So after a rather interesting year filled with ups and downs, a zombie apocalypse and Elvis coming back from the aliens that abducted him, I’ve finally got around to starting the new blog I had been promising to do since forever. I am aware that I said in October 2006 that I would have a new website up soon and that it turns out it’s (checks computers date) the 10th of January 2008. I guess if we’re going to be generous we could describe that as being ‘soon’ if we measure time in terms of decades or centuries. Unfortunately, as with many things that occur, real life and the pressures of working in an actual research laboratory meant that I just didn’t find the time or inclination to do science all day, then come home and write about science even more. None the less, I love what I do and doing actual science in a lab is hard but ultimately rewarding work.
So what will this new blog be about then? Well, pretty much exactly the same thing as my old blog was about with more of a focus on developments in animal immunology, diseases and vaccinations. I’ve still kept a keen interest in the bovine tuberculosis epidemic in Britain involving badgers, been looking into Johne’s disease in several farmed animal species (this is what I do my work on) and have taken an interest in more general diseases of animals (including wild species). I’ll hopefully get around to posting some new material soon that I’ve been squirreling away, but for now here is a post on Mare Reproductive Loss syndrome and how caterpillars were linked to the disease:
This is a story of two organisms and the identification of a really unusual way for bacteria to end up in a place they really should be. One of the organisms involved in this story happens to be a horse:
The other organism involved is the Eastern tent caterpillar (Malacosoma americanum):
You might be thinking at the moment as to how that tiny caterpillar interferes with a horse. After all, the caterpillars in question are a communal bunch that spin little “tents” and horses shouldn’t really have any particular interest in caterpillars. Most remarkably however, these caterpillars are known to be associated with a disorder in pregnant horses called Mare Reproductive Loss Syndrome (MRLS). Most interestingly in this story is the proposed mechanism by which, this tiny (6cm long) caterpillar is responsible for causing a bacteriological infection of the developing fetus and ultimately killing it. But first, it’s time for a bit of background.
What is MRLS?
(Information is from Webb et al. 2004, unless otherwise referenced)
In central Kentucky USA from late-April to May of 2001 an unusually high number of spontaneous equine abortions suddenly started to occur. The rapid rise in abortions among early mare fetuses (a gestation time of around 40-150 days) was particularly prominent, with some farms having up to 60% higher early fetal losses compared with the ‘normal’ rate of 3-5%. As Kentucky prides itself on its horses and they are important in the economy of the area, the news was naturally incredibly disturbing. To illustrate the impact of this disease on the Kentucky economy, it resulted in thousands of mares aborting their fowls and with an estimated loss of over $425 million (!) dollars (Potter et al. 2005). The unknown disease was designated mare reproductive loss syndrome and was rapidly investigated.
Investigation of pregnant mares by ultrasound revealed that the fluids around the fetus were often extremely cloudy. Examination of the dead fetus revealed the presence of numerous bacterial species, but most interestingly no specific pathogens known to be associated with fetal abortions in horses. Instead, the isolated bacteria tended to be associated with commensals such as non-β haemolytic streptococci, actinobacilli, Serratia marcescens and many other bacteria. The majority of these identified with either oral commensals (like the streptococci) or bacteria commonly associated with the gut microbiota such as Enterobacter and Serratia spp.
More curious was the lack of pathology observed in the mother horse itself. Although some horses had some symptoms, such as pericarditis, the majority did not present with a fever or other indication of a systemic bacterial infection (despite this being present in the fetus). This unusual pathology raised the question that the bacteria were a secondary infection and something else, such as an ingested toxin was causing the spontaneous abortions. During the investigation of the outbreak of MRLS, it was found that a caterpillar, the Eastern tent caterpillar (ETS) had a large population explosion at around the same time that the outbreak of MRLS occurred. Could the caterpillar population explosion explain the sudden appearance of MRLS?
Demonstrating a link between caterpillars and MRLS.
The most conclusive paper on the link between MRLS and the ETC was performed by Webb et al., 2004 (See the full reference below). This study performed three experiments that demonstrate ingestion of ETC is associated with MRLS after the results of a pilot study (also performed by these authors) showed promising evidence of a link between MRLS and caterpillars. The three experiments were:
Experiment 1: This compared three groups of pregnant mares in the pasture to replicate what would be ‘natural’ transmission of caterpillars into the horse. One group was on a caterpillar diet, which involved adding a certain density of caterpillars onto a sealed off section of pasture and allowing the horses to basically eat them with their usual feed. Another group was exposed in the same manner to caterpillar frass (see the picture of the caterpillars above, frass is, as far as I understand, what the stuff they spin is called). Lastly, there was the control group that lacked any caterpillars or frass.
Experiment 2: This is very similar to experiment 1 except that this time they fed pregnant mares directly either frozen or autoclaved M. americanum caterpillars or alternatively, another species of caterpillar called Lymantria dispar.
Experiment 3: Finally, they wanted to investigate which part of the caterpillar was specifically involved in causing MRLS. They used saline, whole M. americanum larvae and several different dissections (Such as cuticle, hind-gut and haemolymph*) of the caterpillars and fed these again to pregnant mares.
To put a long story short, their results demonstrated a clear link between the caterpillars and MRLS. Experiment one demonstrated that horses in pasture would ingest the caterpillars and would go on to develop MRLS, while horses fed on caterpillar frass and not fed on anything caterpillar associated did not.
The second experiment demonstrated something very interesting the autoclaved caterpillars did not produce MRLS in any of the 5 horses, while the frozen caterpillars did induce MRLS (in 3 of 5 mares). We’ll come back to this later so bear this in mind for now. The control L. dispar caterpillars did have one abortion in the group but it was not associated with MRLS.
Finally, the last experiment demonstrated that whole caterpillars were able to induce MRLS (as expected) and that MRLS abortions were associated with the outer cuticle (exoskeleton) of the caterpillars most strongly.
Now, the results of this research are incredibly interesting as it hasn’t really been seen before that an insect like a caterpillar could cause such an unusual disease. It’s especially surprising that this disease has only been discovered recently, especially because it’s caused by a common insect that covers a wide area of the United States. The question now remains as to what mechanism could cause MRLS? It’s known that lepidopteran** insects have numerous chemical defense mechanisms and are capable of producing poisons among other toxins. Another potential candidate is the build up of compounds such as cyanide in the cuticle of the insect.
However, perhaps the most intriguing answer to this puzzle isn’t a toxin, poison or similar, but instead a quirk from the caterpillars physical defenses.
Caterpillars with built in bacterial syringes
(The reference for this section comes entirely from Tobin et al., 2004, which is a fascinating paper).
An interesting hypothesis for the potential pathogenesis of MRLS is the “Septic penetrating setae”. Now setae are fairly common structures on a lot of different insects and are basically pointy tubes on the surface of the animals’ exoskeleton. These pointy tubes have numerous functions, but the most obvious is for defense by basically making the insect unpleasant to eat. This is because setae are basically pointy sticks*** and they penetrate into tissues causing considerable irritation. The hairs of the red rump tarantula are basically hollow spikes that are flung into the skin and eyes of an attacker. This causes severe irritation and allows the spider time to flee the scene of the crime.
Image of setae showing their general structure, with the size of these being around 20μm in diameter and demonstrating the pointy ‘barbs’ that cover the structure. The image was acquired from Tobin et al., 2004, and the photos were originally from Henry H. Southgate, University of Kentucky.
So what is the relation between Eastern tent caterpillar setae, mare reproductive loss syndrome and the bacterial infection that induces the abortion? It turns out that the setae are structures that happen to be hollow and they are also liable to fragmenting once they are ingested. Their small size, which is small enough to fit inside a large enough blood capillary (especially when fragmented) and is sharp enough to penetrate tissue, may be able to cause MRLS. The mechanism that is proposed works something like this:
1) The pregnant horse starts to snack down on a nice lunch of caterpillar.
2) A) Setae are broken off the caterpillar, penetrating gum tissue and other areas of the mouth to enter into the bloodstream. This is aided by the ‘spikes’ on the setae that allow the structure to progress more efficiently in moving tissue.
B) Alternatively, the setae make it through to the gut where they penetrate the thin epithelial lining. Again, movement of the intestine and the sharp spikes on the setae enable the fragments to enter into the bloodstream and move around the body.
3) Along with the penetrated setae, bacteria are carried along as passengers inside or alternatively on the surface of the setae until they hit a suitable ‘target’. In this case, certain ‘immunoprivileged’ organs such as the eye, reproductive organs and yes, even the amniotic fluid/uterus where the fetus is developing.
4) Once the setae ‘hits’ one of these organs with its bacterial passengers, or alternatively a critical number of setae have hit, it establishes an infection. This infection then progresses rapidly (less than 38 hours) and leads to the development of MRLS.
This hypothesis for the mode of action that ETCs cause MRLS is actually quite compelling as an explanation. Firstly, it demonstrates the link between the typical commensal bacteria often found in aborted fetuses, recalling they are primarily from the mouth and also some from the gut. It’s unlikely that the setae are punching a hole in the mouth or gut, followed by general septicemia as recall that the mare itself rarely has any associated symptoms. This provides weight to the explanation that the setae (or fragments) carry the bacteria to their target without exposing its passengers to the immune system. Basically a ‘direct delivery’ style system and finds the uterus often (compared to other regions, which is much less frequent) due to its general size, significant movement and shape.
Further, remember how I earlier said to keep in mind the autoclaving experiment from the Webb et al., 2004, study? Autoclaving basically works by heating up stuff to a large temperature, usually about 121-124 degrees. As well as intense heat, there is also a considerable amount of pressure in the chamber during a cycle as well. If you recall, the autoclaved caterpillars were not able to cause MRLS and this could have been the result of the disruption of the internal structure of the setae. Although this concept hasn’t been proven and doesn’t rule out a toxin (which could be deformed as well), it provides a compelling piece of evidence that could corroborate this hypothesis.
What to take from this
This entire line of enquiry shows that diseases and other disorders are not always the simple interaction of X pathogen causing Y disease in a specific host. Sometimes there are some very complicated external factors that may cause a disease but not be directly responsible for it. The association of these caterpillars and an abortive disorder in horses is one such example. Additionally, the implication that setae cause the entire disorder by a simple quirk of their structure is a fascinating finding. It is worth noting, before I get too enthusiastic, that the “Septic penetrating setae” hypothesis has yet to be experimentally confirmed. For my money though, I hope it’s the correct answer because it would provide a fascinating and highly novel mechanism. Microbes gaining entry into vulnerable tissues by using a ‘built-in’ syringe from another organism is just too interesting a concept to be wrong.
*Haemolymph=Insect blood. It’s a combination of interstitial fluid and their oxygen transporting blood vessels. In us, as an example, our interstitial fluid is separated from our blood and not mixed together.
***Yeah, this is a simplification.
Potter D.A., L. Foss, R.E. Baumler and D.W. Held (2005). Managing Eastern tent caterpillars Malacosoma americanum (F) on horse farms to reduce risk of mare reproductive loss syndrome. Pest Management Science, 61:3-15.
Tobin T., J.D. Harkins, J.F. Roberts, P.W. Vanmeter and T.A. Fuller (2004). The mare reproductive loss syndrome and the Eastern tent caterpillar II: A toxicokinetic/clinical evaluation and a proposed pathogenesis: Septic Penetrating Setae. The International Journal of Applied Research in Veterinary Medicine, 2:142-158.
Webb B.A., W.E. Barney, D.L. Dahlman, S.N. DeBorde, C. Weer, N.M. Williams, J.M. Donahue and K.J. McDowell (2004). Eastern tent caterpillars (Malacosoma americanum) cause mare reproductive loss syndrome. Journal of Insect Physiology, 50:185-193.