First record of Burkholderia mallei Turkey 10 strain originating from glanderous horses from Brazil

Abstract

Burkholderia (B.) mallei is the causative agent of glanders in Equidae. This study describes the first record of the Turkey 10 strain of B. mallei in glanderous horses in Northeastern of Brazil. This description should contribute to the future actions of diagnosis, control, and eradication of this disease in Brazil.

Glanders is a zoonotic disease caused by Burkholderia (B.) mallei with compulsory notification to the World Organization for Animal Health (OIE). While glanders was eradicated in Europe and North America during the first half of the last century by restrictive measures to sacrifice infected animals, in Asia, the Middle East, and South America, the disease is still endemic. In recent years, outbreaks of the disease have been reported and continue to grow numbers of countries, including Brazil, Pakistan, Iran, the United Arab Emirates, Lebanon, and India [13]. In Brazil, glanders was described in 1811 by Pimentel [15], and in 1960, the disease was officially considered eradicated. In 2000, Mota et al. registered the emergence of new cases of glanders in the states of Pernambuco and Alagoas. Nowadays, the disease is registered in all Brazilian regions, and little progress has been made in the epidemiological, molecular, and control aspects of this important disease.

The molecular methods have a potential for detecting B. mallei in animals with clinical signs of glanders, differing from Burkholderia pseudomallei infections, which determine similar clinical signs, whereas these Burkholderia species have similarity in their genomes greater than 90% [9, 11, 12, 16, 17, 19, 20]. Polymerase chain reaction (PCR) has been used in the clinical setting and in public health laboratories for the identification of B. mallei [8, 14] and when associated with genetic sequencing, it is possible to reduce time and to improve the efficiency of the reagents used [1, 3, 5].

Due to the high similarity between B. mallei and B. pseudomallei in phenotypic characteristics in microbiological culture, biochemical and molecular tests, and the impossibility of serological differentiation [11], it is necessary to correctly identify this bacterium and the strain involved in suspected cases of glanders. The objective of this study was to know the circulating strains of B. mallei isolated from clinical cases of glanders in horses from Brazil.

The entire experimental procedure is in accordance with the ethical principles adopted by the Ethics Committee for the Use of Animals from the Federal Rural University of Pernambuco, license number 004/2018.

In total, six samples were collected from equine positive in official serological tests for glanders according to the Ministry of Agriculture, Livestock and Supply (MAPA) [2]. Five animals belonged to rural properties located in the Metropolitan Region of Recife (MRR), Pernambuco, and one animal came from the Metropolitan Region of Maceió (MRM), Alagoas. After euthanasia, the animals were submitted to necropsy and fragments of the piogranulomatous lesions were collected in the lung and liver, which were stored in sterile polypropylene bottles at room temperature and transported to the laboratory.

The isolation was done from the purulent material obtained from the interior of the piogranulomatous lesions and processed in a biological safety cabin. The purulent material was cultured using blood sheep agar containing 3% (v/v) glycerol at 37 °C for 48 h [16]. The suspected colonies were selected according to the phenotypic description provided by the OIE [13] and submitted to conventional PCR for confirmation of B. mallei.

DNA extraction was performed in suspected colonies after culturing using the genomic DNA purification Promega® kit for DNA extraction from Gram-negative bacteria following the manufacturer’s recommendation. Conventional PCR was performed on SimpliAmp Applied Biosystems® thermal cycler looking for differences in the fliP sequences of B. mallei strain ATCC 23344^T and B. pseudomallei strain K96243. The oligonucleotides used were Bma-IS407-fliP-f (5′-TCA-GGT-TTG-TAT-GTC-GCT-CGG-3′) and Bma-IS407-fliP-r (5′CTA-GGT-GAA-GCT-CTG-CGC-GAG-3′) in a final reaction volume of 12.5 μL, using Go-Taq green mastermix Promega® and primers at 10 pmol concentration. The thermal profile conditions were an initial denaturation of 94 °C for 3 min, 34 cycles of denaturation at 94 °C for 30 s, annealing at 67 °C for 45 s and extension at 72 °C for 1 min, ending with an extension final at 72 °C for 7 min and as positive control, using standard strain B. mallei ATCC 15310. From the PCR reaction product, 9 μL was subjected to agarose gel electrophoresis (1.5% w/v in TAE buffer) to verify the presence of the 989-bp band [17].

PCR amplified products were purified using the Quiacki purification® kit following the manufacturer’s recommendations and forwarded to the Central Laboratory (LABCEN) of the Center of Biological Sciences (CCB) from Federal University of Pernambuco (UFPE).

It was then performed a bidirectionally sequenced by standard protocols using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems®) following the manufacturer’s recommendations using a 96-well plate and cycled in a Veriti thermocycler (Applied Biosystems®) for 40 cycles of 15 s at 96 °C, 15 s at 56 °C, and 4 min at 60 °C in an automated ABI-PRISM 3130 sequencer (Applied Biosystems®). Subsequently, the sequences acquired were analyzed with aid of software BioEdit® [7] and MEGA 5 to compare with the database found in the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST).

All six samples were submitted to GenBank which provided the following accession numbers: MK947136, MK947137, MK947138, MK947139, MK947140, and MK947141. The sequencing analysis results obtaining all six samples sequences were the same; the Turkey 10 strain of B. mallei then confirms the etiology of glanders (Fig. 1).

Fig. 1.

Specific PCR for B. mallei agarose gel. Line 1, molecular marker; lines 2 and 3, positive controls; and line 4, negative control. Lines 5, 6, 7, 8, 9, and 10 are positive samples

Glanders was described in Brazil in the nineteenth century, and since then, several measures have been suggested and implemented to control this disease [2, 10, 15]. Between 1960 and 1999 in Brazil, glanders was probably underreported and/or new sources of infection may have been introduced with the importation of animals from all over the world [10]. After confirmation of the disease in the country, there were some advances related to bacterial identification, including the use of a strain in the standardization of serological and intradermal diagnostic techniques. Silva et al. [18] studied animals from Pernambuco and Alagoas and observed four groups of genotypes with distinct biochemical characteristics, indicating differences of the circulating strains in the study area. Subsequently, Girault et al. [6] carried out the first draft of the genome of B. mallei strain 16-2438_BM#8 isolated from a mule in Pernambuco and concluded that a comparative genomic analysis of strains from different geographic regions and different hosts is necessary to improve the knowledge about this reemerging disease in the country.

The results obtained in our study indicate the circulation of the same strain of B. mallei in the studied animals. This finding is important from epidemiology aspect of glanders and was probably due to the strong trade in animals between the two states and also the participation of animals from fairs and agricultural events. This strain is also reported in Turkey [4], raising the alert level for the global distribution of this bacterium.

The identification of the same B. mallei strain circulating in different regions is an important finding to develop and to improve serological and molecular diagnostic techniques, with the development of specific molecular markers for strains detection, and also to contribute to vaccine production, since the treatment is not recommended by the OIE.

Funding

This study was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Compliance with ethical standards

The entire experimental procedure is in accordance with the ethical principles adopted by the Ethics Committee for the Use of Animals from the Federal Rural University of Pernambuco, license number 004/2018.