The present study describes, for the first time, the immunological protection from reinfection in cats that overcome CMt bacteremia under well-controlled experimental conditions. Cats that had ostensibly overcome the acute phase of CMt infection without antibiotic treatment were re-inoculated with CMt and showed protection from a second bacteremia. In contrast, the naïve control cats became PCR-positive in blood and tissue samples after CMt inoculation. The mechanisms involved in the protection of these cats have not been completely elucidated by our study, but our evidence suggests a significant role of the humoral and cellular immune responses in this protection.
We evaluated the humoral immune response to CMt to detect possible changes in antibody levels after CMt inoculation. The cats that had overcome a previous bacteremia showed intermediate to high levels of antibodies before CMt challenge. A boost in CMt-specific antibodies could have been expected due to the repeated exposure; however, the majority of cats from group A showed a significant transient decrease in antibodies upon CMt reinfection. A similar decrease in antibody was recently described in a preliminary study investigating repeated CMt exposure . In that study, the authors speculated that the decrease in serum antibodies may be related to the binding of specific free antibodies to the inoculated antigens . Remarkably, one cat in the group of CMt recovered cats was seronegative based on our definition (ELISA signal-to-noise ratio ≤ 1.5); however, this cat maintained protection against reinfection. This observation suggests that specific antibodies against Mhf DnaK are not the sole protective mechanism against reinfection in these cats. Mhf DnaK is a hemoplasma antigen that is largely used in experimental studies to detect seroconversion after hemoplasma infection [12, 13, 15, 16]. Other antigens have been recognized in blood samples from Mhf-infected cats [29, 30], but their immunological potential is unknown. The entire feline hemoplasma genome has been recently sequenced [31, 32]; thus, information regarding additional antigens may soon become available. In serological assays, the combination of different antigens should be considered for a better understanding of the kinetics of the humoral immune response during hemoplasma infection.
In this study, we demonstrated that CMt was present in all analyzed tissues of the naïve control cats upon acute infection, as high tissue loads were found, particularly in the bone marrow. In contrast, the ostensibly recovered cats tested PCR-negative in all tissues analyzed, including the bone marrow. Thus, it appears that the cats were not only protected from bacteremia but were also sufficiently protected from recurrent tissue sequestration to prevent detection based on repeated real-time PCR measurements in the analyzed tissues.
To further characterize the immune response after CMt exposure in naïve control cats and recovered cats, we quantified different cytokine expression levels. Immediately after CMt inoculation, all cats showed a significant increase in TNF-α secretion. This cytokine is the principal mediator of the acute inflammatory response . TNF-α is produced by activated mononuclear phagocytes, and the function of TNF-α is to stimulate the recruitment of neutrophils and monocytes to sites of infection . We suspect that the subcutaneous inoculation of CMt stimulated the release of TNF-α, and it was indeed detected in acutely infected as well as in re-exposed cats.
The Th1/Th2 paradigm has been previously described in cats . Thus, we were able to select cytokines specific for each type of T helper response for the cats in this study. Th1 cells mainly mediate immune responses against intracellular pathogens, whereas Th2 cells are mainly involved in host defense responses against extracellular pathogens and atopic diseases . IL-4 stimulates the expansion of B cells and Th2-associated cells, inhibiting the proliferation of Th1 cells . On the other hand, IFN-γ is secreted by Th1 cells, inducing antimicrobial activity in macrophages . The cats acutely infected with CMt (naïve control cats) showed an early transient increase in IFN-γ expression (Th1 cytokine) that peaked at day 7 after CMt inoculation. In contrast, the cats that had recovered from previous CMt bacteremia did not show an early increase in IFN-γ expression but rather showed a pronounced increase in IL-4 expression (Th2 cytokine) at day 14 pi and prior to the time point corresponding to bacteremia in acutely infected cats. Thus, in the CMt recovered cats, protection from reinfection was associated with an early and pronounced Th2 response, whereas the acutely infected cats responded to CMt challenge with an initial Th1 response and a delayed Th2 response. A switch from a Th1 to a Th2 response in acutely infected cats was observed based on the evolution of the IL-4/IFN-γ ratios. An initial decrease in the ratio of IL-4/IFN-γ was followed by an increase, which was in turn followed by bacteremia. In contrast, the CMt recovered cats did not display skewed Th1/Th2 ratios based on either the IL-4/IFN-γ ratio or the IL-4/IL-12 ratio. Only after the onset of bacteremia were IL-4 levels higher in the acutely infected cats when compared with the CMt recovered cats, and the IL-4 levels were correlated with the bacterial load. Thus, an early Th2 response prior to the onset of bacteremia appears to be beneficial to the protection from CMt reinfection, while a delayed Th2 response seemed to be generated in response to the underlying bacteremia.
Interestingly and in agreement with the trend in Th2 responses, we detected a constant eosinophilia during CMt infection, which was particularly evident in the CMt recovered cats. Eosinophilia is a hallmark of a predominance of the Th2 response [38, 39]. The recruitment of eosinophils, although usually associated with parasitic and allergic inflammation, can also be associated with certain bacterial infections . Eosinophilia has been previously reported during feline hemoplasma infections but was not considered clinically relevant [41, 42]. In addition, a previous study reported an association between eosinophilia and latent Haemobartonella canis infection . In the context of observations by other researchers, our results may suggest that CMt infection leads to an upregulation of Th2 cell populations, which in turn stimulates the recruitment of eosinophils.
Different markers were used to analyze the lymphocyte subsets by flow cytometry after CMt inoculation. The cats that had recovered from previous bacteremia showed higher CD4+ T cell levels when compared with acutely infected cats at several time points after the CMt inoculation. As mentioned earlier in this discussion, our results suggest that the Th2 differentiation of CD4+ T cells occurs during CMt infection to efficiently combat CMt. The Th2 response is characterized by B cell activation and proliferation . Thus, the immune system of CMt recovered cats appears to immediately recognize CMt antigens, and this results in rapid activation of the appropriate T helper cells. T helper cells appear to assist CMt recovered cats to fight subsequent CMt infections and prevent bacteremia by activating secondary mechanisms (e.g., antibody production). Additionally, the activation of CD4+ T cells was most likely responsible for the recruitment of eosinophils .
CD4+CD25+ T cells represent a subpopulation of CD4+ T cells commonly known as regulatory T cells . This subset of cells suppresses the proliferation and cytokine secretion of other T cell populations and suppresses the activation of self-reacting CD4+ and CD8+ T cells . Additionally, regulatory T cells may influence the functional immunity of cats during microbial infection to suppress the immune response to pathogens . For this reason, CD4+CD25+ T cells were monitored during our study to understand their role during infection. CD4+CD25+ T cell levels were higher in the CMt recovered cats when compared with the acutely infected cats following the onset of bacteremia. The downregulation of the immune response induced by regulatory T cells may have promoted the persistence of the CMt infection and may have been instrumental in the establishment of the CMt carrier state. The number of regulatory T cells decreased at peak bacteremia in the acutely infected cats, which may be related to the role of these cells in regulating the immune response. With a decreased number of regulatory T cells, the acutely infected cats showed downregulation of the suppressive function of these cells, which in turn was most likely beneficial for fighting the CMt infection.
Some T cell activation marker levels were higher in the cats that had overcome previous bacteremia than in the acutely infected cats at various time points during the observation period. This could be related to the increased lymphocyte counts detected in the CMt recovered cats, as the lymphocytosis was probably due to chronic CMt antigenic stimulation. A similar phenomenon was identified during a cross-protection study in feline hemoplasmas (unpublished observations, Baumann J, Novacco M, Hofmann-Lehmann R), whereby cats chronically infected with CMt showed significant antigenic stimulation, which led to an increase in γ-globulin (unpublished observations, Baumann J, Novacco M, Hofmann-Lehmann R).
In conclusion, our data indicate that cats that overcome CMt bacteremia possess a persistent immune response skewed towards the Th2 type, which is associated with protection from reinfection with CMt. Further studies are required to better understand the immunological mechanisms involved in this protection. Characterization of the protective immune response against hemoplasmas is an initial step toward the development of effective vaccines against these types of infections.