Physiological Studies of the Rumen ProtozoanOphryoscolex caudatus Eberleini

P. P. WILLIAMS, R. E. DAVIS, R. N. DOETSCH, AND J. GUTIERREZ

Animal Husbandry Research Division, Agriculture Research Service, U. S. Department of Agriculture, Beltsville, Maryland,a,Vd Department of Microbiology, University of Maryland, College Park, Maryland

Received for publication December 14, 1960

ABSTRACTWILLIAMS, P. P. (U. S. Department of Agriculture,

Beltsville, Md.), R. E. DAVIS, R. N. DOETSCH, ANDJ. GUTIERREZ. Physiological studies of the rumenprotozoan Ophryoscolex caudatus Eberlein. Appl.Microbiol. 9:405-409. 1961.-The rumen ciliate Ophry-oscolex caudatus fermented starch with the productionof acetic, butyric, and lactic acids plus CO2 and H2.Cellulose was not significantly metabolized althoughpectin was rapidly attacked in the Warburg apparatus.The protein sources, cottonseed, soybean, and linseed-oil meals, and the amino acids, DL-alanine, DL-valine,and DL-leucine, were utilized by the protozoan, whereasammonia was demonstrated as an end product ofnitrogenous metabolism. Methods for the separationof 0. caudatus from mixed rumen contents are de-scribed.

Ophryoscolex caudatus Eberlein is one of the largerumen ciliates which occasionally occurs in highnumbers when animals are fed diets which are rich instarch. The physiological functions of the protozoanare of some interest both from the standpoint of cellularmetabolism and because of their effect on the host.0. caudatus does not usually swallow cellulose materialsas does Diplodinium, but will ingest starch grains(Hungate, 1955). Little is known of other carbohy-drate or nitrogenous nutrients utilized by the proto-zoan; ammonia production by mixed species of rumenprotozoa has been suggested by the work of Warner(1956), and Blackburn and Hobson (1960) have at-tributed some of the rumen proteolytic activity tothe protozoa. The purpose of the present investigationwas to gain some knowledge of the nutritional habitsof 0. caudatus, along with information of fatty acidand nitrogenous excretory products.

MATERIALS AND METHODS

The inorganic salt solution used in the physiologicalstudies of 0. caudatus contained (w/v): NaCl, 0.5 %;MgSO4, 0.005%; KH2PO4, 0.1%; CaCl2, 0.005 %; and

' Presented in part at the 60th general meeting of the Societyof American Bacteriologists, Philadelphia, Pa. May 1-5,1960.

NaHCO3, 0.1 %. The salts, except sodium bicarbonate,were dissolved in distilled water and sterilized by Seitzfiltration. The sodium bicarbonate was autoclavedseparately in a 10 % solution and added to the filteredsalt solution while the liquid was bubbled with 95 %N2 and 5% CO2 gas for 15 min at a temperature of 39 C.The salt solution had a pH of 6.9.

0. caudatus in counts of approximately 4,000 per mlin rumen samples, removed by stomach tube from asteer 2 hr after the morning feeding, were collected in1-liter Erlenmeyer flasks. The steer's diet consistedof alfalfa hay, 22 %; soybean oil meal, 16 %; barley,61 %; and salt, 1 %. The rumen liquor was kept in awater bath at 39 C while the mixed protozoan popula-tion settled. Fifty milliliters of the sediment wereplaced in 25 ml of inorganic salt solution in a largerubber-stoppered test tube and flushed with 95 %N2 and 5 % CO2 (Stone and Beeson, 1936), and storedat 39 C until used. The plant debris that floated tothe top of the protozoan suspension was removed bypipette and additional salt solution was added to thetube and flushed with 5 % CO2 to maintain anaerobicconditions. The protozoa were allowed to settle outfor approximately 5 min and were then examined micro-scopically. Entodinium, Dasytricha, and Isotricha wereobserved for the most part in the supernatant; Ophry-oscolex and a few Diplodinium species, which were moredense than either Endodinium or Dasytricha, and slug-gishly motile as compared to species of Isotricha, werefound primarily in the grayish-white sediment in thebottom of the tube.To obtain pure samples of 0. caudatus for experi-

mentation, the following techniques of column sedi-mentation were employed: (i) the lighter and moreactive protozoa were drawn off in the supernatantafter the protozoa were allowed to settle for about 5min. The supernatant was replaced with an equalamount of inorganic salt solution. Forms removed bythis process included Dasytricha, Entodinium, andIsotricha; (ii) the remaining sediment was resuspendedin 50 ml inorganic salt solution. The large Dipodiniumsettled out more rapidly than the Ophryoscolex, andremoval of the early sediment eliminated this species.The remaining sediment was microscopically exam-

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ined for purity before (iii) dispensing the suspensioninto a long vertical glass column (dimensions: 146 cmlong, 1.7 cm diameter, 270 ml volume) filled withinorganic salt solution. 0. caudatus settled out in thebottom, and any Entodinium and Dasytricha remainedin the upper portion of the column. The collectedprotozoa were placed in salt solution containing 1 mgper ml each of dihydrostreptomycin sulfate andprocaine penicillin G. One-half milliliter of the proto-zoan sediment from the column in 12 ml of the anti-biotic salt solution gave a protozoan count of approxi-mately 4,000 per ml. Accurate counts were made bybubbling and protozoan suspension rapidly with a0.1-ml pipette by exhaling through it and then suckinga 0.1-ml aliquot rapidly. Aliquots of 0.01 ml were thencounted, averaged, and the total count determinedper ml. Methylene blue in 50 %o (v/v) ethanol was usedto stain wet mounts for morphological observationsand the oligotrich ciliate was identified using the keysof Dogiel (1927) and Becker and Talbott (1927).To check the purity of the protozoan suspensions

which were used in the metabolism studies, Gramstain smears were made of the washed protozoan sus-pensions and microscopic observation showed only afew scattered bacterial cells. As an additional pre-caution against bacterial activity in the Warburgexperiments, a control vessel, containing the antibiotic-treated supernatant fluid free of protozoa, was usedto measure the amount of gas evolved by any con-taminating bacteria. The amount of gas produced bythis final washing fluid was always negligible.

Respiration studies using the Warburg apparatuswere made with 2 ml of the anaerobic salt solutioncontaining 10,000 to 12,000 0. caudatus and 1 mg ofdihydrostreptomycin sulfate and procaine penicillinG per ml. From 8 to 10 mg of the particular substratein 0.2 ml salt solution were provided from the vesselside arm. Aletabolic activity was determined by com-parison with an endogenous vessel and hydrogen pro-duction was measured by exposing the gas to palladiumin the center well of the Warburg vessel, while thetemperature for all the experiments was 39 C and thegas phase 95. %N2 and 5 %c CO2.

In isotopic tracer experiments to determine sub-strate utilization, 0. caudatus were collected andpurified by column sedimentation as previously de-scribed, and suspended in 5 ml of buffered inorganicsalt solution. Cell concentrations ranged from 3,000 to5,000 organisms per ml. To the suspension was added0.5 ,uc of the radioactive substrate which was to betested. A 100-,ul sample of this original culture wasremoved and placed on a planchet for counting. Theprotozoa were then inctubated for 1 hr at 39 C and 5 %oCO2 and 95 7c N2 was used to maintain anaerobicconditions.

After inctubation, the cells were washed twice in 10

ml of tap water to remove most of the extracellularradioactivity, and 100 ,ul of the washed cells wereplaced on a planchet; for a control a planchet wasprepared containing a 100-,ul sample of the final washsupernatant free of protozoa. The planchets were driedunder an infrared lamp and 5-min counts were madefor each sample using a gas flow counter and scaler;samples were plated at less than 0.5 mg per cm2 toreduce self-absorption. Background counts were takenalso on a 5-min basis, and all counts were recorded asaverage counts per min. Substrates2 tested includedstarch-U-C'4 (specific activity 15.2 mc/mM), DL-leucine-2-C14 (specific activity 0.05 mc/mM), DL-valine-4-C14 (specific activity 3.0 mc/mM), and DL-alanine-2-C"4 (specific activity 2.64 mc/mM).Ammonia production by 0. caudatus was determined

by the Conway (1950) technique. Optical densityreadings were made on a Beckman quartz spectropho-tometer model DU3 set at 490 m, and compared to astandard curve in which (NH4)2SO4 was used as a sourceof NH3,. INessler's solution was prepared as describedby Umbreit, Burris, and Stauffer (1957).

RESULTSIdentification of the protozoa. The protozoan dimen-

sions were approximately 200 by 80 A,u depending onithe length of time after cell divisioni. The cell was ovoidand terminated posteriorly in a single spine whichwas longer and broader than the other pre-anal spines.The mouth was observed to be surrounded by anadoral zone of membranelles and a dorsal zone ofmembranelles covered four-fifths of the circumfer-ence of a zone somewhat anterior to the middle of thebody. The protozoan was identified as 0. caudatusEberlein (Fig. 1).

2 Supplied by California Corporation for Biochemical Re-search, Los Angeles, Calif.

3 Becton Instruments, Inc., Fullerton, Calif.

FIG. 1. Ophryoscolex caudatus stained with methylene blue.Bright field. Magnification, 343 X.

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Substrates utilized by 0. caudatus. PreliminaryWarburg apparatus studies indicated rapid utilizationof washed, finely ground rice starch with the produc-tion of H2 and CO2. The gas pressure in millimetersBrodie's solution is plotted against time in Fig. 2.The endogenous line in Fig. 2 refers to protozoa ex-

posed to 0.2 ml of inorganic salt solution minus sub-strate. The adsorption of H2 with palladium is illus-trated by the reduction from 215 mm pressure on theCO2 and H2 curve to the 80 mm pressure noted on theCO2 curve.

The crude substrates, cottonseed oil meal, soybeanoil meal, and linseed oil meal, were utilized in Warburgexperiments, and gave a total of 225 mm pressure as

compared to approximately 150 mm pressure in theendogenous vessel and manometer. The protozoa atthe end of the run were filled with the substrates andactively motile. 0. caudatus did not produce significantamounts of gas from the polysaccharide cellulose

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RIC E STARCH SUBSTRATEC02 AND H2 0 °

240 - ° -

002

po ENDOGENOUS Y.

160

120

80

40

0

0 10 20 30 40 50 60

TIME (MINUTES)

FIG. 2. Gas production from the utilization of rice starch inWarburg experiments by Ophryoscolex caudatus. The protozoawere suspended in 2 ml of 0.5% saline buffered with 0.1% NaHCO3and the gas phase was 5% C02 and 95% N2.

RICE STARCHPECTIN

240 CELLULOSEENDOGENOUS

'= 200Ul)

LiL 160OL

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80

40

0

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0 10 20 30 40 50 60 70 80

TIME (MINUTES)FIG. 3. Comparison of the gas production from the degradation

of rice starch, pectin, and cellulose by Ophryoscolex caudatus.

which followed the endogenous curve closely (Fig. 3).The cellulose curve was also representative of thatobtained with the soluble carbohydrates: maltose,glucose, galactose, cellobiose, and sucrose. However,the colloidal carbohydrate pectin (previously neu-tralized with 0.09 N NaOH) was rapidly attacked andgave a pressure increase of approximately 180 mm in80 min compared to approximately 100 mm pressurein the endogenous vessel.

Since the crude protein sources, cottonseed, soybean,and linseed oil meal, were readily utilized by 0. cauda-tus, tracer amino acids were employed to determine ifthe protozoa could attack a soluble, chemically de-fined nitrogen source. One-hour incubation periodswith C'4-labeled amino acids showed that 0. caudatusreadily concentrated D-leucine, DL-alanine, and DL-valine within the cells. Starch-U-C14 was also incorpo-rated in the protozoa. Table 1 gives the magnitude ofthe radioactivity found in the protozoa in the experi-ments.

Metabolic end products. To determine the fermenta-tion acids of 0. caudatus, the organisms were collectedand purified as described earlier, and placed in twolarge test tubes containing 70 ml of previously gassedsalt solution. To both tubes were added 100 mg washedrice starch and 40 mg each of dihydrostreptomycinsulfate and procaine penicillin G. In one tube, theorganisms were killed at the beginning of the experi-ment by addition of 2 ml of 5 N H2SO ; the second tubewas allowed to ferment the starch for 5 hr at 39 C, atwhich time the culture was killed by adding 2 ml of 5N H2SO4. A count of the number of cells in the experi-mental and control tubes averaged 3,200 cells per mlin 77 ml total volume.The contents of the experimental and control tubes

were steam distilled and the distillate neutralized bytitration with 0.09 N NaOH. The neutralized distillateswere evaporated to dryness and used for chromato-graphic analysis of the volatile acids. The liquid residuefrom the steam distillation was used for the analysis ofthe nonvolatile acids. The volatile acids (0.15 meq)were chromatographed on paper in 20-,ul amountsopposite a spot of mixed known fatty acids, formic,

TABLE 1. Incorporation of labeled amino acids and starch byOphryoscolex caudatus

Radioactivity in counts/min/100 lSubstrate

Initial culture Cells Supernatant*

DL-Valine-4-C04 12,146 1,074 38DL-Leucine-2-C'4 6,171 1,798 170DL-Leucine-2-CI4 6,790 813 18Starch-U-C14 6,822 207 21

* Radioactivity of the final wash water in which the proto-zoa were rinsed. The number of cells contained in 100 Al of thebuffer solution was approximately 16,000.

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acetic, propionic, and butyric, in their sodium salts.The acids were chromatographed ascending in 1 %ethylamine-butanol and the paper was treated with0.04 % bromeresol green in ethanol, giving blue spotson a yellow background (Block, Durrum, and Zweig,1955). The chromatographed volatile portion indicatedthe presence of two of the lower fatty acids, aceticand butyric acids. The occurrence of lactic acid as afermentation product of starch-fed 0. caudatus wasdetermined from the nonvolatile portion of the culture.Only small traces (less than 0.09 meq) of the acid wereencountered when analyzed by methods previouslydescribed (Gutierrez, 1955).

For the detection of ammonia production, 3 ml of asuspension of 0. caudatus (3,000 per ml), which hadbeen deprived of substrate for 4 hr in an antibioticsalt solution, were dispensed in test tubes flushed with5 % CO2 and 95 % N2. Duplicate tubes, containing 10mg casein, 10 mg starch, and an endogenous set, wereprepared. All the tubes were then incubated at 39 Cfor 2 hr and 2 ml of the protozoan suspension fromeach tube were dispensed in the outer well of the Con-way dish and analyzed for ammonia by the methodof Conway (1950). Experiments with 0. caudatusrevealed that 0.8 Amoles of NH3 were released from6,000 protozoa exposed to 6.6 mg of casein after 2 hrincubation at 39 C. With the same experimental con-ditions, only 0.1 ,umole NH3 was produced by theprotozoa from 6.6 mg of starch.

DiscussioNThe rapid breakdown of starch by 0. caudatus

indicates that the protozoa may contribute signifi-cantly to the digestion of the polysaccharide in therumen. As was shown in the current experiments notonly was the starch ingested by the organism, but thedigestion was accompanied by the production of acetic,butyric, and lactic acids, CO2, and H2. An additionalproduct from the breakdown of casein was ammonia.The quantitative experiments on ammonia productionfrom casein by known numbers of 0. caudatus showthat at least for some of the ruminal protozoa thiscan be an important end product, and the protozoaprobably are a factor in the nitrogenous metabolism ofruminants.The possession of proteolytic properties by 0. cauda-

tus, which was demonstrated in the casein and soybeanoil meal experiments, indicates that the protozoa areagents which function in the breakdown of ingestedfood proteins. Ingestion and disintegration of bacterialcells by the protozoa has been demonstrated also(Gutierrez and Davis, 1959; Gutierrez, 1958). A protein-ase for the oligotrichs was suggested by Schlottke asearly as 1936, and Blackburn and Hobson (1960) havereported the ingestion of stained casein particles with

their subsequent disappearance in some species ofoligotrich protozoa. Amino acids can also be assimi-lated to some extent by 0. caudatus as was demonstratedin the tracer experiments; a similar phenomenon hasbeen shown for the ciliate Colpidium campylum (Halland Elliott, 1935). Thus the protozoan competes withsome species of ruminal bacteria which are capable ofattacking amino acids. Bladen, 'Bryant, and Doetsch(1961) have found the ruminal bacterial speciesBacteroides ruminicola can break down DL-serine, L-aspartic acid, and L-cysteine with ammonia produc-tion, and Dehority et al. (1958) have reported C'4-labeled valine and proline were metabolized by mixedrumen contents. The amino acid composition of theproteins in the ration probably influences the rate ofammonia production in the rumen since some aminoacids are preferentially utilized by the protozoa andbacteria (Lewis and Elsden, 1955).

0. caudatus differs markedly from species of theholotrich group which includes Isotricha prostoma, I.intestinalis, and Dasytricha ruminantium. The latterthree species have been shown to rapidly attack solublecarbohydrates such as glucose, fructose, and sucrosewith the production of cellular amylopectin, fatty acids,and gases (Heald and Oxford, 1953; Gutierrez, 1955).In the current experiments with 0. caudatus, glucoseand maltose did not give significant gas productionin the Warburg apparatus, although the possibilityexists that these carbohydrates may be utilized slowly.Manometric experiments with Entodinium have shownglucose is not utilized by the oligotrich protozoan(Abou Akkada, Hobson, and Howard, 1959). Thispoints out one of the major differences in the biochemi-cal properties between the oligotrich and holotrichruminal protozoa, that is the rapid utilization ofsoluble carbohydrates by the latter group which isnot shared by the former.The major fatty acid end products of starch fermen-

tation in washed suspensions of 0. caudatus incubatedwith antibiotics were acetic and butyric acids withtraces of lactic acid. The oligotrich protozoan thusdiffers from the holotrichs Isotricha and Dasytrichasince species of these two genera produce mainly lacticacid plus acetic and butyric acids from the fermenta-tion of glucose (Heald and Oxford, 1953; Gutierrez,1955). Abou Akkada et al. (1959) have analyzed theend products of starch digestion by the small oligo-trich Entodinium; the mixture of acids found wasformic, acetic, propionic, and butyric plus smallamounts of lactic acid. In the ruminal oligotrich proto-zoa, lactic acid is not an important acidic end product,and this characteristic has aided in making the oligo-trichs amenable to culture in vitro (Hungate, 1942;Coleman, 1960). A pH near neutrality was shown tobe an important factor in the culture of Diplodinium

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neglectum (Hungate, 1942); culture of holotrich specieshas been possible only for short periods (Gutierrez,1955).

LITERATURE CITED

ABOu AKKADA, A. R., P. N. HoBSON, AND B. H. HOWARD. 1959.Carbohydrate fermentation by rumen oligotrich protozoaof the genus Entodinium. Biochem. J. (London) 73:44.

BECKER, E. R., AND M. TALBOTT. 1927. The protozoan faunaof the rumen and reticulum of American cattle. IowaState Coll. J. Sci. 1:345-373.

BLACKBURN, T. H., AND P. N. HOBSON. 1960. Proteolysis in thesheep rumen by whole and fractionated rumen contents.J. Gen. Microbiol. 22:272-281.

BLADEN, H. A., M. P. BRYANT, AND R. N. DOETSCH. 1961. Theproduction of isovaleric acid from leucine by Bacteroidesruminicola. J. Dairy Sci. 44:173-174.

BLOCK, R. J., E. L. DURRUM, AND G. ZWEIG. 1955. A manualof paper chromatography and paper electrophoresis.Academic Press, Inc., New York.

CONWAY, E. I. 1950. Microdiffusion analysis and volumetricerror. Crosby Lockwood and Son, Ltd., London.

COLEMAN, G. S. 1960. The cultivation of sheep rumen oligotrichprotozoa in vitro. J. Gen. Microbiol. 22:555-563.

DEHORITY, B. A., R. R. JOHNSON, 0. G. BENTLEY, AND A. L.MOXON. 1958. Studies on the metabolism of valine, proline,leucine and isoleucine by rumen microorganisms in vitro.Arch. Biochem. Biophys. 78:15-27.

DOGIEL, V. A. 1927. Monographie der familie Ophryoscole-scidae. Arch. Protistenk. 59:1-288.

GUTIERREZ, J. 1955. Experiments on the culture and physiologyof holotrichs from the bovine rumen. Biochem. J. 60:516-522.

GUTIERREZ, J. 1958. Observations on bacterial feeding by therumen ciliate Isotricha prostoma. J. Protozool. 5:122-126.

GUTiERREZ, J., A;ND R. E. DAVIS. 1959. Bacterial ingestion bythe rumen ciliates Entodinium and Diplodinium. J. Proto-zool. 6:222-226.

HALL, R. P., AND A. M. ELLIOTT. 1935. Growth of Colpidiumin relation to certain incomplete proteins and amino acids.Arch. Protistenk. 86:443-450.

HEALD, P. J., AND A. E. OXFORD. 1953. Fermentation of solublesugars by anaerobic holotrich ciliate protozoa of thegenera Isotricha and Dasytricha. Biochem. J. 53:506-512.

HUNGATE, R. E. 1942. The culture of Eudiplodinium neglectum,with experiments on the digestion of cellulose. Biol. Bull.83:303-319.

HUNGATE, R. E. 1955. Mutualistic intestinal protozoa. InS. H. Hutner and A. Lwoff [ed.], Biochemistry and physiol-ogy of protozoa. Academic Press, Inc., New York. 388 p.

LEWIS, D., AND S. R. ELSDEN. 1955. The fermentation ofL-threonine, L-serine, L-cysteine and acrylic acid by aGram-negative coccus. Biochem. J. 60:683-692.

SCHLOTTKE, E. 1936. Untersuchungen uber die Verdaungsfer-mente von Infusorien aus dem freien Wasser und aus denRinderpansen. Sitzber. Naturforsch.-Ges. Rostock 3F,6:59-66.

STONE, H. W., AND C. BEESON. 1936. Preparation and storageof standard chronmous sulphate solution. Ind. Eng. Chem.Anal. Ed. 8:188-190.

UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUFFER. 1957.Manometric techniques. Burgess Publishing Co., Minne-apolis.

WARNER, A. C. I. 1956. Proteolysis by rumen micro-organisms.J. Gen. Microbiol. 14:749-762.

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