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Incidental isolation of Setaria equina microfilariae in preparations of equine peripheral blood mononuclear cells
Veterinary Parasitology 161 (2009) 142–145
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Incidental isolation of Setaria equina microfilariae in preparations of equine peripheral blood mononuclear cells Michelle R. Yeargan a, Eugene T. Lyons a, Stephen A. Kania b, Sharon Patton b, Cormac C. Breathnach a, David W. Horohov a, Daniel K. Howe a,* a b
Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546-0099, USA Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 November 2008 Received in revised form 17 December 2008 Accepted 23 December 2008
In the course of a vaccine experiment on horses, microfilariae were observed in cultures of peripheral blood mononuclear cells (PBMCs) isolated from eleven of fifteen study horses. The microfilariae were clearly viable as evidenced by their vigorous movements in the cultures, thus indicating that they had survived the Ficoll gradient purification and the cryopreservation method used for retaining the PBMCs. The microfilariae were identified as Setaria equina, which is a vector-borne filarial nematode that causes a relatively benign infection of equids in which the adult worms reside in the peritoneal cavity. Although it is not possible to definitely state where the infections were acquired, the horses originated from Saskatchewan, Canada and spent a relatively short period of time in the United States prior to blood sampling. Therefore, it is likely that the infections occurred in Canada. Interestingly, assays conducted to determine levels of cytokine mRNA transcripts in the isolated PBMCs seemed to be largely unaltered by the presence of the microfilariae in the cell cultures. These findings demonstrate that a standard method used to purify and cryopreserve PBMCs from blood can result in the unintended co-isolation of worms from microfilaremic animals. Furthermore, the presence of the microfilariae did not appear to alter significantly the results of our immunologic assays, suggesting either that the nematode antigens were not recognized or that immunological tolerance may have developed in these horses. Although notable effects on the assays were not observed in this study, it seems possible that microfilarial contamination could represent a confounding variable for experiments examining cellular immunity. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Microfilariae Setaria equina Peripheral blood mononuclear cells Vaccine Horse
Collection of blood samples for examination of immune responses is common practice during vaccine studies. Peripheral blood mononuclear cells (PBMCs) are often isolated from blood by density gradient centrifugation and then used immediately or cryopreserved for future experiments to assess cell-mediated immunity to vaccination. The PBMCs collected from the vaccinated animals can
* Corresponding author. Tel.: +1 859 257 4757x81113; fax: +1 859 257 8542. E-mail address: [email protected] (D.K. Howe). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.12.020
be analyzed for various phenotypes including cell surface markers, proliferative capabilities, and cytokine production. Importantly, the results obtained from these in vitro assays are assumed to be due to specific immune responses elicited by the vaccine antigen(s). However, inadvertent contamination of the PBMC cultures with molecules that may have immunostimulatory effects (e.g., bacterial endotoxin) can confound the outcome of the assays. In the course of a study examining immunization against equine protozoal myeloencephalitis (EPM), horses were imported from Canada to obtain a population that was naı¨ve to the protozoan parasite Sarcocystis neurona.
M.R. Yeargan et al. / Veterinary Parasitology 161 (2009) 142–145
Fifteen mixed-breed horses were acquired from a ranch in Saskatchewan, Canada, and after a short stay at a facility in Iowa, the horses were transported and pastured in rural Central Kentucky. Over an 8-week period, horses were vaccinated, and blood was periodically collected to examine the immune responses that had been elicited. Whole blood was collected via jugular venipuncture into heparinized blood tubes, and the red blood cells were allowed to settle. The plasma was overlaid onto 4 ml FicollPaque PLUS in 15-ml centrifuge tubes, centrifuged at 500 g for 30 min at room temperature, and the PBMCs were collected at the interface and transferred to clean 15ml centrifuge tubes. The cells were counted on a Beckman Coulter Vi-Cell XR Cell Viability Analyzer, and frozen in cryoprotective medium (RPMI/40% FBS/10% DMSO) at 2 107 cells per vial in Nalgene Cryo 1 8C Freezing Containers. The containers were placed at 80 8C overnight, and the vials were then transferred and stored in the vapor phase of liquid nitrogen until used. After thawing the cryopreserved PBMCs and allowing the cells to settle to the bottom of the wells, the cultures were examined using an Olympus CK30 inverted phase microscope. Unexpectedly, multiple nematode larvae were immediately evident in cell cultures obtained from the horses. Most of the larvae were viable, as evidence by their rapid and energetic movements, and they remained viable in the cryopreserved preparations at greater than 1.5 years post-freezing. In total, PBMC preps from 11 of 15 horses (73%) contained nematode larvae. Based on the volume of blood from which the PBMCs were isolated, the number of worms present in the cultures suggested levels ranging from <1 to 18 larvae/ml of whole blood. The larvae were 250–269 mm in length and appeared to be sheathed (Fig. 1), which are consistent with the phenotypic characteristics of Setaria equina microfilariae (Mf). A fragment of the mitochondrial 12S rRNA gene was amplified from the Mf and sequenced. Alignment of the obtained sequence with sequences available in GenBank revealed highest identity to the S. equina 12S rRNA sequence submission #AF544835 (98% identity), with lower similarity to sequences from Setaria tundra and Setaria digitata (data not shown). Based on their morphologic and genotypic characteristics, the Mf were identified as S. equina. Setaria equina is a common filariid parasite of equids that has been found to occur in North America, Europe, and Asia and is believed to be vectored by Aedes or Culex mosquitoes (Arundel, 1978; Levine, 1968). The prevalence of S. equina in horse populations has not been well documented, but surveys that have been conducted report prevalence ranging from 7% in Kentucky Thoroughbreds (Lyons et al., 1983) to approximately 15% in equids from Louisiana (Coleman et al., 1985) and Turkey (Oge et al., 2003). The adult worms range in size from 5 to 13 cm in length with the males being the smaller of the two, and they normally reside in the peritoneal cavity of the infected horse (Levine, 1968). The ensheathed Mf are approximately 240–280 mm by 7–8 mm in size and are found in the blood, which matches the characteristics exhibited by the Mf in the equine PBMC cultures. Although infections are typically benign, pathologies of the eye and central
143
Fig. 1. Images of a microfilaria present in a culture of peripheral blood mononuclear cells isolated from a horse. (A) Anterior end of microfilaria. (B) Caudal end of microfilaria showing sheath (arrow). Magnification 400.
nervous system have been attributed to S. equina (Frauenfelder et al., 1980). Prepatency of S. equina is 8– 10 months (Arundel, 1978). Therefore, it is probable that the horses used in this study were infected in Canada since they had been in the United States for less than 6 months. It is unknown when the horses were last dewormed, but ivermectin has been found to be highly effective against S. equina adults (Klei et al., 1980) so it is unlikely that these animals had been treated with an avermectin-based anthelmintic for at least 8 months. Although the isolation of S. equina Mf by density gradient centrifugation was unintended and initially surprising, it appears to be relatively congruent with methodology that has been described previously for concentration of other nematode species Mf from blood (Feldmeier et al., 1981; Jones et al., 1975). Approximately 85% of Mf will concentrate into density layers of 1.075 g/ml or less in Percoll gradients (Feldmeier et al., 1981). Therefore, it is predictable that the Mf would be present with the PBMC layer that forms above the Ficoll-Paque PLUS since this medium has a density of 1.077 0.001 g/ ml. As well, the Mf viability that was observed in the PBMC cultures is apparently foreseeable since the method used to freeze the equine PBMC preparations (10% DMSO, 1 8C/min controlled cooling rate) is comparable to protocols that have been described for cryopreserving Mf of Dirofilaria spp., Brugia malayi, and Wuchereria bancrofti (Bartholomay et al., 2001; Lok et al., 1983; Lowrie, 1983). The presence of Mf in the PBMC cultures raised concerns about the reliability of immunologic assays
144
M.R. Yeargan et al. / Veterinary Parasitology 161 (2009) 142–145
immunological tolerance to the nematodes had developed in these horses. The absence of cytokine upregulation by PBMCs, despite the presence of Mf in the cultures, raised questions about the responsiveness of the cells. Since several of the study horses had been infected naturally with S. neurona during the 8-week period that they were maintained on pasture in Kentucky, as evidenced by seroconversion to the SnSAG merozoite surface antigens (Hoane et al., 2005) (data not shown), it was possible to conduct experiments assessing whether stimulatory antigens would elicit immune responses in PBMC cultures containing the Mf. As shown in Fig. 3, PBMCs from microfilaremic horses that had been exposed naturally to S. neurona during the study period exhibited an approximately 35-fold mean increase in IFN-g mRNA after a 24-h incubation with recombinant S. neurona surface antigen SnSAG3 (rSnSAG3). PBMCs from non-S. neurona-infected, microfilaremic horses also exhibited enhanced levels of IFN-g mRNA, which likely represents a mitogenic effect of the rSnSAG3 preparation, but the mRNA increase was only about half of that observed in cultures from the S. neurona-infected horses. These results suggested that the presence of the Mf did not prevent an antigen-specific stimulation of immune cells. In summary, these findings demonstrate that Mf can be unintended co-isolates from microfilaremic animals when using standard methodology to obtain and preserve PBMCs for immunologic studies. Although the assays
Fig. 2. IFN-g and IL-2 mRNA levels in equine peripheral blood mononuclear cell cultures. Cells were incubated for 24 h in media before RNA was extracted with RNA-Stat 60. One microgram of total RNA was reverse transcribed to cDNA, which was used in real-time PCR assays for quantitation of cytokine mRNAs. Semi-quantitative analyses based on the DDCT method of expressing changes in cytokine gene expression (Livak and Schmittgen, 2001) were used to report fold differences relative to one PBMC culture that lacked microfilariae (the calibrator) after normalizing the samples with b-glucuronidase mRNA. No significant differences were observed between the two populations (Mann–Whitney U-test). Results are shown as mean fold increase (S.E.M.) of cytokine mRNA.
conducted with these cells. Specifically, it was speculated that the Mf contamination would stimulate immune cells, thereby altering the level of cytokines expressed in the cultures. To examine this possibility, equine cytokine mRNAs were measured in resting (non-stimulated) PBMC cultures, as described previously (Horohov et al., 2005; Breathnach et al., 2006). As shown in Fig. 2, there was no appreciable increase in mRNA levels for IFN-g and IL-2 in PBMC cultures containing Mf relative to cultures that lacked Mf. In addition, similar results were obtained from assays detecting mRNA for IL-13, IL-4, and IL-5 (data not shown). Taken together, these data indicate that the presence of Mf did not result in an upregulation of cytokines in the PBMC cultures, possibly indicating that
Fig. 3. Upregulation of IFN-g mRNA in PBMCs from microfilaremic horses after stimulation with recombinant S. neurona surface antigen SnSAG3. After incubation of PBMCs for 24 h in the presence of recombinant antigen, RNA was harvested, cDNA was generated, and real-time PCR assays were conducted to quantify IFN-g mRNA. The media control for each horse served as its calibrator for the assay. Microfilaremic horses that had been infected with S. neurona exhibited a 35-fold increase in IFNg mRNA after stimulation with recombinant S. neurona surface antigen, which was nearly twice the IFN-g mRNA increase observed in microfilaremic horses that had not been exposed to S. neurona. This difference was not found to be statistically significant, however (Mann– Whitney U-test). Results shown are mean fold increase (S.E.M.) of cytokine mRNA.
M.R. Yeargan et al. / Veterinary Parasitology 161 (2009) 142–145
described herein did not reveal profound alterations in cytokine levels as a result of the Mf contamination, these experiments were not planned as part of the original vaccine study so, it was not possible to implement all of the proper controls. Consequently, it remains possible that the presence of the Mf foreign antigen in PBMC preparations could represent a confounding variable for experiments examining cellular immune responses. Accordingly, investigators may want to consider deworming experimental animals with an avermectinbased anthelmintic prior to conducting studies that incorporate immunologic assays. Acknowledgements The authors are grateful to Mr. Lynn Ennis for his assistance in securing and handling the horses. This research was partly supported by funds from Fort Dodge Animal Health. Published as Kentucky Agricultural Experiment Station article number 08-14-138. References Arundel, J.H., 1978. Parasitic diseases of the horse. In: Veterinary Review, The University of Sydney, 83 pp. Bartholomay, L.C., Farid, H.A., El Kordy, E., Christensen, B.M., 2001. Short report: a practical technique for the cryopreservation of Dirofilaria immitis, Brugia malayi, and Wuchereria bancrofti microfilariae. Am. J. Trop. Med. Hyg. 65, 162–163. Breathnach, C.C., Sturgill-Wright, T., Stiltner, J.L., Adams, A.A., Lunn, D.P., Horohov, D.W., 2006. Foals are interferon gamma-deficient at birth. Vet. Immunol. Immunopathol. 112, 199–209.
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Coleman, S.U., Klei, T.R., French, D.D., 1985. Prevalence of Setaria equina (Nematode: Onchocercidae) in southeastern Louisiana horses. J. Parasitol. 71, 512–513. Feldmeier, H., Bienzle, U., Schuh, D., 1981. Combination of techniques for concentration and identification of microfilariae from peripheral blood. Trans. R. Soc. Trop. Med. Hyg. 75, 251–253. Frauenfelder, H.C., Kazacos, K.R., Lichtenfels, J.R., 1980. Cerebrospinal nematodiasis caused by a filariid in a horse. J. Am. Vet. Med. Assoc. 177, 359–362. Hoane, J.S., Morrow, J.K., Saville, W.J., Dubey, J.P., Granstrom, D.E., Howe, D.K., 2005. Enzyme-linked immunosorbent assays for the detection of equine antibodies specific to Sarcocystis neurona surface antigens. Clin. Diagn. Lab. Immunol. 12, 1050–1056. Horohov, D.W., Beadle, R.E., Mouch, S., Pourciau, S.S., 2005. Temporal regulation of cytokine mRNA expression in equine recurrent airway obstruction. Vet. Immunol. Immunopathol. 108, 237–245. Jones, T.C., Mott, K., Pedrosa, L.C., 1975. A technique for isolating and concentrating microfilariae from peripheral blood by gradient centrifugation. Trans. R. Soc. Trop. Med. Hyg. 69, 243–246. Klei, T.R., Torbert, B.J., Ochoa, R., 1980. Efficacy of ivermectin (22,23dihydroavermectin B1) against adult Setaria equina and microfilariae of Onchocerca cervicalis in ponies. J. Parasitol. 66, 859–861. Levine, N.D., 1968. Nematode Parasites of Domestic Animals and of Man. Burgess Publishing Company, 477 pp. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2( Delta Delta C(T)) method. Methods 25, 402–408. Lok, J.B., Mika-Grieve, M., Grieve, R.B., 1983. Cryopreservation of Dirofilaria immitis microfilariae and third-stage larvae. J. Helminthol. 57, 319–324. Lowrie Jr., R.C., 1983. Cryopreservation of the microfilariae of Brugia malayi, Dirofilaria corynodes, and Wuchereria bancrofti. Am. J. Trop. Med. Hyg. 32, 138–145. Lyons, E.T., Tolliver, S.C., Drudge, J.H., Swerczek, T.W., Crowe, M.W., 1983. Parasites in Kentucky Thoroughbreds at necropsy: emphasis on stomach worms and tapeworms. Am. J. Vet. Res. 44, 839–844. Oge, S., Oge, H., Yildirim, A., Kircali, F., 2003. Setaria equina infection of Turkish equines: estimates of prevalence based on necropsy and the detection of microfilaraemia. Ann. Trop. Med. Parasitol. 97, 403–409.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Short communication
Incidental isolation of Setaria equina microfilariae in preparations of equine peripheral blood mononuclear cells Michelle R. Yeargan a, Eugene T. Lyons a, Stephen A. Kania b, Sharon Patton b, Cormac C. Breathnach a, David W. Horohov a, Daniel K. Howe a,* a b
Department of Veterinary Science, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY 40546-0099, USA Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 12 November 2008 Received in revised form 17 December 2008 Accepted 23 December 2008
In the course of a vaccine experiment on horses, microfilariae were observed in cultures of peripheral blood mononuclear cells (PBMCs) isolated from eleven of fifteen study horses. The microfilariae were clearly viable as evidenced by their vigorous movements in the cultures, thus indicating that they had survived the Ficoll gradient purification and the cryopreservation method used for retaining the PBMCs. The microfilariae were identified as Setaria equina, which is a vector-borne filarial nematode that causes a relatively benign infection of equids in which the adult worms reside in the peritoneal cavity. Although it is not possible to definitely state where the infections were acquired, the horses originated from Saskatchewan, Canada and spent a relatively short period of time in the United States prior to blood sampling. Therefore, it is likely that the infections occurred in Canada. Interestingly, assays conducted to determine levels of cytokine mRNA transcripts in the isolated PBMCs seemed to be largely unaltered by the presence of the microfilariae in the cell cultures. These findings demonstrate that a standard method used to purify and cryopreserve PBMCs from blood can result in the unintended co-isolation of worms from microfilaremic animals. Furthermore, the presence of the microfilariae did not appear to alter significantly the results of our immunologic assays, suggesting either that the nematode antigens were not recognized or that immunological tolerance may have developed in these horses. Although notable effects on the assays were not observed in this study, it seems possible that microfilarial contamination could represent a confounding variable for experiments examining cellular immunity. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Microfilariae Setaria equina Peripheral blood mononuclear cells Vaccine Horse
Collection of blood samples for examination of immune responses is common practice during vaccine studies. Peripheral blood mononuclear cells (PBMCs) are often isolated from blood by density gradient centrifugation and then used immediately or cryopreserved for future experiments to assess cell-mediated immunity to vaccination. The PBMCs collected from the vaccinated animals can
* Corresponding author. Tel.: +1 859 257 4757x81113; fax: +1 859 257 8542. E-mail address: [email protected] (D.K. Howe). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.12.020
be analyzed for various phenotypes including cell surface markers, proliferative capabilities, and cytokine production. Importantly, the results obtained from these in vitro assays are assumed to be due to specific immune responses elicited by the vaccine antigen(s). However, inadvertent contamination of the PBMC cultures with molecules that may have immunostimulatory effects (e.g., bacterial endotoxin) can confound the outcome of the assays. In the course of a study examining immunization against equine protozoal myeloencephalitis (EPM), horses were imported from Canada to obtain a population that was naı¨ve to the protozoan parasite Sarcocystis neurona.
M.R. Yeargan et al. / Veterinary Parasitology 161 (2009) 142–145
Fifteen mixed-breed horses were acquired from a ranch in Saskatchewan, Canada, and after a short stay at a facility in Iowa, the horses were transported and pastured in rural Central Kentucky. Over an 8-week period, horses were vaccinated, and blood was periodically collected to examine the immune responses that had been elicited. Whole blood was collected via jugular venipuncture into heparinized blood tubes, and the red blood cells were allowed to settle. The plasma was overlaid onto 4 ml FicollPaque PLUS in 15-ml centrifuge tubes, centrifuged at 500 g for 30 min at room temperature, and the PBMCs were collected at the interface and transferred to clean 15ml centrifuge tubes. The cells were counted on a Beckman Coulter Vi-Cell XR Cell Viability Analyzer, and frozen in cryoprotective medium (RPMI/40% FBS/10% DMSO) at 2 107 cells per vial in Nalgene Cryo 1 8C Freezing Containers. The containers were placed at 80 8C overnight, and the vials were then transferred and stored in the vapor phase of liquid nitrogen until used. After thawing the cryopreserved PBMCs and allowing the cells to settle to the bottom of the wells, the cultures were examined using an Olympus CK30 inverted phase microscope. Unexpectedly, multiple nematode larvae were immediately evident in cell cultures obtained from the horses. Most of the larvae were viable, as evidence by their rapid and energetic movements, and they remained viable in the cryopreserved preparations at greater than 1.5 years post-freezing. In total, PBMC preps from 11 of 15 horses (73%) contained nematode larvae. Based on the volume of blood from which the PBMCs were isolated, the number of worms present in the cultures suggested levels ranging from <1 to 18 larvae/ml of whole blood. The larvae were 250–269 mm in length and appeared to be sheathed (Fig. 1), which are consistent with the phenotypic characteristics of Setaria equina microfilariae (Mf). A fragment of the mitochondrial 12S rRNA gene was amplified from the Mf and sequenced. Alignment of the obtained sequence with sequences available in GenBank revealed highest identity to the S. equina 12S rRNA sequence submission #AF544835 (98% identity), with lower similarity to sequences from Setaria tundra and Setaria digitata (data not shown). Based on their morphologic and genotypic characteristics, the Mf were identified as S. equina. Setaria equina is a common filariid parasite of equids that has been found to occur in North America, Europe, and Asia and is believed to be vectored by Aedes or Culex mosquitoes (Arundel, 1978; Levine, 1968). The prevalence of S. equina in horse populations has not been well documented, but surveys that have been conducted report prevalence ranging from 7% in Kentucky Thoroughbreds (Lyons et al., 1983) to approximately 15% in equids from Louisiana (Coleman et al., 1985) and Turkey (Oge et al., 2003). The adult worms range in size from 5 to 13 cm in length with the males being the smaller of the two, and they normally reside in the peritoneal cavity of the infected horse (Levine, 1968). The ensheathed Mf are approximately 240–280 mm by 7–8 mm in size and are found in the blood, which matches the characteristics exhibited by the Mf in the equine PBMC cultures. Although infections are typically benign, pathologies of the eye and central
143
Fig. 1. Images of a microfilaria present in a culture of peripheral blood mononuclear cells isolated from a horse. (A) Anterior end of microfilaria. (B) Caudal end of microfilaria showing sheath (arrow). Magnification 400.
nervous system have been attributed to S. equina (Frauenfelder et al., 1980). Prepatency of S. equina is 8– 10 months (Arundel, 1978). Therefore, it is probable that the horses used in this study were infected in Canada since they had been in the United States for less than 6 months. It is unknown when the horses were last dewormed, but ivermectin has been found to be highly effective against S. equina adults (Klei et al., 1980) so it is unlikely that these animals had been treated with an avermectin-based anthelmintic for at least 8 months. Although the isolation of S. equina Mf by density gradient centrifugation was unintended and initially surprising, it appears to be relatively congruent with methodology that has been described previously for concentration of other nematode species Mf from blood (Feldmeier et al., 1981; Jones et al., 1975). Approximately 85% of Mf will concentrate into density layers of 1.075 g/ml or less in Percoll gradients (Feldmeier et al., 1981). Therefore, it is predictable that the Mf would be present with the PBMC layer that forms above the Ficoll-Paque PLUS since this medium has a density of 1.077 0.001 g/ ml. As well, the Mf viability that was observed in the PBMC cultures is apparently foreseeable since the method used to freeze the equine PBMC preparations (10% DMSO, 1 8C/min controlled cooling rate) is comparable to protocols that have been described for cryopreserving Mf of Dirofilaria spp., Brugia malayi, and Wuchereria bancrofti (Bartholomay et al., 2001; Lok et al., 1983; Lowrie, 1983). The presence of Mf in the PBMC cultures raised concerns about the reliability of immunologic assays
144
M.R. Yeargan et al. / Veterinary Parasitology 161 (2009) 142–145
immunological tolerance to the nematodes had developed in these horses. The absence of cytokine upregulation by PBMCs, despite the presence of Mf in the cultures, raised questions about the responsiveness of the cells. Since several of the study horses had been infected naturally with S. neurona during the 8-week period that they were maintained on pasture in Kentucky, as evidenced by seroconversion to the SnSAG merozoite surface antigens (Hoane et al., 2005) (data not shown), it was possible to conduct experiments assessing whether stimulatory antigens would elicit immune responses in PBMC cultures containing the Mf. As shown in Fig. 3, PBMCs from microfilaremic horses that had been exposed naturally to S. neurona during the study period exhibited an approximately 35-fold mean increase in IFN-g mRNA after a 24-h incubation with recombinant S. neurona surface antigen SnSAG3 (rSnSAG3). PBMCs from non-S. neurona-infected, microfilaremic horses also exhibited enhanced levels of IFN-g mRNA, which likely represents a mitogenic effect of the rSnSAG3 preparation, but the mRNA increase was only about half of that observed in cultures from the S. neurona-infected horses. These results suggested that the presence of the Mf did not prevent an antigen-specific stimulation of immune cells. In summary, these findings demonstrate that Mf can be unintended co-isolates from microfilaremic animals when using standard methodology to obtain and preserve PBMCs for immunologic studies. Although the assays
Fig. 2. IFN-g and IL-2 mRNA levels in equine peripheral blood mononuclear cell cultures. Cells were incubated for 24 h in media before RNA was extracted with RNA-Stat 60. One microgram of total RNA was reverse transcribed to cDNA, which was used in real-time PCR assays for quantitation of cytokine mRNAs. Semi-quantitative analyses based on the DDCT method of expressing changes in cytokine gene expression (Livak and Schmittgen, 2001) were used to report fold differences relative to one PBMC culture that lacked microfilariae (the calibrator) after normalizing the samples with b-glucuronidase mRNA. No significant differences were observed between the two populations (Mann–Whitney U-test). Results are shown as mean fold increase (S.E.M.) of cytokine mRNA.
conducted with these cells. Specifically, it was speculated that the Mf contamination would stimulate immune cells, thereby altering the level of cytokines expressed in the cultures. To examine this possibility, equine cytokine mRNAs were measured in resting (non-stimulated) PBMC cultures, as described previously (Horohov et al., 2005; Breathnach et al., 2006). As shown in Fig. 2, there was no appreciable increase in mRNA levels for IFN-g and IL-2 in PBMC cultures containing Mf relative to cultures that lacked Mf. In addition, similar results were obtained from assays detecting mRNA for IL-13, IL-4, and IL-5 (data not shown). Taken together, these data indicate that the presence of Mf did not result in an upregulation of cytokines in the PBMC cultures, possibly indicating that
Fig. 3. Upregulation of IFN-g mRNA in PBMCs from microfilaremic horses after stimulation with recombinant S. neurona surface antigen SnSAG3. After incubation of PBMCs for 24 h in the presence of recombinant antigen, RNA was harvested, cDNA was generated, and real-time PCR assays were conducted to quantify IFN-g mRNA. The media control for each horse served as its calibrator for the assay. Microfilaremic horses that had been infected with S. neurona exhibited a 35-fold increase in IFNg mRNA after stimulation with recombinant S. neurona surface antigen, which was nearly twice the IFN-g mRNA increase observed in microfilaremic horses that had not been exposed to S. neurona. This difference was not found to be statistically significant, however (Mann– Whitney U-test). Results shown are mean fold increase (S.E.M.) of cytokine mRNA.
M.R. Yeargan et al. / Veterinary Parasitology 161 (2009) 142–145
described herein did not reveal profound alterations in cytokine levels as a result of the Mf contamination, these experiments were not planned as part of the original vaccine study so, it was not possible to implement all of the proper controls. Consequently, it remains possible that the presence of the Mf foreign antigen in PBMC preparations could represent a confounding variable for experiments examining cellular immune responses. Accordingly, investigators may want to consider deworming experimental animals with an avermectinbased anthelmintic prior to conducting studies that incorporate immunologic assays. Acknowledgements The authors are grateful to Mr. Lynn Ennis for his assistance in securing and handling the horses. This research was partly supported by funds from Fort Dodge Animal Health. Published as Kentucky Agricultural Experiment Station article number 08-14-138. References Arundel, J.H., 1978. Parasitic diseases of the horse. In: Veterinary Review, The University of Sydney, 83 pp. Bartholomay, L.C., Farid, H.A., El Kordy, E., Christensen, B.M., 2001. Short report: a practical technique for the cryopreservation of Dirofilaria immitis, Brugia malayi, and Wuchereria bancrofti microfilariae. Am. J. Trop. Med. Hyg. 65, 162–163. Breathnach, C.C., Sturgill-Wright, T., Stiltner, J.L., Adams, A.A., Lunn, D.P., Horohov, D.W., 2006. Foals are interferon gamma-deficient at birth. Vet. Immunol. Immunopathol. 112, 199–209.
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