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A ferret model of synergism between influenza virus and Streptococcus pneumoniae
International Congress Series 1263 (2004) 486 – 490
www.ics-elsevier.com
A ferret model of synergism between influenza virus and Streptococcus pneumoniae Ville T. Peltola a, Jerold E. Rehg b, Jonathan A. McCullers a,* a
Department of Infectious Diseases, St. Jude Children’s Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105-2794, USA b Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA
Abstract. Background: Mouse-adapted laboratory strains of influenza virus have to be used in mouse models of synergism between influenza virus and Streptococcus pneumoniae. Since ferrets support human influenza viruses without adaptation, we sought to characterize a ferret model of synergism between influenza and pneumococcus. Methods: Pairs of ferrets were infected with either influenza virus A/Sydney/5/97 or PBS then challenged 5 days later with either pneumococcus strain D39 or PBS. Clinical and laboratory parameters were monitored during a 48-h follow-up period after the challenge. Results: Ferrets receiving influenza then pneumococcus exhibited more severe clinical signs of illness and larger increases in WBC and segmental cell counts than did ferrets receiving influenza virus or pneumococcus alone. Pneumococcal nasal wash titers were higher in ferrets infected with influenza virus followed by pneumococcus than in those infected with pneumococcus alone. Histopathologic abnormalities in lungs were mild, but differences between sequentially infected and singly infected ferrets were observed. Conclusion: A sequential infection with influenza virus and pneumococcus causes a synergistic increase in morbidity in ferrets. This ferret model will be useful for study of the interactions between non-adapted human influenza viruses and S. pneumoniae. D 2003 Elsevier B.V. All rights reserved. Keywords: Influenza; Pneumococcus; Viral-bacterial synergism; Ferret
1. Introduction Secondary bacterial infections often accompany influenza, and they are responsible for a large portion of mortality from influenza epidemics or pandemics [1,2]. Streptococcus pneumoniae is a major pathogen causing pneumonia and other bacterial complications of influenza. To investigate synergism between influenza and pneumococcus, we have previously developed and characterized mouse models of both sepsis [3] and secondary pneumococcal pneumonia after influenza infection [4]. In those models, mouse-adapted laboratory strains of influenza virus have to be used. Ferrets are susceptible to human influenza viruses without adaptation. They have not been used as an animal model for * Corresponding author. Tel.: +1-901-495-3486; fax: +1-901-495-3099. E-mail address: [email protected] (J.A. McCullers). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.023
V.T. Peltola et al. / International Congress Series 1263 (2004) 486–490
487
bacterial infections, and their susceptibility to pneumococcus is not known. Observations of naturally acquired secondary bacterial infections in influenza-infected neonatal ferrets suggest that viral –bacterial synergism occurs in ferrets [5]. S. pneumoniae has been detected as a cause of pneumonia in mink, which belongs to the same genus of Mustela as ferrets. Pneumococcus has also been used in a chinchilla model of otitis media [6]. Based on these background data, we anticipated that ferrets might be susceptible to sequential infection with influenza and pneumococcus. We sought to characterize a ferret model, which could be used for study of the interaction between non-adapted human influenza viruses and S. pneumoniae. 2. Materials and methods Human influenza virus A/Sydney/5/97 (H3N2) and S. pneumoniae D39, a type 2encapsulated strain, were used for infection of ferrets obtained from Triple F Farms and serologically negative for influenza. 1106 EID50 of influenza virus and 1106 cfu of pneumococcus were diluted in sterile PBS and were administered intranasally, in a volume of 1 ml (500 Al/nostril) to ferrets under anesthesia with inhaled isoflurane 3.5%. Ferrets were infected with either influenza virus or PBS then challenged 5 days later with either pneumococcus strain D39 or PBS (three groups: influenza only, pneumococcus only, and influenza then pneumococcus; two ferrets per group). Clinical signs of infection were monitored, and blood samples and nasal wash samples were collected 0, 6, 24, 36 and 48 h after the challenge. For nasal wash samples, 1 ml of PBS was injected into each nostril of ferrets sedated with 60 mg ketamine i.m., and expelled samples were collected. Nasal washes were titrated for influenza virus in MDCK cells, and for pneumococcal colony counts on tryptic soy agar plates supplemented with 3% (vol/vol) sheep erythrocytes. Ferrets were euthanized 48 h after the pneumococcal (or PBS) challenge. Lungs were removed and fixed in 10% neutral buffered formalin. After 24 h of fixation, the lungs were embedded in paraffin, sliced into 50Am sections, stained with hematoxylin – eosin, and examined microscopically for histopathologic alterations. 3. Results Ferrets infected with influenza only had typical symptoms of influenza (sneezing, decreased playfulness), whereas those infected with pneumococcus alone had no sneezing or other respiratory symptoms, and remained active and playful. Ferrets receiving influenza then pneumococcus exhibited more severe signs of illness than did singly infected ferrets, showing discoordination, refusal to play, and severe lethargy.
Table 1 Nasal wash pneumococcal titers (mean log10 cfu/ml) Hours after challenge
Pneumococcus Influenza then pneumococcus
6
24
36
48
3.4 6.3
1.8 4.0
0.5 3.9
0.5 3.0
488
V.T. Peltola et al. / International Congress Series 1263 (2004) 486–490
The mean WBC count of ferrets infected with influenza then pneumococcus increased by 82% during 36 h, from 8.2109/l to 14.9109/l, and a left shift occurred in WBC differential. No clear change from the pre-challenge baselines occurred in the WBC values in singly infected animals, but values were higher in ferrets infected with influenza (peak 13.7109/l) than in those infected with pneumococcus (peak 5.6109/l). Pneumococcal nasal wash titers were 2 to 3 logs higher at all time points studied in ferrets infected with influenza virus then pneumococcus vs. with pneumococcus alone (Table 1). Influenza viral titers were similar or slightly higher in influenza virus then pneumococcus vs. influenza alone ferrets (4.9 log10 TCID50/ml vs. 4.25 log10 TCID50/ml at 24 h). Lungs of the ferrets were taken for pathologic analysis 48 h after the challenge with pneumococcus or PBS. Pathologic alterations in lungs from all the ferrets were mild, and pneumococcal cultures were negative. However, some findings were detected. Lungs from ferrets infected with influenza and then mock challenged showed interstitial hypercellularity and hyperplasia of the alveolar pneumocytes (Fig. 1b). Lungs from ferrets challenged with pneumococcus 5 days after mock infection showed inflammatory infiltrates consisting predominately of neutrophils (Fig. 1c and d). Lungs from ferrets challenged with pneumo-
Fig. 1. Pathologic analysis of synergism. Normal ferret lung histology at 40 magnification (A). Lungs from ferrets infected with influenza and then mock challenged show alveolar epithelial hyperplasia and interstitial hypercellularity (B). Lungs from ferrets mock infected then challenged with pneumococcus show inflammatory cell infiltrates, at 10 (C) and 40 (D) magnification. Lungs from ferrets challenged with pneumococcus 5 days after influenza infection show epithelial hypertrophy, and hyperplasia, inflammatory infiltrates, and blockade of airways, at 10 (E) and 40 (F) magnification.
V.T. Peltola et al. / International Congress Series 1263 (2004) 486–490
489
coccus 5 days after influenza infection showed hypertrophy and hyperplasia of bronchiolar and alveolar epithelium, attenuation of cilia of epithelial cells, intraluminal inflitrates, and inflammatory cell infiltrates with mononuclear cells and neutrophils (Fig. 1e and f). 4. Discussion Pneumonia and influenza taken together are the sixth leading cause of death worldwide [7]. New strategies for intervention in the process leading from viral to bacterial infection are urgently needed. Animal models can be used for investigation of the mechanisms of interaction between influenza and pneumococcus. We have previously developed a mouse model, where intranasal infection with influenza virus primes mice for lethal pneumonia from intranasal challenge with pneumococcus [3]. Low doses of influenza virus and pneumococcus cause 100% mortality from pneumonia 3 to 7 days after pneumococcal challenge with little or no mortality in singly infected control mice. Synergism between influenza virus and S. pneumoniae has also been demonstrated in a chinchilla model of otitis media [8]. Most human influenza viruses do not grow well in mice without adaptation. Thus, the contribution of particular viruses to synergism cannot be studied in mice. Ferrets are susceptible to infection with non-adapted human influenza viruses, but they have not been used for studies of bacterial infections before. We report here that ferrets are susceptible to pneumococcal infection. Furthermore, sequential infection with influenza followed by pneumococcus 5 days later resulted in synergistic increase in morbidity in ferrets, manifest as more severe clinical signs, higher WBC counts, and higher bacterial titers in nasal washes. The mild pathology in lungs contrasts the moderate clinical signs, and suggests that the ferrets infected with influenza then pneumococcus may have had primarily an upper respiratory infection such as sinusitis instead of pneumonia. This was not assessed except using pneumococcal cultures in nasal washes. Nevertheless, these data suggest the feasibility of the ferret model of synergism between human influenza virus and pneumococcus. Further development of the model is currently under way. The aim is to produce in ferrets a more severe secondary pneumococcal pneumonia that would be easily detectable with either X-rays or by imaging of pneumococcus engineered to express the firefly luciferase with a CCD camera in live anesthetized animals [9]. Acknowledgements This work was supported by NIH (grants AI-49178 and AI-54802), the St. Jude Cancer Center Support (CORE) grant CA-21765, and the American Lebanese Syrian Associated Charities (ALSAC). References [1] L. Simonsen, The global impact of influenza on morbidity and mortality, Vaccine 17 (1999) S3 – S10. [2] A. Klimov, et al., Surveillance and impact of influenza in the United States, Vaccine 17 (1999) S42 – S46. [3] J.A. McCullers, R.G. Webster, A mouse model of dual infection with influenza virus and Streptococcus pneumoniae, in: A.D.M.E. Osterhaus, N.J. Cox, A.W. Hampson (Eds.), Options for the Control of Influenza IV, Elsevier, Amsterdam, 2001, pp. 601 – 607. [4] J.A. McCullers, J.E. Rehg, Lethal synergism between influenza virus and Streptococcus pneumoniae: char-
490
[5] [6] [7] [8] [9]
V.T. Peltola et al. / International Congress Series 1263 (2004) 486–490 acterization of a mouse model and the role of platelet-activating factor receptor, J. Infect. Dis. 186 (2002) 341 – 350. R.H. Husseini, et al., The role of naturally acquired bacterial infection in influenza-related death in neonatal ferrets, Br. J. Exp. Pathol. 64 (1983) 559 – 569. G.S. Giebink, Otitis media: the chinchilla model, Microb. Drug Resist. 5 (1999) 57 – 72. N.C. Office of Statistics and Programming, Programming. Deaths and death rates for the 10 leading causes of death in specified age groups, by race and sex: United States, 1997, Natl. Vital Stat. Rep. 47 (1999) 27 – 37. G.S. Giebink, et al., Experimental otitis media after nasal inoculation of Streptococcus pneumoniae and influenza A virus in chinchillas, Infect. Immun. 30 (1980) 445 – 450. J.A. McCullers, K.C. Bartmess, Role of neuraminidase in lethal synergism between influenza virus and Streptococcus pneumoniae, J. Infect. Dis. 187 (2003) 1000 – 1009.
www.ics-elsevier.com
A ferret model of synergism between influenza virus and Streptococcus pneumoniae Ville T. Peltola a, Jerold E. Rehg b, Jonathan A. McCullers a,* a
Department of Infectious Diseases, St. Jude Children’s Research Hospital, 332 N. Lauderdale St., Memphis, TN 38105-2794, USA b Department of Pathology, St. Jude Children’s Research Hospital, Memphis, TN, USA
Abstract. Background: Mouse-adapted laboratory strains of influenza virus have to be used in mouse models of synergism between influenza virus and Streptococcus pneumoniae. Since ferrets support human influenza viruses without adaptation, we sought to characterize a ferret model of synergism between influenza and pneumococcus. Methods: Pairs of ferrets were infected with either influenza virus A/Sydney/5/97 or PBS then challenged 5 days later with either pneumococcus strain D39 or PBS. Clinical and laboratory parameters were monitored during a 48-h follow-up period after the challenge. Results: Ferrets receiving influenza then pneumococcus exhibited more severe clinical signs of illness and larger increases in WBC and segmental cell counts than did ferrets receiving influenza virus or pneumococcus alone. Pneumococcal nasal wash titers were higher in ferrets infected with influenza virus followed by pneumococcus than in those infected with pneumococcus alone. Histopathologic abnormalities in lungs were mild, but differences between sequentially infected and singly infected ferrets were observed. Conclusion: A sequential infection with influenza virus and pneumococcus causes a synergistic increase in morbidity in ferrets. This ferret model will be useful for study of the interactions between non-adapted human influenza viruses and S. pneumoniae. D 2003 Elsevier B.V. All rights reserved. Keywords: Influenza; Pneumococcus; Viral-bacterial synergism; Ferret
1. Introduction Secondary bacterial infections often accompany influenza, and they are responsible for a large portion of mortality from influenza epidemics or pandemics [1,2]. Streptococcus pneumoniae is a major pathogen causing pneumonia and other bacterial complications of influenza. To investigate synergism between influenza and pneumococcus, we have previously developed and characterized mouse models of both sepsis [3] and secondary pneumococcal pneumonia after influenza infection [4]. In those models, mouse-adapted laboratory strains of influenza virus have to be used. Ferrets are susceptible to human influenza viruses without adaptation. They have not been used as an animal model for * Corresponding author. Tel.: +1-901-495-3486; fax: +1-901-495-3099. E-mail address: [email protected] (J.A. McCullers). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.023
V.T. Peltola et al. / International Congress Series 1263 (2004) 486–490
487
bacterial infections, and their susceptibility to pneumococcus is not known. Observations of naturally acquired secondary bacterial infections in influenza-infected neonatal ferrets suggest that viral –bacterial synergism occurs in ferrets [5]. S. pneumoniae has been detected as a cause of pneumonia in mink, which belongs to the same genus of Mustela as ferrets. Pneumococcus has also been used in a chinchilla model of otitis media [6]. Based on these background data, we anticipated that ferrets might be susceptible to sequential infection with influenza and pneumococcus. We sought to characterize a ferret model, which could be used for study of the interaction between non-adapted human influenza viruses and S. pneumoniae. 2. Materials and methods Human influenza virus A/Sydney/5/97 (H3N2) and S. pneumoniae D39, a type 2encapsulated strain, were used for infection of ferrets obtained from Triple F Farms and serologically negative for influenza. 1106 EID50 of influenza virus and 1106 cfu of pneumococcus were diluted in sterile PBS and were administered intranasally, in a volume of 1 ml (500 Al/nostril) to ferrets under anesthesia with inhaled isoflurane 3.5%. Ferrets were infected with either influenza virus or PBS then challenged 5 days later with either pneumococcus strain D39 or PBS (three groups: influenza only, pneumococcus only, and influenza then pneumococcus; two ferrets per group). Clinical signs of infection were monitored, and blood samples and nasal wash samples were collected 0, 6, 24, 36 and 48 h after the challenge. For nasal wash samples, 1 ml of PBS was injected into each nostril of ferrets sedated with 60 mg ketamine i.m., and expelled samples were collected. Nasal washes were titrated for influenza virus in MDCK cells, and for pneumococcal colony counts on tryptic soy agar plates supplemented with 3% (vol/vol) sheep erythrocytes. Ferrets were euthanized 48 h after the pneumococcal (or PBS) challenge. Lungs were removed and fixed in 10% neutral buffered formalin. After 24 h of fixation, the lungs were embedded in paraffin, sliced into 50Am sections, stained with hematoxylin – eosin, and examined microscopically for histopathologic alterations. 3. Results Ferrets infected with influenza only had typical symptoms of influenza (sneezing, decreased playfulness), whereas those infected with pneumococcus alone had no sneezing or other respiratory symptoms, and remained active and playful. Ferrets receiving influenza then pneumococcus exhibited more severe signs of illness than did singly infected ferrets, showing discoordination, refusal to play, and severe lethargy.
Table 1 Nasal wash pneumococcal titers (mean log10 cfu/ml) Hours after challenge
Pneumococcus Influenza then pneumococcus
6
24
36
48
3.4 6.3
1.8 4.0
0.5 3.9
0.5 3.0
488
V.T. Peltola et al. / International Congress Series 1263 (2004) 486–490
The mean WBC count of ferrets infected with influenza then pneumococcus increased by 82% during 36 h, from 8.2109/l to 14.9109/l, and a left shift occurred in WBC differential. No clear change from the pre-challenge baselines occurred in the WBC values in singly infected animals, but values were higher in ferrets infected with influenza (peak 13.7109/l) than in those infected with pneumococcus (peak 5.6109/l). Pneumococcal nasal wash titers were 2 to 3 logs higher at all time points studied in ferrets infected with influenza virus then pneumococcus vs. with pneumococcus alone (Table 1). Influenza viral titers were similar or slightly higher in influenza virus then pneumococcus vs. influenza alone ferrets (4.9 log10 TCID50/ml vs. 4.25 log10 TCID50/ml at 24 h). Lungs of the ferrets were taken for pathologic analysis 48 h after the challenge with pneumococcus or PBS. Pathologic alterations in lungs from all the ferrets were mild, and pneumococcal cultures were negative. However, some findings were detected. Lungs from ferrets infected with influenza and then mock challenged showed interstitial hypercellularity and hyperplasia of the alveolar pneumocytes (Fig. 1b). Lungs from ferrets challenged with pneumococcus 5 days after mock infection showed inflammatory infiltrates consisting predominately of neutrophils (Fig. 1c and d). Lungs from ferrets challenged with pneumo-
Fig. 1. Pathologic analysis of synergism. Normal ferret lung histology at 40 magnification (A). Lungs from ferrets infected with influenza and then mock challenged show alveolar epithelial hyperplasia and interstitial hypercellularity (B). Lungs from ferrets mock infected then challenged with pneumococcus show inflammatory cell infiltrates, at 10 (C) and 40 (D) magnification. Lungs from ferrets challenged with pneumococcus 5 days after influenza infection show epithelial hypertrophy, and hyperplasia, inflammatory infiltrates, and blockade of airways, at 10 (E) and 40 (F) magnification.
V.T. Peltola et al. / International Congress Series 1263 (2004) 486–490
489
coccus 5 days after influenza infection showed hypertrophy and hyperplasia of bronchiolar and alveolar epithelium, attenuation of cilia of epithelial cells, intraluminal inflitrates, and inflammatory cell infiltrates with mononuclear cells and neutrophils (Fig. 1e and f). 4. Discussion Pneumonia and influenza taken together are the sixth leading cause of death worldwide [7]. New strategies for intervention in the process leading from viral to bacterial infection are urgently needed. Animal models can be used for investigation of the mechanisms of interaction between influenza and pneumococcus. We have previously developed a mouse model, where intranasal infection with influenza virus primes mice for lethal pneumonia from intranasal challenge with pneumococcus [3]. Low doses of influenza virus and pneumococcus cause 100% mortality from pneumonia 3 to 7 days after pneumococcal challenge with little or no mortality in singly infected control mice. Synergism between influenza virus and S. pneumoniae has also been demonstrated in a chinchilla model of otitis media [8]. Most human influenza viruses do not grow well in mice without adaptation. Thus, the contribution of particular viruses to synergism cannot be studied in mice. Ferrets are susceptible to infection with non-adapted human influenza viruses, but they have not been used for studies of bacterial infections before. We report here that ferrets are susceptible to pneumococcal infection. Furthermore, sequential infection with influenza followed by pneumococcus 5 days later resulted in synergistic increase in morbidity in ferrets, manifest as more severe clinical signs, higher WBC counts, and higher bacterial titers in nasal washes. The mild pathology in lungs contrasts the moderate clinical signs, and suggests that the ferrets infected with influenza then pneumococcus may have had primarily an upper respiratory infection such as sinusitis instead of pneumonia. This was not assessed except using pneumococcal cultures in nasal washes. Nevertheless, these data suggest the feasibility of the ferret model of synergism between human influenza virus and pneumococcus. Further development of the model is currently under way. The aim is to produce in ferrets a more severe secondary pneumococcal pneumonia that would be easily detectable with either X-rays or by imaging of pneumococcus engineered to express the firefly luciferase with a CCD camera in live anesthetized animals [9]. Acknowledgements This work was supported by NIH (grants AI-49178 and AI-54802), the St. Jude Cancer Center Support (CORE) grant CA-21765, and the American Lebanese Syrian Associated Charities (ALSAC). References [1] L. Simonsen, The global impact of influenza on morbidity and mortality, Vaccine 17 (1999) S3 – S10. [2] A. Klimov, et al., Surveillance and impact of influenza in the United States, Vaccine 17 (1999) S42 – S46. [3] J.A. McCullers, R.G. Webster, A mouse model of dual infection with influenza virus and Streptococcus pneumoniae, in: A.D.M.E. Osterhaus, N.J. Cox, A.W. Hampson (Eds.), Options for the Control of Influenza IV, Elsevier, Amsterdam, 2001, pp. 601 – 607. [4] J.A. McCullers, J.E. Rehg, Lethal synergism between influenza virus and Streptococcus pneumoniae: char-
490
[5] [6] [7] [8] [9]
V.T. Peltola et al. / International Congress Series 1263 (2004) 486–490 acterization of a mouse model and the role of platelet-activating factor receptor, J. Infect. Dis. 186 (2002) 341 – 350. R.H. Husseini, et al., The role of naturally acquired bacterial infection in influenza-related death in neonatal ferrets, Br. J. Exp. Pathol. 64 (1983) 559 – 569. G.S. Giebink, Otitis media: the chinchilla model, Microb. Drug Resist. 5 (1999) 57 – 72. N.C. Office of Statistics and Programming, Programming. Deaths and death rates for the 10 leading causes of death in specified age groups, by race and sex: United States, 1997, Natl. Vital Stat. Rep. 47 (1999) 27 – 37. G.S. Giebink, et al., Experimental otitis media after nasal inoculation of Streptococcus pneumoniae and influenza A virus in chinchillas, Infect. Immun. 30 (1980) 445 – 450. J.A. McCullers, K.C. Bartmess, Role of neuraminidase in lethal synergism between influenza virus and Streptococcus pneumoniae, J. Infect. Dis. 187 (2003) 1000 – 1009.