Haemonchus contortus: Food of preinfective larvae

EXPERIMENTAL

PARASITOLOGY 29,201~207

Haemonchus

(1971)

contortus:

Food

Guang-Tsan College of Veterinary

Medicine,

of Preinfective

Larvae’

Wang2

University of Illinois,

Urbana, Illinois

(Submitted for publication 3 April 1970) WANG, GUANG-TSAN, 1971. Haemonchus contortus: Food of preinfective larvae. Experimental Parasitology 29, 201-207. Haemonchus contortus eggs were bacterially sterilized and cultivated in three different preparations of Escherichia coli cultures and a nutrient broth at 30 C for 7 days. The following percentages of infective larvae were recovered: supernatant fluid from E. coli culture, 0%; washed E. coli, 71%; heat-killed E. co& 40%; and nutrient broth, 0%. The evidence that preinfective larvae fed on the bacterium per se was substantiated by the autoradiograph of the larva grown on tritiated bacterial cells alone. Radioactivity was found throughout the larval body with most of the activity concentrated in the gut. INDEX DESCRIPTORS: Haemonchus

Thymidine ; Autoradiography

contortus;

Culture ; Escherichia

co&; Tritium

;

; Nematoda; Food.

McCoy (1929a,b) and many others have been able to grow free-living larvae of many species of gastrointestinal nematodes on Escherichia coli cultures. This bacterial culture was also used by Wang (1967) to cultivate Trichostrongylus colubrijormis from egg to infective stage. In 1968, I found that both T. colubriformti and H. contortus could complete their free-living development in monoxenic cultures of many other species of bacteria. Since Glaser and Stoll (1938) succeeded in cultivating H. contortus from egg to infective stage in axenic culture, many researchers have focused on axenic cultivation of the free-living stages, and the method has been extended to the parasitic stages. However, the question of whether the preinfective larvae actually feed on bacteria or their metabolites merits further investigation.

MATERIALS

AND METHODS

Escherichia coli The medium which was used to enhance the incorporation of tritiated adenosine (New England Nuclear) into E. coli consisted of NH&l 5 g, NH4 NOa 1 g, Nas citrate 0.5 g, Na2S04 2 g, MgSO, 0.1 g, KzHPOl 3 g, CaClz 0.01 g, KH2P04 1 g, trace elements, glucose 0.5%, and vitamin-free Casamino acid 1% in 1 liter of distilled water. Approximately 0.1 ml of a 24-hr nutrient broth culture of E. coli and 0.5 mc of tritiated adenosine were inoculated into 500 ml of the sterile medium in an Erlenmeyer flask covered with a stainless-steel cap. The flask was incubated at 37 C in a shaker for 10-12 hr to incorporate the tritiated adenosine into the DNA and RNA of the propagating

Preparation

of Tritiated

E. coli.

After incubation, the tritiated E. coli culture was centrifuged in a Sorvall refrigerated centrifuge at 27,000 g for 10 min. The supernatant fluid was removed for later

’ Supported by US. Public Health Service Grant No. AI-66197. ’ Present address: American Cyanamid Company, P. 0. Box 400, Princeton, New Jersey 08540. 201

202

WANG

assay of specific activity with a liquid scintillation counter (Packard Tricarb) . The mass of E. co& ~11s was washed five times with sterile 0.85% NaCl solution so that the supernatant fluid was free of isotope activity; after each washing, the supernatant fluid was collected separately for later assay. Isolntion

of Eggs and (lultivation

of Larvae

Eggs were collected by sugar flotation from droppings of two lambs infected only with H. cordorbs. They were bacterially sterilized in lOc/, Clorox (a commercial bleach containing 5.25~: sodium hypochlorite’) for 10 min, wabhcd five times with sterile distilled water, and kept in a sterile 50ml screw-capped centrifuge tube. Samples were removed to determine the bacterial sterility, viability, and total number of harvested eggs. The rest of the eggs were kept in a refrigerator before use. Three milliliters of egg suspension containing 20,000 bacteria-free eggs were placed in a small pet’ri dish and t’hen incub&et1 at 25 C. dftcr 1 day, 0.5 ml of washed, packed 3H E. coli cells were added to the petri dish in which first-stage larvae had hatched or were hatching. Petri dishes wcr~ then incubated at 30 C for 2 days. Preparation

of Larvae for Autoradiography

A4t the end of 2 day’s incubation, the larvae were separated from E. coli by washing the bacteria through a Seitz clarifying filter sheet; the filter sheet was then baermannized upside down to collect the larvae. The larvae were further washed three t,imes by gentle centrifugat’ion. The supernatant fluid from the last washing was collected for later assay. The washed larvae were fixed in 70% hot ethanol (70-80 C) and dch$dratcd through 85%, 95%, and two changes of 100% ethanol. Five-tenths percent of eosin-Y was added to the 95% ethanol to stain the larvae to facilitate their detection. The larvae were cleared in xylene before transfer to

0.5% parlodion in absolute methanol. A drop of the suspension containing about 10 larvae was placed on a glass slide. Methanol was spread over the slide, carrying the parlodion to form a thin film which held the larvae on the slide. Immediately after the t,hin film hat1 dried, the slide was coated with Kodak NLIclear Track Emulsion, type iVTR2, either by submerging two slides back to back in t’hr liquid emulsion or by spreading the emulsion with a stainless-steel roller sprcadcr. The emulsion-coated slides were I)lacctl in a plastic slitlc box wit’h the emulsion sick facing the +amc dir&ion and mere dried in a stream of cool air for approximatcaly 30 min. To keep the at’mosphere dry, small bags of Drierit’e were placed in the slitlc box bcforcl thcx 1)0x was sealed with aluminum foil and black tape. The box was stored in a rcfrigcrator for 2 weeks. Processing of the exposed slides was generally tlonc in tlic same manner as processing regular film cxccpt that Kodak Dl9 was used for clcvcloping anal fixation was for 10 niitl in hypo solutjion. All of these procedures w’crc pcrformcd in a dark room by light from a safclamp with a Wratten Series 1 safelight filter and :I 15-w bulb, at’ a minimum distance of 4 ft. Quantitative estimat>ion of grain yicltl was not attcmptcd in t,lic present experiment. In order to dctcrmiue the sl)cGfic activity of tritium present in each step of the expcrimcnt, the supernatant fluid from the E. coli washing, t#hat from the larvae washing, E. coli cells, larrac, and a blank were measured on a Packard “Tricarb” liquid scintillation counter to dcterminc counts per minut*c per ml (count/min/ml) . Cultivation of Free-living 8tngen in Variows Fractions of E. coli Cultwe One-day-old nutrient broth cultures of E. coli were centrifuged with a Sorvall refrigerated ccntrifugc at 27,000 g for 10 min to collect E. coli. The supernatant fluid was further sterilized by Scitz filtration. One-

FOOD OF Haemonchus

FIG. 1. Late second-stage larvae cumulated in the gut (X 490).

of Haemonchus

half of the harvested E. coli were killed by placing the culture tube in an 80 C water bath for an hour. Five-tenths milliliter of bacteria-free egg suspension containing about 20,000 eggs were inoculated into 2.5 ml of each of the preparations described previously, and control eggs were placed in sterile nutrient broth in different small petri dishes (Table II). They were then incubated at 30 C for 7 days. A sterility test was made of each preparation with thioglycolate and nutrient broths at 25 and 37 C. At the end of incubation, duplicate samples were pipetted into a Sedgewick-Rafter counting cell. For each observation, 100 eggs and/or larvae were counted with a compound microscope at 100~ magnification. The percentages of each of the following stages were recorded: eggs, dead preinfective larvae (DL,, DL,), dead infectivc larvae (DLS), live preinfective larvae (L1, Lz) , and infective larvae (L3). RESULTS

Using a light that the larval

microscope, it was found intestines became darker

LARVAE

contortus

203

showing

dark

particles

ac-

(Fig. 1) as the preinfective larvae developed in E. coli cultures and increasingly accumulated particulate material in the body. In Table I, the extremely high radioactivity of E. coli cells showed that most of the TABLE

I

Results of Liquid Scintillation Counting of 3HTagged Escherichia coli, Haemonchus contortus Larvae, and Their Supernatant Culture Fluids Samples

lst, washing of supernatant fluid from E. coli culture 2nd washing of supernatant, fluid from E. coli cult,ure 3rd washing of supernatant fluid from E. coli culture 4th washing of supernatant fluid from E. coli culture 5th washing of supernatant fluid from E. coli culture E. coli cells Last washing of supernatant fluid from larvae Larvae Blank

Activity (count/ min/ml) 37,216

11,198 4,091 752 102 868,536 97 2,856 62

204

WANG

FIG. 2. Autoradiograph of an Haemonckzcs Escherichia coli culture ( X 350).

tritiated adenosine had been incorporated into the multiplying bacteria after lo-12 hr incubation at 37 C. Very little activity remained in the last washings of E. coli and larvae (102 count/min/ml and 97 count/ min/ml, respectively, as compared with 62 count,min/ml for the blank). This result indicated that all of the isotopes present in the larvae should have come from the ingested E. coli cells. This evidence was further substantiated by the autoradiographs of H. contortus larvae (Figs. 2 and 3). AIthough the specific act.ivity of H. contortus larvae was not very high (Table I), good autoradiographs were still obtained from the liquid emulsion with a 2-week exposure. Radioisotopes were distributed throughout the body of the larvae, most of the activity being concentrated in the intestinal areas. Larvae grown on E. coli not tagged with 3H did not. have any silver grains in their autoradiographs. Ta.ble II shows percentage development of H. contortus larval stages in various culture media. When t,he supernatant fluid of the nutrient broth cultures of E. coli was

con!or,l us prcinfrctivc

I:WV:L grown in tritiated

freed of bacteria by Seitz filtration, none of the U. contortus larvae developed to the infective stage, moat dying at a preinfcctive stage. Although 33% were still alive at the end of the 7-day incubat.ion period, their intestines contained little material, and the larvae were sluggish. Also, no infective larvae were recovered from the control nutrient broth cultures. Fifty percent of the eggs did not hatch in the controls. Only the washed or heat-killed E. coli could support larval deveIopmcnt to the infective stage; however, the percent.age of infective larvae rccovcrcd from the washed E. co& (71%) was significantly higher (P < .Ol) than that (40.3%) from the heat-killed E. coli as determinctl by Student’s t test In addition, t’he unusually high percentage of dead preinfective larvae indicated that the heatkilled E. COGcultures did not provide sufficient nutrients to allow most of the larvae to develop to the infective stage. This experiment further illustrated that bacterial cells, rather than their metabolites or their culture media, were the food required for growth of the larvae.

KIOD OF Haemonchus

LARVAE

205

FIG. 3. Autoradiograph of the anterior end of an Haemonchus contortus larva grown in tritiated Escketichia coli culture (X 560). TABLE Percentage

Development

Culture media

of Free-Living

II

Stages of Haemonchus

contortus

in Various

CuZture Media”

Percent recover@

Sterility test

Supernat,ant fluid from Escherichia coli culture Washed E. coli Heat-killed E. coli Nutrient brot.h

preinfective

DI,,. DL2

DL3

Ll, L

I.3

33

0

-

6.7

60.3

0

+ -

19.0 18.3 50.0

3.5 26.5 46.5

6.0 11.9 0

0.5 3.0 3.5

71.0 f 13.1* 40.3 zt 5.7* 0

a Expressed as averages of four experiments. h DL,, DLZ = Dead preinfect,ive larvae. I)La = Dead infective third-stage larvae. L,, Lz = Living third-stage larvae. preinfective larvae. LB = Living infective * The difference between the two means is stat.istically significantly (P< .Ol, Student’s 1 test). DISCUSSION

The preinfective stage larvae (Fig. 1) of H. contortus accumulate dark particles in their guts. Whether these particles are bacterial cells or other particulate substances was hard to determine with the light microscope. Autoradiographs of H. contortus larvae which had grown on washed 3H-E. coli indicated that the preinfective larvae ac-

tually ingested the bacterial cells. Because the 3H-E. coli were washed with 0.85% NaCl solution until the supernatant fluid was free of radioisotope, all the activity present in the larvae should have come from the tritiated E. coli which was the only source of isotopes (Table I). The distribution of tritium throughout the larval body, with most of the activity concen-

206

WAA-;G

tratcd in the gut, indicated that some tritiated E. coli had been digested and assimilated. but most remained undigested in the larval intestine. Weinstein (1953) reported that hookworm larvae fed actively’ on killed bacteria and presented a photomicrograph of a larva with a large accumulation of bacteria in its gut. Results inconsistent’ with mine were rcported by Ciordia and Bizzell (1958). They could not detect any radioactivity in the infectjive larvae of cattle nematodes grown in fecal culture:: containing T-E. coli. Because details of technique were not mentioned in their brief st’atmcnt, it is difficult’ to determine the reason for the discrepancy. In my experiment, t’he larvae used for the autoradiography were reared in a 3H-E. coli suspension. The diffcrencc in larval cuhivation technique may have been the factors which prevented 32P from being detected by Ciordia and Bizzell in the nematode larvae of cattle. That the preinfective larvae fed on bacterial cells was further confirmed by the results in Table II. Infective larvae were recovered from washed E. co& suspensions but never from nutrient broth or sterile supernatant fluid of E. coli cultures. Although some infective larvae were also harvested from heat-killed bacterial cultures, the percentage of recovery was significantly lower than that from the washed E. coli cultures. The results of McCoy (1929a,b), Lapage (1933a,b), and Lawrence (1948) all suggest,ed that preinfective larvae of dog, sheep, and rabbit nematodes ingested bacteria; however, they were able to grow the nematode larvae to the infective stage only on living bacteria but not on heat-killed ones. In the present study, E. coli were concentrated before they were killed by heat. Since the dead E. coli were not actively propagating as did the living ones, the concentration might provide sufficient nutrients for small numbers of larvae to reach the infective stage. This may account for why

previous workers did not harvest any infcctive larvae in heat-killed E. coli culture. In reviewing their previous work, \\:cinstein and Jonezi (1957) stated that both heat-killed and formalin-killed bacterial cells, when supplemented with chicken cmbryo clxt)ract filtrat>o, supported cxcc~llcnt dcvelopment of ~Yippostrongylus nuris larvae to the infective stage. They also found that homogenates of chicken embryo extract would support dcvclopmcnt of the larvae to t,hc infective stage. Howcvcr, when particulates n-cre remov@ from thr embryo cxtract, virtually no dcvelopmcnt to t’he infective stage occurred. The killed bacteria anal particulatcs of chicken embryo extract t,hus contained common growth factors which were important for larval development. Since the free-living st,ages of many gastrointestinal nematodes have been successfully cultivated in bacterial cultures and axenic media, it is clear that bacteria are not t,he only or necessary sources of food for larval development. If the basic components of thcl crilt,urc~ meet t’lie nutritional requirements of the larvae, it makes littlc difference whether the larvae feed on bact’eria or grow in axcnic cultures. Howcvcr, Glascr and St,011(1938:)) and Weinstein and Jones (1957) rcport’cd that the infective larvae rccorcred from axenic cultures were consistently smaller than normal larvae. Whether the small size of the infective larvae has any effect on the subsequent devclopment of their parasitic stages both in vitro and in vivo has not been studied systematically. Although Glascr and Stoll (1938) reported that H. confortus infcctivc larvae from axenic cultures were still able to infect a lamb, they did not determine the percentage of infectivity. Wang (19683 found that there was no significant difference in infectivity of Trichostrongylus colubrifolmis in guinea pigs between larvae grown in feces and those grown in E. coli cultures.

FOOD OF

Haemonchus

REFERENCES CIORDIA, H., AND BIZZELL, W. E. 1958. Relation of Escherichia coli to the development of the preparasitic stages of various cattle nematodes. Journal of Parasitology 44 (supplement), 25. GLASER, R. W., AND STOLL, N. R. 1938. Sterile culture of the free-living stages of sheep stomach worm, Haemonchus contortus. Parasitology

30,32&332. LAPAGE, G. 1933a. Cultivation of parasitic nematodes. Nature, London 131,583-584. LAPAGE, G. 193313. The cultivation of infective nematode larvae on cultures of Bacillus coli. The Third Report of Institute of Animal Pathology. University of Cambridge, pp. 237271. LAWRENCE, J. J. 1948. The cultivation of the freeliving stages of the hookworm, Ancylostoma braziliense de Faria, under aseptic conditions. Australian Journal of Experimental Biology and Medical Science 26,1-8. MCCOY, 0. R. 1929a. The growth of hookworm larvae on pure cultures of bacteria. Science

69,7475.

LARVAE

207

MCCOY, 0. R. 192913. The suitability of various bacteria as food for hookworm larvae. American Journal of Hygiene 10, 140-156. WANG, G.-T. 1967. Effect of temperature and cultural methods on development of the free living stages of Trichostrongvlus colubriformis. American Journal oj Veterinary Research 28, 1085-1090. WANG, G.-T. 1968. Effects of microbes and gases on the free-living stages of Trichostrong$us colubriformis and Haemonckus contortus. Ph.D. dissertation, University of Illinois, Urbana. WEINSTEIN, P. P. 1953. The cultivation of the free-living stages of hookworms in the absence of living bacteria. American Journal of Hygiene F&353-376. WEINSTEIN, P. P., AND JONES, M. F. 1957. The development of a study on the axenic growth in vitro of Nippostrongybus muris to the adult stage. American Journal of Tropical Medicine and Hygiene 6,480-484. WHITMCK, H. V. 1948. Some modifications of the McMaster helminth egg-counting technique and apparatus. Australian Council of Scientific and Industrial Research 21,177-1&I.