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Evaluation of Baermann apparatus sedimentation time on recovery of Strongylus vulgaris and S. edentatus third stage larvae from equine coprocultures
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VETPAR-7622; No. of Pages 3
Veterinary Parasitology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
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Evaluation of Baermann apparatus sedimentation time on recovery of Strongylus vulgaris and S. edentatus third stage larvae from equine coprocultures Jennifer L. Bellaw ∗ , Martin K. Nielsen M. H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA
a r t i c l e
i n f o
Article history: Received 13 February 2015 Received in revised form 30 April 2015 Accepted 4 May 2015 Keywords: Baermann Coproculture Sedimentation Larvae
a b s t r a c t Traditional methods of diagnosing equine Strongylinae infections require culturing feces, sedimenting the culture media in Baermann apparatuses, collecting the sediment, and morphologically identifying recovered third stage larvae. However, this method is plagued by low negative predictive values. This study evaluated sedimentation time within the Baermann apparatus by comparing larval recovery from the traditionally collected sediment, “sediment 1”, and from the usually discarded remaining fluid contents, “sediment 2”, of the Baermann apparatus after 12, 24, and 48 h. A grand total of 147,482 larvae were recovered and examined. Sedimentation time did not significantly influence total larval recovery. At all three durations, significantly more Cyathostominae and Strongylus vulgaris larvae were covered from sediment 1 than from sediment 2. However, less than 60% of all recovered Strongylus edentatus were recovered from sediment 1. As 95% of S. vulgaris larvae were always recovered from sediment 1, the need for collection and examination of the remaining fluid contents of the Baermann apparatus is obviated when performing coprocultures for diagnosis of S. vulgaris infections, and sedimentation for 12 h is adequate. Approximately 70% of Cyathostominae were recovered in sediment 1 at all durations, suggesting that 12 h of sedimentation is adequate, although there is a need for future research to evaluate the risk of selection bias at differing sedimentation times among individual cyathostomin species. In contrast to S. vulgaris, collecting and examining the entire contents of the Baermann apparatus may be necessary when an increased diagnostic sensitivity and negative predictive value is desired in diagnosing S. edentatus infections as only 38–61% of larvae were recovered from sediment 1 portion of the Baermann apparatus. This information will allow researchers and practitioners to make more informed decisions in choosing appropriate larval recovery techniques, balancing recovery, time, and effort. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Twelve species of Strongylinae are known to parasitize the horse with Strongylus edentatus and Strongylus vulgaris being two of the most prevalent (Lichtenfels et al., 2008 Slocombe and McCraw, 1973). The Cyathostominae comprise 40 additional species parasitizing the horse (Lichtenfels et al., 2008). S. vulgaris is the most pathogenic of the Strongylinae, causing a sometimes fatal syndrome known as thromboembolic colic (McCraw and Slocombe, 1976) while S. edentatus infections are rarely pathogenic (McCraw and Slocombe, 1974). Historically, S.
∗ Corresponding author: Tel.: +1 859 218 1145; fax: +1 859 257 8542. E-mail address: [email protected] (J.L. Bellaw).
vulgaris exhibited prevalences surpassing 80% and became the primary target of parasite control (Slocombe and McCraw, 1973). Increased treatment intensities, have since resulted in S. vulgaris prevalences frequently falling below 5% and the detection limit of critical tests and coproculturing techniques (Herd, 1990) in addition to widespread resistance of Cyathostominae to the benzimidazole and pyrantel drug classes with emerging resistance to the macrocyclic lactones (Matthews, 2014). Thus, the Cyathostominae have replaced S. vulgaris as the most pathogenic nematodes of adult horses (Herd, 1990). This shift has resulted in recommending surveillance based parasite control programs to decrease treatment intensities to delay further proliferation of anthelmintic resistance (Nielsen et al., 2014). These programs are based primarily on fecal egg counts. However, the eggs of Strongylinae and Cyathostominae cannot be morphologically differentiated (Hummelinck, 1946). Recent
http://dx.doi.org/10.1016/j.vetpar.2015.05.001 0304-4017/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Bellaw, J.L., Nielsen, M.K., Evaluation of Baermann apparatus sedimentation time on recovery of Strongylus vulgaris and S. edentatus third stage larvae from equine coprocultures. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.001
G Model VETPAR-7622; No. of Pages 3
ARTICLE IN PRESS J.L. Bellaw, M.K. Nielsen / Veterinary Parasitology xxx (2015) xxx–xxx
2
research has illustrated that strict reliance on fecal egg counts and a subsequent inability to diagnose Strongylinae infections has the potential to lead to the reemergence of S. vulgaris, and other Strongylinae, within evidence based programs (Nielsen et al., 2012). Consequently, routine monitoring of S. vulgaris infections is recommended in such programs, and performance of coprocultures is now an established practice for a large proportion of Danish, equine practitioners and are likely to be more frequently implemented in other countries now adopting similar programs (Hertzberg et al., 2014; Nielsen et al., 2014). In evaluation against necropsy data, larval cultures performed for the diagnosis of S. vulgaris and S. edentatus infections were characterized by >95% positive predictive values and low to moderate (37% and 60%, respectively) negative predictive values, allowing for frequent false negatives (Nielsen et al., 2010). However, no studies have evaluated the effect of sedimentation time in the Baermann apparatus on recovery of S. vulgaris and S. edentatus from equine coprocultures for use in diagnosing Strongylinae infections. The primary objective of this study was to evaluate the effect of sedimentation time within the Baermann apparatus on larval recovery of two Strongylus species, providing useful information on which to base recommendations for the performance of coprocultures for the diagnosis of Strongylus spp. infections.
2. Materials and methods 2.1. Feces Feces were collected from two mares, residing in the parasitology research herd at the University of Kentucky’s Maine Chance Farm, the gastrointestinal parasite community of which has been well described (Nielsen et al., 2010). Feces were collected from clean stall floors and divided into several 20 g samples, yielding a total of 27 and 18 samples, respectively.
2.2. Coproculture Individual fecal samples were mixed with equal volumes of vermiculite (Infinity Fertilizers, Inc. Milan, Illinois, USA) and moistened with tap water. Fecal mixtures were suspended in humidity chambers as described by Henriksen and Korsholm (1983). These chambers were incubated at ∼25 ◦ C for 14 days and moistened as necessary. Fifteen of these fecal mixtures, balanced between the two horses, were then sedimented in a Baermann apparatus for each of 12, 24, and 48 h, respectively. Baermann apparatuses consisted of wine glasses containing reservoirs in their stems.
Table 1 Mean percentage ± 95% CI of total harvested Cyathostominae, Strongylus vulgaris, and S. edentatus third stage larvae recovered within sediment 1 of the Baermann apparatus at three sedimentation times. Hours of sedimentation Species Cyathostominae S. vulgaris S. edentatus
12
24
48
70 ± 7 94 ± 4 56 ± 10
67 ± 11 93 ± 4 38 ± 11
74 ± 10 95 ± 2 61 ± 14
p-Values are presented in bold. Significance considered at the 0.05 level.
2.4. Larval identification Each pellet was re-suspended in tap water and transferred to a nematode counting chamber (Chalex Corp. Ketchum, ID, USA). To inactivate larvae, each nematode slide, filled to ∼80% capacity, was then placed on a hot plate at 55 ◦ C for approximately 3 min. Nematode slides were examined at 100×, and all larvae were examined and identified to stage, genera, and species where applicable, according to Russell (1948). Larval counts were recorded separately for sediment 1 and sediment 2 for each replicate. 2.5. Data analysis Statistical analyses were performed using SAS software (version 9.3, SAS Institute, Cary, North Carolina, USA). Prior to analysis, data were evaluated for normal distribution with Shapiro–Wilk and Kolmogorov–Smirnov statistics as well as normal probability plots and were found to fulfill the criteria for normal distribution. Mixed linear models were constructed for analyzing the relationship between the range of total third stage larvae (L3) recovered, total numbers of Cyathostominae, S. vulgaris, and S. edentatus, sedimentation time, and portion of Baermann apparatus collected. The ‘mixed’ procedure with repeated measures was used with ‘Horse,’ as a random effect. ‘Horse,’ sedimentation time: ‘Time’ (12, 24, 48 h), portion of Baermann apparatus collected: ‘1/2’ (Sediment 1/Sediment 2), and range of total L3 recovered: ‘L3 Range’ (Low: 0–2,999; High: 3000+) were kept as class variables, while all other variables were considered continuous. Analyses took into account the influence of ‘Time,’ ‘1/2’, and ‘L3 range.’ For each analysis, the influence of all measured parameters was evaluated using traditional forward and backward elimination of variables. Whenever the variable of ‘1/2’ or ‘L3 Range’ was found significant, a ‘least square means’ analysis was used for a Tukey’s pair-wise comparison and interpreted at the ˛ = 0.05 level of the adjusted ‘least squares means’. Linear correlations between counts of S. vulgaris, S. edentatus, and Cyathostominae were evaluated using the correlation procedure in SAS. All analyses were interpreted at the ˛ = 0.05 level.
2.3. Larval harvest 3. Results After their 12, 24, and 48 h respective sedimentation times, the fecal mixtures were removed from their Baermann apparatuses. The entire sediment at a minimum of ∼5 mL was collected, pipetting until the entire, visible sediment was removed. The sediment was then centrifuged at ∼200 × g for 10 min. These traditionally collected samples, identified as “sediment 1”, were stored at 4 ◦ C until examined within 14 days of harvest. The remaining contents of each Baermann apparatus were centrifuged in 50 mL tubes at ∼200 × g for 10 min. The supernatant was discarded and the pellets transferred to a single 15 mL tube. These tubes, identified as “sediment 2”, were then centrifuged, and stored as described above. This yielded a sediment 1 and a sediment 2 for each given fecal culture.
A grand total of 147,482 larvae were collected and examined during the study. A total of 145,427 L3 were recovered, including 142,725 Cyathostominae; 1194 S. edentatus; 1486 S. vulgaris; 12 Trichostrongylus axei; 6 Triodontophorus spp.; and 4 Poteriostomum spp. larvae. S. vulgaris and S. edentatus comprised ∼ 1% of larvae recovered. The percentage of total recovered Cyathostominae, S. edentatus, and S. vulgaris larvae recovered from the traditionally collected sediment 1 are presented in Table 1 for each sedimentation time. Duration of sedimentation did not have a significant impact on the total number of larvae harvested from the entire Baermann apparatus, nor was there any significant influence of time on the total number of larvae found in sediment 1.
Please cite this article in press as: Bellaw, J.L., Nielsen, M.K., Evaluation of Baermann apparatus sedimentation time on recovery of Strongylus vulgaris and S. edentatus third stage larvae from equine coprocultures. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.001
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J.L. Bellaw, M.K. Nielsen / Veterinary Parasitology xxx (2015) xxx–xxx Table 2 Linear Pearson correlation coefficients calculated between total numbers of Cyathostominae, Strongylus vulgaris, and S. edentatus third stage larvae recovered from the entire Baermann apparatus. p-Values presented in bold. S. edentatus total
S. vulgaris total
Cyathostominae total
S. ede total
1.00
S. vulgaris total
0.15 0.16 0.45 < .0001
0.15 0.16 1.00
0.45 < .0001 0.75 < .0001 1.00
Cyathostominae total
0.75 < .0001
There were significantly more Cyathostominae (p = 0.0061) and S. vulgaris (p < 0.0001) larvae recovered from sediment 1 than from sediment 2 at all sedimentation times. However, there was no significant difference between the numbers of S. edentatus larvae recovered in sediment 1 and sediment 2 at any sedimentation time, nor was there an appreciable trend towards an increased percentage of S. edentatus larvae being recovered in sediment 1 over time (Table 1). Linear correlation analyses between total counts of S. vulgaris, S. edentatus, and Cyathostominae revealed moderate to strong, positive, linear correlations between the total number of both Strongylus species and the total number of Cyathostominae (p < 0.0001) (Table 2). However, there was no linear correlation between the total numbers of S. edentatus and S. vulgaris (p = 0.16) (Table 2). 4. Discussion The vast majority of larvae exited the culture media by the first 12 h, allowing only a negligible increase of total harvested larvae with increased sedimentation time. Therefore, a traditional sedimentation duration of at least 12 h appears more than adequate for total larval recovery of Cyathostominae and both Strongylus species. Longer sedimentation times did not result in higher percentages of total harvested larvae being recovered within sediment 1 rather than sediment 2 (Table 1). Nearly 100% of S. vulgaris larvae completely sedimented and were collected within sediment 1 by the first 12 h, and 70% of the total harvested Cyathostominae larvae were recovered in sediment 1 at all three sedimentation durations (Table 1). Thus, solely collecting the traditional sediment 1 is sufficient for a pragmatic harvest of Cyathostominae and S. vulgaris larvae. However, only 38–62% of S. edentatus larvae were recovered from sediment 1 at any sedimentation time (Table 1). S. edentatus larvae are often observed to be faster and more mobile than other larvae recovered from coprocultures (Cernea et al., 2008). It may be possible that S. edentatus are mobile enough to swim above the pellet of sediment 1 for a considerable amount of time. In order to more accurately diagnose S. edentatus infections and more efficiently collect their larvae, it is recommended that the entire Baermann contents be examined after a minimum of 12 h of sedimentation. Future studies should examine sedimentation times exceeding 48 h to determine when the majority of S. edentatus larvae have completely sedimented. Ultimately, future studies should examine individual Strongylid species harvested from pure cultures and determine the relationship between size, sedimentation time, and sedimentation efficiency in order to fully describe the risk of selection bias in the use of the Baermann technique to recover L3. Linear correlation analyses revealed moderate to strong, positive linear correlations between the total number of both Strongylus
3
species and the total number of Cyathostominae (p < 0.0001) (Table 2). This is expected as the number of Cyathostominae constitutes nearly 98% of the total L3 population and, therefore, when the number of Cyathostominae or total population increases, the total number of Strongylus spp. would also increase. As modifications were made to accommodate the sheer number of harvested larvae, including diluting some samples 10:1 before examination, the total number of larvae is not thought to have interfered with accurately identifying Strongylus larvae. Interestingly, there was no linear correlation between the total numbers of S. edentatus and S. vulgaris (p = 0.16) (Table 2). In summary, the vast majority of S. vulgaris larvae were always recovered from the sediment, obviating the need for collection and examination of the remaining fluid contents of the Baermann apparatus when performing coprocultures for diagnosis of S. vulgaris infections. In contrast, collecting and examining the entire contents of the Baermann apparatus may be necessary when an increased sensitivity and negative predictive value is desired in diagnosing S. edentatus infections. This information should allow researchers and practitioners alike to make more informed decisions in choosing appropriate larval recovery techniques, balancing recovery, time, and effort. Conflict of interest The authors declare no conflict of interest. Acknowledgment The authors are grateful to Dr. L.M. Madeira de Carvalho for his generosity in providing a multitude of useful literature. References Cernea, M., Madeira de Carvalho, L.M., Cozma, V., 2008. Atlas de diagnostic al strongilidozelor la ecvine. Academic Press, Cluj-Napoca, pp. 118. Henriksen, Sv.Aa., Korsholm, H., 1983. A method for culture and recovery of gastrointestinal strongyle larvae. Nord. Vet. Med. 35, 429–430. Herd, R.P., 1990. The changing world of worms: the rise of the cyathostomes and the decline of Strongylus vulgaris. Compend. Cont. Educ. Pract. Vet. 12, 732–734. Hertzberg, H., Schwarzwald, S.S., Grimm, F., Frey, C.F., Gottstein, B., Gerber, V., 2014. Helminth control in the adult horse: the need for a re-orientation. Schweiz. Arch. Tierheilkund. 156, 61–70. Lichtenfels, J.R., Kharchenko, V.A., Dvojnos, G.M., 2008. Illustrated identification keys to strongylid parasites (strongylidae: Nematoda) of horses, zebras and asses (Equidae). Vet. Parasitol. 156, 4–161. Matthews, J.B., 2014. Anthelmintic resistance in equine nematodes. Int. J. Parasitol. 4, 310–315. McCraw, B.M., Slocombe, J.D., 1974. Early development of and pathology associated with Strongylus edentatus. Can. J. Comp. Med. 38, 124–138. McCraw, B.M., Slocombe, J.D., 1976. Strongylus vulgaris in the horse: a review Strongylus vulgaris in the horse: a review. Can. Vet. J. 17, 150–157. Nielsen, M.K., Baptiste, K.E., Tolliver, S.C., Collins, S.S., Lyons, E.T., 2010. Analysis of multiyear studies in horses in Kentucky to ascertain whether counts of eggs and larvae per gram of feces are reliable indicators of numbers of strongyles and ascarids present. Vet. Parasitol. 174, 77–84. Nielsen, M.K., Vidyashankar, A.N., Olsen, S.N., Monrad, J., Thamsborg, S.M., 2012. Strongylus vulgaris associated with usage of selective therapy on Danish horse farms – is it reemerging? Vet. Parasitol. 189, 260–266. Nielsen, M.K., Pfister, K., von Samson-Himmelstjerna, G., 2014. Selective therapy in equine parasite control–application and limitations. Vet. Parasitol. 202, 95–103. Russell, A.F., 1948. The development of helminthiasis in Thoroughbred foals. J. Comp. Pathol. Ther. 58, 107–127. Slocombe, J.O.D., McCraw, B.M., 1973. Gastrointestinal nematodes of horses in Ontario. Can. Vet. J. 14, 101–105.
Please cite this article in press as: Bellaw, J.L., Nielsen, M.K., Evaluation of Baermann apparatus sedimentation time on recovery of Strongylus vulgaris and S. edentatus third stage larvae from equine coprocultures. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.001
ARTICLE IN PRESS
VETPAR-7622; No. of Pages 3
Veterinary Parasitology xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Evaluation of Baermann apparatus sedimentation time on recovery of Strongylus vulgaris and S. edentatus third stage larvae from equine coprocultures Jennifer L. Bellaw ∗ , Martin K. Nielsen M. H. Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY, USA
a r t i c l e
i n f o
Article history: Received 13 February 2015 Received in revised form 30 April 2015 Accepted 4 May 2015 Keywords: Baermann Coproculture Sedimentation Larvae
a b s t r a c t Traditional methods of diagnosing equine Strongylinae infections require culturing feces, sedimenting the culture media in Baermann apparatuses, collecting the sediment, and morphologically identifying recovered third stage larvae. However, this method is plagued by low negative predictive values. This study evaluated sedimentation time within the Baermann apparatus by comparing larval recovery from the traditionally collected sediment, “sediment 1”, and from the usually discarded remaining fluid contents, “sediment 2”, of the Baermann apparatus after 12, 24, and 48 h. A grand total of 147,482 larvae were recovered and examined. Sedimentation time did not significantly influence total larval recovery. At all three durations, significantly more Cyathostominae and Strongylus vulgaris larvae were covered from sediment 1 than from sediment 2. However, less than 60% of all recovered Strongylus edentatus were recovered from sediment 1. As 95% of S. vulgaris larvae were always recovered from sediment 1, the need for collection and examination of the remaining fluid contents of the Baermann apparatus is obviated when performing coprocultures for diagnosis of S. vulgaris infections, and sedimentation for 12 h is adequate. Approximately 70% of Cyathostominae were recovered in sediment 1 at all durations, suggesting that 12 h of sedimentation is adequate, although there is a need for future research to evaluate the risk of selection bias at differing sedimentation times among individual cyathostomin species. In contrast to S. vulgaris, collecting and examining the entire contents of the Baermann apparatus may be necessary when an increased diagnostic sensitivity and negative predictive value is desired in diagnosing S. edentatus infections as only 38–61% of larvae were recovered from sediment 1 portion of the Baermann apparatus. This information will allow researchers and practitioners to make more informed decisions in choosing appropriate larval recovery techniques, balancing recovery, time, and effort. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Twelve species of Strongylinae are known to parasitize the horse with Strongylus edentatus and Strongylus vulgaris being two of the most prevalent (Lichtenfels et al., 2008 Slocombe and McCraw, 1973). The Cyathostominae comprise 40 additional species parasitizing the horse (Lichtenfels et al., 2008). S. vulgaris is the most pathogenic of the Strongylinae, causing a sometimes fatal syndrome known as thromboembolic colic (McCraw and Slocombe, 1976) while S. edentatus infections are rarely pathogenic (McCraw and Slocombe, 1974). Historically, S.
∗ Corresponding author: Tel.: +1 859 218 1145; fax: +1 859 257 8542. E-mail address: [email protected] (J.L. Bellaw).
vulgaris exhibited prevalences surpassing 80% and became the primary target of parasite control (Slocombe and McCraw, 1973). Increased treatment intensities, have since resulted in S. vulgaris prevalences frequently falling below 5% and the detection limit of critical tests and coproculturing techniques (Herd, 1990) in addition to widespread resistance of Cyathostominae to the benzimidazole and pyrantel drug classes with emerging resistance to the macrocyclic lactones (Matthews, 2014). Thus, the Cyathostominae have replaced S. vulgaris as the most pathogenic nematodes of adult horses (Herd, 1990). This shift has resulted in recommending surveillance based parasite control programs to decrease treatment intensities to delay further proliferation of anthelmintic resistance (Nielsen et al., 2014). These programs are based primarily on fecal egg counts. However, the eggs of Strongylinae and Cyathostominae cannot be morphologically differentiated (Hummelinck, 1946). Recent
http://dx.doi.org/10.1016/j.vetpar.2015.05.001 0304-4017/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Bellaw, J.L., Nielsen, M.K., Evaluation of Baermann apparatus sedimentation time on recovery of Strongylus vulgaris and S. edentatus third stage larvae from equine coprocultures. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.001
G Model VETPAR-7622; No. of Pages 3
ARTICLE IN PRESS J.L. Bellaw, M.K. Nielsen / Veterinary Parasitology xxx (2015) xxx–xxx
2
research has illustrated that strict reliance on fecal egg counts and a subsequent inability to diagnose Strongylinae infections has the potential to lead to the reemergence of S. vulgaris, and other Strongylinae, within evidence based programs (Nielsen et al., 2012). Consequently, routine monitoring of S. vulgaris infections is recommended in such programs, and performance of coprocultures is now an established practice for a large proportion of Danish, equine practitioners and are likely to be more frequently implemented in other countries now adopting similar programs (Hertzberg et al., 2014; Nielsen et al., 2014). In evaluation against necropsy data, larval cultures performed for the diagnosis of S. vulgaris and S. edentatus infections were characterized by >95% positive predictive values and low to moderate (37% and 60%, respectively) negative predictive values, allowing for frequent false negatives (Nielsen et al., 2010). However, no studies have evaluated the effect of sedimentation time in the Baermann apparatus on recovery of S. vulgaris and S. edentatus from equine coprocultures for use in diagnosing Strongylinae infections. The primary objective of this study was to evaluate the effect of sedimentation time within the Baermann apparatus on larval recovery of two Strongylus species, providing useful information on which to base recommendations for the performance of coprocultures for the diagnosis of Strongylus spp. infections.
2. Materials and methods 2.1. Feces Feces were collected from two mares, residing in the parasitology research herd at the University of Kentucky’s Maine Chance Farm, the gastrointestinal parasite community of which has been well described (Nielsen et al., 2010). Feces were collected from clean stall floors and divided into several 20 g samples, yielding a total of 27 and 18 samples, respectively.
2.2. Coproculture Individual fecal samples were mixed with equal volumes of vermiculite (Infinity Fertilizers, Inc. Milan, Illinois, USA) and moistened with tap water. Fecal mixtures were suspended in humidity chambers as described by Henriksen and Korsholm (1983). These chambers were incubated at ∼25 ◦ C for 14 days and moistened as necessary. Fifteen of these fecal mixtures, balanced between the two horses, were then sedimented in a Baermann apparatus for each of 12, 24, and 48 h, respectively. Baermann apparatuses consisted of wine glasses containing reservoirs in their stems.
Table 1 Mean percentage ± 95% CI of total harvested Cyathostominae, Strongylus vulgaris, and S. edentatus third stage larvae recovered within sediment 1 of the Baermann apparatus at three sedimentation times. Hours of sedimentation Species Cyathostominae S. vulgaris S. edentatus
12
24
48
70 ± 7 94 ± 4 56 ± 10
67 ± 11 93 ± 4 38 ± 11
74 ± 10 95 ± 2 61 ± 14
p-Values are presented in bold. Significance considered at the 0.05 level.
2.4. Larval identification Each pellet was re-suspended in tap water and transferred to a nematode counting chamber (Chalex Corp. Ketchum, ID, USA). To inactivate larvae, each nematode slide, filled to ∼80% capacity, was then placed on a hot plate at 55 ◦ C for approximately 3 min. Nematode slides were examined at 100×, and all larvae were examined and identified to stage, genera, and species where applicable, according to Russell (1948). Larval counts were recorded separately for sediment 1 and sediment 2 for each replicate. 2.5. Data analysis Statistical analyses were performed using SAS software (version 9.3, SAS Institute, Cary, North Carolina, USA). Prior to analysis, data were evaluated for normal distribution with Shapiro–Wilk and Kolmogorov–Smirnov statistics as well as normal probability plots and were found to fulfill the criteria for normal distribution. Mixed linear models were constructed for analyzing the relationship between the range of total third stage larvae (L3) recovered, total numbers of Cyathostominae, S. vulgaris, and S. edentatus, sedimentation time, and portion of Baermann apparatus collected. The ‘mixed’ procedure with repeated measures was used with ‘Horse,’ as a random effect. ‘Horse,’ sedimentation time: ‘Time’ (12, 24, 48 h), portion of Baermann apparatus collected: ‘1/2’ (Sediment 1/Sediment 2), and range of total L3 recovered: ‘L3 Range’ (Low: 0–2,999; High: 3000+) were kept as class variables, while all other variables were considered continuous. Analyses took into account the influence of ‘Time,’ ‘1/2’, and ‘L3 range.’ For each analysis, the influence of all measured parameters was evaluated using traditional forward and backward elimination of variables. Whenever the variable of ‘1/2’ or ‘L3 Range’ was found significant, a ‘least square means’ analysis was used for a Tukey’s pair-wise comparison and interpreted at the ˛ = 0.05 level of the adjusted ‘least squares means’. Linear correlations between counts of S. vulgaris, S. edentatus, and Cyathostominae were evaluated using the correlation procedure in SAS. All analyses were interpreted at the ˛ = 0.05 level.
2.3. Larval harvest 3. Results After their 12, 24, and 48 h respective sedimentation times, the fecal mixtures were removed from their Baermann apparatuses. The entire sediment at a minimum of ∼5 mL was collected, pipetting until the entire, visible sediment was removed. The sediment was then centrifuged at ∼200 × g for 10 min. These traditionally collected samples, identified as “sediment 1”, were stored at 4 ◦ C until examined within 14 days of harvest. The remaining contents of each Baermann apparatus were centrifuged in 50 mL tubes at ∼200 × g for 10 min. The supernatant was discarded and the pellets transferred to a single 15 mL tube. These tubes, identified as “sediment 2”, were then centrifuged, and stored as described above. This yielded a sediment 1 and a sediment 2 for each given fecal culture.
A grand total of 147,482 larvae were collected and examined during the study. A total of 145,427 L3 were recovered, including 142,725 Cyathostominae; 1194 S. edentatus; 1486 S. vulgaris; 12 Trichostrongylus axei; 6 Triodontophorus spp.; and 4 Poteriostomum spp. larvae. S. vulgaris and S. edentatus comprised ∼ 1% of larvae recovered. The percentage of total recovered Cyathostominae, S. edentatus, and S. vulgaris larvae recovered from the traditionally collected sediment 1 are presented in Table 1 for each sedimentation time. Duration of sedimentation did not have a significant impact on the total number of larvae harvested from the entire Baermann apparatus, nor was there any significant influence of time on the total number of larvae found in sediment 1.
Please cite this article in press as: Bellaw, J.L., Nielsen, M.K., Evaluation of Baermann apparatus sedimentation time on recovery of Strongylus vulgaris and S. edentatus third stage larvae from equine coprocultures. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.001
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J.L. Bellaw, M.K. Nielsen / Veterinary Parasitology xxx (2015) xxx–xxx Table 2 Linear Pearson correlation coefficients calculated between total numbers of Cyathostominae, Strongylus vulgaris, and S. edentatus third stage larvae recovered from the entire Baermann apparatus. p-Values presented in bold. S. edentatus total
S. vulgaris total
Cyathostominae total
S. ede total
1.00
S. vulgaris total
0.15 0.16 0.45 < .0001
0.15 0.16 1.00
0.45 < .0001 0.75 < .0001 1.00
Cyathostominae total
0.75 < .0001
There were significantly more Cyathostominae (p = 0.0061) and S. vulgaris (p < 0.0001) larvae recovered from sediment 1 than from sediment 2 at all sedimentation times. However, there was no significant difference between the numbers of S. edentatus larvae recovered in sediment 1 and sediment 2 at any sedimentation time, nor was there an appreciable trend towards an increased percentage of S. edentatus larvae being recovered in sediment 1 over time (Table 1). Linear correlation analyses between total counts of S. vulgaris, S. edentatus, and Cyathostominae revealed moderate to strong, positive, linear correlations between the total number of both Strongylus species and the total number of Cyathostominae (p < 0.0001) (Table 2). However, there was no linear correlation between the total numbers of S. edentatus and S. vulgaris (p = 0.16) (Table 2). 4. Discussion The vast majority of larvae exited the culture media by the first 12 h, allowing only a negligible increase of total harvested larvae with increased sedimentation time. Therefore, a traditional sedimentation duration of at least 12 h appears more than adequate for total larval recovery of Cyathostominae and both Strongylus species. Longer sedimentation times did not result in higher percentages of total harvested larvae being recovered within sediment 1 rather than sediment 2 (Table 1). Nearly 100% of S. vulgaris larvae completely sedimented and were collected within sediment 1 by the first 12 h, and 70% of the total harvested Cyathostominae larvae were recovered in sediment 1 at all three sedimentation durations (Table 1). Thus, solely collecting the traditional sediment 1 is sufficient for a pragmatic harvest of Cyathostominae and S. vulgaris larvae. However, only 38–62% of S. edentatus larvae were recovered from sediment 1 at any sedimentation time (Table 1). S. edentatus larvae are often observed to be faster and more mobile than other larvae recovered from coprocultures (Cernea et al., 2008). It may be possible that S. edentatus are mobile enough to swim above the pellet of sediment 1 for a considerable amount of time. In order to more accurately diagnose S. edentatus infections and more efficiently collect their larvae, it is recommended that the entire Baermann contents be examined after a minimum of 12 h of sedimentation. Future studies should examine sedimentation times exceeding 48 h to determine when the majority of S. edentatus larvae have completely sedimented. Ultimately, future studies should examine individual Strongylid species harvested from pure cultures and determine the relationship between size, sedimentation time, and sedimentation efficiency in order to fully describe the risk of selection bias in the use of the Baermann technique to recover L3. Linear correlation analyses revealed moderate to strong, positive linear correlations between the total number of both Strongylus
3
species and the total number of Cyathostominae (p < 0.0001) (Table 2). This is expected as the number of Cyathostominae constitutes nearly 98% of the total L3 population and, therefore, when the number of Cyathostominae or total population increases, the total number of Strongylus spp. would also increase. As modifications were made to accommodate the sheer number of harvested larvae, including diluting some samples 10:1 before examination, the total number of larvae is not thought to have interfered with accurately identifying Strongylus larvae. Interestingly, there was no linear correlation between the total numbers of S. edentatus and S. vulgaris (p = 0.16) (Table 2). In summary, the vast majority of S. vulgaris larvae were always recovered from the sediment, obviating the need for collection and examination of the remaining fluid contents of the Baermann apparatus when performing coprocultures for diagnosis of S. vulgaris infections. In contrast, collecting and examining the entire contents of the Baermann apparatus may be necessary when an increased sensitivity and negative predictive value is desired in diagnosing S. edentatus infections. This information should allow researchers and practitioners alike to make more informed decisions in choosing appropriate larval recovery techniques, balancing recovery, time, and effort. Conflict of interest The authors declare no conflict of interest. Acknowledgment The authors are grateful to Dr. L.M. Madeira de Carvalho for his generosity in providing a multitude of useful literature. References Cernea, M., Madeira de Carvalho, L.M., Cozma, V., 2008. Atlas de diagnostic al strongilidozelor la ecvine. Academic Press, Cluj-Napoca, pp. 118. Henriksen, Sv.Aa., Korsholm, H., 1983. A method for culture and recovery of gastrointestinal strongyle larvae. Nord. Vet. Med. 35, 429–430. Herd, R.P., 1990. The changing world of worms: the rise of the cyathostomes and the decline of Strongylus vulgaris. Compend. Cont. Educ. Pract. Vet. 12, 732–734. Hertzberg, H., Schwarzwald, S.S., Grimm, F., Frey, C.F., Gottstein, B., Gerber, V., 2014. Helminth control in the adult horse: the need for a re-orientation. Schweiz. Arch. Tierheilkund. 156, 61–70. Lichtenfels, J.R., Kharchenko, V.A., Dvojnos, G.M., 2008. Illustrated identification keys to strongylid parasites (strongylidae: Nematoda) of horses, zebras and asses (Equidae). Vet. Parasitol. 156, 4–161. Matthews, J.B., 2014. Anthelmintic resistance in equine nematodes. Int. J. Parasitol. 4, 310–315. McCraw, B.M., Slocombe, J.D., 1974. Early development of and pathology associated with Strongylus edentatus. Can. J. Comp. Med. 38, 124–138. McCraw, B.M., Slocombe, J.D., 1976. Strongylus vulgaris in the horse: a review Strongylus vulgaris in the horse: a review. Can. Vet. J. 17, 150–157. Nielsen, M.K., Baptiste, K.E., Tolliver, S.C., Collins, S.S., Lyons, E.T., 2010. Analysis of multiyear studies in horses in Kentucky to ascertain whether counts of eggs and larvae per gram of feces are reliable indicators of numbers of strongyles and ascarids present. Vet. Parasitol. 174, 77–84. Nielsen, M.K., Vidyashankar, A.N., Olsen, S.N., Monrad, J., Thamsborg, S.M., 2012. Strongylus vulgaris associated with usage of selective therapy on Danish horse farms – is it reemerging? Vet. Parasitol. 189, 260–266. Nielsen, M.K., Pfister, K., von Samson-Himmelstjerna, G., 2014. Selective therapy in equine parasite control–application and limitations. Vet. Parasitol. 202, 95–103. Russell, A.F., 1948. The development of helminthiasis in Thoroughbred foals. J. Comp. Pathol. Ther. 58, 107–127. Slocombe, J.O.D., McCraw, B.M., 1973. Gastrointestinal nematodes of horses in Ontario. Can. Vet. J. 14, 101–105.
Please cite this article in press as: Bellaw, J.L., Nielsen, M.K., Evaluation of Baermann apparatus sedimentation time on recovery of Strongylus vulgaris and S. edentatus third stage larvae from equine coprocultures. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.05.001