PATHOGENS AND INFECTIOUS TOXIN PROTEINS IN SLUDGE/BIOSOLIDS
                   Studies show EPA and its partners are destroying public health.
                           1,407 human pathogen species may be in sludge
Jim Bynum and Gail Bynum, Ph.D.         3/19/2006                   Click here  
Reference to studies
This is a continuation of the research studies on Necrotizing bacteria (25) ("fleah eating organisms") in sludge/biosolids.
It shows pathogens (infectious disease causing microorganisms) in sewage sludge/biosolids not only affects  human
health and the environment, but also affects animal health. This section focuses on the fact that EPA Office of Water
and its partners (state environmental departments, municipal wastewater and soil scientists), have known for a very long
time that as bacterial organisms pass from humans to sewage,  to sludge/biosolids, to plants and animals, and back to
humans, genes are transferred to transform them into deadly multi-drug resistant toxin protein producing organisms.
Some of these disease organisms not only create  biological toxin proteins such as enterotoxins proteins (toxic to the
intestinal tract), they create exotoxin proteins (toxic shock -food poisonng) , and neurotoxin proteins (acts on nerve
cells)  while they are alive, but, many organisms create endotoxin proteins (toxic hemorrhagic shock-antigenic
response) while they are active as well as in death. Environmental exposure to endotoxin proteins inhaled in dust can
be just as deadly as direct exposure to bacteria. On top of that, mycotoxin proteins (damage lungs and liver) from fungi
must be contended with by farmers using sludge/biosolids and those who use compost. Yet, for 25 years EPA and its
wastewater partners have promoted sewage sludge/biosolids as a safe and cheap fertilizer for food crops,

Gene  transfer of toxins and  Multi-drug resistant

According to Robert Miller, department of microbiology and molecular genetics at Oklahoma State University, "The study
of gene transfer among bacteria began in 1928, when British bacteriologist Frederick Griffith (46) observed that
nonvirulent pneumococcal (nonpathogenic) bacteria became virulent (pathogenic) when injected into mice along with
dead virulent pneumococcus. Griffith concluded that the initially nonvirulent bacteria picked up a "" agent from the dead
virulent bacteria and thus became potent enough to kill the mice."

In a 1982 EPA study, M. C. Meckes'
(35)  noted,  "In 1959, Wantanabe (31) discovered that some Escherichia coli
strains could transfer antibiotic-senstive strains of shigella spp. Subsequent research has demonstrated that bacteria
carrying transmissible R-factors [genetic transfer]are responsible for the spread of multiple antibiotic resistence among
members of the Entero-bacteriaceae (such as E. coli, Samonella typi, and Shigella dysenteriae) Aeromonas and
Yersinis species (4), Pseudomonas, and Vibro cholerae (34),"

The University of Wisconsin VetMed Center
(36) notes, "The O157:H7 strain is distinguished microbiologically from
other E. coli by its inability to ferment sorbitol and, most importantly, by its production of "shiga-like" toxins (SLT I and II).
(SLTs were so named because of their similarity to the toxin of Shigella- 5 receptor binding B subunits and one active A
subunit create a holotoxin.)"

By 1989,
(25)  Invasive group A streptococcal, Staphylococcus (MRSA), Clostridium, and E. coli 157 were just beginnng
their deadly transformation through the wastewater treatment plants. Only a few people had died from these pathogenic
sludge pollutants during the 1980s. In the past 15 years, Invasive group A streptococcal, Staphylococcus,  Clostridium,
(Necrotizing Fasciitis),  and E. coli 157 have become epidemic

According to ROBERT V. MILLER,
(46)  1998, head of the department of microbiology and molecular genetics at
Oklahoma State University, there are "three common forms of horizontal gene transfer—transduction, conjugation and
transformation—to occur in nature"

He said, "In transduction, bacteriophages (viruses that infect bacteria) pick up genetic material from one bacterial cell
and deposit it in another. As part of their life cycle, bacteriophages attach to bacteria and inject their DNA. This DNA
then serves as a blueprint for making more copies of the bacteriophage, which burst from the infected bacterium and go
on to infect other cells. At times, however, some of the new particles carry bacterial instead of viral DNA. Indeed,
bacteriophages are capable of transferring whole plasmids and pieces of chromosomes between Hosts."

Dr. Miller added, "Until recently, researchers assumed that transformation would not occur in most places, because free
DNA would not be stable in soil or water. But studies by Michael Lorenz and Wilfried Wackernagel of the University of
Öldenburg in Germany, Guenther Stotzky of New York University and others have demonstrated that free DNA can
become stable by associating with soil components and that this DNA can be taken up by competent cells."

Furthermore he added, "The researchers found that conjugation can enable a laboratory strain of Pseudomonas
aeruginosa to pick up a plasmid that naturally provides resistance to mercury toxicity in bacteria that inhabit the polluted
river Taft, near Cardiff, Wales. P. aeruginosa is a common soil and freshwater bacterium that can cause respiratory and
urinary tract infections in humans whose immune defenses are weakened."

According to Dr. Miller, in transformation, "Bacterium undergoing transformation picks up free DNA released from a
dead bacterial cell. As DNA-binding complexes on the bacterial surface take up the DNA  enzymes break down one
strand into nucleotides; meanwhile the other strand may integrate into the bacterium' chromosome." [RNA (83)serves as
the template for translation of genes into proteins, transferring amino acids to the ribosome to form proteins, and also
translating the transcript into proteins]

One study was undertaken by Herbert Marcinek, et al.,
(43) 1998, in which they state, "Since no data on E. faecalis
gene transfer in sewage water treatment plants are available, we performed such studies in the sewage water treatment
plants of two Bavarian cities, Munich (Kla¨rwerk Marienhof: Dietersheim) and Regensburg (Kla¨rwerk Barbing),
Germany. Our data indicate that under the natural conditions of the Regensburg plant ca. 106 to 109 gene transfer
events between different E. faecalis strains should take place per day."

As background they add, "the data for conjugal transfer of genes and genetic elements between different strains of E.
faecalis and between this bacterium and other bacterial species (in both directions [i.e., to and from E. faecalis]) under
laboratory conditions are too numerous to be cited here."

Furthermore they noted, "Resistance plasmid pAMb1 has been shown to transfer among E. faecalis, E. Faecium, and
Lactobacillus reuteri in the digestive tracts of mice (27). (This plasmid also transfers between Lactobacillus curvatus
strains in fermenting sausages [35].) Inducible transfer of conjugative transposon Tn1545 from E. faecalis to Listeria
monocytogenes in the digestive tracts of gnotobiotic mice has also been reported." [Plasmids (79) are (typically) circular
double-stranded DNA molecules separate from the chromosomal DNA]

Marcinek noted, "Gene transfer occurs under natural conditions not only in soil, marine water, and sewage water
treatment plants, but also in other biotopes. For example, transduction via phages [viruses] occurs between
Pseudomonas aeruginosa strains on the surfaces of leaves (21); gene transfer occurs between the fish pathogenic  
bacterium Aeromonas salmonicida and a human Escherichia coli isolate in raw salmon on a cutting board and gene
transfer between physically isolated bacteria is enhanced by the presence of burrowing earthworms as a biological
factor which facilitates cell-to-cell contact."

The study concluded, "Taken together, our data seem to indicate that under severe stress situations very unusual
(high) gene transfer efficiencies can occur." "Heat treatment resulted in enhanced gene transfer from Escherichia coli to
various coryneform bacteria (31), as did other stress situations, such as exposure to organic solvents or detergents and
pH shifts."

Marcinek also warned,  "The argument that the possibility of gene transfer has to be totally excluded in genetically
engineered bacterial strains does not make sense if gene transfer occurs in nature."

An EPA
(56)  study 1997-2002 states, "The release of transgenic plants and bacteria containing genes from subspecies
of Bacillus thuringiensis (Bt) that code for insecticidal proteins poses a potential hazard to the environment. The
continual production of the toxins, especially those encoded by truncated genes that express active toxins rather than
inactive protoxins, by growing transgenic organisms that are indigenous or adapted to the specific habitat may exceed
consumption by insect larvae and biotic and abiotic inactivation. Hence, the toxins could accumulate to concentrations
that may constitute a hazard to nontarget organisms [all living things= bacteria to humans] and that could result in the
selection and enrichment of toxin-resistant target insects. The accumulation and persistence would be enhanced if the
toxins are bound on particles (e.g., clays and humic substances) in the environment and, thereby, are rendered less
accessible for microbial degradation."

Or did EPA mean microbial transformation?

Transformed Infectious Organisms associated with Necrotizing fasciitis (NF)

These are mixed aerobic and anaerobic bacteria species associated with Necrotizing fasciitis:

b-hemolytic streptococci, group A streptococcal - Streptococcus pyogenes, Streptococcus agalactiae,  Staphylococcus
aureus, Staphylococcus intermedius.  Most infections caused by staphylococci are due to Staphylococcus aureus.
However, the incidence of infections due to Staphylococcus epidermidis and other coagulase-negative staphylococci
has been steadily increasing in recent years.  They produce a range of toxins, [proteins] including alpha-toxin, beta-
toxin, gamma-toxin, delta-toxin, exfoliatin, enterotoxins, Panton-Valentine leukocidin (PVL), and toxic shock syndrome
toxin–1 (TSST-1). (12)

Other emerging organisms causing Necrosis are:  Escherichia coli, Peptostreptococcus spp., Bacteroides fragilis spp.,
and Clostridium spp, Clostridium perfringens, Clostridium septicum, or Clostridium histolyticum.  Haemophilus influenzae,
Pseudomonas aeruginosa , Eikenella corrodens, Pasteurella multocida, Capnocytophaga canimorsus), Pseudomonas
aeruginosa, Erysipelothrix rhusiopathiae, Mycobacterium marinum, Noncholera Vibrio, Coxsackievirus B (pleurodynia);
echovirus, Coxsackievirus B, and Legionella, Trichinella spiralis (trichinosis), Taenia solium (cysticercosis), Toxoplasma
gondii (toxoplasmosis). (2)

These pathogens produce proteins that protect the bacteria and are very infectious toxins to the animal or human host.
Proteins
(74) are biology's workhorses -- its "nanomachines." Before proteins can carry out these important functions,
they assemble themselves, or "fold." The process of protein folding, while critical and fundamental to virtually all of
biology, in many ways remains a mystery.

Moreover, when proteins do not fold correctly (i.e. "misfold"), there can be serious consequences, including many well
known diseases, such as Alzheimer's, Mad Cow (BSE), CJD, ALS, Huntington's, Parkinson's disease, and many Cancers
and cancer-related syndromes.

The M protein
(73) has been found responsible for protecting the bacteria from phagocytosis [engulfment and
destruction]  by polymorphonuclear leukocytes [white blood cells] (10). There are over 80 distinct M proteins, but the
two most often isolated in NF are the M-1 and M-3 subtypes of S. pyogenes (8). Another important virulence factor is
the exotoxin. There are five different exotoxin proteins: A, B, C, D, and E. Most commonly noted in NF are the
streptococcal (scarlatina) pyrogenic exotoxins (SPE) types A, B, and C. Exotoxins recruit T cells and increase
production of tumor necrosis factor alpha, interleukin 1-beta, and interleukin 6. The effects are characterized by fever,
shock, edema, and multiple organ failure (11). The streptococcal superantigen (SSA) can also play a role in this
process. Although these proteins are unique to the Streptococcal species, the clinical picture is often difficult to
distinguish from other types of NF (4).

Lawrence Broxmeyer
(80)  Med-America Research, 2004, reported,  "In one study Alzheimer' was misdiagnosed in up to
13% of autopsied patients actually suffering from this Creutzfeldt– (CJD) disease [16]. But the full number of US CJD
patients will never be known until it is proclaimed a reportable disease."

He also notes the affect on the brain is necrotizing

He ponders, "Whether the unthinkable: that Alzheimer' disease and Creutzfeldt– might be caused by the mycobacterial
load in the meat or dairy products of animals with M. bovis caused Mad Cow Disease will require further investigation, a
task made only more difficult by the fact that studies such as Hartley' show pathology identical to '' Cow'' from M. bovis in
cattle, causing a tuberculous spongiform necrotizing encephalitis, without recovery from the central nervous system of
classical forms of bovine TB [43]."

Biological Toxins

According to CDC, Biological Toxins. (66) Biological toxins (also referred to as biotoxins) are nonliving toxic proteins that
are naturally produced by many different types of living organisms. Biotoxins are:

Thousands of times more toxic by mass than chemical warfare agents.

Necrotizing encephalitis affect the brain matter of the young and old, as well as animals. Some studies implicate virial
infection, while other studies implicate toxic proteins.

In reviewing the studies, Dr. Michael Greger,
(81) 1996, found, "An estimated two to three million Americans are afflicted
by Alzheimer's (Scully, 1993); it is the fourth leading cause of death among the elderly in the U.S (Perry, 1995). Twenty
percent or more of people clinically diagnosed with Alzheimer's disease are found at autopsy to not have had
Alzheimer's at all (McKhann, 1984). At Yale, out of 46 patients clinically diagnosed with Alzheimer's, 6 were proven to be
CJD at autopsy (Manuelidis, 1989). In another post-mortem study 3 out of 12 "Alzheimer" patients actually died from a
spongiform encephalopathy (Teixeira, 1995)."

Ravi Shankar Mishra
(75)  et al., Institute of Pathology, Case Western Reserve University,  2004, reports, "The BSE
[bovine spongiform encephalopathy] epidemic is far from over despite the concerted efforts of national, industrial, and
regulatory agencies across the world. An emerging threat is the continual spread of chronic wasting disease in the deer
and elk population in the United States and the uncertainties regarding its transmission to livestock and humans (Miller
and Williams, 2003). As the sources of PrPSc-contaminated food products continue to increase, it has become
increasingly critical to understand the mechanism by which PrPSc, a protein with a protease-resistant core of 27-30 kDa
and a major, if not the only, component of prion infectivity (Prusiner, 1998), maneuvers its way across the impermeable
and highly selective epithelial barrier of the human intestinal tract."

Mishra said,  "a recent report demonstrating the absence of prion infectivity in µMT and RAG1-/- mice orally challenged
with prions despite the presence of M-cells suggests that PrPSc transport across the intestinal epithelial barrier is not
limited to M-cells and that additional pathways must exist (Prinz et al., 2003)."

"Because ferritin shares considerable homology across species, these data suggest that PrPSc-associated proteins, in
particular ferritin, may facilitate PrPSc uptake in the intestine from distant species, leading to a carrier state in humans."
Furthermore, he said, "This report provides insight into the pathway of PrPSc uptake and transport across intestinal
epithelial cells. In particular, our data show that exposure of sCJD [variant Creutzfeldt-Jakob disease] brain homogenate
to DEs generates a C-terminal PrPSc core of 27-30 kDa that is transported across Caco-2 cells in vesicular structures
and that this process is not influenced by the level of endogenous PrPC expression. '

The studies conclusion, "The cotransport of PrPSc with ferritin raises important questions regarding prion uptake from
contaminated food. Although this report uses a homologous experimental setup, ferritin H and L chains are known to
share significant homology across species (Harrison and Arosio, 1996) and may facilitate the transport of PrPSc from
distant species across the intestine. Because PrPSc is notorious for its sticky nature, ferritin may be only one such
carrier protein. The identification and functional role of other proteins associated with DE-treated PrPSc is important for
fully understanding the mechanism of PrPSc uptake from ingested food and preventing a carrier state across species.
Heterologous PrPSc in such carriers may be transported to sites where it may undergo conformational "adaptation" with
time (Hill et al., 2000; Race et al., 2001), or in the case of livestock, lie dormant until ingested by a susceptible host.
Such apparently "healthy" carriers would disseminate PrPSc through a variety of means, posing a potential threat to the
general population."

In a Yale Medical Study, Christopher A. Baker,
(76) et al., 2002, state, "Because our understanding of the molecular
nature of the infectious agent remains incomplete, it is difficult to specify the mechanisms of agent interactions with any
cell type at this time. However, it is intriguing that the pattern of microglial activation is consistent with standard viral
mechanisms that commandeer myeloid cells for invasion and latency, ultimately leading to secondary destructive
changes in the brain."

In another Yale Study, Klaus Radebold,
(77) et al., 2001, states the, "Brain lacks significant lymphatic drainage, and has
little infectivity before 40 days, even after ic inoculation. Thus the infectious inoculum must spread to the gut by a
vascular route, a direction opposite to that generally assumed. This interpretation is consistent with previous studies
demonstrating white blood cell infectivity as well as perivascular PrP accumulations in CJD. Notably, enteric infection at
early as well as later stages of disease, and regardless of the route of agent entry, implicates potential environmental
spread by fecal matter."

Steve Ritter,
(78) reports in Chemical and Engineering News, 2006, Method to detect infectious proteins in soils, that, "A
method to extract and quantitatively detect prions from soil samples has been devised by a team of scientists at two
National Institute for Agricultural Research (INRA) labs in France (Environ. Sci. Technol. 2006, 40, 1497). The
technique could be "a good starting point" to help identify and map prion-contaminated farmland as well as to monitor
the fate of prions over time, notes lead author Peggy Rigou."

"Prions can persist in soil for years"

Furthermore, he adds, "A potential method to detect prions in the blood of live animals was reported last year (C&EN,
Sept. 5, 2005, page 15), but until now, a method to analyze prions in soil had not been reported, Rigou says. The
researchers studied the adsorption and desorption of a recombinant prion protein and other proteins on clay and
natural soil samples to understand prion retention mechanisms. They determined that adsorption occurs mainly via the
N-terminal domain of the protein."

Enterotoxin proteins (toxic to the intestinal tract),

James D. Oliver, (48) Department of Biology, University of North Carolina at Charlotte, 2005, notes, "We have recently
been studying the human pathogen, H. pylori, which is estimated to infect 50% or more of the worlds population
(Lambert et al., 1995) and is associated with the development of chronic human gastritis, peptic ulcers, and gastric
adenocarcinoma (Catrenich and Makin, 1991; Peterson, 1991; Asaka et al., 2001). Molecular methods have detected
the presence of H. pylori DNA in river water, well water, wastewater, as well as surface and shallow groundwater,
suggesting that this organism is waterborne and may be transmitted by the fecal/oral route (Hulten et al., 1996; Hegarty
et al., 1999; Moreno et al., 2003). However, despite its high incidence of infection, the bacterium' reservoir and mode of
transmission remain undetermined." "Finally, we have observed that cells of H. pylori in the VBNC [viable, but
noncultuable] state are resistant to the antibiotics commonly employed for the treatment of ulcers,"

Helicobacter toxins, produced by Helicobacter pylori (inf). These toxins interact with the epithelial cells of the stomach
causing them to produce chemokines [activate proteins] (def) that attract neutrophils [defense proteins] and
macrophages [eater cells that damage surrounding tissue]. This inflammatory response and the resulting extracellular
killing by the phagocytes {engulfment and killing] can lead to tissue damage. [
acid reflux disease?]

D. Strauch
(54) in his 1991 paper, "Survial of pathogenic micro-organisms and parasite in extreta, manure and sewage
sludge" reported that two groups of researchers had found that pathogenic disease organisms will be taken up inside
the food crops."

In 2004, Dr. Muhammad Qasim Khan,
(82) Department of Soil Science, Gomal University, isolated from sewage sludge
fertilizered vegetables  the bacteria, E.coli, Staph.aureus., Staph citreus & Bacillus megatarium. He also isolated the
following Fungi in the vegetables,  Xanthomonas spp., Mucor spp., Rhizopus spp. & Aspergillus spp.

Tao Zhang,
(17) Genome Institute of Singapore (2005), discusses plant viruses in the human gut.
He said, "In addition to bacteriophages [viruses that infect bacteria], the other well-studied human enteric viruses are
the viral pathogens associated with gastroenteritis. They can infect the human small intestine cells, causing damage to
the epithelial lining and the absorptive villi, leading to the malabsorption of water and an electrolyte imbalance"

Zhang said, "Many viral pathogens have been isolated from the feces of gastroenteritis patients, including rotavirus,
astrovirus, calicivirus, hepatatis E virus, certain members of coronavirus and torovirus, and the enteric adenovirus
(serotypes 40 and 41). Except for the adenoviruses, which contain DNA [double strand] genomes, all the others are
RNA [single strand] viruses. Despite intensive studies, many causative agents of human gastroenteritis are still
unknown. [RNA (83) serves as the template for translation of genes into proteins, transferring amino acids to the
ribosome to form proteins, and also translating the transcript into proteins.]

Zhang explains why they are unkown. He said, "Traditionally, discovery of viruses was dependent on culturing the
viruses in host cells in order to propagate and isolate enough pure virions for characterization. However, it is generally
known that the large majority of viruses, including enteric viruses, cannot be cultivated using standard techniques.

"To date" he said, "very little information is available on the human enteric RNA viral flora, despite the fact that many
RNA viruses are known etiologic agents [pathogens] of gastroenteritis."

"In addition" he said, "24 out of the 35 plant viruses detected in these fecal samples were known pathogens of
consumable crops including fruits, vegetables, tobaccos, and cereals suggesting that foods contaminated with plant
viruses might have contributed to the RNA  viral flora in human feces."

Zhang said, "We further provide evidence that the fecal PMMV was viable and could cause infection to a host plant.
PMMV viruses appeared to be stable through the human digestive system." "The discovery of this previously unknown
transmission route for plant viruses is potentially important for a better understanding of certain agricultural practices,
because in traditional agriculture and in developing countries, human and animal refuse are often used as fertilizers for
crops.

He said. "it is conceivable that these viruses might disassociate from the plant tissues and therefore are readily
accessible to host epithelial cells and microbial cells existing in the intestinal environment. Earlier studies showed that
plant viral particles could be assembled in Escherichia coli cells, which leads to the speculation that some feces-borne
plant viruses might be capable of interacting with microbes in human guts. Given that nonpathogenic
commensal intestinal bacteria can significantly alter the immune system of an organism, it is conceivable that viruses
such as PMMV, replicating or not, may also have an effect."

Iin 2005, Alan Godfree and Joseph Farrell (13)  (Retired EPA employee who claimed to be pathogen expert on 503
Peer Review Committee) agreed with the studies and said, "Wastewater contains human, animal, and plant pathogens
capable of causing viral, bacterial, or parasitic infections."

Exotoxin proteins (toxic shock -food poisonng)

3 distinct streptococcal pyrogenic exotoxins (ie, A, B, C) also have been identified. These toxins induce cytotoxicity [toxic
to cells,  and pyrogenicity [producing fever]  and enhance the lethal effects of endotoxins. Recently, the streptococcal
super antigen, a pyrogenic exotoxin, has been isolated from an M-3 strain. (3) staphylococcal enterotoxin B - a form of
staphylococcal enterotoxin that has been used as an incapacitating agent in biological warfare. The genus is defined by
a combination of antigenic, hemolytic, and physiological characteristics into Groups A, B, C, D, F, and G. Groups A and
D can be transmitted to humans via food.

Staphylococci aureus produce a range of toxins, including alpha-toxin, beta-toxin, gamma-toxin, delta-toxin, exfoliatin,
enterotoxins, Panton-Valentine leukocidin (PVL), and toxic shock syndrome toxin–1 (TSST-1). The enterotoxins and
TSST-1 are associated with toxic shock syndrome. PVL is associated with necrotic skin and lung infections.
Staphylococci epidermidis, produce an exopolysaccharide (slime) that promotes foreign body adherence and resistance
to phagocytosis.

According to Clare K. Schmitt,
(66) et al., Uniformed Services University of the Health Sciences, "Many bacterial
exotoxins have the capacity to damage the extracellular matrix or the plasma membrane of eukaryotic cells. The
damage not only may result in the direct lysis of cells but also can facilitate bacterial spread through tissues. Toxins that
mediate this cellular damage do so by either enzymatic hydrolysis or pore formation. Bacterial hyaluronidases,
collagenases, and phospholipases have the capacity to degrade cellular membranes or matrices. Specific examples of
these types of toxins include the -toxin of Clostridium perfringens, which has phospholipase C activity; Streptococcus
pyogenes streptokinase, which can hydrolyze plasminogen to plasmin and dissolve clots; and the clostridial
collagenases. Pore-forming toxins, as the name suggests, disrupt the selective influx and efflux of ions across the
plasma membrane by inserting a transmembrane pore. This group of toxins includes the RTX (repeats in toxin) toxins
from gram-negative bacteria, streptolysin O produced by S. pyogenes, and the S. aureus -toxin."

Schmit said, "S. aureus -toxin can be considered the prototype of oligomerizing pore-forming cytotoxins [proteins]. The -
toxin gene resides as a single copy on the chromosome of most pathogenic S. aureus strains, and its expression is
environmentally regulated at the transcriptional level by the staphylococcal accessory gene regulator (agr) locus. The -
toxin is synthesized as a 319 amino acid precursor molecule that contains an N-terminal signal sequence of 26 amino
acids. The secreted mature toxin, or protomer, is a hydrophilic molecule that lacks cysteine residues and has a
molecular mass of approximately 33 kDa. Recently, the crystallographic structure of the fully assembled -toxin pore was
solved. On the plasma membrane, seven toxin protomers assemble to form a 232 kDa mushroom-shaped heptamer
comprising three distinct domains (Figure 1A). The cap and rim domains of the -toxin heptamer are situated at the
surface of the plasma membrane, while the stem domain serves as the transmembrane channel."

Endotoxin proteins (toxic hemorrhagic shock-antigenic response)

According to CDC, (67) "Endotoxins are a combination of lipid (lipid A) and polysaccharide side chains and are integral
components of the outer membrane of gram negative bacteria. Endotoxins are released into the surrounding
environment during active cell growth or breakdown (lysis), or when bacterial cells are engulfed by immune cells called
phagocytes."

Endotoxins have been known to cause profound inflammation of any tissue exposed to them, including lung tissue.
"Exposure to endotoxins causes an influx of inflammatory cells into the lungs," says NIOSH immunologist Stephen A.
Olenchock. "They bring with them and they release various agents called cytokines [small secreted proteins], which
cause swelling, exudate, or seepage, from blood vessels. These are very potent inflammatory agents."

Occupational inhalation of endotoxins induces fever and constriction of airways. According to Castranova, endotoxins
tend to upregulate the activity of lung phagocytes, encouraging pulmonary inflammation. "Many studies seem to show
that if you put lung phagocytes in a test tube and add endotoxin, not much happens," he explains. "But if you add
endotoxin and then add a second stimulus, the [phagocytic] response to that second stimulus is greater than if the
endotoxin weren't there. The second stimulus could be the dust, the particulate matter."

Initially, the response to endotoxin may seem to be allergic. But unlike allergy, the active component is lipid A, and not
an antigenic protein. "This is not an allergy at all," Olenchock explains. "Allergy involves a type of antibody associated
with a specific antigen. Here, there is an absence of antibody. Endotoxins activate the complement system, which
causes inflammation and then removal of foreign agents."

ENDOTOXIN EXPOSURE FROM DEAD BACTERIAL MATERIAL

In Susan Schiffman's ,(15) 2005, Department of Psychiatry, Duke University Medical Center, study, Science of Odor as
a Potential Health Issue,  there were 3 points made: 1) symptoms can be induced by exposure to odorants (compounds
with odor properties) at levels that also cause irritation or other toxicological effects. That is, irritation—rather than the
odor—is the cause of the health symptoms, and odor (the sensation) simply serves as an exposure marker; 2).  health
symptoms from odorants at nonirritant concentrations can be due to innate (genetically coded) or learned aversions;
and 3) health symptoms thought to be caused by odor might in fact be caused by endotoxins.

In Todar's Online Textbook of Bacteriology, 2002, Endotoxins
(23) are part of the outer membrane of the cell wall of
Gram-negative bacteria. Endotoxins are invariably associated with Gram-negative bacteria whether the organisms are
pathogens or not. Although the term "endotoxin" is occasionally used to refer to any cell-associated bacterial toxin, it is
properly reserved to refer to the lipopolysaccharide complex associated with the outer membrane of Gram-negative
bacteria such as E. coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, and other leading pathogens.

In Ines Niehaus's,
(22) 2002, presentation, Chronic inflammation of the human central nervous system caused by
Salmonella minnesota S-LPS, at the 6th conference of the International Endotoxin Society, Pasteur Institute Paris, she
states, Bacterial endotoxins (lipopolysaccharides) are part of the outer cell wall of Gram negative bacteria.1
Lipopolysaccharides (LPS) elicit multiple acute pathophysiological effects such as fever, lethality, Shwartzman
reactivitiy, macrophage and B-lymphocyte activation, and other activities by animals and humans.2, 3,4, 5Small doses
of LPS injected intravenously induce sepsislike symptoms and activate important inflammatory mediators whereas large
doses of LPS precipitate life-threatening circulatory collapse and multiple organ failure.6 1 mg Salmonella minnesota
LPS leads to lethal septic shock in human beings without medical treatment.7

Gram-negative bacterial infections and toxins (e.g. LPS) have recently been implicated in the pathogenesis of a number
of peripheral neuropathies in humans, including postinfectious polyneuritis or Guillain-Barre syndrome (GBS) and
critical illness polyneuropathy (CIP). Encephalopathy and CIP occur in 70% of septic patients.10 Tumor necrosis factor
and interleukin-1 (IL-1) and IL-2 are important mediators in the septic syndrome.10 The blood-brain barrier (BBB)
becomes leaky in a patchy manner within the first few hours of sepsis.10I.p. injection of LPS at a dose below those
described to disrupt the BBB, cytokine mRNA expression was increased in the spleen, the pituitary, and the brain.11
Disruption of the BBB can occur during a variety of pathophysiologic conditions, including bacterial infections. LPS
increases the permeability of the BBB.12

ENDOTOXIN CASE STUDY OF A LABORATORY WORKER

Ines Niehaus,  a laboratory worker (22) was contaminated with 10 microgram Salmonella minnesota S-LPS through an
open wound. One hour later she showed first signs of illness (fever, flu-like-symptoms, dyspnoea, headache, nausea,
vomiting). Three weeks after her accident she experienced neurological problems (generalized cramps, disturbances of
sensibility, myalgia, tremor, difficulties in learning etc.). The LPS damaged and disrupted the blood-brain barrier. The
LPS caused damages of the cerebral cortex which are proved by positron emission tomography (PET). The LPS,
intercalating in the neuronal membrane, elicited a chronic inflammation of the central nervous system. More than four
years later the LPS began to release from the neurons by the treatment with the quinolone ofloxacin. The patient was
better then but one year later the inflammation of the CNS turns back very heavily. The result of a LAL-test for the CSF
was 6600 pg LPS / ml after a dose of 400 mg ofloxacin ten hour before the spinal tap.

Neurotoxin proteins (acts on nerve cells)
According to Babish,(31) "Cornell University studies (1981-85) on ambient exposure to carcinogenic and mutagenic
compounds included evaluations of sewage sludge from 34 American cities---
--only one sample, Dallas Central failed to demonstrate a dose-related increase in revertants in any of the five tester
strains with or without metabolic activation (S-9). The other thirty-three sludge samples exhibited a positive mutagenic
response with at least one strain; twelve of the thirty-three samples were positive with two or more strains. Seventy-six
percent (25/33) of the positive samples required metabolic activation to demonstrate mutagenicity,..." (pp.56-57)

Extracts from the Dallas sludge was used as a possible non-toxic sludge to test for toxicity in mice versus a Boston
sludge which did test positive for mutagenic effects with metabolic activation. "Both treated and control animals exhibited
signs of discomfort immediately after dosing...fifteen to thirty minutes after dosing, the Boston sludge-treated animals
begin to show sign of effects on the central nervous system. Orientation was effected to some degree in all groups as
animals rotated repeatedly in both clockwise and counter-clockwise directions; one animal (dosed at 32.9 mg/kg) turned
over continuously for a period of four to six minutes.  Additionally, motor coordination and gait were adversely affected
by the Boston sludge extract in nearly all the animals."

"Mean time-to-death for Boston sludge extract ranged from 4.5 to 1.8 days, decreasing with increasing dose. The same
decrease in mean time-to-death with increasing dose was observed for Dallas sludge extract, although the range of 3 to
1 day was somewhat shorter....High-dose groups for both Boston and Dallas sludge extracts did not consume any food
before dying." (p.60)

"Although, the Boston sludge was more toxic, as demonstrated by LD 50, approximately one-half the Dallas sludge, both
of these sludge extracts would be considered extremely toxic." (p.60)

However, the most disturbing part of the study was that, "No gross lesions were observed in any animals which could be
associated with the test materials. Additionally, no treatment effects were seen in gross or relative organ weights.
Hepatic cytochrome P-450 levels were not significantly different from control values." (61)

In effect, there was no documentable evidence indicated to support the cause of death for the mice. In effect, a doctor
would have said these mice died of natural causes.

Babish leaves us with a warning, "it is clear that setting standards of sludge application based on analytical
measurement of one or any number of compounds is inadequate for protection of public health. Moreover, the absence
of information concerning the organic constituents of municipal sewage sludge must not be equated with low risk or
safety. Under conditions of  minimal data, maximal risk must be assumed in order to protect public health." (p. 61)

Mycotoxins (damage lungs and liver)

In a 1979 study, Marsh and Millner (20) (USDA) said, "Spores of Aspergillus fumigatus have been found to be
abundantly present in the outdoor air at a site where large scale experimental composting of sewage sludge is in
progress at Beltsville, Maryland. The health significance of this finding, for that site and for others in the future, is still
only incompletely understood. Further studies are in progress to characterize absolute concentrations of the spores of
the fungus in air at the site, spore dispersal by air from composting operations, and background environmental spore
levels in air."

PATHOGENS AT LARGE

An article in The Guardian, (2)  Monday February 20, 2006, reported that, "Three-quarters of the 38 species of harmful
organisms and viruses identified in the past 25 years are thought to have "jumped" from animals to humans, according
to US and Scottish scientists".

Professor Mark Woolhouse,
(2) from the University of Edinburgh, "-- told the American Association for the Advancement
of Science (AAAS) [at the ] annual meeting in St Louis that the apparent rise in the number of pathogens - agents such
as bacteria that transmit disease - was "too fast" simply to be caused by natural processes such as evolution."

Dr. Woolhouse said,
(4)  "The survey of human pathogens produced a count of 1,407 human pathogen species, with
177 (13%) species regarded as emerging or reemerging. Of all pathogen species, 208 are viruses or prions, including
77 (37%) regarded as emerging or reemerging. For bacteria, the counts were 538 and 54 (10%), respectively; for fungi,
317 and 22 (7%), respectively; for protozoa, 57 and 14 (25%), respectively; and for helminths, 287 and 10 (3%),
respectively."

Dr. Nina Marano,
(3) Centers for Disease Control and Prevention, notes, "new infections reemerging as a result of
antimicrobial resistance developing in existing agents (e.g., emergence of infections caused by multidrug-resistant
strains of Salmonella Newport), or breakdowns in public health measures (e.g., Mycobacterium bovis tuberculosis). She
said, "Strikingly, 75% of emerging infectious diseases have been identified as zoonotic [animal] in origin"

NON-DETECTION  OF BACTERIA DOES NOT EQUAL SAFE SLUDGE,  IT  EQUALS DEADLY Viable but
nonculturable  (VBNC)
PATHOGENS

"According to the article (55) "Pathogen Destruction and Biosolids Composting" in Biocycle of June of 1996, "There is
some evidence that coliforms and Salmonella sp. can survive prolonged exposure to temperatures of 55 C." They cite a
study done by Droffner and Brinton (1995) using DNA gene probes, where they detected E. coli and Salmonella sp. in
samples collected from an in-vessel composting facility after the first 15 days of active composting at a temperature
above 55 C. In Table 5-4 Processes to Further Reduce Pathogens in A Plain English Guide to the EPA Part 503
Biosolids Rule, composting time and temperature requirements for within-vessel composting method was 55 C or higher
for three days! Droffner and Brinton found that it took 56 days and 90 days for the densities of Salmonella sp. and E.
Coli, respectively, to decline below the detection limit...These investigators also "cite evidence of mutant strains of E.
coli and Salmonella sp. resistant to thermal environments in composting." (p. 68)"

Yogita N. Sardessai, 2005,
(47) reported, "in recent years, many studies have revealed the ability of
both Gram-positive and Gram-negative bacteria to go into a viable but non-culturable
(VBNC) state. In this state, bacteria are still viable and show metabolic activity and respiration, but cannot be shown as
colony forming units by the conventional plate counts and hence remain hidden. Microbial ecologists have in the past
stated that plate counts of bacteria in soil, rivers and oceans are typically less than 1% of the total bacteria observed by
direct microscopic methods."

Sardessai adds, "VBNC bacteria are a major concern in public health risk assessments because many pathogenic
bacteria like Vibrio cholerae, Mycobacterium tuberculosis, Campylobacter jejuni, Helicobacter pylori, Vibrio vulnificus
and Escherichia coli have been reported to enter a VBNC state from which they are able to return to the infectious state
after passaging in animal hosts.".

James D. Oliver,
(48) Department of Biology, University of North Carolina at Charlotte, 2005, notes, "Since the original
1982 paper from the laboratory of Rita Colwell (Xu et al., 1982), over 400 papers have appeared which describe various
aspects of the phenomenon most commonly referred to as the " but nonculturable (VBNC) state" A great many
pathogens, as well as nonpathogens, are now known to enter this dormancy state, and its significance in medicine,
bioremediation, the use of bacteria as fecal indicators, and indeed in most microbiological studies where culturability is
employed as the (often sole) indicator of viability, is becoming increasingly Evident."

Oliver said, "a number of studies have found that processes which are normally assumed to be
bactericidal for bacteria may instead result in cells which reside in the VBNC state. These include such treatments as
pasteurization of milk (e.g. Gunasekera et al., 2002) and
chlorination of wastewater (Oliver et al., 2005).

Futhermore, he said, "The number of species described to enter the VBNC state constantly increases, with
approximately 60 now reported to demonstrate this physiological response. Included are a large number of human
pathogens, including Campylobacter spp., E. coli (including EHEC strains), Francisella tularensis, Helicobacter pylori,
Legionella pneumophila, Listeria monocytogenes, Mycobacterium tuberculosis, Pseudomonas aeruginosa, several
Salmonella and Shigella spp. and Vibrio cholerae, V. Parahaemolyticus, and V. Vulnificus."

He adds, "We have recently demonstrated (Adams et al., 2003) that H. pylori is able to enter the
VBNC state as cells are exposed to a natural, freshwater environment, and that this entry is dependent on the ambient
temperature. Cells underwent a transition from culturable rods to predominately nonculturable cocci as they entered the
VBNC state. Further, cells could be demonstrated to be producing DNA transcripts for at least 26 hours after they had
completely entered the VBNC state (Adams and Oliver, unpublished)."

Oliver warned, "These studies suggest that H. pylori is able to persist in the environment in this dormant state until it
enters a suitable host, at which time a new round of infection might be initiated."

Oliver's concluson, "While the importance of VBNC cells in the initiation of human infection is not yet fully clear, it
appears that cells in this state retain virulence, and should be considered by
those investigators and government regulators involved in the public health."

In their study in 1997, Gibbs et al.,
(27)  reported  " In a soil amendment trial biosolids were mixed with sandy soil and
monitored for 37 weeks. In two storage trials biosolids were stored in piles 1m high and monitored for <60 weeks.
Included in the monitoring programme were tests to determine the concentrations of faecal coliforms, faecal streptococci
and salmonellae. In both the soil amendment trials and biosolids storage trials, concentrations of indicator organisms
and salmonellae decreased through an extended hot, dry summer period. Although these organisms were not detected
in the majority of samples taken during the summer, repopulation of faecal coliforms and salmonellae occurred in the
trials following rainfall at the beginning of the winter. In the case of one of the storage trials repopulation occurred
following a period of 50 weeks when salmonellae and faecal coliforms were not detected. When repopulation occurred,
faecal coliform concentrations increased to higher than those at the beginning of the trials. These results suggest that
faecal coliforms and salmonellae were at undetectable concentrations through the summer period but were able to grow
when provided with favourable conditions."

."Straub, Pepper and Gerba
(55) say that the list of pathogens are not constant but keep changing:
As advances in analytical techniques and changes in society have occurred, new pathogens are recognized and the
significance of well-known ones change.Microorganisms are subject to mutation and evolution, allowing for adaptation to
changes in the environment. In addition, many pathogens are viable but nonculturable by current techniques (Rozak
and Colwell 1987), and actual concentrations in sludge are probably underestimated.(p. 58)"

"They add further:

Thus, no assessment of the risks associated with the land application of sewage sludge can ever be considered
complete when dealing with microorganisms. As new agents are discovered and a greater understanding of their
ecology is developed, we must be willing to reevaluate
previous assumptions. (p. 58)"

Pathogens known to be in sludge/biosolids

In 1986, Dr. Charles Gerba, (10) University of Arizona, in his work, EPA,Development of a Qualitative Pathogen
Risk Assessment Methodology for Municipal Sludge Landfilling, noted that a study by,
"Hess and Breer (1975) reported that samonellae on grass treated with sludge could survive for a little less than 16
months in the climate of Switzerland, but most reported times are shorter."

According to Gerba,  "Municipal sludges are known to contain microorganism capable of causing serious illness and
mortality in humans (Evans, 1982). Many of these organisms are responsible for waterborne disease in the United
States today (Craun, 1986). The number of documented case of waterbornes disease in the United States has been on
the increase for almost two decades (Craun, 1986); contaminated groundwater is responsible for almost half of the
outbreaks each year (Keswick and Gerba, 1980). Enteric pathogens are the most common cause of food and
waterborne illness in the United States today (NRC, 1985; Craun, 1986)."

Furthermore, he adds, "Solids-associated viruses in sludge have been shown to be infectious, well protected and able
to survive longer in the environment that free-living viruses in water and wastewater (Gerba, 1984a). Most available
information describes viral survival in soil alone, rather than survival in a sludge-soil matrix".

Gerba does not mention viable but nondetectable bacteria, but he does note the potential for regrowth. He said, "In the
case of indicator bacteria such as coliforms and fecal coliforms, regrowth may occur in buried sludges (Donnelly and
Scarpino, 1984)."  

In conclusion he said, "To determine risks associated with landfilling of sludge, it is necessary to be able to predict
pathogen survival."

As early as  1988,  Yanko (41)  (83) noted in his study on sludge compost for EPA, "Although the use of sludge as a soil
amendment is attractive, it is not without potential health risk. According to Yanko, "The relative public health risk
associated with the beneficial use of sludge is directly related to the extent of public exposure.----risks increases if the
sludge is used on food crops or public access areas. ---The routes of exposure may take various forms, including
handwork in gardens and eating uncooked vegetables, grown in sludge amended soils---Perhaps at the highest risk of
ingesting pathogenic organisms are the very young children playing in yards and gardens that have been treated with
sludge products."

Yanko warned  of the danger posed by fungi found in compost, "such as certain specious of Candida. Cryptocuccus
and Trichosporon, and PATHOGENIC members of some filamentous genera, such as Aspergillus, Phialophora,
Geotrichum, Trichophyton, and Epidermophyton." Also, "Parasitic infections present a potential health risk associated
with home use of sludge due to the existence of highly resistant stages of the organisms and low infectious doses."

In 1993, EPA asked the National Academy of Sciences - National Research Council (NRC)  to review the science
methodology behind the sludge rule. However, there was a serious conflict of interest in the committee chairmanship.
Dr. Al Page (68) of the University of California at Riverside (UCR), was the Chair of the National Science Academy's
National Research Council Committee (NRC).   Dr. Page had just co-chaired the 503  
Peer Review Committee which
approved the sludge science methodology the NRC Committee was being asked to evaluate There he was listed as a
metal bioavailability expert, as was Dr.Andrew Chang, Page's colleague at UCR, another metals bioavailability expert.

In 1996, "the NRC Report (31), "Use of Reclaimed Water and Sludge in Food Crop Production", "assured the public that
Coxsackie Viruses and Echoviruses in sludge only cause "flu-like" symptoms. However, according to Tabor's Cylopedic
Medical Dictionary, the (sixty species `EPA ,(5)') of the two viruses can also cause inflammation of  the heart, spinal
cord and brain".

"The NRC report did add that, "Many of the variables associated with the transmission of infectious  disease from
wastewater and sludge are either not well understood or are unpredictable." (p. 93-94)"

Although both EPA and WEF have been claiming sludge/biosolids are safe from pathogens, the NRC report
(31)  
pointed out the concerns NRC had in regard to pathogens in sludge/biosolids, "There is a great diversity of pathogenic
agents involved in the fecal-oral exposure route, and an equal diversity of the dose-response relationships. Monitoring
for all these agents is impractical; therefore, the use of indicator organisms has been the traditional approach to
estimating sanitary quality. Coliform bacteria have been the most used in this regard...(however, they)...may not
adequately predict the present of viruses, protozoa or helmiths. Many enteric viruses, for example, have a greater
resistance to chemical disinfection and irriation than do most bacterial indicators.....There are instances in sludge
processing, such as composting, in which the coliform levels can not be satisfactorily reduced even though there is
reason to believe that the sanitary level of the material is acceptable (EPA, 1992b;...Many of the parasites of concern
exist in the encysted stage outside the human or animal intestinal track, and are quite resistant to chemical and physical
disinfection in this form. Wastewater reclamation practice relies on the treatment process to control these parasites.
Parasites ova and cysts concentrate in sewage sludge and thus are of most concern for land application of sludge." (p.
94)

The NRC Committee did not review the one human health study (Municipal Sewage Sludge Application on Ohio Farms:
Health Effects)
(9) funded by the EPA and used by the EPA as the ultimate proof of  the safety of sludge." But instead,
used an abstract by a third party (8) re-titled, Demonstration of Acceptable Systems for Land Disposal of Sewage
Sludge. According to the NRC report of the third party abstract which stated, "An epidemiologic study on human
exposure to pathogens in sludge compared health effects in 164 people living on 47 farms which received 2 to 10 tons
of sludge per ha per year for three years to 130 people from 45 farms who formed a control group. Both study groups
were from geologically matched areas of rural Ohio. Study participants answered monthly surveys and had annual
tuberculin testing and serological tests of quarterly blood samples. In addition, monthly surveys included questions
about farm animals' health. It was found that there were no significant differences in the health of those living on farms
where sludge was applied compared to the control group with respect to respiratory or digestive illness or reported
physiological symptoms. Similarly, no differences were reported between domestic animals from sludge-amended
versus control farms (Brown et. al,, 1985). (p. 113)"

"The NRC report failed to reveal the Ohio study's finding that it was never completed and the statement, "There have
been no previous reported studies of the human health effect of land application of treated municipal sewage sludge."

Moreover, the originial study authors [Dorn, Reddy, Lamphere, Gaeuman and Lanese] made it very clear the study was
not to be used in the manner the EPA and NRC are now using it. In the actual study, the researchers state, "The
absence of observed human or animal health effects resulting from sludge application in this study of Ohio farms was
associated with low sludge application rates which were in accordance with Ohio and U.S. Environmental Protection
Agency guidelines. (Not current part 503 guidelines) Caution should be exercised in using these data to predict health
risks associated with sludges containing higher levels of disease agents and with higher sludge application rates and
larger acreages treated per farm than used in this study."

In 2002, " a new NRC Committee
(69) review questioned the safety of sludge/biosolids, "There is a serious lack of
health related information about populations exposed to treated sewage sludge," according to committee chair Thomas
Burke, professor, department of health policy and management at Johns Hopkins University Bloomberg School of Public
Health in Baltimore. "To ensure public health protection, EPA should investigate allegations of adverse health effects
and update the science behind its chemical and pathogen standards."


The 2001 Stefano Dumontet et al.
(29) study. In their study, " The Importance of Pathogenic Organisms in Sewage and
Sewage Sludge" reported in the J. Air & Waste Manage. Assoc., that "Bacterial pathogens in sewage sludge contribute
significantly to health problems, locally and globally."

They state, "Besides the pathogens of past concern (e.g., Bacillus cereus, Clostridium botulinum, Clostridium
perfringens, Salmonella typhi, Shigella, Staphylococcus aureus), Tauxe 35 reported a list of new or emerging
pathogens that have been recognized in the last 20 years as being predominantly foodborne.

They are the Norwalk-like viruses; the bacteria C. jejuni, Campylobacter fetus ssp. fetus, Escherichia coli O157:H7, E.
coli O111:NM, E. coli O104:H21, Listeria monocytogenes, S. enteritidis, Salmonella typhimurium DT 104, Vibrio cholerae
O1, Vibrio vulnificus, Vibrio parahaemolyticus, Yersinia enterocolitica; and the alga Nitzschia pungens (causative agent
of the amnesiac shellfish poisoning). Campylobacter coli and Arcobacter spp.,36,37 V. cholerae O139,38 and
Aeromonas spp. should also be added to the list of emerging bacterial pathogens"

"Technical limitations for detection and isolation procedures can be considered the main difficulties in monitoring sludge
pathogens. In addition, pathogenic bacteria  introduced into a hostile environment may become viable but not
culturable121 without losing their virulence factors. Note also that sludge may contain several xenobiotic compounds,
and composted sludge could facilitate inhospitalities between pathogenic bacteria."

They list the pathogens that have been isolated from sludge/biosolids.

Table 3. Viruses excreted by humans that can be isolated from sewage
sludge.34,43,57,126,127
                                   Virus Diseases or Symptoms Caused
Enteroviruses
Polio virus                                       Poliomyelitis, meningitis, fever
Coxackievirus A                             Herpangina, respiratory disease, meningitis, fever
Coxackievirus B                              Myocarditis, congenital heart anomalies,
                                                            respiratory disease, pleurodynia, rash, fever
Echovirus                                         Meningitis, respiratory disease, diarrhea,
                                                       encephalitis, acute hemorrhagic conjunctivitis, fever
New Enteroviruses
Adenovirus                                       Respiratory disease, eye infection
Parvovirus                                         Meningitis, encephalitis, respiratory disease,
                                      acute hemorrhagic conjunctivitis, fever
Reovirus                                           Not clearly established
Hepatitis A virus                              Infectious hepatitis
Hepatitis C virus                              Infectious hepatitis
Hepatitis E virus                              Infectious hepatitis
Rotavirus                                         Vomiting and diarrhea
Astrovirus                                       Not established
Calicivirus                                       Vomiting and diarrhea
Coronavirus                                     Common cold
Norwalk agent and other                 Vomiting and diarrhea
small round viruses
Adeno-associated viruses               Not clearly established, but associated
                                                 with respiratory disease in children
Polyomaviruses
JC Progressive multifocal leukoencephalopathy
BK Infections of the urinary tract

Table 5. Bacterial pathogens that have been isolated from sewage sludge.43,63,67,73,128

Primary Pathogens                             Opportunistic Pathogens
Motile Aeromonas                             Citrobacter spp.
Arcobacter spp.                                   Enterobacter spp.
Bacillus anthracis                                Escherichia coli
Brucella spp.                                       Klebsiella spp.
Campylobacter coli                             Proteus spp.
Campylobacter fetus ssp.                    fetus Providencia spp.
Campylobacter jejuni                          Serratia spp.
Clostridium botulinum
Clostridium perfringens
Escherichia coli O111:NM
Escherichia coli O157:H7
Escherichia coli O184:H21
Leptospira spp.
Listeria monocytogenes
Mycobacterium spp.
Pseudomonas aeruginosa
Salmonella spp.
Shigella spp.
Staphylococcus (coagulase positive strains) [NF]
Streptococcus (beta-hemolyticus strains)      [NF]
Vibrio cholerae
Vibrio parahaemolyticus
Vibrio vulnificus
Yersinia enterocolitica

Table 6. Pathogenic yeast and fungi that have been isolated from sewage sludge.7,106

Yeast
Candida albicans
Candida guillermondii
Candida krusei
Candida tropicalis
Cryptococcus neoformans
Trichosporon
Fungi

Table 7. Parasites that have been isolated from sewage and sewage sludge.43,109,110

Protozoa                                                      Cestodes                                                            Nematodes
Cyclospora                                   cayetanensis Diphyllobothrium latum                        Ancylostoma duodenale
Cryptosporidium parvum                 Echinococcus granulosus                                       Ascaris lumbricoides
Encephalitozoon intestinalis                   Hymenolepsis nana                                           Necator americanus
Entamoeba histolytica                                  Taenia saginata                                               Toxocara canis
Giardia lamblia                                               Taenia solium                                                   Toxocara catii
Sarcocystis spp.                                                                                                                   Trichiurus trichiura
Toxoplasma gondii
Vittaforma corneae

EPA's list of pathogens in sewage sludge

In 1989, EPA (5) (30) didn't list all of the pathogens that Yanko found in his study of  marketing of sludge compost, EPA
only documented "a list of 25 primary [families of] pathogens in [its proposed sewage sludge rules]:
Among  these were: 1) five bacteria pathogens (Campylobacter, Escherichia, Salmonella, Shigella, and Vibrio
Cholerae), 2) nine virus pathogens (Entroviruses, Poliovirus, Coxsackieviruses, Echovirus, Hepatitis A, Norwalk and
Norwalk like viruses, Reovirus, and Rotavirus), 3) five helminth pathogens (among them are Hookworms, Tapeworms,
and Nematode worms), 4) five protozoans pathogens (Toxoplasma gondii, Balantidium, Entamoeba histolyca, Giardia
lambia, and Crytosporidium), and 5) one fungi pathogen (Aspergillus)."

The bacteria groups Campylobacter jejuni, E. coli, Salmonella, Shigella and Vibrio cholerae cause abdominal cramps,
diarrhea, vomiting, fever, chills, malaise, nausea, headache; Salmonella, Shigella and E. coli infection can lead to death.
Listeria may be even more deadly, but like E. Coli, it was not considered to be a problem in 1989.  [plus,
Staphylococcus and Streptococcal had not picked up the genes to cause Necrotizing fasciitis].

Among the over 120 different viruses are: 1)Entroviruses or Picornaviruses (152 species); many of these will cause
pneumonia, 2) Poliovirus (3 species) causes paralysis, meningitis, fever, 3) Coxsackievirus A (23 species) causes
respiratory illness, fever, meningitis; Coxsackievirus B (6 species) causes same symptoms as A and in addition
myocarditis, congenital heart anomalies and inflammation of heart in newborns, 4) Echovirus (31 species) causes
inflammation of the heart, spinal cord & brain, 5) Hepatitis A virus can cause death , 6) Norwalk and Norwalk like viruses
cause mostly diseases of the gastrointestinal tract, 8) Reovirus, unknown, and 9) Rotavirus causes acute gastroenteritis.

The five Helminths include worms such as roundworms, hookworms, tapeworms and nematode worms. In humans, the
worms may end up in the brain where they are untreatable, as well as in the retina vessels, liver, lung and heart. The
larve cause hemorrhage, inflammation, necrosis in these tissues. This causes myocarditis, endophthalmitis, epilepsy or
encephalitis.

The five Protozoans; 1) Toxoplasma gondii causes pneumonitis, hepatitis and encephalitis, 2) Balantidium is mild, 3)
Entamoeba histolyca forms liver abscess, 4) Giardia lambia is the cause of severe intestinal problems, 5)
Cryptosporidium causes explosive diarrhea and cramps; it was
first described in humans in 1976 [and infected 400,000 in Milwaukee in 1993}

The one fungi considered by EPA was the deadly Aspergillus; it causes inflamed tissues in bronchi, lungs, aural canal,
skin and membranes of the eye, nose or urethra. It may also produce mycotic nodules in the lungs, liver, kidney and
other organs (FR 54-P.5829 & Tabor's Cylopedic Medical Dictionary, 1990).

EPA made no mention of these pathogens in the final sludge rules, nor did EPA mention the 21 known cancer causing
chemicals in sludge listed in the proposed sludge rules. In fact EPA only addressed the 10 metals under the sludge
rules for which treatment plants were authorized to issue removal credits under 40 CFR 403.

However,
(21) in 1993, "EPA concluded that adequate protection of public health and the environment did not require
the adoption of standards designed to protect human health or the environment
under exposure conditions that are
unlikely and where effects were not significant or widespread.
" (Federal Register No. 58, p. 9252)

The theory has been that there are not many people around sludge disposal sites, so the health affects are
not significant or widespread.

CONCLUSION

This data shows EPA and its partners (state environment departments, municipal wastewater and soil scientists), knew
wastewater treatment plants transformed nonpathogenic bacteria into pathogenic multi-drug resistant toxin protein
producing bacteria through gene transfer prior to the creation of the part 503 sludge rules. EPA and its partners have
conspired to keep this data from the people who have been tricked into accepting sludge/biosolids as an unlabelled  soil
amendment or as a cheap fertilizer based on strictly controlled and very limited studies. EPA and its partners have
continued to promote sludge/biosolids as the damage to public health, animal health and the environment became more
and more apparent to the public.