b,c, Regina Cardoso a, Marta Silva Pereira d, Albano Beja-Pereira b, Gordon Luikart b,e, Maria Inácia Corrêa de Sá a a
Instituto Nacional de Recursos Biológicos (INRB), I.P./LNIV, Laboratório Nacional de Investigação Veterinária, Estrada de Benfica, 701, 1549-011 Lisboa, Portugal CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, 4485-661 Vairão, Portugal c ICBAS, Instituto de Ciências Biomédicas, Universidade do Porto, Largo Prof. Abel Salazar, 2, 4099-003 Porto, Portugal d Fullsense, Zona Industrial de Tondela Zim II, Lote 6, 3460-070 Tondela, Portugal e Flathead Lake Biological Station, University of Montana, Polson, MT 59860, USA f Universidade de Lisboa, Faculdade de Ciências, Centro de Biodiversidade, Genómica Integrativa e Funcional (BioFIG), Edificio ICAT, Campus da FCUL, Campo Grande, 1749-016 Lisboa, Portugal b
a r t i c l e
i n f o
Article history: Received 20 May 2011 Accepted 7 September 2011
Keywords: Brucella suis Wild boar Selective medium Bacteriological diagnosis
a b s t r a c t A new selective medium, named LNIV-M, has been developed for isolation of Brucella suis. In this work, we evaluated the growth of B. suis reference and field strains from domestic pigs in different basal media and the susceptibility to different antibiotics contained in the currently used Farrell’s and modified Thayer-Martin media. We also determined the efficacy of LNIV-M and its diagnostic performance for isolating B. suis from wild boar tissue samples. A total of 1649 samples from 918 hunter-harvested wild boars were cultured in LNIV-M, Farrell’s and modified Thayer-Martin media. One hundred and thirty-nine (8.4%) samples from 63 (6.9%) animals resulted in a positive culture. LNIV-M detected 93.6% and 62.6% of positive animals and samples, respectively, while Farrell’s and modified Thayer-Martin media detected, respectively, 92.1% and 79.4% of positive animals and 58.3% and 59.7% of samples. These results confirm the adequate diagnostic performance of LNIV-M in the isolation of B. suis. Ó 2011 Elsevier Ltd. All rights reserved.
In Europe, Brucella suis biovar 2 is the most commonly isolated biovar in domestic pigs. It is also frequently isolated from wild boar (Sus scrofa) and European hares (Lepus europaeus), which are considered its natural reservoirs (Al Dahouk et al., 2005; Cvetnic et al., 2003; Garin-Bastuji et al., 2000; Godfroid et al., 1994; Godfroid and Käsbohrer, 2002). Wild boar seems to be the main source of infection for domestic pigs in the Iberian Peninsula (EFSA, 2009; Muñoz et al., 2010). Due to the lack of specificity of serological tests, the isolation of brucellae from animals is the unique indisputable test to demonstrate the infection (EFSA, 2009). Primary isolation of the slow growing Brucella species requires selective media to inhibit overgrowing contaminants present in field samples. The most widely used media are Farrell’s medium (FAR; Farrell, 1974) and modified Thayer-Martin’s medium (MTM; Brown et al., 1971; Marín et al., 1996a). FAR is an excellent medium but has inhibitory effects on some Brucella species ⇑ Corresponding author at: Instituto Nacional de Recursos Biológicos (INRB), I.P./ LNIV, Laboratório Nacional de Investigação Veterinária, Estrada de Benfica, 701, 1549-011 Lisboa, Portugal. Tel.: +351 217115284. E-mail addresses: [email protected] (A.C. Ferreira), clau [email protected] (C. Almendra), [email protected] a.pt (R. Cardoso), [email protected] (M.S. Pereira), [email protected] (A. Beja-Pereira), [email protected] (G. Luikart), [email protected] tura.pt (M.I. Corrêa de Sá). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.09.004
including B. suis (De Miguel et al., 2011; Jensen and Halling, 2010; Marín et al., 1996a, 1996b). On the other hand, the MTM is suitable for isolating most brucellae, but lacks inhibitory effects on many overgrowing contaminants, and hinders the assessment of colonial morphology by direct observation of culture plates (De Miguel et al., 2011; Marín et al., 1996a, 1996b). The objective of this work was to develop a new selective medium (named herein LNIV-M) for the isolation of B. suis and evaluate its diagnostic performance in the isolation of B. suis biovar 2. To select basal medium for LNIV-M, we used the reference strains, B. suis 1330 (biovar 1, ATCC 23444), B. suis Thomsen (biovar 2, ATCC 23445) and B. suis 686 (biovar 3, ATCC 23446) and 22 B. suis biovar 2 field strains from domestic pigs, identified and typed according to Alton et al. (1988). Blood Agar Base (BAB), GC medium (GC), Tryptic Soy Agar (TSA) (all from Becton–Dickinson, USA), Brucella Medium Base (BMB; Oxoid, England) and Plommet Medium (PM; Plommet, 1991) were evaluated as basal media by colony counting, alone or supplemented with 5% sterile horse serum (-S; Euro-Lone, Canada) or 1% haemoglobin (-H; Becton–Dickinson, USA). BMB, GC and TSA were also supplemented with 0.1% yeast extract (-YE; Becton–Dickinson, USA). Each strain was grown in TSA slants at 37 °C with 10% CO2 for 48 h, harvested in sterile phosphate-buffered saline (PBS) pH 7.2 and spectrophotometrically (S2100 Diode Array Spectrophotometer, WPA) adjusted in the same
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diluent to about 109 CFU/mL (OD600 = 0.170). Then, six 10-fold dilutions of each suspension were performed in PBS and 0.1 mL of each dilution were plated by triplicate in each medium. After incubation at 37 °C for 5 days in 10% CO2 atmosphere, the mean (n = 3) number of CFU/plate was determined in each medium, and statistically compared by ANOVA followed by LSD multiple comparison test, using the PROC GLM procedure of SAS statistical package (SAS/ STATÒ version 9.2; SAS, 2008; Zar, 1984). Our results proved that B. suis growth was significantly (p < 0.0001) inhibited in GC-S and GC-YE with respect to GC-H, BAB-S or TSA-S as described by others (De Miguel et al., 2011). The later three basal media performed similarly and yielded the highest number of CFU/plate (results not shown). Considering the cost of BAB-S and that GC-H is a no-translucent medium and cumbersome to prepared, TSA-S was finally chosen as LNIV-M basal medium. In a second step, the antimicrobial supplement was formulated according to the results of Minimal Inhibitory Concentration (MIC; Sahm and Washington, 1991) and Concentration Enabling Growth (CEG; Marín et al., 1996a) obtained with the 22 B. suis biovar 2 field strains against bacitracin, vancomycin, polymyxin B, nalidixic acid, cycloheximide, nystatin, colistin, nitrofurantoin and amphotericin B (all from Sigma–Aldrich, Germany), in the range of 512–1 mg/L, using Mueller–Hinton broth and agar (Becton–Dickinson, USA). Results are presented in Table 1. The variations in susceptibility to the antibiotics were notorious, except for amphotericin B and cycloheximide antifungal agents which, as expected, did not affect the growth of B. suis bv. 2. In particular, most of the strains (13/22; 59.1%) were inhibited by low concentrations of polymyxin B (MIC 6 4 mg/L and CEG 6 1 mg/L), but all are resistant to higher concentrations of colistin (MIC P 16 mg/L and CEG P 4 mg/L). These results are in agreement with those obtained by De Miguel and colleagues (2011). Considering the MIC and CEG results (Table 1), LNIV-M was formulated with TSA-S supplemented with 20 mg/L vancomycin (bactericidal for most gram positive bacteria), 4 mg/L colistin and 16 mg/L of nitrofurantoin (active against most gram negative bacteria), and 100 mg/L cycloheximide and 16 mg/L (95 000 IU/L) nystatin (active against yeasts and moulds). The efficacy of LNIV-M for
Table 1 Minimal Inhibitory Concentration (MIC) and Concentration Enabling Growth (CEG) for 22 B. suis field strains. Antibiotics
No. of strains
MIC (mg/L)
CEG (mg/L)
Bacitracin
18 4 2 10 8 2 7 12 3 9 11 2 19 3 8 6 6 2 22 21 1 7 9 5 1
512 256 >512 512 512 256 64 32 16 64 4 1 64 32 512 512 512 512 >512 512 256 512 256 128 64
256 128 512 256 128 64 16 8 4 16 1 <1 32 16 256 128 64 32 512 256 128 256 64 32 16
Vancomycin
Colistin methanosulfonate
Polymixin B sulphate
Nalidixic acid Nitrofurantoin
Amphotericin B Cycloheximide Nystatin
culturing B. suis was compared with that of FAR and MTM media (both prepared as described elsewhere; Marín et al., 1996a; Farrell, 1974), using the B. suis reference and field strains described above. The number of CFU/plate grew in LNIV-M, FAR and MTM selective medium and TSA-S (control) was determined and statistically compared as detail above. The overall results are shown in Table 2. No significant differences were found for LNIV-M and MTM with respect to the TSA-S medium used as control, but FAR significantly (p < 0.05) reduced the number of CFU. These data confirm our preliminary studies and the results obtained for other Brucella species (De Miguel et al., 2011; Marín et al., 1996a, 1996b). Moreover, colonial size on LNIV-M and MTM were smaller than on TSA-S but larger than on FAR. Since MTM contains haemoglobin, the translucent LNIV-M has the advantage to allow a better direct identification of Brucella. Finally, to evaluate the relative diagnostic performance of LNIVM in the primary isolation of B. suis with respect to FAR and MTM, we cultured 1649 samples (spleen; liver; lung; peripheral lymph node; reproductive organs) from 918 hunter-harvested wild boars, sampled during the hunting season (November–March) across Portugal. All tissue samples were processed as described by Alton et al. (1988) and 0.2 mL/plate of each tissue homogenate were cultured in duplicate plates on the three media. Samples were considered positive when at least one CFU was isolated after incubation at 37 °C (10% CO2) for up to 10 days. Brucella species and biovars were identified according to Alton et al. (1988). The number of plates rejected due to overgrowing contaminants in LNIV-M was lower than in MTM but higher than in FAR (data not shown). From the 918 wild boars tested, 63 animals (6.9%) were found to be infected with B. suis biovar 2, showing a total of 139 (8.4%) positive samples (63 spleens; 21 livers; 21 lymph nodes; 21 lungs; 13 reproductive organs). LNIV-M detected 59 out the 63 (93.6%) positive animals and 87 out the 139 (62.6%) positive samples, while FAR and MTM detected, respectively, 58 (92.1%) and 50 (79.4%) of animals, and 81 (58.3%), and 83 (59.7%) of samples. The combined use of LNIV-M and MTM detected 95.2% (60) positive animals, whereas LNIV-M and FAR detected 100% positive animals. The results obtained in the diagnostic performance of FAR and MTM are not in agreement with those from efficacy studies. This can be explained by the high inhibitory effect of FAR for most contaminants present in field samples, allowing the growth of Brucella colonies and showing that this medium is more suitable for diagnostic purpose than MTM. The Cohen’s Kappa coefficient (K) and McNemar’s Chi-square test (v2; Zar, 1984) were used to calculate the level of agreement between media. A p less than 0.05 (p < 0.05) was considered statistically significant. Analysis of samples showed a substantial
Table 2 Susceptibility of B. suis reference and field strains to the new LNIV-M selective culture medium, in comparison to modified Thayer-Martin (MTM) and Farrell (FAR) media. Culture media
TSA-S (control) LNIV-M MTM FAR
CFU/plate (mean ± SD) of B. suisa Reference strains
Field strains
Biovar 1 ATCC 23444
Biovar 2 ATCC 23445
Biovar 3 ATCC 23446
Biovar 2 (n = 22)
84.0 ± 7.81B
75.0 ± 15.10B
78.7 ± 5.51B
72.1 ± 21.57B
77.3 ± 8.08B 68.3 ± 15.04B 22.3 ± 2.08A
83.3 ± 6.66B 73.3 ± 7.57B 25.7 ± 3.06A
81.3 ± 1.53B 46.0 ± 14.00B 21.0 ± 1.00A
67.0 ± 20.77B 64.4 ± 23.12B 48.6 ± 18.74A
a Mean and SD (n = 3 from independent experiments with the corresponding reference strain; or n = 22 field strains) of the number of CFU/plate were determined, after triplicate plating of 0.1 mL of a suspension containing around 103 CFU/ mL in each culture medium. TSA-S was used as control. A, B Values with the same letter indicate that means were statistically equivalent (p > 0.05).
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agreement between media, except for lung tissue, where poor recovery of Brucella was achieved. The best value was observed when comparing LNIV-M and FAR (K = 0.699, 95% CI; v2 = 1.25, p P 0.05, data not shown). In conclusion, for an adequate bacteriological diagnosis of brucellosis and to increase its sensitivity, more than one selective culture medium should be used. Data obtained in this study indicate that LNIV-M is a useful selective medium for isolation of B. suis and the combined use of LNIV-M and FAR has shown to improves the performance of the bacteriological diagnosis of brucellosis in swine. Acknowledgments We acknowledge Professor Rogério Tenreiro from Universidade de Lisboa, Faculdade de Ciências, Centro de Biodiversidade, Genómica Integrativa e Funcional (BioFIG) for his detail suggestions and critical review of the manuscript which helped us to improve it. This work was financed by project Grants from FCT (PTDC/CVT/ 69438/2006 and PTDC/CVT/104050/2008). CA was supported by a National Grant (SFRH/BD30737/2006) Fundação para a Ciência e Tecnologia (FCT) and GL was partially supported by Grants from the US National Science Foundation (Grant DEB 074218), the Walton Family Foundation, CIBIO-UP, and Grants from the Portuguese Science Foundation (PTDC/BIA-BDE/65625/2006 and PTDC/CVT/ 69438/2006). References Al Dahouk, S., Nöckler, K., Tomaso, H., Splettstoesser, W.D., Jungersen, G., Riber, U., Petry, T., Hoffmann, D., Scholz, H.C., Hensel, A., Neubauer, H., 2005. Seroprevalence of Brucellosis, Tularaemia and Yersiniosis in Wild Boars (Sus scrofa) from North-Eastern Germany. Journal of Veterinary Medecine 52, 444– 455. Alton, G.C., Jones, L.M., Angus, R.D., Verger, J.M., 1988. Techniques for the Brucellosis Laboratory. Institute National de la Recherche Agronomique, Paris, France. Brown, G.M., Ranger, C.R., Kelly, D.J., 1971. Selective media for the isolation of Brucella ovis. Cornell Veterinarian 61, 265–280.
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