Fetal Bovine Serum - Varicella Vax, some oral polio vax, Merck reported it
was in MMR to Ray Gallup , and in the former Rotavirus vaccine.......

"As reported May 23rd, 2001at the 101st General Meeting of the
American Society   for Microbiology, Nanobacteria has been found to be a
contaminant in previously   assumed-to-be-sterile medical products,
specifically IPV Polio Vaccine. Most   human biologicals and vaccines are
made in fetal bovine serum, a medium that is known to be contaminated
with nanobacteria. In order to prevent this problem in the future,  human
biological products must be made in Nano-Free Culture medium (filtered
first through 20 nanometer filters,Gamma-Irradiated with 150 megarads and
then heated to 90 degrees Centigrade for at least an hour to kill any
nanobacteria present)"

http://www.nanobaclabs.com/PageDisplay.asp?p1=6578

excerpts......
"The  term “Nanobacteria" is short for it’s scientific genus &
species name "Nanobacterium sanguineum", a Latin scientific term for blood
nanobacteria.       Nanobacteria are “nano”-sized in that they are from
20-200 nanometers       in size (a nanometer is 1 billionth of a meter. A
nanometer is the width of ten hydrogen atoms side-to-side!) and are the
smallest known self-replicating bacteria. They are from the Archaea Family
of bacteria, known for their primitive pleomorphic lifestyles"

"Nanobacteria infection by Nanobacterium sanguineum is an “emerging
infectious disease” meaning that it       is newly discovered and that the
diseases it cause are being researched and further described. Its DNA, RNA
and Lipopolysaccaride profiles have been accurately mapped by multiple
scientific researchers at many universities worldwide. Nanobacteria are
not nice bugs and have absolutely no known positive benefits to humans. The
discoverers of nanobacteria, Drs. Ciftcioglu & Kajander developed antigen &
antibody diagnostic blood testing for nanobacterial infections that we
offer as the "NanobacTEST". NanobacLabs has developed safe and effective
nanobiotic prescription treatments"

"Nanobacteria are extremely small, slowly growing bacteria that can be
cultured from       the blood of humans and mammals.</b> Their size is
20-200 nanometers....when compared to “regular” bacteria, Nanobacteria are
1/100 to 1/1,000 the size, allowing them to       easily move around into
other cells and invade them. Nanobacteria cause apoptosis        (cell
death) when exposed to human cells or other bacteria. They can cause
alteration       of RNA and DNA gene-expression patterns of cells they
infect.....this can lead to genetic alteration, abnormal cell growth and
proliferation. When compared to other bacteria, Nanobacteria        grow
very, very slowly, only reproducing every 3 days…..where ”regular”
bacteria reproduce in minutes or hours. Nanobacteria cannot be grown in
  standard culture media and can only be grown in mammalian blood or serum.
      Nanobacteria were discovered in 1988 by Nobel Prize Nominees Dr. Neva
Ciftcioglu,       PhD and Olavi Kajander, MD, PhD as a “contaminant” in
mammalian       cell cultures that kept killing the mammalian cells
(apoptosis) in their       mammalian cell culture research. They have been
researching nanobacterial pathophysiology for nearly 14 years now and are
the worldwide experts on nanobacterial basic science. They are currently
guiding and teaching researchers all over the world. NanobacLabs is the
world leader in the research and development of prescription NANOBIOTICS
that eradicate pathological calcification and nanobacterial infections"


"As reported May 23rd, 2001</b> at the 101st General Meeting of the
American Society   for Microbiology, Nanobacteria has been found to be a
contaminant in previously   assumed-to-be-sterile medical products,
specifically IPV Polio Vaccine. Most   human biologicals and vaccines are
made in fetal bovine serum, a medium that   is known to be contaminated
with nanobacteria. In order to prevent this problem in the future,   human
biological products must be made in Nano-Free Culture medium (filtered
first through 20 nanometer filters,Gamma-Irradiated with 150 megarads and
then heated to 90 degrees Centigrade for at least an hour to kill any
nanobacteria present)"

********
http://www.uku.fi/~kajander/

http://www.uku.fi/~kajander/fatal.html
Mol. Biol. Cell, Suppl., Vol. 7, (1996): 517a
Fatal (fetal) bovine serum: discovery of Nanobacteria
E. Olavi Kajander, Ilpo Kuronen and Neva Ciftcioglu
Department of Biochemistry and Biotechnology, University of Kuopio,
FIN-70211 Kuopio, Finland

A new potential threat for blood and blood prouducts, cell culture, cell
and tissue banking and biotechnology has been discovered: Nanobacterium
sanguineum gen. et sp. nov.. These self-replicating ultrafilterable
bacteria were isolated from over 80% of commercial ÔsterileÕ fetal and
newborn bovine sera and are thus the most common contaminant present in
cell cultures. Growth occured in vitro under cell culture conditions (with
or without mammalian cells) but not under anaerobic conditions. Their
doubling time was 1-5 days. They were culturable also in protein and
lipid-free medium beyond three years with monthly passages. Colony
formation on solid media was poor. The agent multiplied in culture with
serum in a logarithmic mode that could be prevented with aminoglycoside
antibiotics, EDTA, cytosine arabinoside and gamma irradiation. They showed
procaryotic structure with specific antigens detectable by monoclonal
antibodies, were generally mobile, coccoid with a diameter of 200 to 300 nm
in serum, stained poorly with bacteriological stains, were very resistant
to antibiotics and passed through 100 but not 50 nm filters. Their
aminoterminal protein sequences were novel and reproducible. Considerable
evidence suggested presence of nontraditional DNA. They incorporated
radiolabelled uridine into DNA. 16S rRNA gene sequence results place them
in alpha-2 subgroup of Proteobacteria which includes Brucella, also
pathogens of mammalians with preference to the fetus. This new agent causes
cytotoxicity and senescence in many cultured cell lines by apoptotic cell
death and growth arrest.

*******
http://www.uku.fi/~kajander/threat.html
(pictures and graphs at the site)

Vaccines 97, Brown & al ed.,Cold Spring Harbor Laboratory Press, New York,
1997
A New Potential Threat in Antigen and Antibody Products: Nanobacteria
Neva Ciftcioglu, Ilpo Kuronen, Kari Akerman, Erkki Hiltunen, Jukka
Laukkanen and E. Olavi Kajander
Department of Biochemistry and Biotechnology, University of Kuopio,
FIN-70211 Kuopio, Finland.

Several vaccines are currently being produced by using cultured mammalian
cells. Microbiological sterility of such vaccines is of great importance
since several examples indicate potential safety hazards in vaccines
contaminated with unknown organisms. Fetal bovine serum (FBS) used as a
supplement in cell culture is a known safety risk (Hodgson, 1995).
Obviously, not all of the risk factors of FBS are yet known and thus cannot
be controlled. It is commonly known that only about 10% of FBS batches
support cell cloning well (Liddel and Cryer, 1991) but the reasons for this
have remained unclear. As with many other cell culturers, we faced a
problem about 10 years ago of poorly thrivingo cells not attributable to
any known contaminant. In this report, we describe the discovery of a new
bacterium from mammalian blood and blood products, tentatively named as
Nanobacterium sanguineum gen. et sp. nov., and show that this agent is
common and harmful.

DISCUSSION
Culture and Diagnosis of Nanobacteria
The discovery of Nanobacteria came about because we had a problem with cell
cultures namely vacuolized cells (Fig. 1A) and poorly thriving cultures
without any contaminant detectable by standard methods. Transmission
electron microscopy (TEM) made from these poorly thriving cell cultures
indicated the presence of internalized procaryotic organisms (Fig. 1B).
That their source was the commercial "sterile" FBS was proven by
gamma-irradiating all the culture components (Table 1). This experiment
also indicated that sterile culture media for detection of new organisms
can be made by using gamma-irradiated serum as a supplement. The new
organisms passed through 100 nm (but not 50 nm) filters and were called
nanobacteria, since no other bacteria are known that can pass through
filters with such small pores. This ability to pass through such small-pore
filters was most remarkable since they were shown to have a cell wall and
yet were able to surpass the filterablity of cell-wall-less bacteria. They
were unculturable in microbiological media but could be cultured under cell
culture conditions (with or without mammalian cells, CO2 5-10%). These
minute generally coccoid organisms had a diameter of 200 to 300 nm in
serum, and their size increased during the culture due to the production of
a very thick cell envelope (Fig. 1C, D). The thick and calcified envelope
made them visible even by light microscopy. The doubling time of
nanobacteria was 1-5 days (Fig. 2). Their multiplication could be detected
by specific ELISA, optical density, microscopic counting, SDS-PAGE or
methionine and uridine incorporation, and the multiplication could be
prevented with high doses of aminoglycoside antibiotics, EDTA, cytosine
arabinoside and gamma-irradiation. Considerable evidence suggested the
presence of nontraditional DNA. 16S rRNA gene sequence results (data will
be published elsewhere) placed them into the alpha-2 subgroup of
Proteobacteria which includes Brucella(which are also pathogens of
mammalians with preference to the fetus) and Bartonella.
Nanobacteria were isolated from more than 80% of commercial FBS and newborn
bovine sera and are the most common contaminant present in cell cultures.
In addition, we isolated nanobacteria from the blood of 4% of medical
students at our university. Positive identification of nanobacteria
involved growth in cell culture medium with typical growth rate and optical
properties, specific stainability with Hoechst 33258 using the high dye
concentration and positive immunoassay results with immunofluorescence
and/or ELISA using monoclonal anti-nanobacteria antibodies.

Cytotoxicity of Nanobacteria
Nanobacteria are cytopathic in cell cultures and invade mammalian cells in
a distinctive manner: They trigger cells that are not normally phagocytic
to engulf them. These novel organisms are one of the causes for cell
vacuolization, poor thriving and unexpected cell lysis, problems often
encountered in mammalian cell culture. Several mammalian fibroblast lines
were cultured in MEM medium as described previously (Kajander et al.,
1990), and were infected with nanobacteria. Electron microscopy and FITC
staining with specific monoclonal antibodies indicated that nanobacteria
were bound on the surface of the fibroblasts (Fig. 1E-G). We concluded that
they were internalized either by receptor-mediated endocytosis or by a
closely related pathway. After the internalization, fibroblasts showed
apoptotic abnormalities and died if subjected to a high dose (>100
nanobacteria/cell).

Different Growth Phases of Nanobacteria
Washed nanobacteria added to serum-free medium grew very slowly as
evidenced by increase in their numbers and protein level and were firmly
attached to the culture plates. These cultures progressed to large
multicellular formations covered by layers of a firm protective material
several micrometers thick (Fig. 1H). After addition of sterile serum, the
layer disappeared, with typical small coccoid nanobacteria later appearing
in the same cultures with the mobile, larger D-shaped ones (Fig. 1I).
Specific monoclonal antibodies indicated the presence of the same antigenic
sites in both D-shaped and coccoid nanobacteria, and their 16S rRNA gene
sequences were 98% identical.

How can Cell Culture be Possible with Nanobacteria-contaminated Fetal
Bovine Serum?
Although more than 80% of cell culture serum batches are contaminated with
nanobacteria, many cell culturers have not faced this problem with their
cell cultures. We have experienced a major problem with nanobacteria in
cell culture only when they are present at high concentrations relative to
cells. This can occure typically in cell cloning and in long-term
experiments where mammalian cells do not multiply. Internalization of
numerous nanobacteria by a cell results in cytotoxicity. Importantly, most
cell lines multiply faster than nanobacteria. Thus, cytotoxic
concentrations may be avoided.

Why is Nanobacteria a Potential Threat?
Nanobacteria can cause a chronic infection in laboratory animals and in
humans. The agent could be isolated from blood of one peron for 5 years
despite the presence of antibody. When nanobacteria were injected into
rabbits, the agent was initially isolated from urine and then from
cerebrospinal fluid after one year. Nanobacteria multiply very slowly and,
if pathogenic in humans, might cause slow chronic autoimmune-like disorders
(compare with leprosy or brucellosis). Sofar, there are no chronic
bacteraemia known that would not be harmful. Thus, the posibility that
nanobacteria may be present in vaccines made with cell culture, or in
gammaglobulin or other antibody preparations, must be controlled.

SUMMARY AND CONCLUSIONS
Nanobacteria are novel microorganisms that are not detectable with present
sterility testing methods, but they are detectable with new culture and
immunomethods. They are commonly present in bovine and blood products and
thus in cell cultures and antigens, including vaccines derived therefrom,
and may be present in antibody and gammaglobulin products. Nanobacteria are
a potential risk because of their cytotoxic properties and ability to
infect fetuses, and thus their pathogenicity should be scrutinized.

ACKNOWLEDGMENTS
We thank E. Tahvanainen, H. Martikainen, A. Pelttari and T. Ojanen for
their valuable help in microbiological, microscopic, and chemical
techniques. P. Mäenpää, T. Eloranta, J. Kärjä and O. Lindqvist contributed
valuable ideas in discussions. The work was supported by the Academy of
Finland, University of Kuopio, Finland Technology Center, Juho Vainio
Foundation and Savo High Technology Foundation.

REFERENCES
Hodgson, J. 1995. To treat or not to treat: That is the question for serum.
BioTechnology 13: 333.
Kajander, E. O., R. J. Harvima, L. Kauppinen, K.K. Akerman, H. Martikainen,
R. L. Pajula, and S. O. Kärenlampi. 1990. Effects of selenomethionine on
cell growth and on S-adenosylmethionine metabolism in cultured malignant
cells. Biochem. J. 267: 767.
Liddel, J. E., and A. Cryer. 1991. in A practical guide to monoclonal
antibodies, p. 25. Wiley, New York.
Figure 1. Ultrastructure of nanobacteria and their interaction with
fibroblasts.
(A) Perinuclear vacuolization of an infected 3T6 cell under phase-contrast
microscope;
(B) TEM image of a nanobacterium engulfed by a BHK cell;
(C) cultured coccoid nanobacteria (bars 200 nm).
(D) SEM image of nanobacteria attached to culture vessel;
(E) nanobacteria attached to a fibroblast surface (arrow shows the surface
of the fibroblast; bars 1 µm).
(F) Indirect immunofluorescence staining of cultured healthy 3T6 cells with
a monoclonal antibody (8/0) against nanobacteria;
(G) 3T6 cells inoculated with nanobacteria;
(H) TEM of a nanobacterial population in a serum-free culture (arrow shows
a D-shaped nanobacterium in this population);
(I) D-shaped nanobacteria after culture in serum-containing medium (bars 1
µm).
Figure 2. Growth-curve of nanobacteria. As a control, gamma-irradiated FBS
was used. At each time point, samples from triplicate incubations were
taken, frozen and analyzed by turbidometer at the end of the experiment.
Turbidometer units are means of three measurements from 1/6 dilutions of
cultures. 


Table 1. The Effect of 60Co Gamma-Irradiation of Culture Components on
Multiplication of Nanobacteria


Culture Multiplication
FBS
RPMI +
FBS
*RPMI +
*FBS
RPMI -
*FBS
RPMI
nanobacteria or FBS +
*FBS
RPMI
*nanobacteria or * FBS -


The material marked with asterisk (*) received a sterilization dose of 3
megarads during 16 h at room temperature. Cultures were established using
10 % serum and nanobacterial counts were followed for 4 weeks.

********
http://www.idreview.co.uk/pdf/pdf-1/11E1.PDF

"Another intriguing subject is that of the putative nanobacteria studied by
a Finnish group. Present inhuman and bovine sera, they might have
contaminated many biological preparations and have spread in human
populations. As they induce deposition of calcium salts,they may be
involved in diseases involving such depositions, such as renal lithiasis
and atherosclerotic plaques, or even neuro-degenerative diseases. Their
minimal size (200 nM) precludes conventional packaging of a length of DNA
sufficient to code for an autonomous microorganism. But it is possible that
their genetic information is encoded in a modified, more compact DNA.In
conclusion, our fight against emerging diseases has just begun. We should
always be vigilant against the resurgence of known infectious germs and the
emergence of new agents. More than ever, a worldwidenetwork of sentinel
laboratories and a coordinated multidisciplinary effort in biomedical
research are required for our survival. "