Comparison of three different preservatives for ...

Veterinary Parasitology 145 (2007) 361–365 www.elsevier.com/locate/vetpar

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Comparison of three different preservatives for morphological and real-time PCR analyses of Haemonchus contortus eggs Aaron F. Harmon a, Zachary B. Williams a, Larry D. Holler b, Michael B. Hildreth a,b,* a

Department of Biology & Microbiology, South Dakota State University, Brookings, SD 57007, USA b Department of Veterinary Sciences, South Dakota State University, Brookings, SD 57007, USA

Received 19 October 2006; received in revised form 7 December 2006; accepted 11 December 2006

Abstract Despite the development of several recent PCR assays for the egg stages of various trichostrongyles, there have been no protocols described for preserving field samples for PCR without refrigeration. In this study, Lugol’s iodine (LI), sodium azide (SA), and neutral buffered formalin (NBF) were evaluated using Haemonchus contortus eggs to determine their potential as a preservative for trichostrongyle egg samples to be processed with real-time PCR. When egg recovery, embryo development, and egg morphology were evaluated from fecal samples preserved with LI, NBF, and SA, there was equally good recovery and preservation for the first month. Preserved eggs were detectable for 1 month, but after 6 months, none could be recovered. When real-time PCR analysis was performed on eggs isolated from faeces preserved with LI and SA, there was no detectable inhibition compared to fresh, non-preserved eggs; however, NBF significantly inhibited amplification. The results from this study demonstrate that for PCR applications LI and SA are effective preservatives for H. contortus eggs, resulting in good preservation of morphology while allowing for uninhibited PCR. # 2006 Elsevier B.V. All rights reserved. Keywords: Lugol’s iodine; Haemonchus contortus; Egg preservation; Trichostrongyles; Real-time PCR; Sodium azide

1. Introduction The advent of PCR has lead to the development of several diagnostic assays that target the various stages of parasitic nematodes. Several egg-based PCR assays have been designed including those for ruminant trichostrongyles (Zarlenga et al., 1998; Schnieder et al., 1999; Zarlenga et al., 2001; Von Samson-Himmelstjerna et al., 2002; Mochizuki et al., 2006). Though numerous studies have focused on assay development, little work has been

* Corresponding author at: Department of Biology & Microbiology, NPB rm 252 Rotunda Ln, South Dakota State University, Brookings, SD 57007, USA. Tel.: +1 605 688 4562; fax: +1 605 688 5624. E-mail address: [email protected] (M.B. Hildreth). 0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2006.12.011

done to evaluate the appropriate manner in which to preserve eggs prior to PCR or real-time PCR analysis. Furthermore, a recent study (Harmon et al., 2007) has demonstrated that larval development within trichostrongyle eggs can interfere with quantification by realtime PCR (QPCR), emphasizing the need for PCRsensitive preservation protocols. Reported protocols for egg preservation have required either chemical treatment or refrigeration. Both formalin and sodium azide have been described as effective preservatives for strongyle-like eggs (Bundy et al., 1985; Foreyt, 1986; Walhquist and Eberhard, 1991). Alternatively, Lugol’s iodine (LI) has been used to fix trichostrongyle larvae and stain intestinal cells prior to identification (Roberts and O’Sullivan, 1949; van Wyk et al., 2004). It is possible that LI may also

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prevent larval development within the eggshell, allowing it to serve as an effective preservative as well. Though refrigeration is an effective means of preserving nematode eggs (Foreyt, 1986), refrigeration of field isolates is not practical in certain locations; furthermore, development is shown to continue in the eggs of Ostertagia ostertagi even at 4 8C (Christie and Jackson, 1982). To meet the need of current DNA extraction protocols for trichostrongyle eggs (Harmon et al., 2006), preservatives should allow for: preservation of egg morphology, normal egg recovery through fecal flotation, and uninhibited PCR with egg-derived DNA. The purpose of this study was to evaluate the effectiveness of LI, NBF, and SA as preservatives for Haemonchus contortus eggs. The effectiveness of these preservatives was tested in the context of preserving egg morphology, stopping larval development, and allowing for uninhibited real-time PCR.

2.3. Preservation of faecal material

2. Materials and methods

2.4. Chemical preservation impact on QPCR

2.1. H. contortus eggs and preservatives

To compare the impact of these preservatives on QPCR, freshly isolated eggs were divided into five replicates of three groups each containing 25 eggs. Isolated eggs were submerged with 100 ml of the respective preservative in a 2 ml tube and allowed to remain at room temperature for 7 days. An additional group was used immediately as a control with no preservation to determine expected CT values. To prevent loss of DNA from degraded eggs and allow for minor inhibition to be detected, preserved eggs were not washed prior to DNA extraction. QPCR was then performed as described below and CT values used to assess inhibition. During faecal flotation, preservatives would be greatly diluted, potentially reducing any inhibitory effect preservatives may have on QPCR. To determine if egg flotation would diminish any inhibitive effect, 1 g samples of homogenized faecal material were aliquoted into 15 ml tubes so that there would be five replicates for each preservative. Samples were then treated with 2 ml of the respective preservative. After 7 days of storage, flotation was performed followed by DNA extraction and QPCR. Egg numbers were compared to corresponding CT values to determine if inhibition had taken place. Comparisons were made with treated eggs using a standard curve described elsewhere in a study demonstrating real-time PCR quantification with H. contortus eggs (Harmon et al., 2007). DNA extraction was performed as described by Harmon et al. (2006). Extracted DNA was eluted with

The source of H. contortus used in this study came from a previously described lab strain (Harmon et al., 2007), confirmed to be only H. contortus by larval culture and multiplex PCR (Zarlenga et al., 2001). Preservatives evaluated in this study include Lugol’s iodine (LI, 5% iodine and 10% potassium iodide), neutral buffered formalin (NBF, 1:10 dilution of 37% formaldehyde aqueous solution), and sodium azide (SA, 0.15%, w/v, in distilled water). 2.2. Preservation of morphology and floatation tolerance The capacity of each preservative to preserve eggs was first tested using eggs previously isolated through flotation from sheep faeces. For this evaluation, three separate groups each containing 20 H. contortus eggs were submerged into 10 ml of the tested preservatives (LI, NBF and SA) and stored at room temperature with three replicates for each treatment. After 7 days, the recovered eggs from each group were counted, photographed (using an Olympus IZ70 inverted microscope with no phase optics), and put into a clean Petri dish. Sucrose solution (s.g. 1.26) was then added to the eggs, and they were allowed to float for 30 min prior to counting. A control group consisting of three replicates of 20 freshly isolated eggs was also photographed, floated, and counted as described above.

To compare the efficacy of LI as a preservative in comparison to NBF and SA of eggs within faecal samples, fecal material was directly subjected to each preservative for days 1, 7, 14, 30, or 180 before analysis. Additional samples were stored at 4 8C and tested in the same manner, as a control. To minimize sample to sample variability for the evaluation, collected faecal material was first homogenized in a blender, and 5 1 galiquots were prepared for each preservative and for each time period. To determine the approximate number of eggs present in each 1 g aliquot, five additional samples were floated and enumerated utilizing a modified Wisconsin sucrose flotation method (Cox and Todd, 1962). After each time period, fecal flotation was performed, and the recovered eggs were averaged for each treatment group. Representative eggs from each group were also photographed.


100 ml of Qiagen AE buffer, and stored at 20 8C until QPCR. Quantitative real-time PCR was performed on a Stratagene MX3000P (La Jolla, CA), utilizing Brilliant Multiplex QPCR Master Mix (Stratagene) with primers and probe described by Harmon et al. (2007). Amplification was carried out as follows: denaturation at 95 8C for 10 min, then amplification for 40 cycles at 95 8C for 15 s and 62 8C for 1 min. When samples did not amplify, a CT value of 40 was assigned to that sample. 3. Results 3.1. Preservative impact on morphology and recovery of isolated eggs When isolated H. contortus eggs were directly submerged in the preservations for 7 days, morphology was well preserved with NBF and SA, but there was noticeable condensation of the embryonic cells with LI (Fig. 1A). Only SA treated eggs showed a significant decrease in the percentage of remaining eggs after 7 days (75% P < 0.05). When remaining eggs were submerged in sucrose solution, there was a significant loss in floatable eggs with both NBF (70% P < 0.01)

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and SA (66% P < 0.001). There was also a numerical reduction in the number of eggs preserved with LI after sucrose flotation, 82%; however, this was not statistically significant. 3.2. Preservative effects on eggs in fecal samples There was a five- to seven-fold increase in the number of recovered eggs when LI and SA were used to preserve fecal samples for 1 day (P < 0.05). Though NBF resulted in an equally high average of recovered eggs, there was no significant difference from the control due to the high variability of the data from that group. Recovered eggs showed a decreasing trend between days 1 and 180, and there were no eggs remaining after 180 days when LI was used as a treatment (Fig. 2). At 180 days, 38.8% of the NBF preserved eggs, 5.2% of SA preserved eggs, and 1.3% of those refrigerated were recovered compared to the original estimated egg numbers. For the first 30 days, refrigerated eggs demonstrated egg recovery numbers similar to the estimated numbers, except at 7 days, which was not significant. The condensation of cells seen in isolated eggs preserved in LI was not seen when eggs were preserved

Fig. 1. Isolated eggs preserved by saturation with either Lugol’s iodine (A), neutral buffered formalin (B), and sodium azide (C) and eggs floated from faeces preserved for 7 days with Lugol’s iodine (D), neutral buffered formalin (E), and sodium azide (F). Bar represents 50 mm.

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that NBF again inhibited QPCR amplification, despite flotation. 4. Discussion

Fig. 2. Percentage of eggs recovered from faeces preserved 1 day to 6 months with either Lugol’s iodine (LI), neutral buffered formalin (NBF), sodium azide (SA), and 4 8C. Estimated egg numbers (Est) demonstrate the original calculated average. Error bars represent the sample means standard error.

within fecal samples (Fig. 1D). Formalin-preserved fecal samples resulted in slight embryo degradation in some eggs, which was detectable as early as day 7 (Fig. 1E). 3.3. Chemical preservation impact on QPCR When 25 egg samples saturated in LI were amplified with the real-time PCR, four of five samples amplified, resulting in an average CT value of 36.3 (S.D. = 3.03). Only two of the five NBF preserved samples amplified with an average CT value of 39.7 (S.D. = 0.54). The inhibition from LI and NBF was statistically significant (P < 0.001) when compared to 25 egg samples without treatment. Only sodium azide preservation resulted in CT values that were statistically similar to nontreated eggs (27.24, S.D. = 2.83 compared to 28.87, S.D. = 2.43). When eggs isolated by faecal flotation from preserved samples were tested, LI and SA had similar CT values, 20.17 S.D. = 1.54 and 21.87 S.D. = 3.26, respectively. These samples averaged 602 eggs/sample for LI preserved faeces and 537 eggs/sample for SA preserved samples, which corresponded to CT values obtained elsewhere with non-preserved samples between 500 (CT value of 22.8) eggs and 1000 (CT value of 19.8) eggs (Harmon et al., 2007). All five samples preserved with sodium azide and Lugol’s resulted in measurable amplification, but NBF allowed amplification of only three of the five samples. The NBF-fixed samples yielded an average of 142 eggs and had a significantly higher average CT value (38.31 P < 0.001), than 100 egg samples which were unpreserved (CT value of 23.73 S.D. = 1.91), suggesting

All tested preservatives preserved egg morphology and prevented embryo development within the egg when eggs were obtained from preserved faeces by flotation. However, when previously isolated eggs were fixed in LI, there was considerable condensation of the embryonic cells when compared to eggs submerged in NBF or SA, indicating that the tested concentration of LI is not appropriate for preserving eggs which have already been floated from fecal material, especially when morphology is critical. For these eggs, SA would be more appropriate. There appeared to be an increase in the amount of recovered eggs during the first 2 weeks of preservation with fecal samples, but this increase was only significant with LI and SA in the first day of preservation. It is likely that this increase is an artifact due to the high variability in egg counts. High variability with fecal egg counts despite homogenization is not uncommon and has been reported as being problematic with the diagnosis of cattle and sheep trichostrongyles (Levine et al., 1960). The number of recoverable eggs was similar between LI, NBF, SA, and the control sample until 1 month, where then only 33% (S.D. 15.6) of the anticipated egg numbers could be recovered with LI preserved faeces (Fig. 2). The results of the present work suggest that LI preserved eggs should not be stored for more than 2 weeks, but SA and NBF appear to be effective for at least a month. When isolated eggs were amplified after extraction in the presence of 100 ml of SA, there was no significant difference in CT values compared to control eggs. This suggests that SA is superior over LI for preserving eggs for uninhibited PCR; however, flotation reduces this inhibition from LI to an undetectable level. Both formalin and iodine denature proteins, explaining why PCR was inhibited when preserved eggs were not washed prior to extraction (McDonnell and Russell, 1999). Formalin inhibited PCR irrespective of flotation, suggesting that NBF should not be utilized if eggs are to be analyzed by PCR. In summary, results of this study involving H. contortus eggs demonstrate that if LI or SA is added to fecal samples, they can effectively stop intra-egg development while also preserving egg morphology, allow for effective egg recovery, and most importantly, allow for real-time PCR analyses without significant


inhibition. We recommend either LI or SA for field studies which require real-time PCR analysis of trichostrongyle eggs from preserved faeces. Acknowledgements This work was conducted partially using the South Dakota State University Functional Genomics Core Facility supported in part by the National Science Foundation/EPSCoR Grant No. 0091948 and by the State of South Dakota. Financial support was provided in parts by Pfizer Animal Health, South Dakota State University Research Support Grant, and the South Dakota State University Agricultural Experiment Station. This paper was approved for publication as Journal Series No. 3587 by the Director, Agricultural Experiment Station, South Dakota State University. References Bundy, D.A.P., Foreman, J.D.M., Golden, M.H.N., 1985. Sodium azide preservation of fecal specimens for Kato analysis. Parasitology 90, 463–469. Christie, M., Jackson, F., 1982. Specific identification of strongyle eggs in small samples of sheep faeces. Res. Vet. Sci. 32, 113–117. Cox, D.D., Todd, A.C., 1962. Survey of gastrointestinal parasitism in Wisconsin dairy cattle. J. Am. Vet. Med. Assoc. 141, 706–709. Foreyt, W.J., 1986. Recovery of nematode eggs and larvae in deer: evaluation of fecal preservation methods. J. Am. Vet. Med. Assoc. 189, 1065–1067. Harmon, A.F., Zarlenga, D.S., Hildreth, M.B., 2006. Improved methods for isolating DNA from Ostertagia ostertagi eggs in cattle feces. Vet. Parasitol. 135, 297–302.

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