Paper Review: Early-phase transmission of Yersinia pestis by unblocked fleas as a mechanism explaining rapidly spreading plague epizootics written by Rebecca Eisen, Scott Bearden, Aryn Wilder, John Montenieri, Michael Antolin, and Kenneth Gage in the The National Academy of Sciences of the USA in 2006 (as found at pubmedcentral.com)

Background

Yersinia pestis is a gram-negative bacilli bacterium that is transferred with a flea vector to various other animals such as mice, prairie dogs, and humans. This bacterium is the one that causes the Bubonic plague and some studies suggest that it may also be the cause of European Black Death, being a prime reason for this study. This bacterium is considered to be one of the more harmful ones because it induces a blocked response of the vector so that it cannot digest food and starves, wanting to feed more and in doing so builds up a larger quantity of the bacteria in its system to transfer to another host. One previous study on prairie dogs found that Yersinia pestis could not cause an epizootic plague with blocked fleas due to the slight changes in the flea vectors used. Yersinia pestis can be transferred to the skin of a reservoir by contact with contaminated mouthparts and flea feces. This study looks primarily at transfer through flea feeding in vectors that do not block.

Normally studies use Xenopsylla cheopis as the flea vector as it is considered to be the most effective transmitter of the bacterium but it is known to be very susceptible to blocking which in many studies has been considered to be the only possible way for the plague to become epizootic. Oropsylla montana was used as the flea vector in this study because it rarely becomes blocked, allowing normal functions of digestion to occur with no buildup of the bacterium. A second problem with using the vector Xenopsylla cheopis was that it requires a long incubation period to become infectious and they die soon after becoming infectious. Oropsylla montana became infectious immediately after being allowed to feed for one hour and they stayed infectious for up to four days. This study looks at the effect of unblocked fleas to transfer Yersinia pestis to Swiss-Webster mice to determine if blocking is required for epizootic activity.

Model Organism

This study used the flea Oropsylla montana as its insect vector in this study. This was because it is an active transmitter of Yersinia pestis, the bacilli form of the plague being studied, into the Swiss-Webster mice. This flea was used because it is the vector that can introduce the plague to humans in North America. Oropsylla montana was the model organism for this study because it rarely becomes blocked, preventing normal function, when infected with the bacilli, being the focus of the study. They become infectious immediately and can remain infectious for up to four days so that there is ample time to study their transmission of the plague to other organisms.

As discussed above there are many advantages to use this flea than the normal model organism for flea plague transfer in this study. The normal vector used, Xenopsylla cheopis is considered to be a very good transmitter of the plague because it is blocked and the bacteria can build up rather quickly. This causes a problem though because they are not immediately infectious and must take a time to gain the amount of bacteria in their system so that they can infect others. They also do not live very long after gaining the proper amount of bacteria because the bacteria works on them and kills them off with larger concentrations in their system, also forcing them into starvation, killing them even quicker. Oropsylla montana does not have any of these problems and can live up to four days once infectious allowing this study to be carried out over a long period of time, testing at many different points to see the changes in effectiveness at different lengths of time the bacteria had to incubate in its vector.

Why study this?

Today, Yersinia pestis is still a worldwide threat, especially due to its bioterrorist threat because of its rapid spread through the population. It is a large worry of our population to see if an outbreak of the Bubonic or Black Death plague will break out. It is a large fear of the government that it will be used as a biological weapon that we have no way to fight. By studying how this disease is transferred from vector to reservoir we will be better prepared to fight a battle if the bacterium are introduced and become epizootic in our population in the United States. There are places in California where ground squirrels and rock squirrels quickly catch this plague and become epizootic.

We know that it takes a long time for the flea to be able to transmit the disease when the flea is not functioning properly, when its function is blocked, but some how the disease still spreads just as quickly, much quicker in an actual population than in lab studies. It is not very common that fleas will become blocked but that is the main point of study that has been focused upon due to the elevated concentrations of the bacteria that is caused by the formation of a block in the flea’s proventriculus. Because when fleas are blocked it takes a longer time for them to become infectious it is important that in Oropsylla montana it is looked to see if infections at early-phase transmissions are stronger and more likely to cause an epizootic effect, explaining why populations can become epizootic without having a blocked vector that can only transmit for a short time. These fleas, in nature would be able to feed multiple times on infected reservoirs and then infect more reservoirs, easily making the population epizootic. This study was to see if there are alternatives to transmitting the disease for a more rapid spread without the flea vector being blocked due to both timing and amount of bacteria present at the time of infection.

Method Explination

Artificial feeders were made by first isolating Yersinia pestis colonies on blood agar (containing 6% sheep blood and liver from the mouse in the study). This was done so that it could be determined if the specific colonies were virulent, detected by the positive phenotype shown by the en bloc gene, usually showing a different color (not described in experiment). The bacterium was taken from previously infected mice. Once the infectious bacterium colonies had been collected they were contained in a heart infusion broth to grow and replicate. Sprague-Dawley strain rat blood was added to the heart infusion broth to make the final concentration of 1.0 to 3.0×10^9 colony forming units per milli liter of broth. Finally the artificial feeders were covered by prepared mouse skin so the fleas would be more likely to bite into the feeder. Each set of fleas for each time period was allowed to feed on the feeder for an hour to become infectious.

Mice were anesthetized, put under, and a portion of their back was shaved. A capsule was applied to the shaved area with wax-resin with 10-13 fleas inside so that the fleas could feed on the mouse. Before the fleas were added the mouse had blood taken to make sure that it was not immune to the bacteria. Fleas fed for one hour and were separated, after, into those that had fed and hadn’t to then determine the colony-forming units that remained in each flea.

After the fleas had fed on the mice and had been separated into those that had fed and had not the fleas were individually put in 90 micro liters of heart infusion broth to be homogenized, equalized, along with 10 micro liters of glycerol. This solution was then grown on blood agar containing 6% sheep blood to then count the number of colonies growing from each individual bacterium in the flea to determine the colony forming units or the concentration of bacteria in each flea after feeding. A similar process was done using mouse blood after they had shown signs of infection from Yersinia pestis to determine how much bacteria had been transferred to the mouse per flea.

Select Figure Explination

Table 1: Bacterial load and transmission efficiency for O. Montana infected in artificial feeders containing defibrinated rat blood infected with Y. pestis at a concentration of 1.495 to 4.7 x 109 cfu/ml shows many important facts found through this research. Each infected mouse for each time period was given a number. This chart lists the number of fleas that were allowed to feed on that mouse and how many actually fed. Using the data from the colony forming units after each mouse had shown symptoms, it was found how much bacteria, on average and over a range, each flea had transferred to the mouse. It was noted if the mouse became infectious. Finally this table shows, using the data from the amount of bacteria remaining in each flea after transmission, how much bacteria was transferred by each flea during transmission to the mouse (percentage). There was little difference of transmission efficiency over the different time points though there were a few mice who seamed to get less bacteria, percentage wise, from the flea vectors though they still became infectious and had similar total numbers of bacteria in their system. Two interesting points that I saw was that at the three hour point there was no transmission from the flea to the mouse. I had thought that the bacteria was supposed to be immediately infective but it was explained that the three hour point wasn’t very helpful because the bacteria hadn’t been allowed to enter the digestive system enough to be transmitted to the next reservoir. I was also curious about the 6th mouse in the 96 hour time point where transmission did also not occur. I really don’t know how they explained this and I can’t suggest an explanation with the data that is shown.

Fig. 1. Mean maximum number of Y. pestis colony forming units in O. montana flea pools for 3, 24, 48, 72, and 96 h p.i. Data points are means of each replicate. Bacteremia in infectious blood meals ranged from 1.495 to 4.7 x 109 cfu/ml displays the number of colonies formed from testing fleas after feeding on their mice. It is interesting that at three hours there was a below average colony number which then shot up rapidly in the 24 to 48 hour time periods. The colony number then drastically dropped at 96 hours perhaps suggesting that the bacteria had started to die in the fleas.

Personal Interest

It is interesting to say that I found this topic to be interesting due to fictional books that I read in high school. We had a set of books regarding the plague and how different groups of people dealt with it to remove it from their population. I was curious as to how the plague was transmitted through different vectors. I remember one of the books suggesting that it came from a box of cloth, which would be very likely to contain fleas. I am now interested in looking more into alternative ways of transmission because it is strange how blocked fleas, which have a such smaller period of infectious quality are thought to be the better transmitters of this disease because they have larger populations of bacteria in them. This seams unlikely for me because you could not get an exponential effect from one flea infecting one reservoir but you would need it to infect multiple to get a plague to become epizootic in a population.

In class we have talked about many different processes and I was interested by the digestive system of insects because it just seams so simple and perfect. This experiment showed a disadvantage to the system because of describing the frequency that the pathway through the body can be blocked, causing major problems such as starvation of the insect. I am curious to know if there are studies going on to find ways to prevent blocking from occurring in these fleas so that, if transmission of an epizootic plague is due to blocked formation in the fleas then they can somehow prevent blocking from occurring, reducing the number of transmissions of infectious Yersinia pestis.

Scientific Terms Presented

Epizootic is a term often used to describe a disease that is in a large population of animals in a specific region. For example the plague studied here is a plague epizootic, spreading to large numbers of animals in a certain region, rapidly. The scientist’s goal was to find how this bacterium could become epizootic in a population and whether or not it is necessary for the vector to be blocked. It has been found that the transfer of the bacteria by blocked vectors does not always lead to epizootic spread through a population of prairie dogs but with certain flea vectors it can be transferred to an epizootic population of both squirrels and mice.

Blocking of the proventriculus is a build up of bacterium in the vector causing it to starve and feed more generating a larger quantity of bacterium in the vector. This was tested in this experiment to see if having a larger quantity of bacterium would make the bacterium more likely to infect a new host or reservoir of the disease. It was found that blocking of the proventriculus to cause a build up of bacteria was not a necessity for a population to become epizootic.

Changes Due To This Paper

It was commonly held that the plague spread faster by fleas that were blocked because they would regurgitate their blood meals and gain a higher concentration of Yersinia pestis in their gut when they would continue to feed. This experiment proved that that might not have been the case and the fleas are just as good vectors when they are not blocked and can infect an animal after one feeding when the proper distribution of fleas were available in similar numbers to nature. One reason for the exaggeration of blocked studies was due to its finding being so radical, everyone wanted to jump on board. This prevented studies to be done on un-blocked fleas that, as this study shows do transmit in an epizootic fashion to establish the plague in a population. Previous similar studies used more fleas per mouse than is natural and due to this these studies, though they show similar results, must be disregarded.

From all of these results the scientists have suggested that these results of early-phase transmission may be similar to other fleas that usually block and may be able to infect populations early than first thought. Other studies have found early transmission with other fleas such as the cat, dog, and mouse fleas that may be as early as one day, the time when all of the fleas in this study were guaranteed to transmit the plague. There is still the question to the flea that was known to transmit the plague during medieval times, Pulex irritans which in recent studies was found to only transmit as early as three days due to blocking. Could this change if blocking was taken out of the picture and it tried to infect others earlier with early-phase transmission? Overall this experiment opened up new thoughts as to the importance of blocking. This experiment showed that it was not necessary to transmit Yersinia pestis and that without blocking O. montana was able to transmit the plague to more reservoirs of mice allowing for an epizootic effect. This allows for new methods of plague transmission to be studied with new ideas in mind that were previously though to be impossible.

Follow-up

As a follow-up experiment I first considered to attempt to find a way to use the same flea and induce it to become blocked to see if there is some change in the efficiency of this flea. My thinking is that if past studies have used fleas of another species that are blocked that it may be that there is another factor, for example mouth opening that does not allow as much Yersinia pestis to travel into the animal being infected or feeding time is not the same, that will alter the effectiveness of a separate species. If this is not possible to induce a blocked action, perhaps inducing a previously studied flea species to suppress their blocked function so that it can be studied unblocked. So perhaps studying the mechanism that causes blocking to occur in some fleas but not in others would be a good first step to take.

Taking the two flea vectors that were described in this experiment, Xenopsylla cheopis, which has block formation, and Oropsylla montana which does not form blocks I would compare the mechanisms of blood, infected with Yersinia pestis as it passes through the fleas’ digestive systems. A precise amount of bacteria that is required to cause a block formation in X. cheopis would be found by measuring the colony forming units of an artificial feeder and counting the feedings required to induce the regurgitation of blood by the flea, occurs when blood can no longer pass through the digestive tract. Both flea species will be fed on the same concentration for the same number of feedings to see if there is a differing effect with a higher level of bacteria going through the digestive system of Oropsylla montana.

To determine the amount of bacteria remaining in each flea a test to find the number of colony forming units in the flea feces will be done. This is important because it is necessary to know how much bacteria is remaining in the flea to know if the bacteria is having a detrimental effect on the flea that would cause it to die faster other than blocking the digestive system making the flea starve to death. This experiment would rule out many variables of the consistency at which the two different species of flea can be compared with the blocking and non blocking functions to see if there are other effects that the bacteria have on the flea that could cause different levels of bacterial release into a new host reservoir to increase or decrease the epizootic effect the bacteria would have.