1 The fatal illness of the Roman Emperor Antoninus Pius (AD 138-161) Cornelis J. Kooiker, Utrecht NL Summary An unexplained acute illness caused AD 161 the death of the Roman Emperor Antoninus Pius. Three centuries later, his sickbed was described into details in the Historia Augusta. The present paper argues that within the constraints of that text only the rapidly worsening, life-threatening form of staphylococcal food poisoning is able to explain the symptoms and course of the Emperor's illness. This progression of staphylococcal food poisoning can be ascribed to a successive activation of two pathogenic sites on ingested molecules of staphylococcal enterotoxin. First, activation of the emetic site induces vomiting. Subsequent activation of a superantigenic site elicits a massive T cell response that results in the toxic shock syndrome. Progression of the shock in the absence of therapy led to the death of the Emperor. This worsening of staphylococcal food poisoning is rare and said to occur after ingestion of a heavy dose of enterotoxin. Presumably, such a dose was transmitted by the Emperor's favourite Alpine cheese. Mastitis-associated staphylococci, prevalent in the cheese-producing region, could survive in the raw milk cheese of those days, whereas so-called 'starters' that counteract the release of enterotoxin during today's cheese making, were unknown in Antiquity. __________ Cornelis J. Kooiker is a retired clinical pathologist and senior lecturer in human pathology at the State University of Utrecht, The Netherlands. 2 On 7 March AD161, the Roman Emperor Antoninus Pius died on his estate at Lorium (La Bottaccia, Lazio, ITALY) from a hitherto unexplained acute illness. By a reign 'furnishing very few materials for history’, as Edward Gibbon wrote in The History of the Decline and Fall of the Roman Empire, this Emperor is less known than his adoptive father Hadrian (117 -138) and his adoptive son Marcus Aurelius (161 -180). Little attention, too, has been paid to the last days of this ‘amiable as well as a good man’, as Gibbon called him, who for more than twenty-two years ‘diffused order and tranquillity over the greatest part of the earth’. What fatal illness befell this Emperor, aged 74 years, who lived a frugal life ‘in such a way’, according to Marcus Aurelius, ‘that by his own attention he was scarcely in need of the art of healing or of medicaments’.1 Historians paraphrased the historical texts about his death but shed no light on its cause. In this paper, the cause is searched for by interpreting the historical sources in the light of today's medical knowledge. The historical sources It is remarkable that Marcus Aurelius, though living with Antoninus Pius since his adoption, completely ignores in his writings the last days of his adoptive father. Unfortunately, there are no other contemporary sources. Among the sources from later centuries, six only pay attention to the final illness of Antoninus Pius. Five authorsa together tell how Antoninus was seized by a feverish and weakening but not discomforting illness without striking features, by which he after a few days peacefully died a common death. For a diagnosis, this information is too unspecific. Fortunately, a sixth source presents a day-by-day account of the Emperor's sickbed: the Historia Augusta, known also as Scriptores Historiae Augustae. This is a collection of biographies of Roman Emperors, usurpers and pretenders, dating from the twilight years of the Roman Empire. Much debate is still taking place about its reliability but has nothing to 3 do with the last days of Antoninus Pius. His biography in particular is historically highly valued.2, 3 The pertaining text reads as follows: b His death now is told was thus: when he had rather greedily eaten Alpine cheese at dinner, he threw it backc during the night and was enraged by fever next day. On the third day, since he perceived that he was deteriorating, he entrusted in the presence of the [praetorian] prefects the State and his daughter to Marcus Antoninus [Marcus Aurelius] and ordered the golden Fortuna, which was wont to be placed in the bedroom of the Emperors, to be transferred to him [Marcus], then he gave the password 'equanimity' to the commander of the household troops and thus turned to his other side as if he slept, he gave up the ghost at Lorium. Delirious by fever he spoke about nothing else but the State and those kings with whom he was angry. In general, the authenticity of cited words of a dying monarch is rather dubious, but there is no reason to question the reliability of the reported symptoms and course, or to think of wilful murder. First steps towards a diagnosis The text describes an acute illness ushered in during the night by an upper gastrointestinal symptom strikingly coupled to the preceding dinner. Accordingly, the case will be approached as a foodborne disease contracted 4 during dinner and manifesting itself by vomiting during the following night. These facts, together with place and date, are leading in the search for the causative agent. The length of the incubation period of the disease would be an important clue but cannot be estimated. However, the limits on its length are deducible. . The shortest incubation period would have started just before the Emperor finished his meal. Apparently, the first symptom appeared after the Emperor had gone to sleep. Consequently, the shortest incubation period could have been but a little longer than the interval between dinner and sleep. In view of Plutarch's advice 'to let some time intervene between dinner and sleep',5 the length of this period may be estimated at one hour. The longest incubation period would have started shortly after the Emperor had reclined for dinner, and it ended at dawn. What time did the Emperor use to dine? Descriptions of his character make it plausible that he adhered to the Roman custom of dining in the afternoon, and that he opted, like all busy people, for hora decima.6 On the first day of his illness this was equal to 15:43 apparent solar time.d Sunrise next day was at 06:24 apparent solar time,7 14 h 41 min after hora decima. Being somewhat shorter at both ends than this interval, the longest incubation period may be put at 14 h rounded off. The length of the incubation period, then, lay between 1 and 14 hours. Searching for the causative agent Pertinent symptoms together with limits on the length of the incubation period are used to define diagnostic sets of causative agents of foodborne diseases.8 Whenever such a disease manifests itself by vomiting within 1 to 14 hours after a 5 meal, its cause generally is a preformed toxin. This may have been an intrinsic component of the food such as occurs in some fish, shellfish and mushrooms, or it may have been released in the food by contaminating bacteria.8 At a dinner in March in Lorium, 12 miles from Rome and 6.5 miles from the Mediterranean Sea, poisoning by fish may result from bacterial spoilage of tuna fish, mackerel or anchovy,8,9 as well as from toxic fish roe.10 Poisoning by shellfish may be due to toxins concentrated in oysters or mussels feeding on certain dinoflagellates.8,9 Mushroom poisoning may follow ingestion of Amanita, Galerina or Gyromitra species or ingestion of members of the Muscarine and the Gastrointestinal-irritants group.11 The descriptions of the clinical manifestations evoked by each of the mentioned causes of food poisoning are incompatible with the descriptions of Antoninus Pius's illness. The remaining possibility is food poisoning by contaminating bacteria 6 Toxin releasing contaminating bacteria The limits on the incubation period restrict the discussion of toxin releasing contaminating bacteria to Bacillus cereus, Bacillus subtilis and Staphylococcus aureus.8,12 B. cereus is a ubiquitous, spore-forming micro-organism causing an emetic syndrome12 by the highly thermostable toxin cereulide. The illness mainly manifests itself between one and five hours after traditionally prepared Chinese rice dishes since these allow spores in contaminated rice to germinate, grow and release their toxin. The illness usually is mild; its benign course and the close connection with traditional Chinese cookery exclude B. cereus as the cause of Antoninus Pius's fatal illness. B. subtilis is another spore-forming micro-organism that may cause a foodborne disease, but this illness is much less frequent than B. cereus food poisoning as It requires large numbers of bacilli (105 - 108/g).12 These may be found in contaminated food such as pastries and rice dishes kept for a long time between 10 – 60 °C. This allows surviving spores to germinate and the resulting bacteria to multiply13 and release their toxin. After an incubation period from 10 min to 14 hours, vomiting is the predominant symptom. The mean duration of the illness is 1.5 to 8 hours; victims usually recover completely within 24 hours. Lethal cases are not mentioned.12 It is clear that Antoninus Pius's fatal illness cannot be explained by food poisoning by B. subtilis. S. aureus also is widespread in nature, but it does not form spores. It lives as a commensal on the skin of man and warm-blooded animals, in glands of their skin and on nearby mucous membranes. Palaeopathologic findings have given rise to the view that staphylococci have been active pathogenic 7 organisms ever since Man evolved.14 The inflammation and suppuration of wounds described by the Roman author Celsus15 are consistent with staphylococcal wound infection. In 1930 S. aureus was discovered in poisonous food. The cultured bacteria produced a toxic factor that experimentally induced gastrointestinal symptoms of food poisoning16,17 and became known as 'enterotoxin'. First, this factor was thought to be a single substance but immunological analyses revealed several different types of enterotoxin. From 1959 to 1979, seven types were identified and designated SEA, SEB, SEC1,-2,-3, SED and SEE;18 a n eighth type, SEH, was added in 1995. All types of enterotoxin are small proteins (molecular mass 25145 – 28366 Da) consisting of a single polypeptide chain.19 These chains share many amino acid sequences20 and look very alike in their conformations.19 Staphylococci in food do not survive pasteurisation, baking or cooking. Between heating and serving, food handlers carrying S. aureus may contaminate food with living staphylococci.21 An other source is mastitic livestock contaminating its milk.21 Protein-rich food is between 7 and 48 C an excellent medium for staphylococci to multiply.22 When the population density exceeds 106 /g food,23 a quorum-sensing system24 activates between 14 and 45 °C in a large fraction of the strains the release of one or more types of enterotoxin.22 These do not betray their presence in food by any abnormal odour or taste, and they are neither inactivated by pasteurisation nor completely so by the usual way of boiling.21,25 Staphylococcal food poisoning nearly always sets in by vomiting26 between 1 and 13 h after ingestion of poisoned food.27 The length of this interval is 8 inversely dependent on the amount of toxin ingested;21 whereas the severity of the illness depends directly on this amount.18 When ingested together, the effects of different types of enterotoxin are cumulative.25 The illness mostly follows a benign course: victims generally recover within a few days. The elderly are the most vulnerable27 but death is uncommon.21 Even the last toxin under consideration seems unfit to explain the Emperor's delirious and combative fever, rapid deterioration and death. There are, however, a few reports on a quite different course of staphylococcal food poisoning. Deterioration of staphylococcal food poisoning In 1971, a 57-year-old woman died in shock in a hospital in Montgomery (AL, USA), about five hours after eating ham contaminated with SEA producing S. aureus.28 In 1975, a proven outbreak of staphylococcal food poisoning aboard an intercontinental aircraft entailed the hospitalisation of 143 victims. The body temperature of 60 patients exceeded 38 C. Systolic blood pressure was below 100 mm Hg in 14 patients and became immeasurable in one of them. Progressing into severe shock, this patient became anuric and unconscious. Meanwhile his body temperature rose to 39.6 C. Intensive treatment resulted in complete recovery.29 In his review on enterotoxins, Bergdoll mentions a victim of a laboratory accident: within one hour after ingesting an unknown amount of SEB he ‘experienced low blood pressure’, whereas his body temperature rose to 41 C.18 These observations demonstrate that patients ill with staphylococcal food poisoning show by exception a deviating course characterised by fever, 9 disturbed consciousness and severely dropping blood pressure heralding life-threatening shock. The two case reports do not comment on its pathogenesis. Bergdoll,18 stressed in 1983 its occurrence after ingestion of a heavy dose of enterotoxin, in which case the toxin may have entered the circulatory system. Moreover, he pointed out a resemblance to symptoms of a then recently described, but not explained syndrome: the toxic shock syndrome. The toxic shock syndrome In 1978, an acute, life-threatening manifestation of staphylococci was described by the name of 'toxic shock syndrome' (TSS). Two years later a larger study formulated its diagnostic criteria,30 some of which equally characterise the deviating course of staphylococcal food poisoning, namely fever above 38.9 C, alterations in consciousness and hypotension progressing into shock. A toxin responsible for TSS and different from the enterotoxins was isolated from staphylococci in the early 1980s and named ‘toxic shock syndrome toxin-one’ (TSST-1). Next year strains of staphylococci isolated from patients ill with TSS were found to produce enterotoxins (SEA, SEB or SEC) in stead of TSST-1.31 Evidently, enterotoxins can activate quite different pathogenic reactions. The pathogenetic dualism of enterotoxins Ingested enterotoxins interact in the gastrointestinal tract with neural receptors,18 which then send a stimulus to the vomiting centre in the brain.32 This is the emetic activity of enterotoxin (not present in TSST-1 ),33 the exact 10 mechanism of which has not yet been elucidated.34 The minimal effective dose is 139 ± 45 ng.35 In blood or tissue fluid enterotoxins can reveal their much more dangerous superantigenic activity.e Through massive specific activation of T cells it unleashes a stream of cytokines and other mediators. Their action on blood vessels leads to vascular dilation and capillary leakage resulting in hypovolaemia, hypotension and shock: the toxic shock syndrome. In addition, mediators acting on the brain cause fever and combativeness or disturbances of consciousness.19 These two different pathogenic activities of enterotoxin molecules are structurally and functionally independent of each other.39,40 An explanation of the Emperor's illness The symptoms and course of the Emperor's illness can be explained by the two mentioned pathogenic activities of enterotoxins: ● the emetic activity induced the nightly vomiting. ● the superantigenic activity elicited a superantigenic reactionf. It released mediators that caused the Emperor's delirious and angry fever on the second day of his illness. Of paramount importance, however, was the induction of the toxic shock syndrome, for it was the shock that brought the patient in great peril.g Being intrinsically progressive, shock should be treated within two hours lest many vital organs rapidly lose their function.46 But a treatment did not yet exist, hence the Emperor's perception of deterioration on the third day. By 11 subterminal involvement of the brain he presumably slipped into coma,46 described as sleep in two sources.4,47 In this state he died peacefully. The vehicle of transmission The explanation developed for the Emperor's illness rests on his ingesting a heavy dose of enterotoxin. This requires food transmitting such a dose. The Historia Augusta mentions one course: Alpine cheese. Outbreaks of food poisoning, veterinary epidemiologic reports and bacteriologic studies on milk and on cheese-making provide arguments that this cheese may have been the vehicle of the heavy dose of enterotoxin. Poisonous cheese Cheese is an excellent vehicle for staphylococcal food poisoning because of its high protein content. The first medical paper on this subject was induced in 1884 by reports to the Michigan State Board of Health (USA) on poisoning by cheese. From the cheese, a nausea inducing substance was isolated. An analogous finding was published in 1930, but now the toxic factor was shown to be released by S. aureus.17 Later on different enterotoxins were identified in poisonous cheese, including SEA in outbreaks in the UK 48 and SEH in Brazilian cheese.49 Could enterotoxin have been released in Alpine cheese in Antiquity? This first depended on the chance of contamination of the cheese with S. aureus. The chance of contamination The main source of staphylococci in cheese is dairy cows21 suffering 12 from staphylococcal mastitis.50 This illness is generally not recognised until the agent is found in milk or milk-products. Since mastitis is not mentioned in any of the Greek and Latin reference books on farming and veterinary medicine,h.it is plausible that the disease and its consequences were unknown in Antiquity. Nevertheless, there is no reason to doubt that commensal staphylococci were as ubiquitous and active in Antiquity as they are now, when herd infection implies mastitis.14,51,52 On this premise, the prevalence of mastitis associated enterotoxigenic staphylococci among dairy herds in regions resembling the production area of the Emperor's Alpine cheese suggests a similar prevalence in the latter area in Antiquity. Where did the Alpine cheese come from? Texts of Pliny and Galeni point to the Ceutronic Alps, extending from the Isère to the north flank of Mont Blanc.55 The Vatusic from this region was as famous as its 'Gruyère de Beaufort' is now.56 Swiss studies provide data from corresponding Alpine regions. In the middle of last century staphylococci were found in 60% of the bulk milk samples from 1120 dairy herds in the area around Bern.57 In a later study in north-east Switzerland 54% of the S.aureus strains isolated from mastitic milk produced SEA, SEC, SED and TSST-1, separately or in pairs.58 These findings suggest that enterotoxigenic staphylococci were present in the milk of about one third of the Ceutronic dairy herds. Release of enterotoxin in cheese depends on the method of cheese-making. Failings of traditional cheese making In Antiquity, all cheese was raw-milk cheese. This implied that about one third of the Ceutronic dairy farms made cheese from milk containing living 13 enterotoxigenic staphylococci. When, according to traditional cheese making as described by the leading Roman author on farming,59 this milk was kept tepid after addition of rennet, the staphylococci multiplied rapidly during curdling. They would mostly reach their quorum for release of enterotoxin, 23, 24 as substantiated by recent outbreaks of staphylococcal food poisoning due to raw-milk cheese.j As a result the Vatusic from about one third of the Ceutronic dairy farms would have contained enterotoxin. Its concentration can be estimated from some incidents and experiments that recall traditional cheese making by undoing the consequences of pasteurisation.k Two outbreaks of staphylococcal food poisoning in the London area in 1961 were caused by cheese made from dubiously pasteurised milk produced by mastitic cows vigorously treated with penicillin.64 Excreted in the milk, the penicillin killed both the ‘natural microflora’ of the milk and the starter bacteria, thus freeing the way for the penicillin-resistant mastitic staphylococci. Their number increased to an average of 2 x 108/g, amply sufficient to release enterotoxin23 (quantifying and typing of the toxin were still unknown). Suppression of starter activity during cheese making allows added staphylococci to multiply freely and to raise their individual release of enterotoxin, the total production of which may increase more than hundredfold.65 In an Australian experiment on staphylococcal activity in cheese made under virus-induced starter failure, the mean number of added cultured staphylococci increased in 24 hours from 35/ml milk to 3.50 x 107/g cheese; SEA reached in 24 hours an average concentration of 25.9 ng/g cheese.66 The staphylococcal density in the toxic London cheese was 5.71 times the density in the Australian experiment. Apart from time-related differences, this is 14 consistent with the finding that mastitic staphylococci are growing faster during cheese making than added cultured strains.67 If every single staphylococcus in the London outbreaks and in the Australian experiment had produced the same amount of enterotoxin, its unknown concentration in the London cheese would have been 148 ng/g. To the same order of magnitude belongs the 120 ng SEA ∕ g cheese produced in American cheese during the making of which contamination with S. aureus coincided, after pasteurisation, with a malfunctioning starter 68, 69 (the lower concentration may reflect the slower growth of non-mastitic staphylococci). The S. aureus strain isolated from the poisonous Brazilian cheese produced 180 ng SEH/ml culture supernatant.49 These data indicate that the Vatusic from about one third of the Ceutronic dairy farms may have contained around one emetic dose of enterotoxin (139 ± 45 ng) to the gram. Shipping to Lorium would not have lessened its toxicity; on the contrary, enterotoxin increases in concentration during ripening of cheese, and it persists for some years.65 The role of the Alpine cheese Evidently, Vatusic could transmit a heavy dose of enterotoxin, and about one third of the Ceutronic dairy farms may have shipped such cheese. Antoninus Pius reportedly took more cheese at dinner than the amount between one and two ounces (28-57 g) usually eaten with a meal.64 Thus there was a considerable chance that he ingested more than 30 emetic doses of enterotoxin, a quantity bound to elicit the superantigenic reaction (though advanced age lessens T cell mediated immune responses,70 it increases the vulnerability to enterotoxin 27 ). 15 An indirect indication that the Alpine cheese had transmitted a heavy dose of enterotoxin can be deduced from the Latin writer's choice of reiectavit 4 = he threw [it] back violently or repeatedly, 71 to describe the first symptom of the Emperor's illness. Walentowski 3 ignored reiectavit in her commentary but in a medical approach of an unknown illness any unusual wording of a prominent symptom deserves attention. Reiectavit may have been chosen, in stead of vomuit = he vomited, because the cheese reappeared seemingly unaltered. The verb, then, reflects a short incubation period such as may follow the ingestion of cheese containing a heavy dose of enterotoxin.21 It justifies likewise the initial assumption that the illness had been contracted during dinner 16 Conclusions 1. The only known disease found to explain the symptoms and course of Antoninus Pius's fatal illness as described in the Historia Augusta was the rapidly worsening form of staphylococcal food poisoning evoked by both the emetic and the superantigenic activity of staphylococcal enterotoxin. Recent medical reference books 8,72,73 ignore this rare outcome of staphylococcal food poisoning and its pathogenesis. 2. Only heavy doses of enterotoxin are able to evoke the rapidly worsening form of staphylococcal food poisoning, for only then the small fraction of the dose that may cross the gastrointestinal epithelium is large enough to trigger the superantigenic reaction resulting in the life-threatening toxic shock syndrome. 3. The presence of Alpine cheese at the Emperor's dinner supports the diagnosis. This cheese may have been the vehicle of the heavy dose of enterotoxin because of a high prevalence of mastitis associated enterotoxigenic staphylococci among the involved dairy herds, together with serious failings of cheese making in Antiquity. 4. The Latin description of the first symptom can be interpreted as an indication that the Alpine cheese had transmitted a heavy dose of enterotoxin and thus supports the diagnosis. 5. The diagnosis ‘rapidly worsening staphylococcal food poisoning' satisfies the conditions of an adequate retrospective diagnosis formulated by Grmek74 in that it explains, without contradictions, all symptoms and the course of Antoninus Pius's fatal illness as described in the Historia Augusta. The cited commentary of five other classic authors on Antoninus Pius's death is 17 consistent with this retrospective diagnosis. It supports the reliability of the cited text of the Historia Augusta. 6. Bergdoll wrote in 1979 ‘there is no record of when illnesses similar to staphylococcal food poisoning were first observed.’ 75 A survey of diseases in ancient Greece and Rome does not mention them,76 but they cannot have been rare considering the mere risk of cheese. Benign outbreaks, however, will have passed unnoticed by historians whereas incidental fatal cases were unobtrusive, as is evident from the commentary of the five classic authors. The diagnosis derived in this paper suggests that the text of ‘Antoninus Pius’ 12.4-8 in the Historia Augusta attests to such a fatal case, observed AD 161 and recorded because of the status of the victim. Presumably, it is the first report in history of a case of staphylococcal food poisoning. 18 Notes a. Cassius Dio; Epitome de Caesaribus; Orosius; Laterculus Imperatorum ad Iustinum I; Ioannes Malalas. b. Author's translation from the Latin edition of the Historia Augusta by Ernst Hohl.4 c. The Latin writer used 'reiectavit', not 'vomuit', as translations suggest. A ground for this choice is given in the section: The role of the Alpine cheese. d. Measuring time by sundials, the Romans divided in daily live the space of time between sunrise and sunset in twelve equal and successively numbered ‘daily hours’. An expression such as ‘hora sexta’ referred to the end of the sixth ’daily hour’, which coincided with 12:00 apparent solar time of Rome.7 On 5 March AD 161, the first day of the Emperor's illness, sunset at Rome took place 5 h 34.66 min after 12:00 apparent solar time.7 On that day a Roman ‘daily hour’ equalled 55.78 min, and hora decima referred to 15:43 apparent solar time. This held at Lorium as well as at Rome, as their difference in latitude is negligible. e. As confirmed by X-ray crystallography,36 an enterotoxin molecule can form a trimolecular complex with the HLA class II receptor on an accessory immune cell and a T-cell receptor bearing a matching Vβ specificity20 (this is the matching specific amino acid sequence on the variable V-region of the β-chain of the T-cell receptor). For each type of enterotoxin there is a set of 2–9 matching Vβ specificities.19,37 In this way a certain type of enterotoxin can activate all subpopulations of CD4 and CD8 T cells37 bearing Vβ specificities belonging to the set defined by this particular type of enterotoxin; other T-cell receptor regions are not involved. The result is a 19 polyclonal expansion of specifically activated T cells, which may increase from 10% to 70% of the circulating T cells.38 Such an extraordinary T cell stimulating effect marks enterotoxins out as ‘superantigens’.20 f. This reaction is set off by (fragments of) enterotoxin molecules that cross the gastrointestinal border. The reports on deviating courses of staphylococcal food poisoning leave no doubt that such crossings by these small proteins do occur. In addition, trans-epithelial passage of enterotoxins has been demonstrated experimentally.41,42 Intestinal cleavage of enterotoxin molecules by trypsin may facilitate the passage of superantigenic fragments without completely destroying their immunological activity.43 Nevertheless, it is but a small fraction of the total amount of ingested enterotoxin that reaches the subepithelial tissue, since the deviating course of staphylococcal food poisoning has exclusively been observed after ingestion of a heavy dose of enterotoxin.18 g. Shock is another cause of delirium,44 which is common in the elderly or during severe diseases and known for its fluctuating course.45 h. The authors of these books are Cato, Varro, Columella, Pliny, Gargilius Martialis, Cl. Hermeros, Palladius, Pelagonius, Vegetius, and those whose texts were compiled in the Geoponica. i. Pliny writes, ‘the Alps prove the excellence of their forage by two kinds [of cheese]: the Dalmatian [Alps] send the Docleate, the Ceutronic [Alps] the Vatusic’.53 Galen, a contemporary of Antoninus Pius's, calls the Vatusic excellent and popular among wealthy Romans.54 j. The high involvement of cheese in staphylococcal food poisoning in France is ascribed to the preference for raw milk cheeses.60 20 k. Modern cheese making starts by pasteurising the milk, which kills all pathogenic micro-organisms,61 but also the strains of the ‘natural microflora’ of milk which in traditional cheese making effected the initial souring. Today these ‘wild’ strains are replaced by their selectively cultured descendants, the starters.61, 62 They rapidly lower the pH of milk to the optimum for curdling by added rennet. 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