BOVINE LAMIN IllS AND ALLISONELLA HISTAMINIFORMANS

M.R. Garner', A.G. Hay', C.L. Guard' and J.B. Russell 1,3
Department of Microbiology',
Population and Diagnostic Science 2 , Cornell University
and ARS/USDA3

INTRODUCTION

In the last thirty years, the production of American dairy cattle has increased
dramatically, but the average age of a dairy cow in the milking herd has decreased
significantly. Cows are culled for a variety of reasons, but Nelson and Cattell (2001)
stated "cows with a history of laminitis lesions were culled during the current lactation at
a rate 1.77 times higher than cows with no history of laminitis lesions." These same
authors also noted "a history of laminitis increased the likelihood of culling due to
reproduction, mastitis, low production and other reasons." Laminitis is influenced by a
variety of factors common in modern dairy herds (concrete flooring, lack of proper foot
trimming, constant exposure to water and manure, and metabolic stresses), but it has
long been observed that an increased reliance on cereal grains contributes to the
problem and "management of acidosis is critical in preventing laminitis." (Nocek, 1997).
When the ruminal fermentation rate increases and pH declines, ruminal histamine
concentrations increase, the rumen wall becomes inflamed, and bacteria and various
toxins can pass from the rumen into portal blood (Nagaraja, 1998).

Histamine is a powerful inflammatory agent that causes vasodilation of small

capillaries in the laminar corium of the hoof above the hoof. Once the capillaries have
been dilated, circulation stagnates, blood oxygen is depleted, and differentiating
epithelial cells destined to become hoof wall degenerate. The involvement of histamine
in laminitis is complicated by the fact that it is produced endogenously, is sometimes
found in the feed, and can be produced by gut bacteria. Histamine is an amine that is
formed via the decarboxylation of the amino acid, histidine (Figure 1). Rodwell (1953)
isolated histamine producing bacteria from sheep fed an abundance of grain and it had
been generally assumed that lactobacilli were responsible for ruminal histamine
accumulation. However, recent work indicates that a novel bacterium, Allisonella
histaminiformanS also converts histidine to histamine. This latter bacterium was found in
much greater abundance than histamine producing lactobacilli, but its success in the
rumen appears to be highly diet dependent. This paper describes the characteristics,
phylogeny, and unusual growth requirements of A. histaminiformans.

199
 +H \'j_NrH + c02
H 2 H2N

Figure 1. The formation of histamine from the decarboxylation of histidine.

RUMINAL HISTAMINE PRODUCTION

In the 1940's, Dougherty and his colleagues studied acute ruminal acidosis in sheep
and noted that histamine accumulated when the ruminal pH was highly acidic
(Dougherty, 1942; Dougherty and Cello, 1949). With additional research they concluded
that there was "a direct correlation between the level of histamine in the ingesta and the
well being of the sheep" (Dain et al., 1955). Ruminal histamine accumulation has
generally been associated with severe niminal acidosis (Irwin et al., 1979; Wilson et al.,
1975), but cattle fed silages can also have elevated levels of ruminal histamine
(Sjaasted, 1960). Because only 10% of the ruminal histamine could be explained by
histamine in the feed, it appeared that even non-acidotic cattle could have histamine-
producing bacteria.

RUM INAL HISTIDINE ENRICHMENTS

Ruminal fluid from lactating dairy cattle at the Cornell Teaching and Research Center
that consumed a total mixed ration (TMR) had pH values greater than or equal to 6.0,
and we were able to enrich histamine producing bacteria from these cows. When the
ruminal fluid was serially diluted into MRS (a medium selective for lactobacilli)
suplemented with glucose and histidine, histamine was formed at dilutions as high as
10 . However, the highest dilution could be successively transferred even if glucose was
omitted from the medium. This result indicated that the bacteria responsible for
histamine production could not be lactobacilli.

The MRS enrichments had a variety of different cell types. When the histamine
producing enrichments were transferred to a less complex media designed for ruminal
bacteria, there was a dramatic decrease in bacterial complexity, but there was no
decrease in the conversion of histidine to histamine. Agar plates that were streaked
with these latter enrichments had many morphologically distinct colony types, but only
the small translucent colonies produced histamine once they were transferred to broth.
The histamine producing isolates were ovoid shaped cells; this result also supported the
idea that they were not lactobacilli.

CHARACTERIZATION AND IDENTIFICATION

The isolates stained Gram-negative, but 16S rDNA sequencing indicated that they
were most closely related to Gram-positive bacteria (Figure 2). The closest relative was
Dialister pneumosintes, but this bacterium is not a rumen inhabitant, has complex
growth requirements, grows very slowly, and does not produce histamine. Selenomonas
ruminantium and Megaspheara elsdenii were its closest rumen relatives, but neither of
200
these bacteria could utilize histidine as an energy source or produce histamine. Our
isolates were highly resistant to monensin and grew even if the concentration was
greater than 40 pM. Monensin resistance is typically associated with Gram-negative
bacteria, but, it should be noted that S. ruminantium and M. elsdenii, although closely
related to gram-positive bacteria, possess an outer membrane. These latter two
bacteria are highly monensin resistant (Callaway et al., 1999).

The histamine-producing isolates grew rapidly on histidine and could not utilize other
amino acids, carbohydrates or organic acids as an energy source. Because 16S rDNA
gene sequencing indicated that similarities with other bacteria in Genbank were < 94%,
it appeared that our isolates should be assigned to a new genus as well as a new
species. We proposed the name Allisonella histaminiformanS ( Garner et al., 2002). The
genus name Allisonella is derived from the name Allison, honoring M.J. Allison, a
prominent rumen microbiologist who previously isolated Oxalobacter formigenes, a
ruminal bacterium that decarboxylates oxalate. The species name, histaminiformans is
a Latin word that means histamine forming.

100 Mcgasphaera elsdenii

Pfegasphaera rerl'isiae
100100 Dialister pneumosifltes
100 Dialister pneunosinteS
Mlisone!Ia hisft.imin:formans MR2
00 100
uncultured ruminal bacterium 2c228d8
L .j SeIe 0fla5 rwninantu4m
91 Selenwnonas sputie1ia
Rwninococcusflavefaciefls FDI
_—[,6 Ruminococo.is albus
Clostridium cellulolviicurn
Strep weoceus bovis .IB I
71.9 l'eptoslreptOCOCCUS lactolyticus
Bulyrivibriofi&risolVefls
ostridiurn arninophilwn
FIbrobac(er succinogenes
Prevotella ru,ninieola
01

Figure 2. A phylogenetic dendrogram of low G+C, Gram-positive bacteria. The

relationships were based on 16S rDNA gene sequences. The tree was
constructed using the neighbor-joining method and bootstrap values (given
for each branch point) are expressed as a percentage of 1000 replications.
A. histaminiformaflS strain MR2T is shown in bold. The bar designates 0.1%
difference in the 16S rDNA gene sequence.

A. histaminiformanS has simple nutritional requirements and needs only a standard

mixture of salts, butyrate (10 mM), histidine (50 mM), and yeast extract (0.6 g/l) to grow
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(109 cells/ml). A. histaminiformans converts histidine stoichiometrically to histamine (1:1
exchange), and its growth is directly dependent on the amount of histidine present in the
medium until the histidine concentration is greater than 100 mM (Figure 3). Experiments
with washed cell suspensions indicated that A. histaminiformans produced histamine
approximately eight-fold faster than Lactobacillus 30A, the best histidine
decarboxylating strain isolated by Rodwell (1953). Batch culture experiments indicated
that A. histaminiformans initiated growth at pH values as low as 4.5. However, the
histidine decarboxylation reaction consumes a proton and causes a marked increase in
culture pH. When A. histaminiformans was grown in continuous culture and HCI was
added to the medium reservoir, pH could be regulated. Because growth and histamine
production were observed until the pH reached 4.0, it appears A. hisfaminiformans is as
pH resistant as most lactobacilli.

0.8

0.6

o 0.4
a
0
0.2

0 50 100 150 200 250

Histidine (mM)

Figure 3. The effect of histidine on the growth of A. histaminiformans. Media contained 2

gil yeast extract.

ENUMERATION

Proton consumption by the histidine decarboxylation reaction causes a marked

increase in pH, and it was possible to use pH as a preliminary method for enumerating
A. histaminiformans. When mixed ruminal bacteria are serially diluted into MRS medium
that contains glucose and histidine, glucose fermentation causes a decrease in final pH,
but this decrease can be counteracted if a histidine decarboxylating bacterium is
present. The use of pH to enumerate A. histaminiformans is then confirmed by direct
measurement of histamine production (Figure 4).

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 I: I

Optical
Density
iiiiiiiii
pH 6.7 6.5 6.5 6.5 6.5 6.5 6.5 4.2 4.5 6.5
Histamine

Figure 4. A serial (10-fold) dilution of ruminal fluid into MRS medium (pH 6.5)
supplemented with glucose (30 mM) and histidine (50 mM). After 48 of
incubation at 39 C, the pH of each tube was measured with an electrode.
Histamine was assayed by a method employing thin layer chromatography
(Lin et al., 1977).

DIET DEPENDENCY

Lactating cows at the Cornell Teaching and Research Center were fed a total mixed
ration (43% corn silage, 11% alfalfa haylage, 24% high moisture shelled corn, 8.6 %
extruded soybean meal, 5.8% soybean meal, and 4% whole cottonseed on a dry matter
basis, 17% crude protein, 33% neutral detergent fiber, 20 kg per day), and twenty of
them were equipped with ruminal fistulas.

\.. •' ' r20.52

cc 6
. S.

Mn
..\

5.5 6 6.5 7 7.5

pH
Figure 5. Histamine producing bacteria as a function of ruminal pH. Cows fed dairy
ration and timothy hay were sampled approximately 2 h after feeding.
Rumen pH was determined from rumen fluid sample obtained and measured
with a pH electrode. Histamine producing bacteria were enumerated by serial
dilution.

203
 Enumerations indicated that these cows had A. histaminiformans populations that
varied from 104 to 107 per ml, and similar values were obtained when ruminal fluid was
obtained by ruminocentesis from cows in a commercial herd (n = 70). These results
indicated that that A. histaminiformans is found at significant numbers in dairy cattle, but
the numbers of A. histaminiformans and ruminal pH were not well correlated (r 2= 0.52;
Figure 5).

Fistulated non-lactating dairy cows (n=4) on the Cornell campus that are used for in
vitro fiber analysis are routinely fed a diet of timothy hay. Enumerations indicated that
these latter animals had very low numbers of A. histaminiformans (< 101 per ml), and
similar results were obtained with pregnant non-lactating beef cattle (n=12) that were
fed timothy hay and sampled by ruminocentisis. These results indicated that A.
histaminiformans numbers were clearly diet dependent. Because the dairy ration had
an abundance of ensiled materials (corn and hay crop silage), we decided to examine
the effect of silage extracts on the growth of A. histaminiformans in vitro.

Histamine

3
cn >c)
M.
o
0
CO
CD CO
3
CD

Figure 6. The ability of different extracts to stimulate histamine production in A.

histaminiformans in ruminal fluid from a cow fed timothy hay. Garner and
Russell (unpublished results).

A. histaminiformans cannot grow in basal medium that lacks yeast extract, but
growth and histamine production were observed if water soluble extracts of alfalfa and
corn silage were added (Figure 6). Because less histamine was detected when fresh
alfalfa and corn were added, it appears that the growth factor could be by-product of the
ensuing process. To better understand the nature of the growth-stimulating factor, we
subjected the alfalfa silage extract to various treatments and assayed the growth and
histamine production of A. histaminiformans ( Table 1). Alfalfa silage extracts that had
been autoclaved, boiled, de-proteinized with perchloric acid, extracted with chloroform
and treated with an anion exchanger (Dowex 2) retained activity, but activity was lost if
a cation exchanger was used (Dowex 50). These results indicated that the growth
factor was a non-volatile, positively charged compound of small molecular size. The
idea that it is a zwitterionic species was supported by the observation that it was soluble
in methanol as well as water. The methanol extract had an abundance of ninhydrin
reactive material, but a mixture of 20 amino acids could not replace the growth factor.
Because methanol extract that was evaporated and subjected to acid hydrolysis lost
activity, it appears that the growth factor may be a peptide or group of peptides. This
tentative identification was supported by the observation that the growth factor could be
204
replaced by an enzymatic hydrolysate of casein but not a hydrolysate that was
subjected to acid hydrolysis.

Table 1. Treatment of the alfalfa silage extract that either destroyed or did not effect the
ability to stimulate growth

HISTAMINE MEASUREMENTS

A variety of methods have been used to measure histamine (thin layer

chromatography, paper chromatography, high pressure chromatography, fluorometric
determinations and enzyme linked antibody assays), but these methods can be tedious
or insensitive. Recent work indicated that a method employing Gas Chromatography
coupled with Mass Spectrography (GCMS) could be a used to determine histamine in
blood (Pittertschatscher et al, 2002). Because the molecular weight of a derivatized
histamine can be selectively quantitated via an internally labeled (dueterated) histamine
standard, the method is highly selective (Figure 7). GCMS results indicated that cows
fed commercial dairy rations contain at least 30-fold more histamine than those fed
timothy hay, and this finding supported the idea that A. histaminiformaflS is responsible
for much of the ruminal histamine (Figure 7).

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 25001

Iq
Dairy Ration

0
Lim"
HC -C-H*II
a a
1500 2 0
(5 (
a

a
1000;

40 (42 IV 135((5 ((5

044

Hay

1022 1024 I '4

Retention ItIlla

Figure 7. GCMS chromatogram of histamine in rumen fluid from cows fed timothy hay
(<101 Allisonella cells/ml) and dairy ration (106 Allisonella cells/ml). Rumen
fluid from cows fed dairy ration (n=5) contained 10.5 mg/I histamine ± 2.0
while rumen fluid from cows fed hay (n=5) had less than 0.3 ± 0.5 mg/L of
histamine. Inset: Mass spectra of two products from the ethylchloroformate
derivatization of histamine.

HISTAMINE AND LAM INITIS

The potential involvement of histamine in ruminant laminitis is supported by a variety

of observations: 1) ruminants with elevated serum histamine often have sore feet
(Suber, 1979), 2) symptoms of laminitis can be reversed by antihistamine injections
during the early stages (Nilsson, 1963; Jubb and Kennedy, 1970), and 3) histamine
injections into the hooves of acidotic animals caused laminitis (Takahashi and Young,
1981). Our results indicated that dairy cows fed a commercial dairy ration had ruminal
histamine concentrations that were as high as the values seen in animals with severe
laminitis, but none of the animals sampled appeared to have sore feet. This difference
may be related to ruminal pH. Most of the animals that we sampled had ruminal pH
values > 6.0. Recent work from West Germany demonstrated that histamine absorption
from rumen sacs into blood did not increase until the pH decreased (Aschenbach and
Gabel, 2000). These results support the idea that the effect of histamine upon laminitis
in cattle is a multi-factorial process. First, there must be enough histidine
decarboxylation to cause an increase in ruminal histamine. Secondly, ruminal pH must
decrease so the histamine can be transferred from the rumen to the blood.

206
 CONCLUSIONS

While the evidence to make all pathologic conditions affecting the soft tissue within
the hoof capsule attributable to the action of histamine is unproven, it has long been
assumed that histamine was an important factor in bovine laminitis. Bacteria
responsible for ruminal histamine production had not been systematically evaluated, but
our work indicates that a novel bacterium, A. histaminiformanS, appears to play an
an obligate
important role in ruminal histamine production. A. histaminiformaflS is
histidine decarboxylating bacterium that has relatively simple nutritional requirements,
but it needs a growth factor that can be derived from yeast extract in vitro or silages in
vivo. The involvement of A. histaminiformaflS in ruminal histamine accumulation is
supported by the observation that cows fed dairy ration had relatively high numbers of
A. histaminiformanS and high concentrations of ruminal histamine while the converse
was true for cows fed only timothy hay. A. histaminiformaflS is a highly ph-resistant
bacterium, but cows fed commercial rations had high numbers of this bacterium even if
the ruminal pH was near neutral. Cows fed dairy rations had relatively high
concentrations of ruminal histamine without showing overt signs of laminitis, but this
effect may be due to ruminal pH. Other researchers have demonstrated that rumen to
blood histamine flux does not occur until ruminal pH becomes acidic.

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