Introduction

History of re-emergence of glanders in Brazil

Glanders is an infectious zoonotic disease that mainly affects Equidae. From a global perspective, it is still found in some countries in Asia, Africa, the Middle East, and South America [1]. Between 2020 and 2022, cases were reported from China, Iran, Nepal, and Turkey [1]. This disease is caused by a bacterium called Burkholderia mallei and is clinically characterized by fever, lymphangitis, skin nodules, cough, and a purulent runny nose [2].

In Brazil, this disease was introduced in 1811 through the importation of infected horses from Europe [3]. After its eradication from Brazil in 1968, new cases of the disease were described in Equidae in 1999, in the states of Pernambuco and Alagoas, in northeastern Brazil.

The historical background of the re-emergence of glanders in the country was obtained from clinical, epidemiological, serological, and microbiological records that confirmed the circulation of B. mallei in sugarcane mills in the states of Pernambuco and Alagoas. The field and laboratory studies were conducted under the leadership and coordination of researcher Dr. Rinaldo Mota, professor in the Department of Veterinary Medicine at the Federal Rural University of Pernambuco (UFRPE). Professor Marilene de Farias Brito from the Veterinary Institute of the Federal Rural University of Rio de Janeiro (UFRRJ) also participated in the study.

The animals in the studied foci presented a clinical picture of bronchopneumonia, purulent nasal secretions, nodulations, and ulcers in the cutaneous lymphatic chain. After collecting the secretions of cutaneous nodules (abscesses), it was possible to isolate a Gram-negative bacterium that was later confirmed to be B. mallei by biochemical tests. Strauss’s test was also performed on guinea pigs, in addition to anatomic-histopathological and serological tests for antibody research. Lesions compatible with glanders and the presence of anti-B. mallei antibodies were confirmed. After achieving bacterial isolation and confirming the diagnosis, the disease was reported to the Ministry of Agriculture, Livestock and Food Supply (MAPA) of Brazil, and they, in turn, sent a technical team to evaluate the impact of the disease in these states.

This study was conducted in sugarcane mills in the Mata area of the states of Pernambuco and Alagoas, which had cases of a respiratory and lymphatic disease in mules that worked daily for traction of products from the sugarcane industry. The main complaint of the owners of the animals was discharge of nasal phlegm accompanied by ulcerated lesions on the skin, in addition to weight loss and death. Treatment with antimicrobials did not affect the clinical remission.

The team performed a thorough clinical examination of the animals and observed nodular lesions in the nasal mucosa; they harvested purulent material from skin abscesses by aspiration. This material was sent to the Laboratory of Bacterioses of Domestic Animals of the UFRPE for microbiological isolation. First, a blood agar culture medium (non-selective) was used to isolate Gram-positive and Gram-negative bacteria. After 48 h of incubation in aerobiosis at 37 °C, non-hemolytic whitish colonies similar to dew drops were observed. Gram-negative bacteria were detected using Gram’s technique. After performing a battery of biochemical tests, the causative bacterium was suspected to be of the genus Burkholderia spp., hitherto unknown to Dr. Rinaldo Mota of UFRPE.

Insisting on the possibility of it being a new Equidae disease, the researcher searched for information and conducted a detailed survey on bacterial diseases of Equidae across the world, even for those that were already considered to be eradicated. In this bibliographic study, the researcher came across information about glanders, one of the oldest diseases of Equidae, which had a similar clinical and epidemiological description to the current disease presentation in the animals. The researcher then decided to perform other laboratory tests such as Strauss’s test on male guinea pigs. The test results revealed lesions and clinical findings compatible with B. mallei infection. Professor Marilene de Farias Brito of UFRRJ also simultaneously identified the bacterium and confirmed that the histopathological findings were compatible with those of glanders. Soon after, B. mallei infection was also confirmed by serological tests, which, together with previous findings, confirmed the re-emergence of glanders in Brazil.

The first article published in Brazil reporting the clinical, epidemiological, pathological, and diagnostic aspects of glanders in 2000 was a part of this ongoing study [4]. The authors concluded that urgent sanitary measures were needed to prevent the spread of the bacterium to Equidae from other states of the Federation. Research on the different aspects of the disease began that year because, at that time, we already had a scientific lapse of 30 years in the national literature on glanders disease; that is, we did not have robust information to support the control and eradication of the disease. Since the notification of the disease, the country has imposed international restrictions on the Equidae trade as well as on the participation of animals in various events. Altogether, we aim to present and discuss the advances in knowledge regarding glanders in Equidae in Brazil.

Materials and methods

The proposed methodology is an integrative review of glanders disease in Brazil between 2000 and 2022. For this purpose, the SCOPUS and PUBMED databases were used to search for scientific articles, and the Brazilian Digital Library of Thesis and Dissertations (BDTD) was used to search for dissertations and thesis. The combined terms used to search the databases are listed in Table 1. The survey was conducted on March 10, 2022.

Table 1 Search strategy performed in the SCOPUS, PUBMED, BDTD databases on glanders between 2000 and 2022
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The eligibility criterion adopted for the analysis was that the articles, dissertations, and thesis were related to the glanders theme, regardless of the type of study. For research on scientific productions (articles, thesis, and dissertations) of the group of the Federal Rural University of Pernambuco, the aforementioned bases and curriculum lattes of the main researcher (Rinaldo Aparecido Mota) were used. This last tool was included only for the main researcher’s lattes to retrieve important articles, dissertations, and theses on the subject in question that were not indexed in the SCOPUS, PUBMED, and BDTD databases, as they have a historical value. The exclusion criteria were non-participation of Brazilian researchers in the research and records with duplicates in the databases (SCOPUS and PUBMED).

For a better understanding and analysis of the evolution of knowledge regarding glanders in Brazil, a two-part analysis of the data was performed. First, we analyzed the results of the research conducted by the Laboratory of Infectious Diseases of Domestic Animals of UFRPE (they were the pioneers in studying this disease in Brazil). Second, we analyzed the results of other research groups.

Results

The results of database search are shown in Table 2.

Table 2 Results of the search of SCOPUS, PUBMED, BDTD, and UFRPE* databases on glanders disease
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After the preliminary reading of the titles and abstracts of the articles, dissertations, and theses retrieved from the search databases, six articles from the SCOPUS database and nine from the PUBMED database were excluded for data duplication. A total of 41 documents were obtained, including 12 dissertations, five theses, and 24 scientific articles. The UFRPE group contributed 25 documents, four theses, three dissertations, and 18 scientific articles (Tables 3 and 4). During this period, nine dissertations, one thesis, and six scientific articles published by other groups in other states of the Federation were also identified (Table 5).

Table 3 Main advances in the study of glanders from the presentation of theses and dissertations by the UFRPE group
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Table 4 Main advances in the study of glanders based on the scientific articles published by the UFRPE group
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Table 5 Results of research conducted in the SCOPUS, PUBMED, BDTD databases between 2000 and 2022
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aThis thesis was retrieved from personal communication, since there are no records in the databases.

The first publication on the re-emergence of glanders in Brazil, in addition to the first dissertation and thesis, was published by the research group of the Laboratory of Infectious Diseases of Domestic Animals of UFRPE, a pioneer in the study of this disease in Brazil. The timeline of the records of the study is shown in Fig. 1.

Fig. 1
figure 1

Timeline of the records of the studies on the glanders carried out by the research group of the Laboratory of Infectious Diseases of Domestic Animals of UFRPE, a pioneer in the study of glanders in Brazil

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Discussion

In the 22 years since the evidence of the re-emergence of glanders in Brazil, preceded by approximately 30 years of probable eradication of the disease, several scientific studies and training of human resources regarding this disease were conducted. It is important to highlight that after the MAPA was notified about this disease, specific legislation on the disease was published and included in the National Program of Equidae Health, which contains guidelines to prevent, control, or eradicate diseases of Equidae. This normative instruction was updated and published by MAPA on January 16, 2018 [44].

To facilitate understanding, we hereby present a discussion on topics according to the historical evolution of the studies conducted by the UFRPE group led by Dr. Rinaldo Mota and other research groups in the country. Furthermore, we present the clinical, epidemiological, and pathological advances (experimental models). In addition, we also present the microbiological, molecular, and serological diagnosis, as well as future reflections for the advancement in the study of glanders disease in Brazil.

Advances in clinical, epidemiological, and pathological studies

Research on the clinical and epidemiological indicators of glanders was not initiated in Brazil. When the disease re-emerged, previous reports had already demonstrated the signs and some epidemiological indicators of glanders in Equidae. However, with the performance of new research, we have made significant advances in the study of the disease after its re-emergence [4].

Initially, the first thesis of the UFRPE research group was presented in 2003 in the Graduate Program in Veterinary Medicine of UFRPE. The central objective was to describe the frequencies of the clinical findings of glanders in mules working in sugarcane mills in the state of Pernambuco, Brazil. This study contributed in describing the clinical indicators at different stages of the disease. It also contributed to the differential diagnosis of respiratory and lymphatic diseases in Equidae in Brazil. In this study, lesions and clinical signs were restricted to the skin, respiratory, and lymphatic systems. From its re-emergence, glanders was considered as a prominent disease in the country. Furthermore, on conclusive diagnosis, the affected animals were euthanized [7].

Veterinarians working in the field and in the Animal Health Defense service also benefited greatly from this research. Until now, they did not have enough information about the clinical presentation of glanders disease in order to clinically diagnose suspected cases. The studies also allowed us to make photographic records (Fig. 2) of the main clinical findings that were extensively used by the authors for disseminating disease information as teaching material for undergraduate and graduate students in Brazilian universities and other countries. Moreover, it was also used in health defense training in the states of the Federation to identify the disease [4].

Fig. 2
figure 2

Clinical, epidemiological, and pathological findings of glanders’ cases in Equidae in Brazil. Glanders: nodules in lymphatic vessels and skin ulcers (a); respiratory clinical presentation of glanders in mules: unilateral purulent nasal secretion in mules (b); cachexia and purulent nasal secretion in mules (c); nodules and thickening of the lymphatic vessels of the neck in donkeys (d); risk factors for glanders: confinement and feeding in collective troughs (e); pulmonary granuloma in mule necropsy (f); liver nodules were distributed in the liver of mules (g); and proliferative inflammatory reaction in the nasal septum of mules (h)

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The UFRPE team also performed an epidemiological study of the disease, where a low prevalence and high lethality for Equidae (nearly 100%) was observed in positive transit animals. Additionally, they noticed differences in clinical manifestation, the intensity of symptoms, and forms of presentation between each of the studied species (horses, mules, and donkeys), with mules and donkeys having greater susceptibility. Some aspects of the chain of transmission of bacteria in herds were also defined. The disease was characterized as more frequent in animals that were elderly, stabled, or submitted to intensive and/or semi-extensive management, who share food and water in collective troughs and are subjected to inadequate hygienic-sanitary management [4].

In 2003, another thesis in the area of pathology was presented in the Graduate Program in Veterinary Medicine at UFRPE, which dealt with the intraperitoneal inoculation of different field isolates of B. mallei in guinea pigs (Strauss’s test). In this study, the clinicopathological response pattern was defined for different field isolates of B. mallei, in addition to a detailed study of the hematological response of inoculated animals. The pattern of macro- and microscopic lesions revealed differences in the intensity of the response of the animals according to the different bacterial isolates. Moreover, an experimental infection model was established in guinea pigs that contributed to other studies related to the diagnosis of the disease [6].

In 2019, we published a study on space–time analysis of the disease in the country with data from the registration of glanders outbreaks obtained from the World Organization of Animal Health [37]. More recently, in 2022, a dissertation presented in the Graduate Program in Animal Bioscience of UFRPE specifically focused on the study of management factors associated with the occurrence of glanders in Brazil. Variables associated with glanders were identified: the objective of use of the animal (for example: trade, sport, or farm work), lack of information on the forms of transmission of the bacterium, non-periodic examinations for the diagnosis of glanders, requirement of negative test results on the purchase of horses, and use of the extensive rearing system at the lowest frequency of cleaning of the bays. The results should assist in the implementation of management measures that minimize the risk of transmission of the bacterium and reduce cases of the disease in the region [41].

Advances in microbiological, molecular, and serological diagnosis

The beginning of microbiological research involving B. mallei preceded the diagnosis of glanders re-emergence in 2000, when the researcher responsible for proving the existence of the disease in the country isolated the bacterium from closed cutaneous nodules of mules with clinical signs of the disease and performed phenotypic characterization of the bacterium obtained in different culture media. Since then, imperative studies have been conducted that have significantly advanced the microbiological diagnosis through isolation of bacterium and its phenotypic and molecular characterization [4].

The first dissertation on the microbiology of glanders was presented in 2003 at the Graduate Program in Veterinary Medicine at UFRPE. In this study, the biochemical characteristics of B. mallei, different media of bacterial cultivation, phenotypic tests of antimicrobial sensitivity, and molecular characterization of B. mallei isolates recovered from Equidae from the states of Pernambuco and Alagoas were demonstrated. Molecular differences were demonstrated in the circulating strains from northeastern Brazil. The conclusion was that there are no effective treatment protocols for glanders, but the study of antimicrobial resistance of B. mallei isolates can help in the treatment of this disease in humans [5].

The first published study on the microbiological diagnosis of glanders was in 2004. It allowed us to conclude that other bacteria isolated from lesions of the upper airways of Equidae influence the isolation of B. mallei and consequently the microbiological diagnosis of glanders. This study recommended that instead of samples of nasal swabs, samples of closed skin nodules should be collected for microbiological diagnosis purposes, which result in much higher sensitivity and specificity. This study was a hallmark in microbiological diagnosis and decisively confirmed the re-emergence of the disease in the country. The researchers who studied the disease previously did not reach a conclusive diagnosis; this was because they always collected biological material for bacterial isolation of the nasal cavity of sick animals which did not obtain satisfactory microbiological results [8].

In 2009, we participated in a research study where a rapid oligonucleotide microarray assay based on genetic markers was conducted for the precise identification and differentiation of B. mallei and B. pseudomallei, namely, glanders and melioidosis agents, respectively. This study was conducted in partnership with the Institute of Bacterial Infections and Zoonosis of the Federal Institute of Animal Health Research, Germany and allowed the advancement in the differentiation of these species that cause similar clinicopathological diseases in Equidae [14].

In 2019, a study was published on the first record of the Turkey 10 lineage of B. mallei in horses in northeastern Brazil. This study confirmed the circulation of a single line of B. mallei in the northeastern region of the country, and this result will contribute to future improvements in diagnostic techniques [34]. Abreu et al. [38] performed isolation of B. mallei in four animals, in addition to standardizing the PCR to improve the molecular detection of the bacteria in clinical samples from positive animals monitored for glanders. In 2021, another thesis was presented in the Graduate Program in Animal Bioscience at UFRPE, which aimed to genetically characterize B. mallei isolates obtained from glanders affected Equidae in the states of Pernambuco and Alagoas. This research was partnered with the National Food Security Agency of France. Sequencing revealed a correlation between the circulating strains from the two states, in addition to a genetic variation between strains of Pernambuco. This suggested multiple events of the introduction of the bacterium in Brazil. This is the first description of an isolate with genetic similarity with other strains reported in Europe, which suggested more than one event of the introduction of the disease in the country [43].

The identification of the same strain of circulating B. mallei is important for the development and improvement of serological diagnostic techniques and development of specific molecular markers for the detection of Brazilian strains. In addition, the identification of the same strain also contributed to for the development and improvement of serological diagnostic techniques and development of specific molecular markers for the detection of Brazilian strains, in addition to the production of vaccines, since treatment is not recommended by the legislation that is in force in Brazil and the World Organisation for Animal Health (OIE). The results of this research are of great impact to the scientific community because they nullify any hypothesis about the possibility that the causative bacteria of glanders are not present in the country. Some previous discussions in this regard have been put forward over the years by a small group of veterinarians and owners of horses but without a scientific basis. This is in contrast to the vast amount of scientific evidence that proves the occurrence of the disease in the country [42].

Regarding serological and immunoallergic diagnosis, we highlight the contribution of our group with a thesis presented in the Graduate Program in Animal Bioscience of UFRPE in 2010. It aimed to produce and evaluate a national purified protein derivative (PPD) malein using a local B. mallei isolate as an alternative for the diagnosis and control of glanders in Brazil. This PPD-malein was evaluated in guinea pigs and demonstrated good sensitivity and specificity when applied to horses. The horses were experimentally infected with inactivated B. mallei and compared with the control group [16]. In 2012, a dissertation was presented in the same program that aimed to standardize and evaluate an indirect ELISA for the diagnosis of glanders. This test was the first to use an isolate of B. mallei originating from an equine in Brazil [20]. The standardization and evaluation of indirect ELISA was the starting point for new studies that improved this diagnostic technique. In 2015, it was registered in the MAPA of Brazil and included in the National Equidae Health Program as a screening technique for glanders in place of the complement fixation technique (which was used only for animals in international transit). Notably, ELISA technique presented better sensitivity and specificity than the complement fixation test. The PPD-malein produced was not used in the National Program for the control and eradication of glanders, as it was replaced by western blotting as a confirmatory technique. PPD-malein is currently used in Brazil for foals up to 6 months of age [44].

Advances made by other research groups on glanders in Brazil

Ten dissertations on glanders were presented, the first in the Graduate Program in Animal Health at the University of Brasília to understand the epidemiological situation of glanders and equine infectious anemia in traction Equidae of the Federal District in 2011 [17]. In 2012, another dissertation was undertaken at the Veterinary School of the Federal University of Minas Gerais to develop and evaluate a new method for the production of malein for the diagnosis of glanders [19]. In the same year, the dissertation was presented in the Graduate Program in Veterinary Medicine of UFRPE, by another research group, to evaluate the results of the complement fixation test against two commercially available antigens, using two methods of incubation in equine sera from a property in the state of Pernambuco [20]. In 2014, another dissertation was presented in the Graduate Program in Tropical Animal Science of UFRPE to evaluate polymerase chain reaction (PCR) and indirect ELISA as diagnostic methods for B. mallei (Glanders) [25]. In 2016, another dissertation was presented in the Graduate Program in Veterinary Sciences of the Federal University of Paraná to develop and standardize a test for the diagnosis of glanders by plaque seragglutination [27].

In 2017, a dissertation was presented in the Graduate Program in Veterinary Medicine, UFRPE, to evaluate antigens (Biovetech, Recife Brazil) produced with Brazilian strains of B. mallei for the diagnosis of glanders compared to the c.c.pro antigen (GmbH, Oberdorla, Germany), which employs strains from Yugoslavia, India, and Indonesia [29]. In 2018, a dissertation was presented in the Graduate Program in Veterinary Science of the Federal University of Paraíba to study the epidemiological characterization of glanders in Equidae in the state of Paraíba based on secondary data. In 2018, a thesis was presented in the Graduate Program in Tropical Animal Science that phylogenetically identified the strains circulating in the national territory for animals suspected of glanders [31]. In 2019, the last dissertation on this theme was presented in the Graduate Program in Sciences of the University of São Paulo that contributed to the analysis of the critical points caused by this disease’s legal/sanitary threat within the business chain [35].

Training of human resources related to the glanders disease was impressively performed in the northeastern region of Brazil, even for groups that were not part of the team of the main researcher of UFRPE. Only six dissertations were conducted in other states: one in the federal district, one in Paraná, one in São Paulo, one in Minas Gerais, one in Paraíba, and one in Alagoas. Although these dissertations and theses had relevant findings, most were not published as scientific articles, except for a dissertation by the Federal University of Minas Gerais group, which advanced the scientific knowledge in the field. In a previous study, we produced and evaluated PPD-malein for glanders [21]. Another article published by the same group, but which was not part of a dissertation or thesis, presented the results of the validation of three qPCR for the detection of B. mallei in tissue samples of horses. It is noteworthy that knowledge on the subject should be made available to the scientific community in journals after peer review and subsequent publication. Thus, unpublished results are not discussed in depth in this article [39].

In 2017 and 2018, two other important scientific contributions were published together with a group from France. These studies presented the genomic sequence of the B. mallei 16-2438_BM#8 strain isolated from a mule in Pernambuco, northeastern Brazil. They also presented the first molecular characterization based on multiple locus variable-number tandem repeat analysis and single nucleotide polymorphism analysis of a B. mallei strain isolated from a mule found dead in northeastern Brazil in 2016 [28, 30]. In 2020, the findings of a study conducted by the research group of the Biological Institute, São Paulo, in which systematic monitoring of horses infected with glanders was performed by complement fixation test, bacterial isolation, and PCR, were published. This study concluded that the cold complement fixation test with United States Department of Agriculture (USDA) and c.c.pro antigens in combination with PCR increase the sensitivity of diagnosis and may be useful in the diagnosis of chronic glanders [38].

It is noteworthy that the main scientific articles published outside the UFRPE group had the participation of some foreign researchers, who were the main contributors of these studies in the area of microbial genetics and in the diagnosis of glanders.

Challenges for future studies

In recent decades, there have been important advances in the clinical-epidemiological, microbiological, and molecular diagnosis of glanders in Brazil. Notably, relevant training of human resources led to the increase of capillarity and nucleation of new research groups in the country, which may contribute to research in this area. Despite the advances observed in this area, it is still important to nucleate new research groups and approve specific funding lines for research. This is necessary to improve the diagnosis and production of immunizers, because the country will not be able to move forward given the few financial resources and increased competition in the field of research. This disease has a strong impact on the equine production chain and causes severe economic obstacles to the commercialization of animals and their products. Additionally, there are restrictions on the movement of animals in the country and abroad. Moreover, it is an important zoonosis neglected in the country and needs to be better understood and studied. Further study on this is needed because human health professionals do not yet have sufficient knowledge, and the disease may be underreported.

At present, we have a sound scientific base and highly qualified researchers who support research in the area of vaccines. Nevertheless, there are still no investments that further the advances in research in this area. Health education programs based on studies of factors associated with glanders should also be implemented in the country to contribute to disease control and eradication.

In recent decades, the scientific advances of glanders in Brazil have transformed the scenario of knowledge on the subject, even under deficient financial and human resources for the execution of research projects. Much of the knowledge and training of human resources in the area was generated in the northeast region of Brazil, more specifically by the UFRPE group. It is important to highlight that in Brazil, the disease can present with varied clinical and epidemiological characteristics, with a predominance of the acute form in donkeys and mules in the northeast region. However, in the Southeast region, the disease is usually chronic and affects horses more frequently. Even in the face of all the scientific evidence that proves the existence of glanders in Brazil, there continue to be difficulties that lead to legal issues of glanders cases that compromise scientific research and confirmation of the disease by direct methods. International partnerships with important research centers in Germany and France have also been established and are important for the study of glanders in Brazil.

It is important to continue training veterinarians for the clinical identification of the disease. In addition, it is crucial to sensitize the surveillance and control agencies of the Ministry of Agriculture to conduct a seroepidemiological survey on the disease in the country to obtain results in all states of the Federation. This will help to plan and structure control and eradication endeavors.