The use of multilocus variable number of tandem repeat analysis (MLVA) in anthrax and brucellosis zoonotic diseases

Henriette Van Heerden, January 2010
Department of Veterinary Tropical Diseases, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa  

Introduction
Anthrax and brucellosis are zoonotic diseases that can be transmitted to humans through contact with infected animals or animal products. These bacterial diseases primarily affect wildlife and domesticated livestock and therefore have an economic importance. The precise identification of bacterial pathogens at a sub-species level is essential for epidemiological purposes like the delineation of outbreaks and universal tracking of virulent or multi-resistant pathogens. Continuous efforts are undertaken to develop easy to use, low cost and accurate methods which can eventually be applied routinely. Molecular methods based on PCR (Polymerase Chain Reaction) to type the occurrence of different PCR fragments / bands (polymorphisms) in the bacterial genomes (Ochman & Davalos, 2006; van Belkum, 2007) are facilitated by the availability of whole genome sequences. Bacterial genomes contain significant sequences on chromosome (loci) of repetitive DNA that may be variable amongst strains with respect to the number of repeat also called Variable Number of Tandem Repeats (VNTR) loci (van Belkum, 2007; van Belkum et al., 1998). As individual VNTR loci only provide a small amount of information, multiple loci are examined concurrently to further increase its discriminatory power: a multiple-locus VNTR analysis (MLVA) system. Various studies showed that VNTR loci are likely to be a highly significant source of very informative markers for the identification of pathogenic bacteria (van Belkum et al., 1997; Frothingham and Meeker-O’Connell, 1998; Adair et al., 2000; Keim et al., 2000; Supply et al., . 2000). MLVA has been used in brucellosis and anthrax for pathogen identification and strain typing. In this short review some background regarding the aetiology, epidemiology and the use of MLVA for these two bacterial zoonotic diseases will be discussed.

Anthrax
The aetiological agent of anthrax is the spore-forming, Gram-positive rod-shaped Bacillus anthracis. Anthrax is a zoonotic condition and can be transmitted to humans through contact with infected animals or animal products. However, it is primarily a disease which affects wildlife and domesticated livestock. Despite the rare occurrence of human cases, anthrax remains a potential threat because of continued outbreaks of animal anthrax and the importation of contaminated skin or hide products (La Force, 1994). Anthrax is not transmitted directly from victim to victim, but instead is picked up by herbivores, such as wild ungulates, then is spread by scavengers once the infected animal dies (Dixon et al. 1999). Transmissions by stable flies, mosquitoes, louse flies and blow flies have also been reported (Howell et al., 1978; Braack and De Vos, 1990, Turnbull, 2000).

There are three routes to infection: cutaneous, gastrointestinal and inhalation. All three can progress to fatal systemic anthrax without treatment (Dixon et al. 1999), although the inhalational form is considered the most dangerous of the three (Turnbull et al., 1988). Symptoms include fever, shock, multiple organ failure, discharge/bleeding from various orifices, lesions which resemble black eschars, cardiac distress, respiratory distress and swelling of the tongue and throat. The clinical history usually describes the animal to be in good health a few hours before death. If the animal is observed shortly before death, fever up to 42°C (107°F), muscle tremors, dyspnoea, and mucosal congestion are the most common signs (Hugh-Jones and de Vos, 2002). Shortly afterwards, the animal will often have terminal convulsions, collapse and then die (Hambleton et al., 1984; OIE, 2008). Following death, unclotted blood may sometimes be seen to exude from the anus, vulva, nostrils, and/or mouth. Incomplete rigor mortis is also common (Turnbull et al., 1988). Fortunately, B. anthracis is penicillin sensitive and can also be treated with tetracycline, chloramphenicol and streptomycin quite effectively (Odendaal et al., 1991).

Diagnostic identification of anthrax is usually based on the visualisation of the encapsulated bacilli in a blood smear stained with polychrome methylene blue (M'Fadyean reaction) (OIE, 2008). Bacillus anthracis is readily isolated from blood or tissues of animals that died recently of anthrax. Antibody detection in serum from infected animals is rarely used for diagnostic purposes and is essentially a research tool (OIE, 2008). Anthrax disease occurs in many parts of the world, including Asia, central and south America, southern Europe, sub-Saharan Africa and small pockets of Australia, northwest Canada and Haiti. Sporadic cases may occur in other countries, usually traceable to imports of contaminated animal material.

Bacillus anthracis appears to be one of the most monomorphic species known: most techniques available could not distinguish isolates from various sources or geographical locations phenotypically and genotypically. Strain typing using MLVA (Keim et al., 1999; 2000, 2004; Lista et al., 2006; van Ert et al., 2007) made it possible to distinguish outbreaks caused by different strains from those caused by the spread of a single strain, to trace an outbreak strain back to its possible origin (Keim et al., 2001; 2004),) and in future to track the routes of transmission of an outbreak strain within and between animal populations. Initially 8-MLVA markers (Keim et al., 2000) were identified using automated fluorescent DNA sequencer (automated capillary electrophoresis) to determine the size of the PCR fragments. Cluster analysis with the 8 MLVA assay identified two most dissimilar cluster groups (branch A and B) (Keim et al., 2000). Isolates in branch A are found internationally and is therefore responsible for the most epidemics and outbreaks. Branch B strains on the other hand are almost exclusively restricted to Southern Africa. However the resolution achieved with the 8-MLVA was found to be limiting since it did not have enough resolving power to discriminate between two geographically close isolates in natural outbreaks (Keim et al., 2004; Lista et al., 2006). Additional markers were developed and the assay consisted of 15MLVA markers that divided the B. anthracis isolates in 3 major clades (branch A, B and C). This European research group extended the 8-MLVA assay and improved the resolution power of the assay by adding 14 additional markers (Le Fleche et al., 2001). The PCR fragment sizes were determined using standard agarose gel, making this technique more accessible by using basic equipment. Later the resolution power was even further extended to 25 marker loci that enabled the description of two new clades (branch D and E) (Lista et al., 2006).

Brucellosis
Nine species are currently recognized, namely B. abortus (8 biovars), B. melitensis (3 biovars), B. suis (5 biovars), B. ovis, B. canis, B. neotomae, B. pinnipedialis, B. ceti and B. microti (Corbel and Brinckley-Morgan, 1984; Foster et al., 2007; Scholz et al., 2008). Brucella spp are Gram-negative, facultative intracellular coccobacilli that may cause brucellosis in humans and livestock. Classification of Brucella is mainly based on pathogenicity, host preferences, and conventional microbiological tests used for phenotyping (biotyping). General routine identification of Brucella species and biovars relies on phenotyping (Alton et al., 1988). Brucella abortus, B. melitensis, B. suis, and B. neotomae generally occur in the smooth form, while B. canis and B. ovis are rough species. Smooth Brucella species have been described from marine animals (Dawson et al., 2008).

The main economic impact of Brucellosis infection in animals is reproductive failure, whereas in humans it is undulant fever and a debilitating chronic disease. Brucellosis is a important bacterial zoonoses where this bacteria remains the major source of infection through consumption of contaminated foods or occupational exposure. Human brucellosis is caused mostly by B. abortus, B. melitensis, and B. suis and can be acquired by ingestion, inhalation, or contamination of traumatized skin or conjunctiva by infected animal products (Corbel, 1997). Brucellosis in humans is a systemic disease that can manifest with an acute or an insidious onset (Pappas et al., 2005). Treatment in humans is through the course of antibiotics. The disease causing agent is directly and indirectly transmitted from animals to humans. Brucellosis is also transmitted from domestic animals to wildlife. The interface between human, domestic animals and wildlife needs to be addressed especially around game parks and communities surrounding the game parks where contact occur between humans, wildlife and domestic animals.

Some countries have been successful in eradication of the disease, while others, especially in the developing world still live with a huge disease burden. In order to control brucellosis, comprehensive surveillance, pre and post import testing is of paramount importance (Corbel, 1997). Knowledge of the spread and prevalence of the infection is essential when planning control measures. Identification of Brucella is therefore important. Currently, serological screening of potential hosts and isolation and identification of the pathogen from potential hosts are used for the diagnosis of brucellosis. Isolation and identification of Brucella spp. is recognized as the ‘gold standard’ (Alton et al., 1988; OIE, 2008). However, it takes 10-14 days to culture the bacteria and complete 25 phenotypic traits used for species and biovar identification of cultured organisms (Bricker, 2004). The absence of bacterial isolation does not rule out brucellosis since some positive animals yield negative culture results. Therefore serological tests can be used to define the brucellosis status of animals (Jacobson, 1998; Gall and Nielsen, 2004). The OIE (2008) indicates a combination of serological tests suitable for screening livestock for brucellosis. Serological tests include serum agglutination methods (SAT), rapid slide agglutination test (RSAT) (with or without 2-mercaptoethanol), tube agglutination test (TAT) (with or without 2-mercaptoethanol), complement fixation tests (CFT), buffered antigen plate agglutination test (BPAT), fluorescence immunosorbent assays (FPA) and enzyme linked immunoassays (ELISA). The control and diagnosis of brucellosis in small ruminants and cattle requires the use of more than one serological test of which RBT, BPAT, FPA and ELISA are suitable screening test (OIE, 2008). The serological tests are genus and not species specific.

PCR assay is a sensitive, specific, rapid and relatively inexpensive method for detecting Brucella antigen. Various regions have been identified in the Brucella genome and used in PCR assays using blood, milk (Leal-Klevezas et al., 1995) and tissue (O’Leary et al., 2006). Most PCR assays (Baily et al., 1992; Leal-Klevezas, 1995; Romero et al., 1995; Rijpens et al., 1996; Fox et al 1998; Centinkaya et al., 1999; Cortez et al., 2001; Gupta, 2006; O’Leary et al., 2006; Ilhan et al., 2008) is not sensitive enough to identify brucellosis in blood samples and therefore serology is used. PCR can therefore not be used as a diagnostic tool but can be used to identify Brucella cultures and to differentiation Brucella strains at the species level. The AMOS PCR distinguish B. abortus (biovars 1, 2, and 4), B. melitensis (biovars 1, 2, and 3), B. ovis, and B. suis (biovar 1) (AMOS abbreviation based on the initials of the species that it identifies) as well as vaccine strains and wild-type isolates (Bricker and Halling, 1994; 1995) using the unique locations of the repetitive genetic element IS711. Another multiplex PCR Assay (Garcia-Yoldi et al., 2006) identify and differentiate all Brucella species (including the marine mammal isolates) and the vaccine strains B. abortus S19 and RB51 and B. melitensis Rev1. Huber et al. (2009) applied a random amplified polymorphic DNA (RAPD)-PCR in order to develop a multiplex PCR that make use of the AMOS PCR and unique insertions and deletions. This multiplex PCR assay differentiates all Brucella species including B. ceti, B. pinnipedialis and B. microti and accurately differentiates B. abortus biovar 1, 2, 4 from 3, 5, 6, 9 as well as B. suis biovar 1, 3, 4 from 2 and 5.

Bricker et al. (2003) developed an assay based on VNTR composed of octameric sequence (DNA sequence made of 8 nucleotides) AGGGCAGT named "HOOF-Prints" for Hypervariable Octameric Oligonucleotide Finger-Prints. This assay could definitively characterize and differentiate Brucella isolates and discriminate B. abortus biovar 1 field isolates. This assay can therefore be used for epidemiological investigations. The authors indicated that this assay is intended to complement the existing PCR and bacteriological diagnostic tests currently used for Brucella species and biovar identification.

Le Fleche et al. (2006) investigated the use of VNTR loci for molecular subtyping that will enable epidemiologic investigations or tracing back strains to their origin. Fifteen VNTR loci were selected and the 15-MLVA analyses correspond to the classical biotyping scheme of Brucella spp. The 15 markers consist of 7 microsatellite VNTR markers (repeats are up to 8 base pairs in size) that enable species identification and another group of 8 minisatellite VNTR markers (repeats are larger than 8 base pairs in size) with higher discriminatory power (Le Fleche et al., 2006).

Conclusion
The MLVA assay is rapid, highly discriminatory, and reproducible within bacterial, zoonotic isolates. MLVA can significantly contribute to epidemiological trace-back analysis of zoonotic infections and may advance surveillance and control of these important diseases. The advantages of MLVA is its relative simplicity, fast turnaround time, amenability to high-throughput approaches, applicability to nonviable and/or crude preparations, and the ability to easily compare digital results between laboratories.

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