Burkholderia are non-spore-forming, obligately aerobic, rod-shaped, Gram-negative bacteria that are ubiquitously found in plants, animals, soil, and water (Rhodes and Schweizer, 2016). This genus contains some common pathogens, such as Burkholderia cepacia, Burkholderia pseudomallei, and Burkholderia mallei (Foxfire et al., 2021). It is recognized that these bacteria may cause potentially deadly infections in humans and/or ruminants, particularly in immunocompromised individuals (Limmathurotsakul et al., 2014). Generally, transmission of the disease occurs as a result of the exposure to the water or soil where the organisms ordinarily live (Zheng et al., 2021). In 2009, Burkholderia contaminans, an emerging pathogen linked to cystic fibrosis, was included in the B. cepacia complex group (Vanlaere et al., 2009). Burkholderia contaminans has remarkable ability to synthesize antifungal chemicals and survive in a polymicrobial environment (Bernier et al., 2016). After being struck by a cow’s tail in the right eye, a patient developed redness and discomfort for over 20 days, and B. contaminans was isolated from the secretions of that eye. Additional clinical examination revealed the presence of fungal ulcer in the attacked eye of the patient (Lama et al., 2021). Burkholderia contaminans was also considered to be a novel pathogen of bovine mastitis, implicated in many outbreaks in diverse geographic regions (Wang et al., 2022), although it has been usually disregarded. Multidrug-resistant B. contaminans has been identified with multiple sources of antimicrobial resistance genes. The big G + C-rich genome contains a multitude of virulence factors, emphasizing its pathogenicity (Alnoch et al., 2019). This presents new challenges for the prevention and treatment of bovine mastitis.

Bovine mastitis has become the most widespread and expensive production disease in dairy herds worldwide, which is usually caused by intramammary bacterial infection (Seegers et al., 2003). While better dairy herd procedures have helped to eradicate many Gram-positive pathogens that induce mastitis, they have been mostly unsuccessful in reducing the frequency of intramammary infections induced by Gram-negative bacteria (Bannerman et al., 2005). Escherichia coli is the most frequent Gram-negative bacteria that cause mastitis in cattle, and most of our knowledge of the innate immune response to Gram-negative infection comes from previous studies in this bacterium (Zaatout, 2022). In contrast, the adaptive immune response to other widespread Gram-negative bacteria, such as B. contaminans, is far less understood.

Bovine adaptive immune responses consist of cellular and antibody-mediated immune responses that are primarily driven by lymphocytes (Vlasova and Saif, 2021). Different γδ and αβ T-cell subsets are implicated in the protection of the breast against mastitis, and the activation of these T-cell subsets varies among pathogens (Soltys and Quinn, 1999). It was previously found that the proportion and expression of B-cells increased in blood of dairy cows with chronic sub-clinical mastitis after Staphylococcus aureus infection (Grönlund et al., 2006). Since certain pathogens may infiltrate and survive intracellularly, a selective activation of B-cells, indicating the establishment of humoral response, might not be adequate to eradicate intracellular bacteria, which may explain the persistence of infection. Nevertheless, the contributions of diverse lymphoid populations to host defense in spontaneously infected mammary glands of cows remains to be thoroughly investigated. During a bacterial infection, both leukocyte adhesion and the production of cytokines play important roles (Li et al., 2018). However, the proportional contributions of these variables to the pathogenesis of mastitis are unclear and more research is needed.

While most previous studies have been documented the innate immune response to E. coli, less is understood regarding the acquired immunological response of the mammary gland to other Gram-negative bacteria, such as B. contaminans, which has been demonstrated to causes mastitis in cows. We detected an outbreak of cow mastitis caused by single B. contaminans at a dairy farm in Jiangsu province, China, with a total of 20 cases. The purpose of this study was to undertake assessment of T and B cell immune responses against B. contaminans naturally-induced mastitis in dairy cows to develop better strategy against very virulent B. contaminans strains. In the research presented here, we examined the idea that various B and T lymphocyte subgroups are collectively implicated in the udder’s response to a mastitis infection and release pathogen-specific cytokines or antibodies. To test this theory, we have employed a panel of monoclonal antibodies to define the lymphocyte subsets in mastitis and normal peripheral blood in terms of lymphocyte subsets distribution, cytokines, adhesion molecule, and antibody expression. Furthermore, we have presented a comparison of these characteristics for naturally infected cows with B. contaminans, as well as a correlation study between parameters and colony-forming units. These investigations have helped to define the function of the distinct lymphocyte subgroups in the host defense of the cow mammary gland toward infection with B. contaminans, a Gram-negative bacterium that causes mastitis but is clinically significant.

In the present study, an outbreak of clinical mastitis was investigated among a total of 650 lactating cows, microbiological analyses have demonstrated the causative agent of B. contaminans. Mastitis is a disorder of great economic significance, as it reduces both milk quality and quantity and increases the global demand for veterinary antibiotics (Filor et al., 2022). Additionally, the inherent resistance of B. contaminans isolates to the majority of antibiotics restricts treatment options for infected animals, resulting in additional losses from slaughtering infected animals. Attempts have been made to recognize the source of the infection during the epidemic described here. Since no new animals have been recently introduced to the herd, it is probable that the cows contracted the disease from an environmental source. Surprisingly, B. contaminans was unable to be cultivated from any of the environmental samples in the farm. In addition, of B. contaminans was identified from the cows’ milk with at least one prior lactation, but not from the cows’ milk experiencing their first lactation. Undoubtedly, the epidemics of Pseudomonas aeruginosa mastitis in cows is associated with the administration of a possibly contaminated dry-cow antibiotic infusion (Osborne et al., 1981). Therefore, it is probable that antibiotic-resistant bacteria produced by the dry-cow antibiotic medicationmay be a cause of infection. Regardless of the source, Burkholderia strains were probably spread to uninfected animals through the milking process after they were reintroduced into the herd (Berriatua et al., 2001).

In this study, B. contaminans was found to be resistant to many antibiotics tested (penicillin, streptomycin, lincomycin and amoxicillin). Previous studies have extensively investigated antibiotic resistance in B. cepacia. Resistance to β-lactam antibiotics such as penicillin and amoxicillin is caused by class A β-lactamases encoded by B. cepacia organisms, firstly described in the PenA-PenR system of B. cepacia 249, which are now named PenB and PenR (AmpR) (Rhodes and Schweizer, 2016). PenB is a Class A penicillinase with broad spectrum carbapenemase character, which is highly conserved in B. cepacia (Hwang and Kim, 2015). Resistance to aminoglycoside antibiotics such as streptomycin may be caused by AmrAB-OprM efflux pump expression of B. contaminans (Podnecky et al., 2015). In B. vietnamiensis, aminoglycoside resistance emerges during chronic infection or after in vitro exposure to aminoglycosides and is the result of AmrAB-OprM efflux pump expression, which is most likely a homolog of B. cepacia AmrAB-OprA (Jassem et al., 2014). To the best of our knowledge, this is the first report of lincomycin resistance in Burkholderia, and the underlying mechanisms need to be further studied. Similar to numerous other opportunistic infections, it seems that serious illness and mortality may ensue from infection with any B. cepacia species, depending on the clinical condition and propensity of persons with cystic fibrosis at the moment of infection. All B. cepacia species are indeed very resistant to antibiotics (Mahenthiralingam et al., 2008). Hence, treatment of persistent infections is extremely challenging.

During infection and after recurrent pathogen interactions, previously developed mechanisms of the adaptive immune system are activated (den Reijer et al., 2013; Karauzum and Datta, 2017). They include the participation of antibodies generated by B lymphocytes and cell-mediated immune responses governed by T lymphocytes. Typically, a bacterial infection is contained and eliminated after the development of pathogen-specific T and B cell immunity. Moreover, these immune responses are essential for protecting the host from subsequent infections (Kurtz et al., 2017). Remarkably, little is known about the precise function of the humoral immune response in B. cepacia infection. The most notable finding from a previous report (Grönlund et al., 2006) was the enhanced expression and proportion of B-lymphocytes in blood, non-infected and infected quarters in persistent sub-clinical mastitis caused by Gram-positive bacteria, such as S. aureus. The present study identified the reduced total number of B cells in the B. contaminans naturally-induced mastitis cows. Remarkably, we observed a negative correlation between the total number of CD21⁺ B cells and CFU. This indicates that the decrease in B-cell numbers is directly caused by B. contaminans infection. B lymphocytes can serve as antigen-presenting cells, and differentiate into plasma cells that produce immunoglobulins. Antibodies produced are beneficial in the opsonization and phagocytosis of bacteria (Nowicka and Grywalska, 2019). Taken together, this may explain the bad infective consequences of B. cenocepacia. In addition, prior studies have shown that the absolute number of B-lymphocytes in the blood dropped concurrently with the onset of mastitis produced by endotoxin and recovered with the remission of clinical symptoms (Ishikawa and Shimizu, 1983). LPS is a component found in the Gram-negative bacteria’ cell wall that is known as endotoxin. Burkholderia cenocepacia’s lipopolysaccharide (LPS) is likewise highly inflammatory (Govan, 2003) Therefore, we concluded that the decrease in B-cell number in mastitis cattle may be due to the high inflammatory level of LPS in B. contaminans.

Previous studies have shown that IgG, IgM and IgA isoforms of antibodies secreted by B cells are present in the peripheral blood of dairy cows, among which IgG and IgM contents were abundant, while IgA contents were scarce (Enger et al., 2021). As the staple antibody against bacterial cow mastitis, IgG is often used as a diagnostic marker due to its prolonged expression and high sensitivity (Bourry and Poutrel, 1996; Wellnitz et al., 2013; Wall et al., 2016; Drumm et al., 2022; Khatun et al., 2022). The IgM normally precedes IgG antibodies and appears temporarily in an acute infection and functioning in the primary antibody-mediated immune response (Wagner et al., 2008). In the inflammatory response caused by Burkholderia, IgG is a better indicator for early serodiagnosis than IgM with an overall higher sensitivity, specificity, positive/ negative predictive value and likelihood ratios (Hii et al., 2017). In this study, samples were collected at a certain time point, and were subsequently applied to detected the total IgG concentrations. The results showed that no significant difference was observed in total IgG concentration between the healthy cows and cows with mastitis. This suggests that at the sampling time point in this study, the levels B.contaminans-specific IgG may be too low to alter the total IgG concentrations. This result further confirmed our conjecture that humoral immune response may not be adequate to fight intracellular infection of B.contaminans. Therefore, to clarify the protective role of B cells during mastitis in cows infected with B. contaminans, we detected the expression of surface IgG in B cells. Further analysis of B cell subsets revealed that the frequency of IgG⁺ B cells was significantly higher in the B.contaminans-infected mastitis patients than in the healthy cows. IgG is produced by IgG⁺ B cells, which undergo differentiation into IgG-ASC upon activation. IgG⁺ B cell aberrations are associated with autoimmune disorders and persistent infections (Kong et al., 2016). We found that a considerably decreased proportion of CD27⁺IgG⁺ B cells (in total B cells) was observed in the B. contaminans naturally-induced mastitis cows, which was related to B. contaminans infection. Since CD27⁺IgG⁺ B cells belonged to the memory B cell compartments, we examined the prevalence of memory B cells across both groups, and the findings showed that the percentage of memory B cells in total B cells was increased in the B. contaminans-infected cows. This indicated that the elements responsible for the decrease of CD27⁺IgG⁺ B cells may be related to a reduction in memory B cells in B. contaminans infections.

Like B cells, the role of B. cepacia-specific T cells has not been well described. As B. contaminans is an internal pathogen, it is considered that such responses are crucial, especially as antibodies specific to the species have minimal impact on the establishment of immunity (Raupach and Kaufmann, 2001). To better comprehend the host defense mechanism in mastitis, we have evaluated the alterations in T cell subset groups found in the blood of naturally B. contaminans-induced mastitis-stricken cows. We observed a significant increase in the total number of γδ T cells and CD4⁺ CD8⁺ DP T cells in the blood of infected animals compared to the blood of healthy animals. However, there was no difference in the percentage and number of CD4⁺ and CD8⁺ T cells in the blood between the two groups. The function of CD4⁺CD8⁺ DP T cells is mostly unknown. DP T cells were considered to constitute a developmental phase in the thymus prior to maturation into CD8⁺ or CD4⁺ (single positive) mature T cells (Germain, 2002). Consequently, in peripheral tissues and blood, the majority of T cells have maintained expression of just one of such co-receptors with distinct roles, with CD8 T cells directly engaged in cytotoxicity against tumor or infected cells and CD4 T cells orchestrating the immune response. Nonetheless, mature CD4⁺CD8⁺ DP T cells have been identified in peripheral tissues and blood in a variety of conditions, including human malignancies (Overgaard et al., 2015). These cells are diverse and/or exhibit pleiotropic activities, which must be explored in the context of each specific illness. Recent study indicates that DP T cells promote the functional polarization of naive CD4⁺ T cells toward a Th2 phenotype. This ability of DP T cells was detected in healthy donors and was amplified in patients with urologic malignancy, who had higher amounts of circulatory DP T cells (Bohner et al., 2019).

γδ T cells are considered as innate-like T cells with a limited pre-activated phenotype and TCR repertoire (Shiromizu and Jancic, 2018), and they are the major T-cell population in newborn ruminants’ circulation (Jörundsson et al., 1999). They may react promptly to infection or cytokine stimulation in a non-MHC-restricted manner, secreting a broad spectrum of cytokines and exhibiting direct cytotoxicity against infected and altered cells (Shiromizu and Jancic, 2018). It has been suggested that γδ T cells supplementαβ T cells in host defense by giving a fast reaction before theαβ T-cell response has completely formed, i.e., the first line of defense (Williams, 1998). Certain activated γδ T cells are also capable of presenting antigen to CD4⁺ T cells and priming bacterial antigen-specific CD8⁺ T cells. These cells also seem to generate costimulatory molecules in addition to cytokines, suggesting that γδ T cells may really influence the activity ofαβ T cells (Soltys and Quinn, 1999). Thus, the elevated amounts of γδ T cells in the blood of mastitis-affected cows are consistent with their hypothesized involvement in controlling the inflammatory response.

And on this basis we determined the percentages of CD27⁺ T cells and CD44⁺ T cells in the αβ or γδ T cells subsets of the healthy and B. contaminans naturally-induced mastitis cows to assessment the differentiation and activation of T cells. However, we did not find the differences in the percentages of CD27⁺ T cells in either WC1⁺ γδ, CD8⁺, and CD4⁺ T cells between the two groups. CD27, a co-stimulatory molecule, is often used to detect T cell development phases. Potentially, CD27 expression may be utilized to track γδ T cell response to M. bovis infections in cattle (Guerra-Maupome et al., 2019). Our results indicated that T cells from the B. contaminans infected cows shared a similar differentiation phenotype with those from the healthy cows at the early time-points. In addition, we determined the expression of CD44 molecule on the T subsets of the mastitis or healthy cows to investigate the activation of T subsets. We found that the percentages of activated WC1⁺ γδ T cells in peripheral blood were increased in the B. contaminans infected cows. However, there was no difference in the percentage of CD4⁺ CD44⁺ and CD8⁺CD44⁺ T cells in the blood between the two groups. The activation of T cells is one of the initial stages in the host’s response to infections, and the emigration of T cells from the circulation to areas of inflammation requires the progressive interaction of different adhesion molecules released by T cells (Odoardi et al., 2012). CD44, a leukocyte adhesion molecule, interacts with the extracellular matrix’s components and is regarded as necessary for T-cell extravasation in inflammatory locations. This adhesion molecule has been identified as a sensitive indication of T-cell activation (Bosworth et al., 1991). Thus, we conclude that γδ T cells are selectively activated during the host’s defense to B. contaminans in comparison to αβ T cells. And variations in the expression of such adhesion molecules on blood T cells may serve as a possible diagnostic sign of mastitis that can be examined quickly and inexpensively. Our research further supports the notion that γδ T cells supplementαβ T cells in host defense by giving a fast reaction prior to the αβ T-cell response has completely formed, i.e., the first line of defense.

IL-2, TNF-α, and IFN-γ have a vital role in mastitis prevention, according to bovine studies (Alluwaimi, 2004). Recent research on immune responses to infectious disorders has uncovered and characterized a vital function for multifunctional T cells that co-express IL-2, TNF-α, and IFN-γ. It has been hypothesized that multifunctional T cells are a characteristic of protective immunity. For instance, calves typically infected with bovine tuberculosis had a predominance of TNF-α⁺IFN-γ⁺IL-2⁺ and TNF-α⁺IFN-γ⁺IL-2⁻ CD4⁺ T cells with a TEM phenotype (Whelan et al., 2011). It should be noted that, due to the limitation of immunological reagents (the fluorescence of WC1 and TNF-α antibody is in conflict), the expression of activated γδ T inflammatory cytokines was not detected in our study. However, previous studies have shown that γδ T cells in peripheral blood of cows express little IFN-γ and do not express IL-2 in response to superantigen activation (Fikri et al., 2001). Further studies are required to determine the expression inflammatory factors in B. contaminans-specific γδ T cell. But executinging triple-cytokine staining of in vitro stimulation of PBMCs, we observed that many B. contaminans-specific CD4⁺CD8⁺ DP T cells can be activated to produce three of the cytokines TNF-α, IFN-γ and IL-2 simultaneously. This early appearance in regard to the clearance of clinical signs confirms their immediate role in the primary immune response against B. contaminans. After infection with B. contaminans, CD4⁺ or CD8⁺ T cells isolated from blood of cows produce hardly any IFN-γ. This might indicate that IFN-γ is not directly involved in the host immune response in the early stage of mastitis caused by B. contaminans in cows. In this study, individual TNF-α- producing or IL-2-producing CD4 and CD8 cells were a relatively small population. The cows were infected for circa 14 days. It may be not enough time to develop the cell immune response.

In conclusion, we have investigated the lymphocyte subsets distributions and proinflammatory cytokine’s expression on leukocytes isolated from the blood of cows with spontaneously acquired mastitis caused by B. contaminans and healthy cows. Our studies showed a significant reduction in the percentages and absolute number of CD21⁺ B cells in cows with B. contaminans naturally-induced mastitis. And we have demonstrated that γδ T cells and CD4⁺CD8⁺ DP T cells significantly increased at the early stage after infection and displayed differential activation and differentiation. In addition, our results indicated the production of B. contaminans-specific TNF-α⁺IFN-γ⁺IL-2⁺ DP T cells in the immunological response of cows to Gram-negative bacteria infection. This comprehensive analysis of bovine T, B cells responses to infection with B. contaminans will aid in the development of more effective therapies or vaccinations against bovine mastitis.

The data presented in the study are deposited in the GenBank repository, accession number OP890178.

The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Yangzhou University.

ZZ, ZY, and YYang contributed to conception and design of the study. ZZ, YYao, HJ, YM, WC, FZ, and KW performed the experiments. ZZ, JY, CY, JS, and YYang organized the database. ZZ, CY, and JS performed the statistical analysis. ZZ wrote the first draft of the manuscript. YYang revised the manuscript. All authors contributed to the article and approved the submitted version.

This study was financially supported by Seed Industry Vitalization Program of Jiangsu Province (JBGS[2021]117), National Natural Science Foundation of China (32002263 and 31872324), Jiangsu Agriculture Science and Technology Innovation Fund (JATS[2021]486), Basic Research Program of Jiangsu Province (BK20190881), Postdoctoral Research Foundation of China (2019M650126), Young Elite Scientists Sponsorship Program of Jiangsu Province (TJ-2022-031), the 111 Project D18007 and Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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