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A novel isolation method of Brucella species and molecular tracking of Brucella suis biovar 2 in domestic and wild animals
Veterinary Microbiology 150 (2011) 405–410
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
A novel isolation method of Brucella species and molecular tracking of Brucella suis biovar 2 in domestic and wild animals Carlos Abril a,*, Andreas Thomann a, Isabelle Brodard a, Natacha Wu c, Marie-Pierre Ryser-Degiorgis c, Joachim Frey b, Gudrun Overesch a a b c
Centre for Zoonoses, Bacterial Animal Diseases and Antimicrobial Resistance (ZOBA), La¨nggass-Strasse 122, Postfach 8466, CH-3001 Bern, Switzerland Research Unit, Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, La¨nggass-Strasse 122, Postfach 8466, CH-3001 Bern, Switzerland Centre for Fish and Wildlife Health, Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Postfach 8466, CH-3001 Bern, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 January 2011 Received in revised form 22 February 2011 Accepted 28 February 2011
Brucella suis biovar 2 is the most common aetiological agent of porcine brucellosis in Europe. B. suis biovar 2 is considered to have low zoonotic potential, but is a causative agent of reproductive losses in pigs, and it is thus economically important. The multilocus variable-number of tandem repeats genotyping analysis of 16 loci (MLVA-16) has proven to be highly discriminatory and is the most suitable assay for simultaneously identifying B. suis and tracking infections. The aim of this study was to investigate the relatedness between isolates of B. suis biovar 2 obtained during a brucellosis outbreak in domestic pigs and isolates from wild boars and hares collected from proximal or remote geographical areas by MLVA-16. A cluster analysis of the MLVA-16 data revealed that most of the isolates obtained from Switzerland clustered together, with the exception of one isolate. The outbreak isolates constituted a unique subcluster (with a genetic similarity >93.8%) distinct from that of the isolates obtained from wild animals, suggesting that direct transmission of the bacterium from wild boars to domestic pigs did not occur in this outbreak. To obtain a representative number of isolates for MLVA-16, alternative methods of Brucella spp. isolation from tissue samples were compared with conventional direct cultivation on a Brucella-selective agar. We observed an enhanced sensitivity when mechanical homogenisation was followed by host cell lysis prior to cultivation on the Brucella-selective agar. This work demonstrates that MLVA-16 is an excellent tool for both monitoring brucellosis and investigating outbreaks. Additionally, we present efficient alternatives for the isolation of Brucella spp. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Brucellosis Brucella suis Isolation methods MLVA-16 Domestic pigs Wild boars Outbreak
1. Introduction According to the World Organization for Animal Health (http://www.oie.int/wahis/public.php?page=weekly_ report_index), a total of 136 confirmed outbreaks of porcine brucellosis were reported in Europe between January 2007 and December 2010. The causative agent of porcine brucellosis in Europe is usually Brucella suis biovar 2, which is host adapted and does
* Corresponding author. Tel.: +41 31 631 2436; fax: +41 31 631 2634. E-mail address: [email protected] (C. Abril). 0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.02.056
not often infect humans (Garcia-Yoldi et al., 2007; Munoz et al., 2010). B. suis biovars 1 and 3, also cause brucellosis in porcines, but in contrast to biovar 2, they are zoonotic agents that cause severe brucellosis in humans and other animals (Munoz et al., 2010). In Europe, the biovars 1 and 3 have only been reported in Croatia, indicating that they may be restricted to this geographic region (Cvetnic et al., 2005, 2009; Garcia-Yoldi et al., 2007). B. suis biovar 2 is widely spread amongst Eurasian wild boar (Sus scrofa) and European brown hare (Lepus europaeus) populations (Leuenberger et al., 2007; Munoz et al., 2010). As a result, these animals may be responsible for the transmission of B. suis biovar 2 to reared outdoor domestic pigs (Munoz et al., 2010).
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C. Abril et al. / Veterinary Microbiology 150 (2011) 405–410
Multilocus variable-number of tandem repeats analysis (MLVA) is a genotyping method based on the quantification of minisatellite and microsatellite repeats in various loci of a bacterial genome (Kattar et al., 2008). When used for genotyping Brucella spp., the MLVA assay based on sixteen tandem repeat loci (MLVA-16) has enabled the differentiation of the biovars, and distinguished isolates of both widespread and closed, temporal and geographical origins (Garcia-Yoldi et al., 2007; Al Dahouk et al., 2007; Kattar et al., 2008). This study aimed to identify possible sources of a B. suis outbreak that occurred in domestic pigs in Switzerland during 2009 using the MLVA-16 method. To enhance isolation sensitivity and therefore the number of isolates for MLVA-16 analysis, the efficiency of three Brucella isolation methods from tissue samples were tested in parallel. 2. Materials and methods 2.1. Animals and serological tests A culture-confirmed B. suis infection in a fertile curlyhair hog boar originating from the canton of Geneva was tracked back. For that reason, the animals (26) of three herds of domestic curly-hair hogs from which the infected pig originated were tested for Brucella spp. antibodies by the rose bengal (RBT) and complement fixation (CFT) tests according to international standards (Office International des Epizooties O.I.E., 2009b), and reacted positively (RBT ++, CFT 177 IU/ml). Blood and organs (liver, spleen and reproductive organs) from these animals were collected at necropsy. Additionally, spleen and six reproductive organ samples from 41 free-ranging wild boars were analyzed in this study. 2.2. Bacterial isolation and genotypic identification The efficiency of three isolation methods was evaluated in parallel. For method I, approximately 20 mg of tissue was removed with a platinum spatula and transferred directly to a Brucella-selective agar, containing Brucellamedium base (Oxoid, Basingstoke, UK), 5% inactivated horse serum and Modified Brucella Selective Supplement (Oxoid, Basingstoke, UK). For method II, the tissue samples were homogenised before plating on the Brucella-selective agar. One gram of tissue was washed once and suspended in 5 ml of 0.85% NaCl solution. The tissue was then homogenised twice for 30 s in DT-20 tubes at 6000 rpm, using the IKA ULTRA-TURRAX1 tube drive disperser system (IKA, Staufen, Germany). Method III consisted of homogenising tissues in combination with chemical host cell lysis prior to plating on the Brucella-selective agar. The tissue samples were homogenised as in method II. One millilitre of the homogenised tissue was added to an Isolator 1.5 microbial tube (OXOID, Basingstoke, England), and host cells were lysed with saponin to release intracellular bacteria. The Isolator 1.5 tube additionally contains sodium polynetholsulphate which neutralises the bactericidal properties of blood. Finally, in Methods II and III, a total of 300 ml of the homogenised or the
homogenised and lysed suspension was inoculated onto two Brucella-selective agar plates. In a further step, the suspension was spread with a loop, producing a depot followed by single colonies. All cultures were incubated at 37 8C with an atmosphere containing 5% CO2 for 5 days. The blood samples from the curly-hair hogs were analyzed by bacterial isolation using the Isolator 10 microbial tube (OXOID, Basingstoke, England), according to manufacturers protocol and cultivated on Brucella-selective agar using the same conditions as the organ cultures. Additionally, blood samples were tested by a real-time PCR targeting a Brucella spp. specific IS711 insertion element (Hinic et al., 2009). Briefly, 300 ml of EDTA–blood were suspended in 600 ml erythrocyte lysis buffer (1.55 M NH4Cl, 0.1 M KHCO3, 1 mM Titriplex III, 10 conc.) and 300 ml PBS buffer. After 10 min incubation at room temperature, a leukocyte pellet was obtained by centrifugation at 8000 rpm for 2 min. Finally, DNA from the leukocyte pellet was extracted following the tissue protocol of the QIAamp1 DNA Mini Kit (QIAGEN, Hilden, Germany). Brucella identification and species differentiation was accomplished using PCR protocols described by Hinic et al. (2008). 2.3. MLVA-16 MLVA-16 was used to assess the relatedness of B. suis isolates obtained from curly-haired hogs that were involved in the outbreak, along with 15 B. suis isolates from wild boars from the same geographical area (Cantons of Geneva and Vaud), and further 20 B. suis isolates from wild boars and hares from a location in Switzerland 150 km distant from the outbreak (Canton of Bern) (Fig. 1). Sixteen tandem repeat loci (Fig. 1) were screened as previously described (Le Fleche et al., 2006; Al Dahouk et al., 2007). The analysis of PCR products was performed on an Agilent 2100 Bioanalyzer using a DNA 1000 LabChip kit (Agilent Technologies, Waldbronn, Germany), according to the manufacturer’s protocols. Quantification of the tandem repeats was accomplished using a conversion table published by De Santis et al. (2009), which was adapted to process Agilent 2100 Bioanalyzer data. DNA sequencing verified the size of the PCR products and was accomplished using a Sequenator AB 3100 and the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems Norwalk, CT, USA). The categorical MLVA-16 cluster analysis was accomplished using the unweighted pair group method (UPGMA) and BioNumerics software (version 5.1; Applied Math, Sint-Martens-Latem, Belgium). The genetic diversity of each locus was calculated using the Hunter-Gaston diversity index (HGDI) (http://www.hpabioinformatics.org.uk/cgi-bin/DICI/DICI.pl). 3. Results 3.1. Isolation of Brucella spp. from organs Using the bacteriological isolation Methods I and II, Brucella spp. were isolated in three out of 26 curly-hair hogs (Table 1). However, with method III, which included host cells lysis with the Isolator 1.5 microbial system,
C. Abril et al. / Veterinary Microbiology 150 (2011) 405–410
100 93.8
76.5
87.5 100
86.5
73.3
87.5 84.4 93.8 81.3
69.2
81.3 76.6
81.3 75.4
81.3
87.5
75 77.1
70.1
62.9 87.5 81.3 77.1 73.4 81.3 56.9
71.6
2009
2
4
8 14
6
1
5
6
6 21
9 11
8 24
2
4
WS-BE-9
wild boar
2
Switzerland (Bern)
2008
2
4
8 14
6
1
5
6
6 21
9
8
8 24
2
4
ITPA 1033/08
wild boar
2
Switzerland (Bern)
2008
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
ITPA 2225/08
wild boar
2
Switzerland (Bern)
2008
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-165
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-36
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-57
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-61
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-68
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-70
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-71
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-217
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8
9
2
4
WS-GE-155
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9
5
8 15
2
4
WS-GE-234
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9
5
6
7
2
4
WS-GE-208
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
4 21
9
5
8 14
2
4
IMD 1548/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 13
2
9
IMD 1563/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 13
2
9
IMD 1565/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 13
2
9
IMD 1566/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 13
2
9
IMD 1021/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 11
2
9
IMD 1567/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 11
2
9
IMD 1568/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 11
2
9
IMD 836/10
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-135
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-170
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-171
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-178
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-200
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-207
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-228
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-239
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-232
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9 18
6 14
2
8
N 336/03
wild boar
2
Switzerland (Vaud)
2003
2
4
8 14
6
1
5
6
6 21
9 18
6
6
2
7
N 591/03 N 148/03
wild boar wild boar
2 2
Switzerland (Vaud) 2003 Switzerland (Geneva) 2003
2 2
4 4
8 14 8 14
6 6
1 1
5 5
6 6
6 21 6 21
9 18 9 8
6 6 6 24
2 2
7 8
D 1011/98
hare
2
Switzerland (Bern)
1998
2
4
8 14
6
1
5
2
6 21
9 18
9
9
2
4
O/D 1055/08
hare
2
Switzerland (Bern)
2008
2
4
8 14
6
1
5
2
6 21
9 18 12
8
2
4
D 224/98
hare
2
Switzerland (Bern)
1998
2
4
8 14
6
1
5
2
6 21
9 18 10 11
2
4
N 1982/03
wild boar
2
Switzerland (Vaud)
2003
2
4
8 14
6
1
5
6
6 21
9 18 10 11
2
4
ITPA1321/09
hare
2
Switzerland (Freiburg) 2009
2
4
8 14
6
1
5
2
6 21
9
8
6
9
2
4
N 151/03
wild boar
2
Switzerland (Geneva) 2003
2
5
8 14
6
1
5
6
6 21
9
9
6 24
2
9
BCCN 02_50
swine
2
France (72)
2002
2
4
8 15
6
1
5
6
6 21
9
9 10 11
2
9
BCCN 03_6
wild boar
2
France (73)
2003
2
4
8 15
6
1
5
6
6 21
9 12
4 14
2
9
BCCN 00_23
swine
2
France (87)
2000
2
4
8 15
6
1
5
6
6 21
9
6 24
2
6
BCCN 02_23
wild boar
2
France (73)
2002
2
4
8 15
6
1
5
6
6 21
9 12
6
8
2
8
BCCN 02_36
swine
2
France (72)
2002
2
4
8 15
6
1
5
6
6 21
9 11 12
7
2 10
BCCN 03_11
wild boar
2
France (35)
2003
2
4
8 15
6
1
5
6
6 21
9
9
8
7
2 10
BCCN 98_95
swine
2
France (87)
1998
2
4
8 15
6
1
5
6
6 21
9
6
9 16
2 10
BCCN 93_63
swine
2
France (76)
1993
2
4
8 15
6
1
5
6
6 21
9 15
9 13
2
BCCN 02_20
swine
2
France (79)
2002
2
4
8 15
6
1
5
6
6 21
9 13 13
BCCN 03_5
swine
2
France (03)
2003
2
4
8 15
7
1
5
6
6 21
9
6
4 18
BCCN 02_18
hare
2
France (49)
2002
2
4
8 15
6
1
5
2
6 21
9
7
BCCN 02_48
hare
2
France (49)
2002
2
4
8 15
6
1
5
2
6 21
9
9
BCCN 78_6
hare
2
France (57)
1978
2
4
8 15
6
1
5
2
6 21
9 12
BfR 150
swine
4
6
5 16
2
6
5
2
6
2 11
2
Germany (Aulendorf) 1998
2
4
8 15
6
1
5
2
6 21
9 23 12 13
2
4
BCCN 89_155 hare
2
France (57)
1989
2
4
8 15
6
1
5
2
6 21
9 16
7 11
2
6
BCCN 93_24
2
France (03)
1993
2
4
8 15
6
1
5
2
5 21
9 16 11 11
2
8
BCCN 81_287 swine
2
France (67)
1981
2
4
8 15
6
1
5
2
6 21
9 13
9 17
2
5
BfR 138
hare
2
Germany (Nürnberg)
1999
2
4
8 15
6
1
5
2
6 21
9 28
9 11
2
5
BCCN 82_19
hare
2
Poland
1982
2
4
8 15
5
1
5
2
6 21
9 10
9
2
5
BCCN 03_2
hare
2
France (38)
2003
2
4
8 15
5
1
5
2
6 21
9 15
4 17
2
5
ATCC 23445
swine
2
Denmark (Thomsen)
2
4
8 15
6
1
5
2
6 22
9
9
9 18
2
4
BCCN 87_11
hare
2
France (57)
1987
2
4
8 15
6
1
5
2
7 21
9
6
6 23
O/D 1618/08
hare
2
Switzerland (Bern)
2008
2
4
7
7
1
5
2
6 21
9 18 15 11
hare
A2
A
A3
A4
B
7
5
5 13
A1
2 11
2
8
Clusters
Switzerland (Bern)
Bruce 04
2
Bruce 07
wild boar
Bruce 21
WS-BE-44
Bruce 19
Bruce 16
77.9
Bruce 30
100
Bruce 09
93.8
Bruce 18
100
Bruce 45
80.8
Bruce 55
81.7
Bruce 43
81.7
Bruce 42
93.8
Bruce 12
100
Location /Canton Year
Bruce 11
87.5
Host
Bruce 08
93.1
Isolate ID.
Bruce 06
93.8
Biovar
100
90
60
70
80
Genetic similarity
Subclusters
407
Brucella MLVA-16 (loci)
C
87.5 70
67.1
81.3 79.2
9
8
D
2 12 2
4
Fig. 1. MLVA-16-based dendrogram of Brucella suis isolates from Switzerland (non-italicised). Dendrogram calculations were done using the UPGMA method, including data of related isolates from the Brucella2009 database (italics) (http://mlva-u-psud.fr/). Isolate identification, host, biovar, location of sampling sites, year of isolation and number of tandem repeats of each locus are indicated. Isolates obtained during the outbreak are represented in bold.
408
C. Abril et al. / Veterinary Microbiology 150 (2011) 405–410
Table 1 Detection of Brucella ysuis in individual tissue samples by bacterial isolation by three different methods. Animal no
Organs
1
Spleen Bladder Testicle 1 Testicle 2 Epididymis 1 Epididymis 2
2
Spleen Bladder Uterus Uterus (multifocal abscesses)
4
Spleen Bladder Uterus Ovary Spleen Bladder Uterus Ovary Udder abscess
6
7–26
Method II
Method III
+
+
+
+ + +
+ + +
+ + +
Spleen Bladder Uterus Ovary
3
5
Method I
+ + +
+
+
+
+
+
+
Spleen Bladder Uterus Uterus (multifocal abscesses)
+
Spleen Bladder Reproductive organs
Total positive (animals/organs)
Brucella spp. was isolated from 6 out of the 26 animals (Table 1). The bacterial isolates were subsequently identified as B. suis using a previously described protocol (Hinic et al., 2008). Interestingly, blood samples from the pigs that were affected by this outbreak were negative for Brucella spp., both by the culture, as well as by the realtime PCR. A separate comparative bacteriological analysis of the organs from wild boars, including 41 spleens, 3 uteri and 3 testicles with Methods I and III, confirmed the higher sensitivity of method III, which resulted in the detection of 13 positive Brucella cultures from 11 spleens, 1 uterus and 1 testicle. Comparatively, method I detected 9 positive cultures from 9 spleens. 3.2. Characterization of Brucella suis isolates by MLVA-16 The MLVA-16 data from the Swiss isolates were compared with the Brucella2009 database (http://mlvau-psud.fr/). All isolates originating from Swiss wild animals, or from the outbreak in domestic pigs, were closely related to Brucella suis biovar 2 strains from France, Germany, Poland, and Denmark (B. suis biovar 2 reference strain Thomsen, ATCC 23445); indicating that they belong to the biovar 2 group. The 22 most related strains from the Brucella2009 database were included in the cluster analysis (Fig. 1). The most discriminatory loci in this study
3/6
3/6
6/10
were Bruce 04, Bruce 07, Bruce 30, and Bruce 09; which showed a HGDI greater than 0.568. The other loci returned a HGDI lower than 0.246. Using a cut off value of 73% similarity, 4 clusters of B. suis isolates from this study were identified and arbitrarily named clusters A, B, C, and D (Fig. 1). Except for isolate O/D 1618/08, all isolates originating from Switzerland were restricted to cluster A. Isolate O/D 1618/08 represents a single locus that is distinct from the main clusters A, B, C and D. Cluster A contained exclusively Swiss isolates, whereas clusters B, C and D contained isolates originating from France, Germany, Poland and the B. suis biovar 2 reference strain (Fig. 1). Cluster A, which contains 41 out of a total of 42 Swiss isolates, was divided into four subclusters named A1, A2, A3 and A4, using a cut off value of 82% similarity (Fig. 1). Isolates WS-GE-234, WSGE-208, ITPA 1321/09, and N 151/03 from cluster A showed less than 82% similarity with other isolates from the same cluster, and were not associated with any of the four subclusters. Subclusters A1, A3, A4 and the four individual variants comprised all isolates that originated from wild animals (wild boars and hares). Cluster A2 contains solely the isolates from the Brucella outbreak in the herds of domestic curly-hair pigs and no wild boar isolates grouped in this cluster. Although the isolates from the outbreak (A2), and the wild boar isolates from subcluster A3 originated from the same geographic region,
C. Abril et al. / Veterinary Microbiology 150 (2011) 405–410
they are genotypically distinguishable, and can be considered epidemiologically unlinked (Fig. 1). Subcluster A4 contains 3 hare isolates and 1 wild boar isolate. The location of all isolates from wild animals in Switzerland, and isolates from the Brucella outbreak in domestic curlyhair pigs are listed in Fig. 1. 4. Discussion Serological methods are useful for the diagnosis of brucellosis in domestic animals and are standard for surveillance studies (Office International des Epizooties O.I.E., 2009a). Despite this, an unequivocal diagnosis of a Brucella infection can only be made by the isolation of the pathogen (Ewalt, 1989; Pappas et al., 2005; Office International des Epizooties O.I.E., 2009a). Isolation of Brucella spp. is essential for the phenotypic and genotypic characterization of isolates and for subsequent molecular epidemiological investigations. However, the isolation of Brucella spp. from clinical samples remains a difficult task and many drawbacks have been reported (Bricker, 2002; Navarro et al., 2004). Major problems include low isolation rates, delayed diagnosis due to the slow growing nature of Brucella spp. and the risk of infection to laboratory personnel (Pappas et al., 2005; Demirdal and Demirturk, 2008). Cutting the tissue into small pieces and homogenising with a stomacher or a tissue grinder has been shown to improve the isolation rate, and is a recommended procedure by the OIE Manual of Standards for Diagnostic Tests and Vaccines (Office International des Epizooties O.I.E., 2008). In this study, we tested the IKA ULTRA-TURRAX1 system for tissue homogenisation. In contrast to the stomacher and conventional tissue grinders, the IKA ULTRA-TURRAX1 system allows complete and uniform tissue homogenisation, done in hermetically sealed tubes, thus preventing the formation of aerosols. When working with Brucella spp., aerosols represent the highest risk of laboratory acquired infections (Demirdal and Demirturk, 2008). It is known that the lysis of leucocytes from blood samples can increase the recovery and viability of intracellular pathogens, such as Brucella spp., and additionally reduces the incubation time of Brucella cultures (Yagupsky and Peled, 2002; Mantur and Mangalgi, 2004). Here we demonstrated an equivalent effect on the isolation rate of Brucella spp. from tissue samples of domestic pigs and wild boars. Our results indicate that the lysis of host cells with the Isolator 1.5 microbial system enhances the sensitivity of bacterial isolation (method III). Homogenisation of tissue samples alone (method II) is not enough to increase the frequency of isolation, but homogenisation is necessary for the subsequent lysis procedure using the Isolator 1.5 microbial system. In a previous study (Hinic et al., 2009), Brucella spp. were frequently detected in spleen samples from wild boars. Eleven of the 13 Brucella isolates in our study were obtained from wild boar spleen samples. In contrast, the investigation of the outbreak in the curly-hair hog herds revealed that B. suis was mostly restricted to the urogenital tract (9 of 10 isolates), while only one spleen sample was positive for B. suis (Table 1). Brucella spp. could not be
409
detected in the blood samples from the pigs affected by the outbreak neither by bacterial isolation, nor by real-time PCR. These results suggest that during the outbreak in domestic pigs, venereal transmission was the main route of infection, but in natural environments, the ingestion of contaminated materials such as aborted foetuses or membranes, seem to play an important role in transmission of this pathogen. By using MLVA, it was demonstrated that B. suis biovar 2 isolates from wild boars and domestic pigs may share the same genotype, suggesting that the transfer of B. suis biovar 2 from wild boars to domestic pigs may be a frequent event (Garcia-Yoldi et al., 2007). Although all the isolates from the outbreak investigated in this study are included in the Swiss cluster A (i.e., genetic similarity among isolates >73%), they belong to a genotypic subcluster (with a genetic similarity >93.8%), distinct from the isolates obtained from wild boars, both near and distant from the geographical area of the outbreak. Interestingly, the Bruce09 locus was different in three out of seven B. suis isolates of the outbreak (Fig. 1). This locus is considered to be the most polymorphic of those assayed (Garcia-Yoldi et al., 2007; Alvarez et al., 2010). The variation in this locus may have occurred in the herd and may indicate a chronic stage of the infection. Our data suggest that neither the neighbouring wild boars, nor wild boars from a more distant geographic region should be considered as sources of the infection. The infection may rather have been the result of introducing animals from holdings where B. suis was present but had not been detected. Moreover, the relatedness of the subcluster A2 to the other subclusters lets speculate that the outbreak could have originated from an earlier spill-over to domestic pigs from wild boars or from an unknown origin that are not covered in this study. This study demonstrates that sensitive molecular genotyping tools, such as MLVA-16 for brucellosis, are essential for accurate outbreak investigations. Acknowledgements We acknowledge Dr. Patrick Boujon from the Institute Galli-Vale´rio, Dr. Gre´goire Seitert and Dr. Ele´onore Grosclaude from the Service de la Consommation et des Affaires ve´te´rinaires (SCAV); Dr. Vladimira Hinic from the Centre for Zoonoses, Bacterial Animal Diseases and Antimicrobial Resistance (ZOBA) of the Institute of Veterinary Bacteriology (Vetsuisse Faculty, Bern, Switzerland); cantonal hunting offices, hunters and pig owners for their collaboration to this project. This study was funded by a grant from the Swiss Federal Veterinary Office and an internal grant from the Centre for Zoonoses, Bacterial Animal Diseases and Antimicrobial Resistance (ZOBA), Institute of Veterinary Bacteriology, Vetsuisse Faculty, Bern, Switzerland. References Al Dahouk, S., Fleche, P.L., Nockler, K., Jacques, I., Grayon, M., Scholz, H.C., Tomaso, H., Vergnaud, G., Neubauer, H., 2007. Evaluation of Brucella MLVA typing for human brucellosis. J. Microbiol. Methods 69, 137– 145.
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Alvarez, J., Saez, J.L., Garcia, N., Serrat, C., Perez-Sancho, M., Gonzalez, S., Ortega, M.J., Gou, J., Carbajo, L., Garrido, F., Goyache, J., Dominguez, L., 2010. Management of an outbreak of brucellosis due to B. melitensis in dairy cattle in Spain. Res. Vet. Sci.. Bricker, B.J., 2002. PCR as a diagnostic tool for brucellosis. Vet. Microbiol. 90, 435–446. Cvetnic, Z., Spicic, S., Curic, S., Jukic, B., Lojkic, M., Albert, D., Thiebaud, M., Garin-Bastuji, B., 2005. Isolation of Brucella suis biovar 3 from horses in Croatia. Vet. Rec. 156, 584–585. Cvetnic, Z., Spicic, S., Toncic, J., Majnaric, D., Benic, M., Albert, D., Thiebaud, M., Garin-Bastuji, B., 2009. Brucella suis infection in domestic pigs and wild boar in Croatia. Rev. Sci. Tech. 28, 1057–1067. De Santis, R., Ciammaruconi, A., Faggioni, G., D’Amelio, R., Marianelli, C., Lista, F., 2009. Lab on a chip genotyping for Brucella spp. based on 15loci multi locus VNTR analysis. BMC Microbiol. 9, 66. Demirdal, T., Demirturk, N., 2008. Laboratory-acquired brucellosis. Ann. Acad. Med. Singapore 37, 86–87. Ewalt, D.R., 1989. Comparison of three culture techniques for the isolation of Brucella abortus from bovine supramammary lymph nodes. J. Vet. Diagn. Invest. 1, 227–230. Garcia-Yoldi, D., Le Fleche, P., de Miguel, M.J., Munoz, P.M., Blasco, J.M., Cvetnic, Z., Marin, C.M., Vergnaud, G., Lopez-Goni, I., 2007. Comparison of multiple-locus variable-number tandem-repeat analysis with other PCR-based methods for typing Brucella suis isolates. J. Clin. Microbiol. 45, 4070–4072. Hinic, V., Brodard, I., Thomann, A., Cvetnic, Z., Makaya, P.V., Frey, J., Abril, C., 2008. Novel identification and differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae suitable for both conventional and real-time PCR systems. J. Microbiol. Methods 75, 375–378. Hinic, V., Brodard, I., Thomann, A., Holub, M., Miserez, R., Abril, C., 2009. IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serology. BMC Vet. Res. 5, 22. Kattar, M.M., Jaafar, R.F., Araj, G.F., Le Fleche, P., Matar, G.M., Abi, R.R., Khalife, S., Vergnaud, G., 2008. Evaluation of a multilocus variable-
number tandem-repeat analysis scheme for typing human Brucella isolates in a region of brucellosis endemicity. J. Clin. Microbiol. 46, 3935–3940. Le Fleche, P., Jacques, I., Grayon, M., Al Dahouk, S., Bouchon, P., Denoeud, F., Nockler, K., Neubauer, H., Guilloteau, L.A., Vergnaud, G., 2006. Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol. 6, 9. Leuenberger, R., Boujon, P., Thu¨r, B., Miserez, R., Garin-Bastuji, B., Ru¨fenacht, J., Sta¨rk, K.D., 2007. Prevalence of classical swine fever, Aujeszky’s disease and brucellosis in a population of wild boar in Switzerland. Vet. Rec. 160, 362–368. Mantur, B.G., Mangalgi, S.S., 2004. Evaluation of conventional castaneda and lysis centrifugation blood culture techniques for diagnosis of human brucellosis. J. Clin. Microbiol. 42, 4327–4328. Munoz, P.M., Boadella, M., Arnal, M., de Miguel, M.J., Revilla, M., Martinez, D., Vicente, J., Acevedo, P., Oleaga, A., Ruiz-Fons, F., Marin, C.M., Prieto, J.M., de la, F.J., Barral, M., Barberan, M., de Luco, D.F., Blasco, J.M., Gortazar, C., 2010. Spatial distribution and risk factors of brucellosis in Iberian wild ungulates. BMC Infect. Dis. 10, 46. Navarro, E., Casao, M.A., Solera, J., 2004. Diagnosis of human brucellosis using PCR. Expert Rev. Mol. Diagn. 4, 115–123. Office International des Epizooties O.I.E., 2008. Manual of Standards for Diagnostic Tests and Vaccines, 6th ed. Office International des Epizooties O.I.E., Paris. Office International des Epizooties O.I.E., 2009a. Bovine brucellosis (NB: Version adopted in May 2009). In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. pp. 1–35. Office International des Epizooties O.I.E., 2009b. Porcine and rangiferine brucellosis: Brucella suis. http://www.cfsph.iastate.edu/Factsheets/ pdfs/brucellosis_suis.pdf. Pappas, G., Akritidis, N., Bosilkovski, M., Tsianos, E., 2005. Brucellosis. N. Engl. J. Med. 352, 2325–2336. Yagupsky, P., Peled, N., 2002. Use of the Isolator 1.5 microbial tube for detection of Brucella melitensis in synovial fluid. J. Clin. Microbiol. 40, 3878.
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Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Short communication
A novel isolation method of Brucella species and molecular tracking of Brucella suis biovar 2 in domestic and wild animals Carlos Abril a,*, Andreas Thomann a, Isabelle Brodard a, Natacha Wu c, Marie-Pierre Ryser-Degiorgis c, Joachim Frey b, Gudrun Overesch a a b c
Centre for Zoonoses, Bacterial Animal Diseases and Antimicrobial Resistance (ZOBA), La¨nggass-Strasse 122, Postfach 8466, CH-3001 Bern, Switzerland Research Unit, Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, La¨nggass-Strasse 122, Postfach 8466, CH-3001 Bern, Switzerland Centre for Fish and Wildlife Health, Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Postfach 8466, CH-3001 Bern, Switzerland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 January 2011 Received in revised form 22 February 2011 Accepted 28 February 2011
Brucella suis biovar 2 is the most common aetiological agent of porcine brucellosis in Europe. B. suis biovar 2 is considered to have low zoonotic potential, but is a causative agent of reproductive losses in pigs, and it is thus economically important. The multilocus variable-number of tandem repeats genotyping analysis of 16 loci (MLVA-16) has proven to be highly discriminatory and is the most suitable assay for simultaneously identifying B. suis and tracking infections. The aim of this study was to investigate the relatedness between isolates of B. suis biovar 2 obtained during a brucellosis outbreak in domestic pigs and isolates from wild boars and hares collected from proximal or remote geographical areas by MLVA-16. A cluster analysis of the MLVA-16 data revealed that most of the isolates obtained from Switzerland clustered together, with the exception of one isolate. The outbreak isolates constituted a unique subcluster (with a genetic similarity >93.8%) distinct from that of the isolates obtained from wild animals, suggesting that direct transmission of the bacterium from wild boars to domestic pigs did not occur in this outbreak. To obtain a representative number of isolates for MLVA-16, alternative methods of Brucella spp. isolation from tissue samples were compared with conventional direct cultivation on a Brucella-selective agar. We observed an enhanced sensitivity when mechanical homogenisation was followed by host cell lysis prior to cultivation on the Brucella-selective agar. This work demonstrates that MLVA-16 is an excellent tool for both monitoring brucellosis and investigating outbreaks. Additionally, we present efficient alternatives for the isolation of Brucella spp. ß 2011 Elsevier B.V. All rights reserved.
Keywords: Brucellosis Brucella suis Isolation methods MLVA-16 Domestic pigs Wild boars Outbreak
1. Introduction According to the World Organization for Animal Health (http://www.oie.int/wahis/public.php?page=weekly_ report_index), a total of 136 confirmed outbreaks of porcine brucellosis were reported in Europe between January 2007 and December 2010. The causative agent of porcine brucellosis in Europe is usually Brucella suis biovar 2, which is host adapted and does
* Corresponding author. Tel.: +41 31 631 2436; fax: +41 31 631 2634. E-mail address: [email protected] (C. Abril). 0378-1135/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetmic.2011.02.056
not often infect humans (Garcia-Yoldi et al., 2007; Munoz et al., 2010). B. suis biovars 1 and 3, also cause brucellosis in porcines, but in contrast to biovar 2, they are zoonotic agents that cause severe brucellosis in humans and other animals (Munoz et al., 2010). In Europe, the biovars 1 and 3 have only been reported in Croatia, indicating that they may be restricted to this geographic region (Cvetnic et al., 2005, 2009; Garcia-Yoldi et al., 2007). B. suis biovar 2 is widely spread amongst Eurasian wild boar (Sus scrofa) and European brown hare (Lepus europaeus) populations (Leuenberger et al., 2007; Munoz et al., 2010). As a result, these animals may be responsible for the transmission of B. suis biovar 2 to reared outdoor domestic pigs (Munoz et al., 2010).
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Multilocus variable-number of tandem repeats analysis (MLVA) is a genotyping method based on the quantification of minisatellite and microsatellite repeats in various loci of a bacterial genome (Kattar et al., 2008). When used for genotyping Brucella spp., the MLVA assay based on sixteen tandem repeat loci (MLVA-16) has enabled the differentiation of the biovars, and distinguished isolates of both widespread and closed, temporal and geographical origins (Garcia-Yoldi et al., 2007; Al Dahouk et al., 2007; Kattar et al., 2008). This study aimed to identify possible sources of a B. suis outbreak that occurred in domestic pigs in Switzerland during 2009 using the MLVA-16 method. To enhance isolation sensitivity and therefore the number of isolates for MLVA-16 analysis, the efficiency of three Brucella isolation methods from tissue samples were tested in parallel. 2. Materials and methods 2.1. Animals and serological tests A culture-confirmed B. suis infection in a fertile curlyhair hog boar originating from the canton of Geneva was tracked back. For that reason, the animals (26) of three herds of domestic curly-hair hogs from which the infected pig originated were tested for Brucella spp. antibodies by the rose bengal (RBT) and complement fixation (CFT) tests according to international standards (Office International des Epizooties O.I.E., 2009b), and reacted positively (RBT ++, CFT 177 IU/ml). Blood and organs (liver, spleen and reproductive organs) from these animals were collected at necropsy. Additionally, spleen and six reproductive organ samples from 41 free-ranging wild boars were analyzed in this study. 2.2. Bacterial isolation and genotypic identification The efficiency of three isolation methods was evaluated in parallel. For method I, approximately 20 mg of tissue was removed with a platinum spatula and transferred directly to a Brucella-selective agar, containing Brucellamedium base (Oxoid, Basingstoke, UK), 5% inactivated horse serum and Modified Brucella Selective Supplement (Oxoid, Basingstoke, UK). For method II, the tissue samples were homogenised before plating on the Brucella-selective agar. One gram of tissue was washed once and suspended in 5 ml of 0.85% NaCl solution. The tissue was then homogenised twice for 30 s in DT-20 tubes at 6000 rpm, using the IKA ULTRA-TURRAX1 tube drive disperser system (IKA, Staufen, Germany). Method III consisted of homogenising tissues in combination with chemical host cell lysis prior to plating on the Brucella-selective agar. The tissue samples were homogenised as in method II. One millilitre of the homogenised tissue was added to an Isolator 1.5 microbial tube (OXOID, Basingstoke, England), and host cells were lysed with saponin to release intracellular bacteria. The Isolator 1.5 tube additionally contains sodium polynetholsulphate which neutralises the bactericidal properties of blood. Finally, in Methods II and III, a total of 300 ml of the homogenised or the
homogenised and lysed suspension was inoculated onto two Brucella-selective agar plates. In a further step, the suspension was spread with a loop, producing a depot followed by single colonies. All cultures were incubated at 37 8C with an atmosphere containing 5% CO2 for 5 days. The blood samples from the curly-hair hogs were analyzed by bacterial isolation using the Isolator 10 microbial tube (OXOID, Basingstoke, England), according to manufacturers protocol and cultivated on Brucella-selective agar using the same conditions as the organ cultures. Additionally, blood samples were tested by a real-time PCR targeting a Brucella spp. specific IS711 insertion element (Hinic et al., 2009). Briefly, 300 ml of EDTA–blood were suspended in 600 ml erythrocyte lysis buffer (1.55 M NH4Cl, 0.1 M KHCO3, 1 mM Titriplex III, 10 conc.) and 300 ml PBS buffer. After 10 min incubation at room temperature, a leukocyte pellet was obtained by centrifugation at 8000 rpm for 2 min. Finally, DNA from the leukocyte pellet was extracted following the tissue protocol of the QIAamp1 DNA Mini Kit (QIAGEN, Hilden, Germany). Brucella identification and species differentiation was accomplished using PCR protocols described by Hinic et al. (2008). 2.3. MLVA-16 MLVA-16 was used to assess the relatedness of B. suis isolates obtained from curly-haired hogs that were involved in the outbreak, along with 15 B. suis isolates from wild boars from the same geographical area (Cantons of Geneva and Vaud), and further 20 B. suis isolates from wild boars and hares from a location in Switzerland 150 km distant from the outbreak (Canton of Bern) (Fig. 1). Sixteen tandem repeat loci (Fig. 1) were screened as previously described (Le Fleche et al., 2006; Al Dahouk et al., 2007). The analysis of PCR products was performed on an Agilent 2100 Bioanalyzer using a DNA 1000 LabChip kit (Agilent Technologies, Waldbronn, Germany), according to the manufacturer’s protocols. Quantification of the tandem repeats was accomplished using a conversion table published by De Santis et al. (2009), which was adapted to process Agilent 2100 Bioanalyzer data. DNA sequencing verified the size of the PCR products and was accomplished using a Sequenator AB 3100 and the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems Norwalk, CT, USA). The categorical MLVA-16 cluster analysis was accomplished using the unweighted pair group method (UPGMA) and BioNumerics software (version 5.1; Applied Math, Sint-Martens-Latem, Belgium). The genetic diversity of each locus was calculated using the Hunter-Gaston diversity index (HGDI) (http://www.hpabioinformatics.org.uk/cgi-bin/DICI/DICI.pl). 3. Results 3.1. Isolation of Brucella spp. from organs Using the bacteriological isolation Methods I and II, Brucella spp. were isolated in three out of 26 curly-hair hogs (Table 1). However, with method III, which included host cells lysis with the Isolator 1.5 microbial system,
C. Abril et al. / Veterinary Microbiology 150 (2011) 405–410
100 93.8
76.5
87.5 100
86.5
73.3
87.5 84.4 93.8 81.3
69.2
81.3 76.6
81.3 75.4
81.3
87.5
75 77.1
70.1
62.9 87.5 81.3 77.1 73.4 81.3 56.9
71.6
2009
2
4
8 14
6
1
5
6
6 21
9 11
8 24
2
4
WS-BE-9
wild boar
2
Switzerland (Bern)
2008
2
4
8 14
6
1
5
6
6 21
9
8
8 24
2
4
ITPA 1033/08
wild boar
2
Switzerland (Bern)
2008
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
ITPA 2225/08
wild boar
2
Switzerland (Bern)
2008
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-165
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-36
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-57
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-61
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-68
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-70
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-71
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8 24
2
4
WS-BE-217
wild boar
2
Switzerland (Bern)
2009
2
4
8 14
6
1
5
6
6 21
9 18
8
9
2
4
WS-GE-155
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9
5
8 15
2
4
WS-GE-234
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9
5
6
7
2
4
WS-GE-208
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
4 21
9
5
8 14
2
4
IMD 1548/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 13
2
9
IMD 1563/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 13
2
9
IMD 1565/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 13
2
9
IMD 1566/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 13
2
9
IMD 1021/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 11
2
9
IMD 1567/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 11
2
9
IMD 1568/09
swine
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 11
8 11
2
9
IMD 836/10
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-135
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-170
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-171
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-178
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-200
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-207
wild boar
2
Switzerland (Geneva) 2009
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-228
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-239
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9 18
6 17
2
8
WS-GE-232
wild boar
2
Switzerland (Geneva) 2010
2
4
8 14
6
1
5
6
6 21
9 18
6 14
2
8
N 336/03
wild boar
2
Switzerland (Vaud)
2003
2
4
8 14
6
1
5
6
6 21
9 18
6
6
2
7
N 591/03 N 148/03
wild boar wild boar
2 2
Switzerland (Vaud) 2003 Switzerland (Geneva) 2003
2 2
4 4
8 14 8 14
6 6
1 1
5 5
6 6
6 21 6 21
9 18 9 8
6 6 6 24
2 2
7 8
D 1011/98
hare
2
Switzerland (Bern)
1998
2
4
8 14
6
1
5
2
6 21
9 18
9
9
2
4
O/D 1055/08
hare
2
Switzerland (Bern)
2008
2
4
8 14
6
1
5
2
6 21
9 18 12
8
2
4
D 224/98
hare
2
Switzerland (Bern)
1998
2
4
8 14
6
1
5
2
6 21
9 18 10 11
2
4
N 1982/03
wild boar
2
Switzerland (Vaud)
2003
2
4
8 14
6
1
5
6
6 21
9 18 10 11
2
4
ITPA1321/09
hare
2
Switzerland (Freiburg) 2009
2
4
8 14
6
1
5
2
6 21
9
8
6
9
2
4
N 151/03
wild boar
2
Switzerland (Geneva) 2003
2
5
8 14
6
1
5
6
6 21
9
9
6 24
2
9
BCCN 02_50
swine
2
France (72)
2002
2
4
8 15
6
1
5
6
6 21
9
9 10 11
2
9
BCCN 03_6
wild boar
2
France (73)
2003
2
4
8 15
6
1
5
6
6 21
9 12
4 14
2
9
BCCN 00_23
swine
2
France (87)
2000
2
4
8 15
6
1
5
6
6 21
9
6 24
2
6
BCCN 02_23
wild boar
2
France (73)
2002
2
4
8 15
6
1
5
6
6 21
9 12
6
8
2
8
BCCN 02_36
swine
2
France (72)
2002
2
4
8 15
6
1
5
6
6 21
9 11 12
7
2 10
BCCN 03_11
wild boar
2
France (35)
2003
2
4
8 15
6
1
5
6
6 21
9
9
8
7
2 10
BCCN 98_95
swine
2
France (87)
1998
2
4
8 15
6
1
5
6
6 21
9
6
9 16
2 10
BCCN 93_63
swine
2
France (76)
1993
2
4
8 15
6
1
5
6
6 21
9 15
9 13
2
BCCN 02_20
swine
2
France (79)
2002
2
4
8 15
6
1
5
6
6 21
9 13 13
BCCN 03_5
swine
2
France (03)
2003
2
4
8 15
7
1
5
6
6 21
9
6
4 18
BCCN 02_18
hare
2
France (49)
2002
2
4
8 15
6
1
5
2
6 21
9
7
BCCN 02_48
hare
2
France (49)
2002
2
4
8 15
6
1
5
2
6 21
9
9
BCCN 78_6
hare
2
France (57)
1978
2
4
8 15
6
1
5
2
6 21
9 12
BfR 150
swine
4
6
5 16
2
6
5
2
6
2 11
2
Germany (Aulendorf) 1998
2
4
8 15
6
1
5
2
6 21
9 23 12 13
2
4
BCCN 89_155 hare
2
France (57)
1989
2
4
8 15
6
1
5
2
6 21
9 16
7 11
2
6
BCCN 93_24
2
France (03)
1993
2
4
8 15
6
1
5
2
5 21
9 16 11 11
2
8
BCCN 81_287 swine
2
France (67)
1981
2
4
8 15
6
1
5
2
6 21
9 13
9 17
2
5
BfR 138
hare
2
Germany (Nürnberg)
1999
2
4
8 15
6
1
5
2
6 21
9 28
9 11
2
5
BCCN 82_19
hare
2
Poland
1982
2
4
8 15
5
1
5
2
6 21
9 10
9
2
5
BCCN 03_2
hare
2
France (38)
2003
2
4
8 15
5
1
5
2
6 21
9 15
4 17
2
5
ATCC 23445
swine
2
Denmark (Thomsen)
2
4
8 15
6
1
5
2
6 22
9
9
9 18
2
4
BCCN 87_11
hare
2
France (57)
1987
2
4
8 15
6
1
5
2
7 21
9
6
6 23
O/D 1618/08
hare
2
Switzerland (Bern)
2008
2
4
7
7
1
5
2
6 21
9 18 15 11
hare
A2
A
A3
A4
B
7
5
5 13
A1
2 11
2
8
Clusters
Switzerland (Bern)
Bruce 04
2
Bruce 07
wild boar
Bruce 21
WS-BE-44
Bruce 19
Bruce 16
77.9
Bruce 30
100
Bruce 09
93.8
Bruce 18
100
Bruce 45
80.8
Bruce 55
81.7
Bruce 43
81.7
Bruce 42
93.8
Bruce 12
100
Location /Canton Year
Bruce 11
87.5
Host
Bruce 08
93.1
Isolate ID.
Bruce 06
93.8
Biovar
100
90
60
70
80
Genetic similarity
Subclusters
407
Brucella MLVA-16 (loci)
C
87.5 70
67.1
81.3 79.2
9
8
D
2 12 2
4
Fig. 1. MLVA-16-based dendrogram of Brucella suis isolates from Switzerland (non-italicised). Dendrogram calculations were done using the UPGMA method, including data of related isolates from the Brucella2009 database (italics) (http://mlva-u-psud.fr/). Isolate identification, host, biovar, location of sampling sites, year of isolation and number of tandem repeats of each locus are indicated. Isolates obtained during the outbreak are represented in bold.
408
C. Abril et al. / Veterinary Microbiology 150 (2011) 405–410
Table 1 Detection of Brucella ysuis in individual tissue samples by bacterial isolation by three different methods. Animal no
Organs
1
Spleen Bladder Testicle 1 Testicle 2 Epididymis 1 Epididymis 2
2
Spleen Bladder Uterus Uterus (multifocal abscesses)
4
Spleen Bladder Uterus Ovary Spleen Bladder Uterus Ovary Udder abscess
6
7–26
Method II
Method III
+
+
+
+ + +
+ + +
+ + +
Spleen Bladder Uterus Ovary
3
5
Method I
+ + +
+
+
+
+
+
+
Spleen Bladder Uterus Uterus (multifocal abscesses)
+
Spleen Bladder Reproductive organs
Total positive (animals/organs)
Brucella spp. was isolated from 6 out of the 26 animals (Table 1). The bacterial isolates were subsequently identified as B. suis using a previously described protocol (Hinic et al., 2008). Interestingly, blood samples from the pigs that were affected by this outbreak were negative for Brucella spp., both by the culture, as well as by the realtime PCR. A separate comparative bacteriological analysis of the organs from wild boars, including 41 spleens, 3 uteri and 3 testicles with Methods I and III, confirmed the higher sensitivity of method III, which resulted in the detection of 13 positive Brucella cultures from 11 spleens, 1 uterus and 1 testicle. Comparatively, method I detected 9 positive cultures from 9 spleens. 3.2. Characterization of Brucella suis isolates by MLVA-16 The MLVA-16 data from the Swiss isolates were compared with the Brucella2009 database (http://mlvau-psud.fr/). All isolates originating from Swiss wild animals, or from the outbreak in domestic pigs, were closely related to Brucella suis biovar 2 strains from France, Germany, Poland, and Denmark (B. suis biovar 2 reference strain Thomsen, ATCC 23445); indicating that they belong to the biovar 2 group. The 22 most related strains from the Brucella2009 database were included in the cluster analysis (Fig. 1). The most discriminatory loci in this study
3/6
3/6
6/10
were Bruce 04, Bruce 07, Bruce 30, and Bruce 09; which showed a HGDI greater than 0.568. The other loci returned a HGDI lower than 0.246. Using a cut off value of 73% similarity, 4 clusters of B. suis isolates from this study were identified and arbitrarily named clusters A, B, C, and D (Fig. 1). Except for isolate O/D 1618/08, all isolates originating from Switzerland were restricted to cluster A. Isolate O/D 1618/08 represents a single locus that is distinct from the main clusters A, B, C and D. Cluster A contained exclusively Swiss isolates, whereas clusters B, C and D contained isolates originating from France, Germany, Poland and the B. suis biovar 2 reference strain (Fig. 1). Cluster A, which contains 41 out of a total of 42 Swiss isolates, was divided into four subclusters named A1, A2, A3 and A4, using a cut off value of 82% similarity (Fig. 1). Isolates WS-GE-234, WSGE-208, ITPA 1321/09, and N 151/03 from cluster A showed less than 82% similarity with other isolates from the same cluster, and were not associated with any of the four subclusters. Subclusters A1, A3, A4 and the four individual variants comprised all isolates that originated from wild animals (wild boars and hares). Cluster A2 contains solely the isolates from the Brucella outbreak in the herds of domestic curly-hair pigs and no wild boar isolates grouped in this cluster. Although the isolates from the outbreak (A2), and the wild boar isolates from subcluster A3 originated from the same geographic region,
C. Abril et al. / Veterinary Microbiology 150 (2011) 405–410
they are genotypically distinguishable, and can be considered epidemiologically unlinked (Fig. 1). Subcluster A4 contains 3 hare isolates and 1 wild boar isolate. The location of all isolates from wild animals in Switzerland, and isolates from the Brucella outbreak in domestic curlyhair pigs are listed in Fig. 1. 4. Discussion Serological methods are useful for the diagnosis of brucellosis in domestic animals and are standard for surveillance studies (Office International des Epizooties O.I.E., 2009a). Despite this, an unequivocal diagnosis of a Brucella infection can only be made by the isolation of the pathogen (Ewalt, 1989; Pappas et al., 2005; Office International des Epizooties O.I.E., 2009a). Isolation of Brucella spp. is essential for the phenotypic and genotypic characterization of isolates and for subsequent molecular epidemiological investigations. However, the isolation of Brucella spp. from clinical samples remains a difficult task and many drawbacks have been reported (Bricker, 2002; Navarro et al., 2004). Major problems include low isolation rates, delayed diagnosis due to the slow growing nature of Brucella spp. and the risk of infection to laboratory personnel (Pappas et al., 2005; Demirdal and Demirturk, 2008). Cutting the tissue into small pieces and homogenising with a stomacher or a tissue grinder has been shown to improve the isolation rate, and is a recommended procedure by the OIE Manual of Standards for Diagnostic Tests and Vaccines (Office International des Epizooties O.I.E., 2008). In this study, we tested the IKA ULTRA-TURRAX1 system for tissue homogenisation. In contrast to the stomacher and conventional tissue grinders, the IKA ULTRA-TURRAX1 system allows complete and uniform tissue homogenisation, done in hermetically sealed tubes, thus preventing the formation of aerosols. When working with Brucella spp., aerosols represent the highest risk of laboratory acquired infections (Demirdal and Demirturk, 2008). It is known that the lysis of leucocytes from blood samples can increase the recovery and viability of intracellular pathogens, such as Brucella spp., and additionally reduces the incubation time of Brucella cultures (Yagupsky and Peled, 2002; Mantur and Mangalgi, 2004). Here we demonstrated an equivalent effect on the isolation rate of Brucella spp. from tissue samples of domestic pigs and wild boars. Our results indicate that the lysis of host cells with the Isolator 1.5 microbial system enhances the sensitivity of bacterial isolation (method III). Homogenisation of tissue samples alone (method II) is not enough to increase the frequency of isolation, but homogenisation is necessary for the subsequent lysis procedure using the Isolator 1.5 microbial system. In a previous study (Hinic et al., 2009), Brucella spp. were frequently detected in spleen samples from wild boars. Eleven of the 13 Brucella isolates in our study were obtained from wild boar spleen samples. In contrast, the investigation of the outbreak in the curly-hair hog herds revealed that B. suis was mostly restricted to the urogenital tract (9 of 10 isolates), while only one spleen sample was positive for B. suis (Table 1). Brucella spp. could not be
409
detected in the blood samples from the pigs affected by the outbreak neither by bacterial isolation, nor by real-time PCR. These results suggest that during the outbreak in domestic pigs, venereal transmission was the main route of infection, but in natural environments, the ingestion of contaminated materials such as aborted foetuses or membranes, seem to play an important role in transmission of this pathogen. By using MLVA, it was demonstrated that B. suis biovar 2 isolates from wild boars and domestic pigs may share the same genotype, suggesting that the transfer of B. suis biovar 2 from wild boars to domestic pigs may be a frequent event (Garcia-Yoldi et al., 2007). Although all the isolates from the outbreak investigated in this study are included in the Swiss cluster A (i.e., genetic similarity among isolates >73%), they belong to a genotypic subcluster (with a genetic similarity >93.8%), distinct from the isolates obtained from wild boars, both near and distant from the geographical area of the outbreak. Interestingly, the Bruce09 locus was different in three out of seven B. suis isolates of the outbreak (Fig. 1). This locus is considered to be the most polymorphic of those assayed (Garcia-Yoldi et al., 2007; Alvarez et al., 2010). The variation in this locus may have occurred in the herd and may indicate a chronic stage of the infection. Our data suggest that neither the neighbouring wild boars, nor wild boars from a more distant geographic region should be considered as sources of the infection. The infection may rather have been the result of introducing animals from holdings where B. suis was present but had not been detected. Moreover, the relatedness of the subcluster A2 to the other subclusters lets speculate that the outbreak could have originated from an earlier spill-over to domestic pigs from wild boars or from an unknown origin that are not covered in this study. This study demonstrates that sensitive molecular genotyping tools, such as MLVA-16 for brucellosis, are essential for accurate outbreak investigations. Acknowledgements We acknowledge Dr. Patrick Boujon from the Institute Galli-Vale´rio, Dr. Gre´goire Seitert and Dr. Ele´onore Grosclaude from the Service de la Consommation et des Affaires ve´te´rinaires (SCAV); Dr. Vladimira Hinic from the Centre for Zoonoses, Bacterial Animal Diseases and Antimicrobial Resistance (ZOBA) of the Institute of Veterinary Bacteriology (Vetsuisse Faculty, Bern, Switzerland); cantonal hunting offices, hunters and pig owners for their collaboration to this project. This study was funded by a grant from the Swiss Federal Veterinary Office and an internal grant from the Centre for Zoonoses, Bacterial Animal Diseases and Antimicrobial Resistance (ZOBA), Institute of Veterinary Bacteriology, Vetsuisse Faculty, Bern, Switzerland. References Al Dahouk, S., Fleche, P.L., Nockler, K., Jacques, I., Grayon, M., Scholz, H.C., Tomaso, H., Vergnaud, G., Neubauer, H., 2007. Evaluation of Brucella MLVA typing for human brucellosis. J. Microbiol. Methods 69, 137– 145.
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