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Oral application of charcoal and humic acids to dairy cows influences Clostridium botulinum blood serum antibody level and glyphosate excretion in urine

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  • Tierärztliche Praxis Gerlach, Burg,Germany

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The present study was initiated to investigate the influence of oral application of charcoal, sauerkraut juice and humic acids on chronic botulism in dairy cows. A total of 380 Schleswig Holstein cows suffering from chronic botulism were fed daily with 400 g/animal charcoal for 4 weeks (1-4 weeks of study), 200 g/animal charcoal (5-10 weeks of study), 120 g/animal humic acid (11-14s week of study), 200g charcoal and 500 ml Sauerkraut juice/animal (13-16 weeks of study), 200 g charcoal and 100 mL Aquahumin /animal (15-18s week of study), 100 g charcoal and 50 mL Aquahumin (19-22 weeks of study) followed by 4 weeks without any supplementation. Bacteriological and immunological parameters investigated included C. botulinum and botulinum neurotoxins (BoNT) in faeces, C. botulinum ABE and CD antibodies, positive acute phase proteins (APPs) haptoglobin and LPS-binding protein (LBP) using serum ELISA, negative APP paraoxanase by its enzymatic activity and glyphosate in urine by ELISA. Neither BoNT nor C. botulinum was detected in feacal samples. From week six until four weeks before the end of the study, there was a significant reduction in antibody levels. All supplementation, except low doses of charcoal (200g /animal) alone, led to a significant reduction of C. botulinum ABE and CD antibody levels. There also was a significant reduction of glyphosate in urine following supplementation with a combination of 200g charcoal plus either 500 mL sauerkraut juice or humic acid. Haptoglobin, paraoxanase and LBP were significantly increased by the 24th week of the study. The positive APPs and C. botulinum antibodies were significant negative correlations. In conclusion, a charcoal-sauerkraut juice combination and humic acids could be used to control chronic botulism and glyphosate damage in cattle.
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Oral Application of Charcoal and Humic acids to Dairy Cows Influences
Clostridium botulinum Blood Serum Antibody Level and Glyphosate Excretion
in Urine
Henning Gerlach1, Achim Gerlach2, Wieland Schrödl1, Bernd Schottdorf3, Svent Haufe4, Hauke Helm5, Awad Shehata1,6* and Monika Krüger1
1Institute of Bacteriology and Mycology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 29, D-04103 Leipzig, Germany
2Waldstraße 78, D-25712 Burg, Germany
3Carbon Terra GmbH Gutermannstrasse 25, D-86154 Augsburg, Germany
4WH Pharmawerk Weinböhla GmbH, Poststr. 58, D-01689 Weinböhla, Germany
5Südholzring 2, D-25693 Gudendorf, Germany
6Avian and Rabbit Diseases Department, Faculty of Veterinary Medicine, Sadat City University, Egypt
*Corresponding author: Dr. Shehata A, Institute of Bacteriology and Mycology, Veterinary Faculty, University of Leipzig, An den, Tierkliniken 29, D-04103 Leipzig,
Germany, Tel: +49 341 973 8181; E-mail: shehata@vetmed.uni-leipzig.de
Received date: Feb 22, 2014, Accepted date: Mar 26, 2014, Published date: Mar 31, 2014
Copyright: © 2014 Gerlach H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
The present study was initiated to investigate the influence of oral application of charcoal, sauerkraut juice and
humic acids on chronic botulism in dairy cows. A total of 380 Schleswig Holstein cows suffering from chronic
botulism were fed daily with 400 g/animal charcoal for 4 weeks (1-4 weeks of study), 200 g/animal charcoal (5-10
weeks of study), 120 g/animal humic acid (11-14s week of study), 200g charcoal and 500 ml Sauerkraut juice/animal
(13-16 weeks of study), 200 g charcoal and 100 mL Aquahumin/animal (15-18s week of study), 100 g charcoal and
50 mL Aquahumin (19-22 weeks of study) followed by 4 weeks without any supplementation. Bacteriological and
immunological parameters investigated included C. botulinum and botulinum neurotoxins (BoNT) in faeces, C.
botulinum ABE and CD antibodies, positive acute phase proteins (APPs) haptoglobin and LPS-binding protein (LBP)
using serum ELISA, negative APP paraoxanase by its enzymatic activity and glyphosate in urine by ELISA. Neither
BoNT nor C. botulinum was detected in feacal samples. From week six until four weeks before the end of the study,
there was a significant reduction in antibody levels. All supplementation, except low doses of charcoal (200g /
animal) alone, led to a significant reduction of C. botulinum ABE and CD antibody levels. There also was a
significant reduction of glyphosate in urine following supplementation with a combination of 200g charcoal plus
either 500 mL sauerkraut juice or humic acid. Haptoglobin, paraoxanase and LBP were significantly increased by
the 24th week of the study. The positive APPs and C. botulinum antibodies were significant negative correlations. In
conclusion, a charcoal-sauerkraut juice combination and humic acids could be used to control chronic botulism and
glyphosate damage in cattle.
Keywords Humic acids; Peripartual cases;
C. botulinum
Introduction
In recent years, an increased frequency of a new form of bovine
botulism has been observed. This form of botulism differs from
regular food-born botulism by its slow and chronic development with
various unspecific symptoms. This protracted form may develop when
small, sub-lethal amounts of BoNT are taken up and/or absorbed over
several days or are generated in the hind gut [1,2]. Clinical symptoms
of chronic botulism are most often peripartual cases with indigestion
(constipation alternating with diarrhea), non-infectious acute
laminitis, ataxia and stiff stilted gait, impossibility to get up (paralysis),
apathy, engorged veins, positive venous pulse, edema in legs, udder,
and dew-lap, retracted abdomen, forced respiration and unexpected
death. The prevalence of C. botulinum in cattle can be determined by
detection of botulinum neurotoxins (BoNTs) and/or
C. botulinum
vegetative bacteria or spores in the gastrointestinal tract or organs
(liver, kidney, lungs and muscles [1,3,4]. A second way to verify
chronic botulism is with specific antibodies for BoNTs [3,5,6] detected
natural specific antibodies in wild canine species, horses and dairy
cows.
C. botulinum
is an ubiquitous Gram-positive, spore forming,
obligatory anaerobic bacterium that inhabits soil, dust and organic
matter such as feces of animals and man, slaughterhouse wastes,
residues of biogas plants, and bio-compost. It generates eight highly
toxic neurotoxin isoforms (BoNT A-H) that are the most toxic
substances known [7-12]. All isoforms, together with the related
tetanus neurotoxin (TeNT) secreted by
C. tetani
, are Zn2+-
endoproteases. The immunologically distinct neurotoxins (A-H) of
C.
botulinum
are homologous proteins consisting of a heavy and light
chain linked by an essential disulfide bridge. The light chain blocks the
release of acetylcholine at the neuromuscular junction. Human cases
are mostly caused by types A, B, or E, while animal diseases are mostly
caused by types C and D [1,13,14]. Several C. botulinum strains
produce two neurotoxins [11]. Physiological differences are used to
divide
C. botulinum
strains into 4 physiological groups; group I,
consisting of
C. botulinum
A and proteolytic strains of
C. botulinum
B
and F; group II, consisting of
C. botulinum
E and nonproteolytic
strains of
C. botulinum
B and F; group III, consisting of
C. botulinum
Shehata A et al., J Clin Toxicol 2014, 4:2
DOI: 10.4172/2161-0495.186
Research Article Open Access
J Clin Toxicol
ISSN:2161-0495 JCT Volume 4 • Issue 2 • 1000186
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Journal of Clinical Toxicology
C and D; and group IV, consisting of
C. argentinense
(BoNT G).
Neurotoxigenic strains of other Clostridium species such as
C.
butyricum
, (BoNT E, group V) and C. baratii (BoNT F, group VI)
have also been identified [14]. BoNTs are produced as a 150 kDa single
polypeptide chain. The protein is post-translationally proteolyzed to
form a dichain in which the heavy chain (HC, 100 kDa) and light
chain (LC, 50 kDa) are linked through a disulfide bond. HC is
composed of two 50 kDa domains, with the N-terminal half involved
in translocation and the C-terminal half involved in binding with
nerve cells. BoNTs bind specifically to neuronal cells, enter the
cytoplasm, and then cleave core proteins involved in the vesicular
fusion machinery (SNARE proteins) by its metalloprotease activity to
block the release of neurotransmitters. When BoNTs are produced by
the bacteria, the BoNTs are found in complexes associated with
protective proteins (progenitor toxins). These are the nontoxic,
nonhemagglutinin (NTNH, 130 kDa) and several nontoxic
hemagglutinins (NTHAs). BoNTs cannot penetrate intact skin, but the
toxin is absorbed from mucosal surfaces or wounds. In foodborne and
intestinal botulism, botulinum toxins are produced from
C. botulinum
and other BoNT producing Clostridia, which colonize the lumen of
the intestine where the toxins are absorbed from the digestive tract.
BoNT binds through a double-receptor system consisting of a protein
receptor and acidic lipid-gangliosides with its heavy chain domain
[15,16].
The upper or small intestine is the most important site for
absorption of BoNTs [17,18] but other mucous membranes also are
able to absorb BoNTs [19]. BoNT complexes probably do not
dissociate in the digestive tract. The whole toxin complex seems to be
absorbed from the intestine into the lymphatics in the rat ligated
duodenum loop assay. Molecular dissociation occurs immediately
after BoNT complexes, designated progenitor toxins, are absorbed into
the lymphatics [20]. Botulinum progenitor toxins are found in three
forms with molecular masses of 900 kDa (LL toxin for type A), 500
kDa (L toxin for types A, B, C, D and G) and 300 kDa (M toxin for
types A, B, C, D, E and F). The M toxin consists of BoNT and NTNHA
with no hemagglutination activity, whereas the L toxin consists of
BoNT, NTNHA and NTHA. The LL toxin is a dimer of L toxin [21].
NTNHA protects the BoNT from acidity and proteases in the digestive
tract. NTHAs play an essential role in the effective absorption of the
type C progenitor toxins to the small intestine. NTHA exists as
subcomponents; HA-70, HA-33 and HA-17 [21]. The HAs of BoNT/A
and B could disrupt the human epithelial intercellular junction
through species-specific interaction with E-cadherin to presumably
facilitate BoNT transport via the paracellular route [22-24]. NTHAs of
BoNT A may bind to 9 different glycan sites of gut epithelial cells.
Thus NTHA mediated absorption could be prevented by galactose and
its derivates [25].
Treatment of BoNT intoxication is accomplished using specific
polyclonal and monoclonal antibodies. Antitoxins given within 24
hour of the onset of disease can lower the death rate from botulism
and shorten duration of the symptoms [16]. Antibodies only neutralize
non-bounded BoNTs. Other possibilities for treatment could be
peptide based inhibitors that mimic amino acid motives of the SNARE
proteins to inhibit the catalytic part (endopeptidase) of the BoNT [15].
One obstacle for the endopeptidase inhibitors is how to deliver the
molecules into the intoxicated nerve cells. Another way of treatment is
mimicry of receptors. Binding of BoNTs to nerve cells in a two-step
process is generally accepted. Binding to gangliosides is followed by
high affinity binding to a protein receptor(s) [26]. Synaptotagmin has
been proposed as the protein receptor for BoNT/A, /B, /E, and /G
[15,27]. Aptamers, unique oligonucleotides with high affinity for their
(proteins or small molecule) targets, are the newest treatment
possibilities [16]. All of the above mentioned methods are mostly too
expensive for food animals and cheaper methods are necessary.
Neuvonen and Olkkola (1988) used charcoal to treat intoxications in
humans. In the present study,
C. botulinum
types ABE and CD blood
serum antibodies, feacal BoNTs and
C. botulinum
spores from 40
dairy cows in 4 different lactation states (10 each group) were
investigated for 26 weeks [28]. The cows were supplemented with
charcoal and humic acids in different doses to evaluate their BoNT
binding capacity. Parallel haptoglobin [29] and LPS binding protein
[30], as positive acute phase proteins (APPs), and paraoxanase [31,32]
as negative APP were investigated. Urine was tested each 4 weeks for
glyphosate to demonstrate the glyphosate neutralizing capacity of
charcoal and humic acids, and to show a possible connection between
glyphosate and chronic botulism.
Material and Methods
Animals and supplementations
A Schleswig Holstein dairy cow farm of about 380 cows with
average milk production of 9000 L per year showed clinical symptoms
of chronic botulism (flock stiff stilted gait, paresis, apathy, engorged
veins on tarsus, positive venous pulse, mucous saliva, reduced tail
tonus, small wounds in the udder region) in 10-15% of the cows and
60% of the cows suffered from Dermatitis digitalis (mortellaro). The
entire animal population was involved in the various charcoal (CC) (≤
8 mm diameter) and powdery humic acid (WH67) or sauerkraut juice
(SJ) and liquid humic acid (Aquahumin) supplement. Each treatment
represented 10 identical cows of the 1st, 2nd, and 3rd lactation and the
dry cows group for the full time of the investigation. Their last
polyvalent clostridial vaccination (Covexin, Intervet) was on
01.11.2012. The treatment regime with CC, SJ and/or humic acids was
changed every 4 weeks (supplementation periods) in Table 1 and given
as part of the total mixed ratio (TMR).
Date SP1* SP2 SP3 SP4 SP5 SP6 SP7
11.11.2012-08.12.
2012
9.12.2012-20.01.
2013
21.01.2013–
17.02.2013
18.02.2013-17.03.20
13
18.03.2013-31.03.20
13
01.04.2013-14.04.
2013
15.04.2013-14.05.2
013
Supplements 400 g CC1 200 g CC 200 g CC+
500 mL SJ2
120 g HA3 200 g CC+100mL
AH4
100 g CC+50mL
AH
Without
supplements
Citation: Gerlach H, Gerlach A, Schrödl W, Schottdorf B, Haufe S et al., (2014) Oral Application of Charcoal and Humic acids to Dairy Cows
Influences Clostridium botulinum Blood Serum Antibody Level and Glyphosate Excretion in Urine. J Clin Toxicol 4: 186. doi:
10.4172/2161-0495.186
Page 2 of 8
J Clin Toxicol
ISSN:2161-0495 JCT Volume 4 • Issue 2 • 1000186
*Supplementation period, 1CC=Charcoal (Carboligni, Schottdorf, Germany), 2SJ=Sauerkraut juice (KronprinzKonserven, Meldorf, Germany), 3HA=humic acids WH67
(PharmawerkWeinböhla, Germany), 4AH=Aquahumin (Pharmawerk Weinböhla, Germany).
Table 1: Overview at the various times of supplementation.
06.01.2013 20.01.2013 17.02.2013 03.02.2013 03.03.2013 17.03.2013 31.03.2013 01.04.2013
Paresis 2/40 2/40 3/40 3/40 2/40 2/40 2/40 2/40
Cystitis 2/40 2/40 2/40 1/40 4/40 1/40 3/40 3/40
Diahrea 5/40 0/40 3/40 7/40 0/40 5/40 0/40 2/40
Viscus saliva 0/40 0/40 0/40 0/40 1/40 2/40 1/40 4/40
endometritis 0/40 1/40 0/40 0/40 0/40 0/40 0/40 0/40
Ataxia 0/40 0/40 0/40 0/40 0/40 0/40 0/40 1/40
Table 2: Clinical estimation of cows at the various sampling points.
The TMR was composed of grass and maize silage (glyphosate
concentration not tested), concentrated mixed feed (1.93 mg/kg
glyphosate), ground grains (0.51 mg/kg glyphosate), wheat straw (0.03
mg/kg glyphosate) and alfalfa hay (0.02 mg/kg glyphosate). After
31.03.2013, 10 kg draff/cow (0.01 mg/kg glyphosate) was fed. At each
sampling point, each of the 40 treated cows was evaluated for clinical
symptoms (Table 2).
Collection of samples
Blood, faeces and urine were analyzed 7 times at 4 week intervals
with one exception (200 g CC over 6 weeks). Blood specimens were
taken from the Vena coccygenamediana, coagulated blood centrifuged
at 3000 x g for 15 min and the serum samples were stored at -20°C.
Faeces were taken from Ampulla recti and spontaneous urination was
sampled and stored at -20°C. All specimens were quickly cooled and
sent to the laboratory.
Glyphosate testing of urine
Urine samples were diluted 1:20 with distilled water (aqua
distillated, Braun, Germany) and tested for glyphosate by ELISA
(Abraxis, USA) according to the manufacturer’s instructions. Test
validation was done with Gas Chromatography-Mass Spectroscopy
(GC-MS) by Medizinsches Labor Bremen (Germany). The correlation
coefficient between the two tests was 0.96 (Data not shown).
Analysis of free BoNT/A-E and C. botulinum spores in faeces
Preparation of faeces for detection of BoNT/A-E
Faecal samples were diluted 1:3 in PBS (Dulbecco, pH 7.4)
containing 0.1% Triton X-100, 0.1% Tween 20 and 10 mM EDTA. The
samples were thoroughly mixed and frozen at -20°C. After thawing,
the diluted samples were centrifuged at 7000 x g for 15 min and the
clarified supernatants were analyzed with BoNT-ELISA.
Indirect detection of C. botulinum spores
Rumen fluid and faecal samples were diluted 1:10 in RCM (0.5 g in
4.5 ml), vigorously mixed, and heated at 80°C for 10 min. Samples
were incubated at 37°C for 7 d under anaerobic conditions and
subsequently stored at -20°C until tested. After thawing, the sample
was centrifuged at 7.000 x g for 15 min and the clear supernatant was
analyzed for the type-specific soluble antigens of
C. botulinum
types
A-E by ELISA.
BoNT-ELISA
BoNT/A-E were determined by an ELISA developed in our institute
[2]. The standard volume was 100 µl per well and the standard
incubation condition was 1 h at room temperature (1 h at RT) on a
microtiter plate shaker (400 rpm). The coating buffer was 0.1 M
NaHCO3 and the wash solution (WS) was 0.9% NaCl with 0.05%
Tween 20 (Sigma-Aldrich, Taufkirchen, Germany). All washing steps
were done in a Nunc-Immuno-Washer 12 (Nunc, Wiesbaden,
Germany). After coating the ELISA wells with capture antibodies (3
mg/ml, BoNT-immunoaffinity purified-IgG from rabbits against
BoNT/A-E, Institute of Bacteriology and Mycology, University of
Leipzig, Germany) overnight at 4-6°C, they were incubated with 150
ml per well of 1% gelatin from cold water fish skin (Sigma-Aldrich,
Taufkirchen, Germany) in 0.9% NaCl solution for 1 h at RT. The wells
were washed twice with WS and loaded with the prepared faecal
samples diluted 1:2 in 20 mMTris, pH 8.0, assay buffer [adjusted with
1 M HCl] containing 0.9% NaCl, 5 mM EDTA, 1% gelatin from cold
water fish skin, 0.2% bovine serum albumin, 0.1 mg/ml rabbit IgG
from normal serum and 0.2% Tween 20 (chemicals from Sigma-
Aldrich or Fluka, Taufkirchen, Germany). After incubation, the wells
were washed five times with WS and loaded with the detection
antibodies conjugated with HRP and diluted in assay buffer.
C.
botulinum
types A and B were detected with 2.5 mg/ml horse [Fab]2
from IgG against
C. botulinum
A and B (Novartis Vaccines and
Diagnostics Co, Marburg, Germany). Types C and D were detected
with 0.1 mg/ml of IgG from rabbits developed against BoNT/C and D
(Institute of Bacteriology and Mycology, University of Leipzig). Type E
was detected with 2.5 mg/ml IgG from horses against
C. botulinum
type E (WDT, Garbsen, Germany).
After incubation at RT, the plates were washed four times with WS
and the HRP activity was determined by adding 100 µl/well of 3 mM
H2O2 and 1 mM 3, 30, 5, 50-TMB. The substrate reaction was stopped
Citation: Gerlach H, Gerlach A, Schrödl W, Schottdorf B, Haufe S et al., (2014) Oral Application of Charcoal and Humic acids to Dairy Cows
Influences Clostridium botulinum Blood Serum Antibody Level and Glyphosate Excretion in Urine. J Clin Toxicol 4: 186. doi:
10.4172/2161-0495.186
Page 3 of 8
J Clin Toxicol
ISSN:2161-0495 JCT Volume 4 • Issue 2 • 1000186
with 1 M H2SO4 (50 µl/well) and the optical density (OD) was
measured with an ELISA-reader at 450 nm. The sensitivity, specificity,
precision, limit of detection, and range of quantification were
determined previously. Cross reactivity of antibodies with
C. tetani, C.
perfringens, C. sporogenes, C. sordellii, C. novyi, C. butyricum,
Bacillus cereus, Streptococcus agalactiae, Streptococcus
zooepidemicus, Staphylococcus aureus, Staphylococcus epidermidis,
Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas
aeruginosa, Candida albica
ns and
Candida krusei
were all negative.
Evaluation of BoNT-ELISA
The relative units (RU) were calculated from the measured OD
values as follow: (sample-OD minus twice the value of the control-OD
[BoNT-negative sample of bovine faeces]) multiplied by 1000 and
dilution factors per minute substrate incubation time.
Analysis of C. botulinum antibodies using ELISA
Solid phase antigen for ELISAs
C.
botulinum
types A (7272), B (7273), C (2300), D (2301), and E
(2302) obtained from the National Collection of Type Cultures
(NCTC) were used for preparation of ELISA antigens. Culture
supernatant from
C. sporogenes
and
C. perfringens
(Isolated and
identified by the Institute of Bacteriology and Mycology, Faculty of
Veterinary Medicine, Leipzig University) served as a control antigen to
study cross reactivity. All strains were cultured in reinforced
Clostridial medium (RCM; Sifin, Berlin, Germany) and incubated
anaerobically at 37°C for 7 days followed by freezing at 25°C.
Supernatants were checked for BoNT-type with type specific ELISA
[9]. After thawing and mixing, the culture suspension was centrifuged
at 10,000 g for 15 min and the clear supernatant was separated. BoNT-
proteins in the supernatants were detoxified with 20 mM
formaldehyde (four additions weekly) and incubated at 37°C. Active
formaldehyde groups were blocked by the addition of 100 mM lysine
and 100 mM glycine in 100 mMTris/HCl (pH 8.0) solution and
incubated at RT for 24 h. Complete detoxification was verified with
the mouse test by Dr. F. Gessler (Miprolab, Göttingen, Germany), data
not shown. The antigen preparation was washed with PBS (pH 7.4)
and concentrated by ultrafiltration at a molecular weight cut-off of 50
kDa (viva- vivaspin 20, Sartorius Stedim Biotech, Göttingen,
Germany). The protein concentration was measured with a spectral
photometer (MBA 2000) and its integrated software (PerkineElmer,
Norwalk, Connecticut, USA) and adjusted with PBS to 1 mg/ml.
Detection of IgG anti C. botulinum antibodies by ELISA
ELISA plates were coated with 100 ml/well of detoxified antigen
from C. botulinum (1 mg/ml in 0.1 M NaHCO3) and incubated
overnight at 4-6°C. Coated plates were washed twice with 0.9% NaCl
containing 0.05% Tween 20 (Sigma-Aldrich, Taufkirchen, Germany)
followed by 135 µl of blocking solution (1% bovine case) mixed with
15 ml diluted serum sample (1:10 in 50 mMTris buffer, pH 8,
containing 0.9% NaCl, 10 mM EDTA, 1% yeast extract, 1% BSA, 20%
RCM and 1% Tween 20) and incubated for 1 h at RT on a microtiter
plate shaker. After washing four times, IgG from rabbits against
bovine IgG (Fc) conjugated with horse radish peroxidase (HRP)
(Dianova, Hamburg, Germany) diluted 1:20,000 in assay buffer (50
mM Tris pH 7.4, 0.9% NaCl, 0.2% yeast extract, 0.1% BSA, 0.1%
bovine Casein, 2% RCM and 0.1% Tween 20) was added to each well
and incubated 1 h at RT.
HRP activity was determined by adding 100 ml/well of 3 mM H2O2
and 1 mM 3, 3´, 5,´5- tetramethylbenzidine (TMB) in 0.2 M citrate-
buffer (pH 4.0). The substrate reaction was stopped with 1 M H2SO4
(50 µl/well) and the optical density (OD) was measured with an
ELISA-reader at 450 nm. RCM without
C. botulinum
antigen served
as a control antigen to determine the degree of non-specific solid
phase binding of immunoglobulin on each sample (control OD). The
control OD value was subtracted from each antigen specific OD value
to calculate the Anti-
C. botulinum
IgG level relative to an internal
laboratory standard (pooled blood samples from >3000 cows) that was
defined as 100 percent.
Haptoglobin analysis
The Hp concentration in blood serum was determined by ELISA as
described by Schroedl et al. [33]. Briefly, the coating antibody was IgG
from rabbit anti-Hp (DAKO, Hamburg, Germany), which was diluted
1:3000. The standard was bovine plasma in which the Hp
concentration was determined with a standardized colorimetric assay
for bovine Hp (Tridelta Development Ltd., Greystones, Co. Wicklow,
Ireland) and further checked against purified bovine Hp. The standard
concentration ranged from 3 to 200 ng ml-1. The samples were diluted
1:1000 and 1:50000 in assay buffer (50 mMTris-HCl with pH 8.0, 0.15
M NaCl, 10 mM EDTA, 0.1% Tween 20 and 0.2% bovine casein, all
from Sigma-Aldrich, Taufkirchen, Germany). The detection antibody
was polyclonal IgG (rabbit) anti-Hp (DAKO, Hamburg, Germany)
conjugated with horseradish peroxidase. The detection antibody was
diluted 1:10000 in assay buffer. The detection limit, including the
dilution factor of 1000, was 1 µg ml-1.
LBP analysis by ELISA
The LBP coating antibody was affinity purified monoclonal IgG2a
(mouse) anti-LBP-human (mAb-Abi-202) at 1.2 µg/ml. The standard
LBP-range in the ELISA was 0.3 to 20 ng/ml human LBP (LBP-
standard serum). The samples were diluted 1:1,000 and higher. The
assay buffer for dilution of the standard and plasma samples was 50
mM Tris HCl (pH 8.0), 0.15 M NaCl, 10 mM EDTA, and 0.1% Tween
20 (v/v). The detection antibody was affinity purified monoclonal
IgG1 (mouse) anti-LBP-human conjugated with horseradish
peroxidase (mAb-Abi-204) diluted 1:6,000 in assay buffer. The two
mAbs and the standard serum were provided by Prof. Ch. Schuett,
Institute of Immunology, and University of Greifswald, Germany.
Paraoxanase analysis
Paraoxonase/arylesterase activity was measured
spectrophotometrically using paranitrophenyl acetate (PNPA) as a
substrate. A stock solution was prepared using 1M Hepes buffer (pH
7.5), 400 mM p-nitrophenyl acetate in DSMO and 100 mM CaCl2.
The working buffer contained 10 mM CaCl2, 10 mMHepes, and 2 mM
p- nitrophenyl acetate in 50 mL distilled water. Blood serum
specimens (25 µL) diluted 1: 10 in distilled water were applied to
microtiter plates and 200 µL of the working buffer were added. After 3
s shaking, the optical density was measured at 405 nm wave length (t0)
and remeasured 10 min later (t1). The paraoxanase activity in U/mL is
calculated with the following equation:
Paraoxonase activity = (t1-to) x serum dilution x1000 = (t1-t0)
x10x1000 = units/L
Citation: Gerlach H, Gerlach A, Schrödl W, Schottdorf B, Haufe S et al., (2014) Oral Application of Charcoal and Humic acids to Dairy Cows
Influences Clostridium botulinum Blood Serum Antibody Level and Glyphosate Excretion in Urine. J Clin Toxicol 4: 186. doi:
10.4172/2161-0495.186
Page 4 of 8
J Clin Toxicol
ISSN:2161-0495 JCT Volume 4 • Issue 2 • 1000186
Statistical analysis
The statistical analysis was carried out with GraphPad Prism 4
(GaphPad Software, La Jolla, USA). A two-way analysis of variance
followed by unpaired Student t-test was used to identify significant
differences between means.
Results
Effect of supplementation on glyphosate in urine
A significant reduction in glyphosate excretion (P<0.0001) was only
seen at the 14th and 18th week of the study (Figure 1).
Figure 1: Dynamics of glyphosate excretion in urine with the
application of 400 g charcoal daily(CC)the first four weeks (1-4
weeks) followed by 200 g CC daily for weeks 5-10, 200 g CC+500
ml Sauerkraut juice (SJ)daily weeks 11-14, 120 g humic acid
(HA)daily weeks 15-18, 200 g CC+100 mL Aquahumin(AH) daily
weeks 19-20, 100 g CC +50 mL AH weeks 21-22 and without
supplementation weeks 23-26. A significant (P<0.0001) reduction
of glyphosate in urine was detected only in weeks 12 to 19 (4 weeks
daily of 200 g CC+ 500 mL SJ, 4 weeks daily of 120 g HA).
The combination of CC (200 g) and SJ (500 ml) as well as HA (120
g) reduced glyphosate in urine significantly.
Botulinum neurotoxin (BoNT) and C. botulinum in faeces
No BoNT or C. botulinum was detected in feacal samples.
Detection of C. botulinum IgG antibodies in blood serum
The dynamic effects of different supplementations on
C. botulinum
ABE and CD blood serum antibody levels over 24 weeks are shown in
Figure 2.
Daily supplementation with CC and/or humic acids initiated at
week 6 significantly reduced antibody levels (P<0.01 at week 6,
P<0.001 for weeks 8-24, and P<0.05 for week 26. The effect of different
supplements on
C. botulinum
CD blood serum antibody levels over
the 26 weeks is shown in Figure 3.
Supplementation with daily 400 g CC significantly decreased CD
antibody (P<0.01) while a daily application of 200 g CC allowed the
CD antibody level to increase. A highly significant (P<0.001) reduction
in CD antibody was detected only after two weeks supplementation
with 200 g CC plus 500 mL SJ. Antibody reduction was constant from
week 4 to 24; however, four weeks after finishing supplementation
(week 26), CD antibodies increased.
Figure 2: Dynamics of C. botulinum ABE antibodies in blood
serum in relation to the daily application of 400 g charcoal (weeks
1-4), 200 g CC (weeks 5-10), 200 g CC+500 ml sauerkraut juice (SJ)
(weeks 11-14), 120 g humic acid (HA) (weeks 15-18), 200 g CC
+100 mL Aquahumin(AH) (weeks 19-20), 100 g CC +50 mL
Aquahumin(AH) (weeks 21-22), and without supplementation
(weeks 23-26). There was a significant reduction of antibody levels
with a daily supplementation of charcoal or humic acids beginning
from week 6 (P<0.01 for week 6, P<0.001 for weeks 8-24, and
P<0.05 for week 26.
Figure 3: Effect of daily supplementation with 400 g CC (weeks
1-4), 200 g CC (weeks 5-10), 200 g CC+500 ml sauerkraut juice (SJ)
(weeks 11-14), 120 g humic acid (HA) (weeks 15-18), 200 g CCl
+100 mL Aquahumin(AH) (weeks 19-20), 100g CC +50 mL AH
(weeks 21-22) and without supplementation (weeks 23-26) on the
dynamic of C. botulinum CD antibodies in blood serum. There was
a significant reduction in antibody levels from daily
supplementation with charcoal and humicacids (P<0.01 and
P<0.001) for weeks 14-24.
Citation: Gerlach H, Gerlach A, Schrödl W, Schottdorf B, Haufe S et al., (2014) Oral Application of Charcoal and Humic acids to Dairy Cows
Influences Clostridium botulinum Blood Serum Antibody Level and Glyphosate Excretion in Urine. J Clin Toxicol 4: 186. doi:
10.4172/2161-0495.186
Page 5 of 8
J Clin Toxicol
ISSN:2161-0495 JCT Volume 4 • Issue 2 • 1000186
Detection of haptoglobin
Haptoglobin levels in blood serum were not significantly different
with any of the supplements except for week 24 after they were taken
off Aquahumin (Figure 4).
Figure 4: Haptoglobin in blood serum after the daily application of
440 g CC (weeks 1-4), 200 g CC (weeks 5-10), 200 g CC+500 ml
sauerkraut juice (SJ) (weeks 11-14), 120 g humic acid (HA) (weeks
15-18), 200 g CCl+100 mL Aquahumin (AH) (weeks 19-20), 100 g
CC +50 mL AH (weeks 21-22) and without supplementation
(weeks 23-26). A significant (P<0.05) difference was only detected
at week 24.
LBP results
There was a significant increase in LBP in blood serum on week 20
(P<0.001) (Figure 5).
Figure 5: LBP in blood serum in relation to daily oral application of
400 g charcoal (CC) (weeks 1-4), 200 g CC (weeks 5-10), 200 g CC
+500 ml sauerkraut juice (SJ) (weeks 11-14), 120 g humic acid (HA)
(weeks 15-18), 200 g CCl+100 mL Aquahumin(AH) (weeks 19-20),
100 g CC +50 mL (AH) (weeks 21-22) and without
supplementation (weeks 23-26). A significant (P<0.001) increase
inLBP level was seen only at week 24.
Paraoxanase (PON) in blood serum
PON activity increased significantly only at 24-26 weeks (P<0.001)
(Figure 6).
Figure 6: Paraoxanaseactivity in blood serum in relation to daily
supplementation with 400g charcoal (CC) (weeks 1-4), 200g CC
(weeks 5-10), 200 g CC+500 ml sauerkraut juice (SJ) (weeks 11-14),
120 g humic acid (HA) (weeks 15-18), 200 g CC+100 mL
Aquahumin(AH) (weeks 19-20), 100 g CC +50 mL AH (weeks
21-22) and without supplementation (weeks 23-26). Significant
(P<0.05) differences were only detected at weeks 24-26.
Discussion
We investigated the effect of an oral application of CC and humic
acids (HA) alone or in combination with SJ on blood serum
C.
botulinum
ABE and CD antibody levels. Chronic botulism is
characterized by the sub-lethal generation of
C. botulinum
progenitor
toxins in the hind gut. The incorporation of the progenitor toxin and
free BoNT from the gastrointestinal tract (GIT) into the body could
happen via three different routes. Small concentrations of the
progenitor toxin and BoNT bind with hemagglutinins (HA) or the HC
part of the molecule can bind to receptors on the surface of epithelial
cells and transcytosis can occur. Translocated HA disrupts the
epithelial barrier. This is different with type A, B and C progenitor
toxins. Type A and B HAs disrupt the epithelial cell line paracellular
without causing cytotoxic effects in the epithelial cells of their
susceptible hosts while type C HAs possibly evoke cytotoxic-barrier
disrupting activity in the epithelial cells of susceptible animals.
Damaged epithelial cells are not a barrier for progenitor toxins and
BoNTs [20,23,24]. The damaged epithelial barrier permits the toxins to
be distributed throughout the body by blood and lymph vessels. Based
on this knowledge, it is very important to bind these toxins with CC.
The very strong reduction of CD antibodies after the daily application
of 400 g of CC shows this effect. These very high CD antibody levels
without the application of a CD vaccine have not been reported
previously. Such high antibody levels have only been observed in
conjunction with vaccination [3]. Wang et al. showed good sorption of
the hydrophobic herbicide terbuthylacin by CC [35]. Maybe the
hydrophobic surfactant of the commercial herbicide Roundup also
could be absorbed by CC [36]. Graber found that glyphosate can be
absorbed by CC. Our results don`t support these results in animals
[37]. Four weeks daily application of 400 g CC reduced the CD
antibody level dramatically (Figure 3) but did not affect the excretion
of glyphosate in urine (Figure 1). In our own investigation, we only
found neutralization or absorption of a maximum of 300 µg
glyphosate to 1 mg CC (data not shown). The daily application of 200
g CC in weeks 5-10 failed to reduce glyphosate excretion or
C.
botulinum
type CD antibody levels. The mixed application of 200 g
CC and 500 mL SJ significantly reduced the amount of glyphosate
excreted and
C. botulinum
CD antibodies also significantly (P<0.001)
Citation: Gerlach H, Gerlach A, Schrödl W, Schottdorf B, Haufe S et al., (2014) Oral Application of Charcoal and Humic acids to Dairy Cows
Influences Clostridium botulinum Blood Serum Antibody Level and Glyphosate Excretion in Urine. J Clin Toxicol 4: 186. doi:
10.4172/2161-0495.186
Page 6 of 8
J Clin Toxicol
ISSN:2161-0495 JCT Volume 4 • Issue 2 • 1000186
decreased (Figures 1 and 3).
C. botulinum
ABE antibodies were
significantly reduced by all the treatments from week 4 on Figure 2.
The application of HA (WH67) significantly (P<0.001) reduced
glyphosate excretion and
C. botulinum
ABE and CD antibody levels.
Krüger et al. demonstrated that glyphosate reduced the Enterococcus
spp. bacteria that are antagonistic to
C. botulinum [34]
. Shehata et al.
were able to neutralize the antibacterial activity of glyphosate with
different humic acid preparations in vitro [38]. Results from the
application of 200 g CC and 100 mL Aquahumin (liquid preparation)
for 2 weeks compared with 100 g CC with 50 mL Aquahumin for two
weeks showed that a definite amount of these substrates is necessary to
absorb or neutralize glyphosate and/or
C. botulinum
toxins. Mazzei
and Piccolo found that glyphosate may spontaneously and
significantly bind to soluble humic matter by non-covalent
interactions at slightly acidic pH [39]. Binding to matrices such as
soluble fulvic and humic acids could be the reason. Glyphosate
excretion was reduced with the soluble Aquahumin (Figure 1). It was
not anticipated that the combination of 200 g CC and 500 mL SJ per
day would be so very effective. Fermentation of cabbage to SJ is mostly
done by Lactobacillus plantarum [40]. Lactobacilli produce
exopolysaccharides (EPS), homopolysaccharides and
heteropolysaccharides. These biopolymers are widely distributed in
nature and can be the polymers of neutral (pentoses and hexoses) or
anionic sugars (hexoses). They are released into the extracellular
medium by Archebacteria and Eubacteria (both Gram positive and
negative). Approximately 30 species of Lactobacilli are described as
EPS producers. Among them, the best known are
L. casei, L.
acidophilus, L. brevis, L. curvatus, L. delbrueckii, L. bulgaricus, L.
helveticus, L. rhamnosus, L. plantarum
, and
L. johnsonii
.
L. plantarum
generates heteropolymers of glucose, galactose and rhamnose.
Galactose and lactose inhibit the absorption of
C. botulinum
progenitor toxins to the sugar bearing receptors on epithelial cells of
the GIT [41]. The sugar polymer concentrations in nutrient broth
culture of Lactobacilli are in hundreds of mg per liter. EPS may also
interact with proteins, mineral, ions and other compounds [42,43].
Zhang et al. (2013) identified antioxidant effects of
L. plantarum
that
may involve scavenging reactive oxygen species (ROS), up-regulation
of enzymatic and non-enzymatic antioxidant activities, and reduction
of lipid peroxidation [44]. ROS and lipid peroxidation are induced by
glyphosate [45,46]. The neutralization of glyphosate with humic acids
from WH67 was reported by Shehata et al. [33]. The binding
mechanism could be hydrogen bonding to phenolic groups of humic
acid [47]. The positive acute phase proteins (haptoglobine, LBP) only
significantly increased at week 24 and by week 26, both acute phase
proteins (APP) were reduced but
C. botulinum
ABE and CD
antibodies increased. Inflammation indicated by the significant
increase of haptoglobin (P< 0.01) and LBP (P<0.001) may be induced
by proliferation of
C. botulinum
. At week 26, when
C. botulinum
ABE
and CD antibodies were high, the APPs were low. There is a negative
correlation between LBP and
C. botulinum
ABE and CD antibodies
(R2= -0.41 and -0.51, respectively). It is interesting that even though
positive APPs increased, the negative APP paraoxanase also increased
at week 24. This indicates that the anti-oxidative capacity of the cows
increased, but the causes for this are unknown.
Conclusion
Daily oral application of 400 g CC) significantly reduced
C.
botulinum
ABE and CD antibodies by absorption of
C. botulinum
toxins in the gastrointestinal tract but did not reduce glyphosate
excretion in urine. This result was not repeatable with 200 g CC alone
but 200 g CC plus 500 ml SJ reduced both glyphosate excretion and
C.
botulinum
ABE and CD antibodies. The same excellent result was
obtained highly significantly with 120 g humic acids. A certain amount
of CC and/or humic acids are necessary to absorb and/or neutralize
glyphosate and
C. botulinum
toxins.
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Citation: Gerlach H, Gerlach A, Schrödl W, Schottdorf B, Haufe S et al., (2014) Oral Application of Charcoal and Humic acids to Dairy Cows
Influences Clostridium botulinum Blood Serum Antibody Level and Glyphosate Excretion in Urine. J Clin Toxicol 4: 186. doi:
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Citation: Gerlach H, Gerlach A, Schrödl W, Schottdorf B, Haufe S et al., (2014) Oral Application of Charcoal and Humic acids to Dairy Cows
Influences Clostridium botulinum Blood Serum Antibody Level and Glyphosate Excretion in Urine. J Clin Toxicol 4: 186. doi:
10.4172/2161-0495.186
Page 8 of 8
J Clin Toxicol
ISSN:2161-0495 JCT Volume 4 • Issue 2 • 1000186
... Extensive sampling of corn, soybeans, milk and eggs by the US Food and Drug Administration, using a specific analysis method, resulted in residues in a substantial percentage of the plant samples but not above the then current MRLs (FDA, 2017). However, very high concentrations of glyphosate were sometimes found in feed given to farm animals in Denmark that subsequently suffered from diseases such as infertility and malformation of pigs (Krüger et al., 2014b), botulism in cows (Gerlach et al., 2014;Krüger et al., 2013), and pathogenic Salmonella species in chickens Shehata et al., 2014). ...
... In a feeding study with dairy cows, where glyphosatecontaminated feedstuffs (122.7 μg GL/kg BW for 16 weeks) were compared with control feed, no direct effects of glyphosate were detected on various blood and liver parameters (Heymann et al., 2021;Schnabel et al., 2020), as well as the general health condition of the cows (Schnabel et al., 2017). Nevertheless, long-term indirect health effects via changes in the microbiome have been documented (Gerlach et al., 2014;Krüger et al., 2013). Lactic acid producing bacteria generally were negatively affected by Roundup ® (Clair E. et al., 2012;Krüger et al., 2013). ...
... Lactic acid producing bacteria generally were negatively affected by Roundup ® (Clair E. et al., 2012;Krüger et al., 2013). These bacteria normally produce antibiotics and can suppress pathogenic bacteria such as Clostridium botulinum Rodloff and Krüger, 2012) and botulism has increasingly been found in cows that had high concentrations of glyphosate in their feed and urine (Gerlach et al., 2014;Krüger et al., 2013;Krüger et al., 2014a). During in vitro fermentation in bovine rumen fluid, several species of bacteria and protozoa were suppressed after exposure to glyphosate (Ackermann et al., 2015). ...
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... Biochar, wood vinegar (i.e., aqueous solutions of condensed pyrolytic gases) and humic substances can act as redox buffering substances (Husson, 2012;Kluepfel et al., 2014) which may explain why the feeding of biochar, pyrolytic vinegar and humic substances often show similar effects; and why the blending of biochar with wood vinegar or humic substances seems to reinforce the effects (Watarai, Tana & Koiwa, 2008;Gerlach et al., 2014). However, unlike both dissolved organic substances, biochar provides a highly porous framework with high specific surface area, where humic-like substances or pyrolytic vinegar can be adsorbed and unfurl three-dimensionally as a coating of the inner-porous aromatic carbon surfaces of biochar. ...
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... Further research in vivo and/or in vitro in relevant matrixes is necessary, as low pH, for example, in the stomach, could favor glyphosate sorption (Herath et al., 2016). In a study with 380 dairy cows, Gerlach et al. (2014) showed that daily feeding with humic acids (120 g/day) or with a combination of 200 g of biochar and 500 g of sauerkraut juice for 4 weeks significantly reduced the glyphosate concentration in the urine of the cows that were fed with glyphosate contaminated silage. ...
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Biochar, that is, carbonized biomass similar to charcoal, has been used in acute medical treatment of animals for many centuries. Since 2010, livestock farmers increasingly use biochar as a regular feed supplement to improve animal health, increase nutrient intake efficiency and thus productivity. As biochar gets enriched with nitrogen-rich organic compounds during the digestion process, the excreted biochar-manure becomes a more valuable organic fertilizer causing lower nutrient losses and greenhouse gas emissions during storage and soil application. Scientists only recently started to investigate the mechanisms of biochar in the different stages of animal digestion and thus most published results on biochar feeding are based so far on empirical studies. This review summarizes the state of knowledge up to the year 2019 by evaluating 112 relevant scientific publications on the topic to derive initial insights, discuss potential mechanisms behind observations and identify important knowledge gaps and future research needs. The literature analysis shows that in most studies and for all investigated farm animal species, positive effects on different parameters such as toxin adsorption, digestion, blood values, feed efficiency, meat quality and/or greenhouse gas emissions could be found when biochar was added to feed. A considerable number of studies provided statistically non-significant results, though tendencies were mostly positive. Rare negative effects were identified in regard to the immobilization of liposoluble feed ingredients (e.g., vitamin E or Carotenoids) which may limit long-term biochar feeding. We found that most of the studies did not systematically investigate biochar properties (which may vastly differ) and dosage, which is a major drawback for generalizing results. Our review demonstrates that the use of biochar as a feed additive has the potential to improve animal health, feed efficiency and livestock housing climate, to reduce nutrient losses and greenhouse gas emissions, and to increase the soil organic matter content and thus soil fertility when eventually applied to soil. In combination with other good practices, co-feeding of biochar may thus have the potential to improve the sustainability of animal husbandry. However, more systematic multidisciplinary research is definitely needed to arrive at generalizable recommendations.
... El biocarbón también puede incorporarse en las dietas de animales. Esta práctica ha resultado en reducción de bacterias patógenas (Gerlach et al., 2014), reducción de glifosato en la orina (Gerlach et al., 2014) mejorar la ganancia de peso (Leng et al., 2012), reducción en la producción de metano (Leng et al., 2012), aumento en la producción de leche y mitigación de diarreas en ganado vacuno (Geralch, 2012). Asimismo, la misma práctica ha resultado en la incorporación del biocarbón en el suelo por escarabajos estercoleros y mejoras en las características físico-químicas de los mismos (Joseph et al., 2015). ...
... El biocarbón también puede incorporarse en las dietas de animales. Esta práctica ha resultado en reducción de bacterias patógenas (Gerlach et al., 2014), reducción de glifosato en la orina (Gerlach et al., 2014) mejorar la ganancia de peso (Leng et al., 2012), reducción en la producción de metano (Leng et al., 2012), aumento en la producción de leche y mitigación de diarreas en ganado vacuno (Geralch, 2012). Asimismo, la misma práctica ha resultado en la incorporación del biocarbón en el suelo por escarabajos estercoleros y mejoras en las características físico-químicas de los mismos (Joseph et al., 2015). ...
... Existe evidencia no concluyente de que algunos herbicidas perjudica las poblaciones de algunos escarabajos estercoleros (Martínez et al., 2000) que tienen importancia económica por ser descomponedores de estiércol y por introducir nutrientes en el suelo (Lopez-Collado et al., 2017). El biocarbón en el estiércol podría ayudar a mitigar este problema ya que adsorbe glifosato en el sistema digestivo del ganado (Gerlach et al., 2014). ...
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El biocarbón funciona como un mejorador de suelos, especialmente tropicales. Se seleccionaron las materias primas más apropiadas para la elaboración de biocarbón en una localidad del centro de Veracruz, México. Para esto se analizaron participativamente 24 materiales bajo criterios sociales, ambientales y económicos. Antes del análisis se familiarizó a los participantes con la producción de biocarbón por cortina de llama utilizando diversos materiales e integrando su conocimiento local. Además, se hicieron recorridos con los participantes para observar los materiales disponibles. Se seleccionaron al guácimo (Guazuma ulmifolia), tihuixtle (Caesalpinia cacalaco), olotes, caña de maíz (Zea mays) y un conjunto de arbustos espinosos como los materiales más apropiados para la producción de biocarbón. Se evaluaron los costos de producción de los materiales y se midieron los costos de tres modalidades de aplicación. Las aplicaciones se hicieron en diferentes volúmenes (4.59, 29.12 y 39 m3 ha-1) a iniciativa de los participantes. Los costos de producción más los costos de aplicación resultaron de entre 6.5 y 114.5 jornales ha-1 (523 a 9 160 pesos MXN ha-1) dependiendo de la materia prima, cantidad aplicada y modo de aplicación. Se consideró al guácimo como la materia prima más apropiada. La caña de maíz, los olotes podrían ser materiales complementarios en la producción de biocarbón. Por su costo el tihuixtle se utilizaría menos que el guácimo. Los arbustos espinosos tuvieron un costo demasiado alto como para considerarlos apropiados. Palabras clave: agroecosistemas, biocarbón, diagnóstico rural participativo, mejorador de suelos, tecnología apropiada. Biochar works as a soil amendment, especially in tropical soils. Participatory selection was made of the most appropriate feedstocks for biochar production in a town of central Veracruz, México. To that end, 24 materials were analyzed under social, environmental and economic criteria. Before the analysis participants were familiarized with flame curtain pyrolysis biochar production. Beside the analysis, agroecosystems were visited with the participants in order to observe the available feedstocks. Guasimo (Guazuma ulmifolia), tihuixtle (Caesalpinia cacalaco), corn cobs, corn stover and a mixture of thorny bushes were selected as the most appropriate feedstocks for biochar production. Production cost was evaluated for each feedstock and the cost of three application methods was measured. By participants initiative, applications were done in different volumes (4.59, 29.12 and 39 m3 ha-1). Production plus application costs were between 6.5 and 114.5 work days ha-1 (523 to 9 160 mexican pesos ha-1) depending on feedstock, applied quantity and application method. Guasimo threes were considered the most appropriate feedstock. Corn stover and cobs could be used as supplementary feedstocks for biochar production. Because of its cost, tihuixtle will probably be used less than guasimo. Thorny bushes had such hi cost that they were not considered appropriate. Index words: agroecosystems, appropriate technology, biochar participatory rural appraisal, soil amendment.
... Biochar is a non-feed additive in ruminant feed (Gerlach et al., 2013), it's refers to the carbon compounds prepared from the remains of some plants and some animal waste (Yalcin and Arol , 2002). The second step is to activate carbonated substances by exposing them to oxidizing agents such as carbon dioxide (CO2) or water vapor, which helps to burn the carbonate materials that block the pores. ...
... Biochar are currently used to feed ruminants like additive because of their adsorption of gases and to provide a favorable environment for microorganisms in the rumen by increase growth of bacteria (Gerlach et al., 2013) .Biochar today are considered as non-food additives for animal nutrition to improve bowel function by eliminating toxins and impurities in the digestive system, increasing feed intake and improving animal growth (Strahsaker et al., 1997;Villalba et al., 2002). In view of the above, biochar was used in this study as non-feed additives for the Awassi lambs feed to study its effect on the productive performance and the characteristics of its carcasses. ...
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This study was conducted at Department of Animal production farm College of Agriculture University of Tikrit from 6/12/2017 until 21/2/2018 (77days) . Sixteen Awassi lambs were used, aged 4-5 months and average weight 23.38±0.14 Kg . Lambs were divided in to four groups (four lambs at each group) according to their weight, treatments distributed randomly into the groups.Individual feeding was used which included four concentrate diet with different levels of biochar (0% ,1% , 2% and 3% ).Statistical analysis results showed that there was a significant increase (P<0.05) in the final weight for second group (1% biochar) compared with fourth group, also result showed a significant increase (P<0.05) for second group in the average daily gain and final body gain and feed conversion ratio compared with third and fourth groups. Also significant increase (P> 0.05) in the second group in hot and cold carcass weight and in the percentage Dressing based on hot and cold weight, relative to live weight as well as empty body weight and in Rib-eye muscle area compared with the other groups.
... Gerlah et al. [54] fed <8 mm diameter charcoal with other supplements to dairy cows at different doses, and investigated the effect of the treatment on blood serum C. botulinum ABE and CD antibody levels. A daily application of 400 g of charcoal (1-4 week of the experiment) and 200 g of charcoal with 50 mL of sauerkraut juice (11)(12)(13)(14) week of the experiment) per entire herd significantly reduced the antibody levels by about 30% due to the absorption of C. botulinum toxins in the gastrointestinal tract by the charcoal. ...
... Summary of selected data on biochar application to animal feed.Kultu et al. [50] Oakwood 1% to 10% by mass of chicken broilers' and laying hens' feed -Up to 7% better feed conversion ratio -Up to 23% increase in body weight gain -Up to 65% reduction in the number of cracked eggs Nageswara Rao et al.[51] Activated charcoal 1% of goats daily dry matter intake -76% reduction in excretion of aflatoxins -Milk composition was not influenced ThanhVan et al. Up to 1% by mass of chicken broilers' feed -Up to 17% increase in body weight gain -Up to 120% increase in pancreas weight -Up to 20% increase in intestine density -Up to 86% increase in creatinine level -Up to 28% reduction in gizzard mass Gerlah et al.[54] Charcoal Up to ~1 g of charcoal daily per cow -Up to 30% reduction in C. botulinum antibody levelsEvans et al. [55] Poultry litter ~7% of BC by mass of chicken broilers' feed -8% higher feed intake -2% decrease in body weight gain -11% worse feed conversion ratio Evans et al. [56] Poultry litter 2% and 4% by mass of chicken broilers' feed -7% better feed conversion ratio -9% decrease in body weight gain Joseph et al. [57] Jarrah wood BC-molasses mixed at a ratio 3:1, fed daily to cows -Toxin sorption -Easier transfer of nutrients from cow's gut and dung to the soil Prasai et al. [58] Woody green waste 4% by mass of laying hens' feed -1.2% increase in egg productivity -3% increase in eggs weight -8% better feed conversion ratio -2% lower feed intake Prasai et al. [59] Woody green waste 1% to 4% by mass of laying hens' feed -Up to 5% increase in eggs weight -Up to 13% increase in eggs yield -Up to 7% lower feed intake -Up to 14% better feed conversion ratio -Up to 19% increase in shell breaking strength -Up to 26% reduction of N content in excreta Prasai et al. [60] Woody green waste 1% to 4% by mass of laying hens' and chicken broilers' feed -The decreased water content of laying hens excreta -Up to 27% reduction in excreta N content -Up to 45% higher C content in excreta -Up to 47% higher NH3 emission ...
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When it comes to the use of biochar in agriculture, the majority of research conducted in the last decade has focused on its application as a soil amendment and for soil remediation. This treatment improves soil quality, increases crops yields, and sequestrates atmospheric carbon to the soil. Another widely studied aspect connecting biochar with agriculture is the composting processes of various agricultural waste with the addition of biochar. Obtaining the material via the pyrolysis of agricultural waste, including animal manure, has also been investigated. However, given the remarkable properties of biochar, its application potential could be utilized in other areas not yet thoroughly investigated. This review paper summarizes the last decade of research on biochar and its use in crop agriculture and livestock production. Knowledge gaps are highlighted, such as using biochar for the mitigation of odorous emissions from animal manure and by feeding the biochar to animals.
... 5 Die Wasserlöslichkeit des Glyphosats, verstärkt durch das Tensid, ermöglicht die Resorption im Dünndarm, den Abbau zum Glyphosatmetaboliten Aminomethylphosphonsäure (AMPA) in der Leber und die Ausscheidung über Harn und Kot. Somit besteht die Nachweismöglichkeit des Glyphosats im Blut, Harn und in der Duodenalflüssigkeit. 12,33,36 Die akute Vergiftung beim Menschen infolge oraler Aufnahme des Glyphosats in Form von Roundup® verursacht Hypersalivation, gerötet ödematösen Pharynx-Larynx-Bereich mit Schluckstörungen, Magen-Darm-Ulzera mit Erbrechen, Bauchschmerzen und Erosionen, Nekrosen sowie Blutungen im Magen-Darm-Trakt. Weitere Symptome sind Schweratmigkeit, Koma und/oder Herzrhythmusstörungen, Leberschädigung und akutes Nierenversagen. ...
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Introduction: The hay producing plants, concentrate, straw and meadows could be contaminated by the aerosols of glyphosate based herbicide during spraying process of crops and pre-harvest desiccation treatment of cereals. The aim of this study is to investigate the concentration of glyphosate in the duodenal fluid of horses with gastric ulcer syndrome. The stomach and duodenum of referred untreated horse patients (n=92) with colic, weight loss, diarrhoea, anemia or performance intolerance were endoscopically examined right after the admission. Duodenal fluid (40 ml) was collected from the duodenal region where the papilla duodeni major is located. Hematology and clinical chemistry data were examined. The concentration of glyphosate in serum and duodenal fluid samples were analysed using a competitive ELISA and control analysis had also been done with HPLC. Statistical differences between groups were determined by the non-parametric Mann-Whitney-test using a significant level of p≤0,05. Glyphosate was detected in all duodenal fluid (median 12,2 ng/ml; 1st quartile 4,0 ng/ml; 3rd quartile 19,3 ng/ml; min 0,6 ng/ml; max. 192,9 ng/ml) and blood samples (1,79 ng/ml; 1,0 ng/ml; 2,8 ng/ml; 0,2 ng/ml; 3,7 ng/ml) of all horses. Glyphosate concentrations of duodenal fluid samples are significantly higher than in blood samples (Mann Whitney U-test, p≤0,05). The concentration of glyphosate in the duodenal fluid was significantly higher in horses with squamous gastric disease (grade 4/4; n=11/92) compared to horses with normal squamous mucosa (grade 0/4, n=10/92) (median: 19,8 ng/ml versus 8,4 ng/ml). Horses with glandular gastric disease and a grade 4/4 (n=9/92) had higher concentrations of glyphosate in the duodenal fluid than horses with normal glandular mucosa (grade 0/4; n=9/92) (median: 19,2 versus 11,1). The Gamma-Glutamyltransferase (GGT) enzyme activity is significantly higher in the group of horses with lower concentration of glyphosate in the duodenal fluid (≤12,2 ng/ml) compared with the group with higher concentration of glyphosate (>12,2 ng/ml) (median 279,5 versus 101,9 U/L). During autumn the horses had higher concentrations of glyphosate in duodenal fluid (n=18; median 14,3) compared with lower concentrations in spring time (n=34; median 8,1 ng/ml). Horses kept around big cities had significantly higher concentrations of glyphosate in the duodenal fluid in comparison to horses living in the countryside (medians 17,8 ng/ml versus 7,5 ng/ml).
... In our study, these three phyla were also the most dominant in manure in addition to the Actinobacteria. In addition, bactericides like antibiotics (Ji et al., 2018) and herbicides such as glyphosate (Gerlach et al., 2014;Krüger et al., 2013) affect the composition of the microbiome in the gut and manure. ...
Article
Manure inputs into soil strongly affect soil microbial communities leading to shifts in microbial diversity and activity. It is still not clear whether these effects are caused mainly by the survival of microbes introduced with manure or by activation of the soil-borne microbiome. Here, we investigated how the soil microbiome was changed after the introduction of fresh farmyard cattle manure, and which microorganisms originating from manure survived in soil. Manure addition led to a strong increase in soil microbial biomass, gene copies abundances, respiration activity, and diversity. High-throughput sequencing analysis showed that higher microbial diversity in manured soil was caused mainly by activation of 113 soil-borne microbial genera which were mostly minor taxa in not-fertilized soil. Two weeks after manure input, 78% of the manure-associated genera were not detected anymore. Only 15 of 237 prokaryotic genera that originated from manure survived for 144 days in soil, and only 8 of them (primarily representatives of Clostridia class) were found in manured soil after winter. Thus, an increase in microbial biomass and diversity after manure input is caused mainly by activation of soil-borne microbial communities, while most exogenous microbes from manure do not survive in soil conditions after few months.
... Biochar (termed charcoal in the early literature) and activated carbon (AC) have been feed to animals for many centuries for reducing toxic substances such as Clostridium tetani and C. botulinum [340]. Cato the Elder (234-149 BC) mentioned in his classic On Agriculture: 'If you have reason to fear sickness, give the oxen before they get sick 3 pieces of charcoal'. ...
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Biochar is produced as a charred material with high surface area and abundant functional groups by pyrolysis, which refers to the process of thermochemical decomposition of organic material at elevated temperatures in the absence of oxygen. The carbon component in biochar is relatively stable, and, hence, biochar was originally proposed as a soil amendment to store carbon in the soil. Biochar has multifunctional values that include the use of it for the following purposes: soil amendment to improve soil health, nutrient and microbial carrier, immobilising agent for remediation of toxic metals and organic contaminants in soil and water, catalyst for industrial applications, porous material for mitigating greenhouse gas emissions and odorous compounds, and feed supplement to improve animal health and nutrient intake efficiency and, thus, productivity. This article provides for the first time an overview of the multifunctional values and unintended consequences of biochar applications.
... Vor diesem Hintergrund wurde in mehreren Untersuchungen der Einfluss von Pflanzenkohlen und Huminsäuren auf die Neutralisation von Glyphosat im Organismus von verschiedenen Tierarten untersucht. Dabei konnte gezeigt werden, dass Huminsäuren insbesondere im Vergleich zu Pflanzenkohlen besonders starke Neutralisationseigenschaften von Glyphosat aufweisen (Gerlach et al., 2014. Es gibt weiterführende wissenschaftliche Belege, dass Huminstoffe gute Neutralisationskapazitäten auf diverse Schadstoffe haben. ...
... The current study follows a previous on-farm investigation where biochar was given to beef cattle as a feed supplement , In this study, dung beetles (Bubas bison) were used to incorporate the biochar-infused manure to depths exceeding 600 mm . The initial purpose of this study was to trial a costefficient mechanism for carbon sequestration and to apply the results of earlier research that demonstrated animal health benefits resulting from feeding biochar to dairy cattle (Gerlach et al., 2014). This study resulted in an increase in pasture productivity, a reduction in costs, an increase in farmer income and a significant increase in soil carbon and nitrogen content to a depth of 40 cm . ...
Article
The use of biochar in avocado orchard soils has not yet been investigated in rigorous scientific experiments. We determine the effect of wood biochar on avocado growth, fruit production and economic benefit. Biochar was applied at 0%, 5%, 10% and 20% volume by volume basis. Biochar significantly improved the growth of avocado seedlings and increased fruit yield in the first three years after planting. There was an overall increase in soil carbon, fruit yield, tree diameter and height in all biochar treatments relative to the control over the seasons. Trees planted with biochar had 18–26% greater growth rates (in terms of height and stem diameter) than the control. Tree diameter was significantly greater with biochar (145.4 ± 3.3 mm) relative to the control treatment (125.0 ± 2.7 mm). Tree height was also significantly greater with biochar (3.7 ± 0.1 m) relative to the control treatment (3.4 ± 0.1 m). The fruit count from the biochar row was significantly greater (97%) in 2018. Heavy bearing trees typically have a lower yield in the subsequent year but despite this, the 2019 fruit counts were higher in aggregate for the biochar amended trees (20%) relative to the control. A cost-benefit analysis indicated that if yield surplus of fruit trees continued for three years, and assuming avocado prices remain at similar levels, then the discounted net benefit over a hectare would amount to US$8581, or US$105 per metric tonne of biochar applied.
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In the present study, the neutralization ability of the antimicrobial effect of glyphosate by different humic acids was investigated. The minimal inhibitory concentrations of glyphosate for different bacteria such as Bacillus badius, Bifidobacterium adolescentis, Escherichia coli, E. coli 1917 strain Nissle, Enterococcus faecalis, Enterococcus faecium, Salmonella enteritidis and Salmonella typhimurium were determined in the presence or absence of different concentrations of humic acid (0.25, 0.5 and 1.0mgmL(-1)). Our findings indicated that humic acids inhibited the antimicrobial effect of glyphosate on different bacteria. This information can help overcome the negative impact of glyphosate residues in feed and water.
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Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.
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Background: Clostridium botulinum strain IBCA10-7060, isolated from a patient with infant botulism, produced botulinum neurotoxin type B (BoNT/B) and another BoNT that, by use of the standard mouse bioassay, could not be neutralized by any of the Centers for Disease Control and Prevention-provided monovalent polyclonal botulinum antitoxins raised against BoNT types A-G. Methods and results: The combining of antitoxins to neutralize the toxicity of known bivalent C. botulinum strains Ab, Ba, Af, and Bf also failed to neutralize the second BoNT. Analysis of culture filtrate by double immunodiffusion yielded a single line of immunoprecipitate with anti-A, anti-B, and anti-F botulinum antitoxins but not with anti-E antitoxin. A heptavalent F(ab')2 botulinum antitoxin A-G obtained from the US Army also did not neutralize the second BoNT. An antitoxin raised against IBCA10-7060 toxoid protected mice against BoNT/B (Okra) and against the second BoNT but did not protect mice against BoNT/A (Hall) or BoNT/F (Langeland). Conclusion: The second BoNT thus fulfilled classic criteria for being designated BoNT/H. IBCA10-7060 is the first C. botulinum type Bh strain to be identified. BoNT/H is the first new botulinum toxin type to be recognized in >40 years, and its recognition could not have been accomplished without the availability of the mouse bioassay.
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During the last 10-15 years, an increase of Clostridium botulinum associated diseases in cattle has been observed in Germany. The reason for this development is currently unknown. The normal intestinal microflora is a critical factor in preventing intestinal colonisation by C. botulinum as shown in the mouse model of infant botulism. Numerous bacteria in the gastro-intestinal tract (GIT) produce bacteriocines directed against C. botulinum and other pathogens: Lactic acid producing bacteria (LAB) such as lactobacilli, lactococci and enterococci, generate bacteriocines that are effective against Clostridium spp. A reduction of LAB in the GIT microbiota by ingestion of strong biocides like glyphosate could be an explanation for the observed increase in levels of C. botulinum associated diseases. In the present paper, we report on the toxicity of glyphosate to the most prevalent Enterococcus spp. in the GIT. Ingestion of this herbicide could be a significant predisposing factor that is associated with the increase in C. botulinum mediated diseases in cattle.
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Background and Aims Amendment of soil by biochar may reduce efficacy of soil-applied herbicides due to sorption. Methods Bioassays with Green Foxtail (Setaria viridis) tested the influence of two biochars on phytoavailability of S-metolachlor and sulfentrazone under biochar amendment of 0, 13, 26 and 52 Mg ha-1. Results Adsorption of both herbicides was an order of magnitude greater on a high specific surface area (SSA) biochar (EUC-800; SSA 242 m2 g-1) than on a low SSA biochar (BC-1; SSA 3.6 m2 g-1). Herbicide doses near the lowest recommended label rates controlled the weed at 13 and 26 Mg ha-1 of BC-1; sulfentrazone was also effective at 52 Mg BC-1 ha-1. These same herbicide doses controlled weed germination and development only at 13 Mg ha-1 of EUC-800; at herbicide doses near the highest label rates, weed control was also achieved at 26 Mg EUC-800 ha-1, but not at 52 Mg EUC-800 ha-1. Conclusions Increased doses of soil-applied herbicides cannot necessarily offset decreases in herbicide phytoavailability in biochar-amended soils, particularly if the biochar has a high SSA. Considering the long half-life of biochar in soil, pest control needs will be best served by low SSA biochars.
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We sequenced the 2 botulinum toxin gene clusters of Clostridium botulinum strain IBCA10-7060 type Bh. The sequence of bont/H differed substantially from the sequences of the 7 known bont genes for toxin types A–G. The 5′ one-third terminus of bont/H that codes for the botulinum toxin light chain differed markedly from the light chain coding sequences of toxin types A–G. The 3′ two-thirds terminus of bont/H that codes for the botulinum toxin heavy chain contained a novel Hn translocation domain coding sequence and a nonneutralizing type A–like Hc binding domain coding sequence. bont/H was part of an orfX toxin gene cluster that was located at a unique chromosomal site distant from those used by other botulinum toxin gene clusters. The bont/B sequence was similar to that of subtype bont/B2 and was located within its ha toxin gene cluster at the oppA/brnQ site. Our findings further establish that C. botulinum IBCA10-7060 produces novel BoNT/H.
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Lactobacillus plantarum strains isolated and identified from naturally-fermented Chinese sauerkraut were examined in vitro for potential probiotic properties and in vivo for cholesterol-lowering effect in mice. Among 7 isolated L. plantarum strains, strains S2-5 and S4-1 were found to possess desirable probiotic properties including ability to survive at pH 2.0 for 60 min, tolerate pancreatin and bile salts, adhere to Caco-2 cells, produce high β-galactosidase activity and antimicrobial activity against Escherichia coli O157 and Shigella flexneri CMCC(B). In addition, strains S2-5 and S4-1 were susceptible to several antibiotics, and capable of reducing cholesterol level in MRS medium by assimilation of cholesterol at 20.39 and 22.28 μg ml(-1), respectively. The in vivo study with L. plantarum S4-1 showed that feeding with fermented milk containing this strain was able to effectively reduce serum cholesterol level in mice, demonstrating its potential as an excellent probiotic candidate for applications in functional products.