The Role of the Veterinary Surgeons in Developing Theoretical and Clinical Immunology Rolul Medicilor Veterinari În Dezvoltarea Imunologiei Teoretice Şi Clinice

50 Rev Rom Med Vet (2017) 27 | 1: 50-60

THE ROLE OF THE VETERINARY SURGEONS IN DEVELOPING THEORETICAL AND CLINICAL IMMUNOLOGY ROLUL MEDICILOR VETERINARI ÎN DEZVOLTAREA IMUNOLOGIEI TEORETICE ŞI CLINICE

R. IFTIMOVICI1)

For more than 150 years (1850-2010) veterinary De mai bine de 150 de ani (1850-2010), medicii surgeons made a crucial contribution to laying the veterinari au avut o contribuție esențială în punerea basis of and developing immunology. Starting with bazelor şi dezvoltării imunologiei. Pornind de la pri- the first attempts at immunizing bovine pleuropneu- mele încercări de imunizare a pleuropneumoniei bovi- monia (Louis Willems; 1852-1880), and continuing ne (Louis Willems, 1852-1880) și continuând cu apa- with the advent of the germ theory of infectious and riția teoriei germenelui bolilor infecțioase și contagi- contagious diseases of Louis Pasteur and Robert oase ale lui Louis Pasteur şi Robert Koch, medicii vete- Koch, veterinary surgeons like Henri Bouley, Henri rinari precum Henri Bouley, Henri Toussaint, Pierre- Toussaint, Pierre-Victor Galtier and William Schutz Victor Galtier și William Schutz au colaborat îndea- worked closer with doctors and biochemists in giving proape cu medici şi biochimişti pentru conturarea shape to contemporary immunology. imunologiei contemporane. The article also deals with the second generation Articolul de faţă se ocupă de asemenea, cu a doua of veterinary immunologists such as Edmond Nocard generație de imunologi veterinari, cum ar fi Edmond with his research on human and animal tuberculosis, Nocard cu cercetările sale asupra tuberculozei umane Camille Guerin who co-authored the BCG vaccine, și animale, Camille Guerin, care a fost co-autorul vac- Gaston Ramon, the inventor of anatoxin vaccination, cinului BCG, Gaston Ramon, inventatorul vaccinării Romanian Alexandru Ciucă who introduced the diag- cu anatoxină, românul Alexandru Ciucă, care a desco- nosis of aphthous fever serotypes, or his colleague perit diagnosticul serotipurilor de febră aftoasă, sau Alexandru Vechiu, the first researcher to have lapi- colegul său, Alexandru Vechiu, primul cercetător care nized classic swine fever roots. a lapinizat rădăcini de pestă porcină clasică. The tidal wave of cellular biology and molecular Valul de biologie celulară şi genetică moleculară genetics that swept medicine after 1930 brought care a cuprins medicina după 1930 a adus noi gene- forth new generations of veterinary surgeons who raţii de medici veterinari care au deschis noi teritorii opened up new territories governed by widely appli- guvernate de legi aplicabile pe scară largă. Cum ar fi cable laws. Such is chimerism, a concept introduced himerismul, un concept introdus de Ray Owen în 1945 by Ray Owen in 1945 and expanded into immunologi- și care s-a extins în conceptul de toleranţă imunologi- cal tolerance since 1960, or the immune system's dy- că din anul 1960, ori răspunsul dinamic al sistemului namic response to alien cells explained by the 1996 imunitar la celule străine, explicat de Peter Doherty - Nobel Prize winner Peter Doherty. laureatul Premiului Nobel din 1996. Keywords: theoretical and clinical immunology Cuvinte cheie: imunologie teoretică şi clinică

Today immunity is seen as a complex, humoral and mulating the immune system; cellular action of recognition and rejection of the non- c) immunological diagnosis in human and veteri- self. But until 1960 the immune system was under- nary medicine; stood to be exclusively a body response to infectious d) the emergence and development of theoretical germs. The two-millennia-long history of immunology immunology: “the reverse path” from a natural or lab could be divided into at least four stages, each mar- created phenomenon seen with naked eyes to gene- king a revolutionary breakthrough: ralized theory and, finally, to the manufacture of im- a) pure empiricism; munity stimulants and depressants (in cases of auto- b) early accounts of prevention and treatment sti- immune disorders). I. PURE EMPIRICISM 1) Academy of Medical Sciences, Bucharest, Romania e-mail: [email protected] This period, extensively commented by medical Rev Rom Med Vet (2017) 27 | 1 51

historian, can be traced back both in human and vete- scarify sheep ears and smear the incision spot with ca- rinary medicine. prine variola pus or pustular scabs. It started with Mithridates VI Eupator Dionysus, In the 18th and 19th centuries surgeons introduced the legendary King of Pontus in Asia Minor (120 BC to smallpox inoculation to the European cities mostly by 63 BC) who fought three woeful wars against the imitating old practices. However, by the turn of the 18th Romans (88-85 BC; 83-81 BC; 74-66 BC). Fearful of century academic reports could be circulated among being assassinated, the king regularly ingested small surgeons and physicians. non-lethal amounts of various poisons to make himself Lady Mary Wortley Montagu, wife to the British immune. ambassador to Turkey, was brave enough to allow her Human variola (smallpox) and caprine variola children to be “variolated”. The operation was carried (sheep and goat pox) are the best known examples of out by a highly skilled old Greek woman (Lady Mary's prophylaxis and therapy. boy was inoculated in 1721 and her daughter in 1721). As is known, variolation is the first stage, based on In a letter to a friend, in the spring of 1721, an two findings made in antiquity: enthusiastic Lady Mary described the inoculations a) human and caprine variola is a highly conta- done with fresh, powerful smallpox material. Later on, gious and malignant virus; she embarked on a campaign to popularize inoculation b) those who suffer from smallpox will never by writing memoirs and promoting it to the court of acquire it again. They make the best nurses during George I. At her insistence, six condemned felons at variola epidemic. Newgate prison were offered the alternative of han- It did not take long before doctors started to intro- ging or their freedom if inoculated, on 9 August 1721. duce small amounts of viral matter from slightly ill pa- They all preferred to be inoculated and all survived to tients to children and adults in order to prime the be released on 6 September. immune system to recognize and destroy that virus. Greek surgeons Iacob Pilarino (1659-1718) and However, it was a most difficult step that required Emanoil Timoni (1670-1718) were also making efforts great courage. To all appearances, the first attempts to popularize “inoculation” (the term used at that at inoculation were made by the ancient Chinese, the time). Timoni managed to publish a substantial article Koreans, the Japanese and the Indians. on variolation in Philosophical Transactions of the The Chinese used nasal insufflation by blowing Royal Society in London, in 1713, even before Lady powdered smallpox material, usually scabs, up the Mary could plead with Voltaire in favor of the inocula- children's nostrils, with the help of bamboo pipes. tion of children. Pilarino and Timoni spent some time in Indian children under two years of age were the service of Wallachian and Moldavian princes, with wrapped in shirts splattered with pus taken from hu- Pilarino reaching as far high as physician in Peter the man smallpox papules. Scottish children would wrap Great's retinue. their scratched arm skin with wool tissue smeared with By 1720 aristocrats in nearly all of Europe's capital pus. cities sought inoculation for their children as well as for Similar methods of inoculation were reported in themselves. At Versailles, for instance, King Louis XVI the Middle Ages (300-600 AD) in areas now encom- and Maria Antoinette led several noble families to passed by Germany, Austria, the Netherlands, Den- smallpox inoculation in 1774. The king's decision to mark and even Russia, thanks to Viking inroads. submit himself to inoculation came shortly after the The Abyssinians, Berbers and Arabs used to treat Faculty of Medicine's professors had voted 75%-25% pustular scabs with water or milk, while South-Euro- in favor of the saying “Ce qui pouvait etre utile aux pean Slavs (Bulgarians and Serbs) would mildly spank hommes ne pouvait offenser Dieu” (“Things useful their children with pus-smeared twigs. to man will not insult God”). The inoculation saved In the 13th and 14th century, the inhabitants of what Louis XVI, the “king craftsman”, from sharing the fate is now Romania would prick their arm or thigh skin of his predecessor, King Louis XV, who scoffed at the with needles dipped into pustules, wash their children procedure and died of smallpox in 1774. in the water previously used by a carrier of the infec- Finally, British country physician Edward Jenner tion, or attach to their earlobes earrings dipped in (1749-1823) managed to introduce the smallpox vac- smallpox pus. cine (from Variolae vaccinae – smallpox of the cow) at Variolation was also practiced on animals. She- a time when the virus was devastating his country. He pherds living in the Carpathians and the Balkans would made the passage from empiricism to science. 52 Rev Rom Med Vet (2017) 27 | 1

The empirical part of his work was his simple ob- geons graduating from schools in Lyon, Charrenton- servation that milkmaids were generally immune to Alfort, Leipzig, Vienna and Torino were asked to join smallpox. Jenner concluded that the pus filled blisters the process of preparing the smallpox vaccine. Each of acquired by milkmaids from cowpox (a disease similar them worked successfully, improving the method of to smallpox, but much less virulent) made them resis- cultivating the virus on calves' thoracic skin. tant to smallpox. It is true that human and veterinary vaccines came The scientific part came with his realization that to be widely used more than 80 years after Jenner's smallpox vaccination could be put to mass use. As is 1785-1800 discovery set the stage for the eradication known, Jenner fought stoically against the prejudice of smallpox. that mixing human and animal tissue would under- However, we cannot ignore Belgian Flemish doctor mine the divine order of the universe. Jenner pursued Louis Willems (1822-1907) and his attempts at immu- reproducible investigation and proved that the pro- nizing cattle against contagious pleuropneumonia. tective cowpox pus could be effectively inoculated Soon after he graduated from the Leuven Faculty from person to person, not just directly from cattle. He of Medicine (1849), Willems approached veterinary came up with the first example of cross antigenic medicine. His researches conducted into contagious immunity. Besides, he seems to have been a pioneer infectious animal diseases between 1852 and 1880 of virus lyophilization: in 1800 he sent to America re- won him the degree of doctor in veterinary medicine at ceptacles containing cotton threads moistened with Ecole Nationale Veterinaire d'Alfort. lymph and cowpox crusts. The bit of lymph seemed to His notes on cattle pleuropneumonia infection and preserve its properties after a three-week voyage, immunity led to outstanding conclusions: when rehydrated. a) he discovered that the disease could be easily Jenner might have had a few forerunners, in terms transmitted by inoculating serous fluid from the lungs of epidemiological observations. Apparently, one of or from the pleural cavity of an affected animals into them was a German vet who used the “Old Master” the healthy cattle's tip of the tail; alias to publish an article about cow diseases in the b) using his microscope, L. Willems believed he Algemeine Underhandlunden magazine, in 1769. discerned the corpuscles of pathogenic agents that The article noted that milkmaids who acquired cowpox caused pleuropneumonia; buttons on their hands would never catch smallpox. c) only a small number of cattle died after being i- However, there is a long road from simply noticing noculated; following a benign evolution of the disease, something to describing the vaccination method and cattle tended to grow resistant to pleuropneumonia efficiently fighting smallpox. either acquired naturally or inoculated (33; 34). Released in 1798, Jenner's was the first public me- While reading Willems' reports, L. Pasteur (1822- moir in the history of immunology. Though it lined up 1896) complimented him on his achievements, at a amazing cases and impeccable experiments, Jenner's time (1876-1878) when the French doctor himself was 75-page booklet failed to pass peer review for Philo- struggling to promote the germ theory of diseases: “I sophical Transactions of the Royal Society and was re- saw the very interesting corpuscles that you previous- jected (1). Nevertheless, the report found its glory over ly reported it, and have some tests done to them to the next years, being translated into the most spoken cultivate, so far without result. Reportedly are you European languages of that time. there succeeded. I look forward to your publications in No sooner had vaccinogen institutes mushroomed this regard. I thank Mr. Bouley me by you in touch, and everywhere in Europe after Jenner's own establish- by whom I very much appreciated've applied for your ment opened on 2 December 1779 with the help of wonderful discovery of preventive inoculation against renowned doctors (Thomas Keate, Robert Keate, John infectious Pleuropneumonia,” said Louis Pasteur in his Gunning, Francis Rivers, a.o.), that veterinary sur- letter to Willems. Rev Rom Med Vet (2017) 27 | 1 53

II. EARLY ACHIEVEMENTS IN IMMUNOLOGIC 1881 and ending with the apotheotic vaccination PREVENTION AND THERAPY - started with against rabies, conducted between 1885 and 1890. Pasteur's work carried out between 1882 and 1885. Obviously, Pasteur's theory did not come out of the Pasteur was doubtlessly a genius and his paper en- blue. Unfortunately, by the turn of the century all his titled “La theorie des germes et ses applications a la forerunners were put in the shadow by an exuberant medicine et a la chirurgie” (Germ Theory and Its Appli- and biased medical historiography. cations to Medicine and Surgery), presented in 1878, Doctors and veterinary surgeons had stated that changed the world of medicine at the end of the 19th “microbes cause diffusible diseases” well before 1870. century. But their experiments and arguments had failed to Natural immunity helping people fight contagious convince a medical world still indebted to Hypocrites' infectious diseases passed as a curiosity for many theory that contagious diseases, epidemics were ge- centuries. To some this kind of protection appeared to nerated by putrid odors released by decaying organic have been bestowed by heaven. matter, usually found in stale waters (the miasma It was Pasteur who established that organisms theory). (humans and animals) and microorganisms interact, Outstanding medical personalities still clang to the and particularly that immunity is acquired through a miasma theory and their seemingly redoubtable argu- certain process. ments stifled the germ theory. And yet, in 1787, before His studies carried out between 1856 and 1885 Pasteur could elaborate the theory of infectious went through several stages of understanding: microbes, a few pathology pioneers managed to pro- a) fermentations are not “chemical mutations”, mote these very ideas in their writings. Trailblazers in they are caused by the growth of specific micro- human pathology included Ignaz Philipp Semmelweis organisms (1853-1863); (1818-1865), Friedrich Jacob Henle (1809-1885), Phi- b) there is no such thing as spontaneous bioge- lip Klenke (1813-1881), Jean Villemin (1827-1892), nesis (1858-1861). Pasteur's conclusion upset French Pierre Rayer (1793-1867) and Casimir Davaine (1812- biologist Felix Pouchet's claim that organized, complex 1882). life can appear suddenly from non-living matter. Pas- Semmelweis and Henle focused exclusively on hu- teur's firm statement gave a new impetus to surgery: man - related infections. French physician Villemin in 1865 Alphonse Guerin introduced cotton-wool ban- proved (1868) tuberculosis to be an infectious di- dages to prevent wound infections, and two years later sease, transmitted by contact from humans to animals Joseph Lister pioneered antiseptic surgery; and from one animal to another. Reyer and Davaine c) silk cocoon diseases are caused my micro- demonstrated the symptoms of anthrax were accom- organisms (1865-1868); panied by the Bacillus anthracis microbe. That is how d) microbes cause many infectious diseases they opened the zoonosis chapter of comparative pa- in humans and animals. thology. The first two stages of understanding – fermenta- In the preliminary stages of immunology, seen as tion and spontaneous generation, rather pertaining to an offspring of the pathology of infectious diseases, chemistry and general biology – heralded pathology. there were quite a few veterinary surgeons who provi- No further contributions came from either doctors or ded Pasteur and Robert Koch (1843-1910) enough ar- veterinarian surgeons. But medicine and surgery in guments to dispel the miasma theory and enthrone particular benefitted from the introduction of Lister's the germ theory as the etiopathogenesis of infectious antiseptics and the protection of wounds with sterile diseases. bandages. Pasteur embraced veterinary medicine as early as The next two stages developed after 1868 - disea- 1865 while studying the nosemosis and pebrine diseases are caused by microorganisms and the acquired ses in silkworms. He took an increasing interest in immunity – were extensively developed by human and animal infections, looking for solid arguments to found veterinary medicine. Pasteur was meant to become a the germ theory. In the process, he made friends with legend. Heroes, like benefactors of mankind, were several famous veterinarians. badly needed in 1870, when Napoleon III's France First he met Henri Bouley (1814-1885) from the knelt before Bismark in humility. The press raised Alfort-based Faculty of Medicine, then Henri Toussaint hymns to Pasteur, starting with the successful vaccina- (1847-1890) from Toulouse. tion of sheep against anthrax at Pouilly le Fort in May In his early years of research, Pasteur was both 54 Rev Rom Med Vet (2017) 27 | 1

distrusted and despised by the medical elite. Could the Bacillus anthracis have set Pasteur's Renowned professors who were confident in the mind on fire? The “charbon”, as the disease was miasma theory described him as “a petty chemist known, drew his full attention. He had no idea, while unable to find his way in medicine.” writing to Bouley, that he would lose a few battles for But the “petty chemist” received the most en- precedence, or that his victory at Pouilly le Fort in June thusiastic reception from veterinary surgeons. Henri 1880 would actually belong to Toussaint. Bouley became his protector and fiery advocate. At Let there be no misunderstanding: the charbon that time Bouley chaired the French Academy of bacteria was no novelty. Reyer and Davaine identified Sciences and enjoyed huge prestige among scientists it through the microscope as early as 1857, and as well as politicians. He used to say “I support Pasteur German veterinary surgeons Andreas Brauel and Alois with enthusiasm, while our friend Nocard is a veterina- Pollender saw it, too, that same year. There was ry surgeon who supports Pasteur with reason.” nothing new either about the fantastic resistance of Pasteur himself confided in a letter to Bouley, soil spore-forming bacteria or the “cursed fields”. dated 7 September 1877, that the entanglements of Pasteur made haste to release a memoir on the veterinary medicine had amazed him, setting his mind mystery of anthrax's “champs maudits” in 1877, but on fire: Koch had already published a paper on the subject in "Je suis heureux également de trouver l'occasion 1876.The only thing left unsolved, the so-called cherry de vous montrer que je cherche à m'instruire dans la on the cake, was the vaccination against anthrax. médecine vétérinaire puisque c'est en parcourant une Toussaint focused on this subject between 1879 de vos publications que j'ai rencontré le sujet de cette and 1880, but he was competing with the more re- lettre. Si j'étais jeune, et même à mon âge si j'étais nowned and ambitious professor Pasteur. Toussaint valide, j'irais me constituer élève de l'école d'Alfort. defibrinated and filtered anthrax-contaminated blood. Les lectures des ouvrages vétérinaires me mettent la The filtering eliminated pathogenic bacilli and pre- tête en feu". (“I am equally happy to find the occasion served only aggressins. for showing you that I seek to instruct myself in veteri- Pasteur reject Toussaint's approach. Instead, he nary medicine, since it is in reading one of your publi- applied the same method which led to the preparation cations that I encountered the subject of this letter. If I of the vaccine against aviary cholera (aged or heat- were young, or even at my age if I were more fit, I treated cultures). Besides, the chemist had stuck to would make myself a student at Alfort. Reading veteri- the dogma saying that “immunity can only be obtained nary medicine works keeps my mind on fire.”) with live germs. Inoculation will fail if it uses heat- Pasteur failed to quote any specific veterinary killed microbes.” Same as in the case of chicken chole- journal issued in France at that time. It could have ra, Pasteur noticed the inhibiting effect of oxygen on been Recueil de Medicine Veterinaire d'Alfort, founded fermentation. in 1824.Bouley was its editor-in-chief. But then France However, the method did not work for anthrax. His abounded in scientific papers dealing with veterinary hunger for glory made him publish a note saying that medicine, as much as with human medicine. oxygen and aged cultures will solve the anthrax vac- So which were the reports on infectious diseases cine (C.R. a l'Aca. De Sci. 8 Aug, 1880). It was clear and immunology that aroused Pasteur's attention to that, compared with Toussaint's line of action, Pasteur the highest degree? was groping in the dark and was nearing total failure. First of all there were those related to aviary chole- Doctors Charles Edouard Chamberland and Emile ra. Henri Toussaint, a veterinarian who later became Roux, two of Pasteur's apprentices, did their best to physician, sent Pasteur a few chicken cadavers, noting serve their master and keep his fame unaltered. They that they all shared the same germ. He even stated started by spying Toussaint while he was experimen- that in a memoir published in 1880 (18). One year ting with inoculation at Alfort. Later on, both appren- before, Toussaint won the Breant Prize of the Academy tices stated that Toussaint “did not impress them”. (2) of Sciences with a memoir on “the cholera of poultry In fact Toussaint published an encouraging inocu- and its parasite” (he had clearly identified the germ). lation report involving, however, a small number of Pasteur never gave credit to Toussaint's findings and sheep and dogs, as early as 1880. While attending never spoke about the help he had received from the Toussaint's experiments at Alfort, Chamberland and vet professor. Pasteur's followers made sure that the Roux “pinched” a valuable idea: charbon vaccine can germ should be named Pasteurella. be obtained by filtrating defibrinated blood from an in- Rev Rom Med Vet (2017) 27 | 1 55

fected organism where bacilli are killed by an antisep- Medical historians have revealed yet another si- tic (phenol in Toussaint's experimental cases). tuation where veterinary surgeons were robbed of a In other words, Toussaint was the first veterinarian great achievement in the realm of immunology. The who proved that toxins from heat-killed bacteria anti-rabies vaccine turned Pasteur into the benefactor (antigens) are immunogens. of the world, again thanks to the cunning communica- Within a few weeks, Chamberland and Roux pre- tion skills of his apprentices. pared a charbon vaccine following Toussaint's method Pasteur and his disciple Roux (16, 30) started to at the Pasteur Institute in Paris. However, they used work on rabies shortly after they had read an article by potassium bichromate as antiseptic. To be sure, they Pierre Victor Galtier (1846-1908) from Ecole Nationale also applied Toussaint's method, using phenol to kill Veterinaire de Lyon, which was the first higher edu- the bacteria. cation establishment for veterinary medicine in the All this time Pasteur thought that his apprentices world, founded by Claude Bourgelat in 1762. were duly following his orders to prepare the anthrax In his article Galtier announced (13) that he had vaccine via heat attenuation and oxygen. He did not succeeded in having rabies transmitted to rabbits, no- have the slightest idea that the famous test of Pouilly thing that: le Fort between May and June 1881, which marked the a) incubation period in rabbits is shorter than that greatest success since Jenner's inoculation, was based in dogs. Rabbits acquire directly a paralytic, not aggre- on Toussaint's vaccine (Fig. 1). It was the first inocula- ssive, type of rabies, which makes them perfect for ex- tion made with a vaccine obtained from filtered mi- periences, unlike aggressive dogs. crobes, with bacilli annihilated by potassium bichro- b) rabid saliva contains a virulent element (Galtier mate, and a repeat vaccination during which germs transmitted dog saliva to rabbits, and then rabbit sa- were killed by phenol, meaning exactly what Tou- liva to other rabbits). Galtier was the first doctor to ssaint had done. It goes without saying that the two prove that the rabies virus is neurotropic, spreading apprentices kept their mouths sealed while proudly via peripheral nerves; the spinal cord is highly viru- placing the laurel crown on Pasteur's head: any other lent, unlike the blood. As a test, Galtier inoculated gesture or word would have caused a diplomatic scan- rabid saliva in a rabbit's sciatic nerve and infected it. dal. Two years later (1883), Chamberland and Roux c) Galtier made the first anti-rabies vaccination explained in a memoir that their master had the merit attempts (1879-1881) by injecting rabid drool in a of having attenuated the anthrax germ with antisep- sheep's jugular vein. In controlled conditions, he ino- tics. That was how the veterinary professor from Tou- culated rabies virus subcutaneously and intraperito- louse was completely wiped out from the scenery. Late neally, but the results were modest. (14) in 1881 he became mentally ill and died on 3 August The tests performed by Galtier were clandestinely 1890 at the age of 43. copied by Pasteur's disciples. In a memoir devoted to the history of rabies, French medical historian Jean Theodorides said that Pasteur was not fair to Galtier. In 1881 Pasteur referred to the Lyon-based doctor's works as “exceptional”, but then he began to ignore him. He even stated that Galtier “failed to inoculate the rabies virus with rabid nervous matter.” (30) Anyway, Pasteur's disciples made the first use of the anti-rabies virus on 9-year old Joseph Meister in the summer of 1885 (it has never been decided whe- ther the dog that mauled the boy really had the rabies or was just aggressive). Three months later Pasteur received public ovations as a world hero, but nobody stopped to consider Galtier and his fundamental expe- Fig. 1. A myth is created: thousands of visitors from rience, or the genial Roux who actually obtained the everywhere come to admire this mosaic with sheep fixed rabies virus and the vaccination protocol. illustrating the victory against anthrax at Pouilly-le-Fort (1881). The mosaic is laid in a sumptuous crypt built Professor Galtier was nominated for the Nobel under Pasteur Institute in Paris. There is nothing to re- Prize in 1908. He died before he could enjoy it. The No- mind anyone of Henri Toussaint, buried in oblivion. bel Committee then decided that the award should go 56 Rev Rom Med Vet (2017) 27 | 1

to Paul Enrlich and Ilia Mecinikov, two major rivals in per immune sera were tested on dogs (Victor Babeş, immunology. 1887), while anti-diphtheria sera were tried out on We have no intention to damage Pasteur's reputa- guinea pigs (Emil von Behring, 1892). tion or to suggest that the great scientist had moral Camille Guerin (1872-1961) (Fig. 3), a graduate shortcomings. We are all human, we are all selfish from Alfort, worked with Albert Calmette (1863-1933) (more or less honest). If we enlisted his achievements, on the pathogenesis and immu- starting with his first discovery – the racemic acid ex- noprophylaxis of tuberculosis. He periments -, continuing with his studies on bacteria's went down in history by 1922 impact on fermentation and its huge role in surgery, when Benjamin Weill-Halle intro- his own role in dismantling the spontaneous genera- duced the anti-tuberculosis vac- tion theory, the experiments he made on microbial cine created by Calmette and pathogenesis of infectious and parasitic diseases (no- Guerin in 1910 to a lot of new- semosis and flacherie in silkworms, chicken cholera, born babies. The Calmette-Gue- anthrax, puerperal fever, rabies, etc), the principles he Fig. 3. C. Guerin rin bacillus (BCG) is a genome of set for pasteurization, we should conclude that Pas- bovine tuberculosis bacillus which has been modified teur's ethical shortcomings can be thoroughly excused from the “wild type” by being cultivated in the biliary when compared with his gigantic work. duct (the experiments conducted mainly by Guerin In addition, Pasteur had never felt resentful to- started in 1906). Initially meant for treating bovine wards other medical specialities. When times were dif- tuberculosis, the use of BCG vaccine was extended to ficult (1870-1885) and he was frequently subject to humans and has had a crucial importance in fighting criticism and even insults, he looked up to his allies, tuberculosis present in humans. the veterinary doctors. It appears that he assembled Gaston Ramon (1886-1963) (Fig. 4), was proba- the first multi professional teams of chemists, biolo- bly the most famous immunologist of the 20th century: gists, physicians and veterinary doctors. he became both a member of the This scientific strategy continued long after his French Academy of Sciences and death, as Pasteur Institute came to be run by Emile Medicine and the general mana- Duclaux, Emile Roux, Louis Martin, Jacques Trefouel, ger of Pasteur Institute in Paris veterinary doctor Gaston Ramon, a.o. (1940). A graduate from Alfort Romanian followers of Pasteur – veterinary doc- (1906-1910), Ramon was intro- tors Paul Riegler, Alexandru Ciucă, Alexandru Pop, duced to Roux as a resourceful, Francisc Popescu - seemed to have applied this prin- hard-working young man. Roux, ciple at Cantacuzino Institute,while Constantin Surdan who chaired Pasteur Institute, did the same at the Institute of Virusology, which had Fig. 4. G. Ramon hired him at the Garches Annex of been founded by Ştefan Nicolau, yet another of Pas- the Institute which then happened to be testing the teur's disciples. anti-tetanus, anti-diphtheria and anti-gangrene sera Following is a brief enumeration of some veteri- on horses. Ramon made his first important contribu- nary doctors who, guided by Pasteur's achievements, tion in 1921 when he devised a new method of purify- made essential contributions to developing medicine, ing antitoxic sera, which helped to reduce anaphylactic immunology in particular, in the 19th and 20th century. accidents. Later that year, he found that, when in vitro, Edmond Etienne Nocard (1850-1903) (Fig. 2) the diphtheria toxin and the anti-toxin came into con- counted among the closest collaborators of Pasteur tact and caused flocculation reactions of various sizes, and Roux. His main studies dealt depending on the quantities of the two reactants. with the etiopathogenesis of bo- In 1922 Ramon developed an in vitro titration me- vine tuberculosis.Alongside Roux, thod for determining the potency of the diphtheria anti- he experimented the human anti- toxin, then of the tetanus one. What he achieved was an diphtheria serum on horses. easy and safe method of valuing the efficiency of anti- Their tests represented the toxins in both animal and human medicine (before 1922 first attempt (1894) at manufac- titration of anti-toxins was determined only in vivo). turing therapeutic human and ve- Ramon went on to devise a method to prepare terinary sera on industrial scale. anatoxins (1923) by applying small quantities of for- Before their experiments, hy- Fig. 2. E.E. Nocard mol to the diphtheria, tetanus and botulinum toxins, Rev Rom Med Vet (2017) 27 | 1 57

as well as to snake and scorpion venoms (antive- He attended several high specialization courses at noms). Ramon and his laboratory-designed methods Pasteur Institute in Paris, where he conquered the scientific world between 1925 and worked with Amede Borrel and 1926. In 1925 he proposed immune stimulation by Gaston Ramon. Afterwards, he was adjuvants and combined vaccines administered in one appointed head of the therapeutic shot (antigens). In partnership with Christian Zoeller, sera department of Cantacuzino Ramon studied the concomitant immune responses Institute in Bucharest. for at least two associated antigens. In 1929, Ciucă won internatio- As a token of appreciation for his work, Ramon nal fame for introducing the diag- became a member of reputed academies, a Honoris nosis of aphthous fever serotypes Causa graduate of universities around the world, and Fig. 6. Al. Ciucă through a complement fixation test general manager of Pasteur Institute in Paris (1940), (4). This achievement laid the groundwork for specific following Louis Martin, who in his turn had taken immunoprophylaxis in treating a wide range of disea- Roux's place in 1934. Finally, he became the director ses deriving from one root. of the International Office of Epizootics (OIE) in 1948. Robert Royston Amos Coombs (1921-2006) Immunology's hall of fame includes also Roma- (Fig. 7), is another classic immunologist who devoted nian Alexandru Vechiu (1890-1954) (Fig. 5), a pro- his work to improving the diagnosis of human diseases. fessor at the Faculty of Veterinary He graduated from the Edinburg Medicine in Bucharest. He intended University's veterinary medicine to lapinize or attenuate wild virus department. Between 1943 and roots in order to obtain mild roots 1944 he worked as immunologist with unchanged antigens for vac- at the Central Veterinary Labora- cines. The method was introduced tory of Weybridge. Between 1944 by Galtier in 1879 and applied, a- and 1947 he devised the Coombs mong others, by Roux himself for test and presented it in 1945 (5). the preparation of the fixed rabies The test discovered Rhesus anti- Fig. 5. Al. Vechiu virus. What Vechiu had in mind was Fig. 7. R. Coombs bodies in the uterus, helping pre- to create an efficient vaccine against swine fever. As vent accidents typical of the hemolytic disease in the early as 1931 he lapinized “wild” roots of swine fever. newborns. Coombs was also an outstanding allergolo- The result was an attenuated root which only had to be gist. He and Philip George Houthem drew up a compre- processed into vaccine. Eight years later, Vechiu pu- hensive treatise entitled Clinical Aspects of Immunolo- blished his experiments in the prestigious Journal of gy, issued in two editions: 1963 and 1968. Towards the the International Office of Epizootics (33). By 1946 re- end of his life he published his research on anaphylactic searchers J.A. Baker, H. Koprowsky and J. Blach took etiopathogenesis in Sudden Infant Death Syndrome (8). up Vechiu's groundbreaking work and created the vaccine for the control of classical swine fever by em- IV. ASPECTS OF CONTEMPORARY ploying lapinized viruses. Swine vaccination has been THEORETICAL IMMUNOLOGY forbidden for 35 years now, but the vaccines were cru- Humoral and cellular immunity are two antagonis- cial in staving off losses caused by swine fever. tic approaches which have been at odds since the turn of the 19th century. Cellular immunity, based on Ilya III. NEW CONCEPTS AND APPROACHES Mechnikov's discovery of phagocytosis, became the IN IMMUNOLOGICAL DIAGNOSIS banner of Pasteur Institute. Mechnikov and his follo- Hundreds, maybe thousands of veterinary doctors wers considered that phagocytes protect the body by have tried to solve thorny diagnosis-related issues ingesting (phagocytosing) harmful foreign particles, over the last century. However, we shall put the spot- bacteria, and dead or dying cells. light on two outstanding researchers that deserve to Meanwhile Babeş (1888), Behring and Kitasato be called “classics”. (1890), Tizzoni and Cattani (1891) found humoral fac- Alexandru Ciucă (1880-1972) (Fig. 6), studied tors carrying bactericidal properties.Tizzoni and Cattani with Victor Babeş and Ion Cantacuzino, only to be- called them glycoprotein molecules (immunoglobulins). come himself a professor of infectious and contagious These tracks of research triggered fiery disputes which diseases at the Faculty of Medicine in Bucharest. lasted until 1940 when fresh evidence led to the idea 58 Rev Rom Med Vet (2017) 27 | 1

that tissue and blood-derived immunocytes live in per- antigens will be perfectly tolerated if they affect an or- fect harmony between themselves as well as with ganism during embryogenesis (in some animals even immunoglobulin molecules. Moreover, by 1948 A. E. after birth). However, antigens will be annihilated if Fagraeus proved that a certain type of lymphocytes they come into contact with an organism several (plasmocytes) operate like antibody factories. weeks after birth or during its adulthood. The two re- Molecular (and genetic) biology techniques and searchers concluded that while the genetic immune electronic microscopy helped immunology to make system is still in an embryonic stage it scans antigens, huge steps ahead between 1945 and 1970 and particu- allowing them in as its own (self). larly afterwards. Immunology's horizon widened with: This is how Owen's chime- a) theoretical structures this time relying on clear, per- rism led to Medawar's immunolo- fectly reproducible tests; b) a large portfolio of discove- gical tolerance and finally solved ries capable of explaining how immune homeostasis is the great mystery of the 1890 - created, maintained or restored, but also why auto- 1900 period: why do the inner for- immune genetic diseases crop up. ces of an organism make the error WIDELY APPLICABLE THEORETICAL ACHIEVE- of attacking their own structures MENTS include the natural selection theory of Niels (Ehrlich's vague Horror autotoxi- Jerne (1955) and the clonal selection theory of Frank cus) ? Fig. 9. P.B. Medawar Macfarlane Burnet and Frank Fenner (1949-1959), Chimerism thus helped not only explain the self as well as the contributions made to these theories by and nonself concepts which, from 1955 onwards, molecular genetics experts like Joshua Lederberg would become the playground of hundreds of resear- and David W. Talmage. chers into contemporary theoretical immunology, but Immunological tolerance, another law governing also explore a continent unknown before 1950: human homeostasis as well as any transplantation of living and animal autoimmune pathology. cells, tissues or organs from one species to another The portfolio of discoveries lying at the foundation (xenograft), was discovered by American veterinary of contemporary immunology features, among others, doctor and genetics researcher Ray David Owen the discoveries of Australian veterinary surgeon and re- (1915-2014) (Fig. 8). searcher Peter Charles Doherty In 1945, R.D. Owen reported (born 1940) (Fig.10), winner of the that two twin calves fathered by Nobel Prize in 1996, jointly with different sires were immunologi- Swiss professor Rolf Zinkernagel. cally compatible: each calf had its Doherty focused on how the body's own blood cells as well as hematic immune cells protect against hu- cells deriving from its sibling. man and animal viruses. He and Immunological laws known to Zinkernagel managed to deter- genetic experts ruled that alien mine: a) how long it takes for red blood cells should have been de- Fig. 8. R.D. Owen cells to produce an immune reac- Fig. 10. P.C. Doherty voured by recipient immune forces. But that did not tion; b) which are the molecular elements (receptors) happen in the case of the two dizygotic calves. Owen placed on a cell's immunocompetent surface that inter- called this phenomenon chimerism (20). act with antigens. Owen's chimerism lies at the foundation of immu- The two researchers took up forays made into the nological tolerance, a biological reality encompassing little known territory of the immune response dynamics a broad spectrum of fields. British zoologist Rupert and its biochemical stages in the 1960s and 1970s: Everett Billingham (1901-2002) and Peter Brian a) In 1969, Miller and Good discovered the duality Medawar (1915-1987) (Fig. 9) were only two of the (T and B) of lymphocytes and the role of thymus in important researchers who studied immunological to- producing cellular immunity. lerance. In 1960 when Medawar was handed the Nobel b) In 1972, Rosemberg, Herbermann and McCoy Prize, he stressed that Owen should have stood next to discovered the NK cytotoxicity. him to receive the award, because R.D. Owen's tests c) Between 1973 and 1974, Levine and Benacerraf opened this fertile field of knowledge back in 1945. defined the concept of genetic determinism and ex- Further tests made by Billingham and Medawar on plained how the immune response is regulated at phy- consanguineous rooted mice led to the conclusion that siological and pathological levels. Rev Rom Med Vet (2017) 27 | 1 59

d) Equally important was the development of mice fying the different types of virus, J. of Hygiene, consanguinity which provided homogenous responses Cambridge (England), 28: 325-339 to different antigens. 5. Coombs R.R., Mourant A.E., Race R.R. (1945), e) In 1980, Jean Dausset received the Nobel Prize Detection of weak and “incomplete” Rh agglutinins: in Physiology or Medicine in 1980 along with Benace- a new test, Lancet 246 (6358): 15-6. doi: 10.1016/ rraf and George Davis Snell for their discovery of the S0140-6736(45)90806-3 major histocompatibility complex. Dausset went fur- 6. Coombs R.R. (1968), Immunopathology, Br Med J. ther to identify chromosome 6 genes which order the 1(5592):597-602 synthesis of HLA antigens. 7. Coombs R.R. (1998), Historical note: past, present f) In 1987, Japanese scientist Susumu Tonegawa and future of the antiglobulin test, Vox Sang 74 (2): received the Nobel Prize for Physiology or Medicine, for 67-73.doi:10.1159/000030908. PMID9501403 having discovered the genetic mechanism that re- 8. Coombs R.R., Parish W.E., Walls A.F. (2000), arranges itself to form millions of antibodies. Sudden Infant Death Syndrome: Could a healthy Doherty and Zinkernagel described how the T lym- infant succumb to inhalation-anaphylaxis during phocytes recognize their antigenic targets and destroy sleep leading to cot death? Cambridge Publications them. They found that cytotoxic killer T lymphocytes Ltd. ISBN 0-9540081-0-3 (NK) recognize not only a viral antigen hiding in an in- 9. Decourt Ph. (1989), Les vérités indésirables (Ar- fected cell, but also major histocompatibility complex chives internationals Claude Bernard), p.210-239; (MHC). The NK is helped by specialized receptors lo- I 285-297 cated on the lymphocyte's surface. Doherty and Zin- 10. Doherty Peter's Nobel Lecture kernagel went on to prove that MHC is responsible for (http://www.nobelprize.org/nobel_prizes?medicine) the rejection of incompatible transplanted tissue. 11. Dorbec Cécile (1998), Jean Joseph Toussaint, sa A PhD graduate of the John Curtin School of Medi- vie, son oeuvre, teză de doctorat cal Research (the Australian National University Can- 12. Dubos R.J. (1995), Louis Pasteur: franc-tireur de la berra), Doherty focused on the dynamics and molecu- science, P.U.F., Paris lar structure of immunogenetics which obviously 13. Galtier P.V. (1879), Etude sur la rage. Rage du la- stretch beyond the infectionist “Procrustean bed”. pin, C.R.de l'Acad des Sci., Paris, 89, pp.3395-3404 His findings can be applied to human and veterina- 14. Galtier P.V. (1881), Les injections de virus rabique ry pathology. They help explain the molecular accep- dans le torrent circulatoire ne provoquent pas tance of self and the rejection of nonself throughout l'eclosion de la rage et semble confirmer l'immu- the beings' phylogenetic evolution and ontogenetic nité, C.R. de l'Acad des Sci., Paris, 93, pp. 284-285 development. 15. Geison G.L. (1995), The Private Science of Louis There is still much more to learn about molecular Pasteur, ed. Princeton Univ. Press, pp.146-176 immune homeostasis and more discoveries are yet to 16. Gell P.G., Coombs R.R. (1963)¸Clinical Aspects of be made by veterinary immunologists cooperating with Immunology, London: Blackwell human pathology researches, biologist and chemists. 17. Iftimovici R. (2009), Istoria Universală a Medicinei şi Farmaciei, ed. I, Ed. Academiei Române, pp. 436- REFERENCES 444; 528-529 18. Jerne K.N. (1955)¸ The Natural Selection Theory of 1. Cadeddu A. (2005), Les vérités de la science. Antibody Formation, Proc. National Acad of Scien- Pratique, récit, histoire: le cas Pasteur, ed. L.S. ces (USA), 41: 849-857 olschki, pp.127-176 19. Lachmann P., Waldmann H. (2009), „Robert Roy- 2. Chamberland Ch. Ed., Roux E. (1883), Sur l'atté- ston Amos (Robin) Coombs. 9 January 1921 - 25 nuation de la virulence de la bactéridie charbo- January 2006". Biographical Memoirs of Fellows of nneuse sous l'influence des substances antisep- the Royal Society. 55:45-58 tiques, C.R. de l'Acad des Sci, T96, pp 1088-1091 20. Owen D.R. (1945), Immunogenic consequences of 3. Chevallier-Jussiau Nadine (2010), Henry Toussaint vascular anastomoses between bovine twins, et Louis Pasteur. Une rivalité pour un vaccine, Hist. Science, Washington DC, 102: 400-401 des Sci. Med., Paris, 44(1), pp55-64 21. Pasteur L. (1857), Mémoire sur la fermentation 4. Ciucă A. (1929), The reaction of complement fixa- appelé lactique, C.R. de l'Acad des Sci., 45, pp. tion in foot and mouth disease as a means of identi- 913-916 60 Rev Rom Med Vet (2017) 27 | 1

22. Pasteur L. (1861), Animal cules infusoires vivant 30. Toussaint H. (1880), De l'immunité pour la char- sans gaz oxygène libre et determinant des fermen- bon, acquise à la suite d'inoculations preventives tations, C.R. de l'Acad des Sci., Paris, 1857, 45, pp. (Note de Toussaint présentée par M. Bouley), 913-916 Comptes rendus de l'Académie des sciences, 1880, 23. Pasteur L. (1863), Recherches sur le putrefaction, tomul 91, 12 iulie 1880, p. 135 a.n.o. C.R. de l'Acad des Sci., 56, pp. 1189-1194 31. Toussaint H. (1880), Identité de la septicemia 24. Pasteur L., Joubert J., Chamberland Ch. (1878), La expérimentale aiguë et du cholera des poules (Note théorie des germs, C.R. de l'Acad de Med de Toussaint - présentée par M. Bouley), Comptes 25. Pincock S. (2006), Robert Royston Amos (Robin) rendus de l'Académie des sciences, 1880, tomul Coombs, Lancet 367 (9518): 1234.doi: 10.1016/ 91, 2 august 1880, p. 301 a.n.o. S0140-6736(06)68528-0 32. Vechiu Al. (1939) Sur la transmission de la peste 26. Răducănescu H. (1999), Compendiu de imunopato- procine à d'autres espécès animales. Bull. Du Co- logie veterinară, Ed. România de mâine, Bucureşti mité Internat. des Epizooties, pp. 167-209. 27. Richet Ch. (1912), L'anaphilaxie,Masson ed.Paris:73 33. Willems L. (1852), Mémoire sur la pleuropneumo- 28. Stirbu C., Moga Mânzat R. (2005), Boli produse de nie épizootique exsudative du betail et sur le mo- virusuri din familia Picornaviridae, în Boli virotice şi yen de l'en preserver adressé à Monsieur le Ministre prionice ale animalelor, Ed. Brumar, Timişoara: pp. de l'Interieur. Journal d'Agriculteur pratique du ro- 474-490 yaume de Belgique, V: 1852-1852 29. Théodoridès J. (1986), Histoire de la rage, Paris, 87 34. Willems L. (1866), Note relative à la pleurepneu- monie exsudative et à l'inoculation preventive de cette maladie, Overd. Uit BARMB VIII, 1866, nr. 11.