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Physiological and microbiological changes in the abomasum of sheep infected with large doses of Haemonchus contortus
j. Cow.
PA~.H.
1987
VOL.
97
PHYSIOLOGICAL THE ABOMASUM DOSES
C. D. Department
of Pure and Applied
AND MICROBIOLOGICAL CHANGES OF SHEEP INFECTED WITH LARGE OF HAEMONCHUS COflTORl-US
P. R.
NICHOLLS, zoology,
and D. L.
HAYES*
of Microbiology
and *Department
UniversiQ
IN
LEE
of Leeds,
Leeds LSZ 9j’T,
U.K.
INTRODUCTION
Large doses of third-stage larvae of Haemonchus contortus administered to sheep are known to result in an increase in the abomasal pH within 2 or 3 days of infection (Christie, 1970; Coop, 1971; Mapes and Coop, 1973; Christie, Angus and Hotson, 1975). This increase in pH probably causes changes in the physiology of the gastrointestinal tract of sheep similar to those observed during infections with Ostertagia circumcincta, which also causes an increase in abomasal pH. For example, a decrease in the pepsin concentration of the abomasal fluid was observed after infection with 0. circumcincta (Horak, Clark and Botha, 1965) because conversion of pepsinogen to pepsin occurs very slowly above pH 4 (Hirschowitz, 1957). The rumen contains large numbers (about 10” per ml) of viable bacteria, most of which are obligate anaerobes (Hungate, 1975). The abomasum receives fluid and food materials from the rumen; however, little is known about the microbiology of the abomasum, which normally has a low pH (pH 2 to 3). No investigation has been reported on the effect of the induced pH increase, after administering large doses of H. contortus, on bacterial numbers and types. This study was undertaken to confirm the effect of large dosesof H. contortus on abomasal pH and to study any changes in the PO, of abomasal fluid and in the numbers of viable bacteria present and their oxygen sensitivity during the course of infection in sheep. MATERIALS
AND
METHODS
Culture of H. contortus larvae
A pure culture of H. contortus was obtained from the Moredun Institute, Edinburgh, and usedto infect sheepat Leeds. Faecescontaining eggsof H. contortus were collected from infected sheep and cultured in plastic trays at 20°C for 10 to 14days. The third-stage larvae were collected, contaminants removed as described by Rose (1971) and stored for at least 2 weeksat 10°C before use.
0021-9975/87/030299+
10 $03.00/O
0
1987
Academic
Press
Inc.
(London)
Limited
300 Experimental
C.
D.
NICHOLLS
Animals
The 12 sheep used in the experiments parasitic nematodes at the University of with a cannula (18 mm internal diameter, in separate pens. When experimentation 20 months. Experimental
et al.
were Suffolk cross Mules reared free from Leeds Field Station. The sheep were fitted 60 mm long) in the abomasum and housed started, the sheep ranged in age from 12 to
Design
The 12 sheep were divided into matched pairs; one member of each pair was given orally 10” infective third-stage larvae of H. contortus and given access to 1000 g of pelleted food per day plus water ad libitum. The pelleted food was a mineralized dried grass and barley mix containing 7.5 per cent ash, 30 per cent oil, 14 per cent protein and 14 per cent fibre (Lincolnshire Farm Feeds, Grimsby). The other member of each pair was a pair-fed control which was given the same amount of food as that eaten the previous day by the infected partner. Physiological
Anal_vsis qf Abomasal Fluid
Samples of abomasal fluid were obtained via the abomasal cannula by means of a monovette (Sarstedt Ltd., Leicester) before, and at regular intervals during, the infection. The samples were taken at approximately the same time of day on each occasion, and the monovette was completely filled with fluid to reduce exposure of the sample to air. After centrifugation at 1500 g for 5 min to remove food particles and other debris, the pH and the PO, of the supernatant were measured, twice for each sample, with a BMS 3 Mk 2 Blood Micro System (Radiometer, Copenhagen). Readings were obtained within 45 min of sampling and were similar to those obtained from samples analysed, for comparison, within 5 min of sampling. Preparation
and Dispensation
of Media,for
Culture of Anaerobic Bacteria
Diluting fluid was made up as described by Kurihara, Eadie, Hobson and Mann (19683, but modified slightly by altering the concentration of resazurin from 0.0001 g per 100 ml to 0.001 g per 100 m! and omitting the glucose (personal communication, J. Wallace, Rowett Research Institute, Aberdeen). The required ingredients, except the cysteine HCl, were mixed and heated until dissolved; the cysteine HCl was then added to the partially cooled solution. The medium was deoxygenated by bubbling O,-free CO, through the mixture; the gas had been passed over heated copper turnings in a Phoenix furnace (Phoenix Furnaces Ltd., Sheffield), heated to about 350°C to remove any traces of oxygen. Following deoxygenation, a luer-type syringe with a 15 cm long 14-gauge needle attached, was flushed with O,-free CO, and then used to dispense the medium in 9 ml amounts into Hungate tubes (Bellco Glass Inc., New Jersey, U.S.A), preflushed with O,-free CO,. The Hungate tubes possessed a butyl rubber stopper to provide an air-tight seal and a screw-on cap to hold the rubber stopper in place. The medium was autoclaved at 121” C for 15 min and stored at 4” C until required. The growth medium was a non-specific medium on which most, if not all, anaerobic rumen and abomasal bacteria can grow. It was modified from the medium 1 described by Kurihara et al. (1968) by increasing the resazurin as stated above (personal communication, J. Wallace, Rowett Research Institute, Aberdeen). The growth medium was prepared and dispensed as described for the diluting fluid. except that 4.5 ml amounts were dispensed. Chemicals were obtained from B.D.H. Chemicals Limited, Poole; Sigma Chemical Company Limited, Poole or Difco Laboratories, West Molesey. The rumen fluid used in these media was obtained from a healthy sheep, the fluid was strained to remove
ABOMASAL
CHANGES
IN
H. CO.VTORTUS
301
INFECTION
large particles, centrifuged at approximately 20 000 g for 40 min and the supernatant fluid was sterilized at 121°C for 15 min before storage at 4°C. Microbiologzcal
Analysis of Abomasal Fluid
Abomasal fluid samples were obtained by completely filling sterile Bijou bottles via the abomasal cannula. Samples were processed within 45 min of being taken. Appropriate decimal dilutions were prepared from 1.0 ml of the abomasal fluid; a modification of the Hungate technique was employed (Hungate, 1969). This allowed samples to be transferred anaerobically and aseptically between successive tubes by means of sterile 1.0 ml pipettes which had been preflushed with O,-free CO,. During the transfer the tubes were continuously flushed with O,-free CO,. The anaerobic roll tube method (Hungate, 1969) was used to analyse the sample; 0.5 ml of the required dilution was added to 4.5 ml of molten growth medium under anaerobic and aseptic conditions as described above. After mixing bv inversion, the agar was solidified by placing the tubes horizontally on revolving rollers in a perspex tank of cold water. The roll tubes so prepared were incubated at 39°C for 3 days, after which counts were made on the appropriate dilutions. Selected colonies were then subcultured anaerobically onto slopes of the growth medium and, after incubation at 39°C for 3 days, purity checks of the cultures were performed by further subculturing of selected colonies on to plates of the growth medium; this resulted in brief exposure to aerobic conditions prior to subsequent incubation at 39°C for 2 days in an anaerobic jar (Don Whitley Scientific Ltd., Shipley) filled with a mixture of95 per cent CO, and 5 per cent H,.
ofResults
Statistical Anal_yris
For statistical analysis the results obtained for abomasal pH, PO, and number of viable bacteria were divided into four groups, viz. uninfected, pair-fed sheep, before and after- jnfection of their partners, and infected animals, before and after infection (see Table 1). Significant differences between groups were calculated using one-way analysis of variance with linear combination on samples taken between days 3 to 20 after infection, as this was the period when the greatest changes in abomasal pH, PO, and bacterial numbers were observed. Curves for Figs. 1, 2 and 3 were fitted by. the Maximum Likelihood Program (Ross, 1978) on log-transformed X-axes. Regressions for pair-fed control values were fitted by the same program.
TABLE MEAN ABOMASAL
VALUES FLCID
FOR
ABOMASAL FROM
SIX
FLUID PAIR-FED
PH,
PO2
AND
AND
SIX
INFECTED
I
LOG.,”
OFTHE SHEEP
NUMBER BEFORE
OF VlABLE 3 T O 20
AND
BACTERIA
PERML
OF
DAYS AF’TER INFECTIOI,’
WITH 106 THIRD STAGE LARVAE OF N
stageof irzfectm
PH PO, (Pa; Log.,” bacterial count
Before infection of
partners
2.6 * 0.09 (26) 4252 f 187.5 (24) 5.7i~O.25
(16)
jheep
Infected rheep
___
3 to 20 days after infection of
partners
2.6 *O-04 (35) 4279f 160.3 139) 6.0 f 0.28 (14)
3 Before infectzon 2-710.09 5012*312~5* 6~110~15
(26) (241 (23)
to
ajier
20 dazs infection
6.0iO.lt (40, 427f 120.5.f (40) 8.5 5 0.08t
125)
Values given ate mranf S.E. with numbers of samples in parentheses. Group means that are significantly different from the prrvious group meam are indicated by: *Pi 0.0 I, tP< 0.00 1. Days 3 to 20 after infectIon are whrn the major changes were vbserved in ahomasal pH, PO, and bacterial numbers.
302
C. 7
I
D.
NICHOLLS
ef cd.
I
I
I
I
I
I
I
I 0
I IO
20
30
1 40
I 50
I 60
6-
5-
1 a 4-
3-
2f -10
Time
Fig. 1. Mean pair-fed
abomasal sheep.
pH. Values
0 =sheep are given
infected as mean
with lo6 f S.E.M.
70
(days)
infective
larvae
of H.
contort,
0 =uninfected
667
267C
IO
20 Time
Fig. 2. Mean abomasal pair-fed sheep.
fluid PO,. 0 =sheep infected Values are given as mean+
with S.E.M.
(days)
lob infective
larvae
of H. contork,
0 = uninfected,
ABOMASAL
CHANGES
IN
H.
CONTORTUS
INFECTION
303
RESULTS
Abomasal Fluid pH The pH of the abomasal fluid of pair-fed and pre-infected sheep varied between pH 2 and 3 (Fig. I). A rapid increase in pH to 6 to 6.5 was observed within the first 4 days of infection. This elevated abomasal pH was maintained for about two weeks before gradually decreasing to pre-infection values by about 10 weeks post-infection. The increase in abomasal fluid pH, observed between days 3 and 20 inclusive, was significant (PC 0.001; Table 1). The 6 uninfected, pair-fed sheep showed no significant change in abomasal pH during experimentation (Fig. 1; Table 1). Abomasal Fluid PO, Some variation was observed in the PO, of the abomasal fluid of the pair-fed sheep and in sheep before infection (Fig. 2) although it generally remained above 3330 Pa (1 mmHg= 133.3 Pa). About 3 days after infection, a rapid decrease in the PO,, down to less than 600 Pa, was observed in all 6 infected sheep. It remained low for about 2 weeks before it gradually returned to pre-infection values by about 7 weeks post-infection. The decrease in abomasal fluid PO,, observed between days 3 and 20 inclusive, was significant (P
304
C.
D.
et
NICHOLLS
al.
a
Time Fig. 3. Mean infective
number larvae
(log.,,,) of viable bacteria per of H. contortus, 0 = uninfected,
(days) ml of abomasal pair-fed sheep.
fluid. Values
0 =sheep are given
infected as mean
with 10b f S.E.M.
conditions, whereas with isolates obtained during days 5 to 6 of infection, greater losses in viability of cultures were observed. This was presumably due to their exposure to oxygen, either in the air during plating out of these isolates for purity checking, or due to the presence of a small amount of air in the anaerobic jar. These results show that there was an increase in the proportion of oxygen-sensitive bacteria after infection. Similar purity checks carried out on isolates from rumen fluid showed that there was again a high proportion of oxygen-sensitive bacteria present as indicated by the loss of many of the selected isolates (Table 2). Chi-squared analysis was used to determine significant differences between the results; expected values were calculated from the pooled results. The analysis indicated that samples from the uninfected, pair-fed sheep and from sheep before infection, did not show any differences in the proportions of oxygen-sensitive bacteria (P> O-1). However, a highly significant increase in the proportion of oxygen-sensitive bacteria was found in the infected animals, during the first week of infection, compared with that of the pair-fed and pre-infected animals (P < O-00 1) .
ABOMASAL
CHANGES
IN
H.
CONTORTUS 2
TABLE
PROPORTION (WITH
OF OXYGEN-SENSITIVE
lo6
INFECTIVE
‘MEASURED
BY
LARVAE
RECOVERY
OF OF
fluid sheep
Before
from
After
Rumen uninfected *i.e.
IN THE ABOMASAL
CON70R7C:~)
MEDIUM
SHEEP
SELECTED AND
AND
fluid from uninfected
fluid
they
INCUBATION
grow
FLUID
OF 4
RC‘MEN
FLUID
AFTER IN
AN
infection infection
UNINFECTED OF
SUBCULTURE
ANAEROBIC
Total number isolates ongina& selected jrom roll tubes
4
AND
UNINFECTED ONTO
INFECTED SHEEP,
PLATES
of
72
14 (19.4)
110
77 (70) 5 (129)
After infection of partners
40
4 (10)
40
39 (97.5)
jar,
plpbably
due
to exposure
AS THE
jVumber of isolates subsequently lost after subculture (and per cent) *
41
in an anaerobic
OF
JAR
Before infection of partners
from sheep do not
IN
ISOLATES
Stage of infection
Sample
Abomasal pair-fed sheep
H.
ORIGINALLY
GROWTH
Abomasal infected
BACTERIA
305
INFECTION
to air
(see text).
DISCUSSION
All infected sheep exhibited similar physiological and microbiological changes w-ithin the abomasum after infection, namely increases in the abomasal pH, in the number of viable bacteria present and in the proportion of oxygen-sensitive bacteria present in the abomasal fluid, and a decrease in the abomasal PO,. These changes all occurred at about the same time, suggesting that they are correlated. It is probable that the decrease in the PO, of the a.bomasal fluid is primarily due to bacterial activity. There is no obvious explanation for the observed significant difference in the PO, of the abomasal fluid between the infected sheep before infection and the pair-fed sheep; it may be due to variations between individual sheep (some sheep have consistently high or low values compared with other sheep). This could also explain the variation in bacterial counts observed in the pair-fed sheep (see Fig. 3). It is known that on reaching the abomasum, the larvae of H. contortus migrate into the gastric glands, where they moult (Veglia, 1915) and, on emergence from the glands, they continue to cause physical damage to the abomasal mucosa (Nicholls, Lee and Sharpe, 1985). They cause a reduction in the production of HCl in the abomasum, which results in an increase in the abomasal pH (Christie et al., 1975). It is suggested that this increase in abomasal pH provides a more favourable environment for bacteria normally resident in the rumen, where the pH is normally about 6.5. Initially, the facultative anaerobes from the rumen will multiply in the abomasal fluid as it becomes lessacid and the obligate anaerobes will be inhibited or killed by the oxygen present in that fluid. However, with the growth of the facultative anaerobes, oxygen will be utilized so lowering the abomasal PO, which will consequently allow the oxygen-sensitive obligate anaerobes from the rumen to survive, multiply and probably become the predominant group. The PO, of the
306
C.
D.
NICHOLLS
et
d.
abomasal fluid will be kept low by the facultative anaerobes until the gastric glands recover and secrete more HCl so lowering the abomasal pH. This will create a less favourable environment for the bacteria and growth will be progressively inhibited as the pH is lowered, thus allowing the PO, of the abomasal fluid to return to pre-infection values. The sharp rise in the abomasal pH observed 3 days after infection confirms similar observations by previous authors (Christie, 1970; Coop, 197 1; Mapes and Coop, 1973; Christie et al., 1975). A similar rise in abomasal pH has been observed with Ostertagia circumcincta and ‘Trichostrongylus axei in sheep (Anderson, Blake and Titchen, 1976; Ross, Purcell and Todd, 1969) and Ostertagia ostertagi in cattle (Jennings, Armour, Lawson and Roberts, 1966), although it occurred at a later stage of infection. Jennings et al. (1966) observed an increase in the number of viable bacteria in the abomasum of calves infected with 0. ostertagi; however, their analyses were performed with nutrient and MacConkey agar incubated aerobically. These media would only allow recovery of a small proportion of the abomasal bacteria, i.e. those growing under aerobic conditions, but not the obligate anaerobes which are the predominant group in the abomasum of infected sheep. Most bacteria within the rumen are obligate anaerobes and many can be recovered only if strict anaerobic techniques are used, since exposure to oxygen can kill them instantly (Hungate, 1975). In view of these observations, the increase in the number of abomasal bacteria after infection with H. contortus would seem to be due to the greater survival and multiplication of rumen bacteria, rather than any indigenous abomasal flora, since there was a shift towards a greater proportion of oxygen-sensitive bacteria after infection. The anaerobic methods and media used in the present study will recover obligate anaerobes and are therefore better suited to the bacterial types being recovered from the abomasum and this will explain the higher counts obtained in the present study compared to those obtained by Jennings et al. (1966). In addition, some bacteria would be unable to grow on the nutrient and MacConkey agar used by Jennings et al. ( 1966) due to the absence of unidentified growth factors present in the rumen and abomasal fluids; many rumen bacteria are known to require such factors (Hobson, 1969). The shift towards a higher proportion of oxygen-sensitive bacteria suggests that qualitative as well as quantitative changes in the microbial flora of the abomasum occur after administration of large doses of H. contortus larvae. Large dosesof H. contortus alter the abomasal environment, both directly and indirectly, so that it becomes more like that of the rumen, with a near-neutral pH, a very low PO, and supports a greater number of viable bacteria, a higher proportion of which are oxygen-sensitive. It is not clear what effect these microbiological changes in the abomasum have on the host, although the onset of diarrhoea in calves infected with 0. ostertagi has been linked with an increase in the number of viable bacteria in the abomasum CJennings et al., 1966); however, diarrhoea is not a common feature of infection with H. contortus (Fitzsimmons, 1969). More work needs to be done on the identification of the abomasal bacteria before and after infection with H. contortus and other
ABOMASAL
abomasal assessed.
nematodes
CHANGES
IN
H.
of sheep and cattle,
CO,VTORTL:S
INFECTION
307
before their effect on the host can be
SUMMARY
The pH, PO, and the number of viable bacteria per ml of abomasal fluid were recorded before and after administering large doses of infective larvae of H. contortus to sheep. Highly significant increases were observed in the pH, in numbers of viable bacteria and in the proportion of oxygen-sensitive bacteria in the abomasal fluid during the first 10 to 14 days of infection. At the same time a decrease in the abomasal fluid PO, was recorded. Subsequently a slow recovery of pH, PO, and bacterial numbers to pre-infection values ensued. The microbiological and physiological changes occurring in the abomasum of sheep after infection with H. contortusare discussed with reference to these results. ACKNOWLEDGMENTS
We thank Dr D. W. Pickard who carried out the cannulation of the sheep, Dr J. Wallace for help with the microbiological techniques and Mrs S. Eaton, Mr D. Pedley, Miss S. Squires, Mr J. Chappelow and the late Mr E. Hemingway for valuable technical assistance. We also thank Mr M. Robinson for assistance in the modelling of data. This work was supported by grant AG 24/163 from the Agricultural and Food Research Council.
REFERENCES
Anderson, N., Blake, R. and Titchen, D. A. (1976). Effects of a series of infections of Ostertagia circumcincta on gastric secretion of sheep. Parasitology, 72, l-1 2. Christie, M. G. (1970). The fate of very large doses of Haemonchus contortus and their effect on conditions in the ovine abomasum. Journal of Comparative Pathology, 80, 89-100. Christie, M. G., Angus, K. W. and Hotson, I. K. (1975). Manifestations of resistance to Haemonchzcs contortus in sheep: worm populations and abomasal changes in sheep superinfected with 1 000 000 larvae of H. contortus. International Journal for Parasitology, 5, 193-l 98. Coop, R. L. (1971). The effect of large doses of Haemonchus contortus on the level of plasma pepsinogen and the concentration of electrolytes in the abomasal fluid of sheep. Journal of Comparative Pathology, 81, 2 13-2 19. Fitzsimmons, W. M. (1969). Pathogenesis of the trichostrongyles. Helminthological Abstracts, 38, 139-190. Hirschowitz, B. I. (1957). Pepsinogen: its origins, secretion and excretion. Physiological Reviews, 37, 475-5 11. Hobson, P. N. (1969). Rumen bacteria. In Methods in Microbiology, Volume 3B, J. R.
Norris and D. W. Ribbons, Eds, Academic Press,London, pp. 133-149. Horak, I. G.., Clark, R. and Botha, J. C. (1965). The pathological physiology of helminth infestations. 1. Ostertagia circumcincta. Onderstepoort Journal of Veterinary Research, 32, 147-152. Hungate, R. E. (1969). A roll tube method for cultivation of strict anaerobes. In: Methods in Microbiology, Volume 3B, J. R. Norris and D. W. Ribbons, Eds, Academic Press, London, pp. 117-l 32.
308
C.
D.
NICHOLLS
et
cd.
Hungate, R. E. (1975). The rumen microbial ecosystem. Annual Reuiete, of Ecology and Systematics, 6, 39-66. Jennings, F. W., Armour, J., Lawson, D. D. and Roberts, R. (1966). Experimental Ostertagia ostertagi infections in calves: studies with abomasal cannulas. American Journal of Veterinary Research, 27, 124991257. Kurihara, Y., Eadie, J. M., Hobson, P. N. and Mann, S. 0. (1968). Relationship between bacteria and ciliate protozoa in the sheep rumen. Journal of General Microbiology, 51, 267-288. Mapes, C. J. and Coop, R. L. (1973). The effect ofvarious periods ofinfection with the nematode Haemonchus contortus on the development of the nematode Nematodirus battus. Parasitology, 66, 8594. Nicholls, C. D., Lee, D. L. and Sharpe, M. J. ( 1985). S canning electron microscopy of biopsy specimens removed by a colonoscope from the abomasum of sheep infected with Haemonchus contortus. Parasito&y, 90, 357-363. Rose, J. H. (197 1). The isolation and maintenance in viuo of some gastro-intestinal nematodes and lungworms of farm animals. In: Isolation and Maintenance of Parasites in uiuo. Symposia of the British Society for Parasitology, Volume 9, A. E. R. Taylor and R. Muller, Eds, Blackwell Scientific Publications, Oxford, pp 109-121. Ross, G. J. S. (1978). Curve fitting using the Rothamsted Maximum Likelihood Program. In: Numerical Software: Needs and Auailability. D. A. H. Jacobs, Ed. Academic Press, London, pp. 2933323. Ross, J. G., Purcell, D. A. and Todd, T. R. ( 1969). E x p erimental infections of lambs with Trichostrongylus axei; investigations using abomasal cannulae. Research in Veterinary Science, 10, 133-141. Veglia, F. (1915). The anatomy and life history of the Haemonchus contortus. 3rd and 4th Reports of the Director of Veterinar_y Research, Onderstepoort, Union of South Africa, 347-500. [Received for publication,
Januar_
17th, 19861
PA~.H.
1987
VOL.
97
PHYSIOLOGICAL THE ABOMASUM DOSES
C. D. Department
of Pure and Applied
AND MICROBIOLOGICAL CHANGES OF SHEEP INFECTED WITH LARGE OF HAEMONCHUS COflTORl-US
P. R.
NICHOLLS, zoology,
and D. L.
HAYES*
of Microbiology
and *Department
UniversiQ
IN
LEE
of Leeds,
Leeds LSZ 9j’T,
U.K.
INTRODUCTION
Large doses of third-stage larvae of Haemonchus contortus administered to sheep are known to result in an increase in the abomasal pH within 2 or 3 days of infection (Christie, 1970; Coop, 1971; Mapes and Coop, 1973; Christie, Angus and Hotson, 1975). This increase in pH probably causes changes in the physiology of the gastrointestinal tract of sheep similar to those observed during infections with Ostertagia circumcincta, which also causes an increase in abomasal pH. For example, a decrease in the pepsin concentration of the abomasal fluid was observed after infection with 0. circumcincta (Horak, Clark and Botha, 1965) because conversion of pepsinogen to pepsin occurs very slowly above pH 4 (Hirschowitz, 1957). The rumen contains large numbers (about 10” per ml) of viable bacteria, most of which are obligate anaerobes (Hungate, 1975). The abomasum receives fluid and food materials from the rumen; however, little is known about the microbiology of the abomasum, which normally has a low pH (pH 2 to 3). No investigation has been reported on the effect of the induced pH increase, after administering large doses of H. contortus, on bacterial numbers and types. This study was undertaken to confirm the effect of large dosesof H. contortus on abomasal pH and to study any changes in the PO, of abomasal fluid and in the numbers of viable bacteria present and their oxygen sensitivity during the course of infection in sheep. MATERIALS
AND
METHODS
Culture of H. contortus larvae
A pure culture of H. contortus was obtained from the Moredun Institute, Edinburgh, and usedto infect sheepat Leeds. Faecescontaining eggsof H. contortus were collected from infected sheep and cultured in plastic trays at 20°C for 10 to 14days. The third-stage larvae were collected, contaminants removed as described by Rose (1971) and stored for at least 2 weeksat 10°C before use.
0021-9975/87/030299+
10 $03.00/O
0
1987
Academic
Press
Inc.
(London)
Limited
300 Experimental
C.
D.
NICHOLLS
Animals
The 12 sheep used in the experiments parasitic nematodes at the University of with a cannula (18 mm internal diameter, in separate pens. When experimentation 20 months. Experimental
et al.
were Suffolk cross Mules reared free from Leeds Field Station. The sheep were fitted 60 mm long) in the abomasum and housed started, the sheep ranged in age from 12 to
Design
The 12 sheep were divided into matched pairs; one member of each pair was given orally 10” infective third-stage larvae of H. contortus and given access to 1000 g of pelleted food per day plus water ad libitum. The pelleted food was a mineralized dried grass and barley mix containing 7.5 per cent ash, 30 per cent oil, 14 per cent protein and 14 per cent fibre (Lincolnshire Farm Feeds, Grimsby). The other member of each pair was a pair-fed control which was given the same amount of food as that eaten the previous day by the infected partner. Physiological
Anal_vsis qf Abomasal Fluid
Samples of abomasal fluid were obtained via the abomasal cannula by means of a monovette (Sarstedt Ltd., Leicester) before, and at regular intervals during, the infection. The samples were taken at approximately the same time of day on each occasion, and the monovette was completely filled with fluid to reduce exposure of the sample to air. After centrifugation at 1500 g for 5 min to remove food particles and other debris, the pH and the PO, of the supernatant were measured, twice for each sample, with a BMS 3 Mk 2 Blood Micro System (Radiometer, Copenhagen). Readings were obtained within 45 min of sampling and were similar to those obtained from samples analysed, for comparison, within 5 min of sampling. Preparation
and Dispensation
of Media,for
Culture of Anaerobic Bacteria
Diluting fluid was made up as described by Kurihara, Eadie, Hobson and Mann (19683, but modified slightly by altering the concentration of resazurin from 0.0001 g per 100 ml to 0.001 g per 100 m! and omitting the glucose (personal communication, J. Wallace, Rowett Research Institute, Aberdeen). The required ingredients, except the cysteine HCl, were mixed and heated until dissolved; the cysteine HCl was then added to the partially cooled solution. The medium was deoxygenated by bubbling O,-free CO, through the mixture; the gas had been passed over heated copper turnings in a Phoenix furnace (Phoenix Furnaces Ltd., Sheffield), heated to about 350°C to remove any traces of oxygen. Following deoxygenation, a luer-type syringe with a 15 cm long 14-gauge needle attached, was flushed with O,-free CO, and then used to dispense the medium in 9 ml amounts into Hungate tubes (Bellco Glass Inc., New Jersey, U.S.A), preflushed with O,-free CO,. The Hungate tubes possessed a butyl rubber stopper to provide an air-tight seal and a screw-on cap to hold the rubber stopper in place. The medium was autoclaved at 121” C for 15 min and stored at 4” C until required. The growth medium was a non-specific medium on which most, if not all, anaerobic rumen and abomasal bacteria can grow. It was modified from the medium 1 described by Kurihara et al. (1968) by increasing the resazurin as stated above (personal communication, J. Wallace, Rowett Research Institute, Aberdeen). The growth medium was prepared and dispensed as described for the diluting fluid. except that 4.5 ml amounts were dispensed. Chemicals were obtained from B.D.H. Chemicals Limited, Poole; Sigma Chemical Company Limited, Poole or Difco Laboratories, West Molesey. The rumen fluid used in these media was obtained from a healthy sheep, the fluid was strained to remove
ABOMASAL
CHANGES
IN
H. CO.VTORTUS
301
INFECTION
large particles, centrifuged at approximately 20 000 g for 40 min and the supernatant fluid was sterilized at 121°C for 15 min before storage at 4°C. Microbiologzcal
Analysis of Abomasal Fluid
Abomasal fluid samples were obtained by completely filling sterile Bijou bottles via the abomasal cannula. Samples were processed within 45 min of being taken. Appropriate decimal dilutions were prepared from 1.0 ml of the abomasal fluid; a modification of the Hungate technique was employed (Hungate, 1969). This allowed samples to be transferred anaerobically and aseptically between successive tubes by means of sterile 1.0 ml pipettes which had been preflushed with O,-free CO,. During the transfer the tubes were continuously flushed with O,-free CO,. The anaerobic roll tube method (Hungate, 1969) was used to analyse the sample; 0.5 ml of the required dilution was added to 4.5 ml of molten growth medium under anaerobic and aseptic conditions as described above. After mixing bv inversion, the agar was solidified by placing the tubes horizontally on revolving rollers in a perspex tank of cold water. The roll tubes so prepared were incubated at 39°C for 3 days, after which counts were made on the appropriate dilutions. Selected colonies were then subcultured anaerobically onto slopes of the growth medium and, after incubation at 39°C for 3 days, purity checks of the cultures were performed by further subculturing of selected colonies on to plates of the growth medium; this resulted in brief exposure to aerobic conditions prior to subsequent incubation at 39°C for 2 days in an anaerobic jar (Don Whitley Scientific Ltd., Shipley) filled with a mixture of95 per cent CO, and 5 per cent H,.
ofResults
Statistical Anal_yris
For statistical analysis the results obtained for abomasal pH, PO, and number of viable bacteria were divided into four groups, viz. uninfected, pair-fed sheep, before and after- jnfection of their partners, and infected animals, before and after infection (see Table 1). Significant differences between groups were calculated using one-way analysis of variance with linear combination on samples taken between days 3 to 20 after infection, as this was the period when the greatest changes in abomasal pH, PO, and bacterial numbers were observed. Curves for Figs. 1, 2 and 3 were fitted by. the Maximum Likelihood Program (Ross, 1978) on log-transformed X-axes. Regressions for pair-fed control values were fitted by the same program.
TABLE MEAN ABOMASAL
VALUES FLCID
FOR
ABOMASAL FROM
SIX
FLUID PAIR-FED
PH,
PO2
AND
AND
SIX
INFECTED
I
LOG.,”
OFTHE SHEEP
NUMBER BEFORE
OF VlABLE 3 T O 20
AND
BACTERIA
PERML
OF
DAYS AF’TER INFECTIOI,’
WITH 106 THIRD STAGE LARVAE OF N
stageof irzfectm
PH PO, (Pa; Log.,” bacterial count
Before infection of
partners
2.6 * 0.09 (26) 4252 f 187.5 (24) 5.7i~O.25
(16)
jheep
Infected rheep
___
3 to 20 days after infection of
partners
2.6 *O-04 (35) 4279f 160.3 139) 6.0 f 0.28 (14)
3 Before infectzon 2-710.09 5012*312~5* 6~110~15
(26) (241 (23)
to
ajier
20 dazs infection
6.0iO.lt (40, 427f 120.5.f (40) 8.5 5 0.08t
125)
Values given ate mranf S.E. with numbers of samples in parentheses. Group means that are significantly different from the prrvious group meam are indicated by: *Pi 0.0 I, tP< 0.00 1. Days 3 to 20 after infectIon are whrn the major changes were vbserved in ahomasal pH, PO, and bacterial numbers.
302
C. 7
I
D.
NICHOLLS
ef cd.
I
I
I
I
I
I
I
I 0
I IO
20
30
1 40
I 50
I 60
6-
5-
1 a 4-
3-
2f -10
Time
Fig. 1. Mean pair-fed
abomasal sheep.
pH. Values
0 =sheep are given
infected as mean
with lo6 f S.E.M.
70
(days)
infective
larvae
of H.
contort,
0 =uninfected
667
267C
IO
20 Time
Fig. 2. Mean abomasal pair-fed sheep.
fluid PO,. 0 =sheep infected Values are given as mean+
with S.E.M.
(days)
lob infective
larvae
of H. contork,
0 = uninfected,
ABOMASAL
CHANGES
IN
H.
CONTORTUS
INFECTION
303
RESULTS
Abomasal Fluid pH The pH of the abomasal fluid of pair-fed and pre-infected sheep varied between pH 2 and 3 (Fig. I). A rapid increase in pH to 6 to 6.5 was observed within the first 4 days of infection. This elevated abomasal pH was maintained for about two weeks before gradually decreasing to pre-infection values by about 10 weeks post-infection. The increase in abomasal fluid pH, observed between days 3 and 20 inclusive, was significant (PC 0.001; Table 1). The 6 uninfected, pair-fed sheep showed no significant change in abomasal pH during experimentation (Fig. 1; Table 1). Abomasal Fluid PO, Some variation was observed in the PO, of the abomasal fluid of the pair-fed sheep and in sheep before infection (Fig. 2) although it generally remained above 3330 Pa (1 mmHg= 133.3 Pa). About 3 days after infection, a rapid decrease in the PO,, down to less than 600 Pa, was observed in all 6 infected sheep. It remained low for about 2 weeks before it gradually returned to pre-infection values by about 7 weeks post-infection. The decrease in abomasal fluid PO,, observed between days 3 and 20 inclusive, was significant (P
304
C.
D.
et
NICHOLLS
al.
a
Time Fig. 3. Mean infective
number larvae
(log.,,,) of viable bacteria per of H. contortus, 0 = uninfected,
(days) ml of abomasal pair-fed sheep.
fluid. Values
0 =sheep are given
infected as mean
with 10b f S.E.M.
conditions, whereas with isolates obtained during days 5 to 6 of infection, greater losses in viability of cultures were observed. This was presumably due to their exposure to oxygen, either in the air during plating out of these isolates for purity checking, or due to the presence of a small amount of air in the anaerobic jar. These results show that there was an increase in the proportion of oxygen-sensitive bacteria after infection. Similar purity checks carried out on isolates from rumen fluid showed that there was again a high proportion of oxygen-sensitive bacteria present as indicated by the loss of many of the selected isolates (Table 2). Chi-squared analysis was used to determine significant differences between the results; expected values were calculated from the pooled results. The analysis indicated that samples from the uninfected, pair-fed sheep and from sheep before infection, did not show any differences in the proportions of oxygen-sensitive bacteria (P> O-1). However, a highly significant increase in the proportion of oxygen-sensitive bacteria was found in the infected animals, during the first week of infection, compared with that of the pair-fed and pre-infected animals (P < O-00 1) .
ABOMASAL
CHANGES
IN
H.
CONTORTUS 2
TABLE
PROPORTION (WITH
OF OXYGEN-SENSITIVE
lo6
INFECTIVE
‘MEASURED
BY
LARVAE
RECOVERY
OF OF
fluid sheep
Before
from
After
Rumen uninfected *i.e.
IN THE ABOMASAL
CON70R7C:~)
MEDIUM
SHEEP
SELECTED AND
AND
fluid from uninfected
fluid
they
INCUBATION
grow
FLUID
OF 4
RC‘MEN
FLUID
AFTER IN
AN
infection infection
UNINFECTED OF
SUBCULTURE
ANAEROBIC
Total number isolates ongina& selected jrom roll tubes
4
AND
UNINFECTED ONTO
INFECTED SHEEP,
PLATES
of
72
14 (19.4)
110
77 (70) 5 (129)
After infection of partners
40
4 (10)
40
39 (97.5)
jar,
plpbably
due
to exposure
AS THE
jVumber of isolates subsequently lost after subculture (and per cent) *
41
in an anaerobic
OF
JAR
Before infection of partners
from sheep do not
IN
ISOLATES
Stage of infection
Sample
Abomasal pair-fed sheep
H.
ORIGINALLY
GROWTH
Abomasal infected
BACTERIA
305
INFECTION
to air
(see text).
DISCUSSION
All infected sheep exhibited similar physiological and microbiological changes w-ithin the abomasum after infection, namely increases in the abomasal pH, in the number of viable bacteria present and in the proportion of oxygen-sensitive bacteria present in the abomasal fluid, and a decrease in the abomasal PO,. These changes all occurred at about the same time, suggesting that they are correlated. It is probable that the decrease in the PO, of the a.bomasal fluid is primarily due to bacterial activity. There is no obvious explanation for the observed significant difference in the PO, of the abomasal fluid between the infected sheep before infection and the pair-fed sheep; it may be due to variations between individual sheep (some sheep have consistently high or low values compared with other sheep). This could also explain the variation in bacterial counts observed in the pair-fed sheep (see Fig. 3). It is known that on reaching the abomasum, the larvae of H. contortus migrate into the gastric glands, where they moult (Veglia, 1915) and, on emergence from the glands, they continue to cause physical damage to the abomasal mucosa (Nicholls, Lee and Sharpe, 1985). They cause a reduction in the production of HCl in the abomasum, which results in an increase in the abomasal pH (Christie et al., 1975). It is suggested that this increase in abomasal pH provides a more favourable environment for bacteria normally resident in the rumen, where the pH is normally about 6.5. Initially, the facultative anaerobes from the rumen will multiply in the abomasal fluid as it becomes lessacid and the obligate anaerobes will be inhibited or killed by the oxygen present in that fluid. However, with the growth of the facultative anaerobes, oxygen will be utilized so lowering the abomasal PO, which will consequently allow the oxygen-sensitive obligate anaerobes from the rumen to survive, multiply and probably become the predominant group. The PO, of the
306
C.
D.
NICHOLLS
et
d.
abomasal fluid will be kept low by the facultative anaerobes until the gastric glands recover and secrete more HCl so lowering the abomasal pH. This will create a less favourable environment for the bacteria and growth will be progressively inhibited as the pH is lowered, thus allowing the PO, of the abomasal fluid to return to pre-infection values. The sharp rise in the abomasal pH observed 3 days after infection confirms similar observations by previous authors (Christie, 1970; Coop, 197 1; Mapes and Coop, 1973; Christie et al., 1975). A similar rise in abomasal pH has been observed with Ostertagia circumcincta and ‘Trichostrongylus axei in sheep (Anderson, Blake and Titchen, 1976; Ross, Purcell and Todd, 1969) and Ostertagia ostertagi in cattle (Jennings, Armour, Lawson and Roberts, 1966), although it occurred at a later stage of infection. Jennings et al. (1966) observed an increase in the number of viable bacteria in the abomasum of calves infected with 0. ostertagi; however, their analyses were performed with nutrient and MacConkey agar incubated aerobically. These media would only allow recovery of a small proportion of the abomasal bacteria, i.e. those growing under aerobic conditions, but not the obligate anaerobes which are the predominant group in the abomasum of infected sheep. Most bacteria within the rumen are obligate anaerobes and many can be recovered only if strict anaerobic techniques are used, since exposure to oxygen can kill them instantly (Hungate, 1975). In view of these observations, the increase in the number of abomasal bacteria after infection with H. contortus would seem to be due to the greater survival and multiplication of rumen bacteria, rather than any indigenous abomasal flora, since there was a shift towards a greater proportion of oxygen-sensitive bacteria after infection. The anaerobic methods and media used in the present study will recover obligate anaerobes and are therefore better suited to the bacterial types being recovered from the abomasum and this will explain the higher counts obtained in the present study compared to those obtained by Jennings et al. (1966). In addition, some bacteria would be unable to grow on the nutrient and MacConkey agar used by Jennings et al. ( 1966) due to the absence of unidentified growth factors present in the rumen and abomasal fluids; many rumen bacteria are known to require such factors (Hobson, 1969). The shift towards a higher proportion of oxygen-sensitive bacteria suggests that qualitative as well as quantitative changes in the microbial flora of the abomasum occur after administration of large doses of H. contortus larvae. Large dosesof H. contortus alter the abomasal environment, both directly and indirectly, so that it becomes more like that of the rumen, with a near-neutral pH, a very low PO, and supports a greater number of viable bacteria, a higher proportion of which are oxygen-sensitive. It is not clear what effect these microbiological changes in the abomasum have on the host, although the onset of diarrhoea in calves infected with 0. ostertagi has been linked with an increase in the number of viable bacteria in the abomasum CJennings et al., 1966); however, diarrhoea is not a common feature of infection with H. contortus (Fitzsimmons, 1969). More work needs to be done on the identification of the abomasal bacteria before and after infection with H. contortus and other
ABOMASAL
abomasal assessed.
nematodes
CHANGES
IN
H.
of sheep and cattle,
CO,VTORTL:S
INFECTION
307
before their effect on the host can be
SUMMARY
The pH, PO, and the number of viable bacteria per ml of abomasal fluid were recorded before and after administering large doses of infective larvae of H. contortus to sheep. Highly significant increases were observed in the pH, in numbers of viable bacteria and in the proportion of oxygen-sensitive bacteria in the abomasal fluid during the first 10 to 14 days of infection. At the same time a decrease in the abomasal fluid PO, was recorded. Subsequently a slow recovery of pH, PO, and bacterial numbers to pre-infection values ensued. The microbiological and physiological changes occurring in the abomasum of sheep after infection with H. contortusare discussed with reference to these results. ACKNOWLEDGMENTS
We thank Dr D. W. Pickard who carried out the cannulation of the sheep, Dr J. Wallace for help with the microbiological techniques and Mrs S. Eaton, Mr D. Pedley, Miss S. Squires, Mr J. Chappelow and the late Mr E. Hemingway for valuable technical assistance. We also thank Mr M. Robinson for assistance in the modelling of data. This work was supported by grant AG 24/163 from the Agricultural and Food Research Council.
REFERENCES
Anderson, N., Blake, R. and Titchen, D. A. (1976). Effects of a series of infections of Ostertagia circumcincta on gastric secretion of sheep. Parasitology, 72, l-1 2. Christie, M. G. (1970). The fate of very large doses of Haemonchus contortus and their effect on conditions in the ovine abomasum. Journal of Comparative Pathology, 80, 89-100. Christie, M. G., Angus, K. W. and Hotson, I. K. (1975). Manifestations of resistance to Haemonchzcs contortus in sheep: worm populations and abomasal changes in sheep superinfected with 1 000 000 larvae of H. contortus. International Journal for Parasitology, 5, 193-l 98. Coop, R. L. (1971). The effect of large doses of Haemonchus contortus on the level of plasma pepsinogen and the concentration of electrolytes in the abomasal fluid of sheep. Journal of Comparative Pathology, 81, 2 13-2 19. Fitzsimmons, W. M. (1969). Pathogenesis of the trichostrongyles. Helminthological Abstracts, 38, 139-190. Hirschowitz, B. I. (1957). Pepsinogen: its origins, secretion and excretion. Physiological Reviews, 37, 475-5 11. Hobson, P. N. (1969). Rumen bacteria. In Methods in Microbiology, Volume 3B, J. R.
Norris and D. W. Ribbons, Eds, Academic Press,London, pp. 133-149. Horak, I. G.., Clark, R. and Botha, J. C. (1965). The pathological physiology of helminth infestations. 1. Ostertagia circumcincta. Onderstepoort Journal of Veterinary Research, 32, 147-152. Hungate, R. E. (1969). A roll tube method for cultivation of strict anaerobes. In: Methods in Microbiology, Volume 3B, J. R. Norris and D. W. Ribbons, Eds, Academic Press, London, pp. 117-l 32.
308
C.
D.
NICHOLLS
et
cd.
Hungate, R. E. (1975). The rumen microbial ecosystem. Annual Reuiete, of Ecology and Systematics, 6, 39-66. Jennings, F. W., Armour, J., Lawson, D. D. and Roberts, R. (1966). Experimental Ostertagia ostertagi infections in calves: studies with abomasal cannulas. American Journal of Veterinary Research, 27, 124991257. Kurihara, Y., Eadie, J. M., Hobson, P. N. and Mann, S. 0. (1968). Relationship between bacteria and ciliate protozoa in the sheep rumen. Journal of General Microbiology, 51, 267-288. Mapes, C. J. and Coop, R. L. (1973). The effect ofvarious periods ofinfection with the nematode Haemonchus contortus on the development of the nematode Nematodirus battus. Parasitology, 66, 8594. Nicholls, C. D., Lee, D. L. and Sharpe, M. J. ( 1985). S canning electron microscopy of biopsy specimens removed by a colonoscope from the abomasum of sheep infected with Haemonchus contortus. Parasito&y, 90, 357-363. Rose, J. H. (197 1). The isolation and maintenance in viuo of some gastro-intestinal nematodes and lungworms of farm animals. In: Isolation and Maintenance of Parasites in uiuo. Symposia of the British Society for Parasitology, Volume 9, A. E. R. Taylor and R. Muller, Eds, Blackwell Scientific Publications, Oxford, pp 109-121. Ross, G. J. S. (1978). Curve fitting using the Rothamsted Maximum Likelihood Program. In: Numerical Software: Needs and Auailability. D. A. H. Jacobs, Ed. Academic Press, London, pp. 2933323. Ross, J. G., Purcell, D. A. and Todd, T. R. ( 1969). E x p erimental infections of lambs with Trichostrongylus axei; investigations using abomasal cannulae. Research in Veterinary Science, 10, 133-141. Veglia, F. (1915). The anatomy and life history of the Haemonchus contortus. 3rd and 4th Reports of the Director of Veterinar_y Research, Onderstepoort, Union of South Africa, 347-500. [Received for publication,
Januar_
17th, 19861