>>15053899

The Disproved Concept of the Protozoan Etiology of Smallpox*

Frank F. Katz, Ph.D.

Professor Emeritus of Biology

Seton Hall University

South Orange, New Jersey, U.S.A.

*An earlier version of this paper, "The Disproved Concept of the Protozoan Etiology of Smallpox," was read at the Annual Fall Meeting of the Medical History Society of New Jersey, Wednesday, 4 October 2017, The Nassau Club, Princeton, New Jersey.

Keywords: Inclusions, intracellular bodies, protozoa, smallpox, vaccinia, variola, virus

Abstract

In the latter part of the 19th century, European investigators found microscopic objects in cells obtained from cases of smallpox. A. Van Der Loeff reported in 1887 these things had motility; they were considered to be protozoan parasites and the cause of the disease. In 1892, Giuseppe Guarnieri gave them the scientific name, Citoryctes variolae. They, therefore, became known as Guarnieri bodies and, in time, with other undefined intracellular objects, were named inclusion or elementary bodies found in the cytoplasm or nucleus. In the early 20th century, in the United States, the well-known pathologists, William T. Councilman, Walter R. Brinckerhoff, George B. Magrath and Frank B. Mallory, and the parasitologist, Ernest E. Tyzzer, and protozoologist, Gary N. Calkins, also concluded they were protozoans. Moreover, Calkins, in 1904, developed and produced the life cycle of this "organism." The protozoan etiology of smallpox was proven to be incorrect when, in 1905, Adelchi Negri reported the agent of variola to be filterable, an historical characteristic of viruses. With the establishment of techniques for the recovery and more accurate identification of disease-causing agents, it was confirmed that smallpox is due to a virus. Cytoryctes, once thought to be a protozoan, is now considered to be a cellular inclusion and the genus is only of historical interest.

Introduction

In his extensive study of early medicine, Reinhard Hoeppli (1893-1973) noted: "One will easily understand that during the very long periods when the cause of many diseases and pathological conditions were unknown, some of the common parasites were regarded as causative agents … [For example,] sudden death without obvious causes [being ascribed] to an ascaris which had entered the heart".[1]. Associating a disease with a parasitic roundworm such as Ascaris lumbricoides is understandable in view of its large size. There was also a time when certain diseases were thought to be caused by objects that were seen under a microscope and mistakenly identified as parasites. One of those diseases is smallpox.

The story of microscopic objects or bodies suggestive of parasites in histological preparations of tissues from smallpox cases may have begun with Joseph Louis Renaut (1844-1917) in 1881. According to Giuseppe Guarnieri (1856-1918), Renaut, in studying the formation of variola (smallpox) pustulation in stained specimens, observed intracellular spherical globules that he thought might be parasites.[2, p. 404-5]

https://www.priory.com/homol/SmallpoxEtiology.htm

>>15053973

The term, "variola," was derived from the Latin, "varius", to mean "speckled" or "spotty," and was applied to smallpox in 177.[3] However, according to the Oxford English Dictionary, it may have appeared first in print in 1425.[4]

Mervyn Henry Gordon (1872-1953) attributed the first association of Guarnieri or elementary bodies to vaccinia and variola to John Buist (1846-1915) and his publication of 1887.[5, 6] While the Oxford English Dictionary for the term "vaccinia" gives its first date as 1803 [7], it is interesting to note that the terms "Vaccinæ" and "Variolae" are in the title of the classic paper published in 1798 by Edward Jenner (1749-1823) as "Variolae Vaccinae, Or Cowpox".[8] While vaccinia has been commonly called cowpox, there is today some uncertainty about this "big mystery in virology. It is not known whether vaccinia virus is the product of genetic recombination, or if it is a species derived from cowpox virus or variola virus by prolonged serial passage, or if it is the living representative of a now extinct virus".[9]

Buist referred to the cause of smallpox as "This mysterious virus". [6, p. 1] Here he used the Latin word "virus” with a meaning of that time, such as poison [10]. As to a more specific agent, Buist wrote: "In my opinion, which I am quite aware is likely to be disputed, these peculiar bodies are probably resting-stages or fructification forms of micrococci, which may be described as ascococci (bladder-cocci)".[6, p. 161-2]

With regard to intracellular "peculiar bodies" being related to diseases, Fielding Hudson Garrison (1870-1935) stated "It is a remarkable fact that many of the indeterminate diseases allocated to submicroscopic viruses are associated variously with cell inclusions".[11] Smallpox and vaccinia, which are discussed in this paper, are included in his list of 43 such animal and human diseases.[11, pp. 714-15]

From his own studies, Guarnieri named, what he thought were organisms, Citoryctes variolae ("una evidente somiglianza con il Citoryctes vaccinae,") [a clear resemblance to Citoryctes vaccinae].[2, p. 422] He pointed out that A. Van der Loeff (1842-1913) reported such cellular inclusions had movement and were reminiscent of amoeboid protozoans.[2, p. 405] Anna Foà (1876-1944), a collaborator with Giovanni Battista Grassi (1854-1925) at the University of Rome, reported his research on Citoryctes vaccinae did not support the idea that these "corpuscles," presumably referring to intracellular bodies, were alive: "Conchiude che tali corpusculi, per le loro proprieta non possono ritenersi essere vivi" [He concluded that such corpuscles, for their properties, cannot be considered alive]. [12]

The supposition that the etiology of smallpox was a protozoan was reviewed as recently as 2001 by Werner Köhler (1929-).[13] However, he did not include the significant contributions in this area by researchers in the United States. Of a number of such persons, five were pathologists at Harvard Medical School and the sixth, a protozoologist at Columbia University, had a close connection to the work of the Harvard researchers.

The Harvard Investigators

William Thomas Councilman was born on 1 January 1854 in Pikesville, Maryland and died in York Village, Maine on 16 May 1933.[14] At the age of 24, he completed his initial medical training at the University of Maryland where the two-year program "was no better nor worse than most medical schools of that period". [14] This was followed by additional training in Europe and then, in Baltimore at Johns Hopkins University, working with the pathologist, William Henry Welch. In 1892, Councilman became the Shattuck Professor of pathological anatomy at Harvard.

>>15053979

Walter Remsen Brinckerhoff was born in the village of Matteawan, now called Beacon, New York or in New York City on 4 July 1874 and died in the Jamaica Plains section of Boston on 2 March 1911.[15] He received his M.D. from Harvard Medical College in 1901 and in 1903 became an assistant in pathology to William Councilman at Harvard. In 1902, while Brinckerhoff was an intern, there was a smallpox epidemic in Boston.[15] His first-hand experiences with this disease aided Councilman and his associates in their work on it.

George Burgess Magrath was born in Jackson, Michigan on 2 October 1870 and died in Boston on 10 December 1938.[16] Following his graduation from Harvard Medical College with the M.D. degree in 1898, he served as an assistant to Councilman. He was the medical examiner of Boston from 1907 to 1935 in which position he gained fame as a scientific criminologist.

The third assistant to Councilman in this review was Frank Burr Mallory.

He was born in Cleveland, Ohio on 12 November 1862 and died on 27 September 1941.[17] Mallory, too, was a graduate of Harvard Medical School, in 1890, and became an assistant in pathology under Councilman. He undertook additional training in Europe after which he became a member of the Harvard pathology department, assumed Councilman's position when he resigned in 1908, and gained the rank of professor in 1928. Mallory was a pioneer in the development of histological techniques and became well-known for his books on the microscopic anatomy of pathological materials and the preparation of those for diagnosis and study.[18] Papers on protozoa and smallpox with Mallory as an author have not been located. However, in a footnote, he called attention to the work on that subject by his associates at Harvard:

This study of the skin in scarlet fever with the resultant discovery of the peculiar bodies in it is the outcome of the interest taken at the present time by the pathological department of the Harvard Medical School in the subject of pathogenic protozoa in consequence of the investigation of small-pox by Dr. W.T. Councilman and the men associated with him in that work.[19]

Then, regarding scarlet fever, he wrote: "These bodies can be interpreted in various ways, as artifacts, degenerations, or protozoa".[19, pp. 487-88] He argued against the first two possibilities and thought the forms were sporozoan parasites: "If they are protozoa, they may be normal or occasional inhabitants of the skin or have a causal relation to scarlet fever".[19, p. 488] Mallory proceeded to name the organism, cyclaster (sic) scarlatinalis [19, p. 488] but, because of nomenclature priority, changed the name to Cyclasterion scarlatinae.[20]

Another member of this esteemed group of researchers at Harvard was Ernest Edward Tyzzer. He was born in Wakefield, Massachusetts on 30 August 1875 and died there on 23 January 1965.[21] In his second year at the Harvard Medical School, Tyzzer met Councilman who recognized Tyzzer’s abilities in field biology, that is studying organisms in their natural environments, and parasitology and had him carry out special projects such as the possible parasitic cause of tumors in fish. As a senior, Tyzzer joined others in the department studying smallpox and worked on the histopathology of variola and vaccinia.[21, pp. 355-6]

The Harvard Group's Conclusions

In 1903, Councilman, Magrath and Brinckerhoff published preliminary results of their studies on the cellular inclusions found in the skin of smallpox cases.[22] In 1904, they described in great detail the pathology and histology of the disease in materials from 54 autopsies.[23] They started their investigations on smallpox with the belief that "The most important work following that of Guarnieri is that of Wasielewski, which is contained in two publications … He regards the inclusions as parasites and the etiological factor of the disease".[23, p. 120] However, von Wasielewski, as noted by Köhler, “proposed to be cautious in giving a judgement on the role of the parasites".[13, p. 193]

Councilman and his associates studied cytoplasmic as well as intranuclear bodies and concluded they were parts of an organism’s overall life cycle.[22, p. 374] In addition, they stated "we have seen forms of the intranuclear bodies, which seem to show that the intranuclear cycle is sexual in character".[22, p. 375] Tyzzer, however, "never found any of the intranuclear forms" in a rabbit or calf inoculated with vaccinia or variola although the cytoplasmic stages were found.[22, p. 374]

>>15053981

In their 1904 pathology studies, Councilman and his colleagues looked in skin specimens for "some bacterium which could be considered to stand in causal relation to the lesions".[23, p. 56] They reported "Cocci, usually definitely arranged in chains or in pairs, were the organisms usually found." and concluded that "a general invasion of the tissues by cocci seems to be one of the most characteristic features of the disease".[23, p. 56-7] Throughout this 1904 paper, reference is made to parasites but the word "protozoa" does not appear until the section on literature.[23, p. 116] There they noted that "The first reference to the presence of protozoa in vaccinia or variola is contained in an article of Grünhagen".[23, p. 116] They also made this important point: "There is little doubt that the discovery and the general confirmation of the protozoal origin of malaria at this time had a great influence in directing attention to the possible presence of similar bodies in other diseases".[23, p. 116] Perhaps that is an indication that the authors were hesitant about their own conclusions and why they did not say the parasite is definitely a protozoan.

In studies of variola in the skin of monkeys, Magrath and Brinckerhoff "found certain bodies which are identical in morphology and in staining reaction with the structures arranged by Calkins in the life history of Cytoryctes variola". [24, p. 178] Importantly, they were "identical in form and in staining reaction with those in the lesions of small-pox in man".[24, p. 178] However, Magrath and Brinckerhoff referred to protozoa only in their thanks to Gary Calkins "who placed freely at our disposal his knowledge of the protozoa in general and of the forms occurring in the lesions of variola in man".[24, p. 173]

Gary Nathan Calkins

Calkins was born on 18 January 1869 in Valparaiso, Indiana and raised in that state.[25, 26] At the age of 16, he entered the Massachusetts Institute of Technology and was awarded the Bachelor of Science in Biology in 1890.[25] His Ph.D. work was under the eminent cell and developmental biologist, Edmund B. Wilson, at Columbia University and he was awarded that degree in 1897.[26] Calkins remained at Columbia for his entire professional career and became a distinguished protozoologist. In fact, Columbia, in 1906, named him "Professor of Protozoology," the first person in America to have such a title.[25, p. 283] He died on 4 January 1943.[27]

In his extensive study and review of the life history of Cytoryctes variolae [28], Calkins pointed out that while the characteristics of vaccine bodies had been "contested," von Wasielewski's "painstaking experiments and logical deductions [in 1897] left no reasonable doubt as to the nature of these inclusions in vaccinated tissues".[28, p. 137]

Referring to the stages of development a sporozoan passes through, Calkins wrote: "In all probability an analogous series of processes takes place in the parasite causing vaccinia and variola".[28, p. 137] However, this could not be demonstrated through Koch’s postulates and, therefore, "the stages in the life -cycle must be worked out from morphology alone, and without the advantage of the living organism. Dependent thus upon fixed material, there are obviously many possible sources of error, and we cannot hope to fill in every gap, or to avoid errors of interpretation".[28, pp. 137-8]

Calkins, continuing with the genus, Cytoryctes, and not Guarnieri's original Citoryctes,found its life cycle more complicated than the life cycles of other sporozoans.[28, p. 140] He described in great detail the various asexual and sexual stages the parasite goes through and proposed where they might occur in the disease process. For example, with regard to a form being transported in the blood, he wrote: "The one thing certain with regard to this stage of the life history is that the parasite is able to select and to seek out, in some mysterious fashion, the specific organ or tissue which it affects, and which may be situated at a considerable distance from the original seat of infection".[28, p. 155]

>>15053986

In view of its life cycle stages that he had painstakingly studied and described, Calkins also placed this protozoan with the sporozoans but in the family Cytoryctidae, "Genus Cytoryctes, the cause of variola".[28, p. 166]

From 1901 to 1933, Calkins published five textbooks on the protozoa.[29-33] In his 1901book, smallpox, vaccinia, variola, Guarnieri and Cytoryctes are not mentioned. In his 1909 book, four years after the agent of smallpox was reported to be a virus, he stated "the Guarnieri bodies (Cytoryctes variolae) are … constant inclusions in the skin cells of man and apes infected with smallpox. So strong is the morphological evidence of the nature of these inclusions that there is no doubt whatsoever in my own mind as to their protozoan nature and to their affinities with entamoeba and other rhizopods".[30, p. 300 Still, Calkins is uncertain about the organism’s exact taxonomic position: "In the light of our present knowledge, it is much more probable that the Guarnieri bodies are rhizopods and that the complicated changes which were earlier interpreted as pansporoblast formations are phases in the development of the idiochromidia".[30, pp. 308-9] Until there is "more convincing evidence," Calkins held off calling Guarnieri bodies of vaccinia and variola "protozoan organisms of the nucleophaga type".[30, pp. 302-3]

However, Calkins continued, Without going into the controversy again as to whether or not these bodies are organisms, a matter, I may add, which is not yet settled to the satisfaction of pathologists or biologists, I will here give only an interpretation of the questionable structures on the basis of their probable relationship to neurocytes and the other parasitic rhizopods like nucleophaga, a relationship of which I am fully convinced.[30, p. 309]

In Calkins’s 1926 book, there is a section titled "Questionable Protozoa. Chlamydozoa".[32, p. 462] This taxon, Chlamydozoa, was introduced by Stanislaus Josef von Prowazek (1875-1915) in 1907 and referred "to intracellular structures found in human tissues in connection with certain diseases, and regarded by him, as well as by many others, as Protozoa".[32, p. 46]. Calkins noted that "Lipshutz (1912) established the group Strongyloplasmata for organisms" that have the same characteristics as Chlamydozoa and that "Other minute organisms which occur in the form of filterable viruses have been included in this group".[32, p. 463] For organisms "uncertain in systemic position" but which are considered to be Protozoa "largely because they cannot be placed elsewhere," Calkins included in his book the genera Bartonella (Oroya Fever), Rickettsia (trench and typhus fevers) and Dermacentroxenus (Rocky Mountain Spotted Fever).[32, p. 445] He concluded the section on questionable protozoa with "Despite a vast literature on the subject the matter is still unsettled and many regard the etiology of the diseases in question as unknown".[32, p. 463

>>15054001

Pros And Cons On Protozoa In Smallpox

In 1901, M. Funck tried to employ Koch's postulates in identifying the protozoan or bacterial nature of this parasite but was unable to do so.[13, p. 191] From his experiments he concluded: that "cysts full of spores" are the possible aetiological agent of vaccinia (and smallpox)" and "proposed to name the infectious agent of variola and vaccinia Sporidium vaccinale" in appreciation of Ludwig Pfeiffer's studies "in which he classified this interesting living creature as a protozoan".[13, p. 192] K?hler prudently noted "Although filterable agents were already known, he [Funck] never thought about the possibility that his elements (whatever they might have been) could be contaminated by another infectious agent".[13, p. 192]

Alfred Fischer (1858-1913) was an early doubter about the association of protozoa with pox diseases. In 1900, he wrote: "An amoeba-like organism of very doubtful legitimacy has been discovered associated with cow-pox (Cytoryctes variolae), but it is certainly not the cause of the disease. The great prizes for the discovery of the vaccine microbe have yet to be won".[34]

Cytoryctes (= Citoryctes) vaccinae is, of course, the scientific name for cowpox that Fischer should have used, Cytoryctes variolae being the binomial for the supposed protozoan agent of smallpox.

James Ewing (1866-1943, too, was an early sceptic about the protozoan cause of smallpox.[35] In 1904, he stated:

The existence of this [vaccine body] cycle, however, cannot be regarded as distinctly favoring the view that the vaccine body is a parasite, since the various stages, according to the writer's interpretation, present very striking indications of belonging to the cycle of a peculiar degenerative process involving the cytoplasm of epithelial cells.[35, p. 516]

Ewing didn't cite the source for the "cycle" but he included in his bibliography Calkins's 1904 paper without its title; presumably, it is the life cycle in that report [28, p. 157] that Ewing was referring to.

The well-known parasitologist, Asa Chandler (1891-1958), in Chapter X – "Other Sporozoa, and Obscure or Invisible Parasites" – of his 1918 book [36, 168], discussed smallpox and Cytoryctes variolae.[36, p. 170] He called these "problematical organisms" and stated "whether they should be considered Protozoa is open to question".[36, p. 170] Chandler repeated these words in the 1922 edition of this book.[37, p. 170]

In 1933, Calkins still referred to the agent of smallpox (and rabies) as a "so-called" chlamydozoan, but did not have a section on that group.[33, p. 197] In his textbook on protozoa published in 1908 [38], J. Jackson Clarke (1860-1940) wrote: "It is to my mind most probable that the whole vaccine-body is a parasite in the chromidial condition" [38, p. 53] and that "One feature of the virus of small-pox and vaccinia is its power of passing under suitable conditions through a Chamberland filter".[38, p. 52] That suggests, at least, that Clarke was aware that he was dealing with what became known as an infectious entity called virus.

Viral Etiology Of Smallpox

In 1892, Dmitry Ivanovsky (1864-1920), made an extract of diseased tobacco leaves and passed it through a Chamberland (or Pasteur-Chamberland) filter.[39] While he found the filtrate was infectious for other plants, it was Martinus Beijerinck (1851-1931) who, upon repeating Ivanovsky's experiments in 1898, recognized the cause for the disease in the plants was "a new pathogen" and named its kind "virus".[40] Following these reports, Adelchi Negri (1876-1912), in 1905, passed "material freshly collected from a heifer" through a Berkefeld V filter; the filtrate produced "typical Guarnieri bodies in the corneal cells of a rabbit" and material from those cells produced "characteristic vaccinia pustules on the cow’s udder".[41] Thus, Negri established that the etiologic agent for vaccinia is a virus. Then, also in 1905, as acknowledged by Frederick Proescher (1875-1967) [42], he did the same for smallpox:

Since the filterability of variola virus was demonstrated by Negri in 1905, all commonly known micro-organisms have been excluded as its etiological factor … Negri's filter experiments were confirmed by Remlinger and Nouri, Vincent, M. Nicolle, Abid Bey, Carini, and Levi Della Vida.[42, p. 78] Proescher also made the observation that "variola is a difficult virus to filter" and that "It will pass only through Berkefeld filter V".[42, p. 78] However, he noted:

>>15054004

In 1906 Casagrandi [Oddo G. V. Casagrandi (1872-1943)] claimed that the virus can not only be filtered through Berkefeld filters N and V, but also through the common Berkefeld filter and Berkefeld filter W as well as through the Silberschmidt, Chamberlamd F, and Kitasato B.[42, p. 78]

Proescher was of the opinion that "the term ultramicroscopical or invisible virus should be discarded" because he found that the "micro-organisms" of smallpox, poliomyelitis and rabies "can be made visible with certain dyes belonging to one and the same group of aniline dyes"; he believed "that filterability should not be confounded with microscopic invisibility".[42, p. 80]

It is interesting to note that the specimens Guarnieri studied were also stained – carmine, hematoxylin, safranin, Magenta red, etc.[2, p. 407] The fact that the objects seen varied in their shape and size ("Essi hanno forma varia con sporgenze irregolari arrotondate. Hanno un volume assai vario." [They have varied shape with rounded irregular protrusions. They have a very varied volume] may have helped him to believe they were protozoan organisms.[2, p. 407]

Intracellular Inclusions

The story of protozoa being the etiology of smallpox and other diseases later shown to be due to ultrascopic viruses began with finding microscopic bodies within the cytoplasm of cells and identifying them as such organisms. In fact. these objects, identified today as intracellular inclusions, inclusion bodies or elementary bodies, are circumscribed entities in the cytoplasm or nucleus of cells and may be of inorganic materials, such as lead or mercury; organic substances, such as glycogen or insoluble protein; or biological items, such as viruses (herpes or cytomegalovirus) or parasites (Plasmodium or Babesia).[43] Particular stains may be used in their identification and classification as either Type A (eosinophilic or acidophilic) or Type B (basophilic) inclusion bodies.[44]

Jessup M. Shively stated that "Inclusion discovery began in 1786 [by Otto Friedrich Müller (1730-1784)] and their characteristics as well as new discoveries continue today".[45] The discrepancy in dates of discovery and death of the discoverer is due to the publication date of the report.

The Guarnieri bodies, characteristic for smallpox, are classified as Type B inclusions.[46] Paschen bodies, named after Enrique Paschen (1860-1936), are also intracytoplssmic features of variola as well as vaccinia, these being "particles of virus observed in relatively large numbers in squamous cells of the skin".[47] Gordon called Paschen:

The great apostle … of the elementary bodies … who, by employing L?ffler's flagella stain which he improved for the purpose demonstrated them in vaccinia and variola in 1906, and from that time until his recent death, stoutly and consistently maintained that these bodies are the actual causal agent.[5, p. 69]

In time, Paschen was proven to be correct. With the continued development of techniques and instruments for the study of viruses, such as their growth on the chorioallantois of chick embryos [48] and visualizing their morphology by means of electron microscopy [49], it was further established that the etiologic agent of variola is a virus and more of its specific viral morphologic characteristics were learned.[50]

>>15054010

Conclusions

Today, there is no doubt that the etiologic agent of smallpox is a virus. Moreover, it is known that the cellular inclusions or elementary bodies are "sites of viral multiplication in a bacterium or eukaryotic cell and usually consist of viral capsid".[44]

Because of evolutionary relationships, variola, the smallpox virus, is assigned to the genus Orthopoxvirus along with a number of other viruses including those responsible for camelpox, cowpox and horsepox.[51] As a result of this genetic connection, material, believed to contain cowpox (vaccinia), was taken from cows and successfully employed since Jenner's first experiments in 1796 in vaccination for protection against the smallpox disease.[52] An interesting view for the basis of this practice and the results has been expressed by Peter Razzell:

It is possible that the virus Jenner and Woodville found on cows had originated from milkmaids … [who] had been inoculated with smallpox and it is likely that some of them had scratched the itchy pustules" before using their hands in the milking process.[53]

Another and more current view is that "vaccinia may represent a hybrid of the variola and cowpox viruses".[54]

In the history of the actual agent causing smallpox, who and of which nationality would determine the true etiology of this human disease became a concern at least to the author of an editorial that appeared in the Journal of the American Medical Association in 1904. Under the heading of "Protozoan Pathology", the author wrote:

American investigators have been fortunate in being able to enlist the services of Professor Calkins of Columbia University, who, having studied these forms of animal life previously from the standpoint of the zoologist, is able to attack the problems from a different point of view and action than the pathologist. … His conclusions are, therefore, of particular interest, and they are set forth in the "Fourth Annual Report of the Cancer Laboratory of the New York State Board of Health," with which laboratory he is officially connected as consulting biologist. … In his own words, "the question as to the specific organism of smallpox is no longer open to doubt." With this statement, however, there are many pathologists who will take issue, for however weighty the arguments of these observers may be, the story is by no means complete; and until it is, many fairly conservative men will assume the privilege of skepticism, no matter how much and how sincerely they hope that the honors of the successful presentation of the cause of smallpox may fall on an American, and on so deserving a one as Dr. Councilman.[55]

One year later, that editorialist's hopes were dashed with the Italian Negri's discovery that the agent of smallpox is a virus.[42, p. 78]

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>>15054014

References

1. Hoeppli R. Parasites and Parasitic Infections in Early Medicine and Science. Singapore:University of Malaya Press; 1959, 160.

2. Guarnieri G. Ricerche sulla patogenesi ed etiologia dell’ infezione vaccinica e- vaiolosa. Arch Sci Med,. 1892;16(22):404-5.

3. Anonymous. Variola. Online Etymology Dictionary. Available from: https://www.etymonline.com/search?q=variola. Accessed 22 March 2018.

4. Anonymous. Variola. Oxford English Dictionary. Available from: http://www.oed.com.ezproxy.shu.edu/view/Entry/221567?redirectedFrom=

variola#eid. Accessed 22 March 2018.

1. Gordon M. Virus Bodies: John Buist and the Elementary Bodies of Vaccinia. Edinb Med J. 1937; 44:n.s., 70. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5304864/pdf/edinbmedj74878-

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1. Buist JB. Vaccinia and Variola: A Study of their Life-History. London: J & A Churchill; 1887). Available from: https://archive.org/details/b20390750. Accessed 22 July 2018.

2. Anonymous. Vaccinia. Oxford English Dictionary. Available from: http://www.oed.com/ezproxy.shu.edu/view/Entry/220912?redirectedFrom=

vaccinia#eid. Accessed 22 March 2018.

1. Jenner E. An Inquiry Into the Causes and Effects of the Variolae Vaccinae, Or Cowpox, 1798. In: The Harvard Classics, 1909-14.Bartleby.com Great Books Online. Available from: http://www.bartleby.com/38/4/1.html. Accessed 22 March 2018.

2. Anonymous. Vaccinia Virus. Stanford University. Available from: http://virus.stanford.edu/pox/2000/vaccinia_virus.html. Accessed 24 March 2018.

3. Anonymous. Virus. Online Etymology Dictionary. Available from:

http://www.etymoline.com/index.php?term=virus. Accessed 20 September 2016.

1. Garrison FH. An Introduction to the History of Medicine, 4th edition. Philadelphia and London:W.B. Saunders; 1929, 714-15.

2. Foà. Sui Citoryctes vaccinae. Commun sci Monit Zool Ital. 1902; 13(1):34.

3. Köhler W. Zentralblatt f?r Bakteriologie – 100 years ago Protozoa as causative agents of smallpox, or: Cytoryctes and no end. Int J Med Microbiol. 2001; 291:191-5.

etc

etc etc

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