Investigating genetic diversity of Brucella abortus and Brucella melitensis in Italy with MLVA-16

Highlights

• We examined genetic diversity and geographic distribution of Brucella melitensis and Brucella abortus in Italy.

• MLVA-16 distinguished 56 genotypes for B. melitensis, most isolates were in the West Mediterranean lineage.

• MLVA-16 distinguished 32 genotypes for B. abortus but 77.8% of isolates had the same MLVA-11 genotype.

• One B. melitensis from human had an identical genotype as a sheep isolate suggesting a possible epidemiological link.

• Isolates with genotypes not typically found in Italy were readily identified.

Abstract

Despite the eradication of brucellosis from most of Europe, the disease remains relatively common in a variety of livestock in southern European countries. It is therefore surprising that with such high prevalence rates, there have been few genetic characterizations of brucellosis outbreaks in this region. We conducted a genetic assessment of 206 isolates of Brucella abortus and B. melitensis from Italy using Variable Number Tandem Repeats (VNTRs). We determined genetic diversity and geographic distribution of these Brucella VNTR genotypes from 160 farms in eight regions of Southern Italy in a fine-scale analysis using 16 VNTR loci in a MLVA-16 methodology. In a broad scale analysis, we then used a reduced dataset of 11 VNTR loci (MLVA-11) to compare genotypes from Italy to a global database. In the 84 isolates of B. melitensis, there were 56 genotypes using MLVA-16; 43 of these genotypes were found only once. At a broad scale, 81 of these isolates were part of an Italian sub-group within the West Mediterranean group. One of the two B. melitensis isolates from a human patient shared the same genotype as a livestock isolate, suggesting a possible epidemiological connection. In 122 B. abortus isolates, there were 34 genotypes by MLVA-16; 16 of these genotypes were found only once. At a broad scale with MLVA-11, one genotype was predominant, comprising 77.8% of the isolates and was distributed throughout Southern Italy. These data on the current lineages of Brucella present in Italy should form the basis for epidemiological studies of Brucella throughout the country, while placing these strains in a global context.

Introduction

Brucella was first described on the island of Malta in the Mediterranean in 1884 as the causative agent of Malta fever (Nicoletti, 1980). Although several developed countries are believed to be free of the disease, brucellosis widely occurs throughout the world, particularly around the Mediterranean (Pappas et al., 2006). Bovine brucellosis is typically caused by Brucella abortus, while caprine and ovine brucellosis are mainly caused by Brucella melitensis, although cross species infections do occur. Both of these Brucella species are highly infectious to humans and common in livestock worldwide (Godfroid et al., 2011), making brucellosis among the most common zoonoses in the world (Pappas, 2010). Because it is often overlooked, brucellosis is considered a neglected disease of poverty in Europe (Hotez and Gurwith, 2011). The recent socioeconomic problems in the region, particularly Southern Europe, may contribute to new outbreaks; brucellosis is considered a re-emergent disease in Europe (Pappas, 2010).

Currently, European Union member states recognized as Officially Brucellosis-free (OBF) for bovine brucellosis are Belgium, Czech Republic, Denmark, Germany, Estonia, Ireland, France, Latvia, Luxembourg, The Netherlands, Austria, Poland, Slovenia, Slovakia, Finland, and Sweden (EU, 2012). OBF countries for brucellosis in sheep and goats are Belgium, Czech Republic, Denmark, Germany, Estonia, Ireland, Latvia, Lithuania, Luxembourg, Hungary, Netherlands, Austria, Poland, Romania, Slovenia, Slovakia, Finland, Sweden, and the United Kingdom (EU, 2011). In contrast, the disease is still prevalent in countries such as Italy, Spain, Portugal, Greece and Turkey (Avdikou et al., 2005, Ferreira et al., 2012, Kiliç et al., 2011, Marianelli et al., 2007, Valdezate et al., 2010).

In Italy, bovine, caprine, and ovine brucellosis are endemic in the southern part of the country, while the northernmost provinces are considered OBF, with limited numbers of northern cases reported annually (EFSA, 2012). Since 1994, an eradication program based on test-and-slaughter has been ongoing throughout Italy for cattle (Italian Ministry of Health, 1994). By 2012, OBF status for bovine brucellosis was granted to 72 of 110 Italian provinces (EU, 2012). Similarly, since 1992, an eradication program based on test-and-slaughter has also been ongoing throughout Italy for sheep and goats (Italian Ministry of Health, 1992). By (2011), OBF status for caprine/ovine brucellosis was granted to 71 of 110 provinces EU, 2011. Disease prevalence (expressed as the number of herds where at least one animal tested positive for brucellosis, divided by the total number of herds) in 2010 was 2.4% for bovine brucellosis and 2.7% for caprine and ovine brucellosis (EFSA, 2012), with some regions such as Sicily reaching a prevalence of 5.6% for bovine brucellosis (Calistri et al., 2012). Most human cases of brucellosis in Italy are caused by B. melitensis infection, and the disease in humans is more widespread in areas where its prevalence in sheep and goats is also higher (Caporale et al., 1992, De Massis et al., 2005a). In human cases, there has been a clear decrease of disease incidence in the whole country, dropping from 318 cases reported in 2006 to 10 cases in 2010 (EFSA, 2012).

Prevention of human brucellosis depends on control of the disease in animals (De Massis et al., 2005b), as well as proper handling and treatment of animal products. Identification of species and biovars of Brucella field strains isolated from outbreaks, paired with a genotyping approach such as MLVA, is essential to fully understand the epidemiology of the disease and to trace sources of infection, thereby improving the outcome of brucellosis eradication programs. Identifying the presence and identity of Brucella strains in livestock populations is also important to be able to determine importation of any new strains (Di Giannatale et al., 2008).

Molecular epidemiology of Brucella spp. can be challenging due to relatively low genetic variation in its genome (Foster et al., 2009). An approach using variable number tandem repeats (VNTR), in a Multi Locus VNTR Analysis (MLVA) framework is now widely used and often allows for fine-scale resolution of closely related isolates (Huynh et al., 2008, Le Flèche et al., 2006, Whatmore et al., 2006). We chose the MLVA scheme of Le Flèche et al. (2006) due to its widely available database of genotypes, particularly samples from Europe. For some analyses we added an additional locus based on modifications of this methodology by Al Dahouk et al. (2007). These analyses combined allowed for both a regional and global context for the genetic characterization of B. abortus and B. melitensis isolates from Italy.

We genetically characterized 204 isolates from bovine brucellosis and caprine/ovine brucellosis in Southern Italy from cases occurring in 2011 as well as two human isolates from the same time period. B. melitensis has previously been divided into three major lineages around the Mediterranean: East Mediterranean, West Mediterranean and America (Al Dahouk et al., 2007). No prior groupings have been made for B. abortus in the Mediterranean due to relatively few studies. Our goal was to assess the genetic structure of B. abortus and B. melitensis in Italy and compare results to genotypes from isolates from the rest of the Mediterranean.