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raccoon dogIs anyone untouched by the Covid19 pandemic?  Some of our critical research projects are on hold, mostly due to no PPE for personnel to handle study lab animals and also travel restrictions.  We are fully functional at our lab.  We are using any extra time to evaluate data and share our findings with veterinarians and horse owners through Zoom meetings.  Check out the Facebook meeting posts to find the links and join us in our Zoom room.  Until then, here are some facts that help us focus on what is important. The information is up to date as of mid-April.

The SARS-CoV-2 strain of a novel Coronavirus appeared in Asia in 2019 and is known as Covid2019…Covid19 for short.  SARS is an acronym for severe acute respiratory syndrome caused by a betacoronavirus that is transmitted by contact with fomites.  Fomites are infectious materials transmitted by contact with respiratory droplets or body fluids.  Unfortunately for us this virus is transmitted by airborne particles.  Symptoms include fever, headache, body aches, dry cough, hypoxia (lack of oxygen), and usually pneumonia. For you molecular biologists,  SARS-CoV are enveloped, positive-sense, single-stranded RNA virus that infect the epithelial cells within the lungs. SARS-CoV-1 binds the ACE2 (angiotensin-converting enzyme 2) receptor and infects humans, bats, and palm civets. We learned about SARS-CoV-1 in 2003.  There are seven coronaviruses that infect humans. In the 2003 SARS outbreak 9% of patients with confirmed cases died, the hardest hit population was over 60 and over 50% of these people died.

The evolution of  SARS-CoV is interesting.  There were two different strains of SARS-CoV isolated in China in 2003, indicating there were separate species crossing events.  The virus came from wild animals sold as food in a market in Guangdong, China and it was isolated from asymptomatic masked palm civets.  This virus was able to infect humans, raccoon dogs, ferret badgers, and domestic cats. The virus could not be maintained in tissue culture and it did not infect bats until the virus was altered in 2008 in a laboratory to contain a human receptor binding domain. That indicated to researchers that bats could be asymptomatic and serve as a natural reservoir for the virus. And a note for Julie, the raccoon dog, also known as the mangut, tanuki or neoguri, is a canid indigenous to East Asia. It is the only canid species that can climb trees.

The recent ancestor for all coronavirus existed in 8000 BCE. Some say the virus existed 55 million years ago. They coevolved with birds and bats. Bats are the reservoir for alpha and betacoronavirus  while birds serve the same role for the gamma and deltacoronavirus. The global range allowed evolution and dissemination of this virus family.

The path to human infection is from leaf-nose bats to horseshoe bats, civets, and finally to humans. Bovine coronavirus evolved from rodents and crossed species to equids. It was in the 1890’s when bovine coronavirus jumped to people, and the likely cause of the “flu” pandemic that same year.

Of interest to us is that human corona virus (OC43) causes respiratory infections and is suspected of playing a role in neurological diseases. Mouse hepatitis virus (MHV) is a coronavirus that causes epidemic murine illness that has a high mortality.  Prior to the discovery of SARS-CoV, MHV had been the best-studied coronavirus both in vivo (in the animal) and in vitro (in tissue culture) as well as at the molecular level. Some strains of MHV cause a progressive demyelinating encephalitis in mice which has been used as a murine model for multiple sclerosis.

Human coronaviruses vary in risk factors and disease severity.  Some (MERS-CoV) are deadly to more than 30% of those infected and others just cause an irritating, common cold.The human coronavirus discovered in 2003, SARS-CoV-1 has a unique pathogenesis because it causes both upper and lower respiratory track infections.

Six species of human coronaviruses are known.  One species is subdivided into two different strains, making seven strains of human coronaviruses altogether. Four of these strains continually circulate in the human population and produce the generally mild symptoms of the common cold: OC43, HKU1, HCoV-229E, NL63. These viruses cause about 15% of commons colds (the majority of colds are infections caused by rhinoviruses).

Four coronavirus strains have a seasonal incidence occurring in the winter in temperate climates.  There is no preference towards a particular season in the tropics.  Three strains (two species) produce symptoms that are potentially severe; all three of these are β-CoV strains: MERS-CoV, SARS-CoV-1, SARS-CoV-2. The two SARS-CoV strains have occurred in the last 17 years, both from Chinese wet markets.

How does all this affect us? The Center for Disease Control has updated its guidelines for essential workers that have been exposed to people infected with SARS-CoV-2. Workers that do not feel sick are able to return to work so long as they take their temperature before leaving the workplace, wear a face mask at all times, and practice social distancing.

The department of Homeland Security and Health and Human Services outlines how removing shelter-in-place type restrictions after 30 days would lead to a second, very high peak in the number of cases and deaths.  The number of expected deaths is 300,000. The spike in deaths was projected to occur abut 150 days after lifting the stay-at-home restrictions. We are staying put so you can expect more Zoom meetings.

Its concerning that United States hospitals are seeing a shortage of antibiotics, antivirals, and sedatives required by patients on ventilators.  Increased demand and effects of the pandemic has halted production of some drugs. The University of Minnesota has analyzed the supply chain and identified 156 drugs that could go into shortage in the next 90 days, but they have not released the list.

The numerous studies and information on chloroquine on the outcome of COVID-19 patients are so far inconclusive or halted due to a high number of complications. A vaccine trial, called the Solidarity Vaccine Trial will evaluate multiple candidate vaccines at the same time, against a placebo. The expectation is the researchers will have results in 3-6 months due to high enrollment and an adaptive design. We will be in line for that vaccine when it is ready.

Pathogenes laboratory wishes you and your families well.  We will continue to be here to help you with your horses by testing sera, developing new avenues of research and answering your questions about neurodegenerative diseases. Give us a call or join us through Zoom meetings.

It will soon be our 20th Anniversary at Pathogenes!  We’ve paved new ground and published most of our work because I grew up in the “publish or perish” era.  Now days science is all about technology transfer, it’s “patent or perish”! You may have participated in some things we invented that didn’t get off the ground, looking at down-regulation of molecules in response to S. neurona infection. I never cease to be amazed that this assay predicted the degree of illness a horse would have in a few months. It was expensive and cumbersome to run and ultimately discarded.  How about our horse-side antibody assay?  Who could predict that would strike out?  In a mere 15 minutes you knew if antibodies were present in serum or CSF.
I loved that one. Here is what it looked like:Dipstick assay

We took some side trips, identifying a cell that could support the replication of millions of PRRS virus in vitro, we grew 7 species of Eimeria in cell cultures resulting in a vaccine that would eliminate all those hen houses, and developed monoclonal antibodies, one  against S. suis and provided a local University with a much needed pig-herd vaccine. We collaborated with folks in Germany (looking at pigeon sarcocystosis), Norway (an acquired polyneuritis in Norwegian horses) and Canada. Sarcocystosis and Toxoplasmosis in dogs and cats didn’t escape our interest. But our heart belongs to people dealing with neuromuscular diseases in horses. We remember Amy and Ty from years ago, their picture shows the bond we have with our horses.

We have a paper, in press, that blazes a new trail for others to follow.  The paper explains the rationale for detecting S. fayeri toxin in horses, if you want the punch line--we detect cysts in live horses! We aren’t giving up our quest for new treatments or determining how disease progresses in horses, mouse or man. Horses have always led the field in neuromuscular research, spinal nerves were an important source of myelin for research in Myasthenia Gravis and Guillain Barre syndrome.  As most of you know, we got our ideas and direction from the work done in the 1980’s.  Wish we could say it was our idea and our hard work, but really, we  just pieced together a logical story from published literature.

Our current  directions are identifying the inherited genes that predict late onset ataxia in horses and defining the expressed protein environment in response to specific treatments given to animals with neurologic disease.  We are interested in the molecular targets of drugs in the nervous system and why they act for weeks after a dose.  We are surprisingly close to figuring that one out! You'll need special tubes to participate in these studies, contact us to find out if you can be a part of this research.

What stands out in our work is our ability to give clinicians information about their case and how it compares to hundreds of similar cases across the country.  We document all the information derived from our assays and connect the data to the feedback we get from you.  It’s time to catch up and let us know about your horse.  Even if our last contact was years and years ago, you are still in our system and gentle on our minds. Why, we may even have a picture of your horse if you sent one! We’d like to hear from you.  Here are some useful links:

To give us your update please use this form:  https://docs.google.com/forms/d/e/1FAIpQLSc_4HNIEtaCYI4qgt8X99OHypTSOhaJYv0iXO4kXJZ0hqIUPA/viewform?usp=sf_link

A veterinarian can give us information about a horse with neurological disease: https://docs.google.com/forms/d/e/1FAIpQLSeLU8t3ROPE9nrPjhSuReg4C2ZpmsCrYPyzmMQ8yuwVgMfong/viewform?usp=sf_link

A horse owner can give us information about a horse with neurological disease: https://docs.google.com/forms/d/e/1FAIpQLScB0jNG9dHpnNc2AhOIn8KpNVxMFc4QZ2YvSIJHyrh86hZAFQ/viewform?usp=sf_link

If your horse was treated we’d like a post-treatment update: https://docs.google.com/forms/d/e/1FAIpQLSevDKLdGqXFdr_JrH2W8h_4xbgBYJAxXH4Ydt-_vKUjlffy5A/viewform?usp=sf_link

If you suspect polyneuritis equi this form is appropriate:https://docs.google.com/forms/d/e/1FAIpQLScv4wQlg1pW13VPCuZHaG3yGsxQDnF5C2FrHkjgCH7mrT6Swg/viewform?usp=sf_link

We have been a bit quiet lately and that’s because a friend is very sick.  He has amyotrophic lateral sclerosis (ALS). In order to help him, the Pathogenes team needed to catch up on the particulars of this most horrible disease.  There is no cure for ALS and quite frankly, no useful treatments.  It has taken us a few months to read stacks of papers and gather a team of experts. And form a plan.  Our plan isn’t simple and it isn’t easy.  It’s complicated and can change.  As we formulate and fine tune our approach to stopping the progression of ALS our experts review our work for scientific accuracy and feasibility. We don’t mind that no one has heard of what we propose.  We will test our hypothesis and march forward.

You can find a lot of information on ALS on the Web and we won’t repeat it here.  You can check out our ALS tab for some links we found useful. It is worth pointing out that there is familial ALS (fALS) in which genetics plays a big part.  Only 10-15% of patients with ALS (PALS)…these folks are acronym heavy…get fALS.  Most people get spontaneous ALS (sALS), close to 90%.  There are different camps and controversies concerning the pathways involved in disease and how to approach reversing the progress of motor neuron (MN) death, but an overriding theme of those that investigate ALS is the compassion and sharing of information.  Basically, ALS is associated with an enzyme mutation (superoxide dismutase, SOD) and/or other mutations that cause MN death.  Motor neurons make muscles work. You need motor neurons to breathe.

What stirred us to take a break from our bench work and communicate with our EPM-centric following is learning that dogs get spontaneous ALS!   Dog-ALS is associated with the enzyme superoxide dismutase, the mutation is in SOD1.  The onset of dog disease is late in life.  That means there are ALS cases in people, dogs, and genetically engineered mice.  Horsey people realize there are unknown causes of spontaneous neuromuscular disease in horses that cause them to be wobbly.  These horses can progress until they can’t get around and are euthanized.  In some cases there is no diagnosis and no treatment.  We reviewed two of these diseases.

Equine Motor Neuron Disease (EMD) is an acquired neurodegenerative disease in horses affecting the lower motor neurons of adult horses. The disease is characterized by the onset of abnormal nerve function and muscle wasting resulting from the deterioration of motor neurons and myopathy. Horses from 15 months to 25 years old can get EMD. EMD is considered to be a multifactorial disease, however a dietary deficiency in vitamin E is considered to be a major predisposing factor in its development. This is largely related to when horses have a decreased antioxidant capacity leading  to accumulation of free radials and that results in oxidative damage to the ventral motor neuron cells. Could a decreased function of horse-SOD be a factor?

No one knows what causes Equine degenerative myeloencephalopathy (EDM) that is a diffuse, degenerative disease which primarily causes damage to the horse’s spinal cord. EDM is considered to be an advanced form of neuron-axon dystrophy. EDM may have a genetic basis. Horses can develop EDM and equine motor neuron disease (EMD) at the same time and in association with an underlying vitamin E deficiency. Horses with EDM show clinical signs of a general proprioceptive  ataxia-“I don’t know where my feet are” and an abnormal base-wide stance while at rest. Horses will usually start to show signs of EDM when they are 6 to 12 months old. Horses with mild cases of EDM may present as performance-related problems. At first the condition produces subtle signs, being nothing more than  "clumsy" but ataxia slowly progresses as clinical signs are usually slow and insidious. Ataxia signs will become more apparent and worsen over time. Paralysis and spastic muscular movements will become more evident, until late stages where the horse is unable to get up from laying down without assistance. The only way to get a definite diagnosis that a horse has EDM is by conducting post-mortem examination shortly after death.

We’d like to test horses for EMD and EDM for antibody against neurofilaments.  It’s a serum test.   Once we have some results we will share them with everyone.  If we can demonstrate that horses also have a form of ALS, and why shouldn’t they?, we can start looking at treatments in this species.  If you have a horse with a diagnosis of EMD or EDM send us serum and we will test it.  Be sure and have a firm diagnosis.  Not just a “This is on the differential” or “It’s nothing I’ve seen before!”. Testing is expensive and we’re proposing to pay for it.  We need to know and have proof that several diseases have been ruled out.  You can email us for the form to send in a sample for this specific testing until we put a submission form up on our site. We want late cases as well as cases that are early.

The funny thing is that the deeper we delve into ALS and our approach to treatment, some paths seem to converge. As the ALS community gets closer to understanding the pathophysiology of disease we are finding common roads.  All roads seem to be leading to Rome after all.

At some later date when we have some good news for your ALS friends we will ask you to share what we find.  Until then, we are going back to work.

Jim

 

Muscle fasciculations are visible, fine and fast contractions of fine muscle fibers that occur spontaneously and intermittently. Injury to central or peripheral nerves can result in muscle fasciculations. The pathophysiology is different for different sites of the injury. It is thought that most fasciculations have a location away from (distal origin) the motor nerve in normal animals as well as patients with motor neuron diseases.

Fasciculations are known to be associated with hypersensitivity of denervated muscles and they are observed in some diseases such as amyotrophic lateral sclerosis in people. In horses the fine tremor of the face, muzzle, or lips is best associated with West Nile Virus infection.

Other triggers of fasciculations are progressive spinal muscular atrophy, neuromuscular junction disorders, electrolyte disorders, systemic diseases and some medications. Even healthy animals can have fasciculations-these are usually located in the forearm or the eye-lids. Fasciculations were thought to be a prelude to the onset of a progressive or lethal disease that involved the lower motor neuron. However, benign fascicular syndrome has been described in young healthcare professionals.

In normal individuals physical exercise, stress, fatigue, and caffeine abuse can cause or aggravate twitchy muscles. A diagnosis of benign fascicular syndrome can be diagnosed after five years. A benign diagnosis is made when there isn’t a progression to motor neuron disease and that takes 5 years.

Muscle tremors are abnormal and are motor disorders, although fasciculations are not classified as motor disorders. Some genetic diseases of the cerebellum associated with motor disorders are accompanied by fasciculations. A specific type of fasciculation with cramping occurs in peripheral axonal excitability. This occurs when adjacent neurons (to the damaged neuron) begin to re innervate partially denervated muscles. Sometimes in genetic disease, fasciculations affect the tongue as well as the trunk and limb muscles. The lower motor neurons are involved in these cases.

Rare cases of fasciculations occur when muscles fail to relax. Failure of muscle fiber relaxation can occur with neoplasia, immune-mediated disease, heredity, and degenerative disease. In these cases, the pathophysiology is hyperexcitability of the peripheral nerves and consequent continuous muscle fiber activity. Continuous activity occurs when potassium channels are dysfunctional. Potassium channels can be damaged when there is an antibody response against proteins in this structure. Cramps, stiffness, delayed muscle relaxation and excessive sweating can be seen clinically. Motor neuron disease is associated with fasciculations in people. These diseases are detected using EMG’s, electromyography, this machine measures the muscle response or electrical activity in response to nerve stimulation of a specific muscle. During the test, one or more small needles (also called electrodes) are inserted through the skin into the muscle.

Systemic disease, drugs and heavy metal toxicity (like lead) can also induce fasciculations. Low blood levels of phosphate (hypophosphatemia) and calcium disorders (secondary to hyperparathyroidism) sometimes result in fasciculations. Calcium disorders occur with some renal diseases. Neostigmine, a drug, can increase fasciculations in cats by increasing the concentration of acetylcholine (a direct effect) in the concentration of acetylcholine at the neuromuscular junction. Some anesthetic agents work using the same pathway at the neuromuscular junction and have the same results. Mercury toxicity should be considered in peripheral neuropathies of unknown origin that are also accompanied by tremor, ataxia, and depression.

There is no specific treatment for the muscle contractions because fasciculations are a symptom of an underlying condition. It is necessary to identify the origin of the underlying condition and that condition is the therapeutic target.

Recently JAVMA published a letter to the editor and we’d like to give the author kudos!  He was disappointed that after years of using a particular product, a clinical trial revealed the drug was not effective.  He placed his faith on recommendations from trusted and respected colleagues, board certified specialists and continuing education speakers. They didn’t base their opinions on clinical data and that led to years of using a drug for pain in dogs that was just shown to have a complete lack of effect.  We all recognize the importance of new scientific evidence, lack of funding for many projects, and the burden on the people that provide treatment recommendations to know and understand the supporting data- or lack thereof-before dissemination of their opinion.  We concur with the statement in response to the letter that  many clinicians, if they looked at the information used in the daily treatment of patients, would likely be shocked to find out just how little clinical data are truly available to support current recommendations, or how many opinions are not based on experience or understanding-just a conflict of opinion.

We have taken a different path with our work.   Rather than patenting the intellectual property (IP) and  then licensing the technology, on completing research we decided to make our work available to those who needed it, and open the discussion.  Has it cost us in many ways?  Yes.  But then, I grew up in the scientific community at a different time.  Once upon a time, that would be the 1970’s, we shared data and discovery.  I was a lab rat back then, spending many hours with the fluorescent microscope-looking at how Chinese hamster ovary cells responded to ATP stimulation.  OK, I get it.  But the experience was good.  We shared samples and “stuff” that moved knowledge forward.  I used that experience to look at Leptospira and Moon Blindness, another immune mediated condition, for my Masters thesis.  Then off to veterinary school.  While I was practicing veterinary medicine in the field over the next 20 years, things changed.  There was the biochemical revolution that spawned the field of molecular biology, and PCR, and proteomics.  Suddenly things like PCR, had monumental value.  Everything was DNA and  genes.  Heck, whole organisms are patented-Neospora comes to mind, (quick update, the patent office won’t do that anymore).  To use the IP, one has to have a license and pay royalties.  The revolution was in the 80”s to the 90’s, the patent for PCR was filed on June 17, 1987.

When I returned in 1999 to work on my PhD, in molecular biology of course, everything went through the University’s Office of Licensing and Technology (OLT).  Universities recognized the value of IP.  No more sharing.  No more shared discovery.  Heck, recently I wanted to use a video to help veterinarians get CSF taps using a standing procedure.  I was told there would be a largish up front fee followed by a royalty due for each use…one fee for every veterinarian that viewed the tape. It was explained to me the video is like a Beatle’s song-and they wanted to hold my hand.  I made my own video and freely gave it out hoping to get veterinarians to collect CSF and support our Orogin trial. Oh, we were criticized, one veterinarian wanted the video removed and not shared because all those horse owners would be getting CSF taps on their own horses.

We persevered, partially due to the words of one of my PhD professors, Ellis Greiner, “Science is self-correcting”.  If I continued to do work, collaborate with those with true curiosity and an open mind, we would eventually see the day when our ideas would receive acknowledgement and open discussion.  Because that is what science is, open discussion of ideas. To that end we publish our work, in peer reviewed journals, and submit our papers and abstracts for presentation at scientific meetings.  We collected and shared samples, (DNA, RNA, organisms), and imparted ideas, traveled to NIH and Washington and beyond; we teleconferenced, (with Germany, the Netherlands, Japan, and even Kentucky), with those who  were open to the burden of review.  There was plenty of criticism from those who didn’t take the time to understand, review and hold an open discussion.  We carefully persevered, leaving breadcrumbs.  Sure, we patented some IP.  And made some IP  public so that  others can’t patent it.  The IP is available to everyone.

I think the long awaited day is dawning.  The discussion at the upcoming EPM special information session will have a panel discussion on the evidence of the role of S. neurona in infection and disease.  Is it a disease of low parasite burden-host mediated pro-inflammatory response or is disease a reflection of high parasite burden resulting in direct injury to the CNS? they ask.  We are presenting two papers, the most important is our recent data on polyneuritis equi.  Our work is important because our evidence supports, and may identify, one inflammatory pathway for disease.  The story we tell is one of relapsing disease and hope of treatment.  Our story identifies a protagonist molecule and the hero horse heading off with all four legs moving in proper order.  We look forward to SIG participants that will critically review what we have done.  We welcome suggestions or guidance on our errant interpretations. Perhaps some will return to their laboratories and repeat our work. Or collaborate without IP on the mind.  They may claim no funding-we are used to that because we are self-funded.  No big grants, although we’d gladly accept one.*  We are optimistic that this may be the moment.  We’ll let you know Dr. Greiner, and if our time has not come, we will continue to drop those breadcrumbs.

 

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*Disclaimer: We are a for profit company.  We do not accept tax-exempt donations. Nor do we accept generously offered dowry’s from grateful horse-owning newly weds-although we gratefully appreciated the gesture.