Trench Fever: An Ancient Zoonosis

Rhesus macaque (Photo by J.M. Garg, CC)

Trench fever is an ancient disease with a surprisingly short history. Named after its discovery in the trenches of World War I, its case history is only about a century old. Yet, the louse transmitted Bartonella quintana that causes trench fever has been found in human remains as old as 4000 years and is one of the most common infectious organisms found in human ancient DNA (for example). Once thought of as a self-limited moderate ‘five-day’ fever (aka quintana) it is now known that it can also cause endocarditis and a chronic bacteremia. It persists today in most countries anywhere that human body lice are common, often among the homeless.

After the chance discovery of trench fever in captive macaques transported in both directions between the United States and China, a Chinese team investigated the extent of Bartonella quintana in macaques in captive primate centers in China (1). They collected blood from Rhesus macaques at three geographically distant primate centers and cynomologus macaques from one additional primate center. They found Bartonella quintana in macaques from all four centers with enough genetic diversity to suggest multiple sources originating in the wild (1). By Multilocus sequence typing (MLST), a type of genetic fingerprinting, they found more genetic diversity just in the macaques from these centers than from all human isolates analyzed to date around the world. This suggested to the Chinese team that the macaques are the likely original host population of an ancient zoonosis (1). The limited genetic variation in humans suggests that the zoonotic transmission events occurred in the distant past and are not continuing today at a level detected in human populations. Further, the Bartonella quintana sequences fell neatly into three groups that corresponded to the species they were isolated from, suggesting to the Chinese group that B. quintana has co-evolved with species specific exoparasites, mostly lice (1).

Phylogeny of Bartonella quintana. Group 1 is found in humans, group 2 in cytomolgus macaques and group 3 in  rhesus macaques. (Li et al, 2013, Ref. 1)

Although they found B. quintana in a high percentage of macaques in all of these facilities, the conditions at the primate centers could account for the high incidence rate. A related study also found that B. quintana spread very easily within the captive Rhesus macaque centers through the macaque specific louse Pedicinus obtusus (2). They were also able to demonstrate that the macaques developed the chronic bacteremia found in humans suggesting some evolved tolerance (2). Samples from wild macaques will have to be sampled to determine what the natural carrying load of these species are. Rhesus macaques have a range from China to Afghanistan with a large population in India. Other macaque species extend the historic range of possible carriers to the Mediterranean and North Africa.

References:

1. Li H, Bai JY, Wang LY, Zeng L, Shi YS, Qiu ZL, Ye HH, Zhang XF, Lu QB, Kosoy M, Liu W, & Cao WC (2013). Genetic diversity of Bartonella quintana in macaques suggests zoonotic origin of trench fever. Molecular ecology, 22 (8), 2118-27 PMID: 23517327
2. Li H, Liu W, Zhang GZ, Sun ZZ, Bai JY, Jiang BG, Zhang YY, Zhao XG, Yang H, Tian G, Li YC, Zeng L, Kosoy M, & Cao WC (2013). Transmission and maintenance cycle of Bartonella quintana among rhesus macaques, China. Emerging infectious diseases, 19 (2), 297-300 PMID: 23347418

Asymptomatic Plague: Qinghai, China, 2005

Now that we know the Tibet-Qinghai plateau region is where Yersinia pestis originated and the region where subsequent pandemics arose, I think its time to look more closely at regional outbreaks and case studies.

In this region, the marmot (Marmota himalayana) is the primary reservoir for Yersinia pestis. This large communal burrowing rodent is hunted by local Tibetan tribesmen for both meat and pelts. Butchering marmots has long been considered a risk factor for contracting plague via their fleas, aerosols or skin abrasions. To investigate the exposure of marmot hunters to plague, Chinese epidemiologists collected serum from 120 Qinghai villagers, 68 male hunters and 52 female family members, along with 120 negative controls from the non-endemic area of Beijing. None of the villagers or controls reported having a fever within the last two years.

The results are eye-opening and illustrates the importance of occupational exposure. Over a third of the male villagers had an antibody response to Yersinia pestis. Only 2% of their female family members produced an antibody response. Wether two fever-free years are enough time to determine if they had symptomatic plague in the past is an open question. Their letter to Emerging Infectious Diseases does not provide much information on the test subject’s histories or oral reports.

Table 1: Plague antibody assays (Li et al, 2005)

The epidemiologists explained this high level of immune protection to the use of prophylactic antibiotics by marmot hunters. They suggest the presence of tetracycline or sulfamethaoxazole, common prophylactic antibiotics in Tibet, in  their system at the time of exposure would be enough to prevent a symptomatic infection while still giving them an immunizing dose of bacteria.

The use of prophylactic antibiotics is, of course, a double-edged sword. It is clearly preventing symptomatic infections and probably outbreaks. The Chinese epidemiologists credit  most outbreaks in Qinghai to marmot hunters who either a lack of prophylactic antibiotics or have ineffective antibiotics.  On the other hand, the use of antibiotics is possibly encouraging them to harvest the easier to catch, sicker marmots. The use of prophylactic antibiotics also promotes antibiotic resistance in Yersinia pestis.

Li, M., Song, Y., Li, B., Wang, Z., Yang, R, Jaing, L., and Yang, R. Asymptomatic Yersinia pestis, China. Emerging Infectious Disease, 2005, 11 (9): 1494-1496.

Fleshing out Yersinia pestis

Up until a few months ago there were a few representative samples of the Yersinia pestis genome. Important windows into its secrets, but windows none the less. In January a Chinese group remedied this situation by expanding the number of fully sequenced genomes from 15 to 133 (Cui et al, 2013).  China supplied 107 genomes selected from over 900 genotyped specimens collected since 1955 to represent bacterial and host diversity. To these, 11 additional isolates from Mongolia, Myanmar (Burma), the former Soviet Union, and Madagascar were fully sequenced. For the analysis, the previously sequenced 15 genomes were added bringing the total up to 133 including the ancient specimens from 14th century London.

The Core-Genome and the Pan-Genome

Even for a bacterium like Yersinia pestis that is considered to have little genetic diversity, its genome is more elastic than any eukaryote (everything but bacteria). The bacterial genome can be divided into its core genome, found in all members of the species, and the accessory genome, sequences found only in some strains. Plasmids are part of the accessory genome but not all of it. Extra genes are also found on the bacterial chromosome. The core genome is 3.53 Mb long with 3450 genes; the accessory genome has 1.92 Mb with 1249 genes (including 451 on the six known plasmids) (Cui et al, 2013, Table S1). So the accessory genome contains 26% of genes found in the species. This may seem like a lot, but more promiscuous species like Escherishia coli (E. coli) have many more accessory genes than core genes. With E. coli the more specimens that are sequenced, the larger the accessory genome gets with no end in sight.

Combining all of the genes found in Yersinia pestis (core and accessory genome), we have the pan-genome. The pan-genome is 5.46 Mb with 4699 genes (Cui et al, 2013).  No one strain has all of these genes. So different strains do have significant differences in their functions but, as far as I know, there are no significant differences in human prognosis. Hopefully, there will be more study in the future that cross-references strain type  or particular genes with human prognosis, transmission routes (% bubonic vs pneumonic), hosts etc.

Branching Out

Using known and new SNPs, the phylogenetic tree has finally been fleshed out into a healthy looking tree . We couldn’t keep the sickly looking Charlie Brown tree of the past forever! Even so, the tree below represents only the main branches.

Click to enlarge, (Cui et al, PNAS, 2013)

To my mind, the most important aspect of the new tree is that nodes of increased diversity are much more apparent. The authors are the most excited by node 7 where there is a four-way branch, adding two new branches  (3.ANT1 and 4.ANT1) to the main stem of the tree. They refer to this diversity point as the ‘big bang’. This node gains the most attention because the 14th century London genomes are just one step off of node 7 down the 1.ANT1 branch. So it stands the reason that node 7 represents a period of diversity that produced the second pandemic. Yet, looking at their diagram, other locations like node 12 have greater diversity. The 1.IN strains are intermediary on the same lineage between the second the third pandemic. Node 14 is the initial diversity that produced the third pandemic.  Calling node 7 a ‘big bang’ seems to me to have more to do with it producing the second pandemic rather than the diversity at the node itself. The new third and fourth branch (3.ANT and 4.ANT) are concentrated in Mongolia, putting emphasis on the importance of doing such deep sequencing in other Central Asian regions. It is impossible to tell which host species these bursts of diversity occurred within, almost certainly not humans. It’s not that diversity can’t be generated in humans especially during a pneumonic plague, but since it is not endemic in humans,  it must make it back to a reservoir to be preserved anywhere other than in ancient DNA.

Biogeography shows clustering of related strains in regions as would be expected, though they are fairly well mixed within the circled zone in the map above. Samples seem to follow ancient roads, although keep in mind all of these strains have been isolated within the last 60 years.   I do wonder why they were not able to identify a route for the eastern branch two isolates. All of the branch two isolates appear to be running along a fairly straight line from southwest to northeast China (extending trade route III to Manchuria). The 107 Chinese specimens were chosen from > 900 strains identified from 5000 isolates for their diversity revealed by genotyping, host diversity and geography (Cui et al, 2013). It would have been interesting to see a map with all 5000 on it as a measure of abundance (with or without typing).

The oldest strain 0.PE7 is found only on the Qinghai-Tibet plateau in China, an area framed by the ancient trade routes along which most of the western strains are found. This has led Cui et al, 2013 to postulate that the  Qinghai-Tibet plateau as the origin of  Yersinia pestis.

Unsteady Molecular Clocks

Estimating ages from genetics can be a very risky business. To estimate years since the last common ancestor, it requires a steady molecular clock , measured in base changes per unit of time. In theory all of the genes from the core genome should have changed to the same degree from the common ancestor, but that is not the case at all. The number of SNPs in the Yersinia pestis core genome varies greatly. Even excluding the most divergent Angola (0.PE3) strain, there is “a nearly 40 fold difference between the slowest and the fastest evolving branches” (Cui et al, 2013). An unsteady molecular clock was also suggested by previous data from Madagascar, though the discussion was buried in the supplementary material (Morelli et al, 2010, p. S10-s18). Mutator phenotypes do occur (Rajanna et al, 2013), though Cui et al, 2013 assure us that none of these strains are mutators.  On the other hand, a Georgian group suggest that the mutator phenotype, a single point mutation, could naturally reverse (back mutate) altering the predictability of the lineage age (Rajanna et al, 2013). The Chinese group concluded that the faster clock rates for some branches are due to a higher reproduction rate, probably due to more or larger epidemics in the lineage (Cui et al, 2013). The types of genetic changes (SNPs) indicate neutral selection, so the increased reproduction rate is not due to the genetic changes.

While I understand that calculating divergence dates an important exercise to people who focus on phylogenetics, for the understanding of historical plague it is not useful. It is not solid or specific enough to base historical events upon alone. Predictions are just that; all of these groups have been proven wrong, sometimes later by themselves, too often.  Most importantly, it appears that it will eventually be trumped by ancient DNA analysis with an archaeological and/or documentary context. As far as I’m concerned, the Angola strain is a genetic and geographic outlier of uncertain provenance. We don’t know important factors like how long it was kept in active culture before it was made into a stock or the conditions of storage. Both of these can effect mutation rates and the molecular clock (Rajanna et al, 2013).  I’m sure the Angola strain’s story is interesting but unlikely to be useful for understanding the whole species unless it turns up in ancient DNA.

Gaining and Loosing Diversity

Returning to these starburst points on the tree, called polytomys, where multiple lineages share the same ancestor, we have some of the most valuable information in the new phylogenetic tree. Epidemics (and presumably epizootics) are believed to have an increased reproduction rate over enzootic plague. Since the mutation rate is directly tied to the reproduction rate, increased reproduction rates predict an increased mutation rate and, therefore, production of genetic diversity.  The team predicts that “higher clock rates are an indicator of epidemic disease, even in the absence of historical evidence” (Cui et al, 2013). It is unclear how an epidemic can be differentiated from an epizootic by genetics alone. We know from modern observations that not all epizootics spill over into the human population. Yet, major polytomys can at least be used to estimate how many bursts of growth the bacterium has gone through in China. We should see other polytomys with increased sequencing of other Central Asian regions.

While these polytomys show a starburst of new lineages, there is also a loss of diversity during every epidemic. Most of the new lineages produced during an epidemic (or epizootic) will die out (become extinct) when the epidemic ends. If the changes are truly neutral, then which lineage survives to endure in the reservoir will be completely random (as will be the number of surviving lineages). We should also remember that clinical isolates  during an epidemic and ancient DNA can preserve lineages that become extinct (and this is normal). In the four individuals they sequenced from 14th century East Smithfield, they found two different clones, with the second being derivative of the first. Both of these clones may only be found in ancient DNA, not in any living specimen. The more time that passes the greater the likelihood that the minor lineages will become extinct. This tends to make the earlier sections of the pylogenetic tree look cleaner by stripping off side branches.

Another recent study by Vogler et al (2013), supports their scenario on a finer scale during the 9 year epidemic in a port town of Mahajana,  Madagascar from 1991 to 1999. Over a decade we can compare the incidence of plague vs. the genetic diversity. Yersinia pestis evolution can be plotted with great precision. In the lower diagram, clones are color coded to the year of isolation. From 1995 to 1999 it is possible to see the next year’s primary clone emerge in the previous year’s epidemic, which supports local cycling within the city. At the same time, most of the diversity generated is not represented in later outbreaks.

Vogler et al, 2013

Vogler et al, 2013

Host Diversity

Host genus vs Y. pestis strain collected (Cui et al, 2013).

The hosts of these 107 strains give us a glimpse into the host diversity for Yersinia pestis within China (Cui et al, 2013). The figure to the right gives an indication of strain diversity within each host but does not tell us abundance or location within China. What jumps out at me, is that humans and marmots have the most strain diversity. The high strain diversity in humans including 0.PE7, the strain closest to the most recent common ancestor, suggests to the Chinese team that Yersinia pestis has been pathogenic to humans since it evolved (Cui et al, 2013). Thus, at no point in its evolution did it gain the ability to infect humans. The few strains that can not infect humans are hypothesized to have lost their ability to infect humans possibly as a function of purifying selection for voles as hosts. It is interesting that the 1.ORI strains of the third pandemic are only found in humans, rats and mice.  We have to be careful about taking this figure to represent abundance or importance of a particular host. The great gerbil, Rhombomys opimus, is a primary host throughout central Asia is is represented by only one strain in this figure.

Studies published this winter have moved us significantly down the road to fleshing out Yersinia pestis. The genetic survey of Y. pestis in China provides a firm foundation to build on as more ancient DNA becomes available and extensive sequencing is done in other regions. Madagascar continues to be the best laboratory for plague ecology and epidemiology, while the Georgian study begins to address unintended intra-laboratory evolution that may shed light on Y. pestis in the wild. I’ll return to these papers again soon as I continue to examine Y. pestis from different perspectives and ruminate on answers to other questions.

References:

Cui, Y., Yu, C., Yan, Y., Li, D., Li, Y., Jombart, T., Weinert, L., Wang, Z., Guo, Z., Xu, L., Zhang, Y., Zheng, H., Qin, N., Xiao, X., Wu, M., Wang, X., Zhou, D., Qi, Z., Du, Z., Wu, H., Yang, X., Cao, H., Wang, H., Wang, J., Yao, S., Rakin, A., Li, Y., Falush, D., Balloux, F., Achtman, M., Song, Y., Wang, J., & Yang, R. (2013). Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis Proceedings of the National Academy of Sciences, 110 (2), 577-582 DOI: 10.1073/pnas.1205750110

Morelli G, Song Y, Mazzoni CJ, Eppinger M, Roumagnac P, Wagner DM, Feldkamp M, Kusecek B, Vogler AJ, Li Y, Cui Y, Thomson NR, Jombart T, Leblois R, Lichtner P, Rahalison L, Petersen JM, Balloux F, Keim P, Wirth T, Ravel J, Yang R, Carniel E, & Achtman M (2010). Yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity. Nature genetics, 42 (12), 1140-3 PMID: 21037571

Rajanna C, Ouellette G, Rashid M, Zemla A, Karavis M, Zhou C, Revazishvili T, Redmond B, McNew L, Bakanidze L, Imnadze P, Rivers B, Skowronski EW, O’Connell KP, Sulakvelidze A, & Gibbons HS (2013). A Strain of Yersinia pestis With a Mutator Phenotype from the Republic of Georgia. FEMS microbiology letters PMID: 23521061

Vogler, A., Chan, F., Nottingham, R., Andersen, G., Drees, K., Beckstrom-Sternberg, S., Wagner, D., Chanteau, S., & Keim, P. (2013). A Decade of Plague in Mahajanga, Madagascar: Insights into the Global Maritime Spread of Pandemic Plague mBio, 4 (1) DOI: 10.1128/mBio.00623-12

The Great Pneumonic Plague of 1910-1911

The Great Manchurian Plague of 1910-1911: Geopolitics of an Epidemic Disease

by William C. Summers
Yale U Press, 2012

Manchuria was a political mess at the turn of the 20th century. Although it was the homeland of the Qing dynasty, the Chinese considered it a backwater. Japan and Russia on the other hand saw it as potential colonial territory, a beachhead for Japan’s mainland ambitions or access to a valuable ice-free Pacific port for Russia. By 1910 an uneasy truce held giving Russia and Japan corridors to build railroads with stations and towns to support them while technically still in Chinese sovereign territory. The importance of Manchuria to global politics and trade was underscored by the presence of European and American diplomatic representatives in these upstart towns and ports. Into this delicate situation, the discovery that marmot fur could be dyed to pass as ermine brought a flood of primarily Chinese hunters into the region. The drive for furs (and marmot oil) led many to cast aside traditional hunting practices and safeguards, setting the stage for the plague to come.

The beginning of the plague was sparsely recorded. Starting in October 1910 scattered reports were sent that plague had appeared at sites along the railway, but concern was slow in coming. Summers credits the nearly annual outbreaks of plague, low concern for locals, and the temporary camps of hunters for the lack of information on beginning of the plague. Deaths quickly jumped into the thousands in towns along the rail line but never spread very far from the lines. Summers notes that of the over 43,000 recorded cases during the outbreak only one single person survived (p. 19). Some estimates place the mortality closer to 60,000 when early cases and locals away from the rail line are included. Summers notes that this puts the death toll in the range of the great London plague of 1665. By February 1911, the plague was over; blatant proof the plague outbreaks can flourish in winter (as it sometimes did in medieval European winters).

This plague has some unique features. First, there is no report of bubonic plague at all; it was exclusively pneumonic. Most plagues are primarily bubonic with flair ups of pneumonic transmission. Etiology was confirmed by autopsy and the then new bacteriological techniques including culture. American doctor Richard Strong, working with the Chinese under Wu Lien-Teh, did 25 autopsies before the International Plague Conference called by the Chinese in Mukden. There is no doubt the Yersinia pestis caused this epidemic.

Without antibiotics, quarantine and isolation were the only effective means of control. The means were brutal but effective. In the Russian zone the Chinese were crowded into train cars and not let out until there were several days with no one displaying any symptoms. If a case of pneumonic plague was locked in the car with others, the prognosis for the others was predictably terrible. Within this relatively small region, we can also see three different national approaches (China, Russia, and Japan) to controlling the epidemic each always subordinated to their respective political anxieties.

The chapter on origins of the plague begins to move toward was Edmund Russell envisioned as evolutionary history in his book of the same name (2011). The most useful information here is the history of marmot activity and traditional hunting. Summers hypothesizes that traditional shaman-like practices may have aided hunters in only taking healthy animals. Concern for the health of the animal was one of the early traditions abandoned by hunters eager for furs. Following the reports of the time, Summers believes the plague originated in the marmots, and was distinct from the third pandemic lineage. In accordance with modern strain maps, Summers predicts that the 1910-1911 Manchurian outbreak strain will belong to either the antiqua or medievalis biovars (p. 128-9). If this is true, as is likely, it’s a relatively modern challenge to our notion that plague during pandemics has a single lineage and origin. It should be possible to type this outbreak from graves of the epidemic (which should be relatively easy to locate at barely a century old) or even tissue archived from the autopsies (if it was saved and can be located).

Summers is clearly in his element when he discusses the politics of turn of the 20th century politics. This is a region and time period that most Americans know very little about but is still critically important for Asian politics today with the still uneasy relationship between China, Korea, Japan, and Russia. Summers account lays out the different approaches to healthcare and attitudes toward the epidemic as a national and economic threat. This carried over to the International Plague Conference that nevertheless managed to focus on science and medicine. While the scientific politics was interesting I would have liked to hear more about the contents of the wide-ranging Conference report.

Summers does a good job of being very diplomatic will all parties concerned. In doing so, he does overlook a major legacy of the Manchurian plague. There seems little doubt that this plague, witnessed and closely reported on by Russia, the United States and Japan, played a role in plague being developed as a biological weapon. This plague illustrated the deadly efficiency and sustained transmission of pneumonic plague. This book should be read as a prologue to Japan’s biological ‘experiments’ during World War II beginning in Manchuria (see Sheldon Harris’ Factories of Death) and the continuing programs of the United States and Russia during the Cold War.

This book makes a valuable contribution to plague studies, and early 20th century public health practices. Yet there are still unanswered questions on genetics, epidemiology, and ecology. I hope this book along with Myron Eschenberg’s Plague Ports(2007) and others are ushering in a new period of focus on the plague in the 19th-20th century.

Japanese Use of Plague during World War II

I’ve been reading Sheldon Harris’ Factories of Death: Japanese Biological Warfare, 1932-1945, and the American Cover-up. (Rev. ed, 2002), considered the definitive book on biological warfare in the Pacific theater during WWII. My primary interest is in Japanese research and use of plague in their biological warfare program.  Since this blog is, in part, a research tool, this post is a collection of notes taken specifically on plague, though the book covers a much wider program. If you ever wondered why plague is a category A bioterrorism agent, what follows will go a long way in explaining.

Lt. Gen. Shiro Ishii (1892-1959) of Unit 731, the biological warfare unit of Imperial Japan.

Lt. Gen. Shiro Ishii was the primary organizer, promoter and director of the Japanese biological warfare (BW) program. He was involved at all levels from pitch-man to the Japanese military and academia to personally supervising research on human subjects. He began his work research in the potential of biological weapons in the late 1920s.

One of Ishii’s first facilities was called the Zhong Ma Castle in Beiyinhe northern Manchuria. Initially their test subjects were trouble makers among the Chinese population: criminals, communists, and other suspicious persons. Ishii began by focusing on plague, glanders and anthrax. Subjects were injected with the pathogen and the course of their disease was monitored; all were extensively autopsied. (p. 33-34) There are numerous reports of autopsies being carried out on the unconscious, as in  not yet dead.

In 1939 the stressed Japanese military allowed Ishii to send several BW attacks  against Soviet forces in the Nomonhan region. Details of the mission refer to the contamination of water supplies with typhoid but plague, cholera and dysentery effected both Japanese and Soviet troops during the campaign. Harris is unclear whether these were effects of biological weapons operations or naturally occurring outbreaks. (p. 97-98) In 1942 a Soviet defector to Germany claimed that Soviet biological weapons were field tested during combat in Mongolia (/Manchuria) and that a there was a major plague epidemic at that time. (p. 98) With both sides attempting biological warfare and with the level of technology at the time, it is unlikely that it will be possible to unravel outcome of either the Japanese or Soviet efforts.  The Japanese BW program was developed primarily with a future war against the Soviets in mind, as Japan planned to take land north of Manchuria. The intent of the BW program was to give Japan an advantage over the vast population and natural resources of China and the Soviet Union.

Shiro Ishii built an extensive network of facilities in China for the purposes of research and testing. Some details of these units in respect to plague activity follows:

• Unit 731 was the primary unit under Ishii’s command based at his specially built facility at Ping Fan outside of Harbin, Manchuria. The scale of Ping Fan was enormous. Here Ishii built his dream facility complete with four boilers capable of producing one ton of  culture media each sterilized in fourteen autoclaves, ‘Ishii cultivators’ each capable of producing 30 kilograms of bacterial cell mass, and the capacity to maintain  plague, cholera, typhoid and paratyphoid, dysentery, anthrax, gas gangrene, tetnus, and glanders. Ping Fan was also equipped with the capacity to mass produce fleas and its own fleet of airplanes for experiments. (p. 69) After the war Maj. Gen. Kawaashima Kiyochi boasted that Ping Fan could produce 300 kg of plague monthly in addition to other pathogens. (p. 69)

Manchuria, today split between Russia and China. (Public domain image from the CIA’s World Fact Book via Wikipedia Commons.)

• Unit 731 conducted ‘field tests’ throughout northern and eastern China from late 1939 to 1942. They specialized in spreading pathogens by contaminated water and food. They tested cholera, typhoid, paratyphoid, and especially anthrax and plague. They were reported to start epidemics and then enter villages claiming to vaccinate against the epidemic, except that they would inject the pathogen instead. Their experiments involved introducing pathogens by unusual routes such as ‘vaccine’ injections including cholera bacteria. In a Soviet war crimes trial, testimony was given that Ishii’s forces handed out special chocolate bars laced with anthrax to Chinese school children “with unavoidable side effects”‘. (p. 99)

• Unit 731  gave special attention to plague, spreading plague infected rats widely throughout China and experimenting with spreading plague through fomites like contaminated fountain pens or canes. When a full scale epidemic broke out, Japanese soldiers would force an evacuation of the village and burn it to the ground. An American missionary Archie Crouch reported seeing Japanese plains drop odd bombs that spread what looked like wheat over the city of Ningbo and plague erupted just days later. Chinese officials tried to combat the plague with isolation, quarantine and burning the most infected part of the city but over 500 people died of plague and other agents spread by Ishii’s forces. Outbreaks of plague continued in the region of Ningbo until as late as 1959 (p. 101-103).  The city of Quzhou was also subjected to bombs that scattered, soy beans and rags contaminated with plague, cholera, typhoid and possibly anthrax. Bacteriologist Qui Mingxuan lived in the city as a child, and put the death toll for the six years after the first plague outbreak in 1940 at 50,000. Qui noted that there was no history of plague in Quzhou before 1940. (p. 102) In August 1942 plague was sprayed over the village of Congshan in the Zhejiang Province.  Rats began to die in droves two weeks later and over the next two months 392 out of 1200 residents died of bubonic plague. (p. 103) For unexplained reasons, after 1942 Unit 731 stopped large field tests and began to concentrate on more direct human experimentation in controlled environments. Harris estimates that by the end of 1942, “the casualty count in the open tests surely fell into the six figure range” (p. 104).

• Enough credible reports made their way out of China to convince the Allies that Japan was conducting biological warfare. (p. 100-103) In addition to reports coming out of China, American investigators found ampoules of cholera in Burma that locals reported where dropped from planes by the Japanese. In September 1944, Thailand also experienced a plague although there had been no recent plague activity in the area. The Thais and Americans both concluded that these outbreaks in Burma and Thailand were acts of biological warfare by Japan. (p. 226)

• The facility at Nanking, operated by Unit Ei 1644 under the direction of Tomosada Masuda, was a mass production site for bacteria (cholera, typhus, and plague), rodents and vectors. Nanking specialized in flea production for plague experiments. It was also a training site for bacteriologists to conduct biological warfare, producing about  900  from 1941-1943 (p. 142-143)

• Unit 100 in Changchun region worked on plague among other pathogens from 1940 to 1945. Although plague outbreaks had occurred in the region previously, several large suspicious outbreaks that took thousands of lives occurred from 1940 to the end of the war. Unit 100 used these outbreaks as cover for widespread experimentation on villagers.  Injecting slum dwellers with plague under the guise of vaccines against the plague was one of their notable practices. They then relocated about 5000 survivors and burned the slum to the ground. As the war was coming to a close, the order came to destroy all evidence, buildings and people. None of the test subjects or Chinese workers escaped. Some of the infected animals were released into the countryside after the official surrender possibly triggering outbreaks of plague, anthrax and glanders in 1946, 1947 and 1951. (p. 126-133)

• At the Anda test facility in northern Manchuria, Chinese test subjects were tied to open air stakes and bombs containing either an anthrax slurry or plague infected fleas were exploded around them in an attempt to infect them. There is some evidence that anthrax worked slightly better than the plague because the fleas did not handle the exploding bomb well (!). By 1944 they were working on developing means for spreading pneumonic plague and other respiratory pathogens. Fortunately, they were still unsuccessful by the end of the war. (p. 88-90) Ishii would later brag to US war dept. interrogators that he developed a porcelain bomb that successfully disseminated plague (p. 247).

At the beginning of the war, the American biological weapons program was the least developed of all the major combatants. The US military was eager to repair this deficit. American offensive and defensive biological warfare research began at Fort Detrick, Maryland, in 1942. Plague was one of many different pathogens worked on during the war. (p. 210) After the Japanese surrender, American officials were much more interested in extracting intelligence from cooperative Japanese researchers, including Shiro Ishii, than in pursuing justice for the Chinese or even American POWs that were victims of their experiments. To insure cooperation Shiro Ishii was given immunity from prosecution and no one was ever brought to the war crimes trials in Tokyo for biological warfare.  I will leave issues of justice and the cover-up to journalists and political historians.

I do have to take issue with Harris’ assertion that Shiro Ishii was a good microbiologist. He may have been a good pitch-man, organizer and military man but not necessarily a good biologist or physician.  First, I can’t accept that anyone racist enough to mentally justify this work was a competent biologist, much less a physician. Designing and carrying out these ‘experiments’ are the sign of an unstable mind. Methodologically,  ‘try absolutely everything you can think of and something might work’ is not good science, not even in wartime. Even if allied programs did similar things, it is still not good science. He thought more like an engineer than a biologist. The innovation that started his career and gained military attention was the development of a water filtration system to prevent cholera for the military. From a strategic point of view, biological weapons were incredibly risky in the 1940s before the discovery of penicillin and other effective antibiotics. The Japanese military actually had to reign them in out of fears of blow-back. Considering what the Japanese military was willing to do during World War II, this says a great deal.

Generating Immunity to the Plague

Direct fluorescent antibody (DFA) of Yersinia pestis (Source: CDC)

Its pretty amazing that we still don’t have a vaccine against the plague. Work still goes on and it hasn’t been easy by any means, but it really isn’t a priority that you hear about much. Vaccines developed to date have issues with side effects and the need for repeat immunizations to be protective against pneumonic plague. The live, attenuated (weakened) Yersinia pestis vaccine, EV76,  used in China today produces an immunity that lasts only 6 to 12 months. Not an ideal vaccine but it should produce a temporary ring of herd immunity around an outbreak.

Vaccine research is still answering some pretty basic questions like what is the nature of long-term immunity to Yersinia pestis? This is the question a Chinese group led by Ruifu Yang and Zhizhong Song  are seeking to answer (Li et al, 2012). Plague is frequent enough in China for them to be able to assemble a cadre of long-term plague survivors to study their adaptive immunity.

Successful immunity to Yersinia pestis requires both a good humoral (antibody) and cellular (T cell) responses. Li et al (2012) gathered 65 plague survivors from the Yunnan-Guangxi-Fujian endemic plague foci who contracted the infection from 1990 to 2005, of which 23 were over 10 years post-infection. (Serum was collected in 2006.) They collected an additional 48 serum samples from people from the same endemic region of China who had never had a Yersinia pestis infection, and an additional 43 serum samples from people outside of the Y. pestis endemic area without a history of plague to serve as controls. They assayed their antibody response by ELISA and protein microarray and looked at their memory T lymphocyte response to the F1 antigen and LcrV protein.

Their results shed some light on our response to the plague. Of the 65 plague survivors, 78.5% (51/65) still produced a strong antibody response to the F1 antigen; the response rate for those whose infection was within five years was 88% and only decreased to 69.5% in patients infected over a decade earlier. Twelve of the thirteen  longest survivors infected in 1990 were still reactive to the F1 antigen. They did not find a statistically significant difference between genders and there does not appear to be a difference based on age. The youngest age at infection was only three years old in 1997 and still produced a response in 2005. More importantly the antibody titer in 2005 correlated very well with their antibody titer at the time of infection.

The F1 protein is not the only antigen we make a response against in an active infection. They pooled serum for each year of infection for protein microarray testing. They found antibodies to LcrV and YopD in most survivors but the other proteins varied by year of infection (and therefore probably strain of the outbreak). These proteins are potential targets for new vaccines and for detection of F1-negative strains.

They assayed the interferon-gamma production of memory T cells by challenge with F1 antigen and LcrV in 7 plague survivors (infected 4-7 years previously) and 4 controls from non-endemic areas. The interferon-gamma levels produced by survivors and controls was not significantly different. Because Yersinia pestis is an intracellular pathogen early in the infection, the macrophage activation of interferon-gamma should play an important role in the early immune response to plague. Li et al (2012) discuss some possibilities for why they could not detect a difference in interferon-gamma, eventually concluding that the F1 antigen and LcrV proteins are probably not the dominant T cell antigens.

This study provides useful and interesting data for future vaccine development. Li et al (2012) conclude that this study is proof that the best route to an improved vaccine in the near future is an improved live, attenuated vaccine. The F1 antigen and the LcrV protein, the focus of current vaccine efforts, do not appear to be efficient at generating a good cellular immune response necessary to respond to pneumonic plague. With today’s biosecurity concerns, protecting against a bioterrorism event with  aerosolized Y. pestis causing pneumonic plague must be the target of vaccine research.

For the historic plagues, this study illustrates that the immune response can last at least a decade or more. We should presume that the patients in this study were treated with antibiotics and possibly a passive vaccine (antibodies) so that their infection may have been milder and shorter than an untreated historic infection. The longer and more intense the infection, the better the protective immune response should be in survivors. Enduring active immunity could explain lowered mortality rates or altered demographics in successive waves of plague when they occur 10-20 years apart.

Li B, Du C, Zhou L, Bi Y, Wang X, Wen L, Guo Z, Song Z, & Yang R (2012). Humoral and Cellular Immune Responses to Yersinia pestis Infection in Long-Term Recovered Plague Patients. Clinical and vaccine immunology : CVI, 19 (2), 228-34 PMID: 22190397

Did India and China Escape the Black Death?

One of the few things everyone studying the plague can, I think, agree on is the importance of plague dynamics in Asia. Genetic diversity and biogeography suggest that Yersinia pestis evolved in East Central Asia (S. Russia, Mongolia, N. China) and spread along the Eurasian steppe from the Caspian Sea in Kazakstan to the Mongolia very early, perhaps even before it became a human pathogen [1]. The orange labeled clones in the diagram below represent Y. pestis clones that branched off of the main stem before Y. pestis was a human pathogen. These clones only infect voles [2]. They are spread in a wide belt along the Asian steppe but as these are modern clones, we can’t be sure how early this spread occurred. Pandemic Yersinia pestis, ‘the plague’, could have emerged anywhere along this wide Asian belt. Note the red clones (the “Medievalis biovar”) shadow the Silk Road.

Yersinia pestis isolates across modern Asia. (Li et al, 2009) Click to enlarge

The three main pandemics probably arose from different localities  as clones were slowly spread along the Silk Road and endemic foci emerged and expanded [1,2]. The Plague of Justinian is first recorded in Pelusium Egypt, but it probably arrived via canals linking it to the Red Sea and ultimately the Indian Ocean. The Black Death is first recorded at Caffa on the Black Sea. The third (modern) pandemic began in southern China (purple clones on the figure). Not unsurprisingly it is difficult to trace these pandemics back to an endemic site since as a primarily rodent pathogen, Yersinia pestis can move without effecting humans.

The Black Death (1347-1352) draws all the attention because of its scope and scale, the amount of evidence, and the intensity of its legend. In some parts of the world, legend is nearly all we have (or have so far). Although the scientific evidence points toward an Asian origin for Yersinia pestis, there is precious little documentary evidence of it in Asia before modern times (17th century onwards).

George Sussman set out to examine the evidence of the Black Death in India and China in the current edition of the Bulletin of the History of Medicine. What he found in both enlightening and yet mystifying.

Western legends of the Black Death in the Far East go back to contemporary 14th century accounts of the plague in Europe and the Middle East [3]. Witnesses of the Black Death fueled by traveler’s stories imagined that all the known world was stricken, embellishing their writing accordingly. For the most part, modern historians have accepted their accounts of plague in China and India without scientific or historical evidence from China and India themselves. Sussman notes that McNeill’s influential Plagues and Peoples argues that plague foci in the Indian Himalayas and in central Africa are much earlier and more likely to be the source of the first plague pandemic (6th century) than the endemic strip along the Eurasian Steppe that McNeill dated to the 14th century [3]. Modern genetic diversity and biogeography points toward just the opposite with the eastern Asian steppe (Mongolia/N. China) being the original focus and the African focus dating to about the 14th-15th century [1]. There isn’t much evidence that the Indian Himalayan site is very old at all. We clearly need to learn a lot more about the Indian Ocean trade routes in Antique and Medieval periods to understand how the plague reached Pelusium in the 6th century and southern Africa by the 14th century.

So what evidence is there for plague in India before the third pandemic? During the 14th century northern and central India was ruled by Islamic sultans based in Delhi who kept close ties with the Central Asian peoples they came from and with the Middle Eastern centers of Islam [3]. They were well-connected diplomatically, economically and culturally with both Central Asia and the Middle East, areas that were both devastated by the Black Death and its successive waves. Yet there is no evidence of the plague in 14th century India [3]. Origins aside, this is strange, for there to be no record of plague even at ports makes me suspicious of the completeness of the written records. I would expect small local epidemics in ports, even if it couldn’t get traction in the countryside.  Sussman argues that the Indian subcontinent may have been the only area of Eurasia to have population growth during the 14th century [3].

Plague is unambiguously described in the Deccan of India in the early 17th century. It first came to the attention of Emperor Jahangir in Hindustan in 1616 [3]. The annals of the Emperor Jahangir record the third year of the winter plague with mad and dying rodents in January 1619 [3]. The annals include an interesting story of a cat contracting the plague from a mouse and passing it on to a girl who triggered a larger outbreak.

“After this the grain (dana) of the plague (a bubo) appeared in the girl, and from excess of temperature and increase of pain she had no rest. Her colour became changed—it was yellow inclining to black—and the fever was high (tap muhriq gardid). The next day she vomited and had motions, and died. Seven or eight people in that household died in the same way, and so many were ill that I went to the garden from that lodging. Those who were ill died in the garden, but in that place there were no buboes. In brief, in the space of eight or nine days seventeen people became travellers on the road of annihilation”.(Sussman, p. 337-338)

He is describing a case of secondary pneumonic plague that then spread throughout the household. The development of secondary pneumonic plague in a child can be especially damaging because more people will come to care for a child than an adult. In this case, mouse to cat to child to family doesn’t require any fleas at all. The cat got the plague from biting the sick mouse, the child got the plague from playing with the cat and passed it on to her family. The lack of buboes in the last people to die suggest that at some point in the transmission bubonic plague became pneumonic, probably in the child.

So plague is firmly established in 17th century India, but not in the 14th century during the Black Death pandemic. While I expect that we may yet find evidence of small outbreaks, there not does appear to have been a large epidemic. Why that was is unknown. Perhaps a combination of geographic isolation, climate, vector availability and sheer luck.  Turning to neighboring China, the picture becomes more complicated.

As I’ve already mentioned, Yersinia pestis genetic diversity and biogeography suggests that it has been in northern China long before any of the pandemics. With the wide-spread of early clones, the pandemic does not necessarily have to begin where there is the greatest genetic diversity.

Sussman notes that in the third pandemic the large northern outbreak was marmot-derived pneumonic plague while the southern outbreak was rat-derived bubonic plague [3]. This is still the case today. A marmot derived outbreak of pneumonic plague occurred in northern China as recently as 2009. If plague in northern China is usually pneumonic plague from marmots, I’m not surprised that they did not have a specific name for the disease. Pneumonic plague does not produce unique enough symptoms to differentiate from other rapidly lethal respiratory diseases.

The Yuan dynasty controlled China and Mongolia during the first half of the 14th century. This period coincided with a concerted withdrawal from the greater Mongolian world most of whom had by this time converted to Islam [3]. It was a time of great turbulence: famines, epidemics, natural disasters, political unrest, as the last remnants of the Mongol empire in China devolved to regional warlords and the Ming Dynasty began to develop [3]. Record keeping during this devolution is sporadic and uneven, but it does show three rounds of massive epidemic in 1330- 1350 each taking over 60% of at least regional populations. Unfortunately medical descriptions of the disease(s) have not survived [3]. Sussman’s analysis of the overall Chinese population during the early to mid 14th century is that the losses are comparable to the 25-30% loss in Europe that is directly credited to the Black Death [3]. Given the ancient foci of plague in northern China, this is where we should expect it to come from in the 14th century, and so it does appear to. On the other hand, Sussman notes that the first obvious medical description of plague in China dates to 1644.

Sussman questions whether the 14th century epidemics were plague based on some questionable criteria. He is bothered by the apparent lack of spread of the epidemic to the southwest (where the third pandemic began). He thinks a ‘virgin territory’ epidemic in densely populated China should have easily spread throughout China as it spread throughout Europe.  The European pandemic was the unusual behavior for the plague, not a regional epidemic in China where plague was more ancient than in Europe. In other words, I don’t think that Europe and China were equally ‘virgin territory’ epidemics. The importance of ‘virgin territory’ is probably also being over estimated for Yersinia pestis. Also the terrain in southwestern China is unlike northern China or Europe; it is more tropical. We need to let go of the idea that the second and third pandemic must behave the same. With a sample set of only three pandemics, we surely can not say that there must be one pattern that they will all conform. The lack of medical description also makes Sussman question if it was the plague. However, there is apparently no medical description at all to rule plague out or in. He also finds it unlikely that the plague could have traveled the length of the Eurasian steppe because much of it is so sparsely populated. Yet the first epidemic in northern China occurs in the early 1330s, surely enough time to travel the Silk Road west by caravan or Mongol horsemen. It is also possible that this clone spread along the steppe over several decades or even a century before it erupted at multiple points into large epidemics where the conditions were right and into a pandemic in the west.

Noticeably absent in this discussion is archeological evidence in either India or China. Now that we can identify Yersinia pestis aDNA in remains, hopefully this could be investigated in at least northern China. Unfortunately, I rarely hear about any medieval archaeology from India or China.

World Biomes (click to enlarge)

Plague’s normal biome is semi-arid grassland, shown on this simplified biome map as brown and yellow. From Sussman’s information it appears that the Black Death avoided tropical rainforest biomes (light green). This is not really surprising given its endemic regions. It is the opposite of the third and weakest pandemic. The endemic foci produced by the third pandemic are the usual semi-arid grasslands in the American south-west, Madagascar (which has some savanna), and Brazil.

So in conclusion, what are we left with? First, western reports of plague in the east may be more rhetoric than reality. Even if there were small unrecorded outbreaks in India, there doesn’t seem to be much evidence of population decline. For China, it would help to have more evidence of the nature of the northern epidemic. However, the coincidence and lethality of the epidemic support it being the plague. There is still a lot of work to be done on plague history in southern Asia.

References:

[1] Morelli G, Song Y, Mazzoni CJ, Eppinger M, Roumagnac P, Wagner DM, Feldkamp M, Kusecek B, Vogler AJ, Li Y, Cui Y, Thomson NR, Jombart T, Leblois R, Lichtner P, Rahalison L, Petersen JM, Balloux F, Keim P, Wirth T, Ravel J, Yang R, Carniel E, & Achtman M (2010). Yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity. Nature genetics PMID:21037571

[2] Li Y, Cui Y, Hauck Y, Platonov ME, Dai E, Song Y, Guo Z, Pourcel C, Dentovskaya SV, Anisimov AP, Yang R, & Vergnaud G (2009). Genotyping and phylogenetic analysis of Yersinia pestis by MLVA: insights into the worldwide expansion of Central Asia plague foci. PloS one, 4 (6) PMID: 19543392

[3] Sussman GD (2011). Was the black death in India and China? Bulletin of the history of medicine, 85 (3), 319-55 PMID: 22080795