A review of coxiellosis (Q fever) and brucellosis in goats and humans: Implications for disease control in smallholder farming systems in Southeast Asia

Coxiella burnetii and Brucella spp. are pathogenic bacteria that can cause large-scale outbreaks in livestock. Furthermore, these infectious agents are capable of causing zoonotic infections and therefore pose a risk to the close relationship between farm households and their livestock, especially goats. A review of seroprevalence studies of Coxiella burnetii and Brucella spp. in domestic goats demonstrated large differences in the total number of samples tested in different regions and countries. This review aims to provide information on coxiellosis (Q fever in humans) and brucellosis in goats concerning the characteristics of the causative agent, surveillance, and available prevention and control measures at a global level. Implications for Coxiella burnetii and Brucella spp. infections in domesticated goats in Southeast Asia are discussed.


Introduction
Coxiella burnetii (causative agent of Q fever in humans) and Brucella melitensis are important bacterial zoonotic pathogens associated with goats [1], other livestock and humans [2]. These pathogens can cause large-scale outbreaks due to their low infectious dose, environmental resistance and ability of airborne spread via aerosolisation of the pathogens [3]. Furthermore, C. burnetii and Brucella spp. can economically impact rural livelihoods by reducing productivity due to reproductive loss in livestock herds [4,5].
Q fever and brucellosis are challenging to diagnose and treat in humans due to their non-specific clinical signs and intracellular nature [3,4]. They are considered a higher risk for those working with livestock and wildlife through occupational exposure. Both pathogens are potential biothreats classified as "Select Agents" in the USA [6,7] and have long been associated as major causes of laboratory-acquired infections and unintended laboratory releases [8][9][10][11][12].
This review presents characteristics of C. burnetii and Brucella spp., preventive and therapeutic measures. This article aims to provide updated information on coxiellosis and brucellosis in goats, including the detailed characteristics of the causative agent, available prevention and control measures, and the seroprevalence of infection worldwide while focusing on its relevance to Southeast Asia.

Smallholder goat farming in Southeast Asia
In Southeast Asia, smallholder goat farming typically entails raising a small number of goats, fewer than 20, on a small plot of land. These goats are usually raised for their meat and milk and are an essential source of protein and income for numerous rural families in the region [13,14]. Goats are frequently raised in free-range systems in Southeast Asia, where they can graze on natural pasture or browse on shrubs and trees. They may also be fed crop residues, grasses, and other locally accessible feeds [15][16][17]. Goats are browsers requiring minimal inputs and can be grown on marginal land, making them a viable option for small-scale farmers with limited resources.
Additionally, goats are relatively easy to handle and manage, making them a good option for farmers with limited experience with livestock. Lack of market access and veterinary services is among the most significant obstacles facing smallholder goat farmers in Southeast Asia and elsewhere [18]. However, with booming economies throughout the region, there is an increased demand for goat meat in Vietnam and China [19]. In addition, limited access to veterinary services may make managing disease outbreaks difficult and preventing goat herd health issues

Transmission and spread
The primary risk for transmission and spread of C. burnetii is during ruminant parturition [26]. C. burnetii has an affinity for placental tissues, particularly the trophoblast cells of the allantochorion [30]. Infection in livestock is largely asymptomatic: however, it can cause abortion, weak newborns, and decreased milk production [30]. Therefore, in affected animals exhibiting normal parturitions, C. burnetii can be aerosolised, generating a major source of exposure to other mammals, including humans [30]. C. burnetii has been found in blood, lungs, spleen and liver in the acute phase following inoculation of small ruminants hence preparing meat for food in smallholder situations is a significant risk [30]. Further, C. burnetii has been isolated from the milk of small and large ruminants, making consuming unpasteurised milk products an infection risk [30].

Human Q fever
Q fever in humans is likely underdiagnosed due to its nonspecific symptoms and the lack of clinician awareness [31]. During the early stages of Q fever, most patients are asymptomatic or have flu-like symptoms, including fever, fatigue, and muscle pain [26,32]. Human populations with higher seroprevalence of C. burnetii tend to be situated in rural areas, with animals also having a high seroprevalence. In Cyprus, Pakistan and the Netherlands, a high seroprevalence in humans was correlated to areas with high seropositivity of goats and reported goat abortions [33][34][35]. A recent human study on the Queensland (Australia) population indicated similar Q fever seroprevalence rates in metropolitan and rural populations [36], highlighting that Q fever can also affect those in low-risk groups. Human-to-human transmission of Q fever is extremely rare [25].

Prevention
Vaccines are available to prevent Q fever in humans [37,38], although it is not 100% effective, and failures have been recorded [39]. Furthermore, the vaccine is only available in Australia to certain occupations with potential exposure risk [38]. Veterinarians and support staff have a higher potential for Q fever infections via occupational exposure [40,41]. In Australia in 2014, a cross-sectional online study was conducted on two groups, including veterinary doctors (890 responses) and veterinary nurses (852 responses), to ascertain vaccination status and assess knowledge and attitudes regarding Q fever vaccination. According to the findings, veterinarians had higher vaccination rates than veterinary nurses i.e., 74% versus 28%, respectively [31].

Seroprevalence and risk studies in goats worldwide
Goats have been associated with outbreaks of C. burnetii, including in the Netherlands (2007-2020) [23,24], China (2018-2019) [42] and Australia (2012-2014) [43]. Exposure rates vary greatly with geographical location and are summarised in Table 1. Interestingly, from 2020 to 2023, 16C. burnetii seroprevalence studies that examined goat samples (and other livestock) have been published in peerreviewed reports ( Table 1). Many of these studies were performed in developing countries in Africa (Kenya [44], Mali [45], Namibia [46]), South Asia (India [47], Nepal [48], Pakistan [49]) and Southeast Asia (Cambodia [50], Thailand [51]), where smallholder livestock systems are common. One of the likely reasons for the increased number of seroprevalence studies is the availability of accurate screening tests, such as the ELISA [52], for detecting C. burnetii antibodies in a range of species. The studies have revealed significant seroprevalence levels in cross-sectional studies ranging from 0.8% in Nandi County in Kenya [44] to 74.7% in Marsabit County in northern Kenya [53]. Many of the studies were multispecies surveys, including cattle and sheep, in the analysis to better understand the risk factors associated with C. burnetii infection dynamics in smallholder communities.
Various risk factors for seropositive animals have arisen from these studies; in Spain, older animals were at higher risk [54]; in Pakistan and Australia, female animals were at higher risk [33,43]; in Pakistan, goats infested with ticks, history of abortion, retention of foetal membranes, single breeds in herds, and feed mismanagement were all risk factors [33]. In the Netherlands, proximity to other positive farms, dogs or cats in the goat stable, large farms with >800 animals and artificial insemination were all significant risk factors [34].

Coxiella studies in Southeast Asia
A few available studies describe the prevalence of C. burnetii antibodies in Southeast Asia; however, many focus on integrated livestock systems rather than smallholder goat raising.
In Lao PDR, initial investigations of C. burnetii seroprevalence revealed that between 3 and 4% of cattle and buffalos were seropositive [55] with the province of Xayaboury, bordered by Thailand, having increased prevalence [55,56]. Subsequently, studies exclusively on goat serum from five provinces (Vientiane Capital, Xayaboury, Xiengkhuang, Savannakhet and Attapeu) found overall individual C. burnetii seropositivity was 4.1% significant risk factors for seropositivity included where are the animals were located by province (Vientiane Capital, p = 0.05), breed (introduced Boer mixed breed, p = 0.006) and age (goats ≥3 years old, p = 0.014) [19].
In Cambodia, a recent seroprevalence study in six provinces [57] concluded that the overall true prevalence of C. burnetii antibodies in 540 goats was 7.2%. The two risk factors for C. burnetii seropositivity were sex (p-value = 0.0005) and location of the commune (p-value <0.0001), with the odds ratio of C. burnetii seropositive female goat being significant at 9.7 (95% CI 2.7, 35.5) times higher than male. Another seroprevalence study conducted in China indicated that 22% of goats (29% of cattle and 3% of pigs) were C. burnetii seropositive [58] and also found that 25% of humans were C. burnetii seropositive and associated with increasing age [58].

Introduction and history
Brucellosis is an infectious bacterial disease caused by species of the genus Brucella. Brucellosis can be documented as far back as 1600 BCE in Egypt and has continued to cause febrile illness globally. Brucellosis, also known as "Malta Fever", is named after the febrile illness common among British soldiers stationed in Malta during the 19th century [3].

The infectious agent
Bacteria of the Brucella genus are gram-negative coccobacilli of the Proteobacteria phylum. Within the Brucella genus are nine species, of which seven affect terrestrial animals; B. abortus, B. melitensis, B suis, B. ovis, B. canis, B. neotomae and B. microti [2]. Within species are biovars: B. melitensis has three biovars which differ in geographical location rather than pathogenicity.
Brucella spp. is a facultative intracellular pathogen of mammalian hosts that survives and multiplies within phagocytic cells [2,64]. Brucella spp. can enter the host across mucosal membranes [3]. The O-sidechain on the LPS appears involved in invasion mechanisms and protection from oxidative killing, cationic peptides and complementmediated lysis [65]. Brucella spp. avoid destruction by resisting fusion with lysosomes [66]. Brucella spp. organisms ultimately become sequestered within phagocytes of the reticuloendothelial system (RES), such as lymph nodes, liver, spleen and bone marrow, where they multiply in modified phagosomes termed "brucellosomes" [2]. Dissemination to other organs occurs via lymph and blood [67]. A specific tropism exists for reproductive tissues, including the placenta, mammary lymph nodes and ducts, and testicular tissue [1].
Brucella spp. are classified by the US Centers for Disease Control and Prevention as "bioterrorism agents" due to their stability, ability to cause mass disease with a low infectious dose, ability to be aerosolised and potential for mass dissemination of the pathogen [3,64,68]. Brucella spp. can survive long periods in the environment, surviving freezing and thawing and can live for up to 4 months in milk, urine, water, faeces and damp soil [1][2][3]. Cooler conditions favour the survival of Brucella spp. [2]. Brucellosis is a systemic infection that can involve any organ or tissue of the body [2]. Common routes of infection include direct inoculation through cuts and abrasions in the skin, inoculation via the conjunctival sac of the eyes, inhalation of infectious aerosols, and ingestion of infectious milk or milk products [2,3]. The latter is an unlikely aetiology in Southeast Asia as it is the lowest milk-drinking region in the world [69]. The primary source of brucellosis infection in humans is infected birth fluids from animals [3].

Global incidence
Globally, the seroprevalence of Brucella spp. in goats varies from region to region and within countries Table 2. It is difficult to compare studies due to differences in serology methodologies, sampling and analysis. B. melitensis-free status has been given to Sweden, Denmark, Finland, Germany, the UK, Austria, Netherlands, Belgium, France, Luxembourg, Australia and New Zealand [70]. Generally, a higher human incidence of brucellosis occurs where there is a higher Brucella spp. seroprevalence. The true incidence of brucellosis in developing countries may be higher as inadequate medical facilities may lead to underdiagnosis. Several risk factors arise from these serological studies, including exposure to infected animals and trading of animals. Contact with other flocks and free-range grazing has been described as a risk factor for seroprevalence in goat herds [71][72][73][74]. This could explain the general trend of widespread brucellosis occurring in small ruminants in developing nations. These countries tend to have smallholder pastoralists who are either landless or land-poor and move their goats about or allow them to free-range with other goats during the day [67]. High herd numbers and subsequent herd density risk brucellosis as close contact with other animals may make the spreading of the disease more accessible [72,75]. Areas with increased international trade are also at higher risk for expanded seroprevalence [76,77]. In these studies, authors noted that the disease could be introduced from other regions, or trade induces stress on animals and increases herd density, allowing the pathogen to spread [76,77]. "Mis-management" [72] and lack of veterinary care [73] were noted as risk factors. Generally speaking, "mismanagement" was defined as allowing goats to have frequent contact with their manure and lack of appropriate feed [72]. Older animals tend to have higher seroprevalence, probably due to higher exposure to the pathogen [55,56,78]. In some studies, females have higher seroprevalence than males, possibly due to farmers breeding females for longer and having more opportunities for exposure to the pathogen [79].
Breed and genetics may affect seropositivity. In Mexico, native goat breeds adapted to harsh desert conditions were less likely to be seropositive than imported dairy breeds [75]. In Bangladesh and Malaysia, exotic breeds were significantly more likely to have Brucella spp. seropositivity than native breeds [80,81]. Generally, dairy goat breeds are more susceptible to brucellosis than meat breeds as contact with contaminated milk and most reproductive products (i.e., placenta, discharge, amniotic fluid.) allows for more opportunities for goats to be challenged by the pathogen [2]. The Nramp1 gene is critical in innate immunity by enhancing macrophage ability to kill bacteria and positively influences adaptive immunity. Cattle with the Nramp1 gene are correlated with a natural resistance against brucellosis [82,83]. Further, studies of Nelore cattle breeds, which have been adapted to harsh climates, have macrophages that are more effective in controlling intracellular replication of B. abortus than Holstein macrophages, suggesting a higher degree of natural resistance [84].

Brucellosis studies in Southeast Asia and other parts of Asia
Despite limited peer-review articles published regarding brucellosis in South East Asia, evidence suggests that the pathogen is endemic. Five provinces in Lao PDR (Vientiane Capital, Xayaboury, Xiengkhuang, Savannakhet and Attapeu) tested 1458 goat serum samples for Brucella seropositivity resulting in an overall prevalence of 1.4%. They indicated that province (Vientiane Capital, p < 0.001), breed (introduced Boer mixed breed, p < 0.001), production system (commercial, p < 0.001), age (adult, p = 0.004), and farm size (large, 0.001) were all significant risk factors seropositivity for Brucella spp. [19]. Previous studies on cattle in Laos have reported that Brucella spp. is not widespread, with only 0.2% of cattle in 2012 and 0.3% of cattle in 2017 being seropositive to Brucella spp. [55,56].
In Thailand, the first case of brucellosis was reported in 1970 in a 34year-old male farmer, and the second case was documented in 2004 [85]. In 2014, there were five cases of brucellosis; in one case, in 2015, most of the patients were farmers with risks of exposure to contaminated goat parts and drank goat milk [85]. Following an outbreak of undulant fever in 2006 in a village northeast of Bangkok, it was determined that 43.5% of villagers were B. melitensis seropositive with risk factors including recent contact with goats during parturition and eating raw goat meat [86]. The brucellosis situation in Thailand can be attributed to the significant increase in goat farming in the country since 2000 [74]. Brucella seroprevalence in Thailand from 2013 to 2015 in goats and sheep was 12.1% (438/3626) at the herd level; individual goat seroprevalence was 1.4% (1297/94,722), and sheep seroprevalence was 1.6% (139/8658) [74]. At the herd level, only free-ranging animals were substantially associated with brucellosis infection in small ruminants [74]. Another recent study examined 118 samples from goats in northeastern Thailand, including 85 clinical reproductive disorder cases for C. burnetii, Chlamydophila abortus, and Brucella spp. Results showed that 69% (81/118 cases) were seropositive for Q fever (n = 55; 46.6%), brucellosis (n = 8; 6.8%), and chlamydiosis (n = 18; 15.3%). The study highlighted that buck circulation between herds was a risk factor for diseases (Odds ratio = 109.29) [51]. This fact highlights the importance of biosecurity in herd management to prevent the introduction and spread of brucellosis.
In Cambodia, a recent seroprevalence study [57] in six provinces concluded that the overall true prevalence of Brucella spp. antibodies in 540 goats was 0.1% (95% CI 0.0, 1.0). Brucellosis was previously detected in Myanmar, where dairy cows had Brucella spp. DNA identified the milk via PCR [87]. In Vietnam, a report from Binh Thuan province indicated that 14.8% of persons presenting febrile illness using the Rose Bengal Agglutination test for Brucella spp. However, the study suggested that the evidence demonstrated people's exposure to Brucella spp., it did not cause significant health effects on them [88]. Brucellosis has been reported in China, the only region to have developed a human vaccine, although it is not efficacious or safe for use [89]. In one study, rural farmers did not demonstrate antibodies to Brucella spp., yet 14.5% of abattoir workers in the same village were positive [90]. A survey in Assam and Odisha states in Eastern India tested 411 goats, resulting in 22% and 9.8% Brucella seroprevalence, respectively [47]. None of the farmers interviewed were aware of brucellosis. [47].

Prevention and control
Reduction in human incidence is usually coupled with managing animal disease and food hygiene. Pasteurisation effectively kills Brucella spp., and thoroughly cooking meat is advised. Although cultural traditions are difficult to influence, consuming raw placentas, milk and meat is not recommended. To reduce transmission between a herd, parturition material, especially aborted fetuses and placentae, should be removed and destroyed by incineration before other animals, including farm dogs and cats, and children can be exposed to them [1]. Faeces are cleaned up daily. Regular disinfecting more than three times yearly reduces incidence in a herd [71]. Replacement stock should only be purchased from Brucella-free herds [2].
Vaccines are available for both B. abortus and B. melitensis in livestock, and programs have successfully eradicated Brucella spp. from regions. However, entirely satisfactory vaccines are not currently available. Vaccine methods are only recommended after other control measures have failed. The Rev. 1 B. melitensis vaccine for caprine and ovine brucellosis control is created from killed bacteria strains [91]. When appropriately used, it causes long-lasting protection against natural infections for most animals, reducing the zoonotic spread of the disease [92]. The vaccine can induce abortions in pregnant nannies and cause the shedding of the organism in animal milk [2,91]. Hence, only sexually immature females are often included in vaccination programs. The vaccine must be coupled with husbandry, test, and cull methods because it is not absolute. Test and cull methods are challenging to implement as they require a political and financial commitment to authorities and provide compensation to farmers. The World Organization for Animal Health (WOAH, formerly OIE) does not recommend antibiotic treatment of animals [2].
Antibiotic treatment for acute human brucellosis usually involves tetracyclines such as doxycycline for six weeks, with aminoglycosides in addition to the first three weeks of treatment. This combination is prolonged for chronic brucellosis treatment [2]. Human vaccines are only available in China and were used in the Soviet Union, although they are reportedly not efficacious or safe for use [3,89,90]. However, the human vaccine has been demonstrated to induce human brucellosis and severe allergic reactions and is only "recommended for human use in high-risk populations" [89].

Conclusion
C. burnetii and Brucella spp. are important zoonotic diseases across Southeast Asia and elsewhere because they can have significant health and economic effects on humans and livestock. Both diseases can cause illness, disability, and even mortality in humans, while they can reduce livestock productivity and cause reproductive losses.
However, practical considerations for controlling both diseases in goats in settings with limited resources still need to be solved. It is important that global (WHO, WOAH) and regional organisations (ASEAN), including national governments, invest in disease control strategies and increase awareness of Coxiella and Brucella infections as important zoonotic infections. Strategies typically used can mitigate the spread of these bacteria and reduce the risk of zoonotic infections in humans listed below; however, it is difficult to know how practical many of these measures would be in a low-resource setting.
-Biosecurity: The implementation of biosecurity measures can prevent the introduction and dissemination of Coxiella and Brucella. This may involve isolating infected animals from healthy animals, avoiding contact with wild animals, and practising good hygiene, such as washing hands and apparatus between animals. This is especially important following parturition for both diseases. The quarantine of new animals into an existing herd is also a simple, practical measure for biosecurity. -Vaccination: Vaccination is an economical means of preventing the spread of these diseases; however, access to vaccine supplies and administration per the recommended schedule and dosage will be a challenge, depending on the location of the smallholder farmers.
The above-mentioned practical measures focusing on biosecurity and farmer education are likely to have the greatest success; although vaccination remains an option would require government support and infrastructure.
There is an ongoing need for clinical training in the region to include both diseases in the workup for pyrexia of unknown origin and to emphasise to veterinary students and para-veterinarians the zoonotic nature of these pathogens. Additionally, there is a need for improved diagnostic capabilities for Q fever in both animal and human fields [44]. The final additional control measures, 1) testing and culling and 2) treatment with antibiotics, are problematic in a low-resource, developing-country setting due to a lack of compensation for culling, lack of testing and limited access to antibiotic treatment.
To control the spread of both diseases, it is vital to continue human and animal surveillance to understand better the epidemiology of coxiellosis and brucellosis in the South East Asian region and plan and manage control and eradication strategies. Therefore, employing the One Health concept, it is recommended that veterinarians and medical personnel jointly address prevention and control strategies through enhanced surveillance, public sensitisation, and awareness raising.

Funding
This research was funded in whole, or in part, by the Wellcome Trust [Grant number 220211]. For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.

Declaration of Competing Interest
The authors declare no conflict of interest.

Data availability
Data will be made available on request.