Brucellosis is a “tool-deficient” disease that has not yet reached satisfactory levels of disease control (World Health Organization, 2007). The detection of antibodies provides only a provisional diagnosis, but there may also be an exceptionally long incubation period in some infected animals, and individuals may remain serologically negative for a considerable period following infection (Corbel, 2006). The epidemiological studies are based on the serological test to report the significant association between environmental factors, animals' intrinsic characteristics, and bovine brucellosis (Ahasan et al., 2017; Selim et al., 2019). This epidemiological study evaluated factors widely established by serology but associated them with Brucella DNA's presence in dairy cattle using the Probe-qPCR assay.
An unusual increase in the number of seropositive cattle during official surveillance led to the declaration of quarantine for bovine brucellosis in the leading dairy region in Colombia (Instituto Colombiano Agropecuario, 2018b). Although brucellosis is considered endemic in Latin America, there is still a need to evaluate the regional epidemiological situation (World Health Organization and Pan American Health Organization, 2016). The prevalence changes all the time in endemic countries (Godfroid et al., 2002) and it is necessary to update the epidemiological situation analysis to implement effective control and prevention strategies. This study is the first report of molecular prevalence in the region, showing a Brucella-DNA prevalence in concordance with the declared outbreak and higher than the seroprevalence records in the region.
Bovine whole blood as the sample for PCR detection of Brucella spp. has been little considered because Brucella may replicate at low frequency, leading to low and short bacteremia periods (Vrioni et al., 2008). However, under experimental conditions, it has been shown that some Brucella species could invade red blood cells and settle in the cytoplasm without replicating, establishing persistent bacteremia with different bacterial loads (Vitry et al., 2014; Gwida et al., 2016). In humans, a study of three patients infected in the recent Brucellosis outbreak in China confirmed that Brucella persists in peripheral blood (Baoshan et al., 2020). In this study, a high recovery of Brucella-DNA from whole bovine blood was possible. A high Brucella-DNA presence from bovine blood by qPCR, was also obtained in a bovine brucellosis outbreak study in Egypt (Gwida et al., 2016).
The Brucella-DNA presence could indicate the viable bacteria's existence in the cattle blood, but there is also a possibility that the presence of Brucella-DNA could be associated to particle shedding by dead bacteria or vaccine-DNA (Vrioni et al., 2008). The main used vaccine in the studied region is the RB51, however, the presence of the RB51 vaccine strain in the blood should not generate alerts. According to The Animal and Plant Health Inspection Service (APHIS), the RB51 must be rapidly cleared from peripheral blood within three days post-vaccination (National Council of State Legislatures, 2018). The RB51 vaccine's influence was ruled out in the positive samples by the SNP-based qPCR assay, which marked all positive samples as Brucella spp. field strains.
The variable municipality, mainly San Pedro de Los Milagros (SPM), showed a significant association with infection at the animal-level. SPM is considered an essential route for livestock movement, has an important cattle auction, and is close to Medellín (the second-largest city in Colombia), where the state's central slaughterhouse and an important cattle market are located. The preceding increases the animals’ passage of different origins, which may influence the region's spread of pathogens. Infected animals passing through populated areas may produce heavy contamination of streets and places (Corbel, 2006). These circumstances could have contributed to the association between the proximity of the herds to the main roads (≤ 200 m) and the presence of Brucella-DNA in the logistic regression model at the animal-level. Few brucellosis studies have been carried out with spatial analysis involving variables such as proximity to urban and rural areas, altitude, the season of the year, among others (Ibrahim et al., 2010; Dadar et al., 2020), and according to the authors' review, no studies in livestock have evaluated the proximity of herds to main roads. Due to the expansion of the Brucella genus, its evolution, and possible close relationship with environmental bacteria (Ashford et al., 2020), spatial studies are needed to understand the Brucella behaviour in environments.
Animals that belonged to a large herd size (> 50 animals), parlor milking, mechanical milking in pen, high grazing density (≥ 3.5 UA), an animal that belonged to herds with unknown or seropositive brucellosis status were associated with the Brucella-DNA presence. Dias et al. (2009) and Bugeza et al. (2019) attributed the higher risk of Brucella infection in large herds sizes due to the ease of transmission by the proximity between animals (Dias et al., 2009; Bugeza et al., 2019). Management practices in these farms can also influence (Moreno, 2014). Given the low numbers of Brucella organisms that can serve as a source of contamination, prompt detection in field samples and the reinforcement of biosecurity measures are required.
Although the bovine species are the main target of control and eradication programs, other hosts can also be infected by B. abortus or even by another Brucella spp. Some of them can sustain the disease and are considered an infection source for cattle (World Organization for Animal Health, 2018). However, in this study, Brucella-DNA presence was not associated with co-grazing with other animal species. Similar results were obtained in a recent epidemiological study in the country (Cárdenas et al., 2019).
In dairy herds, mainly in large herd sizes, there is a practice of raising calves and heifers in a different property from milk production. Some studies reported that animals' mobilization is a risk factor for introducing brucellosis in herds free of the disease (Cárdenas et al., 2019; Corbel 2006). Since these animals are exposed to different management practices and environmental conditions, this exposure can favor brucellosis infections (Corbel, 2006; Wareth et al., 2015). In this study, no significant association of the presence of Brucella-DNA with the raising of calves in other farms was found, but a significant association was found with the transfer of animals. Raising calves and heifers at a different property from the dairy cows can be appropriate if managed correctly. Separating and keeping the calves and heifers isolated from other herds, then testing them appropriately prior to their return will prevent Brucella introduction into dairy herds (Instituto Colombiano Agropecuario, 2017).
In the logistic regression model, the use of bulls for mating explained the Brucella-DNA presence at the animal and the herd level in the present investigation. Other epidemiological studies also showed a significant association of the bulls with Brucella seropositivity. They considered that sharing a stud bull with neighbors could likely be a potential source of Brucella infection (Muma et al., 2007; Ahasan et al., 2017). Brucella spp. can be found in the testicles and the male genital glands and transmitted by infected semen (Corbel, 2006). However, differences in vaginal acidity, mucosal immunity, and cervical mucus between estruses and anoestrus may affect bacterial survival and kill the bacteria (Uhrig et al., 2013). Therefore, it is more likely that females' infection may occur through environmental contamination with infected seminal discharges (Thomsen, 1943). In Yellowstone National Park, USA, a study showed seroconversion in four of eight bison after intravaginal inoculation suggests that B. abortus transmission by the venereal route in bison can occur. Although routes of transmission are considered like those in cattle (Uhrig et al., 2013), older experiments did not find an essential role in bulls' use for mating in Brucella transmission in cows during copulation (Thomsen, 1943). Updated studies on venereal transmission in cattle are needed. Meanwhile, all bulls from known infected herds should be viewed with suspicion regarding their Brucella spp. infection status (Plant et al., 1976).
Management practices, mainly associated with reproduction, are far more critical in determining the risk of Brucella spp. infection in bovine (Corbel, 2006). Variables such as the history of reproductive problems, pregnant cows, days of gestation, and disposal of birth waste were associated with the Brucella-DNA presence in the animals evaluated. Pregnant cows have been associated with the probability of Brucella-antibody presence in a cross-sectional study in Ethiopia and a study conducted for isolation and subsequent detection by PCR of Brucella spp. in milk samples from seronegative cows in Bangladesh, even showed prevalence almost double in pregnant cows than in nonpregnant cows (Islam et al., 2018). Apart from the last two, these factors were associated with Brucella-DNA presence in the logistic regression models.