Clayton C. Caswell Angela Arenas‐Gamboa and Jeff T. Foster Human domestication of livestock has created numerous opportunities for pathogens to jump from animals to humans. Indeed, zoonotic infections have raised the profile of many pathogenic bacteria of animals, allowing for in‐depth research into pathogenesis. For Brucella spp., this livestock–human association goes back to at least Roman times and likely far earlier (D’Anastasio et al. 2011). The discovery of a novel bacterium in 1887 in British soldiers stationed on the island of Malta was later identified as coming from goats (Godfroid et al. 2005). Initially classified as Micrococcus melitensis (Bruce 1887), an array of microbiologists and taxonomists contributed to describe this species, later reclassified as Brucella melitensis. Unknown at the time was that this discovery was only the beginning of our understanding of a diverse genus of bacteria with many animal hosts, from livestock such as cattle, pigs, and goats, to dogs, rodents, frogs, and fish. However, pathogenesis and basic biology of this genus are largely known from three species and their natural hosts, Brucella abortus in cattle, B. melitensis in goats, and Brucella suis in pigs. Brucella species are Gram‐negative, facultative intracellular bacteria that are non‐motile, non‐spore forming, and extremely small (0.5–0.7 mm × 0.6–1.5 mm) coccobacilli (Alton and Forsyth 1996). The genus Brucella is part of the α‐2 subclass of proteobacteria, and is currently comprised of 12 species, six of which are well‐characterized including B. melitensis, B. abortus, B. suis, Brucella ovis, Brucella canis, and Brucella neotomae, which preferentially infect goats, cattle, swine, sheep, dogs, and desert woodrats, respectively (Moreno 2014). These infections typically result in reproductive pathologies, including abortions and sterility in animals, but Brucella strains are also capable of infecting humans, typically manifesting as undulating fever. Of the “classical” Brucella species, B. melitensis, B. abortus and B. suis are known to cause significant infections in humans, but B. canis and B. neotomae can be zoonotic, while human infection by B. ovis has not been reported (Moreno 2014). Several “atypical” Brucella species have been identified in a variety of wild animals, and while some of these strains have also been isolated from humans, the full zoonotic potential of these Brucella strains remains to be fully elucidated (Whatmore and Foster 2021). Brucella genomes consist of two circular chromosomes, chromosome I is around 2.1 Mb, chromosome II is around 1.2 Mb (López‐Goñi and O’Callaghan 2012). Despite significant variation in host preference, the genome sequences of the Brucella species are highly similar (approximately 97% DNA identity), leading to the initial suggestion that the “species” of the Brucella genus should be considered biovars of a single species, although genomic approaches have upheld species designations of most Brucella species (Whatmore and Foster 2021). However, the genetic determinants of host specificity of different Brucella species are not well defined. Three Brucella species have been traditionally considered most pathogenic to humans, B. abortus, B. melitensis, and B. suis. While there has been a particular focus on these species because of their effects on livestock and human health, other Brucella species are also considered highly infectious in their natural hosts, and all exhibit pathologies in at least one group of animals. As a result, the pathogenesis of other Brucella species has been poorly explored and is often viewed through the lens of human disease. This chapter focuses on the three species, B. abortus, B. melitensis and B. suis, but it should be noted that much remains unknown about host–microbe interactions in Brucella, particularly etiology and host response in recently discovered species. For example, a recent human infection that likely originated in infected frogs or possibly reptiles greatly expanded the breadth of pathogenic species and our understanding of suitable hosts as well as the detection of several atypical strains in humans, presumably of animal origin (Whatmore and Foster 2021). An overview of Brucella species, their target hosts, and mechanisms of transmission is depicted in Figure 16.1. Figure 16.1 Overview of Brucella species, natural hosts, target organs, and mechanisms of transmission. Brucella are intracellular pathogens, with limited evidence of long‐term persistence or survival outside of hosts for most species. Animals thus need to come into contact with bacteria from infected hosts and this primarily occurs during birthing or lactation, although venereal transmission occurs in some species such as B. canis and B. ovis. Brucella was initially thought to target tissues containing erythritol, preferentially use this sugar as a carbon source, with direct implications for transmission and epidemiology (Petersen et al. 2013). Reproductive tissues, including mammary glands, testes, the uterus, and placenta are rich in erythritol, at least in cattle, so milk consumption (by calves or humans) or contact with tissues from birthing events allow for transmission. However, the story is more complicated in other animals, such as goats and pigs, where erythritol is not concentrated in reproductive tissues and is not connected to virulence. Regardless of the molecular target used for tissue tropism, Brucella growing in the placenta of an infected host can induce spontaneous abortion, releasing large amounts of the bacteria. The apparent host preference of Brucella species with specific hosts may at least be partly due to the likelihood of transmission with host species; other individuals of the host species are most likely to contact contaminated milk or be present during birthing or abortions. Humans are one of the few species at these events and infection is relatively common when in close contact with infected livestock. When animals of different species share the same environment, cross‐species transmission can occur, either among livestock or between livestock and wildlife (Godfroid et al. 2014; Moreno 2014). Despite the ability for cross‐species transmission and multiple known hosts for many species, Brucella is relatively genetically homogeneous across the genus. At the same time, Brucella species form distinct lineages that are typically associated with a host species and with virtually no lateral gene transfer (i.e. no sharing of genetic material between lineages or species; Wattam et al. 2014). Unsurprisingly, the best sampled and most fully characterized Brucella come from livestock, with B. abortus from cattle, B. melitensis from goats and sheep, and several B. suis lineages from pigs, being well described genetically (Whatmore and Foster 2021). As detailed below, B. suis is also where these host associations start to break down, with some strains found in rodents, caribou, and hares, in addition to pigs (Godfroid et al. 2014). Traditional epidemiological approaches using biochemical methods and biotyping have been largely replaced by genetic approaches. Brucella are clonal and relatively homogeneous genetically but nonetheless their genomes contain sufficient genetic information for phylogenetic comparisons and molecular epidemiology (Wattam et al. 2014). The two most common approaches, using either variable number tandem repeats from amplified regions of the genome or single nucleotide polymorphisms from whole genomes, have enabled detailed epidemiological studies (reviewed in Whatmore and Foster 2021). Studies have involved cross‐species transmission and historical spread across a broad region among livestock and wildlife (Kamath et al. 2016), traceback studies of infected travelers or livestock (Garofolo et al. 2013) and assessing a countrywide outbreak (Allen et al. 2020). The “classical” Brucella species traditionally associated with disease manifestation in domestic animals are best known to cause reproductive failure in their target hosts. However, cross‐species infections can occur, with most domestic species being susceptible to the bacterium to a certain extent. Brucella has a marked tropism for macrophages and the placenta. However, the pathogen can infect and replicate in a variety of cell types, ranging from osteoblasts, osteoclasts, and fibroblasts (e.g. Khalaf et al. 2020), microglial and endothelial to epithelial cells among others. Owing to this wide array of susceptibility to infection, brucellosis can not only manifest as a reproductive disease, but can also display non‐specific symptoms ranging from osteoarticular to neurological disease in animals. Initial infection of B. abortus in cattle is typically manifested as an abortion storm during the last trimester of gestation in a large percentage of animals present on a farm, followed by a cycle of normal parturitions and random abortions in subsequent pregnancies. Interestingly, some cows can deliver weak infected calves, which typically serve as major sources of infection to other animals. Other clinical signs in cattle include reduced milk production, an increase in the number of somatic cells in the milk (indicative of mastitis), reproductive failure and the development of hygromas (fluid‐filled joint spaces) with or without the presence of any reproductive disease. In bulls, brucellosis can be inapparent with the most prominent feature being the development of chronic epididymitis, seminal vesiculitis and orchitis, the latter being often the result of chronic inflammation. Affected bulls can develop permanent infertility. It must be noted that venereal transmission is not a major route of infection for B. abortus in cattle under natural conditions, which is different from that observed in cases of B. suis, B. ovis or B. canis infection in their natural hosts. However, artificial insemination with contaminated semen can serve as a source of infection. B. abortus has a strong tropism for the pregnant uterus and placenta, including trophoblasts, inducing placentitis and endometritis (Carvalho Neta et al. 2010). Gross pathological changes of the placenta and pregnant uterus are not specific for the infection and are insufficient for an accurate diagnosis (Xavier et al. 2009). Importantly, the placental lesions are non‐uniform within placentomes, with some appearing normal and some demonstrating extensive areas of necrosis. Microscopically, placental changes are characterized by the presence of neutrophils and macrophages admixed with large areas of necrosis, characterized by the presence of fibrin and edema, with or without vasculitis (Xavier et al. 2009; Carvalho Neta et al. 2010). Large numbers of bacterial colonies can sometimes be seen within trophoblasts. In aborted fetuses, pleuropneumonia is the most common lesion (Xavier et al. 2009). Grossly, the pleura is thickened and covered by fibrin typically indicative of a fibrinous pleuritis (Carvalho Neta et al. 2010). The presence of fibrin can also be seen in other fetal body cavities, including the peritoneal and pericardial areas. Reproductive tissues and aborted fetuses should always be collected and examined carefully, as brucellosis must always be differentiated from other diseases that cause abortion in cattle. A definitive diagnosis must be supported by laboratory tests including serology and bacterial isolation. An alternative to isolation is the use of polymerase chain reaction‐based assays, although laboratory intensive approaches are not readily available in all settings. Aborted fetuses and uterine secretions during delivery or abortions are the most important source of infection within a herd. The bacteria can enter the host via inhalation after aerosolization or via ingestion from the gastrointestinal tract, where the infection spreads locally to lymph nodes (the site of intracellular Brucella replication), followed by bacteremia leading to systemic infection. The disease can also be transmitted to calves vertically and through the consumption of contaminated milk, but these routes of infection are less important in cattle. Calves that acquire the infection vertically or by ingesting contaminated milk may remain serologically negative and be asymptomatic. Nevertheless, these heifers may still abort or give birth to infected calves, serving as a disease reservoir within a herd (Olsen and Palmer 2014). B. melitensis is the main etiological agent of brucellosis in goats and sheep and is also the Brucella responsible for most human infections. Goats are more susceptible to infection than are sheep, where the disease is more variable and often self‐limiting (Olsen and Palmer 2014). As in cattle, abortion and infertility are the predominant clinical signs. Abortion occurs in late gestation without retention of fetal membranes, and abortion may be the only clinical sign. Grossly, the placental changes are similar to those described for B. abortus, and aborted fetuses usually appear normal although bronchopneumonia, hemorrhagic pleural fluid and lymphadenopathy may be present (Cutler et al. 2005). Interestingly, mastitis is a more common feature of small ruminant brucellosis as compared to bovines, with grossly evident firm mammary glands that can exude watery, clotted milk (Cutler et al. 2005). As in bovines, the definitive cause should always be investigated by submitting reproductive tissues and aborted fetuses for analysis. Main differential diagnoses in small ruminants should include Coxiella burnetii, Toxoplasma gondii, Chlamydia abortus, Campylobacter fetus, Listeria monocytogenes, and Sarcocystis cruzi, among others. In male goats, B. melitensis can infect the epididymis, testicles, seminal vesicles, and deferent ducts, which often results in decreased fertility. The primary routes of transmission are similar to those described for B. abortus in cattle: infected placenta, vaginal discharges, and fetal fluids during abortion and parturition. Significant differences between B. melitensis and B. ovis infection in sheep are readily apparent. Infection with B. ovis is characterized by a marked tropism of the bacterium for the male reproductive system and to a lesser extent to the uterine tract and placental tissues. The bacterium is predominantly transmitted via the venereal route and is an important cause of infertility in rams as infection is associated with the development of epididymitis and testicular atrophy (Burgess et al. 1981). Grossly, infected animals have either unilateral or bilateral epididymal enlargement or atrophy (chronic cases) 7–12 weeks post‐infection, and the bacterium can be recovered from semen 5–14 weeks post‐infection. Microscopically, there is evidence of epididymitis, testicular degeneration and seminal vesiculitis. Inflammatory cells can be detected in the semen even before the development of epididymitis, which is a valuable method for screening potential carriers of the infection due to the venereal route of transmission (Foster et al. 1987). B. ovis is relatively avirulent in the non‐pregnant uterus, but occasional abortions in ewes, secondary to venereal transmission during the breeding season, can occur. B. ovis is not considered zoonotic. Like other Brucella species, B. canis in dogs has tropism for the reproductive system. Unfortunately, the clinical signs of infection are not specific, and dogs may be subclinically infected or may exhibit signs of reproductive failure. In male dogs, it can cause epididymitis, prostatitis and orchitis, ultimately resulting in testicular atrophy, azoospermia, and infertility (Camargo‐Castaneda et al. 2021). The typical manifestation in females is mid‐ to late‐term abortion, followed by a vaginal discharge that can persist for weeks (Carmichael and Kenney 1968). Another disease manifestation is embryonic death with resorption, which clinically can appear as inability to become pregnant (Olsen and Palmer 2014). As in cattle, it is possible for an infected animal to abort, and subsequently pregnancies can either go to term or abort. In contrast to aborted fetuses in ruminants, aborted pups have non‐specific lesions such as edema, hemorrhage, and congestion. Apparently healthy pups from infected females may be infected in utero and be asymptomatic but can serve as a source of infection to other animals. The disease in neutered or spayed animals may manifest as discospondylitis. Infected dogs have a history of lameness, muscle weakness and neurologic dysfunction, caused by vertebral osteomyelitis and intervertebral disc infection. Uveitis has also been described to occur in a percentage of dogs with or without concomitant reproductive disease (Hensel et al. 2018). Abortions in swine infected with B. suis are much less common than abortions in cattle infected with B. abortus or in small ruminants infected with B. melitensis (Olsen and Palmer 2014). If abortion occurs, it is usually during the second or third month of gestation. Weak and stillborn piglets are common, however. Grossly, placentas from affected sows are edematous and red with multifocal white pinpoint nodules. B. suis has multiple biovars that vary in host specificity and phenotype. Biovars 1, 2 and 3 infect primarily domestic and feral swine and wild boar, with biovar 3 as the most common cause of infection. Biovar 2 can establish a reservoir of infection in hares and can be horizontally transmitted to swine and cattle (Cook and Noble 1984). Although pathogenic in cattle and swine, biovar 2 is not zoonotic. In boars, testicular involvement is frequent. Abscesses and granulomatous inflammation of the head of the epididymis and orchitis has been described (Kernkamp et al. 1946). Lameness secondary to septic arthritis can be seen (Cvetnic et al. 2009). B. suis biovar 4 is an important pathogen in reindeer. However, cattle experimentally infected with this biovar do not develop clinical signs but can be positive via serological assays. In reindeer, Brucella causes abortions, weak calves, retained placenta, orchitis, epididymitis, arthritis, hygromas, and nephritis. The pathology of B. suis biovar 5 infections in rodents are poorly known. At the cellular level, the brucellae are intracellular pathogens of cells of the reticuloendothelial system, particularly macrophages (Gorvel and Moreno 2002). During the course of chronic infection, the organism resides within macrophages where they replicate in a specialized compartment called the Brucella‐containing vacuole (BCV), and the ability of the bacterium to survive and replicate within macrophages is essential to their virulence (Celli and Gorvel 2004). The BCV in which the bacteria reside intracellularly is close to the endoplasmic reticulum, and the membrane of the BCV contains endoplasmic reticulum proteins (Celli et al. 2003
16
Brucella
Introduction
Characteristics of the Organism
Pathogenic Species
Source of Infection: Ecology, Evolution, and Epidemiology
Types of Disease and Pathologic Changes
Brucella abortus
Brucella melitensis
Brucella ovis
Brucella canis
Brucella suis
Virulence Factors
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