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Association of Streptococcus equi with equine monocytes
Veterinary Immunology and Immunopathology 143 (2011) 83–86
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
Association of Streptococcus equi with equine monocytes Catherine Mérant, Abhineet Sheoran, John F. Timoney ∗ Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, United States
a r t i c l e
i n f o
Article history: Received 4 August 2010 Received in revised form 14 December 2010 Accepted 14 June 2011 Keywords: Streptococcus equi Monocytes Phagocytosis/interiorization
a b s t r a c t Streptococcus equi (Se), the cause of equine strangles, is highly resistant to phagocytosis by neutrophils and is usually classified as an extracellular pathogen. Large numbers of the organism in tonsillar tissues during the acute phase of the disease are completely eliminated during convalescence by mechanisms not yet understood. In this study we demonstrate in an opsono-bactericidal assay and by cytometry and confocal microscopy that Se is interiorized and killed by equine blood monocytes. This finding supports the hypotheses that adaptive immune clearance is mediated by tonsillar macrophages and that macrophages monocytes could serve as a vehicle for transport from the tonsil to local lymph nodes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Strangles, a highly contagious suppurative tonsillitis and regional lymphadenitis of equids, is caused by Streptococcus equi (S. equi subsp. equi Se). Infection is characterized by massive neutrophil infiltration of tonsillar tissue and draining lymph nodes and formation of extracellular microcolonies of Se (Timoney and Kumar, 2008). Se, unlike its closely related ancestor, Streptococcus zooepidemicus (Sz), is highly resistant to phagocytosis. Evasion of phagocytosis is mainly due to the effects of a thick hyaluronic acid capsule and the fibrinogen binding SeM which blocks deposition of C3b on the bacterial surface (Boschwitz and Timoney, 1994). During the early stages of infection, Se has been associated with tonsillar epithelial cells and cells that resemble macrophages (Timoney and Kumar, 2008). Thus, although traditionally regarded as an extracellular pathogen, it appears to have the potential to become internalized in cells that include macrophages. Other precedents for this among the pyogenic streptococci include Streptococcus pyogenes in macrophages in the human tonsil, Streptococcus suis in
∗ Corresponding author. Tel.: +1 859 257 4757x8 1106; fax: +1 859 257 5169. E-mail address: [email protected] (J.F. Timoney). 0165-2427/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2011.06.027
porcine tonsillar macrophages and Streptococcus bovis in pigeon macrophages (De Herdt et al., 1995; Osterlund et al., 1997; Thulin et al., 2006; Wilson et al., 2007). However, since each of these streptococci utilize different molecular strategies to resist phagocytosis in their respective hosts, it is probable that Se, given its unusually potent antiphagocytic properties, will exhibit a distinctive interaction with cells of the equine mononuclear phagocytic system. These cells in the tonsil most likely are responsible for convalescent clearance of Se, the efficiency of which may be increased by the activating effect of IFN␥ from antigenspecific helper T cells. Given the potential importance of macrophages in acquired host resistance to Se we have investigated its interaction with equine blood monocytes in a bactericidal assay, by FACS analysis, and by confocal microscopy.
2. Materials and methods Preparation of equine PBMC. Heparinized blood (50 ml) was collected from two 2-year-old and one 15-year-old horses that had had no prior contact with Se. These donors were used throughout the study. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll-Paque (GE Healthcare), washed once at 500 × g and twice at 300 × g for 10 min in PBS and counted in a
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C. Mérant et al. / Veterinary Immunology and Immunopathology 143 (2011) 83–86
Fig. 1. Flow cytometry of CFDA-SE loaded S. equi CF32 and equine PBMC incubated for 1 hr in CRPMI. Separate aliquots of cell suspension were then treated with goat anti-mouse F(ab )2 , and Mabs specific for equine CD4, CD8 and CD172a markers on helper T lymphocytes, suppressor T lymphocytes and macrophages, respectively. Bound Mab was detected with phycoerythrin-labeled goat anti-mouse Ig. The colors identify the gates as follows: red: lymphocytes, blue: monocytes, and green: bacteria. The majority of Se (green) co-cultured with PBMCs in a–d are extracellular. (b) (upper right) shows the small percentage (5.34) of Se internalized based on numbers gated at the same flow cytometry settings as mononuclear leucocytes.
Beckman Coulter automated counter. The cells were then re-suspended at 0.4 × 106 cells/ml in cRPMI: antibiotic-free complete RPMI-1640 supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% glutamine (Gibco). Preparation of bacteria and loading with CFDA-SE. Se CF32 and Sz W60 (Sheoran et al., 1997) were grown overnight in 3 ml Todd Hewitt Broth supplemented with 0.2% yeast extract (THB). Sz was included to control for phagocytic activity because of its much lower resistance to phagocytosis than the closely related Se. Bacteria were washed twice in phosphate buffered saline (PBS) at 300 g at room temperature and diluted 10-fold in PBS. An equal volume of 10 M 5- (and 6)-carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE; Invitrogen, Carlsbad, CA) was added, and the suspension was rotated for 20 min at 37 ◦ C in the dark. The reaction was stopped with an equal volume of FBS and the cells pelleted by centrifugation, washed in PBS-10% FBS and re-suspended in cRPMI. Co-culture of PBMC and bacteria for confocal microscopy. PBMC’s from each horse and Se were cultured either alone after twofold dilution in cRPMI or co-cultured undiluted for 1 h in 24-well flat bottom culture plates at 37 ◦ C in a humidified atmosphere with 5% CO2 .
Flow cytometry. After culture, cells were washed twice in PBS and re-suspended in PBS with 1% BSA- and 0.1% NaN3 (PBA). Aliquots containing 500,000 PBMC were incubated with mAbs against equine CD172a (DH59B), CD4 (HB61A), and CD8 (HT14A) (VMRD, Pullman, Washington) for 30 min. After two 5 min washes at 300 g, the cells were incubated with anti-mouse IgG for 15 min, washed twice and fixed in 1% paraformaldehyde. The secondary Ab for flow cytometry was phycoerythrin (PE) labeled F(ab )2 fragments of goat anti-mouse IgG minimally cross-reactive with equine Ig (Jackson Immunoresearch, West Groove, PA, USA). A Cy5-conjugated donkey anti-mouse Ab (Jackson Immunoresearch) was used for confocal microscopy. All stains were performed at 4 ◦ C. Data were acquired on a FACSCalibur flow cytometer equipped with an Argon laser (BD Biosciences, San Jose, CA). Monocytes and lymphocytes were back-gated on their expression of cell markers CD172a and CD4 or CD8B respectively as recognized by Mabs DH58B, HB61A and HT14A. Bactericidal assay. 20 l of an overnight culture in THB of Se CF32 diluted 10−4 in sterile PBS was incubated at 37 ◦ C with 100 l of convalescent serum for 30 min. This serum was obtained from horse 724 5 weeks after recov-
C. Mérant et al. / Veterinary Immunology and Immunopathology 143 (2011) 83–86
85
Fig. 2. Confocal microscopy showing the intracellular location of S. equi CF32 after culture with equine PBMC. The images of 4 planes of CFDA-SE labeled bacteria (green) cultured with equine PBMC prior to staining with CD172a specific Mab show Se (green) outside and within purple colored monocytes. The secondary antibody was phycoerythrin labeled F(ab )2 fragments of goat anti-mouse IgG.
ery from clinical strangles. A second 20 l aliquot of SeCF32 was incubated with serum from horse 724 before exposure to Se. Aliquots of serum treated bacteria were then added to 1 ml of a suspension of PBMC’s (0.4 × 106 cells/ml) in RPMI1640. 500 l was then placed on ice for a T0 count of viable bacteria. The remainder was rotated for 90 min at 37 ◦ C and placed on ice. Counts of viable Se at T0 and T90 were performed for PBMCs from each horse in triplicate as described previously (Sheoran et al., 1997). Confocal microscopy. CRPMI containing CFDA-SE stained Se and PBMCs labeled with Mab specific for CD172a was placed on a slide beneath a cover slip. Cells were observed with a Leica TCS SP upright confocal microscope using Argon (488 nm) and Helium–Neon lasers (633 nm). Specimens were scanned using a 20× objective with a 50-nm step with 1024 × 1024 resolution. 3. Results and discussion Association of S. equi with equine monocytes. The presence of Se in monocytes was confirmed after 1 h co-culture by flow cytometry by detecting stained organisms in the
same gate as monocytes and by confocal microscopy. Detection of bacteria required a FSC (forward scatter) amplification setting of E01 whereas leukocytes were detected at a lower sensitivity setting (E00). The need for the former setting was more pronounced for Se than Sz (data not shown). Back-gating indicated that most S. equi were outside the lymphocyte gate. Mean % of monocytes (CD172a+ cells) with associated Sz W60 was 50.19 ± 5.34 (n = 3). Mean % of monocytes with internalized Se was 31.89 ± 5.34% (n = 3) (Fig. 1b). Few bacteria were linked to CD4+ (Fig. 1c) or CD8+ (Fig. 1d) lymphocytes. Although the majority of Se co-cultured with equine PBMC’s in the absence of specific antibody remained extracellular based on numbers detected at the same flow cytometry settings as mononuclear leucocytes, phagocytosis of these organisms by mononuclear phagocytes was clearly evident. As expected, larger numbers of the closely related Sz were associated with these cells, a result that reflects its lack of antiphagocytic factors such as SeM, Se18.9 and, in the case of SzW60, a hyaluronic acid capsule (Timoney, 2004).
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Table 1 Survival in PBMC’s of S. equi CF32 opsonized with convalescent and normal horse serum. CFU
S. equi CF32 + convalescent serum (724) S. equi CF32 + normal pre-infection serum of 724 a
T0
T90 a
1680, 1890, 1840
0, 0, 0
2240, 2100, 1900
3600, 3500, 3500
After 90 min rotation at 37 ◦ C.
confocal microscopy show that a few equine monocytes ingest Se in the absence of opsonic antibody it is likely these cells in the tonsils of newly infected horses have a role in initiating acquired protective immune responses. This is consistent with the local and systemic immunogenicity of intranasally applied avirulent Se (Timoney and Galán, 1985). Macrophages or monocytes with intracellular Se may also be a means by which the organism is carried to mandibular and retropharyngeal lymph nodes in newly infected horses. Conflict of interest None of the authors has conflicts of interests.
Intracellular location of S. equi. Confocal microscopy confirmed the intracellular location of Se in monocytes. No CD4+ or CD8+ cells with associated bacteria were seen. Images taken in 4 planes on Fig. 2 illustrates the intracellular location of S. equi (green) in monocytes (purple) in a representative preparation. S. zooepidemicus was also observed within monocytes (data not shown) from each of the 3 donor horses. Bactericidal activity. Bactericidal activity of peripheral blood monocytes in the presence of convalescent horse 724 antibody (Table 1) was surprisingly strong. Therefore, although monocytes are at least 10 times less numerous than neutrophils in horse blood, they have the potential to play a major role in clearance of S. equi. The bactericidal assay as commonly used in studies of immunity to streptococcal infection measures a combination of attachment interiorization and killing of bacteria by phagocytes. No viable bacteria were detected following interaction of Se opsonized with convalescent immune serum with PBMCs. In contrast, >3 × 103 CFU survived when normal serum was used as opsonin. Demonstration of attachment interiorization and killing of Se by mononuclear phagocytic cells has important implications for understanding the interaction of Se with tonsillar macrophages and the role of these cells in clearance of Se during convalescence. Failure of phagocytosis by neutrophils and resident macrophages during the first days of infection results in massive extracellular multiplication of Se in tonsillar follicular tissue and formation of microcolonies (Timoney and Kumar, 2008). Later, as an acquired immune response develops, these bacteria are completely cleared and tonsil tissue becomes culture negative. Se is rarely isolated from healthy equine tonsil (Kasai et al., 1944; Woolcock, 1975). Macrophages abundant in the interfollicular zones of the lingual, palatine and nasopharyngeal tonsils are the most likely effector cells in clearance (Kumar and Timoney, 2005). Also, since flow cytometry and
Acknowledgments The assistance of Lynn Ennis and Lingshuang Sun is gratefully appreciated. References Boschwitz, J.S., Timoney, J.F., 1994. Characterization of the antiphagocytic properties of fibrinogen for Streptococcus equi subsp. equi. Microb. Pathog. 17, 121–129. De Herdt, P., Haesebrouck, F., Charlier, G., Ducatelle, R., Devriese, L.A., Vandenbossche, G., 1995. Intracellular survival and multiplication of virulent and less virulent strains of Streptococcus bovis in pigeon macrophages. Vet. Microbiol. 45, 157–169. Kasai, K., Nobata, R., Rya, E., 1944. On the incidence of Streptococcus hemolyticus in the normal tonsils of horses and the typing of equine tonsillar streptococci. Jpn. J. Vet. Sci. 6, 116–123. Kumar, P., Timoney, J.F., 2005. Histology, immunohistochemistry and ultrastructure of the equine palatine tonsil. Anat. Histol. Embryol. 34, 192–198. Osterlund, A., Poopa, R., Nikkila, T., Scheynius, A., Engstrand, L., 1997. Intracellular reservoir of Streptococcus pyogenes in vivo: a possible explanation for recurrent pharyngotonsillitis. Laryngoscope 107 (5), 640–647. Sheoran, A.S., Sponseller, B.T., Holmes, M.A., Timoney, J.F., 1997. Serum and mucosal antibody isotype responses to M-like protein (SeM) of Streptococcus equi in convalescent and vaccinated horses. Vet. Immunol. Immunopathol. 59, 239–251. Thulin, P., Johansson, L., Low, D.E., Gan, B.S., Kotb, M., McGeer, A., NorrbyTeglund, A., 2006. Viable group A streptococci in macrophages during acute soft tissue infection. PLoS Med. 3 (3), e53. Timoney, J.F., 2004. The pathogenic equine streptococci. Vet. Res. 35, 1–13. Timoney, J.F., Galán, J.E., 1985. The protective responses of the horse to an avirulent strain of Streptococcus equi. Recent advances in Streptococci and Streptococcal diseases. In: Proc. of the IXth Lancefield International Symposium , September 1984. Reedbooks Ltd., Berkshire, pp. 294–295. Timoney, J.F., Kumar, P., 2008. Early pathogenesis of equine Streptococcus equi infection (strangles). Equine Vet. J. 40, 637–642. Wilson, S.M., Norton, P., Haverson, K., Leigh, J., Bailey, M., 2007. Interactions between Streptococcus suis serotype 2 and cells of the myeloid lineage in the palatine tonsil of the pig. Vet. Immunol. Immunopathol. 117, 116–123. Woolcock, J.B., 1975. Epidemiology of equine streptococci. Res. Vet. Sci. 18, 113–114.
Contents lists available at ScienceDirect
Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm
Research paper
Association of Streptococcus equi with equine monocytes Catherine Mérant, Abhineet Sheoran, John F. Timoney ∗ Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546, United States
a r t i c l e
i n f o
Article history: Received 4 August 2010 Received in revised form 14 December 2010 Accepted 14 June 2011 Keywords: Streptococcus equi Monocytes Phagocytosis/interiorization
a b s t r a c t Streptococcus equi (Se), the cause of equine strangles, is highly resistant to phagocytosis by neutrophils and is usually classified as an extracellular pathogen. Large numbers of the organism in tonsillar tissues during the acute phase of the disease are completely eliminated during convalescence by mechanisms not yet understood. In this study we demonstrate in an opsono-bactericidal assay and by cytometry and confocal microscopy that Se is interiorized and killed by equine blood monocytes. This finding supports the hypotheses that adaptive immune clearance is mediated by tonsillar macrophages and that macrophages monocytes could serve as a vehicle for transport from the tonsil to local lymph nodes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Strangles, a highly contagious suppurative tonsillitis and regional lymphadenitis of equids, is caused by Streptococcus equi (S. equi subsp. equi Se). Infection is characterized by massive neutrophil infiltration of tonsillar tissue and draining lymph nodes and formation of extracellular microcolonies of Se (Timoney and Kumar, 2008). Se, unlike its closely related ancestor, Streptococcus zooepidemicus (Sz), is highly resistant to phagocytosis. Evasion of phagocytosis is mainly due to the effects of a thick hyaluronic acid capsule and the fibrinogen binding SeM which blocks deposition of C3b on the bacterial surface (Boschwitz and Timoney, 1994). During the early stages of infection, Se has been associated with tonsillar epithelial cells and cells that resemble macrophages (Timoney and Kumar, 2008). Thus, although traditionally regarded as an extracellular pathogen, it appears to have the potential to become internalized in cells that include macrophages. Other precedents for this among the pyogenic streptococci include Streptococcus pyogenes in macrophages in the human tonsil, Streptococcus suis in
∗ Corresponding author. Tel.: +1 859 257 4757x8 1106; fax: +1 859 257 5169. E-mail address: [email protected] (J.F. Timoney). 0165-2427/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2011.06.027
porcine tonsillar macrophages and Streptococcus bovis in pigeon macrophages (De Herdt et al., 1995; Osterlund et al., 1997; Thulin et al., 2006; Wilson et al., 2007). However, since each of these streptococci utilize different molecular strategies to resist phagocytosis in their respective hosts, it is probable that Se, given its unusually potent antiphagocytic properties, will exhibit a distinctive interaction with cells of the equine mononuclear phagocytic system. These cells in the tonsil most likely are responsible for convalescent clearance of Se, the efficiency of which may be increased by the activating effect of IFN␥ from antigenspecific helper T cells. Given the potential importance of macrophages in acquired host resistance to Se we have investigated its interaction with equine blood monocytes in a bactericidal assay, by FACS analysis, and by confocal microscopy.
2. Materials and methods Preparation of equine PBMC. Heparinized blood (50 ml) was collected from two 2-year-old and one 15-year-old horses that had had no prior contact with Se. These donors were used throughout the study. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll-Paque (GE Healthcare), washed once at 500 × g and twice at 300 × g for 10 min in PBS and counted in a
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Fig. 1. Flow cytometry of CFDA-SE loaded S. equi CF32 and equine PBMC incubated for 1 hr in CRPMI. Separate aliquots of cell suspension were then treated with goat anti-mouse F(ab )2 , and Mabs specific for equine CD4, CD8 and CD172a markers on helper T lymphocytes, suppressor T lymphocytes and macrophages, respectively. Bound Mab was detected with phycoerythrin-labeled goat anti-mouse Ig. The colors identify the gates as follows: red: lymphocytes, blue: monocytes, and green: bacteria. The majority of Se (green) co-cultured with PBMCs in a–d are extracellular. (b) (upper right) shows the small percentage (5.34) of Se internalized based on numbers gated at the same flow cytometry settings as mononuclear leucocytes.
Beckman Coulter automated counter. The cells were then re-suspended at 0.4 × 106 cells/ml in cRPMI: antibiotic-free complete RPMI-1640 supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% glutamine (Gibco). Preparation of bacteria and loading with CFDA-SE. Se CF32 and Sz W60 (Sheoran et al., 1997) were grown overnight in 3 ml Todd Hewitt Broth supplemented with 0.2% yeast extract (THB). Sz was included to control for phagocytic activity because of its much lower resistance to phagocytosis than the closely related Se. Bacteria were washed twice in phosphate buffered saline (PBS) at 300 g at room temperature and diluted 10-fold in PBS. An equal volume of 10 M 5- (and 6)-carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE; Invitrogen, Carlsbad, CA) was added, and the suspension was rotated for 20 min at 37 ◦ C in the dark. The reaction was stopped with an equal volume of FBS and the cells pelleted by centrifugation, washed in PBS-10% FBS and re-suspended in cRPMI. Co-culture of PBMC and bacteria for confocal microscopy. PBMC’s from each horse and Se were cultured either alone after twofold dilution in cRPMI or co-cultured undiluted for 1 h in 24-well flat bottom culture plates at 37 ◦ C in a humidified atmosphere with 5% CO2 .
Flow cytometry. After culture, cells were washed twice in PBS and re-suspended in PBS with 1% BSA- and 0.1% NaN3 (PBA). Aliquots containing 500,000 PBMC were incubated with mAbs against equine CD172a (DH59B), CD4 (HB61A), and CD8 (HT14A) (VMRD, Pullman, Washington) for 30 min. After two 5 min washes at 300 g, the cells were incubated with anti-mouse IgG for 15 min, washed twice and fixed in 1% paraformaldehyde. The secondary Ab for flow cytometry was phycoerythrin (PE) labeled F(ab )2 fragments of goat anti-mouse IgG minimally cross-reactive with equine Ig (Jackson Immunoresearch, West Groove, PA, USA). A Cy5-conjugated donkey anti-mouse Ab (Jackson Immunoresearch) was used for confocal microscopy. All stains were performed at 4 ◦ C. Data were acquired on a FACSCalibur flow cytometer equipped with an Argon laser (BD Biosciences, San Jose, CA). Monocytes and lymphocytes were back-gated on their expression of cell markers CD172a and CD4 or CD8B respectively as recognized by Mabs DH58B, HB61A and HT14A. Bactericidal assay. 20 l of an overnight culture in THB of Se CF32 diluted 10−4 in sterile PBS was incubated at 37 ◦ C with 100 l of convalescent serum for 30 min. This serum was obtained from horse 724 5 weeks after recov-
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85
Fig. 2. Confocal microscopy showing the intracellular location of S. equi CF32 after culture with equine PBMC. The images of 4 planes of CFDA-SE labeled bacteria (green) cultured with equine PBMC prior to staining with CD172a specific Mab show Se (green) outside and within purple colored monocytes. The secondary antibody was phycoerythrin labeled F(ab )2 fragments of goat anti-mouse IgG.
ery from clinical strangles. A second 20 l aliquot of SeCF32 was incubated with serum from horse 724 before exposure to Se. Aliquots of serum treated bacteria were then added to 1 ml of a suspension of PBMC’s (0.4 × 106 cells/ml) in RPMI1640. 500 l was then placed on ice for a T0 count of viable bacteria. The remainder was rotated for 90 min at 37 ◦ C and placed on ice. Counts of viable Se at T0 and T90 were performed for PBMCs from each horse in triplicate as described previously (Sheoran et al., 1997). Confocal microscopy. CRPMI containing CFDA-SE stained Se and PBMCs labeled with Mab specific for CD172a was placed on a slide beneath a cover slip. Cells were observed with a Leica TCS SP upright confocal microscope using Argon (488 nm) and Helium–Neon lasers (633 nm). Specimens were scanned using a 20× objective with a 50-nm step with 1024 × 1024 resolution. 3. Results and discussion Association of S. equi with equine monocytes. The presence of Se in monocytes was confirmed after 1 h co-culture by flow cytometry by detecting stained organisms in the
same gate as monocytes and by confocal microscopy. Detection of bacteria required a FSC (forward scatter) amplification setting of E01 whereas leukocytes were detected at a lower sensitivity setting (E00). The need for the former setting was more pronounced for Se than Sz (data not shown). Back-gating indicated that most S. equi were outside the lymphocyte gate. Mean % of monocytes (CD172a+ cells) with associated Sz W60 was 50.19 ± 5.34 (n = 3). Mean % of monocytes with internalized Se was 31.89 ± 5.34% (n = 3) (Fig. 1b). Few bacteria were linked to CD4+ (Fig. 1c) or CD8+ (Fig. 1d) lymphocytes. Although the majority of Se co-cultured with equine PBMC’s in the absence of specific antibody remained extracellular based on numbers detected at the same flow cytometry settings as mononuclear leucocytes, phagocytosis of these organisms by mononuclear phagocytes was clearly evident. As expected, larger numbers of the closely related Sz were associated with these cells, a result that reflects its lack of antiphagocytic factors such as SeM, Se18.9 and, in the case of SzW60, a hyaluronic acid capsule (Timoney, 2004).
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C. Mérant et al. / Veterinary Immunology and Immunopathology 143 (2011) 83–86
Table 1 Survival in PBMC’s of S. equi CF32 opsonized with convalescent and normal horse serum. CFU
S. equi CF32 + convalescent serum (724) S. equi CF32 + normal pre-infection serum of 724 a
T0
T90 a
1680, 1890, 1840
0, 0, 0
2240, 2100, 1900
3600, 3500, 3500
After 90 min rotation at 37 ◦ C.
confocal microscopy show that a few equine monocytes ingest Se in the absence of opsonic antibody it is likely these cells in the tonsils of newly infected horses have a role in initiating acquired protective immune responses. This is consistent with the local and systemic immunogenicity of intranasally applied avirulent Se (Timoney and Galán, 1985). Macrophages or monocytes with intracellular Se may also be a means by which the organism is carried to mandibular and retropharyngeal lymph nodes in newly infected horses. Conflict of interest None of the authors has conflicts of interests.
Intracellular location of S. equi. Confocal microscopy confirmed the intracellular location of Se in monocytes. No CD4+ or CD8+ cells with associated bacteria were seen. Images taken in 4 planes on Fig. 2 illustrates the intracellular location of S. equi (green) in monocytes (purple) in a representative preparation. S. zooepidemicus was also observed within monocytes (data not shown) from each of the 3 donor horses. Bactericidal activity. Bactericidal activity of peripheral blood monocytes in the presence of convalescent horse 724 antibody (Table 1) was surprisingly strong. Therefore, although monocytes are at least 10 times less numerous than neutrophils in horse blood, they have the potential to play a major role in clearance of S. equi. The bactericidal assay as commonly used in studies of immunity to streptococcal infection measures a combination of attachment interiorization and killing of bacteria by phagocytes. No viable bacteria were detected following interaction of Se opsonized with convalescent immune serum with PBMCs. In contrast, >3 × 103 CFU survived when normal serum was used as opsonin. Demonstration of attachment interiorization and killing of Se by mononuclear phagocytic cells has important implications for understanding the interaction of Se with tonsillar macrophages and the role of these cells in clearance of Se during convalescence. Failure of phagocytosis by neutrophils and resident macrophages during the first days of infection results in massive extracellular multiplication of Se in tonsillar follicular tissue and formation of microcolonies (Timoney and Kumar, 2008). Later, as an acquired immune response develops, these bacteria are completely cleared and tonsil tissue becomes culture negative. Se is rarely isolated from healthy equine tonsil (Kasai et al., 1944; Woolcock, 1975). Macrophages abundant in the interfollicular zones of the lingual, palatine and nasopharyngeal tonsils are the most likely effector cells in clearance (Kumar and Timoney, 2005). Also, since flow cytometry and
Acknowledgments The assistance of Lynn Ennis and Lingshuang Sun is gratefully appreciated. References Boschwitz, J.S., Timoney, J.F., 1994. Characterization of the antiphagocytic properties of fibrinogen for Streptococcus equi subsp. equi. Microb. Pathog. 17, 121–129. De Herdt, P., Haesebrouck, F., Charlier, G., Ducatelle, R., Devriese, L.A., Vandenbossche, G., 1995. Intracellular survival and multiplication of virulent and less virulent strains of Streptococcus bovis in pigeon macrophages. Vet. Microbiol. 45, 157–169. Kasai, K., Nobata, R., Rya, E., 1944. On the incidence of Streptococcus hemolyticus in the normal tonsils of horses and the typing of equine tonsillar streptococci. Jpn. J. Vet. Sci. 6, 116–123. Kumar, P., Timoney, J.F., 2005. Histology, immunohistochemistry and ultrastructure of the equine palatine tonsil. Anat. Histol. Embryol. 34, 192–198. Osterlund, A., Poopa, R., Nikkila, T., Scheynius, A., Engstrand, L., 1997. Intracellular reservoir of Streptococcus pyogenes in vivo: a possible explanation for recurrent pharyngotonsillitis. Laryngoscope 107 (5), 640–647. Sheoran, A.S., Sponseller, B.T., Holmes, M.A., Timoney, J.F., 1997. Serum and mucosal antibody isotype responses to M-like protein (SeM) of Streptococcus equi in convalescent and vaccinated horses. Vet. Immunol. Immunopathol. 59, 239–251. Thulin, P., Johansson, L., Low, D.E., Gan, B.S., Kotb, M., McGeer, A., NorrbyTeglund, A., 2006. Viable group A streptococci in macrophages during acute soft tissue infection. PLoS Med. 3 (3), e53. Timoney, J.F., 2004. The pathogenic equine streptococci. Vet. Res. 35, 1–13. Timoney, J.F., Galán, J.E., 1985. The protective responses of the horse to an avirulent strain of Streptococcus equi. Recent advances in Streptococci and Streptococcal diseases. In: Proc. of the IXth Lancefield International Symposium , September 1984. Reedbooks Ltd., Berkshire, pp. 294–295. Timoney, J.F., Kumar, P., 2008. Early pathogenesis of equine Streptococcus equi infection (strangles). Equine Vet. J. 40, 637–642. Wilson, S.M., Norton, P., Haverson, K., Leigh, J., Bailey, M., 2007. Interactions between Streptococcus suis serotype 2 and cells of the myeloid lineage in the palatine tonsil of the pig. Vet. Immunol. Immunopathol. 117, 116–123. Woolcock, J.B., 1975. Epidemiology of equine streptococci. Res. Vet. Sci. 18, 113–114.