Data Update: Look at the polio fly

by Mike

In our last Data Update, the table that I turned into figures was not a bad table. It was pretty clear. It just contained some unnecessary information, and was spread across two pages, which is always bad. Today, the table in question is really hard to interpret. I could not make heads or tails of it without going over the text, piece by piece. It’s from a 1943 paper (1) in the Journal of Infectious Diseases.

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Like the pictures of cockroach feeding contraptions, this data comes from a laboratory that was using what could be called “experimental epidemiology” techniques, to figure out how much of a public health hazard these bugs really are. To see if bugs could actually ingest, preserve, and spread germs.

This time the bug is the common house fly. Thomas Francis Jr. had just joined the faculty of the University of Michigan from NYU, and with technician Robert Rondtorff he conducted this study in the interest of public health. Soon Francis would be supervising graduate student Jonas Salk, with whom he worked a lot in the 1940s and 1950s, as you might be able to guess from the fact that Dr. Francis now has a Wikipedia page.

By 1943 we knew that polio was spread by filth and tainted water (the fecal-oral route, as we call it). But flies feed on that stuff, and fly around. Does the virus replicate inside the flies, like malaria? This paper established that when flies ingest poliovirus, the virus disappears from the digestive system within 2 days. And therefore, flies probably aren’t making the poliomyelitis epidemic worse. The significance of these findings is indicated by the introduction to a 1950 paper (2) by children’s television character “Herbert Hurlbut”.

Poliomyelitis virus has been isolated from filth-frequenting flies caught in nature during several epidemics in recent years.(3-5) When the Lansing strain of the virus was fed to house flies under experimental conditions, virus was not recovered after 48 hours.(1,6) Recently Melnick and Penner (7) fed virus in human stools to the blowfly Phormia regina and were able to recover it from the flies after about two weeks[.]

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First of all, their experimental system. Feed the flies on polio-infected material. To make sure there was a lot of polio in the flies’ diet, they did not use excrement from polio-infected animals. They used “a 10% suspension of infected [mouse] spinal cords in boiled milk”. In the not-entirely-robotic prose of scientific papers of the era, they say they “offered” this to the flies. Earlier they had just diluted the mashed spinal cords in saline solution, and the flies “did not feed readily”, so they switched to a solution in milk, which the flies found far more appealing.

For this experiment, the flies enjoyed their neuronal polio-milk for 1 hour, after which it was removed and they were allowed to feed on regular milk if they wanted to. The scientists then waited either 0, 2, 7, 13, 25, 49, 120, 240, or 480 hours. After each of these periods, a certain number of flies were killed, and their abdomens were cut away from the rest of the body. Earlier in the paper it had been established that poliovirus does not leave the abdomen (gut) of an infected fly and reach the rest of the body (thorax and head). Now they are looking to see how much polio virus is in the infected flies.

To get an extract containing poliovirus, they took the fly parts and ground them in saline solution using a mortar and pestle. For best results, they ground the samples with alundum (an abrasive preparation of aluminum oxide). They added some ether (diethyl ether, I assume) to the solution, and let it sit in the refrigerator until it was “bacteriologically sterile”. I’m not familiar with the use of ether to remove bacteria, but it looks very suitable to this procedure.

Experimentally and clinically, ether, whether in its vaporous or liquid state, has been proved to have a bactericidal action. Spore bearing organisms, however, are strongly resistant to it. (7)

Not only did ether treatment remove bacteria from the extract, but it also removed most other viruses. Poliovirus, as a non-enveloped virus, is resistant to ether, a substance which destroys the membranes of enveloped viruses. Here’s a table that summarizes, as of 1949 (8), which viruses are ether-resistant.

andrewes-horstmann-ether

from Andrewes and Horstmann (1949)

Note that most viruses are ether-sensitive and should be removed by this sort of treatment, meaning you’re left with polio, papilloma, bacteriophages, and a few other viruses. Including some, but not all poxviruses, for reasons that are unclear to me. Parapoxviruses (myxoma, BPS virus) are ether-sensitive, and orthopoxviruses (smallpox, vaccinia) are ether-resistant, which is still the dogma (see Chapter 21 of Medical Virology). And chordipoxviruses are maybe one or maybe the other. The above table puts sheep-pox and goat-pox (chordipoxviruses) in the resistant group, whereas Plowright and Ferris (9) say that sheep-pox (SP) and lumpy skin disease (LSD) viruses are ether-sensitive.

from Plowright and Ferris (1959)

from Plowright and Ferris (1959)

Anyway, poliovirus is definitely ether-resistant. So this extraction method is useful for studying this particular virus [here’s another example (10)].

It wasn’t possible in 1943 to measure the amount of virus by using a plaque assay, as we do now (applying a solution of virus to a plate of cells and seeing how many plaques [empty spots] form in the cells by being killed by virus). What the authors do instead is infect mice with the fly extract and see if they become paralyzed and/or dead. In Table 1 they established that the virus survives in the flies, but only in the abdomens. Here’s Table 2.

rondtorff-table-2

So one half of the table is data from the flies, and one half is data from the mice, right? That’s what I thought. But no. This table is made up of almost entirely unnecessary information, and only makes one point. I’ve taken the liberty of highlighting the important parts.

rondtorff-table-2-2

In the text, Rendtorff and Francis go into great detail about how they prepared the ground fly mixtures so they could be compared fairly. They weighed the samples before grinding, and added more or less saline depending on how many fly abdomens were in the sample, and what their weight was. In deciding how much saline to add to dilute the sample, they also factored in what the average unfed weight of an abdomen should be, which should be the same for all the flies. This process was to normalize the data, which could be thrown off by the flies’ unequal eating and excreting habits. I feel like pooling together somewhere between 44 and 66 fly abdomens would already take care of the issue of some flies eating more than others, but they did this statistical technique to account for possible variation.

Most of this table is the raw data they used to do the normalization. But… I don’t need to know what the weight of the fly abdomens were. Or how much saline was added. And I definitely don’t need the “Calculated Unfed Weight of Abdomens” column, which is nothing but the “No. of Flies Sampled” column multiplied by the average weight of an unfed abdomen. It’s good to present raw data, I guess. But this data is not important. Maybe it should be separated from the important data.

The least important data of all is the dates on which the experiments were performed. This is something that absolutely never shows up in research articles anymore. In fact, we may have gone too far in the opposite direction, pretending that we did experiments in a certain order because it makes for a better narrative.

All that matters here is two independent variables and one dependent variable. The independent variables are:

  • Temperature of the incubators in which the flies were living, eating and loving life. (25, 30, or 35 °C)
  • Number of hours the flies were allowed to live, between eating the polio-infected meal and being killed. (0, 2, 7, 13, 19, 25, or 49 hours)

The dependent variable is:

  • Time of death (or paralysis) for a mouse injected with fly extract

So all we really need is a bunch of survival curves.

polio-survival

Figure 2: Poliomyelitis virus is destroyed by the fly digestive tract within 2 days, irrespective of temperature. Flies were fed on a suspension of infected spinal cord in milk for 1 hour. Flies were then incubated in a chamber at 25, 30, or 35 degrees Celsius, for 0 (positive control), 2, 7, 13, 19, 25, or 49 hours. At each timepoint, between 45 and 65 flies in each chamber were killed, and abdomen tissue was extracted using ether. After dilution in saline and evaporation of the ether, each sample was used for intracerebral infection of 9 or 10 mice. Twice a day, mice were monitored for death or paralysis.

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1. Rendtorff RC, Francis T Jr (1943). Survival of the Lansing strain of poliomyelitis virus in the Common house fly, Musca domestica L. J Infect Dis 73(3):198-205.

2. Hurlbut HS (1950). The recovery of poliomyelitis virus after parenteral introduction into cockroaches and houseflies. J Infect Dis 86(1):103-104.

3. Trask JD, Paul JR (1943). The detection of poliomyelitis virus in flies collected during the epidemics of poliomyelitis. J Exp Med 77:531-544.

4. Sabin AB, Ward R (1941). Flies as carriers of poliomyelitis in urban epidemics. Science 94:590-591.

5. Melnick JL (1949). Isolation of poliomyelitis virus from single species of flies collected during an urban epidemic. Am J Hyg 49:8-16.

6. Bang FB, Glaser RW (1943). The persistence of poliomyelitis virus in flies. Am J Hyg 37:320-323.

7. Saliba J (1918). Ether therapy in surgical infections and its effect on immunity. New York Med J 107:157-160.

8. Andrewes CH, Horstmann DM (1949). The susceptibility of viruses to ethyl ether. Microbiology 3(2):290-297.

9. Plowright W, Ferris RD (1959). Ether sensitivity of some mammalian poxviruses. Virology 7(3):357-358.

10. Ward R, LoGrippo GA, Graef I, Earle DP Jr (1954). Quantitative studies on excretion of poliomyelitis virus: A comparison of virus concentration in the stools of paralytic and non-paralytic patients. J Clin Invest 33(3):354-357.

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