The one hundred year journey of the genus Brucella (Meyer and Shaw 1920)

ABSTRACT

The genus Brucella, described by Meyer and Shaw in 1920, comprises bacterial pathogens of veterinary and public health relevance. For 36 years, the genus came to include three species that caused brucellosis in livestock and humans. In the second half of the 20th century, bacteriologists discovered five new species and several ʻatypicalʼ strains in domestic animals and wildlife. In 1990, the Brucella species were recognized as part of the Class Alphaproteobacteria, clustering with pathogens and endosymbionts of animals and plants such as Bartonella, Agrobacterium and Ochrobactrum; all bacteria that live in close association with eukaryotic cells. Comparisons with Alphaproteobacteria contributed to identify virulence factors and to establish evolutionary relationships. Brucella members have two circular chromosomes, are devoid of plasmids, and display close genetic relatedness. A proposal, asserting that all brucellae belong to a single species with several subspecies debated for over 70 years, was ultimately rejected in 2006 by the subcommittee of taxonomy, based on scientific, practical, and biosafety considerations. Following this, the nomenclature of having multiples Brucella species prevailed and defined according to their molecular characteristics, host preference, and virulence. The 100-year history of the genus corresponds to the chronicle of scientific efforts and the struggle for understanding brucellosis.

Brucella, brucellosis, Brucellaceae, Alphaproteobacteria, Ochrobactrum, taxonomy, phylogeny

INTRODUCTION

The eminent Costa Rican scientist, Clodomiro Picado-Twight (1887–1944), recounted that during 1915–1916, he isolated ʻMicrococcus melitensisʼ (Brucella melitensis), then the only known etiological agent of Mediterranean fever transmitted to humans by goat's milk (Zeledón-Alvarado 1940). The isolate came from the blood of Mother Superior Nun of the San Juan de Dios Hospital of San José, Costa Rica, who functioned as the director of nursing. This event caused mockery by the physicians who argued that goats were not allowed at the nuns' residence in the hospital. Picado-Twight ignored the mocking and focused on the problem. According to his analysis, the microorganism was M. melitensis, along with isolates from other similar cases, occurring around the same time. He thought of Bang's bacillus (Brucella abortus), the etiological agent of the so-called contagious abortion in cows, as a possible cause of human infections. However, when Picado-Twight talked about it, few scientists believed in the zoonotic transmission of microbes through cow's milk (Zeledón-Alvarado 1940).

Clodomiro Picado-Twight was a knowledgeable microbiologist, educated at the Faculty of Sciences of the University of Paris under the mentoring of the eminent evolutionist Maurice Caullery (1868–1958) and trained at the Institute Pasteur in France (Monge-Nájera and Gutierrez 1989). Alphonse Laveran (1845–1922), Iliá Metchnikoff (1845–1916), Alexandre Yersin (1863–1943), Emile Roux (1853–1933), Alexandre Calmette (1863–1933) and several other eminent microbiologists were part of this renowned institute at the time (Annales de l'Institut Pasteur 1910). During his training, Dr. Picado-Twight became familiar with M. melitensis, the etiological agent of a debilitating, long-lasting pyrexal disease known as Malta fever. This bacterium, discovered in infected soldiers in the island of Malta (also known as Melite, from the Greek meli ʻhoneyʼ or the Latin melite meaning ʻrefugeʼ) in 1887 by a team of scientists working under the supervision of Surgeon Captain David Bruce (1855–1931) (Bruce 1887; Edwards and Jawad 2006). Likewise, Picado-Twight had experience with Bang's bacillus, described in 1897 by the Danish veterinarians Bernhard Bang (1848–1932) and Valdemar Stribolt (1868–1907) in aborted bovine fetuses. Unfortunately, Mr. Stribolt, a brilliant bacteriologist who isolated the microorganism, died young, before achieving proper recogniti Bang 1896, 1897).

At the onset of the 20th century, brucellosis remained endemic and prevalent in Europe, mostly in the Mediterranean countries, including France and its colonies (Cantaloube 1911). The disease was also present in the American continent, Asia and Africa; however, often known by different names (Evans 1947). As a trained microbiologist, Picado-Twight knew that M. melitensis could be transmitted from animal to animal and from animals to humans through secretions or ingestion of raw milk. In infected goats and sheep, the bacterium caused abortions, while in humans, induced a long-lasting and debilitating disease, displaying a broad diversity of clinical symptoms and persisting for many years. He learned at the Institute Pasteur, from the 1908 Nobel prize winner Iliá Metchnikoff (Monge-Nájera and Gutierrez 1989), that some pathogenic bacteria could live inside cells. Before the antibiotic era, brucellosis killed a significant proportion of infected people, and Salvarsan, ʻthe magic bullet,ʼ was an ineffective drug for brucellosis treatment (Hughes 1896). Picado-Twight knew this and, above all convinced that M. melitensis infection acquired by Mother Superior Nun had to come from another source, such as cow's milk. Nevertheless, at the start of the 20th century, European and American bacteriologists believed the pathogens causing Malta fever and Bang's disease were unrelated.

A simple morphological mistake was the reason renowned and famous microbiologists overlooked the close relatedness between M. melitensis and Bacillus abortus. At the end of the 19th century and the beginning of the 20th century, Germany prevailed as the canonical center for microbial taxonomy, and botanists acted as bacterial taxonomists since these microorganisms were part of the plant kingdom (Chester 1901). At that time, morphology remained as the primary tool for classification, following the influence of the Prussian evolutionist Ernst Haeckel (1834–1919). Accordingly, Emil Migula (1863–1938), a respected German botanist and taxonomist, clustered all known bacterial species of the time in three leading morphological families: Coccaceae, Bacillaceae, and Spirillaceae (Migula 1897).

The American botanist and geologist Frederick Chester (1861–1943) from Delaware College modified this classification in 1901 (Chester 1901); still, the morphological criterion remained for two more decades as the fundamental principle for categorizing the bacterial families and the corresponding genera (Buchanan 1916). According to this, Surgeon Major Matthew Hughes (1867–1899), from the Mediterranean Commission directed by David Bruce, described the etiological agent of Malta fever as a round shape ʻcoccusʼ (Hughes 1896). This morphology departed from the shape observed by Bernhard Bang and Valdemar Stribolt, who depicted the microbe causing spontaneous abortions in cows as a rod-shaped ʻbacillusʼ (Bang 1897). In the minds of microbiologists, the former bacteria belonged to the Coccaceae, while the latter to the Bacillaceae and placed in two unrelated genera: Micrococcus and Bacillus (Bc.). This confusion lasted for over 20 years until a new generation of bacteriologists arrived.

SHAPING THE GENUS Brucella

In 1913, Alice Evans (1881–1975), a bacteriologist who graduated from the University of Wisconsin-Madison, worked at the Dairy Division of the US Department of Agriculture Bureau of Animal Industry, in Washington DC. She was the only female researcher working in the division; at that time, this was a rare opportunity for a woman (Evans 1963). Despite this, she did not confront a harsh environment by her superiors. The task given to Evans involved studying bacteria excreted in cow's milk as a sign of udder infection. With the help of the Hungarian/American veterinarian pathologist Adolph Eichhorn (1875-1956), she built up the idea of comparing Bc. abortus with M. melitensis, since both organisms displayed pathogenicity and excreted in the milk of asymptomatic cows and goats (Evans 1947); besides, there were precedents. The British Staff-Surgeon Ernest Shaw (1837-1950), from the Mediterranean Fever Commission, discovered antibodies and M. melitensis in the blood and udder of cows in 1906 (Shaw 1906). Likewise, the medical doctors and bacteriologists Karl Meyer (1884-1974) and Emanuel Fleischner (1882-1926) from the University of California, confirmed the presence of Bc. abortus in cow's milk, in 1917 (Fleischner and Meyer 1917). These findings raised the possibility of cow's milk as a source of human infection. However, at that time, the world lived more concerned about the global war conflict than of a disease transmitted through milk.

After a few days of classical bacteriological work, Evans noted that both bacteria, assigned to different genera, were alike (Evans 1918). Contrary to the coccus and bacillus shape designation, these organisms showed similar coccobacillary morphology when grown under the same conditions. They also showed the same staining pattern, identical biochemical profiles, colony shapes, and displayed cross-agglutination with serum from a Bc. abortus infected cow and with human serum from an M. melitensis sick patient. After exploring their pathogenicity, she found that pregnant guinea pigs aborted to the same extent, regardless of the bacterial strain. A higher agglutination titer observed with the cognate bacteria using the human serum demonstrated a mild antigenic difference between these two strains.

In a classic paper published in 1918, Evans concluded that both M. melitensis and Bc. abortus belonged to the same genus (Evans 1918). She knew the implications of her work because it supported the claims of other investigators who also suggested human infections with Bc. abortus (Mohler and Traum 1911). The close relationship between these two organisms explained the contagion of B. abortus to humans and the Malta fever-like symptoms in human patients infected with the bovine strains. In one of the last paragraphs of her paper published in 1918, she expressed: ʻAre we sure that cases of glandular disease, or cases of abortion, or possibly diseases of the respiratory tract may not sometimes occur among human subjects in this country as a result of drinking raw cow's milk?ʼ (Evans 1918).

The work of Evans remained controversial and faced the skepticism of many microbiologists and pathologists who did not believe the results. Still, few evoked scientific arguments; instead, she received irrelevant and harsh mudslingings such as ʻif there were a close relationship between the two supposed genera, other [male] bacteriologists would have notedʼ (Evans 1963). The proposal claiming that the source of human infections, causing something similar to Mediterranean fever, came from bacteria secreted in cow's raw milk, instigated the hostility of the dairy industry associates. Some alleged that Evans collaborated with companies manufacturing pasteurizing equipment (Evans 1963).

The most hostile and sustained criticism, however, came from the bacteriologist Theobald Smith (1859-1934). This censure was not trivial. Smith prevailed as a prominent and prestigious scientist, who discovered several microorganisms and performed seminal works on the pathogenesis of bovine brucellosis (Smith 1919). Despite this fervent opposition, Karl Meyer and his young medical student Edward Shaw (1895-1986) from the Hooper Foundation at the University of California in San Francisco, confirmed Evans's results regarding the close similarity between these two bacteria. Still, they did not accept the proposal made by Evans to cluster these pathogenic organisms within the genus ʻBacteriumʼ, including ʻBacterium bronchisepticusʼ, since the characteristics of both strains departed from the typhoid-dysentery group. Following this, Meyer and Shaw published a seminal paper in 1920, proposing the genus Brucella, honoring David Bruce, and establishing the names Brucella abortus and Brucella melitensis for the first two species of the genus (Meyer and Shaw 1920), in Meyer's words, ʻa contribution that has stood the test of timeʼ (Piel 1988). In the following years, the close relationship between these two bacteria merged, and the contagion of B. abortus from bovine unpasteurized dairy products to humans accep 1927). Evans herself later championed the pasteurization of milk as a method for preventing brucellosis, and other bacterial transmittable diseases, such as tuberculosis, listeriosis, and salmonellosis (Chung 2010).

Taxonomy is a practical proposal and a tool for understanding close relationships. One pragmatic contribution of joining these two pathogenic bacterial species in one genus concerned the suitable bacteriological methods to isolate and identify Brucella organisms. A second notable contribution was the use of ʻbrucellosisʼ to describe a disease with multiple clinical presentations. Before quoting this name, the syndrome inspired a myriad of original designations around the world. In an earlier paper published in 1896, Surgeon-Captain Matthew Hughes (1867-1899) assigned to the Hospital in Malta, reported 24 unique names for brucellosis (Hughes 1896). Likewise, the British medical advisory mission in India, Dr. Weldon Dalrymple-Champneys (1892-1980), described 34 frequent signs and symptoms documented in thousands of brucellosis patients (Dalrymple-Champneys 1960). As expected, this melange of clnical signs stood as a significant source of confusion.

In medicine, as in other disciplines, names matter and are essential in shaping the minds of those who work with infectious disorders. For instance, Mediterranean fever was stigmatic for Mediterranean countries and confused with other febrile endemic diseases, such as malaria. ʻCrimean feverʼ referred to the 1853–1856 war on the northern coast of the Black Sea, in which many soldiers became infected with B. melitensis. The name ʻundulant feverʼ, used in the United States, describing the wavelike pyrexial curve observed in classical brucellosis, lost its use because of other infectious diseases that also displayed this febrile pattern. Moreover, long-lasting chronic brucellosis seldom coursed with undulant fever. Alternative names such as ʻtyphoid-malarial feverʼ or ʻatypical typhoid feverʼ were somewhat confusing and ascribed to other infectious diseases. The various Brucella species and strains induced the same syndrome in humans; therefore, the term ʻbrucellosisʼ became a proper name for all infections produced by members of the genus (Spink 1956). A name for a disease, caused by two different but related organisms, was also born in 1920.

ADDING MEMBERS TO THE GENUS

In 1914, the Veterinary Inspector of the Animal Pathology Division of the United States, Jacob Traum (1882–1966), with vast experience working with Bang's Disease (Mohler and Traum 1911), isolated a pathogenic microorganism from aborted swine fetuses. Later, other investigators confirmed the bacterium in swine outbreaks and humans working in hog slaughterhouses (Evans 1927). Since antigenically, the bacterium was more closely related to B. abortus than to B. melitensis,Traum initially considered this organism a swine variant of B. abortus (Traum 1914). This misconception lasted for many years, until Irvin Forest Huddleson (1893–1965), from the bacteriological section in Michigan, resolved this issue. In 1929, Huddleson discovered that the two recognized Brucella species and the swine strain displayed different sensitivity to dyes, such as thionin and basic fuchsin, a method still used in the 21st century. Based on this characteristic, Huddleson named the swine isolate as Brucella suis (Huddleson 1929). Since then, the tendency, when naming a Brucella species, is to assign the specific epithet according to the preferred animal host, rather than to a region in which they occur (e.g., ʻmelitensisʼ derived from Melite, a Greek name given to the Malta island) or to the disease they produce (e.g., abortus, from Latin abortiōnem, ʻmiscarriageʼ). Now, we know that most of the B. suis isolates identified by Huddleson in the United States belonged to the so-called biovar 1. Later, other investigators included new biovars (2–5) displaying unique properties as part of the B. suis cluster that slowly turned into a heterogeneous grouping, and proposed new groupings. For instance, the influential British microbiologist Sir Graham Wilson (1895–1987) from the London School of Hygiene and Tropical Medicine proposed three main groups: the bovine-abortus, the porcine-abortus, and the goat/sheep-melitensis, with their subsidiary rough para-abortus and para-melitensis derivatives. He also included in each group several sub-groups containing transitional strains, associated with a particular geographical location (Wilson 1933).

During the next 30 years, the number of ʻatypicalʼ Brucella strains isolated from animals and humans increased due to improvements in the culturing and identification of bacteria (Fig. 1) (Cruickshank and Madge 1954; Meyer and Cameron 1957). Several biovars became part of each species' cluster; though, the genus composed of these classical brucellae remained stable, due to two different but related characteristics apparent to the scientific, medical, and animal husbandry communities. The three bacterial species exhibit strong host preference: B. melitensis for goats and sheep, B. abortus for cattle, and B. suis for pigs. Also, the three Brucella organisms were capable to cross-infect different animal species, such infections remained mostly sporadic and not epizootic. The second property was related to the zoonotic virulence of the various species (PAHO/WHO 1950): B. melitensis characterized as the most dangerous for humans, followed by B. suis, and finally by B. abortus, the least virulent (Evans 1947; Bosseray, Plommet and De Rycke 1982). Nowadays, B. melitensis is still the leading zoonotic bacteria, followed by B. abortus and then by B. suis (Lucero et al. 2008; Liu et al. 2020).

Figure 1.

Discovery of zoonotic and non-zoonotic Brucella species. (A), Timeline of discovery of Brucella species and strains. The dots mark the year in which each species/strain was reported as ʻdifferentʼ from other Brucella species, and not when the species name was given or officially recognized. The dotted lines indicate the dates when the Brucella genus was proposed and when it became part of the Class Alphaproteobacteria. (B), Host preference of zoonotic and not zoonotic brucellae. The most virulent species with a higher zoonotic spectrum are those from domesticated animals, while those displaying lower zoonotic potential are those from wildlife animals. One exception is B. ovis, which is a pathogen for rams and does not infect other hosts. There are only a few human brucellosis cases caused B. neotomae, B. suis biovar 2, Brucella spp. ST27, B. inopinata BO1, Brucella sp. BO2, and frog-type Brucella sp. The Brucella ST27 strains have been detected in cetaceans, but the source of human contagion remains unknown.

Because of their economic, social and public health impact, the host preferences and differential virulence were relevant. The strategies and control measures pursued by the livestock producers and Animal Health authorities improved since they could apply them to different management actions, depending on the bacterial species found. It was possible to trace the infection source and to test the risk of acquiring the disease, according to the virulence displayed by the three Brucella species. For example, while milk pasteurization proved a suitable strategy for controlling B. melitensis and B. abortus zoonotic infections, the control of B. suis concentrated in slaughterhouses and small farms. As an occupational disease of livestock workers, it became possible to identify the focus and the source of infection (PAHO/WHO 1950). Moreover, the economic impact and compulsory slaughtering of the caprine, ovine, and swine industries differed from the management of bovines.

The convenience of this nomenclature was straightforward and suitable from the medical and practical perspective. However, for bacteriologists working with brucellae organisms, it soon became evident the close resemblance of the three species. Unexpectedly, one of the sharpest arguments against the three Brucella species division came from Alice Evans, who expressed that the bacteria were ʻtoo closely related to justify the recognition of three speciesʼ (Evans 1947). She argued for only one Brucella specie with three varieties or subspecies. Evans understood, however, that ʻfor the sake of convenience, the various types of Brucella had been given specific namesʼ. Although she mentioned the subject in several papers and meetings, she did not push beyond that point and worked under the frame of the status quo, primarily composed by veterinarians, clinicians, and animal health authorities.

Through the years, it became clear to those investigators working in the molecular classification of Brucella organisms that all the recognized species were genetically closely related. The monospecific Brucella genus argument persisted and became a battle between ʻlumpersʼ and ʻsplittersʼ, concerning not only the species but also the genus (Moreno, Cloeckaert and Moriyón 2002). A few years before publishing On Origin of Species, Charles Darwin (1809-1882) himself outlined the struggle between these two groups in a letter to British botanist and explorer Joseph Dalton Hooker (1817-1911) in 1857: ʻIt is good to have hair-splitters & lumpersʼ (Endersby 2009; Darwin 2016).

Over the next 40 years, the discovery of atypical Brucella organisms isolated from wildlife and domestic animals became more frequent (Fig. 1). Despite this, the Expert Committee on the Taxonomy of Brucella was reluctant to accept anything but the three existing members (Stabelforth and Jones 1963). The first challenge came in 1953 by the New Zealander veterinarian Malcolm Buddle (1914-1991) for the Department of Agriculture of Wellington (Buddle and Boyes 1953). Dr. Buddle, who had ample experience working in bovine brucellosis, isolated an organism resembling brucellae from various cases of ram epididymitis. Without hesitation, Buddle named this new isolate Brucella ovis, though he also suggested the name ʻBrucella brucei var. ovis australasiaeʼ according to the monospecific genus proposal of various investigators (see below) (Zvirbulis and Hatt 1969). He argued that this new Brucella specie was ʻa stabilized rough mutantʼ, in contrast to the smooth phenotype of the classical three species (Buddle 1956). B. ovis, a pathogen of rams, did not infect humans or displayed virulence for guinea pigs, departing from the most dangerous zoonotic smooth Brucella species. As stated by Buddle in a reply letter: ʻThus, while the aetiological agent of this specific type of ram epididymitis and ewe placentitis may continue to pose fascinating questions for taxonomists, the organism is clearly distinct from B. melitensis on metabolic, pathogenic and epidemiological groundsʼ (Buddle 1965).

About the time of B. ovis discovery, the South African veterinary bacteriologist Govert van Drimmelen (1911–2003) from the Faculty of Veterinary Science at the University of Pretoria described, what he thought, was a new Brucella species isolated from Karakul sheep in South West Africa. Govert van Drimmelen described this strain as ʻB. ovisʼ with characteristics of a rough B. melitensis and, on certain occasions, a strain named ʻBrucella ovigenitalumʼ (van Drimmelen 1953; van Drimmelen et al. 1963). The Subcommittee on Taxonomy of the Genus Brucella of 1963, disregarded both claims due to these inconsistencies, stating that the board fellows were ʻnot satisfied that it [B. ovis] is a member of the genus Brucellaʼ since ʻit bears little antigenic relation to other members of the genus, and its metabolic pattern differs from brucellaeʼ (Stableforth and Jones 1963). Buddle himself recognized South Africa strains as B. melitensis variety karakul, and distinguished them from the New Zealand B. ovis sheep isolates. Despite the overwhelming evidence on the relatedness of B. ovis with the other members of the genus Brucella, the dispute persisted, and the Subcommittee delayed the approval of Buddle's proposal for two decades (Jones and Wundt 1971).

The second challenge to a more limited definition of the genus Brucella came in 1957, from an unexpected source: a desert wood rat named Neotoma lepida. In order to discover new reservoirs for Leptospira organisms (other zoonotic bacteria), Herbert Stoenner (1919–2007) and David Lackman (1910–1988) from the Rocky Mountain Laboratory in Montana, trapped woodrats in the Great Salt Lake Desert of Utah, from which they isolated bacteria that resembled B. abortus since it was a smooth strain displaying predominantly ʻAʼ antigen. At that time, the monospecific sera against ʻAʼ (for abortus) and ʻMʼ (for melitensis) determinants, described by Wilson and Sir Arnold Miles (1904–1988) from the London School of Hygiene and Tropical Medicine, were used as standard reagents to type and distinguish Brucella species (Wilson and Miles 1932). These organisms persisted in woodrats for lengthy periods, without causing the pathological signs observed in experimental murine brucellosis. According to the tradition, they named this bacterium as Brucella neotomae and regarded it as a non-zoonotic species, limited to desert woodrats (Stoenner and Lackman 1957). This finding was not inconsequential since it pointed out wildlife as potential Brucella reservoirs. Despite displaying all the characteristics of the three classical Brucella species, the Subcommittee on Taxonomy of the Genus Brucella of 1963 did not accept B. neotomae as new species since just a few isolates were available, and the bacterium laked relevance from the veterinary, public health, and economic perspective (Stableforth and Jones 1963). Seventy years later, after the first report, a group of investigators found human neurobrucellosis cases caused by B. neotomae (Suárez-Esquivel et al. 2017a), suggesting that, under appropriate circumstances, all Brucella species could become zoonotic bacteria.

The next assessment confronted by the Subcommittee on Taxonomy came from man's best friend, the dog. During the 1960s, several outbreaks of abortions and epididymitis in breeding kennels and packs of field dogs occurred in the United States. The three American veterinarians investigating the canine outbreaks were Cleon Kimberling (1931) from Colorado State University, Donald Luchsinger (1936–1998), from the Department of Agriculture in Iowa, and Robert Anderson (1922–2012) from the University of Minnesota. These veterinarians, who had ample experience in infectious disease, isolated a bacterium resembling Brucella organisms from an aborted canine fetus, in 1966 (Kimberling, Luchsinger and Anderson 1966). The American microbiologist Leland Carmichael (1930–2020) and Dorsey Bruner (1906–1996) from Cornell University, characterized this bacterium and named it Brucella canis (Carmichael and Bruner 1968). The canine isolate lacked the O-chain of the lipopolysaccharide (LPS) molecule, had a stable rough phenotype and, therefore, antigenically related to B. ovis. Still, the biochemical and bacteriological profiles resembled those of B. suis biotype 3 from hogs. Due to its close biochemical resemblance, some investigators claimed that the canine isolate was just another B. suis biotype (Meyer 1969). Gradually, the B. suis cluster became the repository of all atypical Brucella strains that did not fit the standard definitions of the classical species. This tendency, led by the expert bacterial taxonomist Margaret Meyer (1923–2010), from the University of California at Davis, prevailed for many more years (Meyer 1964; 1976; 1969; 1990). On this occasion, however, the controversy did not last long due to substantial bacteriological, biochemical, and immunochemical evidence supporting B. canis as a member of the Brucella genus. Still, the stable rough phenotype and definitive preference for dogs defined this bacterium as a separate Brucella species (Fig. 2) (Díaz, Jones and Wilson 1968). Years later, whole-genome sequence analysis (WGSA) comparison of B. suis (biovar 1 and 4) and B. canis proved that Meyer's claim on the close resemblance between these two species was correct since they were monophyletic compared to the more paraphyletic distribution of other B. suis strains (e.g., Thomsen strain) (Foster et al. 2009). Nevertheless, besides the characteristics mentioned above, B. canis also displayed unique virulent and cell envelope structural properties (Chacón-Díaz et al. 2015; Moreno and Moriyón 2006). Altogether, B. canis and B. suis (biovars 1 and 4) are good examples of genetically closely related species that display distinct phenotypic characteristics, different host preferences and distinct virulence properties.

Figure 2.

Antigenic and DNA relatedness among the various classical Brucella species. (A), Immunoelectrophoresis of soluble cell extracted antigens from smooth B. melitensis (Bm-s) and B. canis (Bc), confronted with rabbit antisera prepared against acetone-killed rough Brucella melitensis (Anti-R-Bm) and rabbit antisera against acetone-killed smooth B. melitensis (Anti-S-Bm), (Adapted from Figs 1 and 2, Díaz, Jones and Wilson 1968). (B), Immunodiffusion of rough-LPSs from B. abortus [a], B. melitensis [m], B. canis [c], and B. ovis [o] against rabbit antiserum prepared to Brucella rough-LPS [Ab]; (adapted from Fig. 1, Moreno, Jones and Berman 1984). (C), immunoelectrophoresis of soluble cell extracts antigens of B. ovis (Bo), rough B. melitensis (Bm-r), and smooth B. melitensis (Bm-s) confronted with antisera prepared against acetone killed smooth B. melitensis (anti-s-Bm); (adapted from Figure 4, Díaz, Jones and Wilson 1968). (D) Competition curves for various bacterial DNAs. In each portion of the figure, radiolabeled, single-stranded DNA fragments were added to homologous DNA-agar. Amounts of unlabeled, single-stranded DNAs were added to separate vials, each containing radiolabeled DNA fragments. The mixture composition and identification of competitors are presented in each part of the figure (adapted from Figure 1, Hoyer and McCullough 1968). In ʻAʼ and ʻCʼ, B. canis and B. ovis antigens react with heterologous antisera prepared against B. melitensis, showing several precipitin bands. The S-LPS antigen line is characteristic of smooth strains. In ʻBʼ, rough LPSs from different brucellae show complete cross-reactivity among them, except B. ovis LPS, showing partial recognition, demonstrating different antigenic structures with B. canis LPS. In ʻDʼ, notice a slight difference with the B. ovis competition curve. The DNA of Francisella tularensis does not compete

During the same decade of the discovery of B. neotomae, B. ovis and B. canis, scientists from the former Soviet Union and Eastern European communist countries working in Siberia, the Caucasus and Kazakhstan, repeatedly reported Brucella strains in squirrels, hares, muskrat, marmots, deer, reindeer, foxes and murine rodents, including common voles (Rementsova and Kusov 1955; Pinigin and Pietuchowa 1960, 1962; Rementsova 1962; Rementsova, Postricheva and Rybalko 1969; Zheludkov and Tsirelson 2010). Reports of Brucella stains in murid rodents in Kenya, Africa, and Australia, came from investigators working in these regions (Heisch et al. 1963; Cook, Campbell and Barrow 1996). All of these strains displayed distinctive bacteriological and antigenic properties (Vana 1980; Davis 1990; Zheludkov and Tsirelson 2010). Of particular interest were the reindeer, murine, and hare isolates. The reindeer strains from Siberia also infected Inuit and Yupik people in the Arctic regions of Alaska, Canada, and Siberia (Pinigin and Pietuchowa 1960; 1962; Meyer 1964). Likewise, the isolates from rodents and lagomorphs caused intense debates on the epidemiology and evolution of the Brucella genus. Indeed, several investigators envisioned them as brucellosis reservoirs for infecting livestock and human (Meyer 1976a).

In order to resolve the taxonomic status of these wildlife-Brucella isolates, Soviet investigators, led by the Professor Anatoly Fedorovich Pinigin (1917-1999), a Soviet World War II hero, working at the Institute for Pest Control of Siberia and the Far East, requested the help from scientists behind the ʻIron Curtain. ʼ One of them was Józef Parnas (1909-1998), a prestigious Polish microbiologist from the Academy of Medicine in Lublin who was an expert in phage typing, then considered a state-of-the-art method in bacterial taxonomy (Parnas, Tuskiewicz and Zalichta 1965). Accordingly to the standard nomenclature, Soviet and Polish investigators proposed the names of ʻBrucella rangiferiʼ, and ʻBrucella muriumʼ for the reindeer and murine strains, respectively (Parnas 1964; Davydov 1965; Parnas, Tuskiewicz and Zalichta 1965; Karol and Parnas 1967). The hare isolates from Eastern and Central Europe were characterized as B. suis biovar 2 (Vítovec et al. 1976). This biovar also infected wild boars and pigs, but seldom humans. Still, there are a few reports of brucellosis cases in hunters or persons in direct contact with wild boars while preparing wild boar meat (Mailles et al. 2017), reveling, again, the zoonotic potential of the various Brucella strains.

However, taxonomy, besides being a conceptual system of classification that helps to understand evolution, is not immune to social issues. After passionate discussions and irreconcilable disagreements, most of the American Subcommittee on Taxonomy members opposed the alternative species names proposed by the Soviet and Eastern European investigators. The disputes held in the Subcommittee on Taxonomy of Brucella meeting in Montreal in 1962, were particularly harsh. The minutes of the meeting published in 1963 point out differences with Soviet scientists and with Parnas. One minute note described a general agreement obtained from all Subcommittee members except Professor Parnas, who disagree, deciding to publish his views elsewhere (Stableforth and Jones 1963).

Ensuing the threat, Professor Parnas published two leading articles on Brucella phage typing and the proposed new species in the prestigious journal Nature (Parnas 1964; Parnas, Tuskiewicz and Zalichta 1965). Because of unfortunate circumstances, Józef Parnas could not pursue his goal in the following years. In 1968, he became a victim of a wave of anti-Semitism during the anti-Jewish Polish campaign crisis of 1967–1968 and ʻsentenced to five years on a charge of spying for Britishʼ. Released after a prolonged hunger strike, Parnas moved to the University of Copenhagen in Denmark, in 1971 (United States Senate 1975). In one of his last manuscripts, Jozéf Parnas still appealed to change the nomenclature of the genus Brucella according to host preference (except for B. melitensis): B. melitensis, ʻB. bovisʼ (instead of B. abortus), B. suis, B. canis, B. neotomae, B. rangiferi, and B. murium (Parnas 1976). He decisively was a splitter.

As expected, the papers published in this high-quality journal provoked some investigators, who took a critical attitude. Margaret Meyer stood as an outspoken opponent. She claimed that the isolates were B. suis biotypes and not new species. In one of her papers, she replied: ʻAll the available evidence indicates that strains of Brucella isolated from rodents are not antique novelties but are biotypes or species currently recognized and classified in the genus Brucella and that the infection has traveled to rather than from the rodentsʼ (Meyer 1976a). In her scientific works, Margaret Meyer included some sharp criticisms against the Soviet and East European proposals. Concerning the reindeer isolates in the United States, she claimed that the primary source of infection for the ʻEskimosʼ was the importation of reindeer from Siberia to Alaska in 1891, blaming Imperial Tsarist Russia (Meyer 1964). Meyer also made sarcastic remarks pointing out that the Soviets ʻare still making a plea to have Brucella suis biotype 4 considered a separate speciesʼ (Meyer 1990). Regarding the rodent brucellosis evolution, she argued: ʻThey [the Soviets] have used an entirely speculative approach in analyzing the possible series of historic events that propelled the species in the genus to their present identities, but have offered no pabulum of substantive evidence to support their viewsʼ (Meyer 1976b). After a long dispute that lasted two decades, Brucella organisms isolated from rodents in the Soviet Union and Eastern Europe just stood as B. suis biovar 5 by the Subcommittee on Taxonomy in the Boston meeting in 1982 (Corbel 1984). Nowadays, we understand the value of various statements put forward by Margaret Meyer; however, not all of them were fortunate, and a few of her pronouncements overstated or wrong, but at that time, they were a source of concern, controversy, and dispute. Above all, Parnas was a good scientist, and later, he tried to smooth things over by giving credit for B. rangiferi discovery to both Meyer and Pinigin (Parnas 1966). The differences, however, were not resolved (Meyer 1990), and the balance of ideas eventually tipped to one side. After all, it was the Cold-War era, and the United States the center of brucellosis research during that period.

The classification of the rodent Brucella as B. suis 5 created some confusion and misunderstandings with bacteria, also called ʻB. suis biotype 5ʼ, isolated by French researchers from cattle and Brucella organisms (Morris 1973). The taxonomic position of Brucella strains isolated from rodents in Queensland, Australia, was a different story since they were ʻdistinct from the cultures isolated from rodents in the USSRʼ, as stated by the Subcommittee on Taxonomy (Corbel 1984). These Australian rodent bacteria remained outside of the B. suis biotype 5 group and were kept in the fridge for more than 50 years until the rise of the genomic era (see below). Based on the metabolic studies of ʻatypicalʼ Brucella organisms performed by Meyer (Meyer 1961), the Subcommittee on Taxonomy decided to rule out other proposals, such as ʻBrucella intermediaʼ, suggested by French investigators (Renoux 1952b); since they identified these bacteria as conventional B.melitensis, B. abortus, and B. suis species.

The balance towards including B. neotomae and B. ovis as a new species came from the ʻhigh-techʼ DNA-DNA hybridization method of the late 1960s that made clear the genetic closeness of these bacteria with the three other Brucella species (Fig. 2) (Hoyer and McCullough 1968). On these grounds, B. neotomae became part of the genus at the Moscow meeting in 1966. Because of some conflicting results in the DNA-DNA hybridization, however, ʻit was decided to defer a decision on the taxonomic positionʼ of B. ovis (Fig 2) (Jones 1967). The bacterium finally became accepted as a species at the 10th International Congress for Microbiology in Mexico City in 1970 (Jones and Wundt 1971).

In the meantime, the Spanish bacteriologist Ramón Díaz (1935) from the University of Navarra, Spain, together with investigators from the University of Wisconsin-Madison, established the protein antigenic relatedness of B. canis and B. ovis with other brucellae (Fig. 2) (Díaz, Jones and Wilson 1967, 1968; Moreno, Jones and Berman 1984). Despite the efforts, B. canis remained a provisional species at the Tokyo meeting in 1974 and just finally accepted at the Munich meeting, in 1978 (Corbel 1982; Osterman and Moriyón 2006). The members of the Subcommittee on Taxonomy agreed in including these three recent bacteria; however, they excluded other Brucella species proposals (Corbel and Brinley-Morgan 1975). Finally, in 1975, appeared the publication of the Subcommittee delegates, describing the minimal standards of the six recognized Brucella species and the respective biotypes (Corbel and Morgan 1975).

THE MONOSPECIFIC Brucella GENUS ERA

For the time being, the ʻechoʼ of Alice Evans's statement on the close resemblance of all members of the genus remained in the environment. Five years after Evan's remark, the French microbiologist Gerard E. Renoux (1915-1998), who later became the Director of the Brucellosis Laboratory at the INRA in Nouzilly, France, published a controversial work, arguing that because of the homogeneous nature of all members of the genus, the Brucella species should be one (Renoux 1952a,b). He proposed the name of ʻBrucella bruceiʼ, assigning the other species and strains as varieties or subspecies, according to his classification: Melitensis, Abortus, Suis, Thomseni and Lisbonnei. Later on, Renoux reasoned that B. melitensis gained preeminence over other names, and then more appropriate for a single species-genus, with all the other brucellae as varieties (Renoux 1958).

Two other scientists, also members of the Subcommittee of Taxonomy on the genus Brucella, supported Renoux's proposal. The German bacteriologist Professor Wilhelm Wundt (1919-1999) from the Institute of Hygiene at the University of Tübingen in Germany, claimed that the differences among the various species were quantitative and not qualitative (Wundt 1958). Likewise, van Drimmelen proposed B. melitensis as the species name for all Brucella organisms followed by the capital letter ʻMʼ, ʻAʼ, or ʻSʼ for B. melitensis, B. abortus, and B. suis, respectively, to distinguish them as varieties. He also suggested epithets for the uncommon types, such as ovis, ovigenitalium, karakulensis, abortusovis, and hominis, among several others (van Drimmelen 1958, 1961). Despite the influence of these three distinguished scientists, most of the Subcommittee members rejected the monospecific genus proposals in all subsequent meetings, from Montreal 1962 up to Boston 1982. Instead, the discussions centered on the Brucella biovars, phenotypic characterization, and atypical strains keeping intact the classical Brucella species classification (Osterman and Moriyón 2006).

About the period that Renoux retired as Director of INRA in 1973, Jean-Michel Verger, a young French researcher from the same institute, was already isolating brucellae from domestic and wildlife animals in France. In the next years, Verger became a leading expert in Brucella taxonomy. In 1985, together with investigators from the Institute Pasteur, Verger pursued the Renoux's monospecific genus proposal by studying the DNA-DNA hybridization parameters of Brucella species and biotypes (Verger et al. 1985). Then, the DNA-DNA hybridization was the best applicable procedure for establishing taxonomic ranks. According to the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics, ʻthe phylogenetic definition of a species generally would include strains with approximately 70% or higher DNA-DNA relatedness and 5°C or lower ΔTm. Both values must be considered, and phenotypic properties should agree with this definitionʼ (Wayne et al. 1987). As expected, the DNA re-association values among the different Brucella species and biotypes were well above 70%. Based on this evidence, the INRA/Institute Pasteur research group made the following statement:

ʻWe believe that taxonomy is a science and that nomenclature should be in accord with this science. Therefore, we propose that only one species, B. melitensis [Hughes 1893] Meyer and Shaw 1920, be recognized in the genus Brucella. B. abortus, B. suis, B. neotomae, B. ovis, and B. canis are thus subjective synonyms of B. melitensis (the type species). We suggest that specific epithets, other than the specific epithet of B. melitensis, formerly associated with the generic name Brucella be used in a vernacular form (i.e., not italicized) to refer to biovarsʼ (Verger et al. 1985).

Some influential investigators in bacterial taxonomy, such as the Belgian bacterial taxonomist Jozef De Ley (1924-1997), who received the Bergey Award of the Bergey's Manual of Systematic Bacteriology, also agreed with the monospecific Brucella genus proposal (De Ley et al. 1987).

In September 1986, during the XIV International Congress of Microbiology, the Subcommittee on the Taxonomy of Brucella met at the University of Manchester, England. Only two members attended to the Subcommittee meeting: Michael Corbel, as Chairman, and Jean-Michel Verger, as a regular member of the Subcommittee. Board members' absences were not justified in the report. However, three members already retired, and the renowned brucellologist Lois Jones (1923-1986) from the University of Wisconsin did not assist because of a long-lasting illness, and she passed away ten days after the Manchester meeting (Corbel 1988). Despite the poor attendance, the two members present in the Manchester meeting discussed the agenda, and on the section corresponding to ʻNomenclature and Classificationʼ, the monospecific Brucella genus proposal postulated, as stated by Verger et al. (Verger et al. 1985) and published in the report (Corbel 1986). In my experience, Lois Jones and Margaret Meyer, both members of the Subcommittee on Taxonomy, enduringly opposed to the monospecific Brucella genus idea (Meyer 1990), previously proposed by Renoux, Wundt, and van Drimmelen; investigators who were also members of the Subcommittee on Taxonomy for several years (Jones and Wundt 1971).

To believe that ʻtaxonomy is a scienceʼ is, however, a tricky business. The threshold standards are arbitrary values. In the genomic era, an arbitrary cutoff value of 95% similarity has been suggested. Why not 96%, or 99%? Even a single nucleotide change can modify the biological behavior of a bacterial strain (Bryant, Chewapreecha and Bentley 2012). No unanimously accepted bacterial species concept exists because it is not a scientific subject, but an epistemological problem (Moreno 1997; Moreno, Cloeckaert and Moriyón 2002). It depends on how human understanding raises a justified belief and consolidates knowledge concerning what happens in the external world (Pollock 1968). From this perspective, the taxonomical endeavor includes two elements that are dialectically inseparable from one another. First, there is the analytical process that comprises empirically observable and measurable characteristics of the subject (e.g., structural, biochemical, genetic properties) that are organized in ranks and arranged in clusters. The second process is the typology (the study of types, symbols, and systematic representations), which conceptually separates a set of items in various dimensions based on the notion of an ʻidealʼ type (e.g., virulent, attenuated, preferred host). This process is a mental construct that stresses specific characteristics related to distinct realities, not strictly linked to quantitative properties. Since typologies create useful heuristics (that do not warrant perfection, precision, or absolute rationality, but they still are sufficient for reaching an immediate goal or a useful approximation), they provide an efficient and practical basis for comparisons. One the one hand, the analytical process depends on the robustness of the method and its resolution (e.g., biochemical, restriction patterns, WGSA). On the other hand, the typological process is a flexible heuristic system that adapts to decision-making (mainly when working with complex data). The cutoff value, however, is and always will be arbitrary, and the parameters and concepts for defining bacterial species are not permanent because they depend on typological decisions (Caro-Quintero and Konstantinidis 2012).

Twenty years were more than enough time to prove that the concept of B. melitensis as the unique species of the genus was neither practical nor of scientific value (Moreno, Cloeckaert and Moriyón 2002; Osterman and Moriyón 2006). From the typological perspective, the concept did not live up to the reality of the animal and public health professionals, scientists, bureaucrats, and other groups that had to deal with the disease (Moreno, Cloeckaert and Moriyón 2002). The proposed scientific truth of the monospecific genus concept was irrelevant, considering the different virulence and host preference of the various species. Likewise, listing all brucellae under one name remained confusing and dangerous for people with little or no experience on Brucella and brucellosis. To complying with the nomen periculosum (dangerous name) rule, biosafety must prevail over other considerations in taxonomic nomenclature (Judicial Commission 1985). Brucella organisms are part of the selected infectious arsenal that could pose a threat to public health, then the monospecific classification was misleading and created significant problems in the databanks and culture collections (Moreno, Cloeckaert and Moriyón 2002). As a corollary, it is worth recalling the statement in the introductory chapter of the 1984 Bergey's edition: ʻa classification that is of little use to the microbiologist, no matter how fine a scheme or who devised it, will be ignored or significantly modifiedʼ (Stanley and Krieg 1984).

In the light of distinctive species discovered in cetaceans and pinnipeds in the 1990s (Ewalt et al. 1994, Ross et al. 1994), the cutoff of 70% DNA-DNA hybridization had limited value because, with upgraded and new molecular techniques, such as DNA restriction patterns, the resolution increased, favoring recognition of the ʻnewʼ species (Michaux-Charachon et al. 1997). These methodologies that distinguished discrete and consistent differences in the genomes broadened the biological species concept for the genus Brucella (Moreno, Cloeckaert and Moriyón 2002). With all these elements at hand, the Subcommittee on Taxonomy of the Genus Brucella unanimously returned to the pre-1986 condition of the six Brucella species, and the status of the monospecific genus decision was taken back (Osterman and Moriyón 2006). Separating the Brucella organisms through the analysis of a variable number of tandem repeats (Le Flèche et al. 2006) and above all, gene sequencing analysis (Whatmore 2009), reinforced the species concept according to the host preference, and dispersion of clusters related to geographic distribution.

The minutes of the Pamplona meeting also included the proposals for ʻBrucella cetaceaeʼ and ʻBrucella pinnipediaeʼ as new species of the genus, first isolated from dolphins and seals in Scotland and the United States, respectively, in 1994 (Ewalt et al. 1994; Ross et al. 1994). Investigators suggested various other names, such as ʻBrucella marisʼ, ʻBrucella delphiʼ, ʻBrucella phocaeʼ, and ʻBrucella phocoenae.ʼ These names were not commensurate with the strict taxonomical name rules or host range, and, therefore, were changed to Brucella ceti and Brucella pinnipedialis, following the preferred host nomenclature of cetaceans (dolphins and whales) and pinnipeds (seals and walrus) (Foster et al. 2007). In time, through genetic analyses, the B. ceti group encompassed three other types: the dolphin, porpoise, and ST27 types being these last two groups phylogenetically closer to B. pinnipedialis (Audic et al. 2011; Guzman-Verri et al. 2012).

THE Brucella GENUS AS PART OF THE CLASS ALPHAPROTEOBACTERIA

Brucella organisms belong to the Phylum Proteobacteria, Subphylum Rhodobacteria, Class Alphaproteobacteria, Order Rhizobiales (illegitimate, according to rule Rule 51b[1], see Euzeby 2006), and family Brucellaceae. The family contains seven genera, according to the polyphasic approach: Crabtreella, Daeguia, Mycoplana, Ochrobactrum, Paenochrobactrum, Pseudochrobactrum, and Brucella (Kämpfer, Wohlgemuth and Scholz 2014). By phylogenetic analysis, other taxonomists recognized just four Brucellaceae genera: Pseudochrobactrum, Falsochrobactrum, Ochrobactrum, and Brucella (Leclercq, Cloeckaert and Zygmunt 2019). This taxonomical assignment and nomenclature, however, required modern sequencing approaches, and for 80 years, the genus Brucella alternated through different bacterial groups (Biberstein and Cameron 1961).

The Manual of Determinative Bacteriology of 1901, anteceding the Bergey's Manual, did not consider M. melitensis and Bc. abortus, as part of the recognized Micrococcus and Bacillus/Bacteria genera (Chester 1901). The first association of the Brucella genus was with the so-called Bacteriaceae family (Mayer and Shaw 1920). The genus remained within this group for several years until the Committee on Bacteriological Nomenclature in Rome wiped out the family (Judicial Commission 1954). Then, unexpectedly, in the 1948 edition of Bergey's Manual of Determinative Bacteriology, the genus became part of the tribe Brucelleae within the family Parvobacteriaceae, with no bacterial type member. Ten years later, the genus changed its status and became a member of its own family Brucellaceae, together with seven other genera, later proven to be unrelated to Brucella (Breed, Murray and Hitchens 1948; Breed, Murray and Smith 1957).

The chaos increased when two distinguished British bacteriologists, Peter Sneath (1923-2011) and Samuel Cowan (1905-1976), published a paper with the remarkable title ʻAn Electro-Taxonomic Survey of Bacteriaʼ, in which using computational methods clustered Brucella in a ʻtaxonomic treeʼ together with Pasteurella, Actinobacilus and Neisseria (Sneath and Cowan 1958). Sneath remained for many years a respected scientist that revolutionized the practice of bacterial taxonomy by inventing numeral taxonomy. Cowan was the curator of the National Collection of Type Cultures in the UK and a well-known scientist for his Manual for the Identification of Medical Bacteria (1965). Because of the prestige of these two bacteriologists and the computational sophistication of that time, very few questioned the suggested taxonomic hierarchy, bringing more confusion to the field. The journey of the phylogenetic association of the Brucella genus around different bacterial groups lasted for many years until an unexpected twist brought light to the confusion.

The first clue came from German investigators led by the lipid chemist Otto Wolfgang Thiele (1917-1998), from the University of Göttingen in 1968. These investigators reported in various papers that the Brucella fatty acids, ubiquinone, and other lipids were very similar to those of Agrobacterium tumefaciens, photosynthetic bacteria, and even to mitochondria (Thiele, Busse and Hoffmann 1968; Thiele Asseline and Lacave1969a, 1969b). The analysis of lipids was state-of-the-art in bacterial taxonomy in the late sixties and seventies (Shaw 1974). Chemotaxonomical studies on fatty acids also allowed clear differentiation of the various Brucella species (Tanaka et al. 1977). Despite this evidence, the Brucella taxonomists ignored the lipids' data, and their taxonomic utility only rediscovered after twenty years (Moreno et al. 1990). Commensurate with the lipid studies, through numerical analysis of a wide range of characters, Sneath and collaborators also found that Brucella organisms were closer to Agrobacterium than to other bacteria. However, they did not discuss the finding and ignored it (Johnson and Sneath 1973).

In the 1980s, a group of German scientists from the Max Planck Institute for Immunology in Freiburg found that the structure of the lipid A of the LPS was a reliable marker for phylogenetic studies (Mayer, Moreno and Weckesser 1986; Weckesser and Mayer 1988). The partial structure of the B. melitensis lipid A revealed the phylogenetic relationship of this bacterium with phototrophic bacteria of the genus Rhodopseudomonas and several members of the Rhizobiaceae family (Mayer, Moreno and Weckesser 1986; Weckesser and Mayer 1988). Later, other investigators extended these findings, showing that Ochrobactrum, Bartonella, Phyllobacterium, and Mesorhizobium, displayed a lipid A structure similar to the Brucella LPS (Fig. 3) (Velasco et al. 1998a; Scheidle, Gross and Niehaus 2005, Malgorzata-Miller et al. 2016; Zamlynska et al. 2017; Conde-Álvarez et al. 2018; Guillotte et al. 2018). These studies also had implications for understanding Brucellapathogenicity, since the lipid A structure connected to the biological activities and endotoxicity mediated by these organisms (Weckesser and Mayer 1988).

Figure 3.

Comparison of predominant Lipid As structures of Alphaproteobacteria organisms. Rickettsia rickettsi and S. meliloti lipid As display a ʻcanonicalʼ structure, with a β(1 → 6)-linked glucosamine disaccharide backbone, phosphorylated at position 1 and 4′ of the aminosugars, and acylated at positions 2 (N-) and 3 (O-) of each monosaccharide. The main difference between R. rickettsi and S. meliloti corresponds to the log chain hydroxylated fatty acids in the latter bacterium. In contrast, the closely related Phyllobacterium triffoli, Bn. henselae and Brucella display a ʻa non-canonicalʼ structure, with a β(1 → 6)-linked diaminoglucose disaccharide backbone substituted at the amino groups with long-chain hydroxylated (up to 30 C atoms), cyclic (e.g., lactobacilic), and unsaturated (e. g., cis-vaccenic) fatty acids. Both, R. rickettsi and S. meliloti LPS are endotoxic. However, the former LPS is more potent than the latter. In contrast, the non-canonical LPSs of P. triffoli, Ba quintana, and B. abortus do not display classical endotoxic activity. Only the predominant average Lipid As structures are represented. The acylation patterns and backbone substitutions may vary. Chemical data on the structures was reported in (Velasco et al. 1998a; Scheidle, Gross and Niehaus 2005, Malgorzata-Miller et al. 2016; Zamlynska et al. 2017; Conde-Álvarez et al. 2018; Guillotte et al. 2018).

Alongside the lipid A studies, a group of bacterial taxonomists headed by Jozef De-Ley studied the RNA cistron similarities of Brucella organisms with other bacteria, and they also found a close phylogenetic relationship of Brucella organisms with various members of the Rhizobiales, such as Agrobacterium and Rhizobium species (De Ley et al. 1987). The RNA cistron comparisons showed a close association with bacteria known as the CDC group Vd, joined within the Alcaligenes/Achromobacter cluster, later known as the Ochrobactrum genus (Holmes et al. 1988). The evolutionary relationship of Brucella organisms with other Alphaproteobacteria members was finally resolved in 1989 by 16S rRNA sequencing, the gold standard method for phylogenetic studies (Dorch et al. 1989; Moreno et al. 1990; Velasco et al. 1998b). With this information in hand, and by comparing the lipids, cell envelope components, genome characteristics, and metabolic patterns, it was possible to broaden the Brucella affiliation with other Alphaproteobacteria and establish an ancestor-descendant relationship (Moreno et al. 1990; Moreno 1992).

The phylogenetic studies revealed the evolutionary connection of Brucella organisms with other eukaryotic cell-associated pathogens and endosymbionts such as the trench-fever Bartonella quintana etiological agent (previously known Rickettsia and Rochalimae), the opportunistic pathogen Ochrobactrum anthropi, the plant endosymbionts of the genera Rhizobium and Sinorhizobium, the plant pathogen A. tumefaciens, and eukaryotic mitochondria (Fig. 4). The closest Brucella relatives were the soil/plant opportunistic Ochrobactrum spp., and the cell-associated animal and human pathogens Bartonella spp. Based on the accumulated evidence, it was proposed an evolutionary path from a hypothetical motile soil/plant bacterial ancestor with broader metabolic alternatives, to the extant Brucella species (Moreno 1990; 1992, 1998). This proposal also included a temporal line of divergence from the hypothetical ancestor to the extant Brucella species by comparing the structural, genetic, and biological properties of the different Alphaproteobacteria (Moreno 1990, 1992, 1998). A significant observation corresponded to differences in genome sizes, structural properties, and metabolic alternatives among the various Alphaproteobacteria (Moreno 1992, 1998). While the plant/soil bacteria displayed large genomes, broad metabolic plasticity, and a complex structural diversity, the animal cell-associated bacteria, such as Brucella, Bartonella, and Rickettsia organisms, had much smaller genomes, fewer metabolic alternatives and less complex structures (Fig. 4) (Moreno 1990, 1992, 1998). Finally, the Subcommittee on the Taxonomy recommended in 1994, incorporating the genus Brucella into the Class Alphaproteobacteria (Corbel and Moriyón 2006).

Figure 4.

16SrRNA phylogeny tree and genome sizes of representative plant-associated, free-living, opportunistic, and animal cell-associated Alphaproteobacteria. (A) The phylogenetic tree was constructed based on the 16SrDNA Alphaproteobacteria sequences available in 1998 (Moreno 1998). The relative genome sizes and the number of replicons that include chromosomes, chromids, and plasmids represented by color ellipses were upgraded to current Genome seizes reported in the GenBank (https://www.ncbi.nlm.nih.gov/genbank/) (B) Comparison of genome sizes of Alphaproteobacteria according to their habitat. Notice that the intracellular pathogens have smaller genomes in comparison with the soil/plant bacteria. Agrobacterium spp., Brucella spp., (except B. suis bv. 3), and O. intermedium have two chromosomes (one genuine chromosome and one chromid). The second chromosome (chromid) of Agrobacterium spp., is linear. Brucella spp., Bartonella spp., A. felis, B. japonicum. R. rickettsi and A. phagocytophilum are devoid of plasmids. Species names from 1998 have been upgraded according to the current taxon (Based in Moreno 1998).

Discussions within the Subcommittee regarding the absence of plasmids in brucellae were recurrent because of the relevance of these elements in antibiotic resistance and virulence (Corbel 1988). It also became clear that the smaller genomes of the animal pathogens such as Brucella, Bartonella, and Rickettsia organisms, correlated with their lower metabolic alternatives and reduced structural plasticity in comparison with the soil/plant-associated Alphaproteobacteria, which had larger genomes and broader metabolic and structural alternatives (Moreno 1990, 1992, 1998). By comparative analyses of bacterial genome sizes and the metabolic pathways available at that time, it was possible to build a hypothetical genome reduction evolutionary route, from an ancestral free-living to an intracellular pathogenic Alphaproteobacteria (Fig. 4) (Moreno 1990, 1992, 1998). The proposal stated that the stringent conditions of the intracellular environment of animal cells could have selected for organisms with smaller genomes and fewer metabolic alternatives, in contrast to the plant and free-living counterparts displaying broader metabolic plasticity and larger genomes. Comparisons by WGSA performed years later gave reliable support to these models (Sällström and Andersson 2005).

The 1990s were exciting times for the genus Brucella. From 1992 to 1998, a group of French researchers from the INSERM in Nimes published several papers showing two chromosomes in various Alphaproteobacteria, including in most Brucella members (Fig. 4) (Michaux-Charachon et al. 1993; Jumas-Bilak et al. 1998). This feature, together with the absence of plasmids, turned out to be a striking discovery. Based on these data and the ancestor-descendant phylogenetic relatedness, it was proposed that the larger Brucella chromosome 1 evolved from an even larger genuine chromosome, while the second smaller chromosome 2 from a megaplasmid, after acquiring housekeeping genes (Moreno 1998). This proposal was known as the ʻplasmid hypothesisʼ, and the second chromosome named ʻchromidʼ, to distinguish it from the genuine and larger chromosome 1 (diCenzo and Finan 2017).

However, the countless ways in which nature expresses itself, deceive the human mind. In 1998, the same French group from INSERM in Nımes found that B. suis biovar 3 stain 686, instead of harboring two chromosomes, had only one megareplicon, which was the sum of the two chromosomes observed in other brucellae (Jumas-Bilak et al. 1998). Whether or not this is a unique characteristic of the B. suis 686 strain or a more general outcome of all B. suis biovar 3 strains, or even present in other Brucella species, remains to be tested. It is likely, however, that this is not an isolated event, considering the odds of finding a sole strain with only one chromosome.

Based on this reliable finding, the INSERM research group proposed that the two chromosomes present in most strains emerged from a hypothetical Brucella ancestor with a single megareplicon that excised into two chromosomes, one larger than the other, through rearrangements of ribosomal sequences in the chromosomal regions. This proposal, known as the ʻschism hypothesisʼ (diCenzo and Finan 2017), implied that the Brucella ancestor had only one large chromosome.

The schism hypothesis was not only in clear opposition to the plasmid hypothesis but against the proposal that a single megareplicon of B. suis biovar 3 emerged through the two chromosomes fusion (Moreno 1998). In time, the plasmid hypothesis gained acceptance over the schism hypothesis (Di Cenzo and Finan 2017). First, the evolutionary evidence showed that B. suis biotype 3 harboring one chromosome was not in a distant phylogenetic branch, but clustered together with other Brucella species possessing two chromosomes. Second, Ochrobactrum spp., the closest relatives of Brucella organisms, also possessed two chromosomes suggesting that the ancestor of these two genera exhibited two chromosomes. Moreover, the smaller Brucella chromosome 2 shared orthologous sequences with the so-called Ti megaplasmid of A. tumefaciens, coding for the type IV secretion system (Moreno 1998). With the advent of WGSA, the corresponding sequences of chromosomes 1 and 2 of Ochrobactrum and Brucella organisms determined an ancestor-descendant relationship, and the origin of replication on chromosome 2 revealed a plasmid-derived origin (Del Vecchio et al.2002; Paulsen et al. 2002; Boussau et al. 2004).

The close association of Brucella with plant and animal pathogens and symbionts also stimulated research regarding the virulence mechanisms. For instance, the Brucella two-component regulatory system BvrS/BvrR essential for shifting from extracellular to intracellular environments was similar to the S. meliloti Chvl/ExoS and A. tumefaciens Chvl/ChvG systems, critical for endosymbiosis and pathogenicity in plants (Sola-Landa et al. 1998). Likewise, the Brucella virB operon coding for the type IV secretion machinery, essential for the intracellular bacterial life, corresponded to the orthologous system coded in the A. tumefaciens pTi plasmid, required for virulence (O'Callaghan et al. 1999). The elements of the bacterial cell cycle and division system of Brucella organisms also resemble those of other Alphaproteobacteria (De Bolle et al.2015). Comparative studies with O. anthropi helped to understand the resistance of Brucella organisms to bactericidal substances of cells (Velasco et al. 2000). Comparisons of putative pathogen-associated molecular patterns with other Alphaproteobacteria, such as the LPS (Fig. 3), flagellum, ornithine containing lipids and lipoproteins, helped to determine the modifications that occurred during evolution to make these molecules almost invisible for pattern recognition receptors of immune cells, explaining in part, the stealthy strategy followed by Brucella organisms (Barquero-Calvo et al. 2009).

THE GENOMIC ERA AND THE DISCOVERY OF NEW Brucella CLASSICAL SPECIES.

The first attempt to differentiate Brucella species and biotypes by DNA molecular tools was through restriction endonuclease fragment analysis in the late eighties (O'Hara, Collins and de Lisle 1985; Allardet-Servent et al. 1988). This strategy distinguished the various species; however, in comparison with other bacterial groups, it had low resolution because of the close genetic relatedness among the brucellae. Restriction-fragment-length polymorphisms introduced in the nineties allowed taxonomical and phylogenetic studies throughout the use of distinct loci, such as the porin omp2a/omp2b, the outer membrane protein omp31, IS711 insertion sequences, and other genomic elements. This technique improved the taxonomic resolution (Fitch et al. 1990; Bricker and Halling 1994; Cloeckaert et al. 1995; Bricker 2002) and resolved the various Brucella species and biotypes in phylogenetic trees (Michaux-Charachon et al. 1997). The most significant advancement came with the analysis of repeated palindromic DNA elements (Halling and Bricker 1994), developed as the multilocus variable-number of tandem-repeat analysis, known as MLVA. The additions or deletions of these repeated units result in a high mutation rate at these loci. Therefore, the MLVA targeted DNA regions are molecular clocks in which ʻtick-tockʼ varies from moderate to fast mutation rate, depending upon the locus. The resolution improved by adding more loci. As expected, the B. melitensis, B. suis and B. abortus complete genome sequences (Del Vecchio et al. 2002; Paulsen et al. 2002; Halling et al. 2005) provided the framework for identifying the tandem repeat candidates for the development MLVA studies (Whatmore et al. 2006; Le Flèche et al. 2006). While some loci were suitable for delimiting species, others remained better for strains (Bricker, Ewalt and Halling 2003; Whatmore et al. 2006). For organisms such as Brucella displaying high DNA similarity, MLVA became an excellent taxonomical alternative for constructing dendrograms.

The first comprehensive MLVAs with numerous strains performed in 2006, revealed that the Brucella species formed discrete clusters, reflecting the traditional taxonomic status based on host preference (Whatmore et al. 2006; Le Flèche et al. 2006). Predictably, B. canisfell in a separate branch, close to theB. suis biovars 1 and 4. Though the B. suis biovar 2 (from hares), biovar 3 (from pigs) and B. suis biovar 5 (from rodents) fell on separate branches and distant from the central B. suis 1 and 4 clusters, suggesting that the B. suis group was heterogeneous and composed of at least two to three distinct species. It soon became clear that the traditional biovar designation did not always agree with the different MLVA groupings. The biovar classification in B. abortus and B. melitensis started losing its meaning and replaced by the MLVA profiles, and the topology of the dendrograms correlated reasonably well with different geographic areas from which the isolates originated (Maquart et al. 2009).

The DNA molecular studies of the first decade of the 21st century readily differentiated the marine Brucella species and types (Le Flèche.et al. 2006; Bourg, O'Callaghan and Boschiroli 2007). MLVA studies distinguished the various B. ceti strains isolated from the Pacific, Atlantic, and Mediterranean dolphin populations, resolving them into the dolphin and porpoise types (Maquart et al. 2009; Guzmán-Verri et al. 2012). Regarding the B. pinnipedialis isolates, they were split into three distinct clusters (Maquart et al. 2009). Through MLVA and other molecular markers, it was possible to differentiate some putative zoonotic brucellae linked to marine strain categories (Fig. 5), such as Brucella spp., genotype 27 (ST27) (Whatmore et al. 2008). After discoverying Brucella organisms from marine mammals at the end of the 20th century, new brucellae species came into the scene.

Figure 5.

Dendrogram based on MLVA16 analysis of various Brucella species and strains representatives (from Fig. 6, Guzman-Verri et al. 2019; creative commons attribution 4.0 international license).

During the second half of the 20th Soviet scientists frequently reported the isolation and characterization of unusual Brucella strains from red foxes and rodents, including cricetid rodents such as common voles (Rementsova 1962; Rementsova and Kusov 1955; Rementsova, Postricheva and Rybalko 1969; Vana 1980). Except for researchers like Margaret Mayer, who was fluent in Russian, few scientists from Western countries captured these data because most of the publications were in hard-to-reach journals in Russian. Then, in 2007, investigators isolated an Ochrobactrum-like bacterium from infected common voles in the Czech Republic (Hubálek et al. 2007). With the help of molecular tools such as omp2a/omp2b locus analysis and MLVA, taxonomists named this new species Brucella microti, following the host nomenclature tradition (Scholz et al. 2008a). Among the classical Brucella, B. microti seemed the most divergent species and in the deepest branch of the phylogenetic tree constructed by WGSA comparisons. Similar to B. suis biovar 5, the biochemical profile of B. microti displayed broader metabolic alternatives and could survive in soil, resembling O. anthropi (Scholz et al. 2008b). Besides, B. microti was virulent in voles and mice (Jiménez de Bagüés et al. 2010) but never reported in humans or domestic animals.

Description of the next classical Brucella species isolated from primates occurred in the second decade of the 21st century. Some antecedents, published in the late 1960s by the primatologist Mary Pinkerton from the Division of Microbiology and Infectious Diseases in Texas, described unusual Brucella organisms in baboons (Papio spp.) from Kenya (Pinkerton 1967). Then, in 2009 there was a report on two Brucella strains from female baboons captured in Tanzania with stillbirths (Schlabritz-Loutsevitch et al. 2009). This strain, named Brucella papionis was phylogenetically closer to B. ovis; however, it contained perosamine sugar in its LPS, fitting with the smooth antigenic types (Whatmore et al. 2014). This finding supported the idea that the ancestor of the classical Brucella species was a non-classical smooth strain (e.g., B. inopinata-like strain) with an LPS molecule built of perosamine sugars.

The last classical Brucella species described came from an atypical smooth strain with ʻAʼ dominant perosamine epitope isolated from a Saint Bernard dog orchiepididymitis case in Costa Rica, in 1984 (Sequeira et al. 1984). First, the strain became confused with B. melitensis, and then with B suis. This organism, characterized 36 years later by bacteriological and DNA molecular tools, including WGSA, was a distinct species provisionally named Brucella sp. BCCN84.3 (Guzman-Verri et al. 2019). Before this report, there were several publications of smooth B. suis-like strains isolated from dogs in the United States, most of them destroyed (Ramamoorthy et al. 2011).

With the present-day molecular technology, taxonomists can describe a ʻnew speciesʼ from a single isolate. Before the genomic era, the international bacteriology code imposed restrictions on the minimum number of isolates required for describing a new bacterial species (Christensen et al. 2001; Janda and Abbott 2002). Even today, there is no consensus on the number of isolates required, and if they should come from various hosts, locations, different time frame periods, or any other matter. The urge to naming new bacterial species depends on the balance between subjective and objective parameters, and it is independent of the taxonomy subcommittees. A proposal that seems pertinent is to create in each taxon a repository for ʻorphan speciesʼ with provisional codes until more isolates become available (Drancourt and Raoult 2005).

EXTENSION OF THE GENUS AND DISCOVERY OF NON-CLASSICAL BRUCELLA SPECIES

Identifying the various Brucella species and hundreds of strains became a straightforward process after MLVA development (Fig. 5). However, the MLVA relationships in the deepest branches do not always agree with the branching generated by WGSA (Suárez-Esquivel et al. 2017b, 2020). With WGSA as the ultimate tool for taxonomic classification, identifying Brucella organisms from one or few isolates was possible, allowing coherent phylogenetic trees based either in core genomes or in single nucleotide polymorphisms (Foster et al. 2009; Wattam et al. 2014) (Fig. 6). WGSA confirmed the close relationships of Brucella species with preferred hosts and with geographic areas (Kay et al. 2014; Suárez-Esquivel et al. 2017b). Combined with historical data, WGSA allowed tracing the introduction and movements of strains from one area to another, in chronological order (Kamath et al. 2016; Suarez-Esquivel 2020). It is worth noting that the overall topology of the first six classicalBrucella species revealed by WGSA fit well with the phylogenetic analysis performed by traditional bacteriological and phenotypic characteristics in the second half of the 20th century (Holmes et al. 1988: Moreno 1992). This event was unexpected, considering the homogeneity of the group. In one cluster, B. melitensis and B. abortus species remained separated from the B. canis and B. suis (biotype 1) cluster. On a more rooted phylogenetic branch stood B. neotomae alone, and in the deepest branch B. ovis, standing O. anthropi as the outgroup of these elementary trees.

Figure 6.

Phylogenetic relationship of classical and non-classical Brucella species with Ochrobactrum spp. The segments of the rooted tree were magnified by the use of Dendroscope version 3.5.8 to increase resolution. The adapted scale is indicated next to each magnified region. A blue square highlights all classical Brucella species, and in red, the non-classical Brucella species. The dotted arrows in each Brucella cluster indicated the direction of the branch linking to the previous group. The Brucella clusters were rooted with Ochrobactrum spp. The Brucella spp. codes correspond to isolates from the Papua grassland mosaic-tailed rat Melomys lutillus (NF 2653), bullfrog Pyxicephalus edulis (10RB9215 and 09RB8910), chameleon Furcifer pardalis (191011898), blue-spotted ribbon tail ray Taeniura lymma (141012304), Argentine horned frog Ceratophrys ornata (B13-0095), dolphin Tursiops truncatus (F5-99), human (BO2), dog (BCNN84-3), and unknown host (83-13). The tree was constructed following the method described by Guzmán-Verri et al. (2019). Metadata of WGS for the phylogenetic reconstruction is presented in (Supplementary file 1).

After developing powerful molecular tools, the field was ready for novel discoveries. A new Brucella species isolated from an infected breast implant in a patient from the United States in 2008, became an unexpected finding (De et al. 2008). The strain, later named Brucella inopinata BO1 (from Latin, paradoxical brucellae), showed broader metabolic activity, and therefore, initially mistaken as Ochrobactrum spp. (Scholz et al. 2010). Brucella inopinata showed significant genetic and phenotypic modifications, including a temperate broad host range phage, similar to that found in Ochrobactrum spp. (Hammerl et al. 2016), departing from the classical Brucella species. Brucella inopinata stood far apart from the classical Brucella cluster in a phylogenetic tree constructed by WGSA (Fig. 6). Soon, a group of investigators described a second ʻBOʼ isolate from a patient in Australia, with chronic obstructive pneumonia. This bacterium, reported in 2010 and named Brucella sp. BO2 shared genetic and phenotypic characteristics with B. inopinata BO1 (Tiller et al. 2010a), but the smooth LPS of the BO2 species lacked perosamine, a hallmark sugar among the classical smooth brucellae (Wattam et al. 2012). Again, the finding suggested that the ancestor of this non-classical Brucella cluster lacked perosamine homopolymers; then, later acquired by the ancestor of the classical Brucella clade. The sources of the BO1 and BO2 human infections remained unknown. Although they formed a distinct phylogenetic clade, they still stood apart from each other. The same year, the non-classical BO1/BO2 lineage received more members; this time, the strains came from the Australian rodent isolates, found back in 1964, but not fully described at that time.

As mentioned before, along the second half of the 20th century, scientists from different continents repeatedly isolated atypical Brucella organisms in murine rodents and foxes (Zheludkov and Tsirelson 2010, Davis 1990; Vana 1980; Szyfres and González-Tomé 1966). However, the influence of Margaret Meyer on Brucella taxonomy prevailed until her last days, and most of the isolates from wildlife animals were stored in the B. suisʻtaxonomical recipientʼ (Meyer 1976aonomical recipientʼ (Meyer 1976a). In 2010, the time was ripe for recognizing new rodent species. Unfortunately, several of the Brucella collections from the various universities and institutes that reported atypical strains from wildlife animals were lost, destroyed, or not accessible (Kutateladze 2004; Snydman, Anaissie and Sarosi 2008; Casadevall and Imperiale 2010). Fortunately, in place and ready for analysis was the 1964 Australian bacterial collection, labeled as B. suis biotype 3, which survived storage for decades (Cook, Campbell and Barrow 1966),

Rodents in Australia are an exciting group since they just arrived from Asia roughly 5.5 to 1.5 million years ago when sea levels remained low (Rowe et al. 2008). The bacteria isolated from rodents in North Queensland in Australia were a homogeneous group that showed some discrete differences in MLVA and had a perosamine LPS. This homogeneity agrees with the narrower genetic diversity of rodents in Australia compared to those in Eurasia (Rowe et al. 2008). The phylogenetic analysis showed a closer relationship with the non-classical BO1/BO2 clade, a finding that linked the human BO2 isolate from Australia with a potential alternative source of infection. Still, the group represented a distinct lineage known as the Australian Brucella-rodent strains (Tiller et al. 2010b; Wattam et al.2012). The same year, it was a report of novel Brucella species in red foxes (Vulpes vulpes) from Eastern Austria (Hofer et al. 2012). This new species, defined as a Brucella vulpis (Scholz et al. 2016a), clustered together with the non-classical Brucella BO1/BO2/rodent group, but in a more basal group. Brucella vulpis displayed a predominant A-smooth antigenic phenotype, and therefore antigenically related to the classical brucellae.

All the extant species of the genus Brucella, so far, were pathogens of mammals. The incidental Brucella isolates from blood-sucking insects or ticks were judged as B. melitensis, B. abortus or B. suis common species transmitted to these insects from the preferred host (Rementsova 1953; Zheludkov and Tsirelson 2010). Likewise, the Brucella organisms isolated from fish, in lungworms that are parasitic for fish, and in humans with the antecedent of having consumed raw fish, were all considered primary bacteria of mammals that occasionally could cycle through fish (Gelev and Gelev 1988; Sohn 2003; El-Tras 2010; Guzamán Verri et al. 2012). Other than these fortuitous examples, cold-blooded animals were nor envisioned as preferred Brucella hosts. Therefore, the discovery of distinct Brucella organisms in anurans and chondrichthyan fish, and reptiles revealed an unknown phenomenon among the Brucella community, a group mostly composed of medically-oriented professionals (Scholz et al. 2016b).

The diversity of Brucella species in Anura is broad, and at least some of them are pathogenic for frogs and humans, and therefore, with zoonotic potential (Rouzic et al. 2020). The new hosts include big-eyed tree frogs (Fischer et al. 2012), African bullfrogs (Eisenberg et al. 2012), Pac-man frogs (Soler-Lloréns et al. 2016), white's tree frogs (Whatmore et al. 2015), Australian green tree frogs (Latheef et al. 2020), among others (Scholz et al. 2016b). Likewise, the report of new Brucella species in a chameleon and a blue-spotted ribbon tail ray were striking and relevant from the biological and evolutionary perspective (Eisenberg et al. 2017, 2020). Several of these new species/strains labeled by codes await complete characterization (Al Dahouk et al. 2017); however, most of them belong to the non-classical Brucella cluster. The most striking feature of the frog and ray strains included a conspicuous flagellum in these two Brucella species, supporting motility for the life cycle of these strains.

The non-classical Brucella cluster shared a nucleotide identity with the classical Brucella cluster of ∼98%. Members of this lineage contained additional information, such as bacteriophages, insertion sequences, and various genes present in soil/plant-associated Alphaproteobacteria, but absent in the classical Brucella cluster. The WGS based phylogeny placed all these novel species well separated from the classical Brucella species and closer to the Ochrobactrum lineage. All the non-classical brucellae displayed broader metabolic alternatives than the classical species, some of them motile and with a distinct O-chain polysaccharide built of sugars different from perosamine. Several species displayed pathogenicity to their host, and at least three of them capable of infecting humans (Al Dahouk et al. 2017; Eisenberg et al.2020). The closest relative to the classical Brucella species was B. vulpis, isolated from a mammal host.

Under the current trend, it is predictable that investigators will discover new Brucella species in the following years. For the time being, we do not have information on the existence of ʻtransitionalʼ forms that may fill the phylogenetic gap between the so-called classical and non-classical Brucella species. Likewise, we do not know if Brucella/Ochrobactrum ʻintermediatesʼ will be discovered. Based on these findings, some taxonomists proposed combining the Brucella and Ochrobactrum genera in a single genus Brucella (Hördt et al. 2020). For example, Ochrobactrum ciceri isolated from chickpea would become ʻBrucella ciceroʼ and Ochrobactrum gallinifaecis isolated from chicken faeces would be ʻBrucella gallinifaecisʼ and suchlike. In any case, we have to be ready to decide where to draw the taxonomical cut-off value based on analytical and typological processes to resolve, to the best of our knowledge and interest, this epistemological problem.

CONCLUDING REMARKS

Brucella organisms exist, behave and have diversified for millions of years regardless of their taxonomical rank and the human endeavor of giving names to everything. For society, however, taxonomy matters because it is a structure of communication, determining how humans attempt to make sense of living systems. The lack of elements of this structure was why the medical personnel did not believe M. melitensis infected Mother Superior Nun, as proposed by Picado-Twight because they associated this bacterium with goats and not with cows. An unusual situation would have occurred if Picado-Twight had described the isolate as Bang's bacillus, the causative agent of bovine abortion. Then, the medical personnel would be surprised, but without questioning the source, because Bang's Disease was prevalent in Costa Rica at the time

For the same reasons, goats infected with B. melitensis or cows with B. abortus go to the slaughterhouse, while voles infected with B. microti or woodrats with B. neotomae live freely. Imprecise or confusing taxonomy may be a source of misunderstandings and dangerous mistakes. Animals infected with B. melitensis display a higher zoonotic threat than those infected with B. abortus. This subject is not trivial and requires proper taxonomical identification since harm occurs when these two bacterial species become confused. Health authorities from countries in which both bacteria coexist in livestock, displaying high zoonotic risk, know this very well (Wang et al. 2020).

Phylogenetics is also relevant for epidemiological surveys, population studies, planning, and decision-making. For instance, B. abortus introduced in the Yellowstone Ecosystem in the United States at least five times in wildlife, became a significant problem, with contagion from bison to elk, and from elk to bison, being free-ranging elk the self-sustaining brucellosis reservoir and the source of livestock infections (Kamath et al. 2016). This finding alerted national health authorities regarding brucellosis transmission in the Yellowstone reserve wildlife and has increased the disagreements between conservationists with other sectors (Conniff 2019). Likewise, through phylogenetics, it was possible to show the introduction B. abortus in Costa Rica five times, following the importation of cattle from the United States, the United Kingdom, and South America (Suarez-Esquivel et al. 2020). This event has alerted authorities on the requirements for the importation of cattle into the country.

The challenge of working with select agents such as the zoonotic Brucella species is a pragmatic issue that does not depend on scientific rationale alone, but also on political decisions and organizational perspectives that relay on bacterial taxonomy (Enquist, Bertuzzi and Atlas 2016). These difficulties occur, most of the time, regardless of the pathogenicity, attenuation, or lack of virulence of the strains. Taxonomists performing numerical analysis should not ignore these problems. In some circles, taxonomy causes serious disputes, because it has implications in conservation and political decisions on dealing with living systems (Garnett and Christidis 2017). This aspect is also relevant in zoonotic infections such as brucellosis: in these diseases always prevails the risk of pathogens crossing the evolutionary boundaries that separate their natural hosts from human populations and provoke an epidemic disease. Although not of epidemiological relevance, various investigators have reported human-to-human Brucella transmissions (Tuon, Gondolfo and Cerchiar 2017).

Over the past 100 years, brucellologists used the principles of biology, bacteriology, biochemistry, inmmunology, ecology, pathology, phylogenetics, epidemiology, and molecular biology to test hypotheses, but not the fundaments of taxonomy. This characteristic does not diminish the value of taxonomy. Many activities essential to the practice of science are not science. Taxonomy matters because scientists, microbiologists, veterinarians, medical doctors, or any other person related to the field of biology need to identify with precision the system under study to communicate the results and to interact with peers and other groups. However, taxonomy is not impartial and never will be, because it does not depend on observable and measurable characteristics only, but also on typological features that create useful heuristics that provide an efficient and practical basis for comparisons. Otherwise, the taxonomy subcommittees would not be necessary because, contrary to the taxonomic proposals, the scientific truths are not subject to voting in a referendum or election by their members. Therefore, taxonomical proposals will always be elusive manifestos subjected to changes and never laws of nature.

Within this perspective and concerning the species of the genus Brucella and the genus itself, some scientists are ʻsplittersʼ and others ʻlumpersʼ (Moreno, Cloeckaert and Moriyón 2002; Ficht 2010; Scholz, Kämpfer and Cloeckaert 2012; Leclercq, Cloeckaert and Zygmunt 2019; Hördt et al. 2020), a particular division shared with many other disciplines. The American historian Jack Hexter (1910-1996), a specialist in 17th-century British history, alleged that, while ʻlumpersʻ chose to reject differences and emphasize similarities by denying any evidence that did not fit their arguments, ʻsplittersʼ, by contrast, highlight the differences, and fought against simple schemes. Lumpers attempt to create coherent patterns; splitters acknowledge and prefer the complexity of life (Chase 2005; Endersby 2009). Still, the balance has tipped in favor of one side, prevailing what seems the more appropriate and pragmatic proposal for scientific, practical, and social purposes.

The history of the Brucella genus during 100 years is, in brief, the chronicle of scientific efforts and the struggle for comprehending animal and human brucellosis. The first attempts to define the Brucella genus were painful due to the economic and public health implications of joining bacterial zoonotic species together in the same group. In time, though, the benefits became evident for the brucellosis control programs and epidemiological and medical purposes. It was not by chance that the first three Brucella species described were the most zoonotic and virulent organisms that affected livestock, while the last species studied have been those that infect wildlife, remarkably cold-blooded animals.

In one century, investigators have incorporated many species and strains within the genus Brucella, and probably more will be discovered. However, the last minutes concerning the decisions of the Subcommittee on Taxonomy of the Genus Brucella were those corresponding to the 2003 Pamplona meeting (Osterman and Moriyón 2006). There are no published minutes of the meetings held in Merida in 2005, London in 2008, Buenos Aires 2010, Berlin 2014, New Dehli 2016, or Bejing 2018. There is only one report by the Secretary of the International Committee on Systematic Bacteriology published in 2010, that has taken into account the impressions and reflections of the Subcommittee members (Ficht 2010).

Taxonomy matters because it is the best instrument we have for comprehending evolution and for establishing evolutionary relationships among species. Understanding the relatedness of all Brucella members with soil/plant Alphaproteobacteria was a significant step in dissecting the biology of brucellosis, discovering virulent systems, and finding new species. Still, analytical procedures must be linked to the biology of organisms. As Margaret Meyer stated, ʻEven though classification purists and numerical taxonomist insist that host not to be considered when ordering a classification hierarchy, there is experimental evidence to indicate, at least in the genus Brucella, that such behavior can provide clues to the lines of evolutionary descentʼ (Meyer 1990).

How brucellologists will define the Brucella genus depends on which characteristics they would think are necessary to pursue. For instance, most old and new microbiology books depict Brucella organisms as ʻfacultative intracellular pathogens. ʼ This description, however, does not give credit to the genuine nature of these bacteria, which is better described as that of facultatively extracellular intracellular parasites of animals (Moreno and Moriyón 2002), since the primary natural reproductive niche of Brucella organism is the intracellular milieu of animal cells, rather than the extracellular environment, where the bacteria mostly survives for some time. The classical notion has to change to fit the biology of the brucellae. Likewise, members of the genus have revealed new characteristics such as a flagellum, broader metabolic alternatives, and new O-chain polysaccharides. Taxonomists will incorporate all these and several other aspects in the upgraded description of the genus. Still, the definition would never be perfect because part of the evolutionary history of the genus was lost, and filling the gaps would depend on the human interpretation.

Despite the advancements and knowledge gained in brucellosis, two significant challenges require a more profound comprehension of the modes in which Brucella organisms vary and adapt. The first task is discovering and understanding the virulence determinants and evolutionary strategies that make some Brucella species more virulent than others. The second assignment is to unveil why Brucella organisms display host preferences. Answering these questions requires complete knowledge regarding the functionality of all Brucella genes, understanding of how the various genomes work and allow the organism to interact with the environment, and comprehending the selective mechanisms for generating diversity within the same and different host species.

After ten decades, the research tools have improved, and the Brucella genus is flourishing in species. We are confident that in the following one hundred years to come, scientists will resolve these and other questions regarding the species of the genus Brucella and the disease named brucellosis.

ACKNOWLEDGEMENTS

I would like to thank Marcela Suarez-Esquivel from Veterinary School, UNA, for her collaboration on the construction of the phylogenetic trees. I would also like to express my sincere gratitude and celebrate David T Berman, Lois M Jones, Paul Nicoletti, Michel Plommet and Ramón Díaz, who have been relevant examples, and Masters in the field of brucellosis.

Conflicts of interest

None declared.