Acid pre-adaptation enhances virulence of Salmonella enterica serovar Typhimurium dam mutant

It is well established that success or failure of bacterial pathogens during infection relies upon its ability to overcome many lethal environments in the host such as acidity, osmolarity and bile salts. In the present study, we have studied the effects of acid adaptation on the virulence of Salmonella enterica serovar Typhimurium dam mutant. Our results indicated that LD(50) of adapted strains were lower than those of control strains. Also, the in vivo assays have shown that the development of a systemic infection is slower for control strains than for adapted strains. In addition, the number of acid-adapted mutants colonizing spleen and liver is higher than control strains. Adhesion and invasion experiments were performed in order to compare the pathogenicity of Salmonella. No significant differences were shown between pre-treated and non-adapted strains. According to these results, we report that acid adaptation of Salmonella enterica serovar Typhimurium dam mutants can increase their in vivo virulence in mice.     

Interplay between MgtC and PagC in Salmonella enterica serovar Typhimurium

In Salmonella enterica serovar Typhimurium, MgtC and PagC are positively regulated by the PhoP-PhoQ two-component system, which is activated under magnesium deprivation. Both MgtC and PagC are of unknown function but have been involved in intramacrophage survival. We have found that the amount of PagC is lowered in a DeltamgtC mutant strain grown in magnesium depleted medium. However, the effect of MgtC on PagC does not account for the growth defect of a DeltamgtC mutant in macrophages since, in contrast to previous reports, our results indicate that PagC does not contribute to intramacrophage survival. In addition, a pagC null mutant is only poorly attenuated in Nramp1-negative or Nramp1-positive mice. On the other hand, a mgtC null mutant is significantly more attenuated with Nramp1-positive than Nramp1-negative mice, suggesting that a functional Nramp1 (Slc11a1) further limits the multiplication of this mutant within the host.     

Caspase-3-dependent phagocyte death during systemic Salmonella enterica serovar Typhimurium infection of mice

Growth of Salmonella enterica in mammalian tissues results from continuous spread of bacteria to new host cells. Our previous work indicated that infective S. enterica are liberated from host cells via stochastic necrotic burst independently of intracellular bacterial numbers. Here we report that liver phagocytes can undergo apoptotic caspase-3-mediated cell death in vivo, with apoptosis being a rare event, more prevalent in heavily infected cells. The density-dependent apoptotic cell death is likely to constitute an alternative mechanism of bacterial spread as part of a bet-hedging strategy, ensuring an ongoing protective intracellular environment in which some bacteria can grow and persist.     

Intracellular growth and survival of Salmonella enterica serovar Typhimurium carrying truncated hemoglobins of Mycobacterium tuberculosis

Two distantly related truncated hemoglobins (trHbs), HbN and HbO, are produced at different growth stages of Mycobacterium tuberculosis. Oxygen and nitric oxide (NO) binding properties of these trHbs suggest their vital role(s) in adaptation of tubercle bacillus under hypoxic and nitrosative stress conditions. Here, we have demonstrated that HbN of M. tuberculosis provides distinct advantage over HbO in supporting intracellular growth and survival of the heterologous host, Salmonella enterica serovar Typhimurium, during macrophage infection specifically against toxicity of NO. HbN and HbO encoding genes of M. tuberculosis have been expressed in a NO-sensitive hmp mutant of S. enterica serovar Typhimurium that exhibits attenuated growth within the macrophages. Presence of HbN and HbO conferred distinct oxygen dependent NO metabolizing activity to the mutant S. enterica serovar Typhimurium. However, the HbN carrying cells exhibited nearly 2-3-fold higher NO metabolizing activity than the isogenic strain having HbO under aerobic condition. More than half of the NO uptake activity of HbN carrying cells was retained when oxygen level dropped to microaerobic condition. In comparison, NO uptake activity of HbO carrying cells of mutant S. enterica dropped drastically (90%) under similar hypoxic conditions. When internalized by mice peritoneal macrophages, HbN carrying cells exhibited 3- and 4-fold higher survival compared to similarly bound and internalized HbO carrying and control cells, respectively. The protective effect of HbN persisted even after activation of macrophages in the presence of IFN-gamma, whereas, HbO did not show any significant effect on survival of the NO-sensitive hmp mutant of Salmonella. These results provide strong experimental evidence in support of the protective role of HbN against nitrosative stress inside macrophages and suggest that intracellular protection conferred by HbN of M. tuberculosis might not be restricted to its native host only.     

A Salmonella enterica serovar Typhi plasmid induces rapid and massive apoptosis in infected macrophages

pR_ST98 is a chimeric plasmid isolated from Salmonella enterica serovar Typhi (S. typhi) that mediates the functions of drug resistance and virulence. Previously, we reported that Salmonella plasmid virulence (spv) genes were present in S. typhi. In our current study, we investigated whether plasmid pR_ST98 exhibits significant cytotoxicity in macrophages. pR_ST98 was transferred into the avirulent Salmonella enterica serovar Typhimurium (S. typhimurium) strain RIA to create the transconjugant pR_ST98/RIA. The standard S. typhimurium virulent strain SR-11, which carries a 100-kb virulence plasmid, was used as a positive control. The bacterial strains were incubated with a murine macrophage-like cell line (J774A.1) in vitro. Apoptosis of J774A.1 cells was examined by electron microscopy and flow cytometry after annexin-V/propidium iodide labeling, and the survival of Salmonella strains in J774A.1 cells was determined. Results showed that macrophages infected with strain pR_ST98/RIA displayed greater levels of apoptosis than those infected with RIA and that pR_ST98 may increase bacterial survival in macrophages. Further studies showed that the pR_ST98-induced death of macrophages was associated with the loss of mitochondrial membrane potential and that pR_ST98 may activate caspase-9 and then caspase-3. The research data indicate that the virulence of bacteria that contain the pR_ST98 plasmid is enhanced; the presence of this plasmid increases the survival of the bacterial pathogen and acts through the mitochondrial pathway to mediate macrophage apoptosis.     

Salmonella enteritidis ghost vaccine induces effective protection against lethal challenge in specific-pathogen-free chicks

Bacterial ghosts (BGs) are empty bacterial envelopes generated by expulsion of the bacterial genome and cytoplasmic contents from bacterial cells, and the process is mediated by lysis protein E encoded on bacteriophage PhiX174. BGs represent a new approach in vaccine development and have been applied to a variety of gram-negative bacterial vaccine candidates. In this study, a BG vaccine generated from Salmonella enteritidis (S. enteritidis) strain DH091 was prepared using the highly efficient plasmid, pBV-mE. The efficacy of the BG vaccine was tested using 75 chicks (Gallus gallus) kept under specific pathogen-free (SPF) conditions. A comprehensive evaluation of the immune response, including humoral and cellular immune responses, interferon-γ (IFN-γ) and interleukin-4 (IL-4) production, and histopathology of various tissues, was performed in BG-vaccinated animals subsequently challenged with S. enteritidis. The results were compared with animals that were immunized with the inactivated vaccine. S. enteritidis ghosts not only promoted the generation of high titer antibodies and IFN-γ and IL-4 production but also stimulated a significant increase in CD8⁺ and CD4⁺ T lymphocytes. In particular, the dramatic increase in CD8⁺ T cells indicated that the vaccine was able to induce clearance of intracellular Salmonella. The protective effects of BG vaccination in SPF chicks against 5 × 10⁹ colony forming units of S. enteritidis were a result of the induction of a more effective immune response than that observed with the inactivated vaccine. These findings demonstrate the potential of S. enteritidis ghosts to be used as effective vaccines.     

Differences in the carriage and the ability to utilize the serotype associated virulence plasmid in strains of Salmonella enterica serotype Typhimurium investigated by use of a self-transferable virulence plasmid, pOG669

Most strains of Salmonella enterica subspecies enterica serotype typhimurium (S. typhimurium) naturally harbour a virulence plasmid which carries the salmonella plasmid virulence (spv) genes. However, isolates belonging to certain phage types are generally found without the plasmid. We have utilized a self-transferable virulence plasmid, pOG669 to investigate the effect of introduction of spv genes into strains of such phage types. The use of the co-integrate plasmid, pOG669, was validated on a diverse collection of strains. pOG669 was transferred into strains of serotypes that are normally associated with the possession of virulence plasmids. All strains maintained the wild type level of virulence in a mouse model, except that introduction of pOG669 restored normal virulence levels in an avirulent, plasmid free strain of S. dublin and resulted in a decrease in virulence in a strain of S. dublin from clonal line Du3. S. gallinarum did not become virulent in mice, but pOG669 was functionally interchangeable with the wild type plasmid when strains were tested in a chicken model. Strains of serotypes not normally associated with the carriage of a virulence plasmid did not increase in virulence upon the introduction of pOG669. An IncX plasmid pOG670 that was included as control was incompatible with the virulence plasmid in a strain of S. dublin, demonstrating that the common virulence plasmid of this serotype is of a different incompatibility group than other virulence plasmids. Strains of S. typhimurium from phage types that do not normally carry a virulence plasmid responded differently to attempts to introduce pOG669. No transconjugants were observed with the strains of DT5 and DT21. The introduction of pOG669 did not alter the virulence of JEO3942(DT10), DT35 and JEO3949(DT66) significantly, while DT1 and DT27 became more virulent. DT27 became as virulent as wild type C5, while logVC(10) of DT1 only increased from 4.1 to 5.7. The ability to express spv-genes was measured by use of an spvRAB'-cat fusion. Expression in S. enteritidis was found to be higher than in other serotypes tested. Only serotypes that naturally carry a virulence plasmid expressed spv-genes. The strain of DT1 expressed spv at a very low level, while expression in the strains of DT10 and DT35 was approximately 2-fold lower than in a control strain of S. typhimurium, while the level in the DT66 strain corresponded to the control strain. The plasmid pSTF9, which carried the fusion gene could not be introduced into the strains of DT5, DT21 and DT27. The RpoS level in the strains was measured indirectly by use of a katE-lacZ fusion. In the DT5 strain the level of expression was low, while the strains JEO3942(DT10), DT21, DT27 and DT35 expressed 4-5 fold the level in this strain. An internal fragment of the rpoS gene was sequenced in three strains. These all showed an identical sequence to a published S. typhimurium rpoS gene.     

gcpA (stm1987) is critical for cellulose production and biofilm formation on polystyrene surface by Salmonella enterica serovar Weltevreden in both high and low nutrient medium

Biofilm formation by Salmonella is a serious concern in the food-processing industry and the persistence of the organism in biofilms becomes a constant source of contamination. Since there is zero tolerance for Salmonella in foods, it is important to understand the mechanism of biofilm formation and to prevent the formation. Therefore, this study aimed at investigating the biofilm-forming ability of seafood isolates of Salmonella enterica serovar Weltevreden (S. Weltevreden) under two different nutrient conditions (normal strength trypticase soy broth (TSB) and 1:100 diluted TSB). The role of cellulose production in biofilm formation and in the expression of multicellular behavior (rough, dark, red morphotype: rdar) was investigated. Fourteen isolates of seafood associated S. Weltevreden were studied for biofilm production in polystyrene microtitre plates. Only one (SW49) of 14 was a strong biofilm former on polystyrene template and was able to produce biofilm in both undiluted TSB and 1:100 diluted TSB at 24 h. All others produced moderate or weak biofilms which was higher in 1:100 diluted TSB compared to undiluted medium. All the isolates except one were positive by PCR for the three genes, gcpA (stm1987), adrA (yaiC) and csgD. Gene expression of gcpA, adrA and csgD was studied by real-time PCR with the one strong (SW49) and one representative weak (SW30) biofilm former. In SW49 at 24 h of incubation, the expression of gcpA from biofilm cells was 33 and 36 times higher than from planktonic cells grown in TSB and diluted TSB respectively and at 72 h the expression from biofilm cells was 57 and 61 times higher than that from planktonic cells. Quantification of gene expression did not reveal any significant difference in the expression of csgD and adrA gene. Deletion of gcpA in SW49 resulted in its inability to produce cellulose and consequent inability to bind calcoflour, inability to form rdar colony on Congo Red-agar plates and failure to produce biofilm on polystyrene substrate. The data indicated that, in case of S. Weltevreden, gcpA is critical for activating cellulose synthesis and biofilm formation both in undiluted and diluted TSB. The results of this study suggest the existence of an alternative biofilm regulatory pathway in S. Weltevreden. Role of adrA in cellulose production in nutrient rich medium is known but role of gepA in the above phenomenon is proved in this study. An understanding of the genes involved would help in looking at strategies of repression of the gene to control formation of biofilm.     

A recombinant adenovirus expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice

Tuberculosis (TB) is caused by an infection of Mycobacterium tuberculosis (Mtb) and remains an enormous and increasing health burden worldwide. To date, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only licensed anti-TB vaccine worldwide, which provides an important but limited protection from the Mtb infection. The development of alternative anti-TB vaccines is therefore urgently needed. Here we report, the generation of Ad5-CEAB, a recombinant adenovirus expressing Mtb antigens of CFP10, ESAT6, Ag85A and Ag85B proteins in a form of mixture. In order to evaluate the immunogenicity of Ad5-CEAB, mice were immunized with Ad5-CEAB by intranasal instillation three times with 2-week intervals. The results demonstrated that Ad5-CEAB elicited a strong antigen-specific immune response, particularly of the Th1 immune responses that were characterized by an increased ratio of IgG2a/IgG1 and secretions of Th1 type cytokines, IFN-γ, TNF-α, IL-2 and IL-12. In addition, the Ad5-CEAB also showed an ability to enhance humoral responses with a dramatically augmented antigen-specific serum IgG. Furthermore, an elevated sIgA were also found in the bronchoalveolar lavage fluid of the immunized mice, suggesting the elicitation of mucosal immune responses. These data indicate that Ad5-CEAB can induce a broad range of antigen-specific immune responses in vivo, which provides a promising and novel route for developing anti-TB vaccines and warrants further investigation.     

Salmonella enterica serovar Dublin strains which are Vi antigen-positive use type IVB pili for bacterial self-association and human intestinal cell entry

Some strains of Salmonella enterica serovar Dublin are Vi antigen-positive. S. enterica serovar Typhi uses Type IVB pili, encoded adjacent to the viaB locus required for Vi antigen synthesis, to facilitate both eukaryotic cell attachment and bacterial self-association under conditions that favour DNA supercoiling. These pilus-mediated events may be important in typhoid fever pathogenesis. A survey of 17 isolates of S. enterica serovar Dublin showed that all strains which carried the viaB region also carried a serovar Typhi-like Type IVB pil operon, and all serovar Dublin Vi antigen-negative isolates lacked the pil operon. The pil operon was completely sequenced from one of the Vi(+) serovar Dublin strains, and was almost identical (4 nt changes; 3 aa changes, in over 10 kb) to that of serovar Typhi. A pilS mutant of one serovar Dublin strain was constructed, and shown to invade cultured human intestinal INT407 cells to an extent only 20% that of the wild-type parent. Purified prePilS protein inhibited INT407 cell entry by serovar Dublin. The wild-type serovar Dublin strain, but not the pilS mutant, self-associated. The data suggest that the serovar Dublin Type IVB pil operon may increase the human-invasiveness of serovar Dublin, compared to pil-free strains.     

Chlamydia pneumoniae infection in IL-10 knock out mice: accelerated clearance but severe pulmonary inflammatory response

In interleukin-10 knock out (IL-10 KO) mice, accelerated clearance of pulmonary Chlamydia pneumoniae infection was observed. On the other hand, the histopathological changes in lung tissue were more pronounced in IL-10 KO mice at all time points after infection and repeated infection than in the wild type mice. Both ex vivo induced antigen-specific proliferation as well as production of proinflammatory cytokines by splenocytes were higher in IL-10 KO mice than in WT mice. Also, intrapulmonary proinflammatory cytokine levels were higher in IL-10 KO mice than in the WT mice. The lack of anti-inflammatory action of IL-10 is likely to contribute to the enhanced clearance but severe inflammation in this experimental model.     

Effect of norepinephrine on colonisation and systemic spread of Salmonella enterica in infected animals: role of catecholate siderophore precursors and degradation products

Norepinephrine promotes the growth of Salmonella enterica in vitro in iron-restricted conditions imposed by the iron-binding proteins serum transferrin and egg-white ovotransferrin by facilitating the release of bound iron and subsequent uptake by the bacteria. Moreover, significantly increased colonisation and systemic spread were observed in mouse and chicken models of S. enterica infection following pre-treatment of animals with norepinephrine. Both ent and tonB mutants showed no growth promotion by norepinephrine either in liquid medium containing serum or on plates containing hens’ egg-white, indicating that the process is dependent both on the ability to synthesise enterobactin and on TonB-dependent uptake of iron. An entS mutant (formerly designated ybdA) and an iroB mutant behaved as wild type in both assays, showing that neither secretion of enterobactin nor conversion of enterobactin to salmochelin S4 is necessary for the effect. On the other hand, the presence of mutations in fes or iroD resulted in loss of growth promotion by norepinephrine in both assays. Since the fes and iroD genes encode enzymes that hydrolyse enterobactin and salmochelin S4 respectively to monomers, these data suggest that excretion of monomeric forms of these siderophores may be important for the uptake of iron released by norepinephrine from transferrin or ovotransferrin. A similar pattern of behaviour was observed with S. enterica serovar Typhimurium in a mouse model of infection; treatment of animals with norepinephrine before intragastric challenge resulted in increased intestinal colonisation and systemic spread of both wild-type and entS mutant strains, while the fes mutant was significantly attenuated in vivo. This suggests that excretion of 2,3-dihydroxybenzoylserine may be essential for norepinephrine-dependent growth promotion in the iron-restricted environment of the infected host. Unlike the situation in vitro, however, tonB mutants of S. enterica serovars Typhimurium and Enteritidis behaved the same as wild type in mouse and chick infection models, respectively, suggesting that norepinephrine-dependent growth stimulation may also occur by TonB-independent uptake of the enterobactin precursor 2,3-dihydroxybenzoic acid.     

Lung lesions in SCID-bo and SCID-bg mice after intratracheal inoculation with wild-type or leucotoxin-deficient mutant strains of Mannheimia haemolytica serotype 1

The purpose of this study was to investigate SCID-bg mice engrafted with bovine haematolymphoid tissues (SCID-bo) as a model for studying bovine Mannheimia haemolytica serotype 1- induced pneumonia, in which leucotoxin (LKT) plays a major role. In experiment A, SCID-bo and SCID-bg mice were inoculated intratracheally with either (1) phosphate-buffered saline (PBS), (2) M. haemolytica wild-type strain 89010807N ("LKT(+)WT"), (3) a M. haemolytica leucotoxin-deficient mutant of strain 89010807N ("LKT(-)mutant"), or (4) the M. haemolytica wild-type Oklahoma strain. Mice were killed for examination at intervals between 20 and 44h after inoculation. Lung lesions consisted of thickened alveolar septa and neutrophil and macrophage infiltrates in the bronchioles and alveoli. Lung lesion scores in the SCID-bo mice inoculated with LKT(+)WT or LKT(-) mutant were significantly (P<0.05) greater than those of the PBS control group, but the two bacterial strains produced results that did not differ significantly. M. haemolytica was isolated from lung, liver and spleen after inoculation but less frequently as time progressed. In experiment B, SCID-bg mice were inoculated intratracheally with live LKT(+)WT or formalin-killed LKT(+)WT and killed 24, 48 or 96 h later. Lung lesions were histologically similar to those observed in experiment A; however, there were no significant differences in the lung lesion scores between groups. It was concluded that the lesions seen in this study were probably not due to LKT, and that the SCID-bo mouse does not provide a good rodent model for bovine pneumonia.     

Characterizing lipopolysaccharide and core lipid A mutant O1 and O139 Vibrio cholerae strains for adherence properties on mucus-producing cell line HT29-Rev MTX and virulence in mice

Components of lipopolysaccharide (LPS), i.e. capsule, O antigen, core oligosaccharide, as well as the toxin-coregulated pili are among the factors which significantly contribute to intestinal colonization by Vibrio cholerae O1 and O139. To further address the contribution of LPS to V. cholerae virulence, we performed in vivo colonization experiments and mucus layer attachment studies with defined LPS and capsule mutants of O1 and O139. We investigated the interaction of V. cholerae strains with the differentiated human intestinal cell line HT29-Rev MTX, and found 3-5-fold reduced efficiencies for attachment by defined LPS and capsule mutants of O1 and O139 in comparison with the wild-type strains. In addition, two O1/O139-specific core oligosaccharide biosynthetic gene products, WavJ and WavD, were characterized and tested for colonization. We demonstrate that single and double knockout mutants in wavJ and wavD have an effect on core oligosaccharide biosynthesis, and that these mutants show an attenuated growth in the presence of novobiocin. Curiously, in the mouse intestinal colonization model, only the O139 wavJ,D mutants demonstrated reduced colonization.     

A Yersinia pestis guaBA mutant is attenuated in virulence and provides protection against plague in a mouse model of infection

There is a need to develop effective countermeasures for Yersinia pestis, the etiologic agent of plague and a potential bioterrorism agent. Salmonella and Shigella spp. deleted in the guaBA genes involved in guanine biosynthesis have been shown to be attenuated in vivo. In this study, we sought to determine whether deletion of the guaBA operon would render Y. pestis auxotrophic for guanine and avirulent; such a strain could serve as a live attenuated plague vaccine candidate. A Y. pestis guaBA mutant was generated by specific deletion of a segment of the guaBA operon, producing a guanine auxotroph that was highly attenuated in a mouse model of Y. pestis infection. Furthermore, mice vaccinated with a single dose of 7 × 10⁴ CFU via the intravenous route were fully protected against subsequent lethal challenge with the Y. pestis parental strain. These findings identify guaBA as a target for deletion to generate a live attenuated plague vaccine.     

A macrophage migration inhibitory factor like gene from scallop Chlamys farreri: Involvement in immune response and wound healing

Macrophage migration inhibitory factor (MIF) is an evolutionarily ancient and highly conserved cytokine with multiple functions. In the present study, a MIF-like gene was cloned from Zhikong scallop Chlamys farreri (designated as CfMIF) based on expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of CfMIF was of 2296 bp, consisting of a 5′ untranslated region (UTR) of 60 bp, a 3′ UTR of 1903 bp with a poly(A) tail and an open reading frame (ORF) of 333 bp encoded 111 amino acid residues with a calculated molecular mass of 12.6 kDa and a theoretical isoelectric point of 5.63. The deduced amino acid sequence of CfMIF shared 27-50.5% similarity with those of other known MIFs. A conserved MIF domain was identified in the deduced amino acid sequence of CfMIF, and conserved proline² and lysine³³ were also found to be present in CfMIF. Phylogenetic analysis revealed that CfMIF is one of MIF members. The tissue distribution and temporal expression of CfMIF in hemocytes of scallop after lipopolysaccharide (LPS), peptidoglycan (PGN) and β-glucan stimulation were detected by real-time RT-PCR. CfMIF gene was ubiquitously expressed in six selected tissues of healthy scallops, with the higher expression levels in hepatopancreas, mantle and gill. In comparison with the control group, the expression of CfMIF mRNA in hemocytes was up-regulated significantly at 6 h, 24 h and 48 h after LPS treatment, and at all time points after PGN and glucan treatment. The cDNA fragment encoding mature peptide of CfMIF was recombined and expressed in Escherichia coli BL21 (DE3) pLysS. The recombinant protein of CfMIF (rCfMIF) promoted sheep fibroblast migration into scraped spaces in vitro. These results generated from the present study encourage us to suggest that CfMIF was a novel member of MIF family, and it was involved in immune response and wound healing by promoting fibroblast migration.     

Mercury exposure as a model for deviation of cytokine responses in experimental Lyme arthritis: HgCl2 treatment decreases T helper cell type 1-like responses and arthritis severity but delays eradication of Borrelia burgdorferi in C3H/HeN mice

Lyme borreliosis is a complex infection, where some individuals develop so-called 'chronic borreliosis'. The pathogenetic mechanisms are unknown, but the type of immune response is probably important for healing. A strong T helper cell type 1 (Th1)-like response has been suggested as crucial for eradication of Borrelia and for avoiding development of chronic disease. Many studies aimed at altering the Th1/Th2 balance in Lyme arthritis employed mice deficient in cytokine genes, but the outcome has not been clear-cut, due possibly to the high redundancy of cytokines. This study aimed at studying the importance of the Th1/Th2 balance in murine Borrelia arthritis by using the Th2-deviating effect of subtoxic doses of inorganic mercury. Ninety-eight C3H/HeN mice were divided into four groups: Borrelia-infected (Bb), Borrelia-infected exposed to HgCl(2) (BbHg), controls exposed to HgCl(2) alone and normal controls. Mice were killed on days 3, 16, 44 and 65 post-Borrelia inoculation. Arthritis severity was evaluated by histology, spirochaetal load determined by Borrelia culture, IgG2a- and IgE-levels analysed by enzyme-linked immunosorbemt assay (ELISA) and cytokine-secreting cells detected by enzyme-linked immunospot (ELISPOT). BbHg mice showed less severe histological arthritis, but delayed eradication of spirochaetes compared to Bb mice, associated with increased levels of IgE (Th2-induced) and decreased levels of IgG2a (Th1-induced), consistent with a Th2-deviation. Both the numbers of Th1 and Th2 cytokine-secreting cells were reduced in BbHg mice, possibly explained by the fact that numbers of cytokine-secreting cells do not correlate with cytokine concentration. In conclusion, this study supports the hypothesis that a Th1-like response is required for optimal eradication of Borrelia.