Brucellosis, which is by Brucella infection, is one of the most important and widespread bacterial zoonotic diseases worldwide. Brucella is a Gram-negative, short rod-shaped bacterium. Currently, six species and 19 biotypes have been identified. Among which Brucella melitensis [1] and Brucella abortus [2] pose the most significant threats to human health. In female animals, Brucella infected can cause abortion, loss of appetite, and difficulty walking, while male animals primarily develop orchitis and epididymitis [3].

According to an analysis of global and regional high-risk populations, approximately 500,000 human cases occur worldwide each year, with under-developed regions in Africa and Asia bearing the brunt of the burden [4]. Previous studies have shown that the host resistance to infectious disease is closely linked to genetic factors [5]. Therefore, identifying genetic markers associated with reduced risk of Brucella infection in goats and expanding disease-resistant populations represnt a fundamental strategy for mitigating the impacts of brucellosis. With rapid advances in genetics, molecular marker-assisted selection (MAS) is now widely used to improve growth and reproductive performance in goats, Among the available markers, SNP and InDels are the most commonly used [6], as they enable the accurate and rapidly identification of superior genotypes in livestock [7].

The ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) gene produces a protein that contains SH3, ANK, and PH domains. This protein participates in cellular processes such as cytoskeletal regulation and vesicle transport [8,9]. ASAP1 has been show to regulate the phagocytic capacity of THP-1–derived macrophages against Mycobacterium tuberculosis H37Ra by remodeling the actin cytoskeleton [10]. The ASAP1 protein is predominantly expressed in specific immune cells, such as dendritic cells (DCs). Notably, infection with M.tuberculosis significantly downregulates ASAP1 expression in DCs [11]. However, the unique structure of Brucella lipopolysaccharide (LPS) distinguishes it from that of typical Gram-negative bacteria like Escherichia coli. Specifically, the lipid A moiety of Brucella LPS has a significantly longer fatty acid chain. This structural difference alters the molecule conformation and impairs its ability to bind effectively to the toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) complex. Consequently, Brucella LPS induces NF-κB mediated immune cell activation to a mch lesser extent than E. coli LPS [12]. This failure to trigger a robust inflammatory response in DCs, a form of molecular mimicry may help Brucella evade detection by the host’s innate immune system.

To date, no studies have linked the ASAP1 gene to resistance against Brucella infection in goats. In this study, we first analyzed the expression profile of the ASAP1 gene in various tissues of Shaanbei white cashmere (SBWC) goats. We then detecte polymorphisms at InDel loci within the ASAP1 gene in goat populations and systematically invesrigated the correlation between these InDel variations and resistance to Brucella in SBWC goats.

Brucella, a facultative intracellular Gram-negative bacterium, invades immune cells such as macrophages, thereby triggering a strong inflammatory response in the host. The ASAP1 gene functions to remodel the cytoskeleton by enhancing F-actin aggregation and increasing the formation of vinculin/paxillin plaques, thereby altering actin remodeling dynamics [10]. Evidence indicates that the endocytosis of M.tuberculosis H37Ra in THP-1 macrophages is achieved through the regulation of actin dynamics by the ASAP1 gene [10]. Notably, both M.tuberculosis and Brucella are intracellular bacterias that evade immune clearance and achieve intracellular proliferation through common mechanisms such as inhibiting phagosome-lysosome fusion and modulating autophagy pathways [15]. Genome-wide association studies have established that specific SNPs in the ASAP1 gene are significantly linked to human susceptibility to tuberculosis. In addition, ASAP1 expression was downregulated and DC migration was severely impaired following infection with M.tuberculosis [11], which prevented the effective activation of early T-cells and lead to a failure in establishing an adaptive immune response [16]. This has been identified as a key mechanism for host susceptibility to tuberculosis. Importantly consistent with findings in tuberculosis [11], our study found a significantly lower expression level of the ASAP1 gene in the testicular tissues of Brucella infected adult bucks compared to healthy controls. It has been reported that during Brucella infection, the effector protein BspF interacts with an Arf6 GTPase-activating protein (ACAP1), interfering with Arf6/Rab8a mediated membrane trafficking and causing abnormal accumulation of trans-golgi network (TGN) derived vesicles on the bacterium-containing vacuole (rBCV), thereby promoting bacterial replication [17]. ASAP1 and ACAP1 belong to the Arf-GAP protein family [17], but ASAP1 is uniquely characterized by an SH3 domain [18], which mediates protein-protein interaction networks by recognizing proline-rich motifs [19], and is widely involved in processes such as cell migration, proliferation, and cytoskeletal remodeling [19,20]. Although the functional importance of ASAP1 had been demonstrated in colorectal cancer [21], gastric cancer [20], and tuberculosis [11], its role in Brucella infection had not been reported.

This study represents the first report to systematically investigate the association between ASAP1 gene variations and Brucella infection risk in goats. The expression of ASAP1 was first analyzed across various goat tissues, This expression profile suggested a potential association of this gene with immune function in animals [22]. As a vital immune organ, the spleen is recognized as a primary defense against bacterial infections through the synergistic actions of innate immunity, adaptive immunity, and mechanical filtration [23]. Therefore, the high expression of the ASAP1 gene in this organ likely indicated a significant role in immune responses and pathogen clearance in goats. Evolutionary genetic analyses have indicated that positive selection pressure on host genes is closely associated with pathogen-driven adaptive evolution [24]. In the SBWC goat population, three InDel loci within the ASAP1 gene were found to display significant population genetic characteristics. We analyzed the genetic diversity and HWE of thise loci, among them, the P2 and P7 loci were predominantly characterized by the I allele, and distinct genotype distributions were observed between populations. The observed deviation from HWE at the control group in the P2 locus could be attributed to breeding practices intrinsic to commercial livestock management. Directed selection for economically important traits and non-random mating schemes (e.g., the extensive use of elite bucks) will systematically alter genotype frequencies, leading to deviations from HWE expectations [25]. Despite these population genetic deviations, the observed associations between ASAP1 InDels and brucellosis susceptibility were further supported by functional evidence from gene expression analyses.

Given that previous studies have often been limited by small sample sizes (N<500), this study provides reliable evidence for the association between ASAP1 gene polymorphisms and resistance/susceptibility of goats to Brucella infection through a large sample population (N>1,000). Due to the large sample size and associated cost constraints, confirmatory ELISA or PCR testing was not performed. However, it is acknowledged that the sole reliance on the RBPT for infection status classification constitutes a methodological limitation. Given that results based on RBPT do not exclude the possibility of false negatives or false positives, to improve reliability in the absence of confirmatory assays, all samples underwent repeated RBPT testing, and only those with consistently positive results were included in the infected group, which has substantially reduced the probability of false positives within the constraints of the available methodology. Prior to the genetic association analysis, the potential associations of age and parity with Brucella infection status were assessed in the selected samples. The confirmation of no significant associations for these covariates strengthened the validity of the genotypic results. It is acknowledged that environmental and management variables, including pen allocation and microenvironment, were not controlled for in this study. Nevertheless, all experimental animals were sourced from farms with standardized management, and the association between ASAP1 genotypes and infection status remained significant after adjusting for age and parity. The association analysis revealed that genotype II at the P2 locus was associated with susceptibility, whereas genotype II at the P7 locus was associated with resistance, suggesting that genotype distribution influences the response of goats to Brucella infection. Previous studies have indicated significant associations between genetic polymorphisms and increased or decreased to Brucella infection. In our earlier work, the cytotoxic T lymphocyte-associated antigen-4 (CTLA4) II genotype was shown to reduce brucellosis susceptibility in goats and enhance secretion of anti-inflammatory factors from PBMCs post-LPS challenge [26]. In our study, although no LD was detected among the three InDel loci, based on previous research in regulatory genomics, it has been proposed that combinations of genetic variants, even in the absence of strong linkage, could encompass multiple cis-regulatory modules (e.g., enhancers or silencers) and that their joint effects might co-regulate target gene expression through mechanisms such as chromatin spatial reorganization [27]. Furthermore, studies suggest that such combinatorial variation may influence transcription-factor networks and protein interactions, thereby potentially contributing to complex phenotypes like disease resistance in a synergistic manner [28]. Accordingly, we continue to explore the non-linked haplotype model from functional and regulatory perspectives. Therefore, PBMCs from goats with different haplotypes were stimulated with LPS. Observations suggested that PBMCs from goats carrying the Hap3 and Hap5 haplotypes tended to exhibit a more rapid initiation and resolution of the immune response following stimulation. These changes appeared to involve activation of the NF-κB pathway, relatively elevated expression levels of pro-inflammatory cytokines (IL-6 and TNF-α), and increased secretion of immunoregulatory factors (TGF-β and IFN-γ). At 24 h post-LPS stimulation, the NF-κB pathway showed indications of downregulation, and the concentrations of pro-inflammatory and immunoregulatory factors decreased. This response profile could potentially contribute to more effective control of infection. Furthermore, the expression level of ASAP1 at 24 h was significantly lower in haplotypes associated with high infection risk compared to those associated with low infection risk, which aligns with the downregulated expression trend of ASAP1 observed in infected animals. In Brucella infected testicular tissue expression was observed aligning directionally with our cellular assay results. However, given that the sample size was limited to n = 1 per group, this observation should be considered preliminary and does not constitute definitive evidence regarding the role of ASAP1 in goat Brucella infection. And the testicular tissue examined in this study serves as a primary reproductive organ target for Brucella infection, offering relevant pathophysiological insights. It should be noted, however, that sample collection from infected animals was highly restricted, and all animals in the main association cohort were female. Therefore, findings from this male tissue analysis should be interpreted as a reference for potential infection effects in reproductive organs. Overall, these three gene loci are genetically independent; based on our exploratory observations, they might collectively influence the immune phenotype when co-present. Nevertheless, the precise mechanisms underlying these phenotypic associations warrant further investigation.

Although the protein-coding region directly determined protein structure, gene expression can be regulated by mutations in non-coding regions through alteration of transcription factor binding affinity, a process that plays a significant role in the evolution of disease resistance traits [29]. Previous studies had shown that intron 3 of the mediator of IRF3 activation (MITA) gene was bound by the RNA binding protein LUC7L2, which leads to intron retention and the subsequent triggers nonsense-mediated mRNA decay (NMD), thereby reducing MITA protein levels and weakening the intensity of the innate immune response to DNA viruses [30]. Importantly, transcription factors including MCM1, CREB, and CPEB play crucial roles in regulating the immune cell cycle, immune gene expression, and cell migration [31–33]. It should be emphasized that the transcription-factor binding sites (TFBS) predicted using AliBaba2 are based solely on in silico sequence analysis; these predictions are preliminary and do not confirm actual regulatory function, their specific mechanisms require further investigation. Considering that cattle, sheep, and humans are all major hosts of Brucella, and given the high expression of ASAP1 was observed in goat spleen in this study, a cross-species homology comparison was conducted. Phylogenetic analysis showed that the goat ASAP1 nucleotide sequence is highly conserved with Ovis aries (98.7%) and Bos taurus (96.3%), but less so with Gallus gallus (71.4%), indicating strong evolutionary conservation among ruminants, as reported in existing comparative genomic studies [34]. In summary, InDel variant in the ASAP1 gene were found to be associated with resistance to Brucella infection in goats, Goats carrying the low-disease-risk haplotype were shown to initiate immune responses more rapidly to combat bacterial infection and clear the pathogen. This outcome may be attributed either to potential functional interactions among the three variant sites or to the differential recruitment of transcription factors (such as MCM1), both of which could ultimately regulate gene function. However, given the high complexity of bacterial invasion and clearance, the precise molecular mechanisms were concluded to require further investigation.

Polymorphisms at three InDel loci within the ASAP1 gene were identified for the first time in the SBWC goat population, and their association with increased or decreased to Brucella infection was established. Under LPS stimulation, PBMCs from goats carrying the low-disease-risk haplotype exhibited a robust immune response and an accelerated termination of the immune response. The high-disease-risk haplotype exhibited lower ASAP1 levels, a pattern that matches the expression trend observed in tissues from Brucella-infected goats. This study elucidates the role of the ASAP1 gene in resistance to Brucella infection and provides potential target loci for the genetic breeding of brucellosis-resistant goats.