Retrospective and prospective perspectives on zoonotic brucellosis

1Brucellosis is a vicious disease caused by facultative intracellular extracellular pathogens of the genus Brucella. The bacterium preferentially replicates within phagocytic cells of the reticuloendothelial system, and in the pregnant animal, inside placental trophoblasts. In domesticated animals, brucellosis is mainly manifested by abortion and epididymitis. Under natural conditions, Brucella is horizontally or vertically transmitted. Horizontal transmission occurs through close contact from host to host by means of secretions, sexual intercourse, and more commonly, through liking of aborted fetuses (Figure 1). Although Brucella has been observed to survive for some time in open environments, the bacterium hardly divides and eventually dies. Likewise, some vectors have sporadically been implicated in brucellosis transmission. However, neither of these two last events plays a significant role in the transmission of brucellosis and they are not of epidemiological relevance.

2In humans, the disease is more severe than in domestic animals, displaying a collection of clinical symptoms (;; Figure 2). While there are a few reports of vertical and horizontal transmission between humans, these are rare events. Therefore, brucellosis in humans is strongly linked to the management of infected animals and ingestion of unpasteurized dairy products (; Figure 1). In this regard, there is a clear connection of brucellosis with the domestication of even-toed ungulates, milking practices, and fabrication of cheese and other dairy products. It is, therefore, not accidental that lactase persistence – a genetic trait that allows adults to digest lactose from raw milk – has been traced to ungulate domestication places and in course with the persistence of brucellosis in ancient pastoral people.

3At no other time in human history have the changes in technology, domestication and environment been more rapid and so extreme. For thousands of years humans have created new ways of living and social actions have emerged to minimize the effects of infectious diseases. However, domestication and clustering of wild life reservoirs with narrower genetic backgrounds have provided a steady supply of emergent pathogenic organisms. In this regard, brucellosis constitutes an utmost example of a how animal pathogens can emerge as public and veterinary health problems. Here I review how humans have fostered the illness we now call brucellosis that has accompanied civilization since ancient times, when the malady was recognized by its main symptoms: abortion and fever.

4The seminal discovery of the causative agent of brucellosis, “Micrococcus melitensis” (later named Brucella melitensis), by the British Surgeon Captain David Bruce, his wife Mary Elizabeth Steele and the Maltese microbiologist doctor Giuseppe Caruana-Scicluna has been eagerly described in many assays. These scientists isolated the bacterium from the liver of diseased soldiers in the Mediterranean island of Malta in 1887, a country that holds prominent megalithic constructions beyond 7000 years old. Following this discovery, the Maltese medical doctor Fioravanti Temistocle Archimede Laurenzo Giuseppe Sammut, better known as “Temi Zammit,” found that the causative agent of Malta fever, Mediterranean fever, Cyprus fever, Neapolitan fever, Gibraltar fever, Crimean fever, Cartagena fever, Rock fever, Barcelonan fever, Corps disease, and undulant fever – just to mention a few names used for this vicious malady – was transmitted from infected goats to humans through contaminated milk. Thereafter, Surgeon Captain M. Louis Hughes and Captain James Crawford Kennedy discovered significant details on the zoonotic transmission of brucellosis, including venereal transmission in both humans and animals.

5Ten years after the isolation of M. melitensis, the Danish scientist Bernhard Bag identified “Bacillus abortus” (later named Brucella abortus) in bovine aborted fetuses. reported the isolation of another organism related to M. melitensis (later assigned as Brucella suis) from aborted pigs in United States. But the final link of these zoonotic bacteria was accomplished in 1918 by the outstanding American microbiologist Alice Catherine Evans. Her achievements helped to understand the epidemiology of brucellosis and contributed to the founding of milk pasteurization as preventive measure. Then, in 1920, Louis Meyer and Wilbur Shaw honored David Bruce and proposed to group these pathogenic bacteria within a single genus named Brucella.

6The events that followed all these inspiring investigations have demonstrated the existence of different Brucella species (Figure 3) that cause brucellosis in domestic animals (cows, sheep, goats, pigs, camels, reindeer, and dogs), wild land animals (bison, elk, hares, muskox, caribou, foxes, and several rodents) and sea mammals (dolphin, whales, seals, and walruses;; ). Despite of this diversity the only species that are linked to human brucellosis are B. melitensis, B. suis, B. abortus, and to minor extent Brucella canis; this last specie being the causative agent of canine brucellosis. Apart from this group there are other Brucella strains (e.g., B. inopinata) that have been rarely isolated from humans; however, no connection between zoonotic transmission and disease has been established.

7Members of the genus Brucella are phyllogenetically related to α-Proteobacteria that live in close association with animal and plant cells. From the genotypic perspective the genus is monophyletic with DNA similarity above 97%. In spite of this, Brucella species can be distinguished by single-nucleotide polymorphism analysis, host preference and conspicuous differences in virulence. In addition, there are several straight forward phenotypic differences, being the most obvious the absence of surface O-polysaccharide chain in naturally occurring rough species such as B. canis and Brucella ovis. One interesting feature of the genus is the absence of plasmids and lysogenic phages, a phenomenon that precludes the horizontal transference of genes through classical routes. Based on this, it has been proposed that the extant Brucella species expand clonally within the host environment and that genetic drift depends almost exclusively on mutation and internal genetic rearrangements.

8Brucellosis is one of the few diseases in which efficient live bacterial vaccines (e.g., B. abortus S19 and B. melitensis Rev1) have been developed. Likewise, through history of microbiology very few diseases have more diagnostic tests than brucellosis. As expected, the isolation of the bacterium stands as the gold standard. However, simple techniques, such as the Rose Bengal test, have survived all challenges and are the most wildly used serological assays. This is not by chance, since by the combination of immunization with smooth vaccines, Rose Bengal serological diagnosis and culling of the animals, brucellosis has been controlled and eradicated in many countries of the world.

9Through coordinated measures, brucellosis was finally eradicated from the island of Malta 90 years after the discovery of the disease. Unfortunately, this has not been the fate of other areas around the Mediterranean Sea, mainly in African, eastern Mediterranean, and Middle East countries, where the disease has been endemic for thousands of years and from which brucellosis was spread around the world (Figure 4).

10Analogous to the island of Malta, Butrint in Albania keeps valuable World Heritage Sites that give testimony on the existence of pastoral inhabitants for millennia. Pathological studies and DNA analysis performed in human remains from graves dated 1260–1020 ya, revealed the presence of Brucella as the causative agent of the disease that affected these Middle Age inhabitants in the ancient city of Butrint. In addition to Albania, other Balkans countries such as Macedonia and Bosnia and Herzegovina still struggle with animal and human brucellosis; a phenomenon that was boosted by the decline of veterinary and health services in these countries during the political and armed conflicts in the 1990s. Human brucellosis outbreaks have also thrived in Balkan neighboring countries such as Greece, Italy, and Turkey. Most likely the disease was endemic in these Mediterranean counties since the beginning of civilization. Remains of cheese buried in Pompeii and Herculaneum have been associated with the transmission of brucellosis in Roman imperial times. Likewise, a critical analysis of Thucydides’ history regarding the plague of Athens (2430–2420 ya) suggests the presence brucellosis. Archeological evidence from 7000 ya in the eastern Mediterranean region of Anatolia demonstrated ancient skills to transport milk and to manufacture yogurt and cheese, all vehicles for brucellosis contagion.

11Presumptive human brucellosis cases in skeletal remains from the Bronze Age (4100–3550 ya) have been found in Palestine and Jordan. It is not coincidental that these places are close to the Fertile Crescent and Taurus Mountains, sites where sheep, goats, cows, and pigs – all known to be preferred Brucella hosts – were domesticated between 12000 and 10000 ya. Brucellosis has been also implicated in Bronze Age sites located in Bahrain, Persian Gulf. This archipelago belongs to a region where the dromedary camel – another common Brucella host – was domesticated about 6000 ya. In this area, human brucellosis acquired through the ingestion of camel dairy products is still endemic, mainly in semi-nomadic Bedouin populations. Analyses of human DNA remains from 5000 to 4500 ya have revealed that late Neolithic Europeans displayed lower frequency of lactase persistence than modern extant populations. This is compatible with evolutionary pressures related to the consumption of raw milk and consequently with higher chances to become infected with Brucella.

12Brucellosis is highly prevalent in Asia. Paleopathological evidence indicates that Lapp people in the Artic area of Northern Eurasia domesticated reindeer (the preferred host for B. suis biovar 4) 3000–2000 ya or even earlier and that these inhabitants also suffered from brucellosis. Bovine and swine were already present in China, Mongolia, and Korea, at least 5000 ya or even before, shortly after their domestication in the Fertile Crescent. It seems that water buffalo (Bubalus spp.) was also domesticated in China about 4000 ya. However, milk and derived dairy products are not commonly found in East Asian cuisines, a culinary activity that is compatible by the lactose intolerance distribution in these populations. Two exceptions are human groups living in the Asian steppes and Mongolia who still consume milk and fermented dairy products; then keeping lactose tolerance and human brucellosis. It is likely that brucellosis was endemic in these areas before imperial times.

13Human brucellosis is highly prevalent in India. Bovine Bos indicus zebu breeds were domesticated in the Indus Valley region (today Pakistan) about 7000 ya. An independent domestication of water buffalo was achieved in India about 5000 ya. Infected water buffalos shed Brucella in the milk; however, these animals are more resistant to Brucella induced abortion than Bos species. An independent domestication of goats also occurred in the Indus Basin in Pakistan already 9000 ya. Considering these events, it is striking that up to 80% of the Indian population is lactose intolerant. It has been determined that the mutation for lactose tolerance was introduced later on to eastward India from the Middle East. This suggests that ingestion of dairy products started later in India than in other regions, and with it, zoonotic brucellosis. Another alternative comes from how Indians prepare their milk: they often ferment it in the form of lassi or paneers, processes that break down the lactose and also kills Brucella organisms.

14Human brucellosis was described in Mediterranean African countries more than 100 ya. It is likely that brucellosis was present in human settlements in Northern Africa already 3000 ya and highly prevalent in Egypt during biblical times (e.g., OT, Isaiah 37:8–9, and 2 Kings 19:8–9). Studies performed in Egyptian archeological sites dated 750 B.C. have revealed several human hip bones with signs of brucellosis in this region. Brucellosis in southern Africa was detected in dairy herds as early 1913 and the first human cases in 1921. It seems that the introduction of Indian and Eurasian bovine, sheep, and goat breeds into Africa occurred rapidly after their domestication in the Fertile Crescent. Nevertheless, it has been established that different African ethnic groups have distinct lactase gene mutations that arose independently in different locations between 6800 and 2700 ya. These data fits well with archeological evidence suggesting that pastoral peoples reached eastern Africa in different migration waves, about 4500–3500 ya.

15It is feasible that brucellosis existed in indigenous African Artiodactyla species (which include a significant number of potential Brucella hosts) long before the introduction of domesticated herds. A paleopathological study has suggested the presence of Brucella infections in australopithecines, already 2.5–2.3-million ya. As expected, this proposal not only has implications on the origin of the disease in local African fauna but, remarkably, also on the feeding habits of these human ancestors. In spite of this, it seems that Brucella infections in indigenous African mammals remain low and only relevant when wildlife ungulates become in contact with infected domesticated cows, goats, or sheep.

16The only indigenous Brucella specie in the American Continent seems to be Brucella neotomae, first isolated in United States from desert wood rats in 1957. B. neotomae is confined to these rodents with no other known hosts. The absence of domesticated ungulate reservoirs before European colonization very likely circumvented the presence of zoonotic brucellosis in the New World. This is revealed by the close to 100% lactose intolerance of adult Amerindians and in Inuit people. Thus, it is unlikely that American inhabitants – who populated the continent between 12000 and 4000 ya – ingested milk from potential Brucella infected wild life ungulates such as bison, muskox, elk, or caribou. Therefore, the various zoonotic Brucella species were likely introduced in America during the last decade of the fifteenth century by the first Spaniards conquerors following the arrival of cattle in the colonies. At that time brucellosis was probably highly endemic in the Iberian Peninsula. This is supported by the discovery of human remains from the late Middle Ages displaying pathological signs of brucellosis and by the description of the disease in Spain. For instance the clinical description of the “lousy fever” suffered by the mystic poet St. Teresa of Jesus – born 20 years after Christopher Columbus opened up the Western Hemisphere to European colonization – is compatible with brucellosis.

17As for other infectious diseases, the spreading of brucellosis from the “Old World” to the “New World” very likely was a significant outcome of the conquests. It has been well documented that during his second voyage to the American Continent in 1493, Christopher Columbus introduced a significant number of cattle and pigs. Very probably by these means the introduction of brucellosis in the continent, including the contamination of indigenous fauna such as bison. Brucellosis was detected in a Yellowstone American buffalo herd already in 1917. Until the first half of the twentieth century, European cows shared with bison herds the same pasture lands making likely cross infection (Figure 1). Indeed, brucellosis in North American bison and elk has been related to cross contamination of bacterial strains (including vaccine strains) from infected European bovine breeds. Furthermore, the same B. abortus biovars (1 and 2) are found in both classes of bovine herds. The disease in the American buffalo is similar to that of domesticated cattle; though it is believed that bison, like water buffalo, may display some resistance to Brucella induced abortion.

18The origin of B. suis biovar 4 infecting Canadian and Alaskan caribou and muskox has been traced to imported reindeer from Siberia, early in the twentieth century. Domesticated reindeer should be also considered a potential source of zoonotic disease since brucellosis – caused by B. suis biovar 4 – has been found in Eskimos. Alternatively, B. suis biovar 4 could have arrived with infected caribou and muskox through the Bering Land Bridge during the last glaciation.

19Human brucellosis was prevalent in Mexico, USA, and Canada for centuries. The first human cases in North America were recognized between 1889 and 1894. With the exception of Mexico, nowadays the presence of human brucellosis has become a rare event in northern hemisphere of the American continent. This was the result of the successful pasteurization of dairy products and the application of control programs based in extensive immunization of herds with smooth Brucella vaccines, diagnostic tests such as Rose Bengal and complement fixation and efficient culling and management of animal flocks during the second half of the twentieth century. In contrast, the absence of coordinated control programs, poor management of animal flocks, and the introduction of vaccines with low efficacy have kept brucellosis highly prevalent in Mexico, Central America, and most South American countries.

20Brucella canis – the last Brucella zoonotic specie described – was discovered in Southern United States in the late 1960s. Dogs were the first animals to be domesticated in the world. The earliest archeological vestiges are from Siberia dated 33000 ya; while in the American continent the oldest known ancient remains date 11000 ya. Then, it was expected to find B. canis in dog’s wild relatives. However, there are no reports of B. canis in wolf or coyote packs and these wild canines seem to display some resistance to smooth Brucella species. Nevertheless, it seems feasible that B. canis evolved in dog’s ancestor after predation of B. suis biovar 4 infected hosts in Asia (e.g., caribou/reindeer), since these two brucellae species are closely related (Figure 3). Moreover, wolves and Artic foxes can become naturally infected with rangiferine brucellosis. As other zoonotic brucellae, B. canis might have penetrated to the American Continent during the European colonization. Alternatively, B. canis could have traveled in infected dogs through the Bering Strait already 12000 ya. Presently, canine brucellosis has spread throughout the American Continent. In any case, the zoonotic potential of B. canis is low and just sporadic human cases have been reported in the world.

21Human and animal brucellosis were very important diseases in New Zealand and Australia as these countries keep large numbers of sheep and bovines. As expected, lactose intolerance occurrence in indigenous people from Oceania is above 95%, a fact that agrees with the absence of indigenous large mammal animals in this region. It is therefore likely that human brucellosis stared with the arrival of infected domestic livestock to Oceania lands in the eighteenth century, through “The First Fleet” and in the ships commanded by Capitan Cook. Before this, the only placental mammals (and potential Brucella hosts) in Australia were bats, some indigenous rats, mice, and the feral dog named “dingo” introduced from Asia 5000 ya. In New Zealand the only placental mammals were bats, kiore rats and the Polynesian dog named kurı-.

22Bovine brucellosis was first recorded in New Zealand in 1893 and eradicated 106 years later by an aggressive program that included S19 vaccination, testing, and slaughter of the infected herds. A comparable control program was followed by Australia with a great success. The first cases of ram epididymitis caused by Brucella ovis were recorded in New Zealand in 1953. Since B. ovis is not pathogenic for humans or other species of animals and mainly affects rams, there are no clear historical records regarding this disease before its discovery. In addition, Brucella strains have been isolated in rodents and foxes in Australia and two unconventional Brucella strains (one in Australia and one in New Zealand) have been detected in humans. In spite of this, no links with the transmission from animals to humans has been established in these cases. Canine brucellosis has just been recently found in domestic dogs in Australia but never reported in dingo or kurı- dogs. Presently, human and animal brucellosis are just sporadic in Australia and New Zealand, remaining feral pigs as the only source of human infections.

23Pathogens and hosts evolve in response to each other and the genetic diversity of both parties represents a pool of possible variants to maintain adaptation via natural selection. Thus, the “arm race” between Brucella and preferred hosts has been driven by genetic adaptation of the bacterium virulent systems confronted with the evolving immune defenses of the host. Domestication, anthropogenic modification of wild life and selection of animals by humans are not neutral phenomena. In each event a concomitant selection of the parasitic microbiota occurs. Consequently, it is expected that the prevalent extant Brucella strains have been selected through “narrow funnels” connected to these processes.

24It does not seem by chance that the most virulent Brucella species with higher zoonotic spectrum are those from domesticated animals; while those that display lower pathogenicity and zoonotic potential are those from wild life animals (Figure 5). Reports of human infections from wildlife reservoirs are scarce. Moreover, within the zoonotic brucellae there are some species that are more virulent than others (e.g., B. melitensis > B. suis biovars 1, 3, and 4 ≥ B. abortus > B. canis;;;; ). In contrast, Brucella ceti and Brucella pinnipedialis preferentially infecting free living cetaceans and pinnipeds, respectively, have seldom been found in other animal groups and their zoonotic potential and overall virulence for other animal species, including bovine and swine, seem low. Likewise, Brucella species and strains (e.g., B. neotomae, B. microti, and B. suis biovar 5) having preference for wild land mammals are confined to their natural hosts and seldom found in domestic animals or humans. Therefore, it is expected that the most prevalent virulent Brucella strains were selected during the domestication of animals.

25The selection of Brucella towards lower or higher virulence has been demonstrated experimentally. Through mutagenesis of genes coding for the so called virulent determinants or regulatory molecules Brucella may become attenuated. Likewise, by means of genetic manipulation or selection through serial passages into animals, Brucella strains can become robust pathogens.

26In addition of displaying host preferences, the various Brucella species and strains also form genetic groups that relate with distinctive geographic origins. This means that Brucella clones rapidly expand and transmit within domesticated groups of animals. In spite of their high DNA similarity, the various bacterial species and strains are selected and form discrete family clusters. These observations parallel those showing that some Brucella strains may have been removed or minimized from the bacterial pool as consequence of the control programs. Indeed, several B. abortus biotypes described decades ago have not been isolated for more than 40 years; instead, predominant variants remain in bovine herds. Therefore, it is feasible that Brucella selection towards higher transmissibility and replication occurs through successive infections in confined hosts, as proposed for the evolution of other diseases.

27One exception is B. ovis (Figure 5). Although this bacterium may have been also subjected to selection processes during the domestication of sheep, it remains non-pathogenic for humans or for other animals. In general, rough brucellae such as B. ovis are less virulent than their smooth counterparts and have narrower ability to infect other hosts. It may be that B. ovis was already selected towards a higher affinity for venereal transmission in sheep before domestication of ovine, as suggested before. Moreover, the basal “deep” phylogenetic location of B. ovis in relation to B. abortus, B. melitensis, B. suis, and B. canis clusters, also suggests earlier adaptation of B. ovis to its host.

28Other trend that has favored the prevalence and dissemination of brucellosis corresponds to the intensive exploitation of productive animals. Humans have taken advantage of the innate social behavior of ungulates and canines and clustered them in small areas. In addition, following anthropocentric purposes, the genetic background of these domestic animals has been narrowed. As in other infectious disease, lower genetic diversity and crowded effect may favor transmission and select for faster replicating organisms with major zoonotic potential. Examples of these were observed in the early days of brucellosis in Malta, and more recently in foodborne outbreaks in Peru and massive outbreaks in Inner Mongolia, threatening hundreds of thousands of people.

29Inner Mongolia, which keeps the largest sheep population (18.2% of the flock), also ranks first in animal and human brucellosis in China. In 2007, new brucellosis cases were reported in 85 out of 102 districts in Inner Mongolia, with positive prevalence remaining in the other 47 districts. From 1996 to 2010, 78246 human cases were detected with 90% of the new cases reported between 2005 and 2010. This accounts for 40% of the near 200000 cases detected in China for this period. In 2010, this figure reached 47.2%. According to various models, this may be just the “tip of the iceberg” and it is expected that the number of human cases will increase dramatically in the following years. Moreover, it has been demonstrated that in endemic areas about 20% of the infected individuals remain undiagnosed. Indeed, family members of the patients with brucellosis are under increased risk of acquiring the disease. Thus, family screening in endemic areas is recommended.

30Novel circumstances for fast transmission of zoonotic brucellosis have also been observed in confined semi-nomadic Bedouins infected from camel’s milk and commercial dog kennels. Camels cohabiting with goats and sheep in small areas are becoming a common practice in Middle East and Arab countries. Dog packs seldom exceed more than a dozen individuals. Consequently, in crowded kennels B. canis spreads rapidly inducing massive abortions in bitches, testicular degeneration in males, and becomes a zoonotic risk. Therefore, intensive exploitation and clustering of animals in poor epidemiological control conditions may favor selection for faster Brucella transmission and zoonotic disease.

31Distinct Brucella clusters have also been identified in wild life animal populations located in areas separated by natural barriers. As with domesticated species, anthropogenic modification of wild life may also have narrowed the genetic diversity, impact host susceptibility and pathogen transmission. A noteworthy event has been the threatening of the American buffalo which was close to extinction. Thus, the prevailing bison herds have been founded by a small group of few surviving individuals. This is relevant since North American bison herds remain infected with B. abortus. The European counterpart of this incident corresponds to the Alpine ibex (Capra ibex). Historically these wild goats were endemic throughout the European Alps. Due to excessive hunting and constrain of their natural habitat, the ibex herds in Central Europe declined to low dangerous numbers. As consequence, the founding of new ibex herds in the Alps come from a pool of few animals, narrowing their genetic diversity. In certain areas ibex herds are infected with B. melitensis strains displaying also high seroprevalence. Therefore, these wild goats may become a source for the reintroduction of B. melitensis in domestic ruminants and humans in Central Europe.

32Another example relates to the hunting of marine mammals, linked to the overexploitation of their natural food resources and contamination of the seas. These negative activities have promoted clustering of different Brucella infected marine mammals in reduced areas where food is available, causing excessive competition, undernutrition, stress, and immunosuppression. As revealed by the increasing brucellosis case reports in some species of cetaceans over others, these unnatural conditions may favor the selection of Brucella organisms with higher transmission rate.

33Brucella divergence seems linked to selective forces within the host environment, and consequently, to the evolution of the host. However, this constrain is not absolute and Brucella species living in wild life or in semi-domesticated hosts may still qualify as potentially pathogens for humans and domestic animals. The phenotypes of B. ceti, B. pinnipedialis, B. microti, and B. neotomae correspond to smooth types equipped with all known “virulent” factors. Up to now “mysterious” subtle differences with the classical zoonotic Brucella have kept these other wildlife species out from causing disease in humans. But the correlation of the various species in relation to host preference is not perfect and phylogenic patters suggest that Brucella organisms are capable to breakdown the species barrier and “jump” from one mammal order to a very differ one (Figure 3). Eventually, this might favor the persistence of a distinct Brucella clone in a different “preferred” host.

34In the presence of brucellosis, management becomes highly demanding. Domesticated animals and humans have coexisted for millennia without significant intervention measures to control the disease. It is likely that a large part of the prevalent Brucella zoonotic species was selected in flocks during this long-lasting initial period. In some regions of the world, mainly in low income countries, these weak control actions are still common. It is likely that a fraction of the genetic background of both humans and animals has been also shaped during the coexistence with Brucella organisms; mainly nearby to the regions where domestication took place.

35After the discovery of Brucella organisms and their mode of transmission, direct measures toward the control and eradication of the disease were taken in several countries. As stated, killing of the bacterium by milk heating was one of the first procedures that prevented the transmission of brucellosis. A second relevant action was the discovery of diagnostic techniques capable to distinguish infected animals. Third, was the development of efficient vaccines for protecting bovine, caprine, and ovine herds. In addition, in some areas systematic slaughtering of the infected animals reduced the density of the bacterium. Though, the control of brucellosis by the sole action of culling the infected animals is extremely expensive and not practical under high disease prevalence conditions. Following this, massive vaccination in combination with serological diagnoses and culling of the infected animals has become the chief strategy for the control of brucellosis. Countries where brucellosis has effectively been controlled have used the following procedures: reliable live vaccines (e.g., S19 and Rev1), adequate immunization protocols (e.g., single dose vaccination, reduced dose), extensive protection coverage (e.g., 100% of the herds at risk), suitable diagnostic tests (e.g., Rose Bengal, RID, Complement fixation, iELISA), sustained removal of the infected animals and restriction in the traffic of animals from infected herds to free herds (e.g., control transhumance herds;;;;; ). Accordingly, these countries have also narrowed the genetic pool of virulent brucellae and succeeded in eradicating human brucellosis.

36The basic reproductive number, also known as R0, is the average number of secondary infections arising from one infected individual in a completely susceptible animal population. That is, for the disease to spread and for an effective animal to animal Brucella transmission it is required that the pathogen’s R0 exceeds 1 (Figure 6). In contrast if R0 < 1, then the disease has the tendency to fade away. Higher the R0 value, higher will be the number of subsequently infected individuals. Concomitantly, larger and denser the population of susceptible individuals higher would be the chances for the pathogen to achieve a steady and successful adaptation in the host. As consequence of a collection of unsuccessful events in many middle and low income countries, the R0 value exceeds 1; thus keeping the disease and the zoonotic potential high.

37Yet, brucellosis is a complex disease and significant political and economic interests are often in play. Of all the problems in control programs, the introduction of low protection rate vaccines stands as a major drawback. Apart from their failure in controlling brucellosis, there are long-term consequences in the use vaccines with low efficacy. In this direction a variety of evolutionary scenarios are possible, including the selection of more virulent Brucella strains.

38Effective vaccination limits Brucella infection, restricts shedding, hampers transmission from animal to animal and diminishes the risk of zoonosis. In addition, when combined with removal of positive infected animals, efficient vaccination may select for breeds with higher resistance against the disease. Immunization with efficient vaccines may replace natural infections by inducing competent immunity; likewise, culling of the infected animals replaces the natural selection of hosts displaying reproduction impartments, such abortion, placenta retention, and infertility. Eventually, these sustained combined strategies establish a R0 < 1 with the concomitant peter out of the disease. Moreover, when R0 < 1 the pathogen evolution rate towards higher virulence may be overcome and virulent field Brucella strains eradicated from domestic flocks.

39In contrast, inefficient vaccines currently used in many countries for the control of bovine, sheep, or caprine brucellosis might work in the opposite direction. Indeed, the protection afforded to non-immune animals by the presence of sufficient numbers of immune individuals, known as “herd immunity” (Figure 6) is threatened if the immune status of the herd is low. That is, inefficient vaccines may promote a fertile niche in weakly immune hosts allowing virulent Brucella to be transmitted though vaccinated animals. In curse this will increase the number of secondary infections. For example, if the anti-Brucella vaccine fails to generate immunity in a fraction p of those animals vaccinated, then to achieve herd immunity we need to vaccinate a proportion of individuals equivalent to R0 - 1/R0(1 - p) (Figure 7). Hence, if p is too big it may be impossible to eradicate brucellosis as it has been the case in many countries where vaccines of low-efficacy have extensively been used.

40In cases in which the relative fitness of competing pathogens depends on the immune status of their host, low-efficacy vaccines inducing responses below the protective threshold may also prompt pathogen evolution towards higher virulence (Figure 8; ). Selection pressures may work in the same direction observed for non-sterilizing antibiotic treatments, in which the surviving microbes may display a higher resistance edge. Furthermore, anti-Brucella vaccines lacking some fundamental virulent molecular determinants or displaying a large collection of mutations, give a competitive advantage to virulent strains possessing full set of these factors, as it has been already shown for rough Brucella strains devoid of O-polysaccharide antigen.

41In certain contexts vaccination induces a “sense of security” in non-specialized general public. This sense of security is sustained in the trust and faith that people have developed on vaccines that successfully prevented and eradicated diseases. If the vaccine is highly efficient, then the faith and trust is justified and not harm is done. However, this complacency is particularly dangerous when vaccines with low efficacy and short-term protective duration are introduced; then, a “false sense of security” may be generated, mainly when the information is not given properly. Generally speaking, the false sense of security lays between the optimal expected efficacy for a given vaccine and the real performance of that vaccine (Figure 8) and it has a direct impact in the assessment of herd immunity. The use of anti-Brucella vaccines displaying low efficacy could generate a false sense of security in the minds of livestock farmers and Veterinary Health authorities, who may believe that herds are fully protected.

42Under low threshold immunity conditions the host becomes a favorable environment for the replication and spread of field bacterial strains and a potential niche for Brucella selection. This is particularly relevant when prevalence is high and surveillance is low to begin with and when the favored virulent microbe emerges within a restricted population. These arguments are supported by several mathematical and epidemiological models.

43In the light of unrestricted use of antibiotics the emergence of antibiotic resistant Brucella clones should not be excluded a priori. However, in contrast to other bacterial pathogens, antibiotics do not seem to play a significant selective role in brucellosis. Due to economical, epidemiological, and public health reasons, treatment with antibiotics has been precluded in productive animals with brucellosis. One exception is canine brucellosis. Pets with brucellosis are frequently treated with antibiotics, not always with success. Antibiotics have also been used in brucellosis research for selecting specific strains displaying antibiotic resistance, This has also important implications in the accidental transmission of Brucella organisms in the laboratory and the potential role of this bacterium as biological weapon. In spite of this, most Brucella clinical isolates remain susceptible to the classical antibiotics used for treatment of brucellosis. In broad terms, people constitute a dead end for Brucella transmission; therefore, antibiotic treatment of infected humans is not epidemiologically relevant (Figure 1). This phenomenon may relate to the absence of plasmids and lysogenic phages in Brucella organisms, as it has been explained elsewhere. In this sense, the short-term emergence of antibiotic resistant Brucella does seem plausible.

44The capabilities of Brucella to infect and propagate in the preferred hosts follow at least five stages: (i) ability to invade; (ii) power to circumvent the initial defenses; (iii) competence to replicate; (iv) capacity to be transmitted; and (v) endurance to be maintained within the host population. How, when, and where pathogens cross the boundaries that separate their natural hosts from human populations and provoke an epidemic disease, is not entirely known. Human-to-human Brucella transmission would require that the pathogen’s R0 exceeds 1. Although Brucella animal pathogens have already achieved the first three stages in humans and in occasions the fourth stage, still the disease in humans is terminal and human mediated transmission is not of epidemiological importance (Figure 1). Thus, Brucella has not yet reached the R0 threshold to emerge as permanent pathogen within human populations and contagion remains dependent on animal reservoirs. However, under these circumstances human brucellosis may display a R0 above the threshold that depends on the zoonotic infection rate. For instance, as consequence of high prevalence in domestic animal reservoirs (sheep) in Inner Mongolia, the R0 for human infection corresponds to 1.8. Under the prevailing control measures and use of low protective vaccines it was predicted that human brucellosis will continue to increase for the next decade in China.

45Ecological factors and human activities may influence and induce changes in the microbial virulence patterns. But to distinguish Brucella clones displaying higher virulence is not an easy task. Brucella organisms lack classical molecular markers commonly used to trace virulence such as toxins, fimbria, plasmids, capsules, antigenic variation or resistant forms. The so called “virulent factors” are intertwined with the overall Brucella structure and physiology and are found in practically all Brucella species examined, independently of their pathogenicity for humans. Moreover, many of the molecular determinants such as cell envelope components, secretion systems, regulatory systems, transporters, and effectors assigned as virulent factors are also found in soil bacteria related to brucellae. As stated before, Brucella species form a compact genetic cluster and display host preference commensurate with their phylogenetic dispersion. Therefore, the major scientific challenges that brucellosis research confronts relate to the identification of those discrete genotypic and phenotypic changes that have favored the adaptation to the preferred hosts and those molecular determinants that have made some Brucella species more virulent than others. In addition, efficient vaccines for dogs, pigs, water buffalo, and camels, as well as for some wild life animals, are required.

46During the first half of the twentieth century through the early 1980s, efficient live Brucella S19 and Rev 1 vaccines for preventing brucellosis were developed together with robust procedures for testing their safety and efficacy. In addition, various inexpensive and straightforward serological tests as well as good management strategies were successfully implemented. Those were the days when brucellosis control programs succeeded in many parts of the world. Circumstances have changed and the global agenda has been modified towards other interests. Taking into account that ignorance persists and economic profits pursue without other considerations, it is difficult to envision what will happen and how biological and cultural evolution will shape brucellosis and human battlement against this zoonotic disease.

47For much of the twentieth century the misuse of antibiotics has taken place with little concern on the evolutionary consequences and selection of antibiotic resistance hypervirulent bacterial strains. It is our contention that we should not repeat that complacency with misuse of poor brucellosis vaccines, dubious immunization protocols, expensive diagnostic tests, and inadequate management procedures.

48Edgardo Moreno wrote and revised the manuscript and made the figures.

49The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.