Integrated Molecular Detection and Epidemiological Investigation of Brucella abortus in Dairy Cattle and Farm Workers During a Brucellosis Outbreak

ABSTRACT

Background

Brucellosis is a highly contagious zoonotic disease caused by bacteria belonging to the genus Brucella that can infect a wide range of domestic and wild animals. A recent brucellosis outbreak was reported at a dairy farm in the peri‐urban region of Khanewal District, Punjab, Pakistan.

Objective

In response, this study aimed to improve understanding of the dynamics of Brucella abortus infection in cattle and dairy farm workers in the local context.

Methods

In November 2024, a total of 83 cattle, representing six different breeds, along with blood samples from 11 farm workers directly involved in handling these animals, were sampled. Relevant epidemiological data were also collected to support the investigation. To detect Brucella infection in cattle, both the Rose Bengal Plate Test (RBPT) and polymerase chain reaction (PCR) targeting the IS711 gene were employed.

Results

All sampled cattle tested seropositive for Brucella antibodies by RBPT, indicating widespread exposure to Brucella. Molecular detection through partial IS711 sequence amplification revealed that 48% of the cattle and 45% of the farm workers (5 out of 11) tested positive for Brucella spp. infection. Subsequent DNA sequencing and BLAST analysis confirmed the presence of B. abortus in both cattle and humans. The obtained IS711 sequences closely matched those previously reported in ruminants and humans in China. Data analysis further showed that cows were more susceptible to B. abortus infection compared to bulls (p = 0.01). However, no significant association was observed between infection status and cattle breed, age, presence or absence of ectoparasites, or history of abortion (for all p > 0.05).

Conclusion

The present study provides evidence of B. abortus detection among cattle at the investigated dairy farm in Khanewal. The findings should be interpreted within the context of the studied farm, as the purposive sampling of a single dairy farm does not permit extrapolation to the wider cattle population of Khanewal District or Pakistan. Further multi‐farm studies involving geographically diverse cattle populations are warranted to determine the broader occurrence and epidemiological pattern of B. abortus.

A brucellosis outbreak was investigated in dairy cattle and farm workers. Among 83 cattle and 11 workers, Brucella abortus was molecularly confirmed using IS711‐PCR and sequencing. Infection was detected in 48% of cattle and 45% of workers, highlighting significant zoonotic and occupational risks.

1. Introduction

Cattle play a pivotal role in Pakistan's economy, serving as a major source of livelihood for millions of rural households. They provide essential commodities such as milk, meat, hides and other byproducts that not only fulfil nutritional needs but also support a range of agro‐based industries (Mohamed 2025). Collectively, these contributions make cattle a significant component of the agricultural sector and a key driver of national GDP (Taalay et al. 2022). The growth of Pakistan's livestock sector is being significantly hindered by a range of parasitic, bacterial and viral infections (Abutalip et al. 2025). Contaminated water, food and soil, as well as various arthropod vectors such as ticks, play a crucial role in the spread of infections like brucellosis to animals and humans (Zhao et al. 2026; Atif et al. 2024).

Brucella abortus is a Gram‐negative, non‐motile, facultative intracellular coccobacillus responsible for brucellosis, a zoonotic disease that poses a serious threat to both animal and human health (Awais et al. 2024). This infection in cattle typically causes late‐term abortions in cows and may lead to orchitis and bursitis in bulls (Zhang et al. 2024; Xu et al. 2024). Among the vectors, ticks such as Dermacentor marginatus and Dermacentor reticulatus have been frequently implicated in the transmission of B. abortus (Moura et al. 2024). Humans typically acquire the infection through direct contact with infected animals or by consuming contaminated animal products (Çelik et al. 2024). Brucellosis can cause a range of symptoms in both animals and humans, from mild flu‐like signs to severe, long‐lasting complications. In humans, the infection is typically managed using a prolonged course of combined antibiotics, most commonly doxycycline and rifampicin (Alavi and Alavi 2013). However, in cattle, effective treatment is lacking due to the persistent risk of bacterial shedding and transmission. As a result, control strategies focus on culling infected animals and implementing preventive vaccination using B. abortus strains RB51 or 19 (Qureshi et al. 2023).

In Pakistan, brucellosis is an emerging veterinary and public health concern, particularly because a large segment of the rural population depends on livestock for their livelihood and remains in close, prolonged contact with animals for rearing and care (Awais et al. 2024). The rising incidence of brucellosis is also attributed to several compounding factors, including poverty, inadequate vaccination coverage, poor awareness about tick‐borne transmission and a general lack of effective tick control measures (Hussain et al. 2021). Addressing these challenges is critical for reducing disease burden and safeguarding public and animal health. Historically, the serum‐based RBPT has been used for diagnosing Brucella infections in both small and large ruminants. However, due to its high false‐positive rate, it has been recommended that its use be discontinued (Tuon et al. 2017). In response, molecular diagnostics such as PCR have increasingly been adopted for detecting Brucella DNA in blood or serum, offering improved sensitivity and a noticeable reduction in false‐positive cases (Becker and Tuon 2021). In the present investigation, we aimed to comprehensively investigate a suspected outbreak of brucellosis at a dairy farm in Khanewal District, Punjab, Pakistan. Specifically, we employed both the RBPT and PCR to detect and confirm Brucella infection in cattle and to compare the diagnostic performance of these methods under field conditions. In addition, we sought to characterize the genetic relatedness of the detected Brucella strains by phylogenetic analysis of IS711 gene sequences to better understand their possible origins and epidemiological links. Furthermore, we evaluated various animal‐ and farm‐level risk factors that may have contributed to the transmission and persistence of infection within the herd.

2. Materials and Methods

2.1. Study Area, Subjects and Study Design

In October 2024, cases of recurrent fever and abortion in cattle were reported from a dairy farm located in Khanewal District (29.85°–30.43°N, 71.5°–72.47°E), Punjab, Pakistan (Figure 1). Based on this observation, all animals on the affected farm were enrolled for Brucella spp. screening using a purposive sampling approach, in which the herd was deliberately selected due to the suspected outbreak. Khanewal, situated between the Sutlej and Ravi rivers, experiences a hot and dry climate, with temperatures ranging from 46°C in summer to 11°C in winter. The region receives an average annual rainfall of approximately 186 mm (7.3 inches) (Aziz et al. 2022).

FIGURE 1.

Map of Pakistan with Punjab province highlighted in sky blue, while Khanewal district is highlighted in dark blue colour. Magnified map shows Khanewal district with cattle and farm worker enrolment sites. Map was created through the QGIS software (https://qgis.org/) using administrative boundary shapefiles obtained from the GeoBoundaries project (via HDX) via link https://data.humdata.org/dataset/geoboundaries‐admin‐boundaries‐for‐pakistan. These data are openly licensed and compatible with CC BY 4.0, and are suitable for reuse in open‐access publications.

A total of 83 cattle, representing six breeds, Holstein Friesian (n = 22), crossbred (n = 17), Sahiwal (n = 17), Cholistani (n = 13), Brahman (n = 7) and Dutch (n = 7), were maintained at the dairy farm. The herd was managed by 11 male workers, aged 16–50 years, including five married individuals with children and six unmarried. Verbal informed consent was obtained from all participating farm workers and the livestock owner before including their animals and personal data in the study. The purpose of the study was explained in the local language, and participants voluntarily agreed to provide information and to be sampled. Written consent was not obtained due to the low literacy levels of many participants. Instead, verbal consent was recorded via the structured questionnaire used to collect basic epidemiological data from livestock owners.

2.2. Blood Sample and Data Collection

All enrolled animals underwent a detailed clinical examination by veterinarians before blood collection. Before sample collection, the vaccination and routine health‐management history of the cattle herd was obtained from the farm management. None of the cattle included in the study had been vaccinated against B. abortus, including the S19 or RB51 strains. The herd was routinely vaccinated against other common cattle diseases, including bovine ephemeral fever (BEF; 3‐day sickness), haemorrhagic septicaemia (HS) and foot‐and‐mouth disease (FMD). The animals were also routinely subjected to anthelmintic treatment (deworming) as part of the farm's routine health‐management practices. From each animal, 5 mL of blood was drawn from the jugular vein. Blood samples collected from each animal were divided for serum‐based Rose Bengal Plate Test (RBPT) and PCR analysis, respectively, allowing both tests to be performed on samples obtained from the same blood draw. Of the collected blood samples, 3 mL was transferred into pre‐labelled gel and clot activator tubes (Samplix, Thailand) for serum separation. In comparison, the remaining 2 mL was placed in EDTA‐coated tubes (Xinle Medical, China) for genomic DNA extraction. For human participants, 3 mL of blood was collected exclusively in EDTA tubes.

A structured questionnaire was used to gather epidemiological data on the enrolled animals, including breed, sex, age, abortion history and presence of ectoparasites, through interviews with the cattle owner and farm workers. Similarly, information from the workers, such as age, sex, marital and fertility status, safety measures taken at the workplace and occupational history, was documented to assess potential associations with PCR positivity for Brucella spp. among both cattle and humans.

2.3. Brucellosis Screening Through Rose Bengal Plate Test

All collected serum samples were initially screened for Brucella species antibodies using commercially available RBPT reagents from the Veterinary Research Institute (VRI), Lahore, Pakistan, following Awais et al. (2024). Testing was performed in accordance with the respective manufacturers' protocols. Briefly, 30 µL of antigen was mixed with an equal volume of serum and gently agitated for 2–4 min. Samples exhibiting distinct agglutination were considered positive; absence of agglutination indicated a negative result. Known positive and negative controls were included in each test batch for quality assurance.

2.4. DNA Extraction and PCR‐Based Detection of Brucella spp

Genomic DNA was extracted from blood samples using a method previously described by Ashraf et al. (2021). The quality of the extracted DNA was evaluated through submerged agarose gel electrophoresis. A pair of oligonucleotide primers (Forward 5′ GGCGTGTCTGCATTCAACG 3′ and Reverse 5′ GGCTTGTCTGCATTCAAGG 3′) was used to amplify the IS711 marker of Brucella species following Bricker et al. (2000). A 50 µL PCR reaction mixture was prepared containing 50 ng of template DNA, 5 µL of 10X PCR buffer, 1.5 mM MgCl₂, 0.125 mM of each dNTP, 0.6 µM of each primer and 1 U of Taq DNA polymerase (AbClonal, USA), with the final volume adjusted using double‐distilled water. The thermal cycling conditions consisted of an initial denaturation at 95°C for 2 min, followed by 30 cycles of denaturation at 95°C for 10 s, annealing at 50°C for 10 s and extension at 72°C for 1 min. A final extension was performed at 72°C for 5 min, as described by Bricker et al. (2000). Positive controls (Brucella species DNA confirmed in a previous study) and negative controls (PCR reaction mixture without DNA template) were included in each run to validate the results.

2.5. DNA Sequencing and Phylogenetic Analysis

All the amplified partial IS711 gene sequences of Brucella spp. were Sanger sequenced for confirmation through a commercial service provider (First Base, Malaysia). Raw sequencing chromatograms were processed using FinchTV (version 1.4.0) to trim low‐quality nucleotides from both ends, and the high‐quality sequences were saved in FASTA format.

For phylogenetic analysis, similar sequences were retrieved from BLAST results and aligned using the ClustalW multiple sequence alignment tool. The best‐fit substitution model was selected based on BIC and AIC criteria using MEGA's integrated model selection tool. A phylogenetic tree was then constructed using the Maximum Likelihood method with 1000 bootstrap replicates to ensure statistical support. The final tree was visualized using the iTOL server. The IS711 sequence of Cetacea (accession number AY174071) was used as the outgroup.

2.6. Statistical Analysis

A significance level of p ≤ 0.05 was considered for all statistical analyses. Data were analysed using Minitab Statistical Software (Minitab, USA). Categorical associations were evaluated using the chi‐square test or Fisher's exact test, as appropriate. For comparisons involving the six cattle breeds, the Fisher–Freeman–Halton exact test was used because several expected cell frequencies were less than 5. In contrast, associations between epidemiological variables and B. abortus infection were assessed using Fisher's exact test. A 95% confidence intervals (CIs) were calculated to compare infection risk.

3. Results

3.1. Rose Bengal Plate Test‐Based Detection of Brucella spp. in Cattle

A 100% RBPT seropositivity rate was observed among the tested cattle, indicating widespread serological reactivity against Brucella antigens.

3.2. PCR Positivity of Brucella spp. in Cattle and Human Blood Samples

PCR successfully amplified a 775 bp fragment of the IS711 gene of Brucella spp. in 40 out of 83 (48%) cattle blood samples collected from a dairy farm in Khanewal District, Punjab. In addition, Brucella spp. were detected in 5 out of 11 (45%) dairy farm workers during the same investigation.

3.3. Sequence Confirmation and Genetic Diversity of B. abortus

As we used genus‐specific primers in this investigation, DNA sequencing of the PCR‐amplified products confirmed the presence of Brucella spp., and BLAST analysis showed that these sequences were 98.93%–99.2% similar to B. abortus. Two representative sequences, one from cattle and one from a human subject, were submitted to GenBank under accession numbers PV545638 and PV545639. Phylogenetic analysis of the partial IS711 gene indicated that the amplified sequences were genetically similar and clustered within the same clade. These sequences were grouped closely with B. abortus strains reported from humans in China (CP030752.1, CP044339.1 and CP107073.1). In addition, the sequences showed genetic similarity with B. ceti (Portugal), B. suis, B. ovis, B. canis (USA), B. melitensis (Syria) and Brucella sp. (Costa Rica), suggesting a conserved IS711 gene structure across multiple Brucella species (Figure 2).

FIGURE 2.

Phylogenetic tree of Brucella abortus based on the partial IS711 marker sequences available in GenBank. The two sequences (PV545638 and PV545639) generated in this study are highlighted in black.

3.4. Risk Factor Analysis

Six cattle breeds were present at the farm and B. abortus was detected across all of them. An exact test showed no significant association between cattle breed and B. abortus detection (exact p = 0.097) (Table 1). Fisher's exact test showed a significant association between sex and B. abortus detection, with a higher proportion of positive animals among cows than bulls (p = 0.01). However, no statistically significant associations were observed between B. abortus detection and age (p = 1) or history of abortion (p = 1). The ectoparasite comparison involved small and unevenly distributed groups (3/9 vs. 37/74), and no statistically significant association was detected (p = 0.5) (Table 2).

TABLE 1.

Overall comparison of Brucella abortus PCR‐based positivity among the enrolled cattle breeds. Percentage prevalence and 95% confidence of interval as range are given in parentheses. The p‐value represents the output of chi‐square test.

Breed	Brucella abortus positive (95% CI)	Brucella abortus negative (95% CI)	Exact p‐value
Crossbred	11/17 (64%, 38.3%–85.8%)	6/17 (34%, 14.2%–61.7%)	0.097
Dutch	3/7 (43%, 9.9%–81.6%)	4/7 (57%, 18.4%–90.1%)
Holstein Friesian	6/22 (27%, 10.7%–50.2%)	16/22 (73%, 49.8%–89.3%)
Brahman	2/7 (29%, 3.7%–71.0%)	5/7 (71%, 29.0%–96.3%)
Sahiwal	9/17 (53%, 27.8%–77.0%)	8/17 (47%, 23.0%–72.2%)
Cholistani	9/13 (69%, 38.6%–90.9%)	4/13 (31%, 9.1%–61.4%)
Grand total	40/83 (48%, 37.1%–59.4%)	43/83 (52%, 40.6%–62.9%)

Note: 95% CI = 95% confidence of interval; p > 0.05 = non‐significant.

TABLE 2.

Association of the studied risk factors with the PCR‐based positivity of Brucella abortus among the cattle enrolled during the present investigation. Bacterial prevalence (%) is given in parentheses. The p‐value represents the results of Fisher's exact test calculated for the studied parameter.

Parameter		N	Brucella abortus positive	Brucella abortus negative	p‐value
Age	Young	9	4/9 (44%)	5/9 (46%)	1
	Adult	74	36/74 (49%)	38/74 (51%)
Gender	Bull	14	2/14 (14%)	12/14 (86%)	0.01**
	Cow	69	38/69 (55%)	31/69 (45%)
Abortion history	Yes	10	5/10 (50%)	5/10 (50%)	1
	No	73	35/73 (48%)	38/73 (52%)
Ectoparasites	Presence	9	3/9 (33%)	6/9 (67%)	0.5
	Absence	74	37/74 (50%)	37/74 (50%)

p > 0.05 = non‐significant. p ≤ 0.01 = significant (**).

4. Discussion

Livestock farming is a cornerstone of Pakistan's rural economy, with cattle serving as a key source of milk, meat and draught power. Despite its importance, brucellosis remains largely underdiagnosed and underreported across many regions of the country (Ullah et al. 2024). To address this knowledge gap, we conducted a comprehensive screening of cattle and farm workers for Brucella species in the peri‐urban areas of Khanewal district using both conventional methods, such as RBPT, and advanced molecular techniques, such as PCR.

The RBPT is a rapid agglutination assay used to screen for Brucella antibodies in humans and animals, valued for its simplicity and speed, often complemented by other diagnostic methods (Díaz et al. 2011). As the RBPT is a widely used and cost‐effective screening tool, we initially employed it to test all animals at the dairy farm. Remarkably, antibodies against Brucella spp. were detected in the serum of all tested cattle, indicating 100% seropositivity. The uniformly positive RBPT results warrant cautious interpretation. Since RBPT is a screening assay, seropositivity does not necessarily indicate active Brucella infection. Cross‐reactive antibodies generated following exposure to antigenically related organisms, particularly Yersinia enterocolitica O:9, may produce false‐positive reactions in conventional Brucella serological tests (Asgedom et al. 2016; Shrimali et al. 2019). Thus, the observed 100% seropositivity may reflect true exposure, cross‐reactivity, or a combination of these factors. This finding contrasts sharply with the results of Awais et al. (2024), who used RBPT in bovines from the Multan district and reported lower prevalence rates of 16% in cattle and 10% in buffaloes. Globally, Brucella seroprevalence in cattle has been reported to range from 2.4% to 56% (Asgedom et al. 2016; Shrimali et al. 2019; Fero et al. 2020; Ntivuguruzwa et al. 2020). Variations in Brucella prevalence across regions may be attributed to multiple factors, including geographical and climatic conditions, sampling strategies, animal breeds, herd sizes and diagnostic methods (Awais et al. 2024). It is well documented that the RBPT, while sensitive for herd‐level screening, may produce false‐positive results under such conditions and has reduced sensitivity during early or chronic infections (Loubet et al. 2024). This discrepancy supports the findings of Tuon et al. (2017) and Loubet et al. (2024), who reported that RBPT often yields a high rate of false‐positive results. Their studies recommend discontinuing RBPT for Brucella detection in both ruminants and humans, in favour of more reliable molecular methods such as PCR. To overcome these limitations and confirm the serological findings, we performed molecular detection using PCR, a method with higher specificity for Brucella DNA. Interestingly, PCR screening revealed that only 48% of the cattle were positive for B. abortus (Table 1), which is considerably lower than the RBPT‐based seroprevalence. This discrepancy suggests that a proportion of the RBPT‐positive animals had residual or cross‐reactive antibodies rather than active infection (Fero et al. 2020; Ntivuguruzwa et al. 2020). As RBPT detects antibodies whereas PCR identifies circulating Brucella DNA, the difference between the two assays may reflect variation in the infection or exposure status of the animals at sampling (Awais et al. 2024). These findings support the use of both serological and molecular approaches for more reliable assessment of herd‐level Brucella infection. Few studies in the literature have employed PCR to detect Brucella species in cattle. For instance, Jamil et al. (2020) utilized a real‐time PCR approach and reported that 3.9% of Pakistani bovines were infected with B. abortus. The prevalence of B. abortus in cattle has been reported as 7.5% in Kenya (Lokamar et al. 2022), 13.8% (Jain et al. 2014) and 19.1% (Kakoty et al. 2017) in different regions of India. These limited data highlight the need for more extensive studies worldwide that utilize molecular tools such as PCR to accurately detect Brucella species. Expanding molecular surveillance will not only improve our understanding of the epidemiology of brucellosis but also play a crucial role in controlling its spread and preventing zoonotic transmission to humans.

The IS711 insertion sequence serves as a useful genetic marker in the phylogenetic analysis of Brucella species, owing to its variable copy number and distinct genomic positions across different species and biovars. This variation enables effective differentiation among Brucella strains and supports investigations of their evolutionary relationships and genetic diversity (Aljanazreh et al. 2023). The genetic diversity of Brucella species has been extensively studied in humans and animals worldwide; however, only a few recent reports from Pakistan have documented the genetic variability of B. melitensis in local cattle and human populations (Ullah et al. 2024; B. Ali et al. 2025). To contribute to this limited body of knowledge, we utilized partial IS711 gene sequences amplified from cattle and human samples in our study for phylogenetic analysis. The resulting phylogenetic tree indicated that the IS711 sequences obtained closely matched B. abortus strains previously isolated from humans (Wang et al. 2020) and large ruminants (unpublished data) in China. However, given the conserved nature and limited length of the IS711 fragment analysed, these similarities should be interpreted as evidence of sequence‐level relatedness rather than evidence of recent transmission or epidemiological connectivity. Establishing potential transmission routes would require more discriminatory genomic markers together with epidemiological information, including animal movements and trade histories (Bricker et al. 2000). Similar patterns of phylogenetic clustering among geographically adjacent countries have been reported previously, highlighting the role of animal movement and trade in the dissemination of Brucella species across borders (B. Ali et al. 2025). The B. abortus sequences generated in this study formed a distinct cluster, clearly separated from other Brucella species, including B. ovis, B. canis, B. suis, B. ceti, B. melitensis and Babesia microti. These reference sequences were previously deposited from a range of countries and isolated from diverse hosts, including humans. The limited data available on the genetic diversity of B. abortus in Pakistan underscores the need for expanded screening of cattle, buffaloes and individuals in close contact with these animals, particularly in remote and understudied regions of the country. Such efforts are essential to enhance our understanding of the bacterium's genetic variability and to inform more effective control and prevention strategies.

Risk factor analysis revealed that PCR positivity of B. abortus was more prevalent in cows than in bulls. The higher PCR positivity observed among females should be interpreted cautiously because the herd was predominantly female, with relatively few males available for comparison. Although a significant association was observed, the present findings do not establish greater biological susceptibility of females to B. abortus infection. Our findings align with those of Saeed et al. (2020), who reported a higher prevalence of brucellosis in cows than in bulls in their study conducted in Central Punjab, Pakistan. Our observation partially aligns with the findings of Warioba et al. (2023), who reported a significantly higher risk of Brucella infection in cows. The increased susceptibility in cows is often attributed to their prolonged retention in herds for breeding and milk production purposes, coupled with inadequate nutritional support relative to their physiological demands. We also found that the presence of B. abortus showed no significant variation with respect to ectoparasite infestation (Table 2). The lack of a statistically significant association between ectoparasite status and B. abortus detection should be interpreted cautiously because the ectoparasite‐positive group was small and unevenly distributed relative to the ectoparasite‐negative group. Thus, the present analysis may have had limited power to detect an association, and the nonsignificant result should not be interpreted as evidence of the absence of a relationship. It was also observed that cattle breed, age or history of abortion was not linked with the presence of B. abortus. These findings align with the study by Jamil et al. (2020), who similarly reported no association between Brucella infection and factors such as breed, age or reproductive disorders, including abortion, among cattle in Pakistan. Similarly, Negash and Dubie (2021) reported that brucellosis showed no significant association with the age or sex of the cattle included in their study, indicating that these demographic factors may not be reliable predictors of infection status. Alqaseer et al. (2023) also reported that the prevalence of Brucella spp. did not significantly differ among cattle of varying ages and breeds in their study conducted in Iraq, suggesting that these factors may not play a major role in the distribution of the infection. In contrast to our findings, Matope et al. (2011) reported a significant association between the prevalence of Brucella spp. and both the age and abortion history of cattle screened in Zimbabwe, indicating that these factors may influence infection rates in certain settings. Several additional risk factors have been reported to be significantly associated with bovine brucellosis. Robi et al. (2021) highlighted that improper disposal of aborted foetuses and birth products, the use of communal bulls, and the sharing of grazing areas and water sources significantly increased the risk of disease transmission within and between herds. Aguiar et al. (2022) identified large herd size (more than 25 cows) and the presence of pigs on the farm as key contributors to Brucella spp. infection. The presence of infected cows, frequent reproductive failures such as repeat breeding, and the birth of weak calves were also noted as important epidemiological variables. This limited and often conflicting epidemiological evidence highlights the need for more comprehensive, large‐scale surveys across Pakistan to better understand the dynamics of bovine brucellosis, which is essential for designing effective control and prevention strategies.

During this investigation, a significant proportion of the farm workers (45%) were also found to be infected with this bacterium. B. abortus is recognized as a significant zoonotic agent and remains a major contributor to human brucellosis globally (Tuon et al. 2017). In Pakistan, molecular evidence of B. abortus has been documented in individuals with high‐risk occupations, including livestock handlers, veterinarians and slaughterhouse workers, highlighting their occupational vulnerability (Ullah et al. 2024; S. Ali et al. 2021; Yousaf et al. 2021). Brucellosis tends to have a higher prevalence among males than females, especially in endemic regions (Soares et al. 2015). Brucellosis can be transmitted to humans through several routes, including inhalation of infectious aerosols and direct contact with the blood, tissues, urine or aborted foetuses of infected animals (Loubet et al. 2024). These modes of transmission are particularly common among individuals working in proximity to livestock or in laboratory and slaughterhouse settings (Khoshnood et al. 2022). Consumption of unpasteurized dairy products is a significant route of zoonotic transmission of Brucella spp., with brucellosis particularly prevalent in rural regions or areas where milk pasteurization is not adequately implemented (Abdali et al. 2020). Analysis of the risk factor data for the farm workers revealed that none of them had any fertility‐related issues, including testicular inflammation. They ranged in age from 16 to 50 years and had been working in this capacity for at least 1 year. Five out of eleven workers were married and had 2–5 kids; their spouses also had no history of abortion. All the farm workers were non‐smokers and never had a urinary tract infection. Brucella infection can persist in a subclinical state for extended periods, even without obvious clinical symptoms, posing a silent risk to both individuals and public health (Qureshi et al. 2023). In Pakistan, the level of knowledge regarding brucellosis and associated biosafety measures among livestock handlers is typically limited to moderate. This is largely because most of them are smallholder farmers with minimal formal education (Ahmed et al. 2024). Moreover, the disease is often mistaken for a simple fever in both humans and animals. At the same time, restricted access to medical and veterinary extension services in rural settings further complicates timely recognition and management. We recommend coordinated cross‐sectoral measures, such as enhancing clinician training for timely recognition of the disease (including PCR‐based screening), strengthening laboratory diagnostic capacity and ensuring the availability of appropriate antibiotic treatments to manage brucellosis effectively. In addition, establishing integrated reporting mechanisms between medical facilities and veterinary services is essential for the rapid detection and control of outbreaks. In parallel, comprehensive awareness campaigns should be launched to educate farm workers and their families about the routes of transmission, early warning signs of the disease and the critical importance of adopting protective measures, such as wearing gloves during animal handling and avoiding unpasteurized milk.

This study has some limitations. The investigation was limited to a single‐farm outbreak, purposive sampling and a small number of human participants, which may restrict the generalizability of the findings. Multicentre studies involving randomly selected farms are needed to obtain more representative epidemiological estimates. Only blood samples were included according to the predefined study protocol; alternative specimens were not part of the sampling design, which may have limited the diagnostic sensitivity during this investigation. Another limitation of this study was the unavailability of additional confirmatory serological assays, such as CFT, ELISA and FPA, which prevented further evaluation of the discrepancy between RBPT and PCR findings. Future studies incorporating multiple confirmatory diagnostic approaches may provide a more comprehensive assessment of B. abortus infection status. Phylogenetic analysis was based on partial IS711 gene sequences, which provide limited resolution for robust phylogeographic and strain‐level inference of B. abortus. Although the sequences obtained were sufficient for molecular confirmation and preliminary genetic comparison, they may not accurately discriminate among closely related strains or elucidate transmission dynamics during the outbreak. Therefore, future investigations should incorporate higher‐resolution molecular approaches, such as multilocus variable‐number tandem‐repeat analysis (MLVA), multilocus sequence typing (MLST), single‐nucleotide polymorphism (SNP)‐based typing, or whole‐genome sequencing (WGS), to achieve improved epidemiological tracing, genetic characterization and phylogeographic analysis of circulating Brucella strains. A formal statistical power analysis was not performed, and the relatively small sample size may have limited the ability to detect meaningful associations, particularly in subgroup analyses. Because multiple epidemiological factors were examined, the significant association observed for sex should be interpreted cautiously, as the possibility of a chance finding cannot be excluded. Confirmation in larger studies with adequate statistical power is warranted. The absence of longitudinal follow‐up and detailed exposure and treatment histories limited our ability to determine the persistence and clinical significance of B. abortus DNA detected in farm workers. Future studies incorporating repeated sampling and comprehensive clinical assessment are needed to clarify the infection status of PCR‐positive individuals.

5 Conclusion

In conclusion, our study identified B. abortus DNA in cattle and dairy farm workers in Khanewal, Pakistan, highlighting the potential zoonotic risk associated with infected dairy herds. Differences in PCR positivity were observed between cattle and sexes, although these findings should be interpreted within the limitations of the study design. No clinical signs of orchitis or testicular inflammation were reported by the participating farm workers at the time of sampling. Effective brucellosis control requires strengthened farm‐level biosafety and biosecurity, including appropriate personal protective equipment, safe disposal of aborted materials and hygienic milking practices. Regular surveillance, appropriate vaccination programs and management of infected animals, together with awareness among farmers and veterinarians regarding zoonotic transmission and safe animal handling, are important for reducing the risk of B. abortus transmission. We strongly recommend conducting large‐scale epidemiological surveys in other regions of Pakistan where cattle populations remain unexplored for B. abortus. The data generated by such studies will support the development of evidence‐based national control strategies and ultimately enhance the productivity and safety of Pakistan's livestock sector.

Author Contributions

Muhammad Asif: writing – review and editing, conceptualization, funding acquisition. Muhammad Kashif Obaid: conceptualization, funding acquisition, writing – review and editing. Muhammad Naeem: formal analysis, investigation, writing – review and editing. Hira Muqaddas: formal analysis, investigation, writing – review and editing. Shakir Ullah: formal analysis, writing – review and editing. Adil Khan: data curation, supervision, resources. Nasir Mahmood: data curation, supervision, resources. Qiaoyun Ren: conceptualization, formal analysis, validation. Fatmah M. Alqahtani: investigation, validation, visualization, writing – review and editing. Musa A. Said: writing – review and editing, investigation, formal analysis. Fakhreldeen Dabiellil: conceptualization, investigation, writing – review and editing. Furhan Iqbal: writing – review and editing, conceptualization, investigation.

Funding

Muhammad Asif was supported by the China Postdoctoral Fellowship Program/Shandong University International Postdoctoral Exchange Program.The authors extend their appreciation to the Deanship of Research and Graduate Studies at King Khalid University, KSA, for funding this work through Large Research Group under the grant number (RGP.2/174/47).

Ethics Statement

The Ethical Research Committee of the Institute of Zoology, Bahauddin Zakariya University, Multan (Pakistan) approved all experimental procedures and protocols used in this study involving both humans and cattle via letter number BZU/Ethics/2024‐38. Ethical approval also included verbal consent from the dairy farm workers for their inclusion in this investigation. All the experiments were performed following ARRIVE guidelines.

Asif, M. , Obaid M. K., Naeem M., et al. 2026. “Integrated Molecular Detection and Epidemiological Investigation of Brucella abortus in Dairy Cattle and Farm Workers During a Brucellosis Outbreak.” Veterinary Medicine and Science 12, no. 6: e71329. 10.1002/vms3.71329

Data Availability Statement

The datasets generated and/or analysed during the current study are available in the GenBank repository, with Accession numbers PV545638 and PV545639.https://www.ncbi.nlm.nih.gov/nuccore/PV545638. https://www.ncbi.nlm.nih.gov/nuccore/PV545639.