Open Access

Genetic diversity of Brucella ovis isolates from Rio Grande do Sul, Brazil, by MLVA16

  • Elaine MS Dorneles1,
  • Guilherme N Freire1,
  • Maurício G Dasso2,
  • Fernando P Poester1 and
  • Andrey P Lage1Email author
Contributed equally
BMC Research Notes20147:447

https://doi.org/10.1186/1756-0500-7-447

Received: 21 March 2014

Accepted: 1 July 2014

Published: 12 July 2014

Abstract

Background

Ovine epididymitis is predominantly associated with Brucella ovis infection. Molecular characterization of Brucella spp. achieved by multi-locus variable number of tandem repeats (VNTR) analyses (MLVA) have proved to be a powerful tool for epidemiological trace-back studies. Thus, the aim of this study was to evaluate the genetic diversity of Brucella ovis isolates from Rio Grande do Sul State, Brazil, by MLVA16.

Findings

MLVA16 genotyping identified thirteen distinct genotypes and a Hunter-Gaston diversity index of 0.989 among the fourteen B. ovis genotyped strains. All B. ovis MLVA16 genotypes observed in the present study represented non-previously described profiles. Analyses of the eight conserved loci included in panel 1 (MLVA8) showed three different genotypes, two new and one already described for B. ovis isolates. Among ten B. ovis isolates from same herd only two strains had identical pattern, whereas the four isolates with no epidemiologic information exhibited a single MLVA16 pattern each. Analysis of minimal spanning tree, constructed using the fourteen B. ovis strains typed in this study together with all nineteen B. ovis MLVA16 genotypes available in the MLVAbank 2014, revealed the existence of two clearly distinct major clonal complexes.

Conclusions

In conclusion, the results of the present study showed a high genetic diversity among B. ovis field isolates from Rio Grande do Sul State, Brazil, by MLVA16.

Keywords

Genotyping Brucella ovis MLVA16 Ovine brucellosis

Findings

Background

Brucella ovis is a rough, Gram-negative, non-spore-forming, non-motile and facultative intracellular bacterium [1]. In rams, the microorganism causes mainly epididymitis [2, 3], whereas in ewes the lesions are characterized by degeneration and inflammation of the endometrium with focal or diffuse lymphoid infiltrations [4].

Infection has been recognized in all countries where sheep are of economic importance and leads to significant losses to animal production [5, 6]. In Brazil, the ovine epididymitis is chiefly described in southern States (Rio Grande do Sul, Santa Catarina, Paraná), where the sheep-raising is more developed [7], having been first reported in 1966 in Rio Grande do Sul State [8]. In 1996, a clinical and serological survey of rams in Rio Grande do Sul State showed prevalence of 13.4% [9]. More recent data, with a broader sampling, (2011/2012) indicates a decrease in this prevalence index to 2.8% of positive animals [10].

Molecular characterization of Brucella spp. achieved by multi-locus variable number of tandem repeats (VNTR) analyses (MLVA) have proved to be a powerful tool to determine relationships among Brucella spp isolates from different animal species and from humans, as well as for epidemiological trace-back studies [1117]. However, data regarding B. ovis genotyping, using MLVA16 or even other techniques are very scarce. Thus, the aim of this study was to evaluate the genetic diversity of B. ovis field isolates from Rio Grande do Sul, Brazil, using MLVA16.

Methods

Fourteen B. ovis field isolates obtained from sheep between 1982 and 1995 were used in this study. They were provided from the collection of Instituto de Pesquisas Veterinárias Desidério Finamor and were isolated (by FPP and MGD) from semen samples collected by electroejaculation from rams in Rio Grande do Sul, Brazil (Santana do Livramento - 10; Uruguaiana - 2; and undefined municipalities - 2). All isolates from Santana do Livramento were from animals of the same herd, whereas the others four B. ovis isolates had not information about herd of origin. All isolates were confirmed to be B. ovis by biochemical and molecular tests [1820]. Approval to use the B. ovis isolates in this study was formally given by the director of IPVDF.

Brucella ovis colonies were inactivated at 85°C for 2 hours and subjected to genomic DNA extraction [21, 22]. DNA from each strain was genotyped by MLVA16, which was divided in: panel 1 (Bruce06, Bruce08, Bruce11, Bruce12, Bruce42, Bruce43, Bruce45, Bruce55); panel 2A (Bruce18, Bruce19, Bruce21); and panel 2B (Bruce04, Bruce07, Bruce09, Bruce16, Bruce30) [11, 15].

From digitalized image of each gel, the band size was estimated and then converted into number of repeat units for each locus by using the software BioNumerics 6.1 (Applied Maths, Belgium) [15]. Brucella melitensis 16M (ATCC 23456T) was used as control for band size estimation of all MLVA16 loci. The genotypes obtained were compared to those deposited in the MLVAbank 2014 (http://mlva.u-psud.fr/brucella/). Clustering analysis was performed using the category coefficient and UPGMA (BioNumerics 6.1) [15]. The Hunter-Gaston diversity index (HGDI) was used [23]. The minimum-spanning tree (MST) was generated using Prim’s algorithm associated with priority rule (eBURST algorithm) and bootstrap resampling [24, 25] (BioNumerics 6.1). The MST presented is the top score tree, the tree with the highest overall reliability score.

Results

Analysis of the MLVA16 loci revealed thirteen distinct genotypes among the fourteen B. ovis strains evaluated (Figure 1) and a HGDI of 0.989. All these MLVA16 patterns represented new genotypes, since no correspondence with those deposited on MLVAbank 2014 was found. However, the comparison of results observed in the eight conserved loci included in the panel 1 (MLVA8) with those available in the MLVAbank 2014 (http://mlva.u-psud.fr/brucella/) revealed that nine among the fourteen isolates had MLVA8 profile identical to profile 1 (Bruce06: 3; Bruce08: 5; Bruce11: 2; Bruce12: 10; Bruce42: 1; Bruce43: 1; Bruce45: 5; Bruce55: 2). The other five B. ovis isolates exhibited two different MLVA8 patterns, which were different of the MLVA8 1 and 2 genotypes (genotype 2 = Bruce06: 2; Bruce08: 5; Bruce11: 2; Bruce12: 10; Bruce42: 1; Bruce43: 1; Bruce45: 5; Bruce55: 2) (the only ones already described for B. ovis) due to polymorphisms in loci Bruce06, 08 and 12. The MST created based on MLVA16 genotypes is shown in Figure 2. Besides the B. ovis strains tested in the present study, all nineteen MLVA16 genotypes of B. ovis available in the MLVAbank 2014 were included in clustering and MST analyses. Analysis of geographical origin in the MST showed that B. ovis strain BCCN 98–46 from Argentina was closely related to a Brazilian B. ovis isolate, strain 241E (Figures 1 and 2). Moreover, MST analysis also revealed the existence of two clearly distinct major clonal complexes (clonal complexes A and B).
Figure 1

Cluster analysis by MLVA16 genotyping of 14 Brucella ovis isolates from sheep of Rio Grande do Sul State, Brazil, 1982 – 1995 plus all 19 MLVA16 genotypes of B. ovis available in the MLVAbank 2014. The cluster analysis was performed using the category coefficient and UPGMA (BioNumerics 6.1). Information on the origin of the isolates was color labeled.

Figure 2

Minimum Spanning Tree (MST) analysis of Brucella ovis isolates from sheep of Rio Grande do Sul State, Brazil plus all 19 MLVA16 genotypes of B. ovis available in the MLVAbank 2014, using the MLVA16 data. The MST analyses included 14 B. ovis field strains tested in the present study (203 L, 252 L, 201 L, S/NL, 266 L, 286 L, 0236., 100 V, 0204., 241E, 31 V, 91AV, 94AV and 5013) and all nineteen B. ovis MLVA16 genotypes available in MLVAbank 2014 (http://mlva.u-psud.fr). The minimum spanning tree presented is the one with the highest overall reliability score and was calculated using Prim’s algorithm associated with the priority rule and the bootstrap resampling. Numbers inside each clonal complex represent the genotype on Panel 1 (MLVA8). Branch length and thickness reflects number of differences between nodes. Information on the origin of the isolates was color labeled in the same way as shown in the Figure 1.

Discussion

Genotyping of microorganism of great veterinary importance, such as B. ovis, is a valuable tool for the control of disease, since it allows the characterization of outbreaks and, the determination of the source of infection and transmission routes [26]. In the present study, molecular characterization of fourteen B. ovis field isolates revealed a high genetic diversity among strains (Figure 1). Interestingly, among ten B. ovis isolates from same herd only two strains had identical patterns (Figure 1). The existence of many different genetic profiles within the same herd has two possible explanations: first, the existence of an intense animal traffic led the introduction of the agent from different origins and second, all B. ovis strains isolated from outbreak were originated from the same B. ovis strain that undergone some changes in loci of MLVA16. Although there are no epidemiological data that can confirm or refute the first explanation, the second hypothesis seems less likely, since the differences observed among the ten B. ovis strains from same herd were not the result of one-repeat unit increase or decrease and were also not restricted to only one MLVA16 locus or panel. Moreover, even though some data had suggested short term evolution particularly among panel 2B loci[27, 28], there was also polymorphism at locus Bruce08 from the most conserved panel (panel 1) (Figure 1). On the other hand, in contrast to smooth strains such as B. abortus, B. melitensis and B. suis that have demonstrated a high stability of all MLVA16 loci under in vivo and in vitro conditions [1214, 29], MLVA16 performed on B. canis, a rough strain, suggesting a hypervariability particularly in some panel 2B loci[30]. Whole genome sequencing of these B. ovis strains from the same herd would be the better way to understand the biological significance of the high genetic diversity observed without any concerns, however it is less practical and much more expensive.

Clustering analysis also showed a large distance between the two isolates from Uruguaiana (Bruce09, 04, 07 and 16), and between the two B. ovis strains from undefined municipalities (Bruce08, 09, 07 and 16), likewise in comparison among all four isolates (Figure 1). These major differences in the MLVA16 genotypic profile and the large difference in the years of isolation of the strains (1982, 1985 and 1995) (Figure 2), together, strongly suggest that no epidemiological relationship exist among these four B. ovis isolates.

Minimal spanning tree analysis revealed the existence of two clearly distinct major clonal complexes (clonal complexes A and B) (Figure 2), one composed by most of Brazilian B. ovis isolates plus French strains and a single strain from Argentina, Australia, Spain and USA (clonal complex A), and a second one with fewer representatives and composed by two strains from France and a single strain from Argentina and Brazil (clonal complex B) (Figure 2). The establishment of these relationships is central to develop a model for evolutionary steps in the difference of the B. ovis MLVA16 genotypes. Nevertheless, more representative sampling is needed for inclusion into this model for a more robust comparison. Therefore, data of present study are especially important, because it expands the universe of B. ovis strains genotyped by MLVA16 in both, amount and origin of strains.

Moreover, since Rio Grande do Sul State is bordered by Argentina, the close relationship between B. ovis strain BCCN 98–46 from Argentina and the Brazilian B. ovis isolate 241E suggests that B. ovis strains were circulating in the Brazilian – Argentinean border. In this context, animal importation could also explain the very close localization of B. ovis isolates from Brazil and B. ovis strains from France and Spain in MST analysis. Although there are no recent records about importation of animals from these countries to Rio Grande do Sul, historical records show that the formation of the sheep flock of this State was mainly achieved through the importation of animals from various countries of Europe and Oceania [31, 32]. Furthermore, the main activity of the flock from Santana do Livramento, RS, from where most B. ovis strains were isolated, was the rearing of Texel breeders, a breed whose origin is in France and the Germany.

In conclusion, the results of the present study showed a high genetic diversity among B. ovis field isolates from Rio Grande do Sul State, Brazil by MLVA16.

Availability of supporting data

The data set supporting the results of this article is available in the Brucella_Brazil at http://mlva.u-psud.fr/brucella/ repository.

Notes

Declarations

Acknowledgments

EMSD, GNF and APL are indebted to Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq – for the fellowships. This study was supported by CNPq, Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Fapemig) and FEP – MVZ Coordenação Preventiva.

Authors’ Affiliations

(1)
Departamento de Medicina Veterinária Preventiva, Escola de Veterinária, Universidade Federal de Minas Gerais
(2)
Instituto de Pesquisas Veterinárias Desidério Finamor

References

  1. Biberstein EL, McGowan B, Olander H, Kennedy PC: Epididymitis in rams. Studies on pathogenesis. Cornell Vet. 1963, 54: 27-41.Google Scholar
  2. OIE – Organization for animal Health: Ovine brucellosis. Manual of diagnostic tests and vaccines for terrestrial animals 2013. 2009,http://www.oie.int/,Google Scholar
  3. Carvalho Júnior CA, Moustacas VS, Xavier MN, Costa EC, Costa LF, Silva TMA, Paixão TA, Borges AM, Gouveia AMG, Santos RL: Andrological, pathologic, morphometric, and ultrasonographic findings in rams experimentally infected with Brucella ovis. Small Rumin Res. 2012, 102 (2–3): 213-222.View ArticleGoogle Scholar
  4. Komissarova LI: Pathological changes in rams and breeding ewes, infected with Brucella ovis. Shornik Rabot Leningrad Veter Instit. 1973, 34: 80-84.Google Scholar
  5. Burgess GW: Ovine contagious epididymitis: a review. Vet Microbiol. 1982, 7 (6): 551-575.PubMedView ArticleGoogle Scholar
  6. Ridler AL, West DM: Control of Brucella ovis infection in sheep. Vet Clin North Am Food Anim Pract. 2011, 27 (1): 61-66.PubMedView ArticleGoogle Scholar
  7. Poester FP, Gonçalves VSP, Lage AP: Brucellosis in Brazil. Vet Microbiol. 2002, 90 (1–4): 55-62.PubMedView ArticleGoogle Scholar
  8. Ramos AA, Mies Filhos A, Schenck JAP, Vasconcellos LD, Prado OTG, Fernandes JCT, Blobel H: Epididimite ovina, levantamento clínico no Rio Grande do Sul. Pesq Agrop Bras. 1966, 1: 211-213.Google Scholar
  9. Magalhães Neto A, Gil-Turnes C: Brucelose Ovina no Rio Grande do Sul. Pesq Vet Bras. 1996, 16 (2–3): 75-79.Google Scholar
  10. Vidor ACM, Santos DV, Kohek Junior I, Machado G, Miranda ICS, Hein HE, Stein MC, Corbellini LG: Estudo epidemiológico para determinar a prevalência da brucelose ovina em machos no estado do Rio Grande do Sul. Acta Sci Vet. 2012, 40: s127-Google Scholar
  11. Al Dahouk S, Le Flèche P, Nockler K, Jacques I, Grayon M, Scholz HC, Tomaso H, Vergnaud G, Neubauer H: Evaluation of Brucella MLVA typing for human brucellosis. J Microbiol Methods. 2007, 69 (1): 137-145.PubMedView ArticleGoogle Scholar
  12. Álvarez J, Sáez JL, García N, Serrat C, Pérez-Sancho M, González S, Ortega MJ, Gou J, Carbajo L, Garrido F, Goyache J, Domínguez L: Management of an outbreak of brucellosis due to B. melitensis in dairy cattle in Spain. Res Vet Sci. 2011, 90 (2): 208-211.PubMedView ArticleGoogle Scholar
  13. Dorneles EMS, Faria APP, Pauletti RB, Santana JA, Caldeira GAV, Heinemann MB, Almeida RT, Lage AP: Genetic stability of Brucella abortus S19 and RB51 vaccine strains by multiple locus variable number tandem repeat analisys (MLVA16). Vaccine. 2013, 31 (42): 4856-4859.PubMedView ArticleGoogle Scholar
  14. Her M, Kang SI, Cho DH, Cho YS, Hwang IY, Heo YR, Jung SC, Yoo HS: Application and evaluation of the MLVA typing assay for the Brucella abortus strains isolated in Korea. BMC Microbiol. 2009, 9: 230-PubMedPubMed CentralView ArticleGoogle Scholar
  15. Le Flèche P, Jacques I, Grayon M, Al Dahouk S, Bouchon P, Denoeud F, Nockler K, Neubauer H, Guilloteau LA, Vergnaud G: Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol. 2006, 6: 9-PubMedPubMed CentralView ArticleGoogle Scholar
  16. Minharro S, Mol JP, Dorneles EMS, Barbosa RP, Neubauer H, Melzer F, Poester FP, Dasso MG, Pinheiro ES, Soares Filho PM, Santos RL, Heinemann MB, Lage AP: Biotyping and genotyping (MLVA16) of Brucella abortus isolated from cattle in Brazil, 1977 to 2008. PLoS One. 2013, 8 (12): e81152-PubMedPubMed CentralView ArticleGoogle Scholar
  17. Miranda KL, Poester FP, Minharro S, Dorneles EM, Stynen AP, Lage AP: Evaluation of Brucella abortus S19 vaccines commercialized in Brazil: Immunogenicity, residual virulence and MLVA15 genotyping. Vaccine. 2013, 31 (29): 3014-3018.PubMedView ArticleGoogle Scholar
  18. Alton GG, Jones LM, Angus RD, Verger JM: Techniques for the brucellosis laboratory. 1988, Paris: INRAGoogle Scholar
  19. Baily GG, Krahn JB, Drasar BS, Stocker NG: Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg. 1992, 95 (4): 271-275.PubMedGoogle Scholar
  20. Bricker B, Halling S: Enhancement of the Brucella AMOS PCR assay for differentiation of Brucella abortus vaccine strains S19 and RB51. J Clin Microbiol. 1995, 33 (6): 1640-1642.PubMedPubMed CentralGoogle Scholar
  21. Pitcher DG, Saunders NA, Owern RJ: Rapid extraction of bacterial genomic DNA with guanidium thyocianate. Lett App Microbiol. 1989, 8 (4): 151-156.View ArticleGoogle Scholar
  22. Sambrook J, Russel D: Molecular cloning: a laboratory manual. 2001, New York: CSHL PressGoogle Scholar
  23. Hunter PR, Gaston MA: Numeric Index of the discriminatory ability of typing systems: an application of Simpson’s Index of Diversity. J Clin Microbiol. 1988, 26 (11): 2465-2466.PubMedPubMed CentralGoogle Scholar
  24. Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG: eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol. 2004, 186 (5): 1518-1530.PubMedPubMed CentralView ArticleGoogle Scholar
  25. Salipante SJ, Hall BG: Inadequacies of minimum spanning trees in molecular epidemiology. J Clin Microbiol. 2011, 49 (10): 3568-3575.PubMedPubMed CentralView ArticleGoogle Scholar
  26. Struelens MJ: Molecular epidemiologic typing systems of bacterial pathogens: current issues and perpectives. Mem Inst Oswaldo Cruz. 1998, 93 (5): 581-585.PubMedView ArticleGoogle Scholar
  27. Maquart M, Le Flèche P, Foster G, Tryland M, Ramisse F, Djonne B, Al Dahouk S, Jacques I, Neubauer H, Walravens K, Godfroid J, Cloeckaert A, Vergnaud G: MLVA-16 typing of 295 marine mammal Brucella isolates from different animal and geographic origins identififies 7 major groups within Brucella ceti and Brucella pinnpedialis. BMC Microbiol. 2009, 9: 145-PubMedPubMed CentralView ArticleGoogle Scholar
  28. García-Yoldi D, Le Flèche P, Marín CM, De Miguel MJ, Munõz PM, Vergnaud G, López-Goñi I: Assessment of genetic stablility of Brucella melitensis Rev 1 vaccine strain by multiple-locus variable number tandem repeat analysis. Vaccine. 2007, 25 (15): 2858-2862.PubMedView ArticleGoogle Scholar
  29. Whatmore AM, Shankster SJ, Perrett LL, Murphy TJ, Brew SD, Thirlwall RE, Cutler SJ, MacMillan AP: Identification and characterization of variable-number tandem-repeat markers for typing of Brucella spp. J Clin Microbiol. 2006, 44 (6): 1982-1993.PubMedPubMed CentralView ArticleGoogle Scholar
  30. Kang SI, Heo EJ, Cho D, Kim JW, Kim JY, Jung SC, Her M: Genetic comparison of Brucella canis isolates by the MLVA assay in South Korea. J Vet Med Sci. 2011, 73 (6): 779-786.PubMedView ArticleGoogle Scholar
  31. Viana JGA, Waquil PD, Spohr G: Evolução histórica da ovinocultura no Rio Grande do Sul: Comportamento do rebanho ovino e produção de lã de 1980 a 2007 [abstract]. Revista Extensão Rural UFSM. 2010, XVII: 20-Google Scholar
  32. Ribeiro LAO: Risco de introdução de doenças exóticas pela importação de ovinos. Boletim do Laboratório Regional de Diagnóstico – UFPEL. 1993, 13: 39-44.Google Scholar

Copyright

© Dorneles et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Advertisement