Dealing with disease

28 February 2021 - By: Alex Evans

Emerging infectious diseases have certainly been making headlines in recent years but they are far from a rare occurrence in the animal kingdom. There are countless animal species currently at threat of local and global extinction due to the rapid spread of harmful parasites and virulent pathogens, many of which can be traced back to humanity’s actions. To even the odds, researchers around the world are working hard to further our understanding of these diseases and develop applications for practical epidemiology. From fungal syndromes to viral genomes, let us take a careful peek into the world of emerging infectious disease research.

Bats - bw

SURVIVAL IS ONLY SKIN DEEP

Emerging infectious diseases come in many forms and can target a wide range of animal organs and tissues. One particularly devastating infectious disease, chytridiomycosis, is believed to play a major role in the global decline of amphibians by wreaking havoc on the skin of the animals it infects. Nicholas Wu, an ecophysiologist currently at the University of Sydney, Australia, has spent years unravelling the mysteries of how this disease functions and how it has become such an effective killer.

“Chytridiomycosis is an amphibian disease caused by the Batrachochytrium dendrobatidis (Bd) fungus, which attaches to the skin of animals and feeds on their keratin,” says Nicholas. “Since amphibians rely on their skin for key physiological functions such as drinking, breathing, and salt regulation, this fungus can disrupt these key processes, which results in very high chances of mortality for infected individuals.”

Freshwater frogs are susceptible to salt loss from their bodies, with salt leaking out into their environment as water moves in, but healthy frogs are able to maintain their internal salt levels using salt channels in their skin. Unfortunately, chytridiomycosis can seriously hamper the ability of frogs to maintain this balance. “There was an important study in the journal Science showing that the main cause of death for Bd-infected frogs was cardiac arrest, brought on by low salt in the blood,” says Nicholas. “The mechanisms that connected skin disruption to low salt in the blood weren’t particularly clear, which is where our work came in.”1

Using an integrated approach to the investigation, Nicholas was able to address this question from multiple angles and identify exactly what was causing this lethal imbalance. “We looked at different levels of physiological disruption, from the genes expressing the production of these salt channels and the abundance of channel proteins through to the whole tissue and organismal level responses,” he explains. “At the protein level, we found reduced abundance of salt transport proteins in infected frogs, matching the higher salt leakage out of the body. Surprisingly we also found increased gene expression of those same proteins, showing that the infected frogs’ immune response is actively trying to upregulate these transport channels to regain their salt balance.”1

Thanks to Nicholas’s research, we now have a better understanding of how infection leads to mortality and we can even identify the threshold of fungal load at which physiological disruption becomes a death sentence. “We hope that this research will be used in amphibian conservation efforts by informing people which individual frogs can be treated and which are beyond the threshold for recovery,” he adds.

Although Nicholas’s work has focused on the impact of Bd on salt imbalance, there is another related fungus, Batrachochytrium salamandrivorans (Bs), that causes chytridiomycosis in salamanders. However, much less research has been conducted on this more recent threat. There are salamanders in the world, particularly in North America, that do not have lungs and rely solely on their skin to breathe. “For these salamanders, instead of salt disruption leading to death, perhaps asphyxiation or lack of oxygen transport across the skin might be more of an immediate threat,” explains Nicholas. “No one has yet looked into this in detail and all we know so far is that the salamanders’ skin is affected the same way as frogs, but it is uncertain if the cause of death by Bs is the same.”

THERE MITE STILL BEE HOPE

It is no secret that the 21st century has not been kind to the world’s pollinators, with many bee species facing global decline caused by human threats. Individually, these threats can challenge the survival of a bee population, but when combined, they can cause an overwhelming cascade of lethality. Thankfully, Alan Bowman, a researcher of parasitic pests at the University of Aberdeen, UK, is working to tackle one of the smallest but deadliest foes of the European honey bee (Apis mellifera), called Varroa destructor, and its pathogenic payload. “I’ve been working on ticks and the pathogens that they carry for 30 years,” says Alan. “I first heard about these Varroa mites in 2008 and put together a proposal to investigate this species and its ability to transmit disease.”

V. destructor is an ectoparasitic mite of bees originating from Asia, and while it lives in a relatively stable equilibrium with Asian honey bees, the Varroa mite is currently regarded as one of the biggest contributors to bee colony deaths in the western world. First making its way across Europe in the late 1960s and reaching the UK in the early 1990s, Varroa has had a devastating effect on managed and feral honey bees. To make matters worse, not only is Varroa a harmful parasite to European honey bees in its own right, but it is also a vector for a number of debilitating diseases including deformed wing virus (DWV).

“On its own, DWV is actually fairly benign with a low level of infection and covert symptoms,” Alan explains, “but in the presence of Varroa, DWV is able to replicate to a much larger degree, resulting in crumpled wings and leaving the bees incapable of flight.” Needless to say, a hive of flightless bees does not forage particularly well and, without enough food to survive the winter, most hives infested with Varroa are dead within 2 years. To halt this devastating parasite–pathogen combination, Alan and his team are focusing on finding genetic interventions that hamper the impact of Varroa mites on honey bee hives and restrict their ability to transfer DWV.

“Although gene therapy is unlikely to be used as a control method itself, it does show us which genetic pathways can be disrupted to successfully kill off the mites,” says Alan. “This can make future insecticide development much more streamlined and likely to succeed.” Brain tissue is a common target for insecticides because the disruption of neural processes is likely to have a rapid and lethal result,2 but Alan’s group has also successfully identified the genes controlling the mite’s saliva and experimented with knocking these out. “We’ve found that mite saliva can lower the host bee’s immune system and allow the virus to affect its host more profoundly,” says Alan. “The usual route of transmission for the virus is through the oral–faecal route, with only a small proportion of the virus infecting the host. But, with Varroa, the virus is able to get straight into the body cavity and run riot.” 

Alan is also interested in exploring ways to manipulate the mite’s behaviour to reduce its ability to reproduce and spread DWV to new hosts. One such method is to disrupt the breeding patterns by kicking up a stink. “We’ve been able to identify a chemical scent that the bees produce which acts as the cue for the Varroa to lay eggs,” he explains. “We realised that we can’t yet stop the mites laying eggs on their hosts, but we can encourage the mites to lay eggs out of season when there are no young bees to feed on.”

Working with parasites certainly has its difficulties and keeping experimental populations alive in the absence of hosts is a big one, but Alan’s group has been working on vital solutions to this issue. “I’m very proud of the artificial feeding system that we have created for our Varroa mites, which has allowed us to look at the virus transmission and dynamics, and now we are able to sustain two generations of mites in our system and this has helped us to find some very effective Varroa-killing compounds which are completely safe to bees.” However, Alan and his team’s efforts are far from over and he has plans that will help to further our ability to investigate this growing threat. “We also wish to develop a long-term artificial breeding system for Varroa mites, not just for our own research, but for all the other researchers in this field,” he says. “Keeping populations of Varroa alive for experiments can be very challenging as they require beehives which are only operational during the warmer half of the year!”

 

SPEEDY GENOMES SAVE LIVES

Throughout history, there have been many zoonotic diseases that have made the jump from animal to human with disastrous consequences. Tuberculosis, Zika, Ebola and, of course, COVID-19 are all recent examples that have been in the public eye. Less well known are the people working to discover how these diseases spread and how these insights can translate to life-saving responses. Josh Quick, a specialist in the field of infectious disease genomics at the University of Birmingham, UK, focuses on the development of rapid genome sequencing for disease outbreaks around the world. “I really got interested in infectious disease via genomics by learning how to apply new and exciting genomics techniques to emerging infectious diseases,” says Josh. “I started my career in the commercial sector but ended up working at Birmingham with Nick Lowman, who had an established research programme studying infectious disease, and we collaborated to work on genetic epidemiology.”

For Josh, the crux of his work is the constant drive to develop more rapid and robust techniques for sequencing the genomes of pathogens. “I’m very much a practical scientist and my focus is on the application of research to real-time epidemiology,” he explains. Josh’s work has taken him around the world, using the latest technologies and techniques to bring fast response epidemiology to where it is needed most. “After nanopore technology became available in 2014, I was able to travel to West Africa, Central Africa, Brazil and other places to study emerging diseases on site.”

By collecting a large amount of high-quality genetic data from clinical samples, Josh and other epidemiologists have been able to provide detailed insights into how these emerging infectious diseases spread and, crucially, provide them quickly enough to help inform response efforts in real time. “In 2015, we sequenced 175 Ebola genomes (almost 5% of all known cases), which was considered to be quite a big achievement at the time,” says Josh, “but as of October 2020, the UK’s coronavirus genomics consortium has already sequenced over 81,000 SARS-CoV-2 genomes responsible for COVID-19 outbreaks,3 demonstrating how incredibly far the technology has come in just a few years!”

According to Josh, the demand for genetic surveillance data in epidemiology has become so high in recent years that there has been a huge collaborative effort to analyse results and bring about responses to infectious disease in real time. “CogUK encapsulates this whole system in action,” he explains. “It’s a decentralised network of 15 labs producing fast turnaround genome data that can feed into the national picture and help to identify transmission clusters and outbreaks.” The insights of CogUK into the COVID-19 outbreaks in Wales pointed to specific genetic lineages coming from England, which led to real impacts on government policy regarding cross-border travel. “In terms of numbers of genomes produced and open collaboration, the UK has the best genomics surveillance network in the world,” says Josh. “Although, as we’re learning at the moment, a world-leading surveillance network alone isn’t enough to control the spread of disease !”

“There is a lot of experimental testing that goes into the development and deployment of these techniques,” says Josh. “We start with PCR primer pools to amplify overlapping fragments of the viral genome, which you can then sequence and replicate the viral genome for mass production.” This testing is essential because these genomes will be used by many laboratories for different projects, so they need to be robust in real-world conditions and work with clinical samples of varying quality, rather than just synthetic test samples. “We spend a lot of time testing and optimising to ensure that the products are robust,” says Josh.

One of the reasons that Josh’s work has been so successful is his emphasis on rapid response. “Speed is absolutely of the essence, which is why fast-sequencing approaches are our number one priority,” he explains. “I was able to post an early version of the SARS-CoV-2 sequencing protocol very early in the outbreak4 (January 2020) thanks to a genome sequence from Fudan University in China. It’s a great example of how rapid data sharing can benefit everyone, and my protocol has now been downloaded over 70,000 times (as of October 2020).” Josh concludes by emphasising the importance of the collaborative process in handling widespread infectious diseases such as COVID-19: “I really enjoy seeing international cooperation between scientists where impact doesn’t just end with papers and grants, but in a global effort towards this pandemic response.”

FOR A BAT-TER TOMORROW

While there are many serious emerging infectious diseases raging across the animal kingdom right now, perhaps one of the most pressing is white-nose syndrome (WNS), an infectious fungal disease that is responsible for widespread bat losses across North America. Yvonne Dzal is an ecophysiologist (and LeBron James fanatic) based at the University of Winnipeg, Canada, under the mentorship of Craig Willis. Together, they are directing their research towards improving our understanding of this devastating disease. “I am a bat enthusiast that has been studying these fascinating animals for over a decade,” says Yvonne. “It saddens me that if bat populations continue to decline at current rates, we stand to lose a key component of biodiversity, as well as the many ecological and economic benefits that bats provide.”

WNS is a skin disease caused by the fungus Pseudogymnoascus destructans (Pd) that has been responsible for catastrophic declines in hibernating bats across the USA and Canada since its discovery in New York in 2007. “WNS has now been confirmed in 18 bat species, with the fungus detected in 39 US states and 7 Canadian provinces,” Yvonne explains. “Several species of endangered bats are already at risk and predicted to suffer range-wide extinction, including three formerly common and widespread bat species.”

With her research into WNS, Yvonne hopes to mitigate the effects of this disease before it is too late. “Since WNS emerged in North America over a decade ago, millions of dollars have been devoted to research on chemical and biological interventions to treat bats with WNS,” she says. “While some promising approaches are being tested, to date, no effective treatment is ready to be deployed.” Thankfully, Yvonne and her team are using experimental methods to help identify the most worthwhile routes for recovery. By integrating physiological and ecological data, Yvonne is able to create predictive models of population impacts based on WNS pathophysiology. These models can then be used to influence disease management in the field. “My research on the differences in species susceptibility and the physiological mechanisms underlying fungal resistance or tolerance is critical for evaluating the safety and efficacy of treatments in the wild.”

One of Yvonne’s experimental lines of enquiry revolves around the effects of WNS on the ability of certain bat species to breathe through the skin of their wings, known as cutaneous respiration. “We hypothesised that WNS-susceptible little brown bats (Myotis lucifugus) rely more on cutaneous respiration than the WNS-tolerant or -resistant big brown bats (Eptesicus fuscus),” she explains. Using a divided respiration chamber, Yvonne and her team were able to separately measure the gas exchange taking place at the wings and the pulmonary respiration taking place at the lungs and respiratory system. They discovered that the WNS-susceptible species did indeed rely more on cutaneous respiration as predicted. They also found that higher Pd fungal loads reduced their ability to effectively perform cutaneous respiration and led to harmful effects such as increased energy expenditure and water loss. Based on these findings, Yvonne and her team recommend that conservation efforts with these most susceptible species should reconsider applying topical anti-fungal treatments to the wings because it may interrupt healthy bat physiology such as gas exchange.

Over the next few years, Yvonne will be working on a number of WNS projects as part of her postdoctoral Liber Ero Fellowship.5Experimental work is vital to our understanding of emerging infectious diseases such as WNS,” says Yvonne. “Investigating the physiology of imperilled bat populations is the only way that we can come up with scalable solutions that aid in the recovery and conservation of bats in the face of pathogenic invasions”. However, Yvonne is keen to emphasise that time-sensitive work of this nature does not exist in a vacuum and the clock is steadily ticking down for North American bats. “This research may well be the last opportunity to address these questions as WNS invades western North America, where bat diversity is the highest on the continent,” she explains. “Given the steady spread of the fungus, behavioural, ecological, and physiological data on bats are urgently needed while we still have enough uninfected bats to study .”

 

References:

1. Gauberg J, Wu N, Cramp RL, et al. 2019. A lethal fungal pathogen directly alters tight junction proteins in the skin of a susceptible amphibian. J Expl Biol, 222 (3).

2. Campbell E, Budge G, Watkins M, et al. 2016. Transcriptome analysis of the synganglion from the honey bee mite, Varroa destructor and RNAi knockdown of neural peptide targets. Insect Biochem Mol Biol, 70, 116-126.

3. https://www.cogconsortium.uk/news_item/commentary-cog-uk-report-12-15th-october-2020/

4. https://www.protocols.io/view/ncov-2019-sequencing-protocol-single-sample-bdbfi2jn

5. https://news-centre.uwinnipeg.ca/all-posts/uwinnipeg-researcher-chosen-as-a-liber-ero-fellow/
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