Allisonella histaminiformans, a novel histamine-producing bacterium that may play a role in rumen disorders and bovine laminitis
In the 1940s and 1950s, Dougherty and his colleagues (Dougherty, 1942; Dain et al., 1955) studied the impact of ruminal acidosis, and noted that grain feeding also promoted ruminal histamine production. Histamine is a powerful inflammatory agent, and they concluded that there was direct correlation between “the histamine level of ingesta and the well being of the animal.” Histamine is formed from the decarboxylation of the amino acid, histidine, and even a small conversion of histidine to histamine can be toxic (Suber et al., 1979).
In the 1950s, Rodwell (1953) isolated histidine decarboxylating lactobacilli from sheep and horses fed grain-based rations. The potential involvement of lactobacilli was consistent with the observation that lactobacilli are highly pH-resistant bacteria that accumulate in the rumen when animals are fed an abundance of cereal grain. However, recent work indicated that histamine-producing lactobacilli could not be isolated from cattle that were fed 90% cracked corn or a commercial dairy ration (Garner et al., 2003)
Ruminal histidine enrichments from dairy cattle yielded a new bacterium that was classified as Allisonella histaminiformans (Garner et al., 2003).
A. histaminiformans is an obligate histidine fermenting bacterium that does not utilize other energy sources, produces histamine 3-fold faster than even the best lactobacilli and grows at pH values as low as 4.0. A. histaminiformans is not found in cattle consuming hay, but it is found at high numbers in cattle fed dairy rations.
Enrichment and isolation of Allisonella histaminiformans
Based on the prior work of Rodwell (1953), we prepared ruminal histidine enrichments that employed MRS, a medium that is often used to select lactobacilli (deMan et al., 1960). MRS is a poorly buffered medium and glucose fermentation causes a marked decrease in pH. When ruminal fluid from dairy cattle was inoculated into MRS supplemented with 50 mM histidine, most of the histidine was converted to histamine. The enrichments fermented glucose, but subsequent work indicated that glucose was not essential for histidine decarboxylation. This observation indicated that bacteria other than lactobacilli were producing histamine (Garner et al., 2003).
When the histidine enrichments were plated in an anaerobic glove box, several colony types were observed, but only small translucent ones produced histamine (Garner et al., 2003). The isolates were ovoid-shaped cells and this observation supported the assumption that they were not lactobacilli (Figure 1). They had relatively simple nutritional requirements and could be sub-cultured in a carbonate-based medium that had small amounts of yeast extract and butyrate so long as histidine was present.
Figure 1. Differential Interference Contrast (DIC) image showing morphology of A. histaminiformans strain MR2.
The isolates grew rapidly (1 hr doubling time) with histidine, but they did not utilize carbohydrates, organic acids or other amino acids as an energy source for growth. The idea that the isolates were obligate histidine decarboxylating bacteria was supported by experiments employing washed cell suspensions. Washed cells did not utilize arabinose, cellobiose, citrate, fructose, fumarate, galactose, glucose, inositol, lactose, malate, maleonate, maltose, mannitol, mannose, melibiose, melezitose, pyruvate, raffinose, rhamnose, ribose, succinate, sucrose, trehalose, or xylose, and volatile fatty acids were not produced.
Classification of Allisonella histaminiformans
Standard tests indicated that the isolates were novel, and this conclusion was supported by modern molecular techniques (Garner et al., 2003). Genes encoding ribosomes evolve slowly and microbiologists have shown that differences in the 16S fragment can be used as taxonomic tools (Woese, 1994). 16S rRNA gene sequencing indicated that isolates obtained from different cows (n = 6) were virtually identical. When the sequences were aligned and compared to those in GenBank, the isolates grouped with low G+C Gram-positive bacteria (Figure 2).
Figure 2. A phylogenetic dendrogram of low C+G, Gram-positive bacteria. The relationships were based on 16s rRNA gene sequences. The tree was constructed using the neighbor-joining method and bootstrap values are expressed as a percentage of 1000 replications. A. histaminiformans strain MR2 is shown in bold. The bar designates 0.1% difference in the 16s rRNA gene sequence. Taken from Garner et al. (2003)
Our isolate’s closest relative was Dialister pneumosintes, a bacterium first isolated from people that died during the influenza epidemic of 1918 (Olitsky and Gates, 1921). Dialister does not grow in defined laboratory medium, but experiments with chopped meat broth indicated that it could not convert histidine to histamine. The second closest relative in the database Genbank was an uncultured ruminal bacterium. The ruminal bacteria, Megasphaera elsdenii and Selenomonas ruminantium, were also found in this same grouping, but they did not produce histamine.
Based on the observation that bacteria in GenBank were genetically and physiologically distinct from our isolates, we proposed a new genus and species, Allisonella histaminiformans (Garner et al., 2003). The genus name Allisonella honors Milton J. Allison, a prominent rumen microbiologist. Dr. Allison previously isolated Oxalobacter formigenes, a ruminal bacterium that decarboxylates oxalate (Allison et al., 1985). The species name is a Latin word that means ‘forming histamine’.
Some other anaerobic bacteria can ferment histidine and grow, but these organisms employ glutaconyl-CoA decarboxylase and produce acetate and butyrate rather than histamine (Chen and Russell, 1989; Gottschalk, 1986). Histidine decarboxylase, the enzyme that produces histamine, has been studied in great depth and is widely distributed in bacteria (Schelp et al., 2001).
However, this latter reaction is not directly coupled to ATP formation, and intracellular proton consumption was not sufficient for growth of Lactobacillus buchneri (Molenaar et al., 1993).
To our knowledge, A. histaminiformans is the first histamine producing bacterium that can utilize histidine as its sole source of energy.
Enumeration of Allisonella histaminiformans
In vitro studies indicated that A. histaminiformans was a highly pH resistant bacterium, but histidine decarboxylation consumes a proton and leads to a marked increase in culture pH. The ability of A.
histaminiformans to increase the pH provides a straightforward method of enumeration in dairy cattle fed commercial rations. If mixed ruminal bacteria are inoculated into MRS medium, the final pH is less than 4.5, but A. histaminiformans can counteract this decline in pH if histidine is present (final pH 6.5). By serially diluting ruminal fluid into MRS that contains histidine and measuring the pH of each dilution, it is possible to estimate the numbers of A. histaminiformans in ruminal fluid (Figure 3).
Figure 3. 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 hrs of incubation at 39° C, the tubes were opened. pH was measured with an electrode and histamine was assayed by a method employing thin layer chromatography.
The use of pH to enumerate A. histaminiformans is confirmed by direct measurement of histamine production (thin layer chromatography).
Diet effects
Early experiments indicated that A. histaminiformans could not be isolated from fistulated cows fed timothy hay, but dairy cattle fed a commercial ration had large populations (>106 cells per ml) of A. histaminiformans (Garner et al., 2003). This diet-dependent difference could not be solely explained by the amount of histidine in the diet or the pH of the ruminal fluid. When stationary phase A. histaminiformans cultures were serially diluted into autoclaved ruminal fluid from cattle fed hay, histamine was not detected at dilutions greater than 10-2 even if histidine (50 mM) was added. In contrast, histamine was detected in the 10-9 dilution if the autoclaved ruminal fluid was obtained from cattle fed the commercial ration and supplemented with histidine (50 mM).
The dairy ration had large amounts of silage, and in vitro experiments supported the idea that silages stimulated the growth of A. histaminiformans.
Silages were mixed with water and blended to make water-soluble extracts. The extracts were boiled under N2 gas and autoclaved (121°C, 15 min).
Because A. histaminiformans could not grow in ruminal fluid from cows fed timothy hay, we could then assay the ability of these extracts to stimulate growth (Figure 4).
Figure 4. The ability of various extracts to stimulate the histamine production of A. histaminiformans in ruminal fluid from a cow fed timothy hay. Garner and Russell (unpublished results).
All of the extracts stimulated growth to some degree, but the alfalfa silage extracts were clearly the most potent. Alfalfa extracts that had been sterile filtered (0.2 µm) had the same activity as those that were autoclaved. The effect of alfalfa extracts could be assayed by measuring the optical density of A. histaminiformans. When the extract was increased in stepwise fashion, optical density increased, and extract from only 5 mg of alfalfa dry matter promoted maximal histamine production in 1 ml of culture medium (Figure 5). Based on these results it appeared alfalfa silage was promoting the growth of A. histaminiformans in cattle fed the dairy ration.
Figure 5. The effect of alfalfa silage extracts on the growth (optical density) of A. histaminiformans MR2. The amount of extract is expressed as dry matter equivalent per ml culture medium. Garner and Russell (unpublished results).
Nature of the growth factor
Alfalfa silage extracts that were treated with perchloric acid and KOH still stimulated histamine production by A. histaminiformans, and this result indicated that the growth factor was not a protein.
When the de-proteinized extracts were treated with chloroform, activity remained in the aqueous fraction. Conversely, chloroform fractions that were evaporated to dryness did not stimulate the growth of A. histaminiformans. These results indicated that the growth factor was not a lipid soluble compound.
De-proteinized extracts that were treated with Dowex 2 (anion exchanger) were as active as those that were not treated, but no activity was observed if Dowex 50 (cation exchanger) was added. This result indicated that the growth factor was a small positively charged compound, but further work will be needed to define more precisely its exact nature.
Other histidine-utilizing bacteria A. histaminiformans is not the only histidine utilizing bacterium in the rumen. In the 1980s, a group of bacteria since called ‘hyper-ammonia producing bacteria’ were isolated, and Clostridium aminophilum was able to decarboxylate and deaminate histidine at a very rapid rate. In this case, histidine was converted to acetate, butyrate and ammonia rather than histamine. Continuous culture studies indicated that the growth of C. aminophilum was inhibited at acidic pH values (Chen and Russell, 1989), and preliminary experiments indicate that even modest decreases in ruminal pH in vitro had a profound impact on the conversion of histidine to histamine versus non-toxic end products. If the pH was less than 6.5, histamine production by A. histaminiformans was enhanced, but little histamine accumulated when the pH was greater than 6.5.
Conclusions
The role of histamine in the etiology of rumen acidosis has not been definitively described, but a variety of studies have shown that histamine can decrease rumen motility and increase the severity of laminitis (Aschenbach and Gabel, 2000; Takahashi and Young, 1981). The involvement of histamine in laminitis is supported by the observation that anti-histamines can be used as a treatment (Nilsson, 1963). Oral administration of histamine to cattle that were not acidotic did not induce laminitis, however, histamine is not absorbed rapidly from the rumen unless the pH is acidic (Aschenbach and Gabel, 2000).
Recent work indicates that the rumen has a previously unrecognized bacterium that produces histamine, A. histaminiformans. A. histaminiformans is a highly specialized bacterium that is only able to convert histidine to histamine. Because A. histaminiformans is a highly pH-resistant bacterium, it is better able to compete with other histidine-utilizing bacteria when the pH is low. A. histaminiformans could not be isolated from cattle fed hay, but it is found at high numbers in cattle fed dairy cattle rations. The ability of A. histaminiformans to grow in the rumen is promoted by acidic pH and a nutritional factor derived from silages (particularly alfalfa silage).
The nutritional factor appears to be a small positively charged molecule, but further work will be needed to define more precisely its exact nature. The observation that the growth factor can be derived from alfalfa silage could have practical significance. Logue et al. (2000) noted that dairy cattle fed grass silage had a significantly greater incidence of laminitis and foot lesions than cattle that were fed non-fermented dry forage.
References and suggested reading
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Agricultural Research Service, USDA and Department of Microbiology, Cornell University, Ithaca, NY, USA
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