posted
I made mention of these findings and I have been asked to post the text here. It looks like these results were published in the August 2006 issue of the Journal of Parasitology. Heavy reading for the weekend. What I glean from this is that 50% of the mice transfused with contaminated blood were infected. All this without the need for ticks to bite anyone! ________________________________________________ Transfer of Borrelia Burgdorferi s.s. Infection via Blood Transfusion in a Murine Model Elizabeth S. Gabitzsch, Joseph Piesman, Marc C. Dolan, Christine M. Sykes, and Nordin S.Zeidner,
Centers for Disease Control and Prevention, Div. of Vector-Borne Infectious Diseases, Bacterial Zoonoses Branch, Foothills Campus, Fort Collins, Colorado 80522. e-mail: [email protected]
ABSTRACT: Without antibiotic treatment, the Lyme-disease-causing bacterium, Borrelia burgdorferi can be cultured from the peripheral blood of human patients nearly 6 wk post-tick bite. To determine if Lyme disease spirochetes can be transmitted from a spirochetemic donor mouse to a naive recipient during blood transfusion, blood taken from immunocompetent infected mice was transfused into either immunodeficient (SCID) mice, inbred immunocompetent animals (C3H/HeJ), or outbred mice. Nine of 19 (47.7%) immunodeficient mice, 7 of 15 (46.8%) inbred immunocompetent mice, and 6 of 10 (60.0%) outbred mice became infected with B. burgdorferi after transfusion. Our results indicate that it is possible to acquire B. burgdoferi infection via transfused blood in a mouse model of Lyme borreliosis.
Lyme disease is a systemic, tick-borne disease with clinical manifestations, which include dermatologic, rheumatologic, neurologic, and cardiac abnormalities. The causative agent of Lyme disease in the United States is the spirochete Borrelia burgdorferi sensu stricto, which has been isolated from cultures of blood from infected individuals early in the course of infection (Nadelman, Pavia et al., 1990). Depending upon the strain of B. burgdorferi, peak spirochetemia of the organism is reached between 7 and 10 days post-tick exposure (Dolan et al., 2004), but can be cultured from the blood of humans for close to 6 wk posttick infestation (Schmidt et al., 1989). Previous studies have demonstrated that whole blood inoculated with B. burgdorferi (strain B31), separated into red cell frozen plasma and platelet fractions, and stored under blood banking conditions, had cultivable spirochetes in both the red cell (4 C) and fresh-frozen plasma fractions (_18 C) for up to 45 days, and in platelet concentrates (20-24 C) over a 6-day period. (Badon et al., 1989) Multiple investigators have found that B. burgdorferi was able to survive the standard blood processing procedures applied to transfused blood in the United States (Baranton and Saint-Girons, 1987; Nadelman, Sherer et al., 1988). In this study, we examined whether B. burgdorferi can be transfused successfully from a spirochetemic donor to a naive recipient via citrated blood transfusion in a mouse model of Lyme borreliosis.
Infected donor mice were generated by placing 5 laboratory-reared nymphal Ixodes scapularis ticks infected with B. burgdorferi (strain B-31), raised as previously described (Piesman, 1993), on each C3H/HeJ (Jackson Laboratories, Bar Harbor, Maine) or Imperial Cancer Research Fund (ICR) mouse. The ticks were allowed to feed until repletion. Ten days post-tick infestation, a time point previously shown to be peak spirochetemia in C3H mice (Dolan et al., 2004), 400 l of sodium citrated blood was collected from donor mice by cardiac puncture. The citrate solution was prepared according to the American Association of Blood Banks technical manual and combined with blood at 14% (v/v) concentration (Brecher, 2005); 200 ul of the donor blood was placed in a microcentrifuge tube containing 20 ul of heparin for quantitative PCR (qPCR) analysis, and 200 ul (equivalent to 1 unit of blood transfused into a 150-kg patient) was transfused immediately into a recipient mouse via intravenous injection. In individual mice where intravenous inoculation was unsuccessful, an intraperitoneal injection was utilized. To quantify spirochetes, DNA was isolated from whole, heparinized donor blood with the use of a QIAGEN (Valencia, California) blood kit, and qPCR was run with the use of FLA primers and probe as previously described (Zeidner et al., 2001).
To test whether recipients with different levels of innate immunity could resist a spirochete transfusion, whole blood transfusions were performed in 3 study groups: (1) C3H/HeJ (Jackson Laboratories, Bar Harbor, Maine) infected donor mice transfused to severe combined immunodeficient (SCID-C3H/HeJ) recipient mice (Jackson Laboratories, Bar Harbor, Maine); (2) C3H/HeJ infected donor mice transfused to na�ve C3H/HeJ recipient mice; and (3) Imperial Cancer Research Fund (ICR) (obtained from a pathogen-free colony maintained at the Centers for Disease Control and Prevention [CDC], Fort Collins, Colorado) infected outbred donor mice to naive ICR outbred recipient mice.
Four weeks post-transfusion, ear biopsies were obtained from recipient mice and cultured in Barbor-Stoenner-Kelly medium (Barbour, 1984) to determine infection prevalence of B. burgdorferi (Sinsky and Piesman, 1989). Ear biopsies were obtained from donor mice 4 wk posttransfusion to confirm that recipient mice were transfused with infected blood. Recipients that received blood from an uninfected donor were removed from the study. Cultures of biopsied ears, hearts, and bladders were checked for the presence of spirochetes every 7 days for 4 wk by dark-field microscopy.
Blood transfusion from C3H/HeJ infected donor mice to SCID recipient mice resulted in 9 of 19 (47.7%) recipient mice becoming infected (Table I). Transfusion from infected C3H/HeJ donor mice to naive C3H/HeJ recipient mice resulted in 7 of 15 (46.6%) mice acquiring infection and transfusion from ICR donor mice to naive ICR recipient mice resulted in 6 of 10 (60%) becoming infected with B. burgdorferi (Table I). The number of spirochetes/milliliter delivered to the recipient mice did not affect successful transmission. The average number of bacteria/ milliliter of donor blood delivered to mice that acquired infection was only slightly higher (n 225) than that of donor blood delivered to mice who did not acquire an infection (n 212). Although a true threshold of spirochetes needed for successful transfusion could not be calculated in this study, as few as 22 spirochetes/milliliter blood were successful in adoptively transferring infection in C3H/HeJ mice (Table I). The method of delivery of blood, intravenous versus intraperitoneal injection did not appear to impact the likely success of transfusion (data not shown). _________________________________________________
TABLE I. Infectivity of blood transfused from tick-exposed donor mice to recipient mice.
Study Donors* Recipients* Spirochete load� 1(C3HtoSCID) 10/10 2/10 Average 177/ml (127-227) 2(C3HtoSCID) 9/10 7/9 Average 557/ml (368-968) 3(C3HtoC3H) 6/10 4/6 Average 160/ml (88-216) 4(C3HtoC3H) 9/10 3/9 Average 122/ml (22-543) 5(ICRtoICR) 10/10 6/10 NA�
* Number positive by culture and PCR. � Spirochete number determined by qPCR. � Spirochete load in outbred donors was below detectable level by qPCR, possibly due to their low sensitivity to hematogenous dissemination of spirochetes. _________________________________________________
Since the CDC initiated Lyme disease surveillance in 1982, there have been no documented cases of acquiring Lyme disease from blood transfusion and follow-up studies of patients who received units from donors that had previously been diagnosed with the disease have not documented that transmission occurred (Halkier-Sorensen et al., 1990; Bohme et al., 1992). Several explanations have been proposed for the apparent lack of blood transfusion acquired Lyme disease. Lyme borreliosis is characterized by a wide variety of nonspecific symptoms that often go unrecognized. The first obvious sign of infection is usually a circular rash, erythema migrans (EM), which appears 3-30 days postinfectious tick bite and occurs in only 70-80% of infected persons (CDC, 1997). It is possible that the introduction of B. burgdorferi intravenously may not result in the formation of an EM lesion, thus lessening the chances of successful diagnosis of disease. In addition, the short amount of time in which spirochetes remain at peak levels in the blood limits the likelihood of transmission by blood transfusion. The American Red Cross and the U.S. Food and Drug Administration request that persons with chronic illness due to Lyme disease refrain from donating blood as a precautionary measure. It is recommended that patients who have been treated with antibiotics, and who have recovered, can donate blood beginning 12 mo after the last dose of antibiotics.
Inherently, the risk of acquiring Lyme disease from a blood transfusion may be extremely low, given that hematogenous spread of the bacterium would occur very early in infection during a spirochetemic phase. Either this phase would pass without recognition, or symptomatic patients would probably seek treatment for ailments associated with the disease and, therefore, would not donate blood, minimizing the risk of transfusion-associated Lyme disease.
We are aware that our studies have been conducted only within an animal model of Lyme borreliosis and may not completely reflect what occurs in humans during acute infection via tick bite. However, given the positive transfusion data obtained in our murine model, especially the positive transfer of spirochetes to immune competent mice, the underlying mechanisms for successful B. burgdorferi transfusion warrants more extensive study.
The authors would like to thank Gabrielle Dietrich for expert technical assistance with tick feedings.
[ 09. December 2006, 04:27 PM: Message edited by: Roy Goodloe - Texas ]
Posts: 10 | From Lewisville, TX | Registered: Nov 2006
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dmc
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wow... Now need demonstrations in front of Red Cross blood drives.... Could have signs "I've got lyme, want my blood?"
and with the Red Cross's slogan, "Give the Gift of Life - Give Blood, comes with little critters at no extra charge."
Posts: 2675 | From ct, usa | Registered: Jan 2004
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Beverly
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Informative post Roy.
Yeah dmc, I had a blood transfusion in 1984, and I got much sicker afterwards..I know I got something from the blood.
My sister also gave blood a few times without knowing she had lyme. Whole thing makes me upset.
Posts: 6639 | From Michigan | Registered: Jun 2001
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-------------------- Neil Posts: 697 | From Tucson, AZ USA | Registered: Apr 2002
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mojo
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I've always wanted to be an organ donor but I made sure my family knows NOT to donoate my organs - I am not "CDC" positive so some dumb doctor may actually want them! I would hate to give this dreaded disease to anyone else, even if it means denying them an organ.
Posts: 1761 | From USA | Registered: May 2006
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Greatcod
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This is a very important piece of research, and justifies the caution the blood donation agencies applied to "Lymed" blood donors. The first experiemental evidence of infection by transfusion that I have seen. Thank you.
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quote:Originally posted by mojo: I've always wanted to be an organ donor but I made sure my family knows NOT to donoate my organs - I am not "CDC" positive so some dumb doctor may actually want them! I would hate to give this dreaded disease to anyone else, even if it means denying them an organ.
Same here!
-------------------- --Lymetutu-- Opinions, not medical advice! Posts: 96227 | From Texas | Registered: Feb 2001
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posted
Just to update everyone, I have contacted CDC and gotten a name of Hari Nakasi at the FDA as a contact to determine what they will do with this finding. CDC does research and studies but ANOTHER AGENCY (the kind of communications disconnect that makes me turn up the heat) namely the FDA, is responsible for the quality of our blood supply. No answer yet, but I have asked Dr. Gabitzsch (key contact on this CDC study) about further studies. Curious if they have looked at contaminated blood to see how long these little buggers exist under refrigeration.
To explain my background, I am not a Lyme victim. I work for a company selling biomedical microscopes. I showed a darkfield scope to a Hyperbaric lab that treats Lyme patients in Richardson, Texas. We looked at each patient's blood and saw live images of LOTS spirochetes wiggling inside of the blood cells. You talk about spooky! The blood cells were also clumped togther unlike those of us with normal blood. Yes, we had a poking fest taking blood samples from anyone who would sit still!
One patient had been diagnosed with Fibromyalgia years ago with no improvment. She was finally diagnosed with Lyme and the antibiotics were starting to help. My brain started wondering if she could donate blood. Inquiry at Carter Bloodcenters and American Red Cross found that she would be allowed since Fibramyalgia is not a blood-borne disease.
My concern is for myself and anyone who might have to go in for a procedure where a blood transfusion is necessary. It sounds weird but I would take one of my darkfield scopes in and inspect any blood they planned to use. Blood banks are not required to scope their blood, but as you can see in this research (last sentience in paragraph 5), they confirmed infection using DARKFIELD MICROSCOPES.
The chemistry tests being developed I've read are very inaccurate and are still under development. If this tool and can prevent the spread of this disease thru our blood supply, then the FDA needs to instruct all blood centers across the nation to start screening ALL of their blood packets.
I'm planning to attend a support group meeting in January where I hope to capture live images of spirochetes. Hopefully I'll be able to post these images to this website so people can see what is causing their suffering.
Posts: 10 | From Lewisville, TX | Registered: Nov 2006
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tdtid
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Roy,
Pictures would be wonderful.
You're able to see the spirochetes? Could this be used as a test for lyme disease?
Cathy
-------------------- "To Dream The Impossible Dream" Man of La Mancha Posts: 2638 | From New Hampshire | Registered: Oct 2006
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My doctor has been using Darkfield microscopy to see the buggers for ages. They are visable in most people, but the authorities say they are artefacts. Since when do artefacts move around quickly, enter cells, change shape, and grow over a period of days??
IMO, It can be a helpful tool to confirm a spirochetal infection.
Why is it allowed to be used for Syphillis, but not Lyme?
Have you tried leaving the blood over night? From what I have heard they multiply over night and are supposed to be more visable.
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Lyma Bean
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Hi Roy!
I sent you a private message about someone I think would love to assist you.
Take Care....Terri
-------------------- Posts: 1405 | From Plano, Texas | Registered: Dec 2001
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TX Lyme Mom
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Here's more on blood microscopy from the MP website. It shows what amateur patients can do with second-hand microscopes bought cheap on E-Bay.
Remember, other bacteria besides Borrelia can persist in CWD (cell wall deficient/divergent) forms though, so try not to get too hung up on only just one species of pathogens.
Consider also the mycoplasmas and the mycobacteria, to name just a couple of other bacterial species.
It's when they start hiding out intracellularly that they cause the most trouble though, because then they tend to "paralyze" the WBCs of the immune system at the same time as they are "parasitizing" it. That's why the MP program works so exceptionally well for a variety of chronic illnesses, besides just Lyme disease, such as sarcoidosis, the rheumatic diseases, etc. -- despite negative rumors to the contrary by uninformed folks who seek to discredit it.
TX Lyme Mom
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posted
quote:Originally posted by ChrisBtheLymie: Have you tried leaving the blood over night? From what I have heard they multiply over night and are supposed to be more visable.
Chris, It's not just leaving it overnight which makes the "keets" come out of hiding. You must use Andy Wright's technique of excluding oxygen. They come out of the RBCs and the WBCs when the oxygen content drops down into the anaerobic range.
What one does is to seal out oxygen from the sample by using a bit of vaseline on the edge of the slide and then using another slide as a cover slip. The critters come out as soon as the oxygen is depeleted in the tiny smear of blood. They come out as "pearls on a necklace", bound up in a biofilm, according to Andy Wright's motion picture slides.
Those slides are available on the DVDs of the Chicago conference via the MP website. (Click on the icon in the upper left hand corner of the homepage to request a copy. Fee charge for DVDs helps to support the non-profit ARF's educational fund.)
Similar microscopy photoes of this "chain of pearls necklace" are also available for free via the links in my post above.
Posts: 4563 | From TX | Registered: Sep 2002
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TX Lyme Mom
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quote:Originally posted by tdtid: Roy,
Pictures would be wonderful.
You're able to see the spirochetes? Could this be used as a test for lyme disease?
Cathy
Cathy, Yes, you can also see the spirochetal forms too, in addition to the more ubiquitous CWD forms, but you have to know how to find them. Andy Wright has photographed the spriochetal forms, but they aren't easy to capture.
I posted the info on how to obtain the DVDs, which include Andy Wright's motion picture photoes, from the Chicago MP conference in my message to Chris, directly above this post.
Sorry for the redundancy. I didn't see your question until I reviewed this entire topic, after posting my previous message to Chris.
Posts: 4563 | From TX | Registered: Sep 2002
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Lyma Bean
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Up for Roy
-------------------- Posts: 1405 | From Plano, Texas | Registered: Dec 2001
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bettyg
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Wonderful post and archives of blood links!
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treepatrol
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-------------------- Do unto others as you would have them do unto you. Remember Iam not a Doctor Just someone struggling like you with Tick Borne Diseases.
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Roy, can the spirochetes be specifically detected without confusing them with other organisms? If so, is this a good test then for seeing them and would it then be diagnostic for the disease? Also, can any other co-infections be seen?
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