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Ecological observations and laboratory experiments on the free-living stages of Cooperia oncophora
J.
COMPo
PATH.
1963. VOL. 73.
ECOLOGICAL OBSERVATIONS AND LABORATORY EXPERIMENTS ON THE FREE-LIVING STAGES OF COOPERIA ONCOPHORA By
J. H.
ROSE
Ministry qf Agriculture, Central Veterinary Laboratory, W rybridge INTRODUCTION
A programme of research in progress at the Central Veterinary Laboratory is concerned with parasitic gastro-enteritis of cattle. One aspect of this work deals with the biology of the free-living stages of the different nematode species associated with the disease. The present paper is an account of both ecological observations and laboratory experiments with one of these species-Cooperia oncophora. METHODS
Ecological Observations
Observations were made on the rate of development of the free-living stages in faecal pats at different times of the year, and on the migratory activity and longevity of larvae in faeces, on herbage and in soil. The relationship between the form of the faeces and development of the freeliving stages was also investigated. The methods used have been described in detail in previous papers dealing with similar observations on other nematode species (Rose, 1955; Rose 1961; Rose 1962) and so will be dealt with only briefly in the present paper. Development if the free-living stages. Faeces, freshly collected from a stalled calf, were deposited in the form of a pat on to a plot of short perennial rye-grass growing in a galvanised iron box and exposed to the local climatic conditions. A replicate was set up each month throughout the year from July 1959 to June 1960. Samples of faeces were examined at frequent intervals using methods described by Michel and Rose (1954) and the development of the free-living stages was observed. Migratory activities and longevity
if larvae
in faeces, on herbage and in soil.
Two series of experiments were carried out. The first was set up in June 1959 during a dry summer, and the second in July 1960 during a wetter summer. Faeces collected from artificially infected stalled calves were thoroughly mixed to ensure, as far as possible, an even distribution of C. oncophora eggs. One pound of faeces was deposited in the form of a pat, approximately 6" in diameter and I" in depth, on to the centre of an 18" square plot of short perennial rye-grass. Replicates were set up on a number of plots. The faeces used in the first series of experiments contained approximately 107 e.p.g., while those used in the second series contained 240 e.p.g. Samples of faeces from different vertical and lateral levels of the pat, all of the herbage cut at different vertical and lateral levels (Rose 196 I) and the uppermost one inch layer of soil from a single plot were examined for larvae. The number of larvae in the residue of faeces after sampling was also determined, separation being effected with a Baermann type apparatus. A fresh plot was examined in this way each week for the first month, then subsequently at intervals of 4 weeks. In the
EXPERIMENTS ON
Cooperia oncophora
first series of experiments, plots were examined until larvae were no longer recovered, but in the second series the experiment was terminated in November 1961 although small numbers of larvae were still present on the plots. Relationship between the form of the faeces and viability and development of the free-living stages. A comparison was made between the viability and development of the free-living stages in faecal pats, in diarrhoeic faeces thinly spread over plots of short herbage in a series of criss cross lines and exposed to the local climatic conditions, and in similarly spread faeces kept continuously moist by placing plots under an automatic mist control apparatus (Bean, Trickett and Wells, 1957). Samples of faeces were examined daily and the viability and development of the free-living stages were observed. This experiment was repeated several times during the dry summer of 1959 and during the wetter summer of 1960.
Laboratory Experiments The effects of temperature and humidity on the rate of development of eggs and larvae in faeces, and on the longevity of infective larvae separated from faeces were studied using methods described by Rose (1955, 1961 , 1962 ). The relationship between age oflarvae and infectivity was determined by feeding infective larvae, which had been kept in water at 10 to 11°C and at 4 to 6°C for different periods of time, to worm free lambs. These were subsequently autopsied and examined for C. oncophora. RESULTS
Development of the Free-living Stages Only a small proportion of the eggs in faecal pats deposited outof-doors during the winter reached the infective stage. The majority died while still non-embryonated. The remainder became embryonated within a few days, but subsequent development was retarded and several weeks elapsed before infective larvae were recovered from the faeces (Table I). During the remainder of the year mortality was not so heavy and the rate of development was more rapid, increasing as the temperature rose (Tables 1 and 2, Fig. 3). The greatest numbers of infective larvae were recovered from the faecal pats during the summer, despite the dry weather prevailing. Adequate moisture was present in the pats to allow development to be completed. Faeces spread over the grass plots became hard and dry within three and four days during the summer of 1959, however, and none of the contained eggs reached the infective stage (Table 3). During the wetter summer of 1960, when on a number of occasions the spread faeces remained moist for a week or more, mortality was not excessive and many of the eggs developed into infective larvae, as did eggs in the artificially moistened faeces (Table 3). Comparable results were seen in the laboratory experiments. Larvae developed to the infective stage in thin layers of faeces kept moist. In faeces which dried out within 3 to 4 days, the extent of development varied according to the temperature (Table 4), but the infective stage was not reached.
J.
H. ROSE
Migration of Larvae Infective larvae were most numerous in the outer surface of the faecal pat (Table 5), even when this was hard and dry, suggesting a very active migration to this region from the remainder of the pat. The rate of migration from the faecal pat on to the herbage differed as between the two series of experiments. In the first series, started in June 1959, development from egg to infective larva took between 6 and 14 days yet few larvae were recovered from the herbage until August (Fig. I). During this time many infective larvae were TABLE
I
RATE OF DEVELOPMENT OF THE FREE·LIVING STAGES OF C. ONCOPHORA IN FAECES DEPOSITED OUT·OF·DOORS AT DIFFERENT TIMES OF THE YEAR
Infected faeces deposited out of doors in:-
Time for development to srd stage larvae (weeks) Minimum
Maximum
2 3 3 6 10 12
1959 July August September October November December January February March April May June
7
10
1960
8 7 3 2i Ii
TABLE
II
9 8 3 3 2
2
EFFECT OF TEMPERATURE ON THE RATE OF DEVELOPMENT OF THE FREE·LIVING STAGES OF C. ONCOPHORA IN MOIST FAECES
Temperature (0C)
22-23 14- 16 10-11
Time taken for development to 3rd stage larvae (days) Minimum
Maximum
3
9 21 56
4
21
EXPERIMENTS ON
Cooperia oncophora
TABLE 3 EFFECT OF THE FORM OF FAECES ON DEVELOPMENT
The numbers of infective larvae recovered three weeks after the start of each experiment.
Irifective larvae per g. offaeces
-----
Spread faeces
Date
Faecal pat
Watered
Non-watered
1959 July September
143 15 8
0 0
176 141
1960 July
211
198
184
------ ----
TABLE 4 DEVELOPMENT OF EGGS AND LARVAE IN THIN LAYERS OF 'WET' AND 'DRY' FAECES KEPT AT DIFFERENT TEMPERATURES
Extent of development
Temperature °C
-1--------
'Dry'
-
'Wet'
Infective larvae per g 'Dry'
tiffaeces
'Wet'
22-23
2nd stage larva
Infective larva
0
25 2
14-16
I
st stage larva
Infective larva
0
216
Embryonated egg Infective larva
0
78
10-11
TABLE 5 VERTICAL DISTRIBUTION OF INFECTIVE LARVAE IN FAECAL PATS
Date of sampling
Aug. 17th Sept. 7th Oct. 12th Jan. 18th
Number of irifective larvae from Top
Middle
Bottom
1352 1010 29 6 78
48 9 33 9 0
4 4 0 0
Top - 2/ION Middle - 4/ ION Bottom - 4/ION (4 typical results are tabulated)
J.
289
H. ROSE
recovered from the faeces (Fig. 2). There was no further increase in the herbage infestation until the late autumn, a peak being reached in November (Fig. r), with a corresponding decrease in the numbers oflarvae recovered from the faecal pats. In the second series, started inJuly r960, the rate of development was the same as in the previous year, but larvae were recovered from the herbage a few days after becoming infective and continued to be recovered in increasing numbers throughout August, a peak being reached in September (Fig. I). Fig.
I.
24
20
16
j
12
. o
-"
E
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o
28 28 Nov. 1961
16
23 June 1959
Weeks
Longevity of larvae on herbage. Each point represents all the larvae recovered from the herbage of a single plot.
Fig.
2.
20
16
1 ~
12
.5
I
8
J
4
0
0
2 Jon. 1959
16-
32
48
64
80
96 23 May 1961
112
----. 128
Persistence of larvae in faeces. Each point represents all of the larvae remaining in the faecal pat.
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60
80
100
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10
15
0
20
25
30
35 40
45 50
55
60
WEEKS
65
70
75
80
85
90
95
100
105
110
"'
120
125
28 NOV. 196\
IJ
Mean weekly minimum and maximum ground temperatures, total weekly rainfall and number of rain days per week from June 23rd 1959 to November 28th 1961.
I
I[l3 JUNE 1959 •
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130
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J.
H. ROSE
These differences in the rates of migration on to the herbage can be correlated with differences in the climatic conditions prevailing during the summers of 1959 and 1960 and the resultant effects on the state of the faecal pats. The summer of 1959 was dry and sunny (Fig. 3). As a result the outer surface of the faecal pats became hard and dry within a few hours of being deposited out-of-doors, and after about 3 weeks the whole pat was completely dry and remained in this condition throughout June and July. There was little rain during this period except during a thunderstorm in July, when I ·9" of rain fell, but this did little more than wet the surface of the pats for a short time. During August it rained on five consecutive days (Fig. 3) thoroughly wetting the pats. Subsequently there was an appreciable increase in the herbage infestation (Fig. 1). The fine weather returned and continued throughout September, the pats became hard and dry again and no further increase in the herbage infestation was seen Fig. 4· 20
Inner area
16
12
~
." C
~
8
:0
0
.;
.s ~
0
>
~
.
"0 G;
.0
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0
8 Outer area
.. o
27 uly 1960
Weeks
28 Nov. 1961
Vertical and lateral distribution oflarvae on herbage. The histogram represents the results of Experiment 2. The distribution of the larvae on the herbage was similar in Experiment I. Key. The herbage was first cut 2" above the soil surface to give the Upper herbage It was then cut at the soil surface to give the Lower herbage. Inner area-Extended 0" - 2" from faecal pat. Outer area-Extended 2" - 6" from faecal pat.
EXPERIMENTS ON
Cooperia oncophora
until October. This corresponded with a change in the weather (Fig. 3) and a change in the condition of the pats which became soft and moist again. The weather during the summer of 1960 was quite different. Rain fell frequently (Fig. 3), the faecal pats remained soft and moist for most of the time and there was a continuous increase in the herbage infestation throughout the summer. It was evident from these observations that the rate of migration on to the herbage depended on the condition of the faecal pats. The infective larvae were virtually unable to migrate from the hard dry pats but migrated readily from those which were soft and moist. The condition of the pats was in turn determined principally by the effects of climatic factors, and in particular by the amount and frequency of rainfall. Most larvae were recovered from the 'inner' area of the grass plots (Fig. 4), immediately around the pats, having migrated less than two inches laterally. Some did migrate farther, however, between two and six inches on to the 'outer' area (Fig. 4). The larvae also migrated vertically on the grass blades. Some were recovered from the 'upper' herbage at all times of the year, sometimes in appreciable numbers, but the majority were found on the 'lower' herbage (Fig. 4). Few larvae were recovered from the soil.
Longevity and Infectivity of the Infective Larvae Infective larvae survived on the herbage of the experimental plots for almost two years (Fig. I) and in the faeces until such time as the pats had disintegrated which was 6 to 16 months (Fig. 2, Table 6). The herbage infestation remained high for 7 to 8 months, TABLE 6 TIME TAKEN FOR
Infected faeces deposited out of doors
1959
July August September October November December
1960
January February March April May June
COMPLETE DISINTEGRATION OF FAECAL PATS
Time for complete disintegration qf faecal pat (Months)
Length qf time during which larvae were recovered from faecal pat (Months)
12 13 8 9 8 8
12 7 9 7 7
7 7 12 10 12 13
I I
6 6 II
9
II
12
J.
293
H. ROSE
many larvae surviving from the summer until the following spring, but only a few survived for between one and two years. (Fig. I). The infective larvae did not appear to be adversely affected by the climatic conditions prevailing during the course of the experiments. Many survived in the hard dry faecal pats throughout the summer of 1959 (Fig. 2). They survived well on herbage also during dry weather, suggesting a marked degree of resistance to desiccation. This was confirmed in laboratory experiments when infective larvae dried on glass slides survived for as long as 17 weeks (Fig. 5). The ability of the infective larvae to overwinter suggests a good degree of tolerance to low temperatures. This was demonstrated in the laboratory when larvae survived for more than 2 years at low temperatures, whereas at higher temperatures longevity was appreciably less (Table 7). Even under continuous freezing infective larvae Fig. 5. Relative humidity
100
,
j
.g'
80
60
~
~
~
40
0
i
l
20
o
3
6
9 Week.
12
15
18
Effect of desiccation on infective larvae.
TABLE 7 MAXIMUM LONGEVITY OF INFECTIVE LARVAE IN WATER KEPT AT DIFFERENT TEMPERATURES
Temperature (0C)
F
Maximum longevity (Months)
24-25
13
15-16
IS
10-11
>24
6-7
>24
294
EXPERIMENTS ON
Cooperia oncophora
survived for up to 24 weeks (Fig. 6). Larvae which had been kept in the laboratory at low temperatures for periods of 22 and 25 months respectively gave rise to patent infections when fed to worm-free lambs reared indoors (Table 8), demonstrating that they had retained their infectivity. FifS· 6. 100
80
•> D
j
I
60
~
8.
2c 3
.i!
40
20
o
5
10
15
Weeks
25
20
30
Effect of continuous freezing on the longevity of infective larvae suspended in water and kept at -4° to -6°C. TABLE 8 EFFECT OF AGE OF LARVAE ON INFECTIVITY
Lamb
Date infected
-
Approx. age of larvae (months)
Approx. no. of larvae administered
Worms recovered
I
July 1962
22
600
94
2
July 1962
22
600
301
3
Oct. 1962
25
600
22
4
Oct. 1962
25
I
600
23 \
DISCUSSION
The results of the present observations have a number of implications relevant to translation, the process whereby eggs in faeces become infective larvae on herbage available to the grazing animal, in areas where climatic conditions are comparable with those studied.
J.
H. ROSE
295
During the winter, conditions will not be conducive to translation. The majority of the eggs in faeces deposited on pastures at this time of the year will fail to develop into infective larvae. During the remainder of the year mortality will be less and appreciable numbers of eggs will become infective larvae, but the extent of translation is likely to vary with the weather. Wet weather will be conducive to translation as the faecal pats will remain soft and moist so that the infective larvae will be able to migrate readily on to the herbage. In dry weather, however, translation will be limited by the inability of the larvae to migrate from the dry faecal pats. As the larvae are able to survive for long periods in dry faeces, the pats can act as reservoirs of infection until a change in the weather makes migration possible, so that eggs in faecal pats deposited on pastures during a dry summer can complete their development into infective larvae which may then give rise to heavy herbage infestations in the autumn and early winter when migration from the pats can take place. The dissemination of faeces over pastures may either limit or facilitate translation, the precise effect being determined principally by the weather. In ~eneral when freshly deposited faeces are spread over the pastures in dry weather mortality of the pre-infective stages will be heavy so that translation will be limited. In wet weather, except in the winter, mortality will not be heavy and the development of eggs in faeces into infective larvae on herbage will be favoured. The dissemination of faecal pats which have been on the pastures for some time and in which the larvae are at the infective stage, will generally aid translation as the infective larvae are able to survive well under a wide range of climatic conditions, providing the condition of the faeces permits adequate dissemination. Although translation is only one of the factors involved in the transmission of infection, nevertheless the present observations do contribute to an understanding of certain aspects of transmission . . Outbreaks of infection are frequently encountered during warm wet summers. These would appear to be directly related to the climatic conditions being conducive to translation. From time to time heavy outbreaks occur also in the autumn and winter following a dry summer. The source of these outbreaks has been a matter of conjecture in the past. It would seem from the present observations that due to the slow rate of development and heavy mortality of the preinfective stages, eggs in faeces deposited on the pastures during the autumn and winter cannot be the source of infection. It was also noted that during a dry summer there was no appreciable build up of the herbage infestation. It is unlikely therefore, that larvae can survive on the herbage in numbers sufficient to give rise to such outbreaks. It was observed, however, that faecal pats can serve as reservoirs of infection throughout the summer, larvae migrating on to the herbage when the dry weather ends. In this way it is possible for the herbage infestation to be built up in the autumn or early
EXPERIMENTS ON
Cooperia oncophora
winter sufficient to give rise to outbreaks of infection in susceptible stock. Although the heaviest outbreaks of infection would be expected to coincide with periods of high translation, owing to the ability of larvae to survive for up to 2 years on pasture herbage, it should be possible for infection to be picked up at any time of the year when susceptible animals are turned out to graze. American workers (Baker, 1939; Goldberg and Rubin, 1956; Drudge, Leland, Wyant and Rust, 1958; Goldberg and Lucker, 1959) have, in fact, demonstrated that infection can be picked up from pastures rested for from 7 to 9 months. The present observations would suggest that infection can be picked up from contaminated pastures rested for longer periods. Although heavy outbreaks of clinical disease are unlikely to result directly from grazing such rested pastures, this could be a means of perpetuating infection in a herd and providing the source of infection for subsequent clinical disease. The ability oflarvae to survive well on pastures for 7 or 8 months is of particular practical significance with regard to control by grazing management. It would seem that resting contaminated pastures for a few weeks, as is often advised, will serve no useful purpose in the control of C. oncophora infection. CONCLUSIONS
Outdoor observations were made on the rate of development of the free-living stages of C. oncophora at different times of the year and on the migratory activity and longevity of the larvae in faeces, on herbage and in soil. The relationship between the form of the faeces and development was also investigated. The effects of temperature and humidity on the survival and development of eggs and larvae in faeces, and on the longevity of infective larvae separated from the faeces, were studied in the laboratory. The results of these observations are discussed in relation to translation and the transmission of infection in the field. REFERENCES
Baker, D. W. (1939). Cornell. Vet., 29, 45. Bean, G., Trickett, E. S., and Wells, D. A. (1957). ]. agric. engng. Res., 2,44· Drudge, J. R., Leland, S. E., Wyant, Z. N., and Rust, J. W. (195 8 ). ]. Parasit, 44, 434. Goldberg, A., and Rubin, R. (1956). Proc. helm. Soc. Wash., 23, 65. Goldberg, A., and Lucker, J. T. (1959). Ibid., 26,37· Michel, J. F., and Rose, J. H. (1954).], compo Path., 64, 195· Rose, J. R. (1955). Ibid. 65, 370; (1961). Parasitology, 51, 295; (1962 ). ]. compo Path .• 72, I I. [Received for publication, March 1st, 1963]
COMPo
PATH.
1963. VOL. 73.
ECOLOGICAL OBSERVATIONS AND LABORATORY EXPERIMENTS ON THE FREE-LIVING STAGES OF COOPERIA ONCOPHORA By
J. H.
ROSE
Ministry qf Agriculture, Central Veterinary Laboratory, W rybridge INTRODUCTION
A programme of research in progress at the Central Veterinary Laboratory is concerned with parasitic gastro-enteritis of cattle. One aspect of this work deals with the biology of the free-living stages of the different nematode species associated with the disease. The present paper is an account of both ecological observations and laboratory experiments with one of these species-Cooperia oncophora. METHODS
Ecological Observations
Observations were made on the rate of development of the free-living stages in faecal pats at different times of the year, and on the migratory activity and longevity of larvae in faeces, on herbage and in soil. The relationship between the form of the faeces and development of the freeliving stages was also investigated. The methods used have been described in detail in previous papers dealing with similar observations on other nematode species (Rose, 1955; Rose 1961; Rose 1962) and so will be dealt with only briefly in the present paper. Development if the free-living stages. Faeces, freshly collected from a stalled calf, were deposited in the form of a pat on to a plot of short perennial rye-grass growing in a galvanised iron box and exposed to the local climatic conditions. A replicate was set up each month throughout the year from July 1959 to June 1960. Samples of faeces were examined at frequent intervals using methods described by Michel and Rose (1954) and the development of the free-living stages was observed. Migratory activities and longevity
if larvae
in faeces, on herbage and in soil.
Two series of experiments were carried out. The first was set up in June 1959 during a dry summer, and the second in July 1960 during a wetter summer. Faeces collected from artificially infected stalled calves were thoroughly mixed to ensure, as far as possible, an even distribution of C. oncophora eggs. One pound of faeces was deposited in the form of a pat, approximately 6" in diameter and I" in depth, on to the centre of an 18" square plot of short perennial rye-grass. Replicates were set up on a number of plots. The faeces used in the first series of experiments contained approximately 107 e.p.g., while those used in the second series contained 240 e.p.g. Samples of faeces from different vertical and lateral levels of the pat, all of the herbage cut at different vertical and lateral levels (Rose 196 I) and the uppermost one inch layer of soil from a single plot were examined for larvae. The number of larvae in the residue of faeces after sampling was also determined, separation being effected with a Baermann type apparatus. A fresh plot was examined in this way each week for the first month, then subsequently at intervals of 4 weeks. In the
EXPERIMENTS ON
Cooperia oncophora
first series of experiments, plots were examined until larvae were no longer recovered, but in the second series the experiment was terminated in November 1961 although small numbers of larvae were still present on the plots. Relationship between the form of the faeces and viability and development of the free-living stages. A comparison was made between the viability and development of the free-living stages in faecal pats, in diarrhoeic faeces thinly spread over plots of short herbage in a series of criss cross lines and exposed to the local climatic conditions, and in similarly spread faeces kept continuously moist by placing plots under an automatic mist control apparatus (Bean, Trickett and Wells, 1957). Samples of faeces were examined daily and the viability and development of the free-living stages were observed. This experiment was repeated several times during the dry summer of 1959 and during the wetter summer of 1960.
Laboratory Experiments The effects of temperature and humidity on the rate of development of eggs and larvae in faeces, and on the longevity of infective larvae separated from faeces were studied using methods described by Rose (1955, 1961 , 1962 ). The relationship between age oflarvae and infectivity was determined by feeding infective larvae, which had been kept in water at 10 to 11°C and at 4 to 6°C for different periods of time, to worm free lambs. These were subsequently autopsied and examined for C. oncophora. RESULTS
Development of the Free-living Stages Only a small proportion of the eggs in faecal pats deposited outof-doors during the winter reached the infective stage. The majority died while still non-embryonated. The remainder became embryonated within a few days, but subsequent development was retarded and several weeks elapsed before infective larvae were recovered from the faeces (Table I). During the remainder of the year mortality was not so heavy and the rate of development was more rapid, increasing as the temperature rose (Tables 1 and 2, Fig. 3). The greatest numbers of infective larvae were recovered from the faecal pats during the summer, despite the dry weather prevailing. Adequate moisture was present in the pats to allow development to be completed. Faeces spread over the grass plots became hard and dry within three and four days during the summer of 1959, however, and none of the contained eggs reached the infective stage (Table 3). During the wetter summer of 1960, when on a number of occasions the spread faeces remained moist for a week or more, mortality was not excessive and many of the eggs developed into infective larvae, as did eggs in the artificially moistened faeces (Table 3). Comparable results were seen in the laboratory experiments. Larvae developed to the infective stage in thin layers of faeces kept moist. In faeces which dried out within 3 to 4 days, the extent of development varied according to the temperature (Table 4), but the infective stage was not reached.
J.
H. ROSE
Migration of Larvae Infective larvae were most numerous in the outer surface of the faecal pat (Table 5), even when this was hard and dry, suggesting a very active migration to this region from the remainder of the pat. The rate of migration from the faecal pat on to the herbage differed as between the two series of experiments. In the first series, started in June 1959, development from egg to infective larva took between 6 and 14 days yet few larvae were recovered from the herbage until August (Fig. I). During this time many infective larvae were TABLE
I
RATE OF DEVELOPMENT OF THE FREE·LIVING STAGES OF C. ONCOPHORA IN FAECES DEPOSITED OUT·OF·DOORS AT DIFFERENT TIMES OF THE YEAR
Infected faeces deposited out of doors in:-
Time for development to srd stage larvae (weeks) Minimum
Maximum
2 3 3 6 10 12
1959 July August September October November December January February March April May June
7
10
1960
8 7 3 2i Ii
TABLE
II
9 8 3 3 2
2
EFFECT OF TEMPERATURE ON THE RATE OF DEVELOPMENT OF THE FREE·LIVING STAGES OF C. ONCOPHORA IN MOIST FAECES
Temperature (0C)
22-23 14- 16 10-11
Time taken for development to 3rd stage larvae (days) Minimum
Maximum
3
9 21 56
4
21
EXPERIMENTS ON
Cooperia oncophora
TABLE 3 EFFECT OF THE FORM OF FAECES ON DEVELOPMENT
The numbers of infective larvae recovered three weeks after the start of each experiment.
Irifective larvae per g. offaeces
-----
Spread faeces
Date
Faecal pat
Watered
Non-watered
1959 July September
143 15 8
0 0
176 141
1960 July
211
198
184
------ ----
TABLE 4 DEVELOPMENT OF EGGS AND LARVAE IN THIN LAYERS OF 'WET' AND 'DRY' FAECES KEPT AT DIFFERENT TEMPERATURES
Extent of development
Temperature °C
-1--------
'Dry'
-
'Wet'
Infective larvae per g 'Dry'
tiffaeces
'Wet'
22-23
2nd stage larva
Infective larva
0
25 2
14-16
I
st stage larva
Infective larva
0
216
Embryonated egg Infective larva
0
78
10-11
TABLE 5 VERTICAL DISTRIBUTION OF INFECTIVE LARVAE IN FAECAL PATS
Date of sampling
Aug. 17th Sept. 7th Oct. 12th Jan. 18th
Number of irifective larvae from Top
Middle
Bottom
1352 1010 29 6 78
48 9 33 9 0
4 4 0 0
Top - 2/ION Middle - 4/ ION Bottom - 4/ION (4 typical results are tabulated)
J.
289
H. ROSE
recovered from the faeces (Fig. 2). There was no further increase in the herbage infestation until the late autumn, a peak being reached in November (Fig. r), with a corresponding decrease in the numbers oflarvae recovered from the faecal pats. In the second series, started inJuly r960, the rate of development was the same as in the previous year, but larvae were recovered from the herbage a few days after becoming infective and continued to be recovered in increasing numbers throughout August, a peak being reached in September (Fig. I). Fig.
I.
24
20
16
j
12
. o
-"
E
:i
o
28 28 Nov. 1961
16
23 June 1959
Weeks
Longevity of larvae on herbage. Each point represents all the larvae recovered from the herbage of a single plot.
Fig.
2.
20
16
1 ~
12
.5
I
8
J
4
0
0
2 Jon. 1959
16-
32
48
64
80
96 23 May 1961
112
----. 128
Persistence of larvae in faeces. Each point represents all of the larvae remaining in the faecal pat.
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til
~
z
~
0
20
40
60
80
100
·5
1<>
1'5
,0
2·5
I
i
10
15
0
20
25
30
35 40
45 50
55
60
WEEKS
65
70
75
80
85
90
95
100
105
110
"'
120
125
28 NOV. 196\
IJ
Mean weekly minimum and maximum ground temperatures, total weekly rainfall and number of rain days per week from June 23rd 1959 to November 28th 1961.
I
I[l3 JUNE 1959 •
,W!
Fig. 3.
130
~
i:l
i5"
~ .g,
'"
i:i'
~
.g,
~
~
~
~
~
~
~
J.
H. ROSE
These differences in the rates of migration on to the herbage can be correlated with differences in the climatic conditions prevailing during the summers of 1959 and 1960 and the resultant effects on the state of the faecal pats. The summer of 1959 was dry and sunny (Fig. 3). As a result the outer surface of the faecal pats became hard and dry within a few hours of being deposited out-of-doors, and after about 3 weeks the whole pat was completely dry and remained in this condition throughout June and July. There was little rain during this period except during a thunderstorm in July, when I ·9" of rain fell, but this did little more than wet the surface of the pats for a short time. During August it rained on five consecutive days (Fig. 3) thoroughly wetting the pats. Subsequently there was an appreciable increase in the herbage infestation (Fig. 1). The fine weather returned and continued throughout September, the pats became hard and dry again and no further increase in the herbage infestation was seen Fig. 4· 20
Inner area
16
12
~
." C
~
8
:0
0
.;
.s ~
0
>
~
.
"0 G;
.0
E =>. Z
0
8 Outer area
.. o
27 uly 1960
Weeks
28 Nov. 1961
Vertical and lateral distribution oflarvae on herbage. The histogram represents the results of Experiment 2. The distribution of the larvae on the herbage was similar in Experiment I. Key. The herbage was first cut 2" above the soil surface to give the Upper herbage It was then cut at the soil surface to give the Lower herbage. Inner area-Extended 0" - 2" from faecal pat. Outer area-Extended 2" - 6" from faecal pat.
EXPERIMENTS ON
Cooperia oncophora
until October. This corresponded with a change in the weather (Fig. 3) and a change in the condition of the pats which became soft and moist again. The weather during the summer of 1960 was quite different. Rain fell frequently (Fig. 3), the faecal pats remained soft and moist for most of the time and there was a continuous increase in the herbage infestation throughout the summer. It was evident from these observations that the rate of migration on to the herbage depended on the condition of the faecal pats. The infective larvae were virtually unable to migrate from the hard dry pats but migrated readily from those which were soft and moist. The condition of the pats was in turn determined principally by the effects of climatic factors, and in particular by the amount and frequency of rainfall. Most larvae were recovered from the 'inner' area of the grass plots (Fig. 4), immediately around the pats, having migrated less than two inches laterally. Some did migrate farther, however, between two and six inches on to the 'outer' area (Fig. 4). The larvae also migrated vertically on the grass blades. Some were recovered from the 'upper' herbage at all times of the year, sometimes in appreciable numbers, but the majority were found on the 'lower' herbage (Fig. 4). Few larvae were recovered from the soil.
Longevity and Infectivity of the Infective Larvae Infective larvae survived on the herbage of the experimental plots for almost two years (Fig. I) and in the faeces until such time as the pats had disintegrated which was 6 to 16 months (Fig. 2, Table 6). The herbage infestation remained high for 7 to 8 months, TABLE 6 TIME TAKEN FOR
Infected faeces deposited out of doors
1959
July August September October November December
1960
January February March April May June
COMPLETE DISINTEGRATION OF FAECAL PATS
Time for complete disintegration qf faecal pat (Months)
Length qf time during which larvae were recovered from faecal pat (Months)
12 13 8 9 8 8
12 7 9 7 7
7 7 12 10 12 13
I I
6 6 II
9
II
12
J.
293
H. ROSE
many larvae surviving from the summer until the following spring, but only a few survived for between one and two years. (Fig. I). The infective larvae did not appear to be adversely affected by the climatic conditions prevailing during the course of the experiments. Many survived in the hard dry faecal pats throughout the summer of 1959 (Fig. 2). They survived well on herbage also during dry weather, suggesting a marked degree of resistance to desiccation. This was confirmed in laboratory experiments when infective larvae dried on glass slides survived for as long as 17 weeks (Fig. 5). The ability of the infective larvae to overwinter suggests a good degree of tolerance to low temperatures. This was demonstrated in the laboratory when larvae survived for more than 2 years at low temperatures, whereas at higher temperatures longevity was appreciably less (Table 7). Even under continuous freezing infective larvae Fig. 5. Relative humidity
100
,
j
.g'
80
60
~
~
~
40
0
i
l
20
o
3
6
9 Week.
12
15
18
Effect of desiccation on infective larvae.
TABLE 7 MAXIMUM LONGEVITY OF INFECTIVE LARVAE IN WATER KEPT AT DIFFERENT TEMPERATURES
Temperature (0C)
F
Maximum longevity (Months)
24-25
13
15-16
IS
10-11
>24
6-7
>24
294
EXPERIMENTS ON
Cooperia oncophora
survived for up to 24 weeks (Fig. 6). Larvae which had been kept in the laboratory at low temperatures for periods of 22 and 25 months respectively gave rise to patent infections when fed to worm-free lambs reared indoors (Table 8), demonstrating that they had retained their infectivity. FifS· 6. 100
80
•> D
j
I
60
~
8.
2c 3
.i!
40
20
o
5
10
15
Weeks
25
20
30
Effect of continuous freezing on the longevity of infective larvae suspended in water and kept at -4° to -6°C. TABLE 8 EFFECT OF AGE OF LARVAE ON INFECTIVITY
Lamb
Date infected
-
Approx. age of larvae (months)
Approx. no. of larvae administered
Worms recovered
I
July 1962
22
600
94
2
July 1962
22
600
301
3
Oct. 1962
25
600
22
4
Oct. 1962
25
I
600
23 \
DISCUSSION
The results of the present observations have a number of implications relevant to translation, the process whereby eggs in faeces become infective larvae on herbage available to the grazing animal, in areas where climatic conditions are comparable with those studied.
J.
H. ROSE
295
During the winter, conditions will not be conducive to translation. The majority of the eggs in faeces deposited on pastures at this time of the year will fail to develop into infective larvae. During the remainder of the year mortality will be less and appreciable numbers of eggs will become infective larvae, but the extent of translation is likely to vary with the weather. Wet weather will be conducive to translation as the faecal pats will remain soft and moist so that the infective larvae will be able to migrate readily on to the herbage. In dry weather, however, translation will be limited by the inability of the larvae to migrate from the dry faecal pats. As the larvae are able to survive for long periods in dry faeces, the pats can act as reservoirs of infection until a change in the weather makes migration possible, so that eggs in faecal pats deposited on pastures during a dry summer can complete their development into infective larvae which may then give rise to heavy herbage infestations in the autumn and early winter when migration from the pats can take place. The dissemination of faeces over pastures may either limit or facilitate translation, the precise effect being determined principally by the weather. In ~eneral when freshly deposited faeces are spread over the pastures in dry weather mortality of the pre-infective stages will be heavy so that translation will be limited. In wet weather, except in the winter, mortality will not be heavy and the development of eggs in faeces into infective larvae on herbage will be favoured. The dissemination of faecal pats which have been on the pastures for some time and in which the larvae are at the infective stage, will generally aid translation as the infective larvae are able to survive well under a wide range of climatic conditions, providing the condition of the faeces permits adequate dissemination. Although translation is only one of the factors involved in the transmission of infection, nevertheless the present observations do contribute to an understanding of certain aspects of transmission . . Outbreaks of infection are frequently encountered during warm wet summers. These would appear to be directly related to the climatic conditions being conducive to translation. From time to time heavy outbreaks occur also in the autumn and winter following a dry summer. The source of these outbreaks has been a matter of conjecture in the past. It would seem from the present observations that due to the slow rate of development and heavy mortality of the preinfective stages, eggs in faeces deposited on the pastures during the autumn and winter cannot be the source of infection. It was also noted that during a dry summer there was no appreciable build up of the herbage infestation. It is unlikely therefore, that larvae can survive on the herbage in numbers sufficient to give rise to such outbreaks. It was observed, however, that faecal pats can serve as reservoirs of infection throughout the summer, larvae migrating on to the herbage when the dry weather ends. In this way it is possible for the herbage infestation to be built up in the autumn or early
EXPERIMENTS ON
Cooperia oncophora
winter sufficient to give rise to outbreaks of infection in susceptible stock. Although the heaviest outbreaks of infection would be expected to coincide with periods of high translation, owing to the ability of larvae to survive for up to 2 years on pasture herbage, it should be possible for infection to be picked up at any time of the year when susceptible animals are turned out to graze. American workers (Baker, 1939; Goldberg and Rubin, 1956; Drudge, Leland, Wyant and Rust, 1958; Goldberg and Lucker, 1959) have, in fact, demonstrated that infection can be picked up from pastures rested for from 7 to 9 months. The present observations would suggest that infection can be picked up from contaminated pastures rested for longer periods. Although heavy outbreaks of clinical disease are unlikely to result directly from grazing such rested pastures, this could be a means of perpetuating infection in a herd and providing the source of infection for subsequent clinical disease. The ability oflarvae to survive well on pastures for 7 or 8 months is of particular practical significance with regard to control by grazing management. It would seem that resting contaminated pastures for a few weeks, as is often advised, will serve no useful purpose in the control of C. oncophora infection. CONCLUSIONS
Outdoor observations were made on the rate of development of the free-living stages of C. oncophora at different times of the year and on the migratory activity and longevity of the larvae in faeces, on herbage and in soil. The relationship between the form of the faeces and development was also investigated. The effects of temperature and humidity on the survival and development of eggs and larvae in faeces, and on the longevity of infective larvae separated from the faeces, were studied in the laboratory. The results of these observations are discussed in relation to translation and the transmission of infection in the field. REFERENCES
Baker, D. W. (1939). Cornell. Vet., 29, 45. Bean, G., Trickett, E. S., and Wells, D. A. (1957). ]. agric. engng. Res., 2,44· Drudge, J. R., Leland, S. E., Wyant, Z. N., and Rust, J. W. (195 8 ). ]. Parasit, 44, 434. Goldberg, A., and Rubin, R. (1956). Proc. helm. Soc. Wash., 23, 65. Goldberg, A., and Lucker, J. T. (1959). Ibid., 26,37· Michel, J. F., and Rose, J. H. (1954).], compo Path., 64, 195· Rose, J. R. (1955). Ibid. 65, 370; (1961). Parasitology, 51, 295; (1962 ). ]. compo Path .• 72, I I. [Received for publication, March 1st, 1963]