Malaria Near the Arctic Circle

Malaria study area (Hulden et al, 2005)

When I think of Finland, malaria just doesn’t normally come to mind. Although northern climes often have swarms of mosquitoes, its hard to imagine mosquito-borne infections gaining much traction in the short summer season. Yet defying imagination, malaria has thrived in northern Finland, Sweden and Russia near the arctic circle in the past. In the late 19th and early 20th century, Plasmodium falciparum and Plasmodium vivax caused outbreaks in northern Europe. Despite the outbreak of P. falciparium at Archangelsk in the 1930s, P. vivax is believed to be the primary malarial species in northern Europe.

Finnish researchers Lena Huldén, Larry Huldén, and Kari Heliövaara focused on the 1800-1870 period in southern Finland as having the ideal demographic, medical and temperature records before the advent of quinine to study malaria transmission in cold climates.

Medical records are available for Finland from annual reports of ‘district physicians’ and local ministers for most of the 19th century. Doctors were stretched thin across Finland but in the fifty years between 1826 to 1870 there were 542 reports of malaria. Ministers were required to record the cause of death of their parishioners from 1749.  Digitization of parish records by the Finnish Genealogical Society has made this data available online for 1800-1850. Terms used for malaria were specific enough that general fever terms in the records did not correlate with malaria outbreaks or temperatures.

Malaria isn’t recorded in Finland until the 17th century, probably brought by migrant workers and gained traction among people gathered for summer infrastructure projects in southern Finland in the 18th century. Death records and physician reports indicate that during mid-19th century epidemics the mortality rate reached as high as 3% of the population with 7-20% infected. The worst epidemic occurred in 1862.

There are three Anopheles mosquito species found in Finland. All are believed to have been present in Finland since prehistory.  It had been thought summer temperatures of 16 C (60.8 F) were required to maintain endemic malaria, but malaria has been recorded areas in of northern Sweden and Finland that don’t reach 16C in the summer. Males die shortly after mating and female Anopheles must hibernate from late summer until well into spring. Therefore, the female spends most of its life indoors hibernating with humans and sheltered domestic livestock. The female will take sporadic nocturnal blood meals over the winter but won’t lay her eggs until spring.

Huldén,  Huldén, and Heliövaara correlated malarial deaths with annual, seasonal and monthly temperatures. The only significant correlation occurred between summer temperatures of the previous year, but not at all with annual or seasonal temperatures of the same year. Malarial deaths peaked in the spring rather than the usual late summer or autumn. So how does this work with a temperature correlation to the previous summer? Winter infections. The previous summer temperatures effect how many mosquitoes will be hibernating over the winter in homes. Sporadic blood meals over the winter in the confined space of the home spreads the infection to most of the humans and other hibernating mosquitoes causing infections that peak in the spring. Humans are the primary reservoir of infection in cold climates. It doesn’t matter that the malarial sporozoites won’t mature outdoors during the cool summer because they will mature in the cozy confines of winter homes. Fatal spring infections in 40-50 infants born in the winters from 1750 to 1850 supports the theory that the female mosquitoes were capable of transmitting malaria for the entire winter.

Age distribution among the malarial deaths was very similar to the total population indicating that all ages groups were equally vulnerable to infection. Huldén,  Huldén, and Heliövaara interpret this as indicating infection occurred at a time when the entire family would be together in heated buildings, in the winter rather than in summer when  occupations cause families to live apart by age and gender often in unheated buildings.

Epidemiological data can usually be explained but not necessarily predicted. They have provided another example of why epidemiology can’t always be definitive in ruling in or ruling out the diagnosis of a historic epidemic. Based on outdoor temperatures malaria should have never been endemic in Finland at all. This study highlights the importance of the indoor environment for malaria (and other zoonoses).

Reference

Huldén L, Huldén L, & Heliövaara K (2005). Endemic malaria: an ‘indoor’ disease in northern Europe. Historical data analysed. Malaria journal, 4 (1) PMID: 15847704

Black Death Genome Fished Out of East Smithfield

Fishing just isn’t what it used to be, and neither is DNA sequencing. Reconstructing the ancient plague genome required the development of new technology that was able to enrich the sequencing sample by concentrating the Y. pestis sequence fragments from the brew of human DNA and contaminants in all aDNA extracts.

Using an Agilent Capture Array (above), a large international group led by Johannas Krause and Hendrik Poinar [1] fished ancient, degraded fragments of Yersinia pestis out of bone and teeth extracts from the East Smithfield Black Death cemetery using lures (probes) composed of short fragments of a modern Y. pestis strain. These lures (probes) are attached to the slide and the extracts are washed over the slide. Complementary ancient fragments in the extract will hybridize with the single stranded probes while the remainder of the extract is washed away. (The match does not have to be exact for these probes to hang on to the extract fragment allowing them to pull out fragments with minor sequence variants.) The fragments can then be released from the slide and sequenced. Using two of these slides (one in Canada and one in Germany?) they captured over two million unique fragments. Overlapping regions of sequence were lined up to reconstruct “93.48% of the targeted regions” (complementary to the modern CO92 strain of Yersinia pestis). All genes of the modern Y. pestis strains appear to be accounted for, although the existence of any part of the ancient genome that is completely foreign to modern strains can not be ruled out.

When they analyzed the ancient sequence they found that it is surprisingly similar to modern Y. pestis strains. It differed from modern strains at only 97 positions all of which matched the ancestral genes from Yersinia psuedotuberculosis. The most important information in this paper is that the genes of the Black Death clones do not have significant genetic differences that would make the ancient clone(s) more virulent than modern strains.

They also went on to place their reconstructed genome on the phylogenetic tree of Y. pestis relative to public sequences of modern Y. pestis strains. They placed the East Smithfield Black Death clone at the node where modern Y. pestis strains branch from the ancestral stem leading to Yersinia pseudotuberculosis, its parent species. Actually it is the primary East Smithfield clone because they also found another derivative clone in the extracts from only four people at East Smithfield. Placing the Black Death strain at the branch point is basically the same finding as Haenesh et al, 2010 [2] that I’ve discussed in a previous post. Apart from having much more sequence to compare, the finding really isn’t new. It also doesn’t really tell us anything new about the earlier, first pandemic known as the Plague of Justinian. Despite their assertion that the Plague of Justianian was “distinct from all currently circulating strains commonly associated with human infections, or it was another disease all together” [1], they can not rule out that the Black Death strain itself is not identical to or a descendant of the Justinian strain. I see no reason to think that it was another disease. Two previous groups, Drancourt’s group in Marseille [3] and Wiechmann and Grupe in Bavaria [4], have found Yersinia pestis in 6th century remains.  Further, the speed, virulence, and signs and symptoms of the Plague of Justinian match descriptions of the Black Death.

The similarity of the Black Death strain with modern Yersinia pestis strains validates modern public health and biosecurity concerns over the plague. Although this ancient strain would be susceptible to modern antibiotics — if they are administered in time, we will need all the information we can get for a potential arms race with the plague.

[1] Bos, K., Schuenemann, V., Golding, G., Burbano, H., Waglechner, N., Coombes, B., McPhee, J., DeWitte, S., Meyer, M., Schmedes, S., Wood, J., Earn, D., Herring, D., Bauer, P., Poinar, H., & Krause, J. (2011). A draft genome of Yersinia pestis from victims of the Black Death Nature DOI: 10.1038/nature10549

[2] Haensch, S., Bianucci, R., Signoli, M., Rajerison, M., Schultz, M., Kacki, S., Vermunt, M., Weston, D., Hurst, D., Achtman, M., Carniel, E., and Bramanti, B. (2010). Distinct clones of Yersinia pestis caused the Black Death. PLoS Pathogens, 6 (10)

[3] Drancourt M, Signoli M, Vu Dang L, Bizot B, Roux V, Tzortzis S, et al. Yersinia pestis Orientalis in remains of ancient plague patients. Emerg Infect Dis [serial on the Internet]. 2007 Feb [date cited]. Available from http://wwwnc.cdc.gov/eid/article/13/2/06-0197.htm

[4] Wiechmann I, & Grupe G (2005). Detection of Yersinia pestis DNA in two early medieval skeletal finds from Aschheim (Upper Bavaria, 6th century A.D.). American journal of physical anthropology, 126 (1), 48-55 PMID: 15386257

DNA of the Black Death at East Smithfield, London

It seems as though every couple of months a new paper is published reporting Yersinia pestis DNA from ancient remains. This week brought the latest installment from London’s East Smithfield Black Death cemetery. This cemetery holds a special place in the scientific investigations of the Black Death because it is so well documented as being specifically for the first wave of plague in 1348-1350 and the  recovery of so many well-preserved skeletons. This cemetery has been the subject of several bioaracheological studies, primarily by former plague skeptic Sharon DeWitte, making this one of the best characterized set of Black Death victims yet to be discovered. DeWitte is also one of the co-authors of this study.

Using a new method of ‘targeted enrichment’  and high through-put sequencing an international group led by Hendrik Poinar was able to clone and sequence relatively long stretches of Yersinia pestis DNA from recovered remains. They were given access to 100 samples (53 bones and 47 teeth) from which they found 20 positive results for Y. pestis. Unfortunately they don’t indicate how many individuals these samples represent. Although the bone yielded more aDNA, the teeth had far more positive results; 37% of teeth to only 5.7% of bones. Poinar’s group believes this is consistent with a blood-borne pathogen because the tooth pulp is more vascular than bone.

Poinar’s group has reconstructed more of the ancient genome than any group to date. They had the advantage of knowing that this burial pit was open for only a short time and specifically for plague victims. They worked under the assumption that all victims of the plague died from the same strain and were therefore able to construct a composite organism. They could not have made this assumption in a churchyard cemetery that could be open for centuries. They were able to reconstruct 99% of the pPCP1 plasmid (95% with five fold coverage) and showed that its sequence matches 11 of 14 known strains today. They were also able to reconstruct a portion of the pMT1 plasmid that contains genes for the F1 antigen and a small portion of the bacterial chromosome. The Black Death strain seems to be a variant of the Medievalis biovar, but its exact placement is unclear. This has led to premature claims that the Black Death strain is extinct. Given that they haven’t shown a single mutation/polymorphism that makes a functional change, there is no evidence yet that the medieval strain was intrinsically more virulent than modern strains.

They took great pains to close all of the possible technical questions. They obtained human remains from the cemetery of St Nicholas Shambles also in London that dates from about a century earlier to serve as negative controls. These 10 specimens remained negative throughout. They analyzed the types of DNA damage found in human mtDNA from both East Smithfield and St Nicholas Shambles, and compared the level and types of damage to the Y. pestis aDNA to prove the Y. pestis DNA was original to the remains. They followed all of the isolation and contamination prevention procedures recommended for aDNA and sent their clones to two independent high-throughput sequencing facilities to confirm the sequence. By comparing the sequences of the two facilities they were able to resolve DNA damage from true polymorphisms/point mutations.

Why is this study important? First and foremost, it confirms that a well-known Black Death burial pit was due to Yersinia pestis. They developed a method to reconstruct more of the ancient genome than has been done before that should improve our phylogenetic analysis of Y. pestis. They answered all of the technical questions  that should finally bring consensus on the cause of the plague. This does not mean that other pathogens didn’t co-circulate or that every epidemic labeled as the plague really was. Now its time to dig into the epidemiology and get to the really important questions!

Schuenemann, V., Bos, K., DeWitte, S., Schmedes, S., Jamieson, J., Mittnik, A., Forrest, S., Coombes, B., Wood, J., Earn, D., White, W., Krause, J., & Poinar, H. (2011). PNAS Plus: Targeted enrichment of ancient pathogens yielding the pPCP1 plasmid of Yersinia pestis from victims of the Black Death Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1105107108

DNA Detective Work Identifies Black Death Culprit, NPR: Science Friday, September 2, 2011. Ira Flatow interviews Hendrik Poinar and Michael McCormick. (podcast)

Ancient Remnants: Biomolecules in Paleomicrobiology

Ancient DNA is not the only method of detecting and identifying ancient pathogens. Survival challenges for ancient DNA place very real limitations on its usefulness and sensitivity as a detection method. The main advantage of aDNA is that it can be genotyped to compare with modern species. For archaeological purposes, other biomolecules may be detected better and cheaper than aDNA.

Sources of Ancient Biomolecules

Proteins and lipids can be extracted from a variety of materials. Bone tissue and especially teeth are still the primary source for non-nucleic acid biomolecules. Hair and mummified skin, muscle, and internal organs have all been successfully used to extract usable proteins. Both proteins and DNA can be extracted old paraffin embedded human tissues previously used for past histological examination. Frozen tissue can yield protein, lipid and DNA.

Materials and methods for nonnucleic acid biomolecule detection in ancient material (Fig 2, Tran, Aboudharam, Raoult, and Drancourt, 2011)

Methods of Detection

• Immunohistochemistry: Immunohistochemistry combines antibody detection of microbial components with cellular morphology. These methods are similar, if not identical to, standard immunohistochemical assays done on modern patient tissue. Although protein and tissue degradation can be a problem, immunohistochemistry has been done successfully on mummified tissue, detecting 16th century smallpox and syphilis.
• ELISA and Immunochromography: Extracted protein can be assayed by immunochromatography (potentially as a dipstick) or ELISA (enzyme linked immunosorbent assay). Both immunochromatography and ELISA has been used to detect Yersinia pestis‘ F1 antigen in late medieval and early modern specimens. Pusch et al (2004) does a good comparison of the sensitivity of Y. pestis F1 antigen immunochromography versus ancient DNA amplification on the same specimens.  Immunodetection methods have been used to detect Plasmodium falciparum  (malaria) in natural mummies from ancient Nubia and Egypt, as well as in skeletal tissue from the 16th century Medici family of Italy.
• High Performance Liquid Chromatography (HPLC) can be used to differentiate ancient lipids extracted from specimens. It has been used primarily for the detection of mycolic acids and mycocerosic acids found in the cell wall of Mycobacterium tuberculosis. Mycolic acids have been used to confirm tuberculosis in remains as old as 9000 years before present.
• Mass Spectrometry: Mass spectrometry has been successfully used on ancient proteins and peptides. Studies on collagen proteins have been able to distinguish ancient animal species and tuberculosis. Mass spectrometry can also identify microbial products like tetracycline identified and analyzed in Late Antique Nubian bones (Nelson et al, 2010).  Their analysis was able to prove that the tetracycline was deposited in the living bone tissue rather than infiltration from soil bacteria.
• Late Serology: Active immunoglobulins (IgG) have been extracted from ancient bones. When exposed to “pathogen-specific antigen” the antibody still reacts. Antibodies against syphilis reacted from extracts of ancient bones that also showed osteological lesions of syphilis and PCR confirmation of Treponema pallidum (Tran et al, 2011). I haven’t read any studies using this technique yet.
• ImmunoPCR: ImmunoPCR is a somewhat odd technique that detects protein, not DNA. The DNA is essentially an amplified reporter of the antigen-antibody reaction monitored by real-time PCR. I haven’t read any studies using immunoPCR yet either.

Any of these methods can be used in conjunction with aDNA analysis to confirm a diagnosis on an ancient specimen. A developing consensus for diagnosing ancient infectious disease calls for the identification of species specific aDNA and some species specific non-nucleic acid biomolecule. Like ancient DNA studies, consensus standards for non-nucleic acid biomolecules have yet to be agreed upon. Determining what the appropriate positive and negative controls are is vital.The entire field is still rapidly evolving and it is likely to take years to develop. The one thing we know for sure is that these new molecular techniques will revolutionize archaeology over the next generation.

Tran TN, Aboudharam G, Raoult D, & Drancourt M (2011). Beyond ancient microbial DNA: nonnucleotidic biomolecules for paleomicrobiology. BioTechniques, 50 (6), 370-80 PMID: 21781037.
[Review article] This review article is available free online.

Pusch CM, Rahalison L, Blin N, Nicholson GJ, & Czarnetzki A (2004). Yersinial F1 antigen and the cause of Black Death. The Lancet infectious diseases, 4 (8), 484-5 PMID: 15288817

Nelson ML, Dinardo A, Hochberg J, & Armelagos GJ (2010). Brief communication: Mass spectroscopic characterization of tetracycline in the skeletal remains of an ancient population from Sudanese Nubia 350-550 CE. American journal of physical anthropology, 143 (1), 151-4 PMID: 20564518

Hunting Pathogens in Siberian Permafrost Graves

Burial sites in Yakutia, Siberia in the Russian Republic. (Source: Theves et al, 2011)

The Yakut community of Eastern Siberia has gained some attention from anthropologists because it culturally stands out from other Siberian populations. Their Turkic language, unique burial practices, and horse-breeding culture is not native to Siberia. Recent genetic analysis of 58 bodies preserved in permafrost from the last five centuries and 166 current members of the community, along with historical records, has produced a well characterized Central Asian population. Crubézy et al (2010) report that this community originated from a small group of horse riders from the Lake Baikal region who settled as the northern most incursion into Siberia of the Steppe horse cultures. By the 15th century these male riders had intermarried with local Siberian women creating a genetic profile and community that has remained consistent from the 15th century to today (Crubézy et al, 2010).

Figure S1: Male grave from Boulgouniak 1 (Source: Theves et al, 2011)

Recent success at extracting DNA from bodies in the Siberian permafrost inspired an ambitious project to survey all of the microbes found in tissue samples from a small set of Yukat remains.  Contact with European Russian settlers exposed the Yukat to new pathogens. They were decimated by smallpox and measles carried by Russians in the 17th to 19th century. Thèves et al (2011) extracted DNA out of permafrost graves dating from the late 17th to 19th century to survey their microbiome.

Thèves et al (2011) collected samples from five sets of remains, two single graves of a mummified man (Boul. A, Fig. S1) and mummified woman (Boul B., Fig. S2) and a triple grave (OY A, B, C, Fig. S3) of skeletons. Teeth were collected from all five and mummified lung tissue samples were taken from Boul A and Boul B. They extracted  DNA from all specimens for human DNA analysis and microbial DNA screening.

They used a two-step process to identify pathogens in these five people. Bacterial species were initially identified by amplification of 16S rRNA sequences that have been characterized for each genus. This process identified many of the environmental microbes in the remains. Potential pathogens were further confirmed by amplification of  rpoB gene sequences that are more species specific. Each amplification product was cloned and sequenced. A 95% sequence match with sequences in the microbial databases was required for a match to be considered valid. They also amplified human DNA sequences (mtDNA, autosomal short tandem repeats (STRs) and Y chromosome STRs) to evaluate DNA quality, check for PCR inhibitors and confirm that these remains are consistent with the Yakut lineages.

Fig. S2: Boulgouniak. 2 grave of a Siberian woman enclosed in ice. (Source: Theves et al, 2011)

These graves are not exactly what I imagine when I think of remains in permafrost. As you can see from the photo to the left and even more so in the photos below, various levels of decomposition have occurred.When I think of permafrost graves, I think of a Mastodon coming out of the ice nearly completely intact. As we will see, not only do these pictures suggest significant thawing, but perhaps that the remains were waterlogged for much of their thawed time. I am a little puzzled why they chose these particular remains for this study, but we don’t always get the research material that we would like.  I really have to wonder if the skeletonized remains below are any better preserved than complete skeletons found outside of permafrost. Ice lined graves do not guarantee good DNA recovery.  OYC samples did not produce any human nuclear DNA and only environmental bacteria, mostly Clostridium species. Thèves et al (2011) suggest that all DNA in the OYC tooth was destroyed by bacterial invasion through a cavity in the tooth. It is unusual to use a tooth with cavities or traumatic damage for aDNA extraction.  Thèves et al (2011) simply state that this skeleton had cavities in the teeth. This is a good example of why teeth with cavities are unsuitable for aDNA extraction. From the five remains they got 67 usable clones that passed all of their quality control measures.

Figure S3: Multiple grave from Oyogosse site. (OY A, B, C) (Source: Theves et al, 2011)

The identity of the clones was established by comparing their sequences to the NCBI reference genomic database. The majority of clones were from environmental bacteria. This is particularly unsurprising considering they appear to have ground the whole tooth to powder for DNA extraction, rather than using only the protected dental pulp.  They identified many species common to Arctic and the Antarctic including Xanthomonas sp., Pseudomonas sp., Azotobacter sp. and Clostridium sp. An unexpected find in OYB was a single clone of Myobacterium marinum, a species usually found in fresh and salt water globally. Thèves et al (2011) suggest that M. marinum may be present in the permafrost environment. Considering that these remains thawed enough to decay down to skeletons, perhaps this indicates this grave was waterlogged.

Evidence of three pathogens were found. Shigella dysenteriae  16S rRNA was found in two out of three members of the Oyogosse site (OYA & OYB). Considering the OYC tooth only produced environmental microbial DNA, this could indicate that this multiple grave came from an outbreak of dysentery.  Streptococcus pneumoniae was found in OYB, although the match was only 92% and therefore below their threshold for confirmation. Further investigation with rpoB primers failed to amplify either S. dystenteriae or S. pneumoniae in either specimen and no further investigation was done on either pathogen.  Bordetella pertussis (Whooping Cough) was identified in Biol 1 (Fig S1 above) by 16S rRNA and the rpoB gene sequences. Thèves et al (2011) note that this is the first evidence of B. pertussis among the Yukat. No pathogens were identified in the female single grave (Fig S2, biol 2), although only its lung tissue was screened for pathogens.

Amplifying the universal microbial genes is probably the only way to find these common pathogens in an archaeological setting. There is no doubt that the process is more complicated that suicide PCR which looks for individual pathogens. I do wonder about the efficiency of detection when grinding the entire tooth to a powder. This type of work is also prone to contamination with environmental bacteria. It is also possible that the environmental microbes identified in remains could help reconstruct the environmental history of the grave, especially important for much older graves.

Thèves, C., Senescau, A., Vanin, S., Keyser, C., Ricaut, F., Alekseev, A., Dabernat, H., Ludes, B., Fabre, R., & Crubézy, E. (2011). Molecular Identification of Bacteria by Total Sequence Screening: Determining the Cause of Death in Ancient Human Subjects PLoS ONE, 6 (7) DOI: 10.1371/journal.pone.0021733

Crubézy E, Amory S, Keyser C, Bouakaze C, Bodner M, Gibert M, Röck A, Parson W, Alexeev A, & Ludes B (2010). Human evolution in Siberia: from frozen bodies to ancient DNA. BMC evolutionary biology, 10 PMID: 20100333

Tools of Paleomicrobiology

Studying ancient microbes requires creativity. Contamination and  preservation are the primary problems, dealing with limited and degraded tissues. We don’t find corpses in permafrost every day! Most of the time tissue is confined to bones and mummies kept in a wide variety of environments. This post will review some of the major tools I have found for investigating ancient microbes. I do not have experience working with ancient materials, so this is based completely on what I have read.

With all of these methods, not detecting the microbe does not mean that it’s not there. It may be simply too degraded for the method to have worked. These methods also can’t determine when the microbe entered the material.

Tools for hunting ancient microbes:

1. Sampling and cultures: Culturing ancient microbes is limited to specimens that have been kept frozen. It might be possible that some bacterial spores could endure long enough to culture (this being both a risk and boon).
2. Light Microscopy depends on cellular and tissue preservation. Since most bacterial stains rely on an intact cell wall, I am not very confident about the effectiveness of the standard stains. The gram stain becomes unreliable on old living bacterial cultures much less material that is centuries old. Light microscopy should be able to give at least preliminary identification of parasites like lice, ticks, fleas, worms, etc.
3. Immunology:
   

   Direct fluorescent antibody (DFA) of Yersinia pestis (CDC, PHIL #1918)

   Antibody stains of tissue sections for specific bacterial components like the Yersinia pestis fluorescent antibody stain to the right may be more effective than more traditional stains if preservation is good enough. It has been done for tissue stored in old paraffin blocks (the way  tissue that has been used for histological purposes is stored). However, loss of antigenic properties in decayed tissue makes immunohistological methods difficult on archaeological remains (Lepidi, 2008). Plague F1 antigen has also been detected from pulverized tissue with an antibody-based dipstick. Antigen detection has been showed to be significantly more sensitive than DNA  for Yersinia pestis (Carsten et al, 2004). This makes a lot sense when you consider the relative amounts of antigen vs. the specific region of DNA to be amplified. If this works for Y. pestis, it should also work for other species specific proteins. The presence of microbes in a population can also be  detected by a specific host response against it, essentially by detecting antibodies that can still bind antigen.

4. Electron Microscopy: Both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are useful for paleomicrobiology. The three-dimensional image produced by SEM can better identify the dried remains of parasites like the human lice identified among Napoleon’s soldiers (Raoult et al, 2006). TEM is particularly useful for finding viruses in fresh tissue; smallpox has been identified in a 16th century Italian mummy and old formalin fixed tissues (Lepidi, 2008). Syphilis was also identified in another Italian mummy by TEM (Lepidi, 2008). However, TEM is particularly sensitive to preservation conditions and the quality of the tissue. Molecular methods are more likely to securely identify bacteria and viruses.
5. Molecular methods: There are two basic types of molecular detection: detection of unique biomolecules specific to a species like the F1 antigen of Yersinia pestis, or analysis of microbial DNA. Microbes produce an array of biomolecules that are genus if not species specific such as tetracyclines produced by Streptomyces species. However, the primary molecular method is the amplification of specific microbial DNA. It can be done by looking for specific microbes, usually by the suicide PCR technique, or by attempting to amplify all bacterial species in a specimen by amplifying well characterized ribosomal genes, identifying species by their specific sequences. Both DNA-based methods have their pros and cons. I will come back to the DNA based methods in posts on the method and with specific examples in the near future.
6. Osteology – I don’t know much about osteology and paleomicrobiology sources don’t write about osteology as a tool of paleomicrobiology but it seems to me that there are indications in the bone of a few pathogens. To be fair, the bones reveal a minority of major pathogens. Tuberculosis, leprosy, and syphillis-like pathogens are the only agents I know of off-hand that leave distinctive bone lesions. (I have to chuckle every time I read that because there is no sign of these three diseases that the population seems healthy, or some similar gross exaggeration.) Otherwise, the bones can give non-specific signs of infection that can be followed up on by other methods.

So these are the basic tools I have come across in my reading. I’ll be back with discussions of some of the major considerations and things to look out for in paleomicrobiology projects soon.

References:

Drancourt, M., & Raoult, D. (2005). Palaeomicrobiology: current issues and perspectives Nature Reviews Microbiology, 3 (1), 23-35 DOI: 10.1038/nrmicro1063

Lepidi, H. “Histologic detection of past pathogens”, pp. 69-72 in Paleomicrobiology: Past Human Infections. D. Raoult & M. Drancourt, Eds. Springer, 2008.

Raoult D, Dutour O, Houhamdi L, Jankauskas R, Fournier PE, Ardagna Y, Drancourt M, Signoli M, La VD, Macia Y, & Aboudharam G (2006). Evidence for louse-transmitted diseases in soldiers of Napoleon’s Grand Army in Vilnius. The Journal of infectious diseases, 193 (1), 112-20 PMID: 16323139

Carsten M Pusch, Lila Rahalison, Nikolaus Blin, Graeme J Nicholson, and Alfred Czarnetzki. (2004) Yersinial F1 antigen and the cause of the Black Death. The Lancet Infectious Disease, 4, 284-284.

Trench Fever in German Mass Burial

Trench fever seems to be all the rage these days in paleomicrobiology. It seems as though every time Bartonella quintana is added to a panel of pathogens for aDNA screening its found at some level. So far its been found in in a tooth from 4000 before present, in late medieval Venice, 14th century France, and Napoleonic Europe.

Construction at the University of Kassel in Germany discovered a mass grave  revealing “most of the individuals had been males of the age classes juvenis and adultas“. Grumbkow et al (2011) report that local historians, anthropologists and medical examiners concluded that it was a military cohort that died of epidemic disease in the late 18th to early 19th century. They focused on a reputed outbreak of typhoid fever in the winter of 1813/14 that had been linked with regional epidemics started by Napoleon’s troops fleeing from the battle of Leipzig. However, Grumbkow et al (2011) do not present any evidence to directly link their sample of remains to any army that took part in the battle, nor the reasoning behind dating the remains to late 18th to early 19th century for that matter.

To investigate whether these people were victims of a typhoid fever outbreak, Grumbkow et al obtained samples of 18 skeletons for DNA screening. They note that typhoid fever was a common diagnosis for any fever that caused red spots, but could include typhus, parathyroid fever and trench fever. Therefore they screened DNA extracted from 16 femurs and 2 humeri for Salmonella species, Salmonella enterica typhi, Bartonella quintana, Rickettsia prowazekii, and for the bacterial 16S rDNA (ribosomal DNA). As you might have guessed, they only found Bartonella quintana in three bones, that is 16.6.%. This is roughly what Roualt et al (2006) found at Vilnius at 20% for Bartonella quintana.

However, I do have a few concerns with this paper. First, the work is incomplete. They mention in their discussion that they need to screen for more Bartonella quintana genes to confirm their results. This is especially important because the samples they did amplify were all 100% identical to the genebank sequences (making contamination more likely). They list the 16S rDNA primers along with the others but never present any data for these primers. They used diluted “bacterial-positive control DNA” as a control for PCR inhibitors from the soil, which may be what the 16S rDNA primers were used for but it is not explicit. Contamination concerns usually preclude the use of positive controls but they write that “amplification with positive controls and spiked samples always showed the expected results”. The near standard ‘suicide PCR’ protocol for aDNA was not used. Brief communication or not, more information is needed.

Grumbkow, P., Zipp, A., Seidenberg, V., Fehren-Schmitz, L., Kempf, V., Groß, U., & Hummel, S. (2011). Brief communication: Evidence of Bartonella quintana infections in skeletons of a historical mass grave in Kassel, Germany American Journal of Physical Anthropology DOI: 10.1002/ajpa.21551  [Epub ahead of print]