THE JOURNAL OF INFECTIOUS DISEASES. VOL. 135. ,,",0. 3 • MARCH 1977 © 1977 by the University of Chicago. All rights reserved.
EDIT'ORIAL Swine Influenza and Lungworms
Received for publication September 7,1976, and in revised form December 10, 1976. Please address requests for reprints to Dr. Gordon D. Wallace, Pacific Research Section, P.O. Box 1680, Honolulu, Hawaii 96806. 490
of an influenza virus from humans. He noted that experimental infection with the swine influenza virus alone produced a very mild, sometimes asymptomatic infection that he referred to as "filtrate disease" [7]. However, if the inoculum included H. suis or if H. suis was already established in the experimental animal, the disease was clinically typical and often severe. The role of the bacterium in natural disease has not been established [8]. In Shope's first H. suis immunization experiment [5], a group of "normal" swine were inoculated im every eight days with the bacterium. Heat-killed bacteria were given to four swine, and four others were given live bacteria. Two to four days after the third injection, all four swine in the group injected with the living organisms developed clinically typical influenza that was confirmed virologically and serologically (rise in titer of neutralizing antibody). The four swine in the other group exhibited illness at the same time, but these animals had a much milder disease that was similar to "filtrate disease." All eight animals developed neutralizing antibody to swine influenza virus. More than a month later, four more swine from the same source were injected with killed H. suis. This time the onset of influenza (clinically mild) was observed after the second injection of the bacterium. Influenza virus was isolated from two animals, and a rise in neutralizing antibody titer was demonstrated in the other two. Although Shope usually used swine from the Rockefeller Institute's farm, the animals used in the experiments described above had come from another farm. They had been purchased, 27 in all, from a herd in which clinical influenza had never been observed. Sera from all animals had been tested by a neutralization test in mice [9]. Three animals had low titers of antibody to swine influenza virus, thought to be "natural antibody" [5]. These three were not used in the experiment. Later, Shope repeated the experiment, first with swine from the Rockefeller Institute's farm and then with swine from the other farm, but of later farrowings. However, influenza could not be induced. No explanation for the negative findings was given.
Downloaded from http://jid.oxfordjournals.org/ at New York University on June 6, 2015
The well-known and biologically fascinating hypothesis that swine lungworms (Metastrongylus) are involved in the transmission and natural history of swine influenza virus was first proposed by the late Dr. Richard Shope of the Rockefeller Institute (New York, N~Y.) [1]. He reported that lungworms residing in their earthworm intermediate hosts can harbor a masked form of swine influenza virus which becomes infectious when the lungworm is acquired by a pig. Since the first recorded epidemic of influenza in swine in 1918 [2, 3], observers have noted the almost simultaneous appearance of the disease annually in numerous herds throughout the midwestern United States. Epidemics usually commence in late autumn with the first cold weather. This phenomenon led Shope and others to suspect that outbreaks on individual farms were autochthonous and had been triggered by stress, possibly cold [3]. Particularly intriguing to early investigators was the question of what happens to the virus between seasonal epidemics in "closed herds," into which animals are not introduced from the outside. (Evidence that swine influenza virus is transmitted at times other than the epidemic season has been obtained recently [4].) This question led Shope to the lungworm transmission hypothesis. He became interested in lungworms because some observers suspected that earthworms (the natural intermediate host of swine lungworms) were involved in the transmission of hog cholera [1, 3]. Some very interesting and unexpected developments in 1936, during a routine experiment on the immunization of swine against the bacterium Haemophilus suis, initiated Shope's extensive investigations on transmission of influenza virus by swine lungworms [5]. At that time H. suis was thought to be important in the pathogenesis of swine influenza. Shope established a viral etiology in 1930 [6, 7], three years before the first isolation
Editorial
Results of experiments with lungworms from farms where influenza appeared annually seemed to support this belief [12]. For example, when laboratory-raised swine were infected with such lungworms and subjected to the usual provocations, some developed acute influenza. Although widely accepted, the lungworm hypothesis also has been viewed with considerable skepticism [13]. Nevertheless, it seems obvious from Shope's data that influenza virus can remain latent for relatively long periods in swine, followed by active replication, shedding into the respiratory tract, and an immune response. However, the role played by lungworms in this interesting phenomenon is open to question. For example, Mensik [14] reported isolation of influenza virus from pigs for as long as 135 days after birth to sows experimentally infected during pregnancy. After being inoculated with virus intranasally during one pregnancy, a sow delivered three successive litters of infected pigs [IS]. There was no mention of lungworms in these reports. Also Blaskovic et a1. [16] presented circumstantial evidence of latency or chronic influenza virus infections in swine not infected with lungworms. Furthermore, it now appears that swine influenza virus is endemic in Hawaii in swine herds not infected with lungworms (author's unpublished observations). Shope did not present convincing evidence that the swine used in his experiments were free of virus before they were infected with lungworms. In fact, using a mouse neutralization test, he found preexisting antibody to swine influenza virus in three of 27 experimental swine. Because the antibody titers were low (I :2), Shope assumed that the antibody was nonspecific. Perhaps if a more sensitive serologic test had been available at the time, antibody might have been detected in more than three of the experimental swine in that particular group. Controls were not mentioned in most of Shope's experiments. In retrospect, it seems that inclusion of swine infected with lungworms not exposed to virus would have been wise. It is conceivable that lungworms only activated influenza in swine already latently infected with the virus. As mentioned earlier, in some experiments swine developed acute influenza after lungworm infection alone. It appears that in only one experiment (in which exposure to cold weather was used as the provoking stimulus) was a group of swine not
Downloaded from http://jid.oxfordjournals.org/ at New York University on June 6, 2015
Using lungworms reared and exposed to influenza virus under experimental conditions, Shope conducted more than 100 experiments involving over 250 swine during a period of several years. Earthworms were reared in barrels filled with soil in which lungworm ova from feces or from bronchial exudate of swine with acute influenza were mixed. Mature third-stage lungworm larvae developed in the earthworms four to six weeks later. Earthworms from a single such exposure were used as a source of "infected" lungworms for more than two years [10]. Pigs fed such earthworms were considered to. be "prepared" and were ready for "provocation." Shope tried a variety of procedures to provoke acute influenza, in addition to injections of H. suis as described above. These methods included ascaris infection, intrapleural inoculation of CaCI 2 , spraying with cold water, administration of alcohol in toxic doses, and injection of a variety of microorganisms, hormones, and chemical compounds [10]. Of special interest was the development of influenza in swine simply after the initial lungworm exposure, which occurred in 15 swine in seven experiments. In one article [10] Shope reported 98 separate experiments, involving 216 swine, conducted during the course of three years. Influenza, usually symptomatic but sometimes asymptomatic, was provoked in 40% of the swine. One or more cases were seen in about one-half of the experiments. Shope reported that im injection of live H. suis was by far the most effective provocative stimulus, although he did not mention the proportion of successful experiments with this method. Ascaris infection provoked influenza in 16% of 53 prepared swine, and later he reported some success with exposure to cold [II], but usually he was unsuccessful with other stimuli. When H. suis was used as the provoking stimulus, injections were given at eight-day intervals. Shope found that influenza followed the second or third injection, usually on the third day, but occasionally more injections were required. He hypothesized that the provoking stimulus from this method was an allergic reaction. Of considerable concern to Shope was his inability to demonstrate virus in lungworms. Numerous attempts employing the best technology of the time were singularly negative. However, he never swayed from his belief that virus was present in a "masked" form in exposed lungworms.
491
Wallace
492
GORDON W. WALLACE
Laboratory of Parasitic Diseases Pacific Researcli Section National Institute of Allergy and Infectious Diseases National Institutes of Health Honolulu, Hawaii
References
1. Shope, R. E. The swine lungworm as a reservoir and intermediate host for swine influenza virus. II. The transmission of swine influenza virus by the swine lungworm. J. Exp. Med. 74:49-68,1941. 2. Dorset, M., McBryde, C. N., Niles, W. B. Remarks on "hog flu." J. Am. Vet. Med. Assoc. 62:162-171, 1922.
3. Shope, R. E. The epidemiology of the origin and perpetuation of a new disease. Perspect. BioI. Med. 7: 263-278, 1964. 4. Nakamura, R. M., Easterday, B. C., Pawlisch, R., Walker, G. L. Swine influenza: epizootiological and serological studies. Bull. W.H.O. 47:481-487, 1972. 5. Shope, R. E. The swine lungworm as a reservoir and intermediate host for swine influenza virus. I. The presence of swine influenza virus in healthy and susceptible pigs. J. Exp. Med. 74:41-47, 1941. 6. Shope, R. E. The etiology of swine influenza. Science 73:2 14-2.I5, 1931. 7. Shope, R. E. Swine influenza. III. Filtration experiments and etiology. J. Exp. Med. 54:373-385, 1931. 8. Easterday, B. C. Swine influenza. In H. W. Dunne [ed.]. Diseases of swine. Iowa State University Press, Ames, 1970, p. 127-157. 9. Francis, T., Jr., Shope, R. E. Neutralization tests with sera of convalescent or immunized animals and the viruses of swine and human influenza. J. Exp. Med. 63:645-653, 1936. 10. Shope, R. E. The swine lungworm as a reservoir and intermediate host for swine influenza virus. III. Factors influencing transmission of the virus and the provocation of influenza. J. Exp. Med. 77:111-126, 1943. II. Shope, R. E. The swine lungworm as a reservoir and intermediate host for swine influenza virus. V. Provocation of swine influenza by exposure of prepared swine to adverse weather. J. Exp. Med. 102:567-572, 1955. 12. Shope, R. E. The swine lungworm as a reservoir and intermediate host for swine influenza virus. IV. The demonstration of masked swine influenza virus in lungworm larvae and swine under natural conditions. J. Exp. Med. 77:127-138, 1943. 13. Easterday, B. C. Animal influenza. In E. D. Kilbourne [ed.]. The influenza viruses and influenza. Academic Press, New York, 1975, p. 464-468. 14. Mensik, J. Intra-uterine infection and persistence of swine influenza virus in the organism of infected piglets and sows (in Czechoslovakian). Sb. Ces. Akad. Zemedelsk. Ved. Vet. Med. 4:797-806,1959. 15. Mensik, J. Experimental infection of pregnant sows with influenza suis virus. I. Proof of virus in placental tissue and in organs of newborn piglets(in Czechoslovakian). VCd. Prace. Ustavu. Vet. 2:31-47, 1962. 16. Blaskovil, D., Jamrichova, 0., Rathova, V., Kol:iskova, D., Kaplan, M. M. Experimental infection of weanling pigs with A/swine influenza virus. 2. The shedding of virus by infected animals. Bull. W.H.O. 42: 767-770, 1970. 17. Sen, H. G., Kelley, G. W., Underdahl, N. R., Young, G. A. Transmission of swine influenza virus by lungworm migration. J. Exp. Med. 1I3:517-520, 1961. 18. Shotts, E. B., Jr., Foster, J. W., Brugh, M., Jordan, H. E., McQueen, J. L. An intestinal threadworm as a reservoir and intermediate host for swine influenza virus. A confirmation and amplification of Shope's syndrome. J. Exp. Meel. 127:359-369, 1968.
Downloaded from http://jid.oxfordjournals.org/ at New York University on June 6, 2015
infected with Iungworms included as a control [11]. None of the animals in this group developed influenza, whereas some of those exposed to lungworms did, but the number of control animals probably was too small to be significant. The only data from experiments in swine other than Shope's that appear to support the lungworm transmission hypothesis firmly were published by Sen et al. [17]. Unfortunately, many essential details were missing from this report, and it is difficult to evaluate the results. For example, the authors mentioned that swine infected with lungworms of "noninfluenza origin" remained normal. The number of such swine and how they were provoked, if at all, were not stated. Shotts et al. [18] reported nematode transmission of swine influenza virus in a model system using a rat nematode, Strongyloides ratti, and rats and mice. These workers did not present convincing data that virus was present in the progeny of the nematode, rather than simply being present in the feces of rats undergoing an acute influenza infection while shedding nematode larvae. In conclusion, latency of influenza virus in swine may be an important method by which the virus is maintained and perpetuated in this species. The role of the lungworm in this phenomenon remains to be determined but does not seem to be essential. The mechanisms influencing latency and reactivation of influenza virus in swine are virtually unknown. It is interesting to speculate on the possibility that influenza virus may also become latent in other species, including humans.
EDIT'ORIAL Swine Influenza and Lungworms
Received for publication September 7,1976, and in revised form December 10, 1976. Please address requests for reprints to Dr. Gordon D. Wallace, Pacific Research Section, P.O. Box 1680, Honolulu, Hawaii 96806. 490
of an influenza virus from humans. He noted that experimental infection with the swine influenza virus alone produced a very mild, sometimes asymptomatic infection that he referred to as "filtrate disease" [7]. However, if the inoculum included H. suis or if H. suis was already established in the experimental animal, the disease was clinically typical and often severe. The role of the bacterium in natural disease has not been established [8]. In Shope's first H. suis immunization experiment [5], a group of "normal" swine were inoculated im every eight days with the bacterium. Heat-killed bacteria were given to four swine, and four others were given live bacteria. Two to four days after the third injection, all four swine in the group injected with the living organisms developed clinically typical influenza that was confirmed virologically and serologically (rise in titer of neutralizing antibody). The four swine in the other group exhibited illness at the same time, but these animals had a much milder disease that was similar to "filtrate disease." All eight animals developed neutralizing antibody to swine influenza virus. More than a month later, four more swine from the same source were injected with killed H. suis. This time the onset of influenza (clinically mild) was observed after the second injection of the bacterium. Influenza virus was isolated from two animals, and a rise in neutralizing antibody titer was demonstrated in the other two. Although Shope usually used swine from the Rockefeller Institute's farm, the animals used in the experiments described above had come from another farm. They had been purchased, 27 in all, from a herd in which clinical influenza had never been observed. Sera from all animals had been tested by a neutralization test in mice [9]. Three animals had low titers of antibody to swine influenza virus, thought to be "natural antibody" [5]. These three were not used in the experiment. Later, Shope repeated the experiment, first with swine from the Rockefeller Institute's farm and then with swine from the other farm, but of later farrowings. However, influenza could not be induced. No explanation for the negative findings was given.
Downloaded from http://jid.oxfordjournals.org/ at New York University on June 6, 2015
The well-known and biologically fascinating hypothesis that swine lungworms (Metastrongylus) are involved in the transmission and natural history of swine influenza virus was first proposed by the late Dr. Richard Shope of the Rockefeller Institute (New York, N~Y.) [1]. He reported that lungworms residing in their earthworm intermediate hosts can harbor a masked form of swine influenza virus which becomes infectious when the lungworm is acquired by a pig. Since the first recorded epidemic of influenza in swine in 1918 [2, 3], observers have noted the almost simultaneous appearance of the disease annually in numerous herds throughout the midwestern United States. Epidemics usually commence in late autumn with the first cold weather. This phenomenon led Shope and others to suspect that outbreaks on individual farms were autochthonous and had been triggered by stress, possibly cold [3]. Particularly intriguing to early investigators was the question of what happens to the virus between seasonal epidemics in "closed herds," into which animals are not introduced from the outside. (Evidence that swine influenza virus is transmitted at times other than the epidemic season has been obtained recently [4].) This question led Shope to the lungworm transmission hypothesis. He became interested in lungworms because some observers suspected that earthworms (the natural intermediate host of swine lungworms) were involved in the transmission of hog cholera [1, 3]. Some very interesting and unexpected developments in 1936, during a routine experiment on the immunization of swine against the bacterium Haemophilus suis, initiated Shope's extensive investigations on transmission of influenza virus by swine lungworms [5]. At that time H. suis was thought to be important in the pathogenesis of swine influenza. Shope established a viral etiology in 1930 [6, 7], three years before the first isolation
Editorial
Results of experiments with lungworms from farms where influenza appeared annually seemed to support this belief [12]. For example, when laboratory-raised swine were infected with such lungworms and subjected to the usual provocations, some developed acute influenza. Although widely accepted, the lungworm hypothesis also has been viewed with considerable skepticism [13]. Nevertheless, it seems obvious from Shope's data that influenza virus can remain latent for relatively long periods in swine, followed by active replication, shedding into the respiratory tract, and an immune response. However, the role played by lungworms in this interesting phenomenon is open to question. For example, Mensik [14] reported isolation of influenza virus from pigs for as long as 135 days after birth to sows experimentally infected during pregnancy. After being inoculated with virus intranasally during one pregnancy, a sow delivered three successive litters of infected pigs [IS]. There was no mention of lungworms in these reports. Also Blaskovic et a1. [16] presented circumstantial evidence of latency or chronic influenza virus infections in swine not infected with lungworms. Furthermore, it now appears that swine influenza virus is endemic in Hawaii in swine herds not infected with lungworms (author's unpublished observations). Shope did not present convincing evidence that the swine used in his experiments were free of virus before they were infected with lungworms. In fact, using a mouse neutralization test, he found preexisting antibody to swine influenza virus in three of 27 experimental swine. Because the antibody titers were low (I :2), Shope assumed that the antibody was nonspecific. Perhaps if a more sensitive serologic test had been available at the time, antibody might have been detected in more than three of the experimental swine in that particular group. Controls were not mentioned in most of Shope's experiments. In retrospect, it seems that inclusion of swine infected with lungworms not exposed to virus would have been wise. It is conceivable that lungworms only activated influenza in swine already latently infected with the virus. As mentioned earlier, in some experiments swine developed acute influenza after lungworm infection alone. It appears that in only one experiment (in which exposure to cold weather was used as the provoking stimulus) was a group of swine not
Downloaded from http://jid.oxfordjournals.org/ at New York University on June 6, 2015
Using lungworms reared and exposed to influenza virus under experimental conditions, Shope conducted more than 100 experiments involving over 250 swine during a period of several years. Earthworms were reared in barrels filled with soil in which lungworm ova from feces or from bronchial exudate of swine with acute influenza were mixed. Mature third-stage lungworm larvae developed in the earthworms four to six weeks later. Earthworms from a single such exposure were used as a source of "infected" lungworms for more than two years [10]. Pigs fed such earthworms were considered to. be "prepared" and were ready for "provocation." Shope tried a variety of procedures to provoke acute influenza, in addition to injections of H. suis as described above. These methods included ascaris infection, intrapleural inoculation of CaCI 2 , spraying with cold water, administration of alcohol in toxic doses, and injection of a variety of microorganisms, hormones, and chemical compounds [10]. Of special interest was the development of influenza in swine simply after the initial lungworm exposure, which occurred in 15 swine in seven experiments. In one article [10] Shope reported 98 separate experiments, involving 216 swine, conducted during the course of three years. Influenza, usually symptomatic but sometimes asymptomatic, was provoked in 40% of the swine. One or more cases were seen in about one-half of the experiments. Shope reported that im injection of live H. suis was by far the most effective provocative stimulus, although he did not mention the proportion of successful experiments with this method. Ascaris infection provoked influenza in 16% of 53 prepared swine, and later he reported some success with exposure to cold [II], but usually he was unsuccessful with other stimuli. When H. suis was used as the provoking stimulus, injections were given at eight-day intervals. Shope found that influenza followed the second or third injection, usually on the third day, but occasionally more injections were required. He hypothesized that the provoking stimulus from this method was an allergic reaction. Of considerable concern to Shope was his inability to demonstrate virus in lungworms. Numerous attempts employing the best technology of the time were singularly negative. However, he never swayed from his belief that virus was present in a "masked" form in exposed lungworms.
491
Wallace
492
GORDON W. WALLACE
Laboratory of Parasitic Diseases Pacific Researcli Section National Institute of Allergy and Infectious Diseases National Institutes of Health Honolulu, Hawaii
References
1. Shope, R. E. The swine lungworm as a reservoir and intermediate host for swine influenza virus. II. The transmission of swine influenza virus by the swine lungworm. J. Exp. Med. 74:49-68,1941. 2. Dorset, M., McBryde, C. N., Niles, W. B. Remarks on "hog flu." J. Am. Vet. Med. Assoc. 62:162-171, 1922.
3. Shope, R. E. The epidemiology of the origin and perpetuation of a new disease. Perspect. BioI. Med. 7: 263-278, 1964. 4. Nakamura, R. M., Easterday, B. C., Pawlisch, R., Walker, G. L. Swine influenza: epizootiological and serological studies. Bull. W.H.O. 47:481-487, 1972. 5. Shope, R. E. The swine lungworm as a reservoir and intermediate host for swine influenza virus. I. The presence of swine influenza virus in healthy and susceptible pigs. J. Exp. Med. 74:41-47, 1941. 6. Shope, R. E. The etiology of swine influenza. Science 73:2 14-2.I5, 1931. 7. Shope, R. E. Swine influenza. III. Filtration experiments and etiology. J. Exp. Med. 54:373-385, 1931. 8. Easterday, B. C. Swine influenza. In H. W. Dunne [ed.]. Diseases of swine. Iowa State University Press, Ames, 1970, p. 127-157. 9. Francis, T., Jr., Shope, R. E. Neutralization tests with sera of convalescent or immunized animals and the viruses of swine and human influenza. J. Exp. Med. 63:645-653, 1936. 10. Shope, R. E. The swine lungworm as a reservoir and intermediate host for swine influenza virus. III. Factors influencing transmission of the virus and the provocation of influenza. J. Exp. Med. 77:111-126, 1943. II. Shope, R. E. The swine lungworm as a reservoir and intermediate host for swine influenza virus. V. Provocation of swine influenza by exposure of prepared swine to adverse weather. J. Exp. Med. 102:567-572, 1955. 12. Shope, R. E. The swine lungworm as a reservoir and intermediate host for swine influenza virus. IV. The demonstration of masked swine influenza virus in lungworm larvae and swine under natural conditions. J. Exp. Med. 77:127-138, 1943. 13. Easterday, B. C. Animal influenza. In E. D. Kilbourne [ed.]. The influenza viruses and influenza. Academic Press, New York, 1975, p. 464-468. 14. Mensik, J. Intra-uterine infection and persistence of swine influenza virus in the organism of infected piglets and sows (in Czechoslovakian). Sb. Ces. Akad. Zemedelsk. Ved. Vet. Med. 4:797-806,1959. 15. Mensik, J. Experimental infection of pregnant sows with influenza suis virus. I. Proof of virus in placental tissue and in organs of newborn piglets(in Czechoslovakian). VCd. Prace. Ustavu. Vet. 2:31-47, 1962. 16. Blaskovil, D., Jamrichova, 0., Rathova, V., Kol:iskova, D., Kaplan, M. M. Experimental infection of weanling pigs with A/swine influenza virus. 2. The shedding of virus by infected animals. Bull. W.H.O. 42: 767-770, 1970. 17. Sen, H. G., Kelley, G. W., Underdahl, N. R., Young, G. A. Transmission of swine influenza virus by lungworm migration. J. Exp. Med. 1I3:517-520, 1961. 18. Shotts, E. B., Jr., Foster, J. W., Brugh, M., Jordan, H. E., McQueen, J. L. An intestinal threadworm as a reservoir and intermediate host for swine influenza virus. A confirmation and amplification of Shope's syndrome. J. Exp. Meel. 127:359-369, 1968.
Downloaded from http://jid.oxfordjournals.org/ at New York University on June 6, 2015
infected with Iungworms included as a control [11]. None of the animals in this group developed influenza, whereas some of those exposed to lungworms did, but the number of control animals probably was too small to be significant. The only data from experiments in swine other than Shope's that appear to support the lungworm transmission hypothesis firmly were published by Sen et al. [17]. Unfortunately, many essential details were missing from this report, and it is difficult to evaluate the results. For example, the authors mentioned that swine infected with lungworms of "noninfluenza origin" remained normal. The number of such swine and how they were provoked, if at all, were not stated. Shotts et al. [18] reported nematode transmission of swine influenza virus in a model system using a rat nematode, Strongyloides ratti, and rats and mice. These workers did not present convincing data that virus was present in the progeny of the nematode, rather than simply being present in the feces of rats undergoing an acute influenza infection while shedding nematode larvae. In conclusion, latency of influenza virus in swine may be an important method by which the virus is maintained and perpetuated in this species. The role of the lungworm in this phenomenon remains to be determined but does not seem to be essential. The mechanisms influencing latency and reactivation of influenza virus in swine are virtually unknown. It is interesting to speculate on the possibility that influenza virus may also become latent in other species, including humans.
