Category Archives: molecular biology

Microbial Forensics of a Natural Pneumonic Plague Outbreak

For bioterrorism agents like Yersinia pestis it is necessary to identify the strain and its source specifically enough for forensic use. Categorizing an epidemic isolate and tracing its source is always important for public health measures, but the level of precision is far higher for legal uses. Developing forensic techniques to characterize and parse very similar strains of a species and trace it to a specific location robs terrorists (and states) of the ability to deny responsibility for an attack (Koblentz & Tucker, 2010). The ability to launch a secret and deniable attack on an enemy has been viewed as one of attractive advantages of biological warfare.

A Chinese group led by Ruifu Yang and Yujun Cui recognized that only whole genome sequencing could adequately parse the strains of the monomorphic species Yersinia pestis but that the computing power necessary to compare entire genomic sequences as the database enlarges is impractical (Yan et al, 2014). Unlike most pathogens, typing only specific regions of the genome are just not enough to get a unique genetic fingerprint for low genetic variability pathogens like Yersinia pestis. This is yet another indication of the genomic similarity of all Yersinia pestis strains.

The Chinese group developed a two stage method of classification detailed enough for forensic work.  They took a twelve person outbreak of pneumonic plague contracted from a dog in 2009 in the Qinghai area of Tibet / western China, specifically at Xinghai as their test case (Wang et al, 2010). In the first step they took six cases including the two dogs who died in the outbreak and compared them to 24 strains representing the 23 phylogroups of the phylogenetic tree. This comparison selected which branch of the phylogenetic tree the outbreak belonged. There were no SNP (single nucleotide polymorphisms) different between the seven isolates confirming a common source, one of the dogs based on outbreak narratives. The seven isolates were all the same strain belonging to branch 1.IN2 of the tree. The second step was to then compare the isolates to all known strains of 1.IN2 shown below. Since these strains all come from the Qinghai-Tibetan plateau, they were able to add other strains historically isolated from this region.

Distribution of 1.IN in Qinghai  (site source)
Distribution of 1.IN2 in Qinghai (Yan et al, 2014, click to enlarge)

The results localized the new isolates (r) as being from the same focus as strains g, r, s, t. u plus, interestingly, the 0.PE7 strain (green b) that is over 300 SNPs different from the 1.IN2 strains. All of these other strains from this branch are scattered around the Qinghai region near Lake Qinghai. The polysomy (branch point) that produced all of the 1.IN2 in Xinghai (g,r,s,t,u) is located closer to the eastern end of Lake Qinghai, where the Chinese team hypothesizes this these strains began. The new outbreak isolates did not match any previous isolates from Xinghai which is testimony to the degree of movement of these strains around the region. Without the case narrative, they would not have been able to identify the specific foci at Xinghai, but would have got it to the region of east Qinghai lake. This illustrates how important sampling all of these foci are because a biological attack is likely to be far from its site of environmental isolation. Characterization of all laboratory strains, obviously, needs to happen as well for forensic tracing.

Reconstructing the historical epidemiology of this region will be an area of continuing research. The location of 0.PE7, the most genetically ancestral strain ever found — the closest the common ancestor of all Yersinia pestis, plus the likelihood that the ‘big bang’ epidemic (or epizootic), that produced the third pandemic, represented by node 12, was also in this region. (Each of the nodes represents a bang of evolutionary diversity, with all major branch points in the lineage probably representing large epidemics or epizootics.) The full diversity of strains in this region (unrelated to the outbreak isolates) are not shown in the figure above. This same group lead by Ruifu Yang  produced the primary phylogenetic tree of Yersinia pestis in China that noted that the molecular clock is not constant (Cui et al, 2012), here calculates that N12 is about 212 years old (95% confidence being 116 to 336 years ago) (Yan et al, 2014).  They note that in the history of Qinghai, there was a major human outbreak in the year 1754 CE linked to a Buddhist missionary working in Qinghai and Gansu provinces (Yan et al, 2014). Its is unclear if we can trust this narrative at all; scapegoats are common in plague narratives. Linking the 1.IN2 strains from Qinghai to four of the five o.IN2 isolates from Tibet suggest that the epidemic moved from Qinghai to Tibet in one ancient epidemic, though remaining isolate from Tibet looks like a more recent transmission from Qinghai. Regardless of the movements of 1.IN2, this area is believed to have been a site of long-term survival of Yersinia pestis, potentially over a thousand years, so that it has a lot to teach us about enduring foci.

Microbial forensics has already been used in criminal investigations, court cases and intelligence operations, such as the ‘Amerithrax’ (anthrax) attacks of 2001, anthrax spores sprayed over Japan by a cult, and suspicious plague cases in New York City (Yan et al, 2014). Phylogenetic microbial forensics was successfully used to show the intentional transmission of HIV from Dr Richard Schmidt to his girlfriend in his 1998 trial. This was the first successful use of microbial forensics in a court case (Koblentz & Tucker, 2010). In these cases, isolates are taken from the accused, the victim, other sexual partners, and the local population so show phylogenetic linkage between the accused and victim in the context of the local epidemiology.  The United States, United Kingdom, Sweden, the Netherlands, Japan, Canada, Germany, Australia, Singapore, and now China are involved in the development of microbial forensics (Koblentz & Tucker, 2010; Yan et al, 2014).

Reference

Koblentz, G. D., & Tucker, J. B. (2010). Tracing an Attack: The Promise and Pitfalls of Microbial Forensics. Survival, 52(1), 159–186. doi:10.1080/00396331003612521

Yan Y, Wang H, Li D, Yang X, Wang Z, et al. (2014) Two-Step Source Tracing Strategy of Yersinia pestis and Its Historical Epidemiology in a Specific Region. PLoS ONE 9(1): e85374. doi:10.1371/journal.pone.0085374

Wang, H., Cui, Y., Wang, Z., Wang, X., Guo, Z., Yan, Y., et al. (2010). A Dog-Associated Primary Pneumonic Plague in Qinghai Province, China. Clinical Infectious Diseases, 52(2), 185–190. doi:10.1093/cid/ciq107

Cui, Y., Yu, C., Yan, Y., Li, D., Li, Y., Jombart, T., et al. (2012). 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/-/DCSupplemental/sd01.xls

Historians Chronicling Plague Genetic Discoveries

After my last post critiquing Cohn’s scientific interpretations, I think its only fair to write about all the historians who are actively engaging and incorporating scientific findings in their work. I’ve communicated with a lot of historians who are following the scientific work on the plague and I know there will be some articles and books coming out over the next year or so that incorporate some of new genetics in historical analysis.

So for science folks, these two articles give us some insight into how historians see plague genetics unfolding. Little concentrates on the early drama over plague genetics. Bolton covers that material also, but also looks at newer information on transmission dynamics too.

Little, L. K. (2011). Plague Historians in Lab Coats. Past & Present, 213(1), 267–290. doi:10.1093/pastj/gtr014

Bolton, J.L. ‘Looking for Yersinia pestis: scientists, historians and the Black Death’ in L. Clark and C. Rawcliffe (eds.), Society in an Age of Plague, The Fifteenth Century XII (Woodbridge: Boydell, 2013), publication date 15 August 2013, ISBN 9781843838753. (In the same book/issue as Cohn’s paper discussed in the last post.)

Overall, I am really optimistic about the interdisciplinary work that can be done on the plague.

Academic Plague Identity Wars Continue

Just when you think the academic wars over the identity of the medieval plague are over, another volley is cast by Samuel Cohn. In the past I haven’t mustered the energy to respond to his papers and books because there are just so many scientific misunderstandings, but its time to respond. Obviously, scientific studies that cover all the bases aren’t enough, so for now I’ll to correct some of his misinformation (leaving most of his historical analysis to the historians to critique).

Samuel K. Cohn, Jr. (2013) “The Historian and the Laboratory: The Black Death Disease” pp. 195- 212 in Society in the Age of Plague. The Fifteenth Century XII. Clark, L and Rawcliffe, C. Eds. Boydell Press.

Cohn portrays the discovery of Yersinia pestis at the turn of the 20th century as yet another French vs British competition in a partisan British manner. While trying to undercut the importance of French findings, he does not include the political context of Victorian India and particularly English trade interests in his discussion of the Indian outbreak. He does blame harsh British tactics in India on their recognition that Yersinia pestis was the pathogen (which he questions). This whole section needs a good going over by an modern historian.

In his criticism of the aDNA work, he claims that most researchers had “negative findings” but Gilbert et al is the only paper he cites that really failed to find plague. If he is referring to the number of negative specimens, then he doesn’t understand how rare the survival of aDNA is in general and the importance of the more sensitive protein methods. Like most of the press, he makes way too much out of the observation that the East Smithfield isolates represent an ‘extinct’ clade. It would be far more surprising if a Black Death isolate were identical to a modern strain. Evolution does not stop, especially not in the accumulation of polymorphisms (neutral mutations). He ignores the fact that the Third Pandemic strains are descendants of the Black Death isolates. He doesn’t seem to understand that genetic diversity is produced in every epidemic and that most of it is lost at the end of the epidemic. This is especially true of Yersinia pestis diversity generated in humans because it must be transmitted to a reservoir species to be preserved.

Not for the first time, I wonder if he understands what natural immunity is and he uses references from the 1950s or before on human immunity to the plague. All medieval immunity is natural. They did not have the means to generate artificial immunity. Natural immunity can be passive (mother to child) or active (generated after exposure).   He misrepresents Li et al (2012) as an indication that immunity is short-lived when in fact the study shows the antibody response is strong (69.5% at 10+ years) and correlated with the strength of their response at the time of the initial infection. Just because we are having problems generating a vaccine that can repel pneumonic plague doesn’t mean that a response generated against an active infection couldn’t repel bubonic plague.  It takes a much stronger response to cope with an aerosol exposure.  When plague epidemics are coming about every 10-15 years, an immunity that lasts 10-20 years would be enough to produce an age differential in the mortality rate. I will leave his analysis of the mortality from historic sources to historians to comment upon. While childhood mortality rates in plague epidemics are clues toward immunity, they are only one variable in comparing the epidemics. We also have to look at what else is occurring among the children such as normal childhood mortality, co-infection and other co-morbidity.

He makes the leap of logic that decreases in total mortality rates equals changes in human immunity. There are many variables that effect the intensity of an epidemic. Decreases in human mortality may suggest changes in the rodent population and/or rodent immunity. If epidemics occur too closely spaced the rodent population will not have recovered enough to generate a large outbreak. Of course, other environmental changes can alter the rodent population and exposure of humans to rodents.

He makes assertions on vector transmission that are not referenced and uses a reference from 1913 (!) to assert that the septicemia in humans is not high enough to allow human-to-human flea transmission. He seems to be assuming that transmission would need to be accomplished by a single flea or louse, which is unlikely. He gives no reference for his assertion that the bacterial load in plague is lower than insect vector transmitted typhus or Lyme disease. He seems to think that only one vector could be at work in the second pandemic rather than rat fleas, human fleas, and lice all transmitting Y. pestis in the same epidemic. Pathogens will take any opportunity available to transmit.

He starts reaching for straws in the conclusions:

There are statements like this: “The ancestor of this family, Yersinia psuedotuberculosis, which geneticists argue gave birth to this new strain of Yersinia, perhaps as late as the eve of the Black Death” (p. 210) Yersinia pestis is not a new strain of Yersinia pseudotuberculosis! It is a species in its own right. A strain is a distinctive subpopulation of a species.  Emerged as late as the eve of the Black Death? Nonsense. There is the little thing of the Plague of Justinian about 800 years earlier, with aDNA and protein evidence. This emergence involving genome rearrangement, loss of genes, gain of chromosomal genes and plasmids would likely have taken at a minimum centuries before 541.

“Could an earlier variety of the ancestor Yersinia suddenly have developed pathogenic factors such as plasmids or, on the level of protein biosynthesis, abilities form a capsule or to release endotoxin, thus suddenly transforming the benign pseudotuberculosis into a new and vicious pathogen, but without diminishing its ability to spread effectively from person to person?” (p. 211)

This one is easy…. NO! Bacteria do not suddenly develop plasmids; they acquire them from other species. In Y. pestis’s case, all of these plasmids are significantly modified from the ancestral plasmids they received. It also takes more than one gene or even plasmid to produce Y. pestis virulence from Y. pseudotuberculosis. He seems to also be implying that a change to increased virulence in humans is the species differentiating event for a primarily rodent pathogen.  Then he strangely follows this (a few sentences down) with the speculation that the “modern bacillus may actually be more toxic than that of the pathogen of the historic plague.”(p. 211) Huh? What happened to his speculation above that “a new and vicious pathogen” was at work?

“As regards Black Death and the ‘Third Pandemic, when and by what criteria does ‘a strain’ of a pathogen come to be reckoned as the causal agent of another ‘disease’, which has to be classified differently from that caused by a related pathogen of the same genetic family, as is currently recognized in the case of Yersinia pestis and its older relative, Yersinia pseudotuberculosis? Even if scientists thought that a pathogen is the equivalent of the disease it in part causes, that is the only pertinent defining feature? Even if scientists thought that the pathogens of the ‘Second’ and ‘Third’ Pandemics were identical (and now they do not), should we then return to the strict reductionism of Koch circa 1890, that a pathogen is the equivalent of the disease it in part causes, that it is the only pertinent feature?” (p. 212)

What? Now we have to reargue germ theory? Pathogens can have different presentations and different epidemic dynamics; some transmit by a variety of means. Co-infections and other co-morbidities certainly matter, but you don’t have the disease without the pathogen.  This is not a type of disease like pneumonia where multiple pathogens cause similar effects. Cohn is grasping at straws and bending scientific concepts to suit his purposes.