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Veterinary Parasitology 158 (2008) 329–335 www.elsevier.com/locate/vetpar
Assessing the efficacy of Duddingtonia flagrans chlamydospores per gram of faeces to control Haemonchus contortus larvae Nadia Florencia Ojeda-Robertos a,*, Juan Felipe de Jesus Torres-Acosta a, Armando Jacinto Aguilar-Caballero a, Armı´n Ayala-Burgos a, Ligia Amira Cob-Galera a, Carlos Alfredo Sandoval-Castro a, Roberto Carlos Barrientos-Medina a, Pedro Mendoza de Gives b a
Campus de Ciencias Biolo´gicas y Agropecuarias, Universidad Auto´noma de Yucata´n. Carretera Me´rida-Xmatkuil (km 15.5), Me´rida, Yucata´n, Mexico b Centro Nacional de Investigaciones Disciplinarias en Parasitologı´a Veterinaria, INIFAP. Carretera Cuernavaca-Cuautla (km 11.5), No. 8534, Col Progreso, Jiutepec, Morelos, C.P. 62550, Mexico Received 24 June 2008; received in revised form 27 August 2008; accepted 28 August 2008
Abstract The aims were (a) to quantify the number of Duddingtonia flagrans chlamydospores per gram of faeces (CPG) recovered from sheep administered with different oral doses and, (b) to describe the relationship between CPG and eggs per gram of faeces (EPG) on the efficacy to reduce Haemonchus contortus infective larvae. Three doses of chlamydospores per kg BW were orally administered during seven days: (T1) non treated control group, (T2) 1 106, (T3) 2.5 106 and (T4) 5 106. Three lambs, infected with H. contortus, were used per group. Faeces were obtained from the rectum of each lamb during the fungal administration period (days 0–6) and for six days after that period. Four coproculture replicates were made from each animal in days 2, 4, 6, 8 and 10. A higher chlamydospore dose produced higher CPG in faeces ( p < 0.05), but a clear dose dependent effect was not found either in the larvae reduction or in the CPG:EPG ratio. When ratios were re-analyzed, independently of the treatment groups of origin, a better efficacy was obtained with a ratio from 5 to 10 CPG:EPG and a higher ratio (>10 per egg) showed a lower reduction efficacy ( p < 0.05). The binomial analysis showed that for each unit of increment in CPG:EPG ratio there was a reduction of larvae number until a point (between 5 and 10 CPG:EPG) where no further reduction was detected. The surface response test indicated that the number of larvae was reduced by CPG until possible saturation. The highest CPG:EPG ratios did not necessarily improve efficacy of D. flagrans. # 2008 Published by Elsevier B.V. Keywords: Duddingtonia flagrans; Faecal chlamydospores count; Haemonchus contortus; Sheep
1. Introduction The nematophagous fungi Duddingtonia flagrans may represent a valuable tool to reduce the quantity of gastrointestinal nematode infective larvae in the pasture. The orally administered spores are meant to reach the faeces * Corresponding author. E-mail address:
[email protected] (N.F. Ojeda-Robertos). 0304-4017/$ – see front matter # 2008 Published by Elsevier B.V. doi:10.1016/j.vetpar.2008.08.022
and develop their nematode trapping structures to trap the infective larvae before they leave the faeces and reach the surrounding vegetation. To achieve that goal, a high spore density that reaches the faeces is required (Rahmann and Seip, 2007), i.e. using inundative protocols (Jaffee, 1993; Sanyal et al., 2008). According to this approach, an increased density of trapping fungi results in an increased probability of trapping parasitic nematodes (Sanyal, 2004; Sanyal et al., 2008). Thus, a considerable effort has
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been invested to define the optimal number of chlamydospores dosed per os to achieve the maximum reduction of larvae emerging from faeces of sheep (Pen˜a et al., 2002; Terril et al., 2004) or goats (Paraud et al., 2005). A higher dose was expected to produce a stronger larvae reduction effect due to a higher quantity of chlamydospores in the faeces (Faedo et al., 2002). However, previous dose titration trials could not determine the number of chlamydospores reaching the faeces. A technique to quantify chlamydospores per gram of faeces (CPG) has been recently described (Ojeda-Robertos et al., 2008). This technique provides the opportunity to study the impact of different chlamydospore doses on the number of CPG reaching the faeces and the effect of CPG on the efficacy of larvae reduction in faeces. Furthermore, it can test the relationship between CPG and the number of eggs per gram of faeces (EPG) on the reduction efficacy. Thus, the aims of this experiment were (a) to quantify the number of D. flagrans CPG recovered from sheep administered with different oral doses, and (b) to describe the relationship between CPG and EPG on the efficacy to reduce Haemonchus contortus infective larvae. 2. Materials and methods 2.1. Duddingtonia flagrans chlamydospore production The Mexican strain of D. flagrans FTHO-8 (CENIDPAVET, INIFAP) was used. Chlamydospores were produced and harvested according to Llerandi-Jua´rez and Mendoza-de-Gives (1998). Chlamydospore doses were prepared for each lamb according to the experimental group and the body weight. The doses were placed into separate plastic tubes for each animal, for each day and were kept at 4 8C, until use. 2.2. Experimental animals Twelve male hair sheep (18 kg 3.5 BW), raised nematode free, were individually housed in metabolic crates. Sheep were offered a complete diet on the basis of body weight (27 g of dry matter per kg BW). The feed consisted of star grass hay (34%), soybean meal (32%), maize meal (21%), sugar cane molasses (9%) and minerals (4%). The apparent dry matter digestibility of the diet was 70%. Absence of other nematophagous fungi in the sheep faeces was confirmed as described by Llerandi-Jua´rez and Mendoza-de-Gives (1998). Animals were artificially infected with 400 infective larvae of H. contortus per kg BW. The trial was performed when all the animals showed a patent infection.
2.3. Experimental groups On day 22 post-infection, animals were grouped according to the quantity of individual EPG and were randomly distributed to four experimental groups (n = 3). The chlamydospore doses were offered daily to sheep in each group as follows: T1 (0 chlamydospores per kg BW), T2 (1 106 chlamydospores per kg BW), T3 (2.5 106 chlamydospores per kg BW) and T4 (5 106 chlamydospores per kg BW). Four coproculture replicates were made for each animal in each treatment group (n = 12 coprocultures per group) on days 2, 4, 6, 8 and 10 of the trial. 2.4. Oral administrations of D. flagrans chlamydospores The chlamydospore suspension for the individual animal dose per day was placed in a 5 g of oat-molasses mix as described by Ojeda-Robertos et al. (2005). The doses were administered per os daily for seven days (days 0–6). During the treatment period the animals were fed as previously described and had access to fresh water. The animals in the control group (T1) received the same oat-molasses mix without chlamydospores. 2.5. Qualitative determination of chlamydospores in sheep faeces During the experimental period (days 0–12), the presence of viable chlamydospores in sheep faeces was confirmed. For this purpose, a faecal pellet was obtained daily directly from the rectum of each lamb and was placed in a petri dish with agar-water (2%). On the following day (second day of incubation) 300 H. contortus infective larvae were introduced as fungal bait to the respective petri dishes. After eight days of incubation, the surface of each petri dish was observed under the microscope (10). The criteria to declare a positive dish were: the presence of tri-dimensional fungal nets and/or trapped nematodes. A dish was declared negative when trapping structures were not present and/ or no larvae were trapped. 2.6. Quantification of nematode eggs and chlamydospores in sheep faeces Faecal samples were obtained directly from the rectum of each lamb on days 2, 4 and 6 (during the chlamydospore administration) and days 8 and 10 (postadministration period). A total quantity of 40 g of faeces was collected on the respective sampling days from
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each animal. Faeces from each lamb on the respective day were mixed and randomly divided into four replicates of 9 g. Seven grams of faeces were deposited in petri dishes (13 cm diameter) to make coprocultures (12 dishes per day per group). The other 2 g of faeces were used to determine the EPG and CPG in the faeces as described by Ojeda-Robertos et al. (2005). 2.7. Coprocultures Faeces in petri dishes were slightly crushed and stirred. The coprocultures were moistened and aerated every day for the period till incubation (eight days). Coprocultures were maintained under laboratory conditions (mean temperature 25.4 8C, mean humidity 80%). The H. contortus infective larvae were collected using a Baermann technique for 24 h. After harvesting, the infective larvae were maintained at 4 8C until further counting. Larvae counting was performed using ten aliquots of 5 ml from each culture according to the technique described by Fontenot et al. (2003). The number of larvae per gram of faeces (LPG) from each petri dish was obtained dividing the total number of larvae in each coproculture by the 7 g of faeces used. 2.8. Efficacy of three chlamydospore oral doses The efficacy of larvae reduction in each day was determined as the reduction of larvae population from the respective treated groups compared to the control group (Pen˜a et al., 2002; Paraud et al., 2005). The EPG mean number of each group and their respective LPG mean were determined on days 2, 4, 6, 8 and 10 of the experiment. The percentage of larval development (larvae yield) in each group was determined by the following formula: (LPG/EPG) 100 (Paraud et al., 2005). The larval reduction percentage was determined each day with the formula described by Terril et al. (2004): 100 (yield in the treated group (T2, T3, T4) 100/yield in the control group (T1)). The larvae yield and the larvae reduction percentage were also estimated for two periods: (a) during the chlamydospores administration (days 2, 4, and 6) and (b) after the chlamydospores administration (days 8 and 10). 2.9. Relationship between the number of CPG and EPG, and their effect on larvae reduction During the period of chlamydospore administration (days 2, 4 and 6) the number of CPG per treatment group was recorded. The CPG and H. contortus EPG, were used to determine CPG:EPG ratios, using the formula: CPG/
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EPG. The ratios calculated for each treatment group were used to explore their effect on the larvae recovery. 2.10. Data analyses 2.10.1. Efficacy of three chlamydospore oral doses Before the efficacy of the three doses was assessed, the quantity of EPG shed by animals in the different dose groups was compared. Attempts to normalize the variance of the EPG data (log transformation log10(EPG + 1)) were not successful. Thus, a Kruskal–Wallis test was used to compare the medians between groups of EPG on days 2, 4, 6, 8 and 10. The difference in the number of LPG between the control group (T1) and the treated groups (T2, T3 and T4) was analyzed with a General Linear Model procedure. This was performed on days 4, 6 and 8. Analyses included the log10(EPG + 1) as co-variable. For the analyses, the variables EPG and LPG were transformed using the Boxcox procedure (Minitab–Inc. release 12, 1998) to homogenize variances. Data of LPG on days 2 and 10 were not normalized in spite of using different approaches of data transformation. Thus, data were compared on those days using the Mann–Withney test. The difference in the number of LPG between the control group (T1) and the treated groups (T2, T3 and T4) was also compared per period: (i) during the chlamydospores administration (days 2, 4, and 6) and (ii) after the chlamydospores administration (days 8 and 10). For this purpose the same GLM procedure was used, including log10(EPG + 1) co-variable. 2.10.2. Relationship between the number of CPG and EPG, and their effect on larvae reduction CPG output and CPG:EPG ratio in the groups. The log transformed CPG data (log10(CPG + 1)) of the treated groups were compared with one-way ANOVA. Differences between group means were determined with Tukey post hoc test. The difference between the median of the ratios CPG:EPG during the days 2, 4 and 6 in the treated groups (T2,T3 and T4) were analyzed using a Kruskall–Wallis test, followed by the Dunn test for multiple comparisons (Hollander and Wolfe, 1973). Efficacy of different CPG:EPG ratios. In order to explore the effect of different CPG:EPG ratios on the reduction efficacy, four group categories were compared: zero CPG:EPG (control non treated), low CPG:EPG (from 0.09 to 4.99), medium CPG:EPG (from 5.00 to 10.00) and high CPG:EPG (from 10.10 to 55.90). The LPG obtained from the different CPG:EPG ratios were log transformed (log10(LPG + 1)) before ANOVA test was performed. This included the log
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transformed EPG data (log10(EPG + 1)) as co-variable. Post hoc comparisons were made between groups using the Tukey test. A negative binomial model was used to describe the relationship between different CPG:EPG ratios and the LPG. Only ratios different from zero were considered for the analysis. The fitted model had the following expression:
3. Results 3.1. Efficacy of three chlamydospore oral doses The presence of viable chlamydospores in the faeces of lambs dosed with chlamydospores was observed from day 1 (second day of administration) until day 9 (three days after the last chlamydospore administration). This was confirmed with the presence of tridimensional fungal nets and/or trapped nematodes in the petri dishes (Table 1). The EPG between treated groups were not statistically different in all sampling days (Table 2). The larval yields and the reduction efficacies did not show a clear dose dependent effect. The larvae yield (and the subsequent reduction efficacy) varied between sampling days (Table 2). On days 2 and 6, the larvae yields found in groups T2 and T3 were significantly lower than that of the control group. On day 4, only the T4 group showed a significantly lower larvae yield compared to the control group. On days 8 and 10 of the trial (posttreatment period) the larvae yield of the treated groups and the control were similar. In spite of the variation described above, the LPG during the chlamydospore administration period was lower in the treated groups compared with the control group ( p < 0.05). Such difference was not found during the post-treatment period as the LPG increased when the administration of chlamydospores was ceased.
y ¼ c þ expðb0 þ b1 x1 þ b2 x2 . . .Þ where y is the LPG; c the constant; b0 the Intercept; b(1,2. . .) the slopes; x(1,2. . .) is the CPG:EPG ratios. Interaction CPG, EPG and infective larvae. The interaction CPG EPG infective larvae was explored using a surface response test (Minitab–Inc. release 12, 1998). The data (CPG, EPG and LPG) from the chlamydospore administration period were used after log10(n + 1) transformation. A full cubic surface response model was initially fitted. However, only the quadratic components were significant and thus they were selected for further analyses. The final model included the main effects (EPG and CPG) quadratic effects (EPG2 and CPG2) and the interaction of main factors (EPG CPG). The fixed model with this data was: y ¼ b0 þ b1 EPG þ b2 CPG þ b3 EPG2 þ b4 CPG2 þ b5 CPG EPG; u
3.2. Relationship between the number of CPG and EPG, and their effect on larvae reduction
where y = LPG; b0 = Intercept; b1. . .5 = regression coefficients (slopes) for each effect; EPG is the Eggs per gram; CPG is the Chlamydospore per gram. All statistical analyses used an alpha of 0.05 to determine significant differences and were made with the statistical package Minitab–Inc. release 12 (1998).
3.2.1. CPG output and CPG:EPG ratio in the groups The CPG in the D. flagrans treated groups showed a clear dose dependent effect: the CPG in T2 group was
Table 1 Presence of viable Duddingtonia flagrans chlamydospores in sheep faeces dosed with three different oral doses administered during seven days (days 0–6). Chlamydospores oral doses per kg BW
Sheep
Sampling days 0
1
2
3
4
5
6
7
8
9
10
11
12
1 2 3
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ +
+ +
+
+
2.5 10 6
4 5 6
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ +
+ +
+ +
+
5 10 6
7 8 9
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ +
+ + +
+ + +
+ + +
1 10
6
(+): Presence of tri-dimensional fungal nets and/or trapped nematodes. (): Trapping structures were not present and/or no larvae were trapped.
significantly lower than that of T3. The CPG in T4 group was the highest ( p < 0.05) (Table 2). However, the CPG:EPG ratio did not reflect the dose dependent effect found with CPG (Table 3). In this case, the lowest CPG:EPG ratio was found in the T3 group, which was lower than that of T4 ( p < 0.05). The T2 showed the medium level and the T4 group had the highest CPG:EPG ratios (Table 3).
3.2.2. Efficacy of different CPG:EPG ratios Table 4 shows the reduction efficacies, compared to the control, of the four categories of CPG:EPG ratios irrespective of treatment group of origin. The reduction efficacies were 76.6, 84.5 and 32.0% for groups with low, medium and high ratios respectively. The number of larvae in the low and medium groups was lower than those in the control cultures ( p < 0.05). Surprisingly, the larvae in the control and high groups were not significantly different. This experiment showed a negative binomial relationship between the CPG:EPG ratio and the LPG: for each unit of increment in the CPG:EPG ratio there was a reduction in the number of larvae (LPG) (b1 = 0.21; p = 0.016). The fixed equation for these data was: LPG = 10.3993 + exp(6.8233 + (0.2177) CPCPG:EPG ratio).
3.2.3. Interaction CPG, EPG and infective larvae The shape of the surface response analyses formed with the CPG, EPG and LPG data predicted a quadratic increase in LPG as a result of increasing EPG. Meanwhile, increasing quantities of CPG caused a quadratic reduction in LPG. The LPG showed little change along CPG axis when the EPG was low. However, LPG decreased rapidly with CPG increments when EPG was high. The LPG data suggested a saturation effect of CPG. The LPG obtained is similar for all EPG when CPG is at high concentration. The fixed model for these data was:
Larvae number
¼ 2:1408ð1:74146Þ 0:9034ð0:9906Þ ðEPGÞ
þ 0:7793ð0:2552Þ ðCPGÞ þ 0:3202ð0:1460Þ
ðEPG EPGÞ0:0822ð0:0289Þ ðCPG CPGÞ
0:1947ð0:0720Þ ðEPG CPGÞ
ðadjusted r 2
¼ 0:402; p ¼ 0:0001Þ 156 200b 276 458a 391 548b 274 427b
LPG
1.9 2.5 4.0 9.1
0.45 7.8 3.0 3.3
38.7 0.0 0.0
91.9 44.4 79.0 83.7
CPG
Yieldb Reductionc
0.0 14.6 19.3
701 1052a 14.8 0.0 287 486b 3.5 74.9 440 363a 5.9 59.0 462 673c 9.72 56.4
LPG
7338 6773a 3953 2710 c 1654 1838a 22.5 5890 312a 163 98b 1458 1300a 24.8 6675 5408 2216 2749 1563 1584 22.7
4750 3250a 27575 6678c 8187 6395a 47008 15600c 7429 5633a 27709 4744 c 6909 5403 34392 13879
Yield b Reductionc EPG
8121 6756a 1845 2374b 968 975a 11.9 6577 7670a 27 41a 3391 5508a 51.6 7382 7084 976 1918 2127 3974 28.6
8004 5405a 20371 13209b 36 29c 12975 8368a 21292 6057b 1009 1573a 12513 7728a 17613 4657b 380 445c 11164 7425 19758 8701 474 1004 b
LPG
T4,5 106a (n = 12)
EPG, CPG: Different letters (a, b) in the same row (each day) indicate significant difference ( p < 0.05); LPG: Different letters (a, b, c) in the same row indicate significant difference compared to the mean of the control group ( p < 0.05). a Doses of chlamydospores per kg BW during seven days. b Yield: (LPG/EPG) 100. c Percentage reduction = 100 (treatment yield 100/control yield).
0.0 0.0 0.0
61.2 80.5 72.4 58.9
CPG
T3,2.5 106a (n = 12) Yieldb Reductionc EPG
850 657a 1580 2126a 24.8 39 65a 1626 3750a 49.0 518 644 1599 2818 41.8
6384 8188a 3316 5950a 5129 7361
CPG
Alter chlamydospore administration 8 15046 22810a 2874 4351a 19.1 10 13908 19427a 4033 7263a 29.0 Period 2 14477 20728 3453 5885 28.2
Yieldb EPG
8038 11519a 4913 1359a 10946 14815a 10000 6687a 9754 14021a 5650 2755a 9579 13188 6854 4712
LPG
During chlamydospore administration 2 13029 18219a 640 651a 4.9 4 16100 21278a 2252 3126a 14.0 6 18191 24677a 2631 4874a 14.5 Period 1 15693 20906 1805 3330a 22.3
EPG
T1 (n = 12)
Day
Treated groups
T2,1 106a (n = 12)
Control group
Table 2 Means (SD) of eggs per gram of faeces (EPG), chlamydospores per gram of faeces (CPG), larvae per gram of faeces (LPG), larvae yield (development larvae) and reduction percentage using three different Duddingtonia flagrans chlamydospore oral doses.
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Table 3 Arithmetic means (SD), median and range of CPG:EPG ratios in faeces of sheep treated orally during seven days with 1 106 (T2), 2.5 106 (T3) and 5 106 (T4) chlamydospores per kg BW. Treatment group
Mean ()
Median
Range
T2 (n = 36) T3 (n = 36) T4 (n = 34)
7.8 8.2 3.6 3.3 13.2 14.8
6.155a 1.460b 6.465a
0.09–31.67 0.73–13.85 1.82–55.9
Different letters (a, b) in the same column indicate significant difference ( p < 0.05).
4. Discussion Previous dose-titration trials attempted to determine the best chlamydospore oral dose to be offered to animals for the control of infective larvae in the faeces (Pen˜a et al., 2002; Paraud et al., 2005). However, those trials did not show a conclusive dose dependent effect in the efficacy to control GIN in the faeces (Ketzis et al., 2006). The present work confirms these previous results, since no clearly defined dose dependent effect pattern was observed. The present trial demonstrated that the number CPG is clearly related to the dose of chlamydospores offered per os: animals in group T2 had the lowest CPG, significantly lower than T3 and T4. Also, T3 had significantly less CPG than T4. This result confirms previous suggestions that a higher chlamydospore oral dose could result in a higher quantity of chlamydospores reaching the faeces of treated animals (Larsen et al., 1998). In spite of the clear dose dependent effect on CPG found in this experiment, the effect was not translated into a clear dose dependent effect on the reduction efficacy. This evidence is contrary to recent in vitro studies where a clear dose dependent effect was observed when chlamydospores (not exposed to digestion process) were added to faecal cultures containing nematode eggs (Sanyal, 2004; Sanyal et al., 2008). Thus, if the CPG does not seem to directly affect the number of larvae
recovered, then alternative approaches may be used to explain the chlamydospore dose response. With that in mind, CPG:EPG ratios were generated for each group. Once again, the CPG:EPG ratio from the different groups did not show a clear dose dependent effect (Table 3). The CPG:EPG ratio of the group with the lowest dose (T2) was significantly higher than T3. The reason behind this inconsistency was the variability in the number of nematode eggs (EPG) in the different groups, even when the faecal egg output of the groups was not different (the groups were adjusted for EPG to perform the trial). In order to further explore the effect of the CPG:EPG ratios on the reduction efficacy, the ratios generated from the different doses were distributed into four groups with increasing ratios. This approach made evident that a low CPG:EPG ratio significantly reduced the quantity of LPG achieving considerable larvae reduction compared to the control level. The medium CPG:EPG ratio showed a significantly lower production of larvae and a better percentage reduction. However, the highest CPG:EPG ratio did not affect the number of larvae compared to the control group. This is again different to recent studies where higher quantities of chlamydospores per egg (more than ten chlamydospores per nematode egg) resulted in a lower larvae production (Sanyal, 2004; Sanyal et al., 2008). The difference between the present results and other trials might be due to the status of chlamydospores (digested in this case vs. undigested in other studies). The results above are consistent with the regression analysis used: the CPG:EPG ratio and the larvae production showed a negative binomial relationship. This indicated that for each unit of increment in the CPG:EPG ratio, there was a reduction of larvae number until a point (between 5 and 10 CPG:EPG) where no further reduction was detected. The surface response test suggested that the number of larvae recovered was not influenced by the number of
Table 4 Larvae per gram of faeces (LPG), larvae yield (%) and larvae reduction (%) in four different ratio levels of chlamydospores per gram and eggs per gram (CPG:EPG) in sheep faeces. Group
CPG:EPG ratio
LPGa mean SE
Yield larvae (%) b
Reduction (%) c
Zero Low Medium High
0 (n = 34) 0.09 to 4.99 (n = 55) 5.00 to 10.00 (n = 25) 10.10 to 55.90 (n = 26)
411 0.33a 87 0.29b 34 0.39c 194 0.43a
22.3 5.2 3.5 15.2
76.6 84.5 32.0
Different letters (a, b, c) in the same column indicate significant difference. a Back transformed means Standard error adjusted for EPG. b Yield = (LPG/EPG) 100. c Reduction = 100 (yield with chlamydospores 100/control yield).
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eggs in the coprocultures nor by the number of CPG, but by an interaction between both variables as shown in previous trials (Sanyal, 2004; Sanyal et al., 2008). However, in the present study a new phenomenon was evident: the number of larvae was reduced in the coprocultures until a likely saturation effect of CPG was observed. This experiment suggests that it would be desirable and feasible to target the chlamydospore dose according to the quantity of EPG found in the faeces of sheep. The dose should be aimed to achieve a proportion around 5– 10 CPG per EPG in the faeces. To achieve this, it is necessary to estimate daily faecal output on any given diet and the digestibility of chlamydospores. This is important because the total faecal output varies for each diet. Also, the digestibility of chlamydospores has been estimated to be around 10% (Grønvold et al., 2004; Ojeda-Robertos et al., 2008). This trial suggested that the number of CPG can be a valuable addition towards understanding the larvae reduction achieved by D. flagrans. However, the complex relationship between EPG, CPG and LPG warrants further studies. Acknowledgments The financial support for this trial was provided by CONACYT-SAGARPA-COFUPRO, Mexico (project no. 12441). This work was part of the PhD project of Nadia F Ojeda-Robertos at the FMVZ-UADY. We also thank the technical assistance of Guilmar Ceh-Santos, Rosendo Canul-Garcı´a, Ivan Pe´rez-Herrera, Alejandra Franco-Peraza, Mario Borges-Massa, Yaremi CruzRodrı´guez, Belen Uitzil and Rosa Ofelia Valero-Coss. We are deeply indebted to Allison Singer and Andre´s Escalante-Tio´ for their help with the language corrections of the text. References Faedo, M., Larsen, M., Grønvold, J., 2002. Predacious activity of Duddingtonia flagrans within the cattle faecal pat. J. Helminthol. 76, 295–302. Fontenot, M.E., Miller, J.E., Pen˜a, M.T., Larsen, M., Gillespie, A., 2003. Efficency of feeding Duddingtonia flagrans chlamydospores to grazing ewes on reducing availability of parasitic nematode larvae on pasture. Vet. Parasitol. 118, 203–213.
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