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Evaluation of a concentration method for counting sarcocystis gigantea sporocysts in cat faeces
Veterinary Parasitology, 26 (1988) 207-214 Elsevier Science Publishers B.V., A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
207
E v a l u a t i o n o f a C o n c e n t r a t i o n M e t h o d for C o u n t i n g Sarcocystis gigantea S p o r o c y s t s in Cat Faeces P.B. M c K E N N A
Palmerston North Animal Health Laboratory, P.O. Box 1654, Palmerston North (New Zealand) W.A.G. C H A R L E S T O N
Department of Veterinary Pathology and Public Health, Massey University, Palmerston North (New Zealand) (Accepted for publication 13 J a n u a r y 1987)
ABSTRACT M c K e n n a , P.B. a n d Charleston, W.A.G., 1988. Evaluation of a concentration m e t h o d for counting Sarcocystis gigantea sporocysts in cat faeces. Vet. Parasitol., 26: 207-214. A technique for d e t e r m i n i n g t h e n u m b e r s of S. gigantea sporocysts in cat faeces using a concentration procedure a n d h a e m o c y t o m e t e r was evaluated. T h e results showed t h a t it was more accurate t h a n a modified M c M a s t e r m e t h o d a n d h a d a m e a n recovery rate of 73% at four levels of infection ranging from about 2000 to over 20 000 sporocysts per gram of faeces.
INTRODUCTION
Estimates of the intensity of helminth and coccidial infections are frequently made from the numbers of eggs or oocysts present in faeces. Such estimates are usually based on counts in small representative faecal samples, and are normally expressed in terms of their concentration per gram. A number of procedures have been described for obtaining these counts of which the M c M a s t e r technique (Whitlock, 1948) is the most commonly employed. In this dilution procedure, a specified weight of faeces is mixed with a known volume of saturated aqueous NaC1 and a representative sample introduced into a special chamber. The heavy faecal debris falls to the b o t t o m of the chamber and the eggs and/or oocysts float free to the underside of the cover glass were they can be counted. Similar counts of the numbers of eggs or oocysts in faeces could also be obtained using a modification of the concentration procedure described by
0304-4017/88/$03.50
@) 1988 Elsevier Science Publishers B.V.
208 M c K e n n a and Charleston (1987), which involved the use of a haemocytometer to estimate the numbers recovered. In assessing the usefulness of any faecal analysis procedure, the reliability and accuracy of the technique and the time and effort involved are of prime importance. While the M c M a s t e r method is simpler and faster than a concentration/haemocytometer procedure, initial attempts to use it for counting Sarcocystis gigantea sporocysts in cat faeces revealed two main problems. Firstly, the top side of the chamber was too thick to permit the use of an objective lens of sufficient magnification to enable the small sporocysts to be readily seen. Secondly, the large amount of debris floating up with the sporocysts obscured them. Accordingly, the present study was undertaken to evaluate the reliability and accuracy of both a modified M c M a s t e r method and a concentration/haemocytometer procedure for estimating the number of S. gigantea sporocysts in cat faeces. MATERIAL AND METHODS
Sporocysts The sporocysts used in all studies were obtained from experimentally infected cats. The cats, raised and maintained free of extraneous Sarcocystis infection on a diet of tinned fish, were infected by feeding them S. gigantea cysts excised from the oesophagi of naturally infected sheep. Sporocysts from some of these cats were recovered from faeces by crude sugar floatation and stored in tap water at 4 ° C until used. An estimate of the concentration of sporocysts in this suspension was obtained by removing five 1-ml samples while mixing with a magnetic stirrer and counting the numbers in each using a haemocytometer. The desired number of sporocysts per millilitre for any one experiment was adjusted either by dilution with water or by concentration.
McMaster counting technique The M c M a s t e r technique used was essentially as described by Whitlock (1948) except that (a) the top side of the standard chamber was removed, and replaced by two 0.4 to 0.6 mm thick cover glasses on which were etched 1-cm-square grids; (b) after mixing the faecal sample, and before introducing a representative aliquot into the chamber, the sample was sieved.
Concentration/haemocytometer counting technique This technique was based on the procedures decribed by M c K e n n a and Charleston (1987). Five grams of faeces in 50 ml of water were homogenised
209 for 2 minutes using an electric beater. The homogenised material was then washed sequentially through a 250- and 53-#m sieve with an additional 50 ml of water, and the filtrate was adjusted to a final volume of 100 ml. Following agitation with an air pump, a 20-ml (20%) subsample was removed from this filtrate, centrifuged at 6000Xg for 5 min, and the top 18 ml of supernatant removed. The remaining 2 ml of sediment were then resuspended by mixing with 18 ml of saturated aqueous NaC1 ( SG 1.2 ), and centrifuged again as above. After centrifugation, the top 2 ml of material were carefully removed, using a flask fitted to a water vacuum pump, and 26 ml of water added to it. This material was then sedimented by centrifugation, and reduced to a final volume of 500 #l by removing the supernatant. The number of sporocysts in the sediment was then determined by examining six 0.9-]~1 replicates in a haemocytometer (improved Neubauer).
Experimental Procedures Comparison of counting techniques Known numbers of sporocysts were added to eight 5-g samples of faeces from
Sarcocystis-free cats. To four of these samples sporocysts were added to produce concentrations of approximately 22 264 + 849 sporocysts per gram (s.p.g.) and to the other four, 2 226 + 85 s.p.g.. Water was added to all samples which were then homogenised and sieved as described for the concentration/haemocytometer counting technique. From each filtrate of each sample,£wo 20% subsamples A and B (each containing the equivalent of 1 g of faeces) were removed, sedimented, and reduced to final volumes of 2 and 3 ml, respectively. The number of sporocysts in subsample A was determined by adding 18 ml of NaC1 solution (1.2 SG) and by proceeding according to the concentration/ haemocytometer technique, while the number of sporocysts in subsample B was determined using the McMaster procedure. For the latter purpose, the 3 ml of faecal sediment were thoroughly mixed with 27 ml of NaC1 solution ( SG 1.2 ), and a representative aliquot was introduced into the modified McMaster chamber. In both cases, therefore, the faecal sediment was suspended in 9 volumes of NaC1 solution, with similar faeces to NaC1 solution proportions (5 and 3.33 % respectively) and comparable sensitivities (each sporocyst counted = 100 s.p.g. McMaster, 92.6 s.p.g, concentration/haemocytometer).
Errors within counting technique The concentration/haemocytometertechnique described above has two main sources of potential sampling error: the error introduced in removing a 20% subsample from the homogenised and sieved sample, and the errors associated with examining only small volumes (six 0.9 ttl) of the final preparation (500 ~1). The significance of such potential errors was examined in the following experiment.
210
Eight 5-g faecal samples from Sarcocystis-free cats were divided into two lots of four. To one lot, sporocysts were added to produce concentrations of approximately 3 065 + 236 s.p.g, and to the other 12 668 + 612 s.p.g.. From the filtrates of each homogenised and sieved sample, two 20% subsamples were removed and processed in the usual manner. These subsamples were reduced to final volumes of 500/~l, and the numbers of sporocysts determined, using six 0.9-/~1 replicates for each of the two subsamples. Data from both lots of four samples were then each subjected to a three-level nested analysis of variance.
Reliability o/faecal sampling method Estimates of the numbers of S. gigantea sporocysts per gram could be obtained by processing all the faeces available by the concentration/haemocytometer technique, and then dividing the number of sporocysts recovered by the weight of the original material. Alternatively, similar estimates could be obtained by processing only a 1 g sample. In practice it has been found more convenient to start with an initial 5-g sample and to process a 20% (i.e. 1 g) subsample as in the previous experiments. In these experiments, care was taken to ensure that each lot of 5-g samples contained equivalent numbers of sporocysts by adding the same numbers to each. In acquired infections, however, sporocysts may not be evenly distributed throughout the faecal mass, and the numbers in a 5-g sample taken from it may not, therefore, be representative of TABLE I Faecal sporocyst counts obtained using the c o n c e n t r a t i o n / h a e m o c y t o m e t e r a n d modified McMaster methods in cat faeces at two levels of sporocyst concentration* Sporocyst Concentration
22 264± 849
2 2 2 6 ± 85
Sample Number
Counting m e t h o d / s p o r o c y s t counts* McMaster
Concentration/ haemocytometer
1 2 3 4
7800 9800 6600 4400
18 705 16 853 14 631 13 520
M e a n ± se
7150 ± 1130
15 9 2 7 ± 1156
1 2 3 4
1000 800 700 700
M e a n ± se *Sporocysts per gram ( s.p.g. ) of faeces.
800±
1 852 1 019 1 574 1 482 71
1 482±
173
211
their concentration in the whole. The following experiment was undertaken to investigate this possibility. One day's total faecal output (86 g) from an eight-month-old cat, infected 13 days previously with S. gigantea cysts, was thoroughly mixed for three minutes using a bowl and spatula and six 5-g samples removed. Each sample was then processed as previously described, and estimates of their sporocyst concentrations per gram so obtained were examined for statistically significant differences by analysis of variance. RESULTS
Comparison of counting techniques The results are presented in Table I. The numbers estimated by both p r o T A B L E II
Sources of sampling error within the concentration/haemocytometer counting technique. Figures represent actual numbers counted: expected counts 3065 _+236 s.p.g. Samples
A
Subsamples
B
A~
A2
C
BI
B2
D
C1
C2
DI
D2
Replicates
A'~
A';
A~
A~
Bi
B';
B,~
B~
Ci
C';
C,~
C~
Di
Di
D~
D,~
Readings
7 10 2 3 1 5
4 6 2 4 4 6
4 4 7 7 4 1
9 8 4 5 2 6
6 3 3 7 5 4
5 4 6 5 3 4
7 4 7 4 3 2
3 0 3 6 6 6
6 4 0 4 2 3
7 3 5 7 5 1
2 4 2 5 5 5
2 5 3 3 3 2
5 4 5 3 4 5
5 2 3 4 6 4
4 3 5 9 4 3
5 4 4 1 6 8
28
26
27
34
28
27
27
24
19
28
23
18
26
24
28
28
Replicate total Subsample
total Sample total
54
61
115
55
51
106
47
41
88
50
56
106
Sample
mean
28.8
26.5
22.0
26.5
Conversion factor
92.6
92.6
92.6
92.6
s.p.g. (sample
means)
2667
2454
2037
2454
212
cedures were always lower than the expected counts. At both levels of infection, however, the concentration/haemocytometer technique was significantly better than that of the McMaster procedure (paired t-test; P < 0 . 0 1 , t = 10.63; P < 0.05, t-- 4.384, 3 df at concentrations of 22 264 and 2 226 s.p.g., respectively ).
Errors within counting techniques The results (Tables II and III ) showed no significant differences ( P > 0.05) between subsamples within samples, nor between replicates within subsamples, at either of the two levels of infection. T A B L E III
Sources of sampling error within the concentration/haemocytometer counting technique. Figures represent actual numbers counted: expected counts 12 668 + 612 s.p.g. Samples
A
Subsamples
Replicates Readings
Replicate total
B
Ai A'I
A2
A';
A~
C
B1
A2
B'I
B2
D
C1
B';
B,~
B:_;
21 17 16 14 20 17
18 13 20 16 13 24
22 17 18 16 16 17
22 19 18 16 11 17
22 20 15 16 15 19
13 19 11 12 21 9
12 15 20 21 16 15
15 19 21 18 22 12
105
104
106
103
107
85
99
107
C'~
C2
C';
35 18 16 20 13 19
C,~
D1
C~
D't
D~
D';
D2
D,~
21 21 23 26 15 16
15 12 21 25 13 17
22 20 16 15 17 20
18 18 18 12 17 16
18 17 24 19 19 ll
24 12 17 16 15 17
19 9 22 14 14 11
121 122
103
ll0
99
108
101
89
Subsample
total Sample sums
209
209
418
192
206
398
243
213
456
207
190
397
Sample
mean Conversion factor s.p.g. ( sample
means)
104.5
99.5
114
99.3
92.6
92.6
92.6
92.6
9677
9214
10 556
9195
213 T A B L E IV Variations of S. gigantea sporocyst counts between six 5 gram samples obtained from a single faecal mass Samples A Readings
Total Conversion factor s.p.g,
B
C
D
F
25 21 26 19 18 18
14 22 20 28 25 31
21 27 27 24 25 14
15 28 14 18 25 21
16 22 25 29 10 21
23 23 17 22 28 15
127
140
138
121
123
128
92.6 l l 760
92.6 12 964
92.6 12 779
92.6 11 205
92.6 11 390
92.6 11 853
Reliability of faecal sampling method The results, which are presented in Table IV, showed that there were no significant differences ( P > 0.05) between the estimated numbers of sporocysts per gram for any of the six faecal samples examined. DISCUSSION
Although the present study is based on limited data, the results suggest that the concentration/haemocytometerprocedure is more accurate than a modified McMaster method for estimating the numbers of S. gigantea sporocysts in cat faeces. This is rather surprising since others (Levine et al., 1960; Dorney, 1964; Egwang and Slocombe, 1981 ) have found the McMaster technique to be generally superior to most tested for the counting of eggs and oocysts. The reasons for this apparent disparity are not entirely clear, but they probably relate, at least in part, to the small size of the sporocysts under study and to the nature of cat faeces. Even after sieving, the samples still contain large amounts of fine faecal debris and are frequently very turbid. Because of this, and because of the depth of the McMaster chamber, light penetration is poor and many of the sporocysts present in these preparations could be obscured or over!ooked. When counting sporocysts by the concentration/haemocytometer technique, however, much of the faecal debris has already been eliminated and, because only 0.1 mm depth of material is examined, light penetration is not a problem. In addition, any uncertainties in identification can be resolved by the
214
use of a higher magnification objective lens, a process which is not possible with the McMaster chamber, even when the top slide is replaced with a thin cover glass. Investigation of the potential sources of error within the counting and sampling procedures adopted in this study suggests that the concentration/haemocytometer method offers a reasonably consistent and reliable means of estimating the level of S. gigantea infection in the cat definitive host. Using this technique our results indicate that at four levels of infection ranging from about 2000-20 000 s.p.g., a mean percentage recovery of approximately 73% might be expected (data from Tables I to III, inclusive). An even more accurate estimate of the numbers of S.gigantea sporocysts in faeces could, therefore, be obtained by multiplying the numbers counted by 1.4.
REFERENCES Dorney, R.S., 1964. Evaluation of a microquantitative method for counting coccidial oocysts. J. Parasitol., 50: 518-22. Egwang, T.G. and Slocombe, J.O.D., 1981. Efficiency and sensitivity of techniques for recovering nematode eggs from bovine faeces. Can. J. Comp. Med., 45: 243-8. Levine, N.D., Mehra, K.N., Clark, D.T. and Aves, I.J., 1960. A comparison of nematode egg counting techniques for cattle and sheep faeces. Am. J. Vet. Res., 21: 511-15. McKenna, P.B. and Charleston, W.A.G., 1987. The recovery of Sarcocystis gigantea sporocysts from cat faeces. Vet. Parasitol., in press. Whitlock, H.V., 1948. Some modifications of the McMaster helminth egg-countingtechnique and apparatus. J. Counc. Sci. Ind. Res. Aust., 21: 177-80.
207
E v a l u a t i o n o f a C o n c e n t r a t i o n M e t h o d for C o u n t i n g Sarcocystis gigantea S p o r o c y s t s in Cat Faeces P.B. M c K E N N A
Palmerston North Animal Health Laboratory, P.O. Box 1654, Palmerston North (New Zealand) W.A.G. C H A R L E S T O N
Department of Veterinary Pathology and Public Health, Massey University, Palmerston North (New Zealand) (Accepted for publication 13 J a n u a r y 1987)
ABSTRACT M c K e n n a , P.B. a n d Charleston, W.A.G., 1988. Evaluation of a concentration m e t h o d for counting Sarcocystis gigantea sporocysts in cat faeces. Vet. Parasitol., 26: 207-214. A technique for d e t e r m i n i n g t h e n u m b e r s of S. gigantea sporocysts in cat faeces using a concentration procedure a n d h a e m o c y t o m e t e r was evaluated. T h e results showed t h a t it was more accurate t h a n a modified M c M a s t e r m e t h o d a n d h a d a m e a n recovery rate of 73% at four levels of infection ranging from about 2000 to over 20 000 sporocysts per gram of faeces.
INTRODUCTION
Estimates of the intensity of helminth and coccidial infections are frequently made from the numbers of eggs or oocysts present in faeces. Such estimates are usually based on counts in small representative faecal samples, and are normally expressed in terms of their concentration per gram. A number of procedures have been described for obtaining these counts of which the M c M a s t e r technique (Whitlock, 1948) is the most commonly employed. In this dilution procedure, a specified weight of faeces is mixed with a known volume of saturated aqueous NaC1 and a representative sample introduced into a special chamber. The heavy faecal debris falls to the b o t t o m of the chamber and the eggs and/or oocysts float free to the underside of the cover glass were they can be counted. Similar counts of the numbers of eggs or oocysts in faeces could also be obtained using a modification of the concentration procedure described by
0304-4017/88/$03.50
@) 1988 Elsevier Science Publishers B.V.
208 M c K e n n a and Charleston (1987), which involved the use of a haemocytometer to estimate the numbers recovered. In assessing the usefulness of any faecal analysis procedure, the reliability and accuracy of the technique and the time and effort involved are of prime importance. While the M c M a s t e r method is simpler and faster than a concentration/haemocytometer procedure, initial attempts to use it for counting Sarcocystis gigantea sporocysts in cat faeces revealed two main problems. Firstly, the top side of the chamber was too thick to permit the use of an objective lens of sufficient magnification to enable the small sporocysts to be readily seen. Secondly, the large amount of debris floating up with the sporocysts obscured them. Accordingly, the present study was undertaken to evaluate the reliability and accuracy of both a modified M c M a s t e r method and a concentration/haemocytometer procedure for estimating the number of S. gigantea sporocysts in cat faeces. MATERIAL AND METHODS
Sporocysts The sporocysts used in all studies were obtained from experimentally infected cats. The cats, raised and maintained free of extraneous Sarcocystis infection on a diet of tinned fish, were infected by feeding them S. gigantea cysts excised from the oesophagi of naturally infected sheep. Sporocysts from some of these cats were recovered from faeces by crude sugar floatation and stored in tap water at 4 ° C until used. An estimate of the concentration of sporocysts in this suspension was obtained by removing five 1-ml samples while mixing with a magnetic stirrer and counting the numbers in each using a haemocytometer. The desired number of sporocysts per millilitre for any one experiment was adjusted either by dilution with water or by concentration.
McMaster counting technique The M c M a s t e r technique used was essentially as described by Whitlock (1948) except that (a) the top side of the standard chamber was removed, and replaced by two 0.4 to 0.6 mm thick cover glasses on which were etched 1-cm-square grids; (b) after mixing the faecal sample, and before introducing a representative aliquot into the chamber, the sample was sieved.
Concentration/haemocytometer counting technique This technique was based on the procedures decribed by M c K e n n a and Charleston (1987). Five grams of faeces in 50 ml of water were homogenised
209 for 2 minutes using an electric beater. The homogenised material was then washed sequentially through a 250- and 53-#m sieve with an additional 50 ml of water, and the filtrate was adjusted to a final volume of 100 ml. Following agitation with an air pump, a 20-ml (20%) subsample was removed from this filtrate, centrifuged at 6000Xg for 5 min, and the top 18 ml of supernatant removed. The remaining 2 ml of sediment were then resuspended by mixing with 18 ml of saturated aqueous NaC1 ( SG 1.2 ), and centrifuged again as above. After centrifugation, the top 2 ml of material were carefully removed, using a flask fitted to a water vacuum pump, and 26 ml of water added to it. This material was then sedimented by centrifugation, and reduced to a final volume of 500 #l by removing the supernatant. The number of sporocysts in the sediment was then determined by examining six 0.9-]~1 replicates in a haemocytometer (improved Neubauer).
Experimental Procedures Comparison of counting techniques Known numbers of sporocysts were added to eight 5-g samples of faeces from
Sarcocystis-free cats. To four of these samples sporocysts were added to produce concentrations of approximately 22 264 + 849 sporocysts per gram (s.p.g.) and to the other four, 2 226 + 85 s.p.g.. Water was added to all samples which were then homogenised and sieved as described for the concentration/haemocytometer counting technique. From each filtrate of each sample,£wo 20% subsamples A and B (each containing the equivalent of 1 g of faeces) were removed, sedimented, and reduced to final volumes of 2 and 3 ml, respectively. The number of sporocysts in subsample A was determined by adding 18 ml of NaC1 solution (1.2 SG) and by proceeding according to the concentration/ haemocytometer technique, while the number of sporocysts in subsample B was determined using the McMaster procedure. For the latter purpose, the 3 ml of faecal sediment were thoroughly mixed with 27 ml of NaC1 solution ( SG 1.2 ), and a representative aliquot was introduced into the modified McMaster chamber. In both cases, therefore, the faecal sediment was suspended in 9 volumes of NaC1 solution, with similar faeces to NaC1 solution proportions (5 and 3.33 % respectively) and comparable sensitivities (each sporocyst counted = 100 s.p.g. McMaster, 92.6 s.p.g, concentration/haemocytometer).
Errors within counting technique The concentration/haemocytometertechnique described above has two main sources of potential sampling error: the error introduced in removing a 20% subsample from the homogenised and sieved sample, and the errors associated with examining only small volumes (six 0.9 ttl) of the final preparation (500 ~1). The significance of such potential errors was examined in the following experiment.
210
Eight 5-g faecal samples from Sarcocystis-free cats were divided into two lots of four. To one lot, sporocysts were added to produce concentrations of approximately 3 065 + 236 s.p.g, and to the other 12 668 + 612 s.p.g.. From the filtrates of each homogenised and sieved sample, two 20% subsamples were removed and processed in the usual manner. These subsamples were reduced to final volumes of 500/~l, and the numbers of sporocysts determined, using six 0.9-/~1 replicates for each of the two subsamples. Data from both lots of four samples were then each subjected to a three-level nested analysis of variance.
Reliability o/faecal sampling method Estimates of the numbers of S. gigantea sporocysts per gram could be obtained by processing all the faeces available by the concentration/haemocytometer technique, and then dividing the number of sporocysts recovered by the weight of the original material. Alternatively, similar estimates could be obtained by processing only a 1 g sample. In practice it has been found more convenient to start with an initial 5-g sample and to process a 20% (i.e. 1 g) subsample as in the previous experiments. In these experiments, care was taken to ensure that each lot of 5-g samples contained equivalent numbers of sporocysts by adding the same numbers to each. In acquired infections, however, sporocysts may not be evenly distributed throughout the faecal mass, and the numbers in a 5-g sample taken from it may not, therefore, be representative of TABLE I Faecal sporocyst counts obtained using the c o n c e n t r a t i o n / h a e m o c y t o m e t e r a n d modified McMaster methods in cat faeces at two levels of sporocyst concentration* Sporocyst Concentration
22 264± 849
2 2 2 6 ± 85
Sample Number
Counting m e t h o d / s p o r o c y s t counts* McMaster
Concentration/ haemocytometer
1 2 3 4
7800 9800 6600 4400
18 705 16 853 14 631 13 520
M e a n ± se
7150 ± 1130
15 9 2 7 ± 1156
1 2 3 4
1000 800 700 700
M e a n ± se *Sporocysts per gram ( s.p.g. ) of faeces.
800±
1 852 1 019 1 574 1 482 71
1 482±
173
211
their concentration in the whole. The following experiment was undertaken to investigate this possibility. One day's total faecal output (86 g) from an eight-month-old cat, infected 13 days previously with S. gigantea cysts, was thoroughly mixed for three minutes using a bowl and spatula and six 5-g samples removed. Each sample was then processed as previously described, and estimates of their sporocyst concentrations per gram so obtained were examined for statistically significant differences by analysis of variance. RESULTS
Comparison of counting techniques The results are presented in Table I. The numbers estimated by both p r o T A B L E II
Sources of sampling error within the concentration/haemocytometer counting technique. Figures represent actual numbers counted: expected counts 3065 _+236 s.p.g. Samples
A
Subsamples
B
A~
A2
C
BI
B2
D
C1
C2
DI
D2
Replicates
A'~
A';
A~
A~
Bi
B';
B,~
B~
Ci
C';
C,~
C~
Di
Di
D~
D,~
Readings
7 10 2 3 1 5
4 6 2 4 4 6
4 4 7 7 4 1
9 8 4 5 2 6
6 3 3 7 5 4
5 4 6 5 3 4
7 4 7 4 3 2
3 0 3 6 6 6
6 4 0 4 2 3
7 3 5 7 5 1
2 4 2 5 5 5
2 5 3 3 3 2
5 4 5 3 4 5
5 2 3 4 6 4
4 3 5 9 4 3
5 4 4 1 6 8
28
26
27
34
28
27
27
24
19
28
23
18
26
24
28
28
Replicate total Subsample
total Sample total
54
61
115
55
51
106
47
41
88
50
56
106
Sample
mean
28.8
26.5
22.0
26.5
Conversion factor
92.6
92.6
92.6
92.6
s.p.g. (sample
means)
2667
2454
2037
2454
212
cedures were always lower than the expected counts. At both levels of infection, however, the concentration/haemocytometer technique was significantly better than that of the McMaster procedure (paired t-test; P < 0 . 0 1 , t = 10.63; P < 0.05, t-- 4.384, 3 df at concentrations of 22 264 and 2 226 s.p.g., respectively ).
Errors within counting techniques The results (Tables II and III ) showed no significant differences ( P > 0.05) between subsamples within samples, nor between replicates within subsamples, at either of the two levels of infection. T A B L E III
Sources of sampling error within the concentration/haemocytometer counting technique. Figures represent actual numbers counted: expected counts 12 668 + 612 s.p.g. Samples
A
Subsamples
Replicates Readings
Replicate total
B
Ai A'I
A2
A';
A~
C
B1
A2
B'I
B2
D
C1
B';
B,~
B:_;
21 17 16 14 20 17
18 13 20 16 13 24
22 17 18 16 16 17
22 19 18 16 11 17
22 20 15 16 15 19
13 19 11 12 21 9
12 15 20 21 16 15
15 19 21 18 22 12
105
104
106
103
107
85
99
107
C'~
C2
C';
35 18 16 20 13 19
C,~
D1
C~
D't
D~
D';
D2
D,~
21 21 23 26 15 16
15 12 21 25 13 17
22 20 16 15 17 20
18 18 18 12 17 16
18 17 24 19 19 ll
24 12 17 16 15 17
19 9 22 14 14 11
121 122
103
ll0
99
108
101
89
Subsample
total Sample sums
209
209
418
192
206
398
243
213
456
207
190
397
Sample
mean Conversion factor s.p.g. ( sample
means)
104.5
99.5
114
99.3
92.6
92.6
92.6
92.6
9677
9214
10 556
9195
213 T A B L E IV Variations of S. gigantea sporocyst counts between six 5 gram samples obtained from a single faecal mass Samples A Readings
Total Conversion factor s.p.g,
B
C
D
F
25 21 26 19 18 18
14 22 20 28 25 31
21 27 27 24 25 14
15 28 14 18 25 21
16 22 25 29 10 21
23 23 17 22 28 15
127
140
138
121
123
128
92.6 l l 760
92.6 12 964
92.6 12 779
92.6 11 205
92.6 11 390
92.6 11 853
Reliability of faecal sampling method The results, which are presented in Table IV, showed that there were no significant differences ( P > 0.05) between the estimated numbers of sporocysts per gram for any of the six faecal samples examined. DISCUSSION
Although the present study is based on limited data, the results suggest that the concentration/haemocytometerprocedure is more accurate than a modified McMaster method for estimating the numbers of S. gigantea sporocysts in cat faeces. This is rather surprising since others (Levine et al., 1960; Dorney, 1964; Egwang and Slocombe, 1981 ) have found the McMaster technique to be generally superior to most tested for the counting of eggs and oocysts. The reasons for this apparent disparity are not entirely clear, but they probably relate, at least in part, to the small size of the sporocysts under study and to the nature of cat faeces. Even after sieving, the samples still contain large amounts of fine faecal debris and are frequently very turbid. Because of this, and because of the depth of the McMaster chamber, light penetration is poor and many of the sporocysts present in these preparations could be obscured or over!ooked. When counting sporocysts by the concentration/haemocytometer technique, however, much of the faecal debris has already been eliminated and, because only 0.1 mm depth of material is examined, light penetration is not a problem. In addition, any uncertainties in identification can be resolved by the
214
use of a higher magnification objective lens, a process which is not possible with the McMaster chamber, even when the top slide is replaced with a thin cover glass. Investigation of the potential sources of error within the counting and sampling procedures adopted in this study suggests that the concentration/haemocytometer method offers a reasonably consistent and reliable means of estimating the level of S. gigantea infection in the cat definitive host. Using this technique our results indicate that at four levels of infection ranging from about 2000-20 000 s.p.g., a mean percentage recovery of approximately 73% might be expected (data from Tables I to III, inclusive). An even more accurate estimate of the numbers of S.gigantea sporocysts in faeces could, therefore, be obtained by multiplying the numbers counted by 1.4.
REFERENCES Dorney, R.S., 1964. Evaluation of a microquantitative method for counting coccidial oocysts. J. Parasitol., 50: 518-22. Egwang, T.G. and Slocombe, J.O.D., 1981. Efficiency and sensitivity of techniques for recovering nematode eggs from bovine faeces. Can. J. Comp. Med., 45: 243-8. Levine, N.D., Mehra, K.N., Clark, D.T. and Aves, I.J., 1960. A comparison of nematode egg counting techniques for cattle and sheep faeces. Am. J. Vet. Res., 21: 511-15. McKenna, P.B. and Charleston, W.A.G., 1987. The recovery of Sarcocystis gigantea sporocysts from cat faeces. Vet. Parasitol., in press. Whitlock, H.V., 1948. Some modifications of the McMaster helminth egg-countingtechnique and apparatus. J. Counc. Sci. Ind. Res. Aust., 21: 177-80.