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Plague cases reported by country to World Health Organization (WHO)
from 2000 to 2009. Source
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The causative agent of the Plague of Justinian (542-546 AD), the Black Plague (1347-1350), and a third pandemic of plague in Asia (1850-1935) may be in the belly of a flea that’s ready to grab its next blood meal from your dearest pets or even you. According to the CDC, in the United States, 999 human plague cases occurred between 1900 and 2010. Many more cases were and are still seen throughout the world.
Who’s the culprit? The bacterium Yersinia pestis, with its unwitting accomplice, the flea. A flea commonly associated with the plague is Xenopsylla cheopis, the oriental rat flea. Though it’s named for its tendency to feed on rodents, the rat flea may venture to other mammalian hosts, such as humans and common house-pets like cats and dogs. Dogs aren’t likely to become sick from plague but cats, like humans, can contract plague from Y. pestis-infected fleas and subsequently spread the pneumonic version of the plague through coughing. Lucky for people and their pets, the plague is not anywhere near as threatening as it was in the past. For instance, we don’t have to rely on techniques, such as bloodletting and medicinal leeching, used by doctors of the Middle Ages (pictured on the right). The plague is now treatable with common antibiotics.
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| "Doctor Beak of Rome." This engraving by Paul Fürst depicts the leather clothing and beak-like mask worn by plague doctors during the Black Plague pandemic. Source |
Of course, it is still important to understand transmission so infection can be avoided in the first place and so the use of antibiotics can be kept to a minimum. Thus, experiments take place to investigate Y. pestis in various flea species.
Much of the research on this bacterium investigates Y. pestis interactions with the oriental rat flea, a big player in plague transmission. However, Y. pestis can be spread by other flea species too. This spreading can occur through various mechanisms, including early-phase transmission and regurgitative transmission (Vetter et al., 2010; Hinnebusch, 2012). Early-phase transmission occurs when a flea ingests the blood of a Y. pestis-infected host and then, shortly after, feeds on another host, thereby spreading the bacteria. Regurgitative transmission occurs when the blood of an infected host causes the Y. pestis bacteria to form a blockage in the flea’s foregut (also known as the proventriculus); this starves the flea and causes it to seek out a new host, which it then infects by regurgitating a significant amount of bacteria.
The blockage of the fleas foregut is accomplished by the formation of Y. pestis biofilms, collections of bacteria held together by components of their extracellular matrix. It is known that in rat fleas Y. pestis are capable of forming biofilms that cause regurgitative transmission, but, until a study in January 2014 by Tam, et al. little was known about how, or if, Y. pestis could form and sustain these important biofilms in the guts of Ctenocephalides felis, cat fleas (fleas that can feed on house pets, rodents and humans).
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| C. felis, as seen by electron microscopy, next to its primary host. Source and source |
To investigate this biofilm formation Tam, et al. infected cat fleas with Y. pestis and found that biofilms do form in the proventriculus. Studies involved specific Y. pestis genes, including two from the four gene operon, hmsHFRS, as well as three genes, ypo2150, ypo2458 and ypo3682 that each encode a LysR-type transcriptional regulator. Of the the latter three only ypo2150 was found (through testing colonization of mutant strains in fleas and in vitro cultures) essential for biofilm formation. ypo2150 was thus deemed yfbA (Yersinia pestis flea biofilm regulator A). As for the hmsHFRS operon, one of its gene products, poly-(β1-6)- N-acetylglucosamine (PNAG), is an extracellular polysaccharide involved in the formation of Y. pestis biofilms (Erickson et al., 2008). By testing for colonization of hmsF mutants and hmsR mutants, it was established that both genes help determine biofilm formation in cat fleas. The necessity of the hmsHFRS operon for biofilm formation had already been shown in oriental rat fleas (Hinnebusch, 2012).
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| Biofilm formation in gut of cat fleas infected with wild-type, hmsR mutant, yfbA mutant, yfbA (pyfbA) mutant. Arrows depict biofilm formation. |
Shown below is the formation (or lack there of) of biofilms in cat flea proventriculi by wild-type Y. pestis and hmsF mutants. The bacteria were modified to express GFP by the insertion of pGFP. Hardly any green fluorescence can be seen after seven days (d7) in fleas infected by hmsF mutants (A-C), indicating that substantial Y. pestis biofilms were not present. Contrastingly after only three days (d3) green fluorescence can be seen in fleas infected by wild-type Y. pestis (D-F) and the green fluorescing biofilms are even more apparent seven days after infection (G-I). The lack of biofilm formation in images A-C suggests that the hmsF gene is important in Y. pestis colonization of the gut.
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| Biofilm formation in gut of cat fleas infected with GFP-expressing wild-type, and GFP-expressing hmsF mutant Y. pestis. d7 denotes 7 days after infection, and d3 denotes 3 days after infection.) As a control, scanning electron microscopy images of the proventriculi of fleas infected with wild-type Y. pestis were compared to the proventriculi of uninfected fleas (J,K). Biofilm formation was observed in the wild-type infected fleas and not in uninfected. |
The experiments revealed not only that Y. pestis is capable of colonizing in cat fleas, but also that yfbA, hmsR and hmsF are required for colonization and biofilm formation in cat fleas. A previous study suggests that cat fleas have a low potential to transmit Y. pestis from one host to another. Tam, et al. referred to this study and noted that in their own experiments around 50% of infected fleas developed Y. pestis biofilm blockages in their proventriculi, possibly hinting that regurgitative transmission may be more feasible than previously thought. Tam et al proposed further investigations into transmission by cat fleas due to the distribution of this flea species throughout the world, its tendency to seek domesticated animals and humans as hosts, and its ability to survive off of hosts, making it a possible off-host vector for Y. pestis. Future studies may also examine how YfbA, the LysR-type transcriptional regulator, influences biofilm formation, by investigating what gene(s) the regulator controls.
Notably, studies on Yersinia pestis are particularly relevant, because of the recent outbreak of plague in Madagascar, with 119 cases recorded since August. 98% of those cases were of the bubonic form, spread by fleas. The more we study Y. pestis and the fleas that act as vectors for transmission of plague to humans, the closer we will get to combating the disease and truly leaving pestis in the past.
Kenesha, Erica, and Judene
References
Tam C, Demke O, Hermanas T, Mitchell A, Hendrickx APA, Scheewind O. (2014). YfbA, a Yersinia pestis Regulator Required for Colonization and Biofilm Formation in the Gut of Cat Fleas. Journal of Bacteriology. 196(6), 1165-1173.
Erickson DL, Jarrett CO, Callison JA, Fischer ER, Hinnebusch BJ. (2008). Loss of a Biofilm-Inhibiting Glycosyl Hydrolase during the Emergence of Yersinia pestis. Journal of Bacteriology. 190(24), 8163-8170.
Hinnebusch BJ. (2012). Biofilm-Dependent and Biofilm-Independent Mechanisms of Transmission of Yersinia pestis by Fleas. In A.M.P. de Almeida and N.C. Leal (Eds.), Advances in Yersinia Research (237-243). New York, NY: Springer.
Vetter SM, Eisen RJ, Schotthoefer AM, Montenieri JA, Holmes JL, Bobrov AG, Bearden SW, Perry RD, Gage KL. (2010). Biofilm formation is not required for early-phase transmission of Yersinia pestis. Microbiology. 156(7), 2216-2225.
Wheeler CM, Douglas JR. (1945). Sylvatic Plague Studies: V. The Determination of Vector Efficiency. The Journal of Infectious Diseases. 77(1), 1-12.
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