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DOI: 10.1016/j.ram.2020.06.012
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Disponible online el 9 de Noviembre de 2020
Brucella canis Group 2 isolated in Argentina
Brucella canis Grupo 2 aislado en Argentina
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Eduardo Jorge Boeria,
Autor para correspondencia
eduardoboeri@gmail.com

Corresponding author.
, María Julia Madariagaa, María Luz Domingueza, María Luisa Teijeiroa, Natalia Mercedes Fernandeza, Sebastián Alejandro Elenab, Marcos David Trangonic
a Instituto de Zoonosis Luis Pasteur, Av. Diaz Velez 4821 (1405), Ciudad Autónoma de Buenos Aires, Argentina
b Laboratorio de Referencia de la OIE para Brucelosis, Dirección General de Laboratorio y Control Técnico (DiLab), Servicio Nacional de Sanidad y Calidad Agroalimentaria (SENASA), Martínez, 1640 Buenos Aires, Argentina
c Laboratorio de Brucella, Campylobacter y Microbiota, Instituto de Biotecnología-IABIMO, INTA-CONICET, CICVyA-CNIA INTA Hurlingham, Argentina
Recibido 12 septiembre 2019. Aceptado 26 junio 2020
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Table 1. Description of B. canis strains and techniques used for their characterization and differentiation in groups 1 and 2.
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Abstract

The aim of this study was to estimate the diversity and prevalence of both groups of Brucella canis 1 and 2 with and without deletion respectively in different areas of Argentina. A total of 104 bacterial cultures were typed as B. canis strains using the classical biotyping method. Two PCR assays were performed to confirm that all isolates were B. canis and not Brucella suis. The differentiation between groups 1 and 2 was achieved using another PCR assay and the diversity of B. canis isolates was assessed with four MLVA_16 markers. All strains belonged to Group 2. Bruce 09 marker (MLVA_16 assay) showed the greatest diversity. Only Group 2 of B. canis was identified among the strains evaluated. The markers chosen from the MLVA_16 allowed us to detect genetic diversity among the strains of B. canis studied.

Keywords:
MLVA
Diversity
Deletion
BMEI1435
Brucella canis Group 2
Resumen

El objetivo de este trabajo fue estimar la diversidad y la prevalencia de ambos grupos de Brucella canis 1 y 2 (con y sin deleción, respectivamente) en diferentes áreas de Argentina. Un total de 104 cultivos bacterianos se tipificaron como cepas de B. canis usando biotipado clásico. Se realizaron dos ensayos de PCR para confirmar que todos los aislamientos eran B. canis y no Brucella suis. La diferenciación entre los grupos 1 y 2 se logró con otro ensayo de PCR, y la diversidad entre las cepas de B. canis se obtuvo mediante el empleo de cuatro marcadores del ensayo de MLVA_16. Todas las cepas pertenecieron al grupo 2. El marcador Bruce 09 (ensayo MVLA-16) mostró la mayor diversidad. Sólo se halló el Grupo 2 de B. canis entre las cepas estudiadas. Los marcadores del MLVA_16 permitieron detectar la presencia de diversidad genética entre las cepas de B. canis analizadas.

Palabras clave:
MLVA
Diversidad
Deleción
BMEI1435
Brucella canis Grupo 2
Texto completo

Brucella canis is an etiological agent of canine brucellosis that has been found in many countries, such as the US, China and Mexico as well as in countries of South America, Asia and Europe. This zoonotic disease causes abortion in female dogs and epididymitis in male dogs as the main symptom. In addition, this disease causes important losses in breeding kennels because of reproductive failures5.

Bacterial isolation is the “gold standard” for the definitive diagnosis, despite the low sensitivity of this procedure regarding clinical samples2. In addition, the global scientific community uses the process of phenotypic characterization from isolates to distinguish the different species of the genus Brucella1.

Researchers have employed the polymerase chain reaction assay (PCR) in many studies for the molecular typing of Brucella species and biovars from bacteriological cultures10,14. For example, the Bruce-ladder assay (a multiplex PCR assay) is the only analysis accepted by the World Organization for Animal Health (OIE) for identification and typing Brucella species13. However, some B. canis strains can be identified erroneously as B. suis10. The researchers that reported this finding identified two groups of B. canis based on the presence or absence of a region of the BMEI1435 polysaccharide deacetylase gene in B. canis. Indeed, Group 1 presents a deletion (PCR amplifies a 607-bp fragment), whereas Group 2 lacks the deletion (PCR amplifies a 1674-bp fragment). López Goñi et al. replaced two primers of the PCR original protocol and obtained the Bruce-ladder v2.0 test14. This new test allows the differentiation of strains of B. canis, five biovars of B. suis and B. neotomae. In parallel, Kang et al.9 also developed new primers from a specific region to avoid the problem of the original Bruce-ladder.

Another research group found that only Group 2 is present in Medellín city15. More recently, another study reported both groups in China4.

Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) is a method that has been performed for the typing of bacterial species including Mycobacterium tuberculosis, Bacillus anthracis and others. A set of eight microsatellite loci has been proposed because they are highly discriminatory and efficient for distinguishing strains within a local outbreak. Although we cannot correctly predict the biovar or even the species of an isolate, this method is a useful tool especially in outbreaks. To date, different MLVA assays have been developed for typing Brucella strains with different kind of outcomes and utilities12.

To the best of our knowledge, in Argentina, there are no studies published on groups of B. canis isolates or any publication on B. canis MLVA studies. With this in mind, the aim of this study was to estimate the prevalence of both groups of B. canis in different areas of Argentina from microbiological isolates of clinical dog samples. Other objectives were to compare the performance of two PCR assays in discriminating B. canis and B. suis strains and, finally, to estimate the diversity of B. canis using four markers of the MLVA_16 assay.

The analyzed samples consisted of 104 B. canis isolates from clinical dog samples obtained between August 2009 and December 2018. The samples were collected from the Autonomous City of Buenos Aires and different areas of Argentina. Strains were typed following the recommendations described elsewhere1. Molecular typing confirmation of all B. canis strains and not B. suis was performed using two PCR assays9,14. DNA was extracted using a commercial kit, ROCHE High Pure PCR Template Preparation Kit (Roche Diagnostics, GmbH Roche Applied Science, Germany), following the manufacturer's instructions. A concentration between 50 and 70 ng/μl was used for further analyses after measuring DNA with ND-1000 NanoDrop. As a negative control of DNA extraction, we used ultrapure water. Two PCR assays (PCR1 and PCR2) were carried out to differentiate B. canis and B. suis. PCR1 and PCR2 were performed according to Lopez Goñi et al.14 and Kang et al.9 respectively. For both PCRs, B. canis strain RM6/66 and B. suis biovar 1 were used as positive controls. In addition, we used ultrapure water as a negative control. A third PCR (PCR3) was conducted according to Koylass et al.10B. canis strain RM6/66 (Group 1) and Brucella melitensis biovar 1 (Group 2) were used as positive controls, respectively. All the products from PCR1, PCR2 and PCR3 were analyzed by electrophoresis in 1.5% agarose gel stained with ethidium bromide (0.5μg/ml) and UV light visualization.

The markers used for the MLVA assay in this study were Bruce 04, Bruce 07, Bruce 09 and Bruce 1612. The selection of these markers was based on their greatest diversity among species of B. canis4,8 (Table S1). A total of 15 strains were selected from various areas of Argentina: San Miguel de Tucumán (Province of Tucumán); Rio Cuarto (Province of Córdoba); Rosario (Province of Santa Fe); Río Grande (Province of Tierra del Fuego); General Pico (Province of La Pampa); Corrientes (Province of Corrientes); Ituzaingó, Tortuguitas, Paso del Rey, Del Viso (two strains), Villa Ballester, Banfield, Verónica (Province of Buenos Aires) and Villa Lugano (Autonomous City of Buenos Aires). This allowed an approximate representation of the different alleles (number of repeats) present in different areas of the country.

DNA amplification was conducted in tubes with a final volume of 25μl per reaction. Each reaction contained DNA (50μg), each primer (forward/reverse; 0.5μM), each of the four deoxynucleotide triphosphates (dNTPs; 200μM), 1X buffer with magnesium chloride (MgCl2, final concentration of 1.5mM (PROMEGA 5X Green GoTaq® Flexi Buffer Migration Pattern) and 1 U of Taq DNA Polymerase. B. canis strain RM6/66 and B. melitensis 16M were used as positive controls. Ultrapure water was used as negative control. The thermocycling conditions were as follows: a first denaturation at 94°C for 3min and 30 cycles of 94°C for 30s, 60°C for 30s and 72°C for 50s with a final extension of 72°C for 5min. Bionumerics software version 3.5 (Applied Maths, St-Martens-Latem, Belgium) was used to estimate the size of the PCR bands of the agarose gels. The quantification of the number of repeats of each marker was performed following the protocol described by Le Flèche et al.12 The dendrogram was generated through the unweighted pair group method following arithmetic averages (UPGMA) and using the PAST software6. The Hunter-Gaston Diversity Index (HGDI)7 was calculated using Epicompare software version 1.0 (www.ridom.de/epicompare) to elucidate the discriminatory power of the genotyping methods. Number 0 and 1 stand for “without diversity” and “extreme diversity”, respectively. These results reflect the total of detected alleles. The PCR products were analyzed in 2% agarose gel in Tris acetate and EDTA (ethylenediaminetetraacetic acid) 1X buffer (TAE) with the addition of 0.5μg/ml of ethidium bromide. An Applied Biosystems Veriti™ Thermal Cycler was used for the PCR reactions.

All 104 strain isolates were typed as B. canis according to the classical biotyping method. In this study, we evaluated two PCR assays for molecular typing of the evaluated bacteria that had been confirmed as B. canis strains by the classical method.

The two PCRs used to assess the species (PCR1 and PCR2) confirmed that all strains were B. canis (Table 1). Thus, both techniques yielded consistent results. PCR1 shows the molecular patterns between strains of B. canis and B. suis. PCR2 displays a 776-bp differential amplicon between species, i.e. B. suis contains the amplicon, whereas B. canis lacks it. In this study, all the evaluated strains were B. canis Group 2 (without deletion; 100% (CI 95% 0.95–1.00); Table 1, Fig. 1).

Table 1.

Description of B. canis strains and techniques used for their characterization and differentiation in groups 1 and 2.

Order  Isolation  Biotype  Bruce-ladder v2.0  Kang et al., 766pb  Amplicon  Deletion  Year  Place 
70150  B. canis  B. canis  B. canis  1674  No  2014  Recoleta 
70152  B. canis  B. canis  B. canis  1674  No  2014  Recoleta 
76531  B. canis  B. canis  B. canis  1674  No  2015  P. del Rey& 
55420  B. canis  B. canis  B. canis  1674  No  2013  V. Lugano& 
87447  B. canis  B. canis  B. canis  1674  No  2016  La Pampa& 
23653  B. canis  B. canis  B. canis  1674  No  2009  Caballito 
91224  B. canis  B. canis  B. canis  1674  No  2016  Villa Crespo 
23671  B. canis  B. canis  B. canis  1674  No  2009  Caballito 
10  27269  B. canis  B. canis  B. canis  1674  No  2010  Almagro 
11  23502  B. canis  B. canis  B. canis  1674  No  2008  Caballito 
12  47937  B. canis  B. canis  B. canis  1674  No  2007  Almagro 
13  25962  B. canis  B. canis  B. canis  1674  No  2007  P. Chacabuco 
14  51545  B. canis  B. canis  B. canis  1674  No  2013  V Ballester& 
16  92151  B. canis  B. canis  B. canis  1674  No  2016  Barracas 
18  47247  B. canis  B. canis  B. canis  1674  No  2009  Caballito 
19  51080  B. canis  B. canis  B. canis  1674  No  2009  Mataderos 
20  100887  B. canis  B. canis  B. canis  1674  No  2017  Belgrano 
21  48502  B. canis  B. canis  B. canis  1674  No  2010  P. Madero 
22  35241  B. canis  B. canis  B. canis  1674  No  2011  Villa Lugano 
24  1865  B. canis  B. canis  B. canis  1674  No  2015  R. Grande 
25  1789  B. canis  B. canis  B. canis  1674  No  2014  Rosario& 
26  1863  B. canis  B. canis  B. canis  1674  No  2012  R. Grande 
27  1864  B. canis  B. canis  B. canis  1674  No  2012  R. Grande& 
29  1790  B. canis  B. canis  B. canis  1674  No  2012  Belgrano 
31  C28  B. canis  B. canis  B. canis  1674  No  2016  Pompeya 
32  C32  B. canis  B. canis  B. canis  1674  No  2016  Del Viso& 
33  C36  B. canis  B. canis  B. canis  1674  No  2016  Del Viso 
34  C40  B. canis  B. canis  B. canis  1674  No  2016  Del Viso 
35  C42  B. canis  B. canis  B. canis  1674  No  2016  Del Viso& 
36  C1  B. canis  B. canis  B. canis  1674  No  2015  Barracas 
37  C5  B. canis  B. canis  B. canis  1674  No  2010  Caballito 
39  C9  B. canis  B. canis  B. canis  1674  No  2010  Verónica & 
40  C14  B. canis  B. canis  B. canis  1674  No  2010  Tucumán 
41  C25  B. canis  B. canis  B. canis  1674  No  2016  Tortuguitas& 
42  C26  B. canis  B. canis  B. canis  1674  No  2016  Del Viso 
43  C27  B. canis  B. canis  B. canis  1674  No  2016  Tortuguitas 
44  C31  B. canis  B. canis  B. canis  1674  No  2016  Del Viso 
45  C33  B. canis  B. canis  B. canis  1674  No  2016  Del Viso 
46  C34  B. canis  B. canis  B. canis  1674  No  2016  Del Viso 
47  C37  B. canis  B. canis  B. canis  1674  No  2016  Del Viso 
48  C38  B. canis  B. canis  B. canis  1674  No  2016  Del Viso 
49  C39  B. canis  B. canis  B. canis  1674  No  2016  Del Viso 
50  C41  B. canis  B. canis  B. canis  1674  No  2016  Del Viso 
51  C43  B. canis  B. canis  B. canis  1674  No  2016  Del Viso 
55  C10  B. canis  B. canis  B. canis  1674  No  2009  Castelar 
57  C15  B. canis  B. canis  B. canis  1674  No  2009  Tucumán& 
58  C16  B. canis  B. canis  B. canis  1674  No  2009  Ramos Mejía 
61  C21  B. canis  B. canis  B. canis  1674  No  2010  San Martín 
62  C22  B. canis  B. canis  B. canis  1674  No  2010  Vicente López 
63  C24  B. canis  B. canis  B. canis  1674  No  2010  Olivos 
67  C13  B. canis  B. canis  B. canis  1674  No  2009  Caballito 
70  49080  B. canis  B. canis  B. canis  1674  No  2012  Caballito 
71  74873  B. canis  B. canis  B. canis  1674  No  2016  Almagro 
72  35241  B. canis  B. canis  B. canis  1674  No  2011  Villa Lugano 
73  47214  B. canis  B. canis  B. canis  1674  No  2008  Caballito 
74  47937  B. canis  B. canis  B. canis  1674  No  2007  Almagro 
75  74532  B. canis  B. canis  B. canis  1674  No  2016  Barracas 
76  70218  B. canis  B. canis  B. canis  1674  No  2015  Villa Urquiza 
77  77794  B. canis  B. canis  B. canis  1674  No  2016  Belgrano 
78  47246  B. canis  B. canis  B. canis  1674  No  2009  Caballito 
79  57327  B. canis  B. canis  B. canis  1674  No  2012  Barracas 
80  79207  B. canis  B. canis  B. canis  1674  No  2017  Banfield& 
81  79341  B. canis  B. canis  B. canis  1674  No  2017  Banfield 
82  79894  B. canis  B. canis  B. canis  1674  No  2017  V.del Parque 
83  47131  B. canis  B. canis  B. canis  1674  No  2009  P. del Rey 
84  47224  B. canis  B. canis  B. canis  1674  No  2009  Caballito 
85  C48  B. canis  B. canis  B. canis  1674  No  2011  Caballito 
86  C49  B. canis  B. canis  B. canis  1674  No  2012  Caballito 
87  C50  B. canis  B. canis  B. canis  1674  No  2012  Caballito 
88  C51  B. canis  B. canis  B. canis  1674  No  2011  Caballito 
89  C52  B. canis  B. canis  B. canis  1674  No  2013  Caballito 
90  C53  B. canis  B. canis  B. canis  1674  No  2014  Caballito 
91  C54  B. canis  B. canis  B. canis  1674  No  2011  Caballito 
92  C55  B. canis  B. canis  B. canis  1674  No  2012  Caballito 
93  C57  B. canis  B. canis  B. canis  1674  No  2013  Caballito 
94  C58  B. canis  B. canis  B. canis  1674  No  2012  Caballito 
95  C61  B. canis  B. canis  B. canis  1674  No  2013  Rio cuarto& 
96  C64  B. canis  B. canis  B. canis  1674  No  2014  Verónica 
97  C70  B. canis  B. canis  B. canis  1674  No  2013  Barracas 
98  82175  B. canis  B. canis  B. canis  1674  No  2018  Ituzaingó 
99  82122  B. canis  B. canis  B. canis  1674  No  2018  P. Patricios 
100  81296  B. canis  B. canis  B. canis  1674  No  2018  P. Patricios 
101  83961  B. canis  B. canis  B. canis  1674  No  2018  Balvanera 
102  Cepa 12  B. canis  B. canis  B. canis  1674  No  2018  Corrientes& 
103  82543  B. canis  B. canis  B. canis  1674  No  2018  Ituzaingó& 
104  RM6/66  B. canis  B. canis  B. canis  607  YES  2018  Reference 

&: strains analyzed by MLVA.

Figure 1.

Electrophoresis on agarose gel of PCR products showing representative strains of Group 2. Lane 1: Negative control of PCR. Lane 2: Negative control of DNA extraction. Lane 3–7: profile of five strains evaluated. Lane 8: positive control of Group 2 (1674bp). Line 9: positive control of Group 1 (607bp). Line 10: molecular marker Dangsheng Biotech 100bp DNA ladder plus.

(0,12MB).

Subsequently, we performed an evaluation of the strains by MLVA_16 by using four markers. For this purpose, we analyzed 15 strains from different regions of Argentina. The overall Diversity Index (HDGI) of the technique was 1.0 (CI 95% 1.0–1.0). The HDGI coefficient ranged from 0.705 to 0.895. Bruce 09 marker displayed the greatest diversity (0.895 CI 95% 0.816–0.974), followed by Bruce 16 (0.876 CI 95% 0.803–0.949), Bruce 07 (0.848 CI 95% 0.805–0.890) and Bruce 04 (0.705 CI 95% 0.550–0.859) (Table S2). Additionally, we identified 15 different molecular patterns (Bruce 04, Bruce 07, Bruce 09, Bruce16 respectively: 4-8-6-9, 6-5-8-5, 6-5-11-8, 6-5-11-9, 6-5-6-5, 6-6-8-9, 6-8-10-11, 6-9-8-8, 7-7-10-5, 8-6-7-9, 8-6-8-8, 8-8-4-12, 8-9-9-11, 8-9-5-5, 11-7-10-7) (Table S3). The molecular pattern detected in Del Viso (6-5-8-5) and the ones found in Banfield (6-8-10-11) were similar to those described in the city of Beijing and the Autonomous Region of Guanxi, Republic of China, respectively4. In addition, the molecular pattern found in La Pampa (6-6-8-9) was similar to those in Paju city, Gyeonggi Province, South Korea8.

In this study, we evaluated various B. canis strains from different regions of Argentina to assess the groups of strains existing in Argentina. All strains were identified using the classical biotyping method and molecular techniques. Among all the strains analyzed in this study, we only detected B. canis type 2 (without deletion of the BMEI1435 gene). This finding is consistent with a previous report from Colombia15. Similarly, most of the isolates analyzed in China belong to this type, although three isolates exhibited a deletion4. By contrast, Koylass et al., found both groups circulating in equal proportion in samples evaluated from Europe, South America and the United States. Specifically, the strains with the deletion came from Peru, Germany and the United States10. The pathogenicity of the strains with and without this deletion has not been studied yet. These data could trigger future research regarding the characteristics of the BMEI1435 gene. This gene has a hydrolase function and participates in the metabolism of carbohydrates. The variation of this gene may have an impact on virulence in the host, but this is still unclear. A recent study compared different Brucella genes and their virulence but did not include BMEI1435 in the analysis, perhaps due to the lack of consideration regarding pathogenicity at the date of the cited study3.

In the present study, we used four markers presented at panel 2 of the MLVA_16 (Bruce 04, Bruce 07, Bruce 09 and Bruce 16) and based on the polymorphism found by other researchers4,8. We did not use the complete panel of MLVA_16 and therefore we believe we cannot speak of “genotypes”. Therefore, we defined them as molecular patterns instead. As a preliminary study, we used 15 strains from different regions of the country, which provided data on the diversity among circulating B. canis strains in specific areas of Argentina. It should be noted that the remaining markers of the full panel of the MLVA_16 in the research of Di et al.4 and Kang et al.8 showed similar repeat numbers. These data suggest high homology of B. canis for these gene regions. Thus, the four markers used in the present study are suitable for evaluating B. canis genetic diversity. However, the use of the complete panel is very useful for other Brucella species such as B. melitensis, B. suis and B. abortus11.

The results from the dendrogram (Fig. 2) suggest great similarity between the strains of Villa Ballester and Del Viso (Province of Buenos Aires) and this result could be explained by the closeness of the locations (36km). Although other strains, such as the strains of the city of Rosario (Province of Santa Fe) and Ituzaingó (Province of Buenos Aires) are somewhat further away (300km), they also showed high similarity. In addition, the similarity between the strains of Rio Grande (Province of Tierra del Fuego) and the town of Banfield (Province of Buenos Aires), which are separated by 2800km, is of particular interest. These findings suggest that dogs may have circulated from one location to another. This is quite usual as people often migrate with their dogs.

Figure 2.

Dendrogram based on the different copy numbers of the tandem repeats of the 25 alleles found in the 15 strains studied by MLVA. The Euclidean similarity measure was used for the construction of the phylogenetic tree.

(0,09MB).

Finally, as a first preliminary study in Argentina, our results indicate the existence of a genetic diversity among circulating B. canis strains. In addition, the development of the complete MLVA_16 panel should be done in the near future in order to broaden the knowledge of the circulating genotypes in Argentina.

Our results suggest that the only group of B. canis circulating in Argentina is Group 2 (without BMEI1435 gene deletion). The two PCRs used for molecular typing yielded consistent results. Therefore, both are useful to discriminate between B. canis and B. suis. However, PCR1 should be used to evaluate biovars of B. suis. The use of the four markers chosen from the MLVA_16 allowed us to identify the genetic diversity among the strains of B. canis circulating in Argentina. Thus, as a preliminary test, MLVA_16 is useful for the genetic discrimination of this bacterium.

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgment

We thank Dr. Gabriela Escobar for the provision of strains, Dr. Zumarraga Martín for processing agarose geles and Dr. Julia Sabio y García for critical reading of the manuscript. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Appendix A
Supplementary data

The following are the supplementary data to this article:

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