|Home | About | Journals | Submit | Contact Us | Français|
Sixty-three Brucella isolates from water buffaloes and cattle slaughtered within the Italian national plan for brucellosis control were characterized by multiple-locus variable-number tandem repeat analysis (MLVA). Genotyping indicated a strong influence of geographic origin on the Brucella abortus biovar distribution in areas where brucellosis is endemic and highlighted the importance of rigorous management procedures aimed at avoiding inter- and intraherd spreading of pathogens.
Brucellosis is the most widespread zoonosis worldwide and is of major public health and economic significance (1). The disease is caused by Brucella spp., which can infect several important livestock species, including cattle, water buffaloes, goats, sheep, and pigs (1, 2). 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 humans, 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 that affects various organs and tissues (3). The genus Brucella includes 9 species (2) characterized by more than 90% DNA/DNA homology (4, 5). In the last few years, the characterization of the variable number of tandem repeats (VNTR) by multiple-locus VNTR analysis (MLVA) was effectively used for typing of Brucella spp. in humans and animals, including bovine and ovine species, wild boars, hares, water buffaloes, and marine mammals (2, 4, 6, 7). Such typing of Brucella can be useful for epidemiological studies and may advance control of human and animal brucellosis.
The two species most commonly involved in human infections are B. abortus, which is epizootic in cattle, and B. melitensis, which is more virulent and more diffused in sheep and goats (3). 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 (1). The available strategies to control brucellosis are based on very strict management procedures, slaughter of all seropositive animals, and, where allowed, vaccination (8).
The aim of this study was to evaluate the distribution of Brucella biovars in both cow 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 allow better management of the surveillance program (9).
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 program, based on a test-and-slaughter approach, animals were not vaccinated and all subjects positive by serological tests (Rose Bengal and complement fixation tests) were culled and processed for microbiological isolation of Brucella spp. (10). The animals included in this study were collected from 17 cow 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 2,639 km2 and includes 1,891 cow herds (44,550 animals) and 929 water buffalo herds (176,308 animals), while the province of Salerno covers an area of 4,918 km2 and contains 3,947 cow herds (61,596 animals) and 447 water buffalo herds (86,784 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, United Kingdom) for 3 to 5 days at 37°C under 5% CO2. Bacterial DNA was extracted from fresh cultures using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. All isolates were first identified as Brucella spp. on the basis of positivity for agglutination to specific antisera and biochemical tests performed with the Vitek 2 instrument (bioMérieux, Craponne, France). Furthermore, molecular typing was performed by the Italian Reference Centre for Brucellosis (Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise, Teramo) with the AMOS (Abortus-Melitensis-Ovis-Suis) PCR assay (11) for species identification and PCR-restriction fragment length polymorphism (RFLP) analysis of the genes omp2a, omp2b, and omp31 for biovar identification (5, 12). These genes code for Brucella major outer membrane proteins (OMPs) strongly associated with peptidoglycan but with low or no immunogenicity or protective activity against B. abortus and B. melitensis in host infection (13). Final biovar identification was performed by growth in the presence of thionine and basic fuchsin using the slide agglutination test with Brucella A- and M-monospecific antisera (Veterinary Laboratories Agency, Weybridge, United Kingdom) (2). The same DNA samples were analyzed by the MLVA-16 typing technique, as described elsewhere (14, 15), with some modifications. The 16 primer pairs were divided into two groups: panel 1 (loci Bruce06, Bruce08, Bruce11, Bruce12, Bruce42, Bruce43, Bruce45, and Bruce55) was more conserved and was characterized by moderately variable minisatellites, and panel 2 (loci Bruce04, Bruce07, Bruce09, Bruce16, Bruce18, Bruce19, Bruce21, and Bruce30) constituted highly discriminatory microsatellites (14, 15). These markers were chosen because their stability was already assessed (15), and they are widely employed for characterization of Brucella spp. (4, 6, 7, 14, 16). 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 of the forward primers were labeled with either the fluorophore 6-carboxyfluorescein (FAM) (Bruce06, Bruce42, Bruce18, and Bruce07), VIC (Bruce08, Bruce43, Bruce19, and Bruce09), NED (Bruce11, Bruce45, Bruce21, and Bruce16), or PET (Bruce12, Bruce55, Bruce04, and Bruce30). (VIC, NED, and PET are fluorescent dyes with chemical structures currently not publicly available and are proprietary to Life Technologies, Hawthorne, NY.) PCR products were mixed together in a 1:1:1:1 ratio to obtain four different mixtures, each containing 4 amplicons labeled with 4 different fluorophores. The mixtures were then denatured for 5 min at 95°C in the 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 QIAXcel instrument (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 data set using the BioNumerics software. Clustering analyses used the categorical coefficient and the unweighted-pair group method using average linkages (UPGMA) algorithm. The 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% versus 7% in cattle), while B. abortus bv. 3 was significantly associated with cattle (59% versus 41% in water buffalo) (two-tailed P value of <0.0001 by Fisher's exact test). The two isolates of B. melitensis bv. 3 were isolated from cattle and a water buffalo, respectively. These results are in agreement with previous reports on B. abortus biovars in cattle and water buffaloes in Italy (2). The B. abortus biotypes exhibited a sectorial geographic distribution, as the biovar 3 isolate was found only in the province of Salerno (SA), while the biovar 1 isolate mostly (37/39) was found in the province of Caserta (CE).
The MLVA typing assay of the Brucella isolates indicated the presence of 52 different genotypes (Fig. 1), thus discriminating 30 strains out of 39 isolates within B. abortus bv. 1, and 20 strains out of 22 isolates within the B. abortus bv. 3. The most polymorphic loci were Bruce04, with 5 allelic types, and Bruce07, Bruce09, and Bruce30, exhibiting 7 allelic types (diversity index [DI] values of 4.0, 4.1, 3.1, and 3.5, respectively). The less polymorphic loci were Bruce06 and Bruce42, with two allelic types, and Bruce45, with one allelic type (DI values of 1.2, 1.2, and 1.0, respectively). Clustering analysis using UPGMA grouped the Brucella isolates into clusters with 90% similarity (Fig. 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 a 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 the defining of 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, and 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 (http://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 geographically dependent distribution is clearly depicted in Fig. 2. The only exception was represented by two related strains (456 and 49839) that were identified on two farms separated by a road distance of about 135 km. These results indicate that longer-distance transmission of Brucella may occur, although it is uncommon in the studied area, probably favored by the ability of the microorganism to survive for weeks or months in water, urine, feces, damp soil, manure, and slurry under favorable conditions (cool, dark, and damp) (9, 17, 18).
Maximum parsimony analysis showed that the major genotypes appeared closely connected with the provinces (Fig. 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 neighboring herds. Since 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 biosecurity measures, including all of the logistical, structural, management, and personnel requirements necessary to avoid the entry and spread of pathogens into the herd. Indeed, most of these farms could allow a high probability of direct animal contact between neighboring herds; this is due to the lack of adequate enclosures, which are often made of barbed wire, and the presence of 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 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 (see Table S1 in the supplemental material), likely originating from a common strain. Indeed, variations at loci coding for tandem repeats (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 the succeeding infections of different subjects (6, 19). Related MLVA genotypes within the same herd indicate the persistence of a strain, mostly referable to bacterial resistance to environmental conditions and/or the presence of carrier animals. These farms therefore appear inadequate in biocompartmentalization measures aiming at controlling spreading of the pathogen within the herd to avoid animal contagion (20). By inquiry of local veterinary services, these farms were mostly deficient in cleaning and disinfection procedures, often overcrowded, and not provided with spaces dedicated for quarantine. Nine herds (farms 1, 4, 5, 10, 12, 13, 15, 18, and 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 coexistence of several Brucella strains, belonging either to the same cluster or different clusters, with variable numbers of mutated loci (see 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 regarding personnel qualification, entrance of risky visitors, and means of transport.
Interestingly, two B. abortus bv. 3 isolates of cow and water buffalo origins, respectively, obtained from two herds located at a road distance of about 50 km in the same area displayed the same MLVA profile, indicating the possibility of interspecies 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 that avoid animal exchange or contacts between different farms to prevent intra- and interherd pathogen spreading.
MLVA analysis proved to be an appropriate method for bacterial characterization and effective epidemiological analyses aimed at supporting specific plans for control and eradication of brucellosis in water buffalo and cow herds.
Published ahead of print 26 November 2012
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02887-12.