To our knowledge, this is the first study to utilize both RBPT and qPCR for the detection of Brucella spp. in Nigeria. Our findings revealed Brucella could be easily missed in pyretic patients if unscreened. Hence, a public health challenge and a recipe for its underreporting. We do not have evidence to support whether any of the patients fall under the clinical definition of fever of unknown origin (FUO)¹³. We do know that brucellosis is not screened in clinics across the country and available data on the human population are from research and academic studies. Hence, routine screening of at-risk patients should be instituted across the country.

The RBPT has been reported to have a diagnostic sensitivity of 100% and a specificity of 98.69%¹¹. Hence, it has maintained its scientific application and acceptance in the diagnosis of infectious diseases. In comparison to culturing or nucleic acid amplification methods, it is inexpensive and technically simple. Despite this, qPCR could also offer equally 100% diagnostic sensitivity and specificity for many infectious diseases^14,15. This is primarily because qPCR is based on the extracted genomic materials usually present in active infections whereas seroprevalence may not show current infection or shedding of the pathogen since animals may seroconvert without detectable shedding and can remain seropositive for years post-infection or sometimes may not seroconvert during active shedding of infection¹⁶. In 2019, a study reported the diagnostic usefulness of qPCR in early diagnosis of infectious diseases in patients as opposed to the use of serological testing which requires antibody formation approximately 2 to 3 weeks after onset of symptoms before they could be detected¹⁷. Here, we considered all RBPT as true positives and the discordance between RBPT and qPCR (only in domestic animals) could be due to the stage of disease progression.

As there was 100% concordance between RBPT and qPCR in clinical samples, we advocate for routine clinical screening of pyretic patients for brucellosis. The 5.8% brucellosis prevalence detected in pyretic patients in this study was higher than the prevalence previously reported in Sokoto (0.7%)¹⁸, and Borno (3.8%)¹⁹ but lower than those previously reported in Kwara state (22.2%)⁷, and the national pooled prevalence of 17.6%⁶. The prevalence in humans is also within the range of 0–55.8% reported by Qureshi et al.²⁰ and Djangwani et al.¹⁰ in the sub-Saharan African region, highlighting the significant presence of brucellosis infection in this area. These differences in prevalence could be due to the differences in disease burden across the country and sampling strategy. Our findings highlight the substantial health burden of brucellosis and the potential for misdiagnosis as malaria or other febrile illnesses²¹. The burden of brucellosis in humans could be attributed to risk factors such as close contact through occupational exposure²², the consumption of raw dairy products^23,24,25, and religious beliefs that denounce practices that include the test and slaughter of positive livestock^26,27.

Among the 150 cattle samples screened, the RBPT revealed a prevalence of 18% which was higher than the national pooled prevalence of 12.2% in cows⁶ and the regional pooled prevalence of 8.6% from the 3213 cattle screened in the North Central region between 2001 and 2021⁶. This prevalence was also higher than the 9.6% reported in cattle slaughtered at the Ilorin Metropolitan abattoir in 2020⁷ and the 14.2% reported in samples collected from cattle farms in 2017⁸. According to a recent global brucellosis review, the prevalence of brucellosis in cattle in Sub-Saharan Africa ranged from 0.2–43.8%²⁰. The RBPT also revealed a prevalence of 25% in goats which was higher than the North Central pooled prevalence of 19.2%, the national pooled prevalence of 10.2%⁶, and the range of 0.0–20.0% in goats²⁰.

Generally, controlling brucellosis in humans involves controlling the disease in its reservoir animal hosts. However, small-scale livestock keepers have been known to avoid diagnostics for lack of compensation and fear of consequences (e.g., the slaughter of their entire herd) if a single animal were to test positive for brucellosis^2,22. Typically, brucellosis treatment is discouraged, and the affected animals are usually culled² because of the carrier status, the emergence of antibiotic-resistant strains, and the need for continued treatment to avoid recurrence. The chronic nature of brucellosis, combined with the capacity of Brucella to reside within host cells and sequester at difficult-to-reach sites, can contribute to treatment relapse²⁰. The relapse rate in uncomplicated cases is estimated to be 5–15%. The cause of these relapses is unclear because of the emergence of antimicrobial resistance (AMR) or the inability to eradicate germs at the infection sites. However, studies on Brucella in endemic middle-income countries have generally shown that bacteria remain susceptible to doxycycline and rifampicin, which are commonly used antibiotics for brucellosis treatment^20,28,29.

The qPCR assay revealed 100% concordance in human clinical samples but significant discordance in the samples from cattle and goats. The qPCR revealed a prevalence of 11.3% in cattle (as against 18% by RBPT) and 15.3% in goats (as against 25% by RBPT). The discordance in the diagnostic or analytical sensitivity could be attributed to the proven lack of serological assay to detect ongoing infections with the potential for detectable antibodies to persist for a long. Since pyretic clinical samples were collected, antibodies may coincide with the presence of an ongoing infection. Despite the discordant diagnostic sensitivity between these two highly reliable tests, the diagnostic specificity was 100% as there were no false negative samples detected. This qPCR was previously reported to be a highly sensitive and specific diagnostic tool for brucellosis with a low CT value¹².

As an endemic, under-reported, and neglected zoonoses, the surveillance of Brucella spp. could be improved using one health approach and a combination of RBPT and qPCR assay. The evidence of exposure to brucellosis is worrisome because, aside from its negative effect on reproductive performance such as abortion, stillbirth, and reduced productivity of livestock, it is a persistent and highly transmissible pathogen to humans^16,30. In addition, the disease is associated with significant economic losses^3,31. One key intervention needed to reduce the burden of brucellosis in Nigeria is vaccination which was reported to be cost-effective for brucellosis control instead of the test-and-slaughter approach³². In addition, Nigeria must work to improve animal identification systems including animal tagging and movement control. Some countries have achieved eradication through a combination of efficient diagnosis, vaccination, and test-and-slaughter³³. Hence, mass animal vaccinations using the B. abortus S19 or the RB51 vaccine should be instituted across brucellosis hotspots across the country³³.

As our analysis revealed that none of the sociodemographic variables is associated with the occurrence of brucellosis in humans or cattle, it implies that any public health intervention should be channeled towards all age groups, gender, and level of education across the state with a focus on reducing identified risk behaviours. Generally, more emphasis should be placed on public awareness on reducing the consumption of raw milk particularly in persons that were observed to be drinking raw milk directly from the udder of the cow, since B. abortus has been isolated from raw and sour milk of Fulani cattle in Nigeria²². While there was no statistically significant association between the different sampling points in humans and cattle, there were significant variations between the burden of brucellosis in each of the three LGAs with more cases in Ifelodun than the other two LGAs. This was in agreement with the report of Ogugua et al.²³ who reported that herd location significantly impacted the occurrence of brucellosis in Southwestern Nigeria.

Since infected ruminants can shed Brucella spp. and other zoonotic pathogens, the Codex Alimentarius recommends the pasteurization of raw milk³⁴. It is, therefore, essential to educate farmers on the need for pasteurization at the appropriate temperature as well as post-pasteurization handling of dairy products to reduce the burden of this disease. In addition, the various initiatives aimed at improving the dairy value chain in Nigeria must intensify screening for zoonotic milk-borne pathogens such as Brucella spp. Furthermore, other important endemic zoonotic pathogens such as Coxiella burnetii, Mycobacterium bovis (zoonotic tuberculosis), Salmonella spp., Listeria spp., E. coli, Campylobacter spp., and Clostridium spp. could be transmitted through the consumption of unpasteurized milk.

One of the main strengths of this study is its uniqueness and novelty in the study area. It is the first published work that utilized both RBPT and qPCR for the detection of Brucella spp. in Nigeria in pyretic patients and domestic animals in their proximity. Despite this, the following limitations may affect its widespread replication in Nigeria: sampling bias, small sample size, and the study’s participants were limited to specific LGAs of the state, so the observed awareness and practices may not be representative of the entire state.

This study established that brucellosis should be considered as a differential diagnosis in pyretic patients in Kwara State. In addition, its high prevalence in cattle and goats could be a source of infection for humans as the bacteria is shed in milk which is consumed unpasteurized in certain parts of Nigeria. We advocate for mass screening for brucellosis, public enlightenment with emphasis on the identified risk factors, and mass vaccination of domestic animals. More molecular studies are needed to further study the genotypic relatedness of human and animal genotypes.