Sunday, April 27, 2014

0 Part 2: Sexual Transmission Of Lyme Disease - Is There Evidence?

Syrian hamsters mating. From How to Breed
Syrian Hamsters
on wikiHow.
This article is the continuation of "Part 1: Sexual Transmission Of Lyme Disease - Is There Evidence?", our series on sexual transmission and contact studies involving Borrelia burgdorferi.

To review the content of the first article briefly: We discussed three studies which are often used online to support the idea that Lyme disease could be a sexually transmitted disease between human patients. While two of those studies provided some evidence patients had spirochetal DNA in their bodily fluids, none of the studies provided evidence that Lyme disease is sexually transmitted by people. Setting aside the yet-to-be available Middelveen et al study, there are no studies to date which provide evidence that Lyme disease can be sexually transmitted in humans and there are very few studies which investigate this method of transmission in animals. We also had an introduction to Dr. Elizabeth Burgess' Lyme disease transmission studies, the criticism from Dr. Burgdorfer which they received, and took a glance at her study findings.

In Part 2 of this series, we'll be looking more closely at the results of  Dr. Burgess' studies and that of other researchers on contact transmission and the potential for sexual transmission of Lyme disease in animals.

The Tally So Far: Studies On Lyme Disease Sexual Transmission And Contact Transmission

So far, I have not located any studies on Lyme disease and sexual transmission in humans, and found only a few studies about human sexual secretions containing Borrelia burgdorferi DNA.

Is this lack of research on this method of transmission in people an oversight by researchers who are more focused on observing tickborne transmission? Or has the dominant view of researchers been that based on Lyme disease's pathogenesis, human sexual transmission of Borrelia burgdorferi is highly unlikely if not impossible - so why conduct more studies on it? Either way one answers, there is very little research on different transmission methods of Lyme disease in people.

What we do have are early animal studies which were designed to investigate the nature of Lyme disease transmission through various methods - methods such as: how much more effective needle inoculation was compared to tick bites in transmitting infection; injecting mice with the urine of infected cattle to see if they would get infected; a few studies where it was hoped animals would copulate and that one could determine if sexual transmission could occur.

To date, there are more animal studies which are about the potential for contact transmission of Borrelia burgdorferi -which means any kind of contact between animals either directly or indirectly via exposure to their blood, saliva, feces, urine, milk, or colostrum - than there are studies specifically about sexual or venereal transmission. It is the results of these early animal studies which were hoped would give one a better idea of whether or not other non-tickborne methods of transmission might also be possible in people, too.

Given the wide range of data available from different animal model transmission studies over a period of nearly fifteen years, I've decided to place them into a table to view them all in one place.

In general, the study data in the table below fall under the following five categories:
  1. Evidence of the presence or absence of Borrelia burgdorferi in different bodily fluids of animals. These are not transmission studies. These are studies which demonstrate spirochetes or spirochetal DNA can be found in various secretions, but they do not show that such spirochetes or infection can be passed on to another host animal.

  2. Evidence that the use of needle inoculation/subcutaneous injection of infected bodily fluids from animals can transmit Borrelia burgdorferi to uninfected animals and a tick was not required.

  3. Evidence pointing to oral inoculation as a potential route of transmission. These are studies where the researcher deliberately tried to infect an animal with Borrelia burgdorferi (or a substance assumed to contain it) either orally or oronasally, then looked at the results.

  4. Evidence supporting or rejecting sexual (venereal) transmission of Borrelia burgdorferi between studied animals.

  5. Evidence suggesting that an uninfected animal became infected somehow by being housed in the same space as an animal already infected with Borrelia burgdorferi. These studies suggest that contact transmission may have occurred because no direct action was taken by the researcher to infect the control/uninfected animal, and no ticks were deliberately introduced to the control/uninfected animal.
For the purpose of focusing discussion, the table below mostly displays information related to the possibility of oral, contact, and sexual (venereal) transmission and not much transmission data via needle inoculation (though some of the studies also focus on that method of transmission and may be mentioned in passing).

Table 1: Evidence For The Presence of Borrelia Burgdorferi Infection Via Different Forms of Inoculation and Potential Contact Transmission

Year Publication Oral
Inoculation

Contact

Spirochetal
DNA
Whole Cell
Spirochetes

Antibody
Test
Culture/
PCR
Status
1986 Experimental inoculation of dogs with Borrelia burgdorferi N


Y


n/a


N


IFA+ n=1/1
contact 
exposed dog
no PCR
completed
1986 Experimental
inoculation of Peromyscus spp. with Borrelia burgdorferi:
evidence of contact transmission
N


Y


n/a


+ n=1/1 blood,
contact mouse


Exp 1: IFA+
n=2/2 contact mice;
4 log2, 6 log2
Exp 2: +n=10/10 contact mice
4 log2-6 log2

Burgess speculates local IgA reaction possible instead of systemic IgG
no PCR
completed


1986 The prevalence
and significance of Borrelia burgdorferi in the urine of feral reservoir hosts


n/a


n/a


n/a


+ n=2 urine,
via darkfield
+n=21/22 kidneys, positive correlation w/ Babesia presence
n/a


no PCR
completed


1986 Suspected borreliosis in cattle Y
cat fed
infected milk


n/a


n/a


+ n=2/6 urine, via darkfield


IFA+

+n=1/1 cat fed infected milk had 1:164 titer
+n=5/5 mice 1:8-1:32 titers with sc inoculation of milk
+n=5/5 mice 1:32-1:64 titers with sc inoculation of urine
- cultures
negative in blood, urine, and milk


no PCR completed


1987 Oral infection of Peromyscus maniculatus with Borrelia burgdorferi and subsequent transmission by Ixodes dammini Y


N


n/a


+ n=1/10 blood,
+ n=1/10 organs
+n=1/10 blood (tick-fed)
+n=1/10 organs (tick-fed)


IFA+
+n=10/10
orally infected mice
+n=10/10 mice from ticks that fed on orally infected mice; 6 orally infected mice developed symptoms
no PCR completed





1988 The urinary bladder, a consistent source of Borrelia burgdorferi in experimentally
infected white-footed mice (Peromyscus leucopus)
n/a


N


n/a


- 0/15 urine samples
+ 2/15 blood samples
+ tissue samples: bladder, kidney, spleen
n/a


+ tissue culture, no PCR


1988 Clinical and serologic evaluations of induced Borrelia burgdorferi infection in dogs. n/a


Y


n/a


n/a


IFA antibody +
ELISA +
n=1 control dog elevated + IgG
no PCR completed


1988 Borrelia burgdorferi
infection in Wisconsin horses and cows
Y
n=3 mice inoculated with cow urine
n/a


n/a


+ n=2/10 cow urine
+ n=2/3 colostrum
- all milk samples
IFA+ IgG
n=2/3 colostrum titer, n=1/3 cows ~1:512
n=2/3 mice 1:64; 1:28
samples cultured,
no PCR completed


1989 Experimental inoculation of mallard ducks (Anas
platyrhynchos) with Borrelia burgdorferi

Y
n=4 ducks

n/a


n/a


+ n=1/4 cloaca secretion,
+ n=1/4 kidney
IFA+ n=3/4 kidneys of orally inoculated ducks; +n=1/4
mesentery
positive cultures, no PCR completed
1990 Experimental infection of the white-footed mouse with Borrelia burgdorferi Y

n/a

n/a

+ n=1 blood

IFA+
all asymptomatic mice
tissue culture, no PCR completed
1991


Experimental infection of dogs with Borrelia burgdorferi N

Y


n/a


+ n=1 urine, blood of contact dog IFA+
+n=1 uninoculated dog near 1 infected dog
both asymptomatic
no PCR completed




1991

Relative infectivity of Borrelia burgdorferi in Lewis rats by various routes of inoculation Y
oronasally




N


n/a


- n=0/13 oronsally infected
- n=0/6 males venereally
- n=0/7 females venereally
only needle inoculated rats + n/a
1992 Experimentally induced infection of cats with Borrelia burgdorferi Y
n=2

n/a

n/a

+ n=2 blood smear;
n=1 lung
n=2 orally inoculated IFA+
n=2 ocularly inoculated IFA+
no PCR completed

1993 Detection of Borrelia burgdorferi in Urine of Peromyscus leucopus by Inhibition
Enzyme-Linked Immunosorbent Assay
N


n/a


+n=57/87
whole cells or subunits in urine

+ n=57/87 whole cells or
subunits in urine


+IFA
+ELISA
+n=47/75 serum
+n=57/87 antigens in urine
+n=50/87 tissue culture;
+n=36/50 infected bladders
no PCR completed
1994 Experimental infection of dogs with Borrelia burgdorferi N

Y

n/a

+ n=1/4 infected dog blood, urine

IFA+
+n=1/1 control dog, 1:128 (max titer)
+n=2/2 guinea pigs inoculated with urine, 1:128
no PCR completed




1994

Distribution of Borrelia burgdorferi in host mice in Pennsylvania

n/a




Y
see notes part 3
n/a + n=112 isolations/1619 mice (from ear tissue)

IFA+ in tick-bitten mice
see notes
part 3
no PCR completed
1996 

Lyme Borreliosis in the laboratory mouse



-


- - - - see below -
1996 Dissemination of Borrelia burgdorferi after experimental infection in dogs N n/a
- n=0/6 urine culture
- culture bladder
PCR completed
- urine
1997

Tick-raccoon associations and the potential for Lyme disease spirochete transmission in the coastal plain of North
Carolina
n/a Y
see notes part 3


n/a n/a IFA+
see notes
part 3
no PCR was completed
1998 Viable Borrelia burgdorferi in the urine of two clinically normal horses n/a

n/a

n/a

+n=2/5 horses, urine +


+PCR FA on urine samples
1999 Investigation of venereal, transplacental, and contact transmission of
the Lyme disease spirochete, Borrelia burgdorferi, in Syrian
hamsters
N


N


n/a


-n=0/6 female hamsters,
-n=0/6 male hamsters infected
venereally
no mice contact infected
- IFA in 6/6 venereal test hamsters
and all contact mice
- tissue samples, no PCR was
completed


Table Key:

+ positive - negative

n= #/# number of samples or study animals infected or not infected out of the total studied

n/a not applicable to study or no data available in given paper


Notes On The Above Table

While every effort was made for this table to be comprehensive, it may not be complete. (If you know of any additional publications and data which apply, please comment below so that I may add the paper to this table.)

Note that certain papers which have been cited online pointing to different methods of contact transmission or evidence of Borrelia burgdorferi spirochetes or spirochetal DNA in bodily fluids were not included in the above table. This is because these specific papers were either review or editorial papers which cited more than one study already included in the above table, and I did not want to give the impression that additional studies were completed where the studies and findings were reproduced by another researcher. (I do, however, mention these papers in part 3 of this series on sexual transmission.)

Note also that in certain columns data is lacking or relies on older methods:

In some studies, antibody testing was not completed on animals because the research goal was to focus on finding evidence of Borrelia DNA and/or spirochetes in a given sample and not to measure antibodies in the host animal.

In the 1980's and early 1990's, PCR studies were not performed on many samples. This is because at the time PCR was a relatively recent invention, was at first a slow and labor intensive process, and not many researchers had access to it. Also, there was some discussion over what the best method was to use on specific samples, and it hadn't been completely determined yet.

Immunofluorescent antibody (IFA) assay testing was the primary method of testing samples for the presence of antibodies, rather than ELISA or Western blot. This method has been considered less sensitive than others, but given studies listed use one or more monoclonal antibodies specific to Borrelia burgdorferi (such as H5332, specific to North American OspA) the positive results obtained are noteworthy.

So What Do These Studies Indicate or Suggest?

1.  Contact transmission of Borrelia burgdorferi could occur between specific animals under specific conditions.

While there is some evidence that some animals can be experimentally infected with Lyme disease through an oral or ocular route, it is unclear if animals end up infected through these routes in the natural world. It's important to note that dosages and the method used in a laboratory experiment may not have their parallel in real life conditions. Optimally, observing how infections occur in the wild would be best - but it is difficult to study animals in the wild to determine exactly when and how they get infected.

What the data in this table reflects is that there is some evidence to suggest that contact transmission via urine may be a concern with some animals - particularly cattle and mice. One study on ducks points to the possibility of oral transmission, though no one has duplicated it thus far or extended additional transmission research to other kinds of birds.

Based on the studies reviewed, if a form of contact transmission can occur in animals, it appears to happen inconsistently and in low numbers of animals - and so far appears to happen more frequently in specific species. Perhaps repeat studies with larger groups of animal subjects would be helpful - or perhaps they would demonstrate that contact transmission still only occurs in relatively few subjects under narrowly defined conditions. We just don't know.

There is also some evidence that certain animals are highly unlikely to contract Lyme disease via contact transmission via urine, such as Lewis rats and Syrian hamsters.

Additional autopsy studies in such cases that demonstrate where spirochetes are found in infected animals can be useful as they can indicate why spirochetes were not likely to be found in urine based on which tissues spirochetes colonized.

2.  In some cases, it is not exactly clear how contact animals were infected in individual studies. All we know is they had positive antibody tests and some showed signs of infection.

Based on the studies within the above table, the following studies resulted in at least a positive IFA result in a contact animal, and in some cases evidence of spirochetal infection:
  1. Experimental inoculation of dogs with Borrelia burgdorferi.
  2. Experimental inoculation of Peromyscus spp. with Borrelia burgdorferievidence of contact transmission.
  3. Clinical and serologic evaluations of induced Borrelia burgdorferi infection in dogs.
  4. Experimental infection of dogs with Borrelia burgdorferi.
  5. Distribution of Borrelia burgdorferi in host mice in Pennsylvania.
  6. Tick-raccoon associations and the potential for Lyme disease spirochete transmission in the coastal plain of North Carolina.
Of these studies, the last two listed above contained speculation by the researchers about whether or not mice and raccoons could contract Lyme disease through other methods outside of a tick bite - and neither of those two studies were completed under lab conditions and had control animals.

As for the remaining four studies, there is no indication exactly how uninfected control animals ended up with positive antibody tests and signs of infection when in the presence of infected animals. Apparently it has happened - but how and why it happened in a number of these studies even when animals are in captivity is unclear.

It is the unknown method of contact transmission in such studies which has led to speculation by others that sexual transmission may have occurred between animals. However, without a definite confirmation that sexual contact was the route of transmission, what happened remains a mystery.

We do not have documentation in a number of cases of whether or not infected and contact animals were housed together indoors or outdoors, or if they were isolated from birds or other environmental factors which may have introduced infected ticks or Borrelia burgdorferi. Without more data, it is difficult to determine whether or not animals may have been infected via a different route.

All the same, there is research above which indicates point #1 - that animals can be infected by other animals' secretions, although how often it happens and how is a good question. So far, urine and raw milk ingestion appears to be a plausible mode of transmission between some animals.

3.  There haven't been many animal studies specifically focused on sexual or venereal transmission.

Based on the studies within the above table, the following studies were specifically focused on sexual or venereal transmission and their results:

Title    Sexual or Venereal Transmission?
Investigation of venereal, transplacental, and contact
transmission of the Lyme disease spirochete,
Borrelia burgdorferi, in Syrian hamsters
    - negative
Relative infectivity of Borrelia burgdorferi
in Lewis rats by various routes of inoculation
    - negative
Lyme Borreliosis in the laboratory mouse    - negative

Of three papers where experiments were specifically designed to see if venereal transmission occurred between animals, the results of all three were negative.

Three seems like a very small number of studies, and they were only completed on rats, Syrian hamsters, and mice. If you, the reader, knows of any additional studies on sexual or venereal transmission of Lyme disease in animals which were not included here - please comment below with the title(s) and link(s) to the paper(s).

Some Researchers Weigh In On Non-Tick Methods Of Transmission

More has been said about the methods of transmission in mice than any other animal because mice are used more extensively in a lab environment for the study of Lyme disease than any other species.

In 1996, Dr. Stephen Barthold wrote "Lyme Borreliosis in the laboratory mouse"in the Journal of Spirochetal and Tick-Borne Disease. On pages 23-24, he states:
"There has been no evidence of contact transmission or detection of viable spirochetes in urine of laboratory mice. Lung, bladder, and kidney are frequently infected, but spirochetes in these tissues are present in the connective tissue of the serosa, subserosa, submucosa (bladder), and periarterial connective tissue (lung, kidney), rather than lumina of tubules, ureters, bladder, or airways."
Based on his research up to that point in time, mice were not transmitting spirochetes through their urine to uninfected mice, and spirochetes which were found in autopsies were deeply embedded in connective tissue and not lining the vessels or surface of ureters, bladders, or bronchial tubes. In other words, if live spirochetes were present, they would be highly unlikely to be found in urine.

His findings differ from those of Dr. Burgess, and those of Dr. Magnarelli.

Magnarelli et al in the paper, "Detection of Borrelia burgdorferi in Urine of Peromyscus leucopus by Inhibition Enzyme-Linked Immunosorbent Assay" determined that 57 out of 87 mice had either subunits or whole cell Borrelia burgdorferi in their urine.

Despite this high number, Magnarelli et al cited their own difficulties in detecting evidence of Lyme disease infection in mice consistently:
"For more than half of the field-collected mice tested, there was concordance in the results of the serum antibody, culture, and urine analyses. Similar results were records for four of five laboratory-bred mice inoculated with B. burgdorferi. However, there were discrepancies. B. burgdorferi antigens sometimes were detected in urine from field-collected mice without supportive data from antibody assays or culture work. This was particularly noticeable in animals captured during October and November, a period after peak nymphal I. scapularis population levels had been reached."
The authors also pointed out that antibody-positive serum and titers can be low especially during early infection, successful culturing of B. burgdorferi depended partially on the number of spirochetes present in host tissues, and that occasionally, serum antibody analyses and culturing results were positive, while urine antigen test results were negative - antigens may not always be released into urine.

In 2001, we have what may have been the last published words from Dr. Burgess on contact transmission in the book, "Infectious Diseases of Wild Mammals" published by Iowa State University Press, chapter 26. Authored by Dr. Richard Brown and Dr. Elizabeth Burgess, the chapter contains this quote:
"Although direct transmission may occur in some situations, it has not been easily substantiated (Mather et al 1991) and has yet to be shown as epidemiologically important." 
The authors supported the idea that direct transmission of spirochetes may occur between animals under certain circumstances - but it's not easy to provide evidence of what exactly happened and it occurs in only a very small number of cases.

More recently, in 2010, in the book, Borrelia: Molecular Biology, Host Interaction and Pathogenesis by Horizon Press, on p. 381, the most recent note on transmission stated:
"Mice are also susceptible to infection following intragastric inoculation of very high doses of B. burgdorferi N40, but there is no evidence for contact transmission (Barthold, 1991) or in utero (placental) transmission, although maternal infection may cause fetal death (Silver et al, 1995; Weis et al, 1997)."
Since our focus here is on contact, venereal, and oral transmission and not maternal/in utero (a topic worthy of a post in itself), it's notable that mice can be infected intragastrically based on Barthold's studies - though it takes high doses to do so, and it is a question how likely it is such doses would be found in nature. According to Burgess, Peromyscus are apparently susceptible to oral inoculation with B. burgdorferi, since oral infection with ~400 spirochetes resulted in sufficient spirochetemia to infect Ixodes (previously dammini) scapularis larvae.

That there has been a variety of study outcomes regarding mice, transmission, and inoculation routes leads to questions about whether or not it is not just species that matters when it comes to how Lyme disease affects the host - but also the individual genetic background of the host animal.

One should take note that anyone studying Borrelia burgdorferi in wild type mice should make sure they do not use mice which are naturally resistant to infection with Lyme disease due to a particular genetic variant of the antigen receptor TLR2, because such mice can greatly affect study outcome.

Could it be that the discrepancies Magnarelli et al found in their research was due in part to the presence of mice in their study group which had this genetic variant?

Future Directions

One question this raises for some reading along is how likely is it that Borrelia burgdorferi spirochetes can be found in human urine, and is there evidence of non-sexual transmission of Lyme disease between people? This is a question that will be discussed in an upcoming blog post, as there have been a number of studies on testing human urine samples for the presence of Lyme disease.

But the main question which kicked off this post is this one: Will we have any evidence in the near future that Borrelia burgdorferi can be sexually transmitted between partners?

So far, we do not, though a recent paper by Middelveen et al, "Isolation and Detection of Borrelia burgdorferi from human vaginal and seminal secretions" is the first publication to make the claim in its abstract that live motile spirochetes have been found in vaginal and seminal secretions, and that because two partners in the study share the same strain of Borrelia burgdorferi in their secretions, odds are greater than random chance that one partner passed Borrelia burgdorferi to the other.

For these claims, I currently have no evidence. And unfortunately, my ability to give the paper a thorough review is limited without access to the full text of the paper. Once the Middelveen et al paper is out of embargo and I have read the entire paper, it deserves its own future blog entry given how important this topic has become for many patients.

To sum up: Sexual transmission of tickborne diseases - is there evidence? Answer: Not so far. But existing studies to date provide evidence that oral inoculation and urine transmission may occur in a few cases in animals.

Questions For Further Discussion:
  • Why did different researchers have different results when it comes to the issue of contact transmission?

  • How much did animal species play a role in the outcome?

  • Why is it in a number of cases, uninfected animals exposed to infected animals had positive antibodies for Borrelia burgdorferi but were culture negative and no spirochetes could found in tissues post-mortem?

  • How did duration of untreated infection in the host relate to the odds of finding spirochetal DNA or a spirochete in a given sample?

  • How did the timing of testing of the host animal relate to the odds of finding spirochetal DNA or a spirochete in a given sample?

  • How do methods and materials and test conditions affect outcomes (e.g. urine acids can lyse cells) ?

  • How did the researchers demonstrate they had ruled out or eliminated other potential methods of transmission via experimental design?

  • What - if any - connection is there between nonsexual modes of Lyme disease transmission and a sexual one?

Coming up next:

Part 3: Sexual Transmission Of Lyme Disease - Is There Evidence?

The next post will include notes on some of the above studies mentioned in the table and
additional studies which have been cited when mentioning transmission methods of
Borrelia burgdorferi. The temperature at which Borrelia burgdorferi dies
off will also be discussed in relationship to pasteurization.



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