Link between geographical origin and occurrence of Brucella abortus biovars in cattle

AEM Accepts, published online ahead of print on 26 November 2012 Appl. Environ. Microbiol. doi:10.1128/aem.02887-12 Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 2 3 SHORT-FORM PAPER Link between geographical origin and occurrence of Brucella abortus biovars in cattle and water buffalo herds 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Running title: Genotyping of Brucella abortus by MLVA Giorgia Borriello 1*, Simone Peletto 2, Maria G. Lucibelli 1, Pier L. Acutis 2, Danilo Ercolini 3, Giorgio Galiero 1 Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, Italy 1 ; Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d Aosta, Turin, Italy 2 ; Dipartimento di Scienza degli Alimenti, Università degli Studi di Napoli Federico II, Portici, Italy 3 Corresponding author. Mailing address: Istituto Zooprofilattico Sperimentale del Mezzogiorno, Dipartimento di Sanità Animale, Via Salute, 2, 80055, Portici (NA), Italy. Phone: 0039 081 7865385. Fax: 0039 081 7865273. E-mail: giorgia.borriello@izsmportici.it 1

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 ABSTRACT Sixty-three Brucella isolates from water buffaloes and cattle slaughtered within the Italian National plan for brucellosis control were characterized by MLVA. Genotyping indicated a strong influence of geographic origin on B. abortus biovars distribution in areas where brucellosis is endemic and highlighted the importance of rigorous management procedures aiming at avoiding inter- and intra-herd pathogen spreading. Brucellosis is the most widespread zoonosis worldwide, of major public health and economic significance (16). The disease is caused by Brucella spp., which can infect several important livestock species, including cattle, water buffalo, goats, sheep and pigs (7, 16). The principal symptom of the infection in all animal species is abortion or premature expulsion of the fetus. The pathogen can be transmitted to humans through consumption of contaminated and untreated milk or dairy products, or by direct contact with infected animals. In man the disease can induce undulant fever, malaise and myalgia, sometimes associated with serious complications such as encephalitis, meningitis, peripheral neuritis, spondylitis, suppurative arthritis and vegetative endocarditis. The disease can also occur in a chronic form affecting various organs and tissues (8). The genus Brucella includes 9 species (7) characterized by more than 90% DNA/DNA homology (11, 19). In the last years, the characterization of the variable number of tandem repeats (VNTR) by Multiple-Locus VNTR Analysis (MLVA) was effectively used for Brucella spp. typing in humans and animal species including bovine, ovine, wild boar, hare, water buffalo and marine mammals (7, 9, 11, 13). Such typing of Brucella can be useful for epidemiological studies and may advance control of human and animal brucellosis. 2

50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 The two species most commonly involved in human infections are B. abortus, epizootic in cattle, and B. melitensis, more virulent and more diffused in sheep and goats (8). Brucellosis has been successfully eradicated in most developed countries, even though it is still endemic in many developing and some developed countries in Latin America, Southern Europe, Africa, Southeast Asia, and the Middle East (16). The available strategies to control brucellosis are based on very strict management procedures, slaughtering of all seropositive animals and, where allowed, vaccination (2). The aim of this study was to evaluate the distribution of Brucella biovars both in cattle and water buffalo herds in a region of brucellosis endemicity, to create a model of epidemiological trace-back analysis useful to determine the origin of the contamination and consequently to allow a better management of the surveillance programme (17). Sixty-three Brucella isolates obtained from lymph-nodes of 46 water buffaloes and 17 cattle slaughtered within the Italian National plan for the control of brucellosis were investigated. As indicated by the Italian control programme, based on a test-and-slaughter approach, animals were not vaccinated and all the subjects positive to serological tests (Rose Bengal and complement fixation tests) were culled and processed for microbiological isolation of Brucella spp. (15). The animals included in this study were collected from 17 cattle and 28 water buffalo herds located in two provinces, Caserta (CE) and Salerno (SA), of the Campania region during the year 2008. The province of Caserta covers an area of 2639 km 2 and includes 1891 cattle herds (44550 animals) and 929 water buffalo herds (176308 animals), while the province of Salerno covers an area of 4918 km 2 with 3947 cattle herds (61596 animals) and 447 water buffalo herds (86784 animals). The two districts are characterized by intense exchanges of animals (mainly water buffaloes), food and commercial goods. The Brucella isolates were cultured on Brucella agar (Oxoid, Hampshire, UK) for 3 to 5 days at 37 C, under 5% CO 2. Bacterial DNA was extracted from fresh cultures using the 3

75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 DNeasy blood and tissue kit (QIAGEN, Hilden, Germany), according to the manufacturer s instructions. All isolates were firstly identified as Brucella spp. on the basis of positivity to agglutination to specific antisera and biochemical tests performed with the instrument VITEK 2 (BioMérieux, Craponne, France). Furthermore, a molecular typing was performed by the Italian Reference Centre for Brucellosis (Istituto Zooprofilattico Sperimentale dell Abruzzo e del Molise, Teramo) by the AMOS (Abortus-Melitensis-Ovis-Suis) PCR assay (3) for species identification and PCR-RFLP analysis of the genes omp2a, omp2b and omp31 for biovar identification (4, 19). These genes code for Brucella major outer membrane proteins (OMPs) strongly associated with peptidoglycan but with low or no immunogenic or protective activity against B. abortus and B. melitensis in host infection (5). Final biovar identification was performed by growth in presence of thionin and basic fuchsin using the slide agglutination test with Brucella A- and M-monospecific antisera (VLA, Weibridge) (7). The same DNA samples were analyzed by the MLVA-16 typing technique, as elsewhere described (1, 12) with some modifications. The 16 primer pairs were divided into two groups: panel 1 (loci Bruce06, Bruce08, Bruce11, Bruce12, Bruce42, Bruce43, Bruce45, and Bruce55), more conserved, characterized by moderately variable minisatellites, and panel 2 (loci Bruce04, Bruce07, Bruce09, Bruce16, Bruce18, Bruce19, Bruce21, and Bruce30) constituted by highly discriminatory microsatellites (1, 12). These markers where chosen because their stability was already assessed (12) and they are widely employed for Brucella spp. characterization (1, 6, 9, 11, 13). Amplifications were performed as follows: denaturation for 3 min at 94 C, followed by 30 cycles at 94 C for 30 s, 60 C for 30 s, and 72 C for 50 s, with a final extension at 72 C for 7 min. All the forward primers were labelled with either the fluorophore FAM (6- carboxyfluorescein) (Bruce06, Bruce42, Bruce18, Bruce07) or VIC (Bruce08, Bruce43, Bruce19, Bruce09) or NED (Bruce11, Bruce45, Bruce21, Bruce16) or PET (Bruce12, Bruce55, Bruce04, Bruce30) (VIC, NED and PET fluorescent dyes with chemical structure 4

100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 currently not publicly available and proprietary to Life Technologies, Hawthorne, NY). PCR products were mixed together in a ratio 1:1:1:1 to obtain four different mixtures each one containing 4 amplicons labelled with 4 different fluorophores. The mixtures were then denatured for 5 min at 95 C in presence of Hi-Di formamide and analyzed by capillary electrophoresis with a 310 Genetic Analyzer (Life Technologies) equipped with a 47 cm long and 50 μm section capillary filled with the separation medium POP-4 polymer. PCR products relative to the loci Bruce06, Bruce11 and Bruce42 were also resolved by automated electrophoresis performed with the instrument QIAXcel (QIAGEN) to visualize eventual amplicons longer than 500 bp. Statistical analysis was carried out using the GraphPad QuickCalcs software available online (http://www.graphpad.com/quickcalcs/index.cfm). Band size estimates were converted to a number of units within a character dataset using the BioNumerics software. Clustering analyses used the categorical coefficient and the UPGMA algorithm. Maximum parsimony tree was calculated using BioNumerics, treating the data as categorical and giving the same weight to all loci. The molecular characterization performed to assess the species and the biovar of the Brucella strains indicated the presence of 39 isolates of B. abortus bv. 1 (62%), 22 isolates of B. abortus bv. 3 (35%) and 2 isolates of B. melitensis bv. 3 (3%). In particular, B. abortus bv. 1 was significantly associated with water buffalo species (92% vs. 7% in cattle), while B. abortus bv. 3 with cattle (59% vs. 41% in water buffalo; Fisher's exact test two-tailed P value < 0.0001). The two isolates of B. melitensis bv. 3 were isolated from a cattle and a water buffalo, respectively. These results are in agreement with previous reports on B. abortus biovars in cattle and water buffalo in Italy (7). The B. abortus biotypes exhibited a sectorial geographic distribution, as the biovar 3 was isolated only in the province of Salerno (SA), while the biovar 1 mostly (37/39) in the province of Caserta (CE). 5

124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 The MLVA typing assay of the Brucella isolates indicated the presence of 52 different genotypes (Figure 1), thus discriminating 30 strains out of 39 isolates within the biovar abortus 1, and 20 strains out of 22 isolates within the biovar abortus 3. The most polymorphic loci were Bruce04 with 5 allelic types and Bruce07, Bruce09 and Bruce30, exhibiting 7 allelic types (Diversity Index DI values were 4.0, 4.1, 3.1, 3.5, respectively). The less polymorphic loci were Bruce06 and Bruce42 with two allelic types, and Bruce45 with one allelic type (DI values were 1.2, 1.2, 1.0, respectively). Clustering analysis using UPGMA, grouped the Brucella isolates into clusters with 90% similarity (Figure 1). The 2 B. melitensis isolates belonged to two different clusters, while the 61 B. abortus isolates were classified into 40 clusters, among which 31 included one single isolate, while 9 grouped closely related isolates corresponding to 22 different genotypes. Isolates belonging to a defined cluster are presumed to be recently evolved from one common ancestor and defining clusters can therefore be useful to trace transmission routes. Isolates with the same genotype were revealed in restricted areas, as for genotype 22 in the Monte San Giacomo-Sacco area, genotype 26 in the Grazzanise-Santa Maria la Fossa area, genotype 34 in the Grazzanise-Falciano del Massico area, genotype 33 in the Baia e Latina-Pietramelara area. In our study, when isolates from the above mentioned 9 clusters were mapped on a chart by using Google Map (https://maps.google.it/maps/ms?msid=209780122068490664721.0004c868c55cfd59a9cc7& msa=0&ll=41.3397,15.430298&spn=2.272386,5.410767) we observed correspondence of geographical clustering with the results of cluster analysis. This geographical dependent distribution is clearly depicted in Figure 2. The only exception was represented by two related strains (456 and 49839) which were identified in two farms at about 135 km of road distance. These results indicate that longer distance transmission of Brucella may occur, although uncommon in the studied area, probably favoured by the ability of the microorganism to 6

148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 survive for weeks or months in water, urine, faeces, damp soil, manure and slurry under favourable conditions (cool, dark and damp) (10, 14, 17). Maximun parsimony analysis showed that the major genotypes appeared closely connected with the provinces (Figure 2), with epidemiological connections in the local areas or districts. In 9 cases, strains belonging to the same cluster were recovered from different farms, likely indicating the possibility of a direct or indirect transmission of a strain to neighbouring herds. Since the Italian law prohibits animal trading from infected herds, the presence of closely related strains in restricted areas and close farms suggests the lack of adequate bio-security measures, including all the logistical, structural, management and personnel requirements necessary to avoid the entry and spreading of pathogens into the herd. Indeed most of these farms could allow high probability of direct animal contact between neighbouring herds; this is due to the lack of adequate enclosures, often made of barbed wire, moats or tiny streets. In 10 cases, more than one Brucella isolate (2 to 5) was collected from the same farm during the surveillance period. MLVA analysis indicated that three herds (farms 1, 4 and 9) were characterized by the occurrence of closely related genotypes, belonging to the same cluster and exhibiting differences at a maximum of three loci (Table S1), likely originating from a common strain. Indeed, variations at loci coding for TRs (especially for loci with high DI values) can be generated during the course of bacterial replication in the host, spreading in the environment through abortion, adaptation to external conditions and succeeding infections of different subjects (9, 20). Related MLVA genotypes within the same herd indicate the persistence of a strain, mostly referable to bacterial resistance to environmental conditions and/or presence of carrier animals. These farms therefore appear inadequate in biocompartimentation measures, aiming at controlling spreading of the pathogen within the herd to avoid animals contagion (18). By inquiry to local veterinary services, these farms resulted mostly deficient in cleaning and disinfection procedures, often overcrowded and not provided 7

173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 with quarantine dedicated spaces. Nine herds (farms 1, 4, 5, 10, 12, 13, 15, 18, 36) displayed instead the presence of more than one major genotype, thus suggesting different sources of contamination. Among these, two farms (farms 1 and 4) exhibited the co-existence of several Brucella strains, belonging either to the same cluster or different clusters, with variable numbers of mutated loci (Table S1), likely originating from both different sources of contamination and reiterated infections within the stall. The feedback on these farms indicated that they had several issues to address on biosecurity measures, not only related to inadequate disinfection, enclosure and overcrowding, but also to deficient care for personnel qualification and entrance of risky visitors and means of transport. Interestingly, two B. abortus bv. 3 isolates of cattle and water buffalo origin, respectively, isolated from two herds located at about 50 km road distance in the same area, displayed the same MLVA profile, indicating the possibility of inter-species contagion between different farms. These data indicate a specific distribution of B. abortus biovars in restricted geographical areas of Southern Italy where brucellosis is endemic. This evidence highlights the importance of rigorous herd management procedures avoiding animals exchange/contacts between different farms to prevent intra- and inter-herd pathogen spreading. MLVA analysis proved to be an appropriate method for bacterial characterization and effective epidemiological analyses aiming at supporting specific plans for control and eradication of brucellosis in water buffalo and cattle herds. REFERENCES 1. Al-Dahouk S, Le Flèche PL, Nöckler K, Jacques I, Grayon M, Scholz HC, Tomaso H, Vergnaud G, Neubauer H. 2007. Evaluation of Brucella MLVA typing for human brucellosis. J. Microbiol. Methods 69:137-145. 8

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247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 19. Vizcaíno N, Verger JM, Grayon M, Zygmunt MS, Cloeckaert A. 1997. DNA polymorphism at the omp-31 locus of Brucella spp.: evidence for a large deletion in Brucella abortus, and other species-specific markers. Microbiology. 143:2913-2921. 20. Whatmore AM, Shankster SJ, Perrett LL, Murphy TJ, Brew SD, Thirlwall RE, Cutler SJ, MacMillan AP. 2006. Identification and characterization of variablenumber tandem-repeat markers for typing of Brucella spp. J. Clin. Microbiol. 44:1982-1993. Figure legends FIG. 1. UPGMA clustering analysis of 63 Brucella spp. isolates corresponding to 52 genotypes. MLVA-16 profiles for each strain are shown. In the columns, the following data are presented: DNA batch (key), genotype, province (CE=Caserta; SA=Salerno), species (WB=water buffalo; BOV=cattle), farm, village, Brucella species and biovar. The colour code highlights identical values in each column. FIG. 2. Maximum parsimony analysis of 61 Brucella abortus isolates. Each coloured circle corresponds to one farm from the studied area. Green and red colour correspond to Caserta and Salerno provinces, respectively. Numbers in black indicate MLVA-16 genotypes. Circles that are highlighted in purple indicate Brucella strains isolated in water buffalo. 11