Effects of Mycoplasma gallisepticum vaccination on serum α 1 -acid glycoprotein concentrations in commercial layer chickens 1, 2

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1 Effects of Mycoplasma gallisepticum vaccination on serum α 1 -acid glycoprotein concentrations in commercial layer chickens 1, 2 E. D. Peebles,* 3 R. Jacob,* S. L. Branton, and P. D. Gerard * Department of Poultry Science, Mississippi State University, Mississippi State 39762; USDA-Agricultural Research Service, Poultry Research Unit, Mississippi State, MS 39762; and Department of Mathematical Sciences, Clemson University, Clemson, SC ABSTRACT Increases in circulating acute phase protein (APP) levels occur in reaction to systemic infections in animals. However, no previous research has been conducted to monitor possible changes in APP levels of birds in response to prelay vaccinations of various live attenuated Mycoplasma gallisepticum vaccines in conjunction with their subsequent use as an overlay vaccine during the production period. Serum concentrations of the APP, α 1 -acid glycoprotein (AGP), were determined on d 0, 1, 3, 7, 14, and 28 after subjecting commercial laying hens to one of the following treatments at 10 wk of age (woa): 1) control (no vaccination); 2) ts-11 strain M. gallisepticum (ts11mg) vaccination; 3) M. gallisepticum-bacterin (MGBac) vaccination; and 4) ts11mg and MGBac combination (ts11mg & MG- Bac) vaccination. Furthermore, at 45 woa, the birds in half of the units assigned to each treatment group were vaccinated with high-passage F-strain M. gallisepticum (HpFMG). Birds in treatment 1 that were (single control) and were not (double control) vaccinated with INTRODUCTION Mycoplasma gallisepticum is a bacterium that can lead to respiratory complications in laying hens (Ley and Yoder, 1978; Branton et al., 1984). Live attenuated vaccines (LAV) derived from M. gallisepticum strains such as F-strain M. gallisepticum (FMG) and ts11- strain M. gallisepticum (ts11mg), which exhibit low to moderate virulence, may be used to protect flocks HpFMG, and birds in treatments 2, 3, and 4 that were vaccinated with HpFMG were further tested during lay on d 0, 1, 3, 7, 14, and 28 after vaccination. On d 7, 14, and 28 postvaccination at 10 woa, the ts11mg & MGBac, ts11mg, and MGBac group AGP concentrations were not different from one another, but all were higher than those in the control group. Similarly, on d 3, 7, and 14 postvaccination, the single control, and the MGBac ts11mg, and ts11mg & MGBac treatment groups that were later vaccinated with HpFMG at 45 woa, were not different, but all were higher than that in the double control group. In conclusion, elevated circulation AGP concentrations may be used to detect and confirm subclinical infections in pullets up to 28 d after having been vaccinated with ts11mg, MGBac, or their combination. Furthermore, in association with depressed performance, elevated serum AGP concentrations in layers may be used to confirm HpFMG infections up to 28 d after its use as a vaccine. Key words: α 1 -acid glycoprotein, acute phase protein, Mycoplasma gallisepticum, layer, vaccine 2014 Poultry Science 93 : Poultry Science Association Inc. Received January 14, Accepted February 20, This is Journal Article Number J from the Mississippi Agricultural and Forestry Experiment Station supported by MIS Use of trade names in this publication does not imply endorsement by Mississippi Agricultural and Forestry Experiment Station of these products, nor similar ones not mentioned. 3 Corresponding author: dpeebles@poultry.msstate.edu against infections by more virulent field strains of M. gallisepticum (Levisohn and Kleven, 1981; Cummings and Kleven, 1986; Kleven et al., 1990). An inactivated M. gallisepticum bacterin (MGBac) has also been reported to afford notable, although limited, protection against infections by field strains of M. gallisepticum (Kleven et al., 1984; Yoder and Hopkins, 1985). The inoculation of laying hens with high-passage FMG (HpFMG) at 12 wk of age (woa) has been reported to delay initiation of lay by 1 wk and to reduce total egg production (EP; Burnham et al., 2002), and Branton et al. (1988) showed that EP was reduced subsequent to the eye-drop inoculation of layers with HpFMG at 45 woa. Branton et al. (1997) later showed that lymphopenia, heterophilia, monocytosis, eosinopenia, and basopenia along with increased numbers of lymphoid foci occurred in the bone marrow and spleens of 6 woa commercial layers for 4 wk after they had 1396

2 ACUTE PHASE PROTEIN RESPONSE TO VACCINATION received HpFMG by eye-drop inoculation. Burnham et al. (2003) also showed that plasma protein and serum triglyceride concentrations were increased in laying hens during lay at 22 woa in response to an HpFMG inoculation at 12 woa. However, the vaccination of layers with MGBac has been reported to either have no subsequent effect on EP (Khan et al., 1986) or to afford protection against EP losses due to field strain M. gallisepticum challenges (Yoder and Hopkins, 1985; Sasipreeyajan et al., 1987). Furthermore, the ts11mg vaccine is capable of inducing a mild serological response and subsequently providing protection against airsacculitis that is commonly associated with virulent M. gallisepticum infections in chickens (Whithear et al., 1990; Evans and Hafez, 1992; Abd-el-Motelib and Kleven, 1993). Barbour et al. (2000) have indicated that the vaccination of broiler breeder hens with ts11mg was capable of displacing field strain M. gallisepticum tracheal and infraorbital sinus infections. However, other work in layers has shown that, unlike HpFMG, ts11mg is unable to completely displace some more virulent M. gallisepticum infections (Kleven et al., 1998) and may not provide continued protection against field strain M. gallisepticum infections throughout lay (Yoder, 1978; Mohammed et al., 1987; Yoder, 1991). Peebles and Branton (2012) have suggested that when used in conjunction with an overlay of HpFMG during lay, a prelay ts11mg inoculation may be part of an effective regimen for the continual protection of layers against field strain M. gallisepticum infections while avoiding the adverse effects that a prelay Hp- FMG inoculation can have on performance. Upon testing for the effects of a prelay inoculation of ts11mg at 10 woa in conjunction with an overlay inoculation of HpFMG at 45 woa, Vance et al. (2008a) reported that layer performance, including EP, was not adversely affected. However, eggshell pimpling and internal egg blood spot incidences were consequently increased in the birds at 56 woa (Vance et al., 2008a). Furthermore, the ts11mg vaccination at 10 woa alone increased serum calcium levels at 22 woa (Peebles et al., 2009), increased yolk lipid percentage at 32 woa (Vance et al., 2008b), decreased Haugh unit scores throughout postpeak lay (Vance et al., 2008b), and altered relative vaginal length at 58 woa (Vance et al., 2009). In a companion article in which the performance of these birds was reported, Jacob et al. (2014) found that between 21 and 45 woa, the separate and combined vaccination of pullets with ts11mg and MGBac at 10 woa had no effect on their performance, and that total percentage EP across lay (18 through 52 woa) was also not affected by treatment. However, when overlaid with HpFMG at 45 woa, birds that had either received no vaccination or that received an MGBac vaccination at 10 woa exhibited a decrease in EP. In addition, all the vaccinated birds, irrespective of their prelay and overlay vaccination treatments, exhibited a decrease in BW. Because acute phase responses are associated with depressed performance in chickens, the detection of increased circulating levels of acute phase proteins (APP) may be used to identify performance-related subclinical diseases (Klasing and Johnstone, 1991; Klasing, 2004; Iseri and Klasing, 2013). Infectious bursal disease (Inoue et al., 1997), bronchitis, and laryngotracheitis (Nakamura et al., 1996) viruses have been observed to be associated with increased levels of the APP, α 1 -acid glycoprotein (AGP), in chickens. Furthermore, Curtis and Thornton (1973) have used heat-killed M. gallisepticum and M. meleagridis to induce the synthesis of APP in chickens, and Takahashi et al. (1994, 1997) have used Escherichia coli to induce AGP synthesis in broiler chickens. Iseri and Klasing (2013) also observed that plasma AGP concentrations in White Leghorn roosters increased significantly by 18 h and peaked at 24 h after birds were intravenously injected with dead E. coli. It was also observed that through 48 h after challenge, AGP remained at levels intermediate to those at 18 and 24 h. However, no information is available concerning possible changes in the circulating APP concentrations of layers in response to their subjection to prelay LAV in combination an LAV overlay during the EP period. Therefore, the objective of the current investigation was to determine the serum concentrations of AGP in layers in response to the vaccination regimen previously suggested by Peebles and Branton (2012) that is administered alone or in combination with a prelay MGBac vaccine. MATERIALS AND METHODS Pullet Housing and Management 1397 One-day-old Hy-Line W-36 commercial laying chicks were obtained from a certified M. gallisepticum- and Mycoplasma synoviae-free commercial source. The chicks were placed in a conventional house on clean dry pine shavings until 9 woa and were fed with diets formulated to meet or exceed NRC (1994) guidelines. Feed and water were provided ad libitum for the duration of the study. The experimental birds were transferred into an environmentally controlled disease isolation facility with temperature-controlled negative-pressure fiberglass isolation facilities at 9 woa. Eleven pullets were randomly selected and placed into each of 16 isolation units with 4 replicate units assigned to each of 4 different treatment groups. When the total daily EP of the birds in each unit reached 10%, hen numbers were reduced to 10 birds per unit, for a total of 40 birds per treatment group (Jacob et al., 2014). The units were housed in a previously described poultry disease isolation facility (Branton and Simmons, 1992), which were maintained under the same conditions as those described by Evans et al. (2012). Four birds per unit were bled from the ulna cutaneous vein for the detection of M. gallisepticum infection via serum plate agglutination (SPA) test while being placed in the isolation units. At the same time, choanal cleft swabs were collected from all the birds in each

3 1398 Peebles et al. unit to confirm their Mycoplasma-free status by PCRbased DNA detection techniques (Kleven, 2008). Choanal cleft swab samples were also taken to confirm the relative retention of the applied vaccines in vaccinated birds and the absence of M. gallisepticum in nonvaccinated birds at 43, 45, and 52 woa. The DNA from the swabs was used for M. gallisepticum-specific PCR using primers described by Lauerman (1998). All the procedures were approved by the USDA-Agricultural Research Service Institutional Animal Care and Use Committee (Mississippi State, MS). Data Collection Blood was collected from 3 tagged birds per treatment replicate unit on d 0, 1, 3, 7, 14, and 28 after vaccination at 10 and 45 woa to determine serum AGP concentrations. On d 0 at 10 and 45 woa, blood was collected immediately before vaccination. Furthermore, blood was collected from the same 3 tagged birds per treatment replicate unit at 9 and 43 woa to determine baseline concentrations of serum AGP before treatment. Layer Housing and Management Beginning at 18 woa, the artificial lighting schedule was increased by 15 min/d until a cycle of 16 h 15 min light and 7 h 45 min dark was achieved. Layers were maintained on that schedule through the reminder of the experiment. At 24 and 48 woa, feed samples were analyzed for moisture, ash, CP, crude fat, crude fiber, and Ca, according to the methods of the Association of Official Analytical Chemists (AOAC, 1990). Treatments At 10 woa, birds in treatment 1 (control) received no vaccinations. Birds in treatment 2 (MGBac) were vaccinated with M. gallisepticum-bacterin (MG-Bac, Fort Dodge Animal Health, Overland Park, KS) via an intramuscular (breast) injection route. Birds belonging to treatment 3 (ts11mg) were eye-drop-vaccinated in the left eye with ts11-strain M. gallisepticum (Mycoplasma Gallisepticum Vaccine, Merial Select, Gainesville, GA). In treatment group 4, birds were vaccinated with a combination of both ts11mg and MGBac (ts11mg & MGBac). At 45 woa, the birds in half of the replicate pens in each of the previous treatment groups were challenged with a laboratory stock of HpFMG (99th passage above the unknown passage level). A 24 h broth culture of HpFMG was generated in Frey s broth medium (Frey et al., 1968) and 20 μl of the overnight culture was applied via eye-drop inoculation. Birds in treatment 1 (control) that were and were not vaccinated with HpFMG, and birds in treatments 2, 3, and 4 that were vaccinated with HpFMG were examined for the remainder of the study. Therefore, after HpFMG vaccination at 45 woa, the following treatment groups consisted of 2 isolation units containing 10 birds each, for a total of 20 birds per treatment group. Birds in treatment 1 (double control) received no vaccination at 10 or 45 woa. Birds in treatment 2 (single control) received no vaccination at 10 woa, but received Hp- FMG at 45 woa. Birds in treatment 3 received MGBac at 10 woa and HpFMG at 45 woa. Birds in treatment 4 received ts11mg at 10 woa and HpFMG at 45 woa. In treatment 5, birds received ts11mg & MGBac at 10 woa and HpFMG at 45 woa. Handling procedures were implemented that minimized the risk of cross-contamination. α 1 -Acid Glycoprotein ELISA A solid phase ELISA kit was used for the determination of chicken AGP concentrations. The assay used affinity purified anti-chicken AGP antibodies for solid phase immobilization and horseradish peroxidase conjugated anti-chicken AGP antibodies for detection. Standard preparation, sample preparation, and the materials and methods of the assay were performed as described by the manufacturer (Life Diagnostics Inc., Westchester, PA). Each sample was tested in duplicate. Serum AGP concentrations were expressed as milligrams per milliliter. Statistical Analysis A completely randomized experimental design was used. The data at 10 and 45 woa were analyzed separately. Within each of wk 10 and 45, the data examined at the multiple time periods (day) were subjected to repeated measures analysis. The fixed effects in the analyses were day, treatment, and day treatment. A spatial power correlation structure was used to account for unequally spaced repeated measures on individual birds. The baseline data across 9 and 43 woa were subjected to ANOVA with treatment as the fixed effect. Replicate, treatment replicate, and bird (treatment replicate) were considered as random effects in both analyses. Duplicate sample data for each individual bird were averaged before analysis. All data were analyzed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC). Least squares means were compared in the event of significant global effects, and both global effects and differences among least squares means were considered significant at P RESULTS AND DISCUSSION The initial SPA and PCR results at 9 woa were negative for all the birds, which established their M. gallisepticum-free status. At 43 and 45 woa, PCR results showed that control and MGBac-vaccinated birds were negative, whereas ts11mg- and ts11mg & MGBacvaccinated birds were positive for the presence of M. gallisepticum DNA. These results confirmed the lack of any cross-contamination by ts11mg. Furthermore, at

4 ACUTE PHASE PROTEIN RESPONSE TO VACCINATION 1399 Table 1. Mean serum α 1 -acid glycoprotein concentrations (mg/ml) in layer pullets on d 0, 1, 3, 7, 14, and 28 after having received no vaccination (control), or after being vaccinated with Mycoplasma gallisepticum bacterin (MGBac), ts11 strain M. gallisepticum (ts11mg), or a combination of both ts11mg and M. gallisepticum bacterin (ts11mg & MGBac) at 10 wk of age 1,2 Day Control MGBac ts11mg ts11mg & MGBac c b ab a c b b a b a a a b a a a b a a a a c Means within a row with no common superscript differ significantly (P 0.05). 1 Two duplicate samples from each of 3 birds in each of 4 replicate units were used to calculate each mean. 2 Pooled SEM = woa, all birds that were administered the HpFMG vaccine overlay were positive for M. gallisepticum PCR, whereas the double control birds were negative. Mean baseline AGP concentrations at 9 woa (before vaccination treatment at 10 woa) in birds assigned to the control, MGBac, ts11mg, and ts11mg & MGBac treatment groups were 0.470, 0.486, 0.438, and 0.506, respectively (pooled SEM = ). Mean baseline AGP concentrations at 43 woa in birds assigned to the double control treatment group, and in the single control, MGBac, ts11mg, and ts11mg & MGBac treatment groups that were later vaccinated with HpFMG at 45 woa were 0.492, 0.481, 0.548, 0.493, and 0.553, respectively (pooled SEM = ). No significant (P = 0.562) differences in baseline AGP concentrations were observed among the various vaccine treatments across 9 and 43 woa. The baseline values observed at 9 and 43 woa were close to those observed by Nakamura et al. (1998) in 2 woa male specific-pathogen-free (SPF) White Leghorn chickens, 12 h after being shaminoculated with PBS. These current values were also at the upper range of baseline values reported by Iseri and Klasing (2013) for 18 to 24 woa SPF White Leghorn roosters. At 10 woa, there was a significant (P ) day treatment interaction for AGP (Table 1). Immediately before birds received 1 of the 3 previously described vaccine treatments on d 0 at 10 woa, there were no significant differences in serum AGP concentrations in the birds belonging to the different treatment groups. Conversely, on d 1 (24 h) after birds had been vaccinated, serum AGP concentrations in birds that belonged to the ts11mg & MGBac treatment group were higher than those in the MGBac treatment group, which in turn, along with the ts11mg-treated birds, were higher than that in the controls. Serum AGP levels in the ts11mg group were intermediate to those in the MGBac and ts11mg & MGBac treatment groups. However, at 3 d postvaccination, the AGP concentrations of birds in the ts11mg & MGBac group were higher than those in both the ts11mg and MGBac groups, and both of those were likewise higher than the controls. Treatment differences for AGP were similar on d 7, 14, and 28 postvaccination. At those times, AGP concentrations in the ts11mg & MGBac, ts11mg, and MGBac groups were not different from one another, but all were higher than those in the control group (Table 1). At 10 woa, the results indicated that circulating AGP concentrations on d 1 and 3 postvaccination were increased by both the MGBac and ts11mg vaccines, but that the greatest increase resulted in response to the ts11mg & MGBac treatment combination. The increase in AGP in response to the ts11mg & MGBac treatment was significantly greater than that for the MGBac treatment on d 1, and for both the MGBac and ts11mg treatments on d 3. However, on d 7, 14, and 28, although the 3 vaccination treatments increased AGP concentrations in comparison with those in the control group, the ts11mg & MGBac treatment no longer exerted a stronger effect than it did compared with the MGBac on d 1 and both the MGBac and ts11mg on d 3. Similar to the current AGP results in pullets at 10 woa, Inoue et al. (1997) observed that plasma AGP levels in SPF White Leghorn chickens at 3 to 7 woa were significantly increased 2 d after being inoculated with either of 2 virulent strains of the infectious bursal disease virus. Maximum AGP levels were reached at 4 to 5 d postinoculation. A vaccine strain of the virus also caused slight, but nonsignificant, increases in AGP at 4 d postinoculation. By 10 d postinoculation, plasma AGP levels in those birds inoculated with the virulent strains returned to normal levels, whereas those administered the vaccine strain returned to normal levels by 6 d postinoculation. Likewise, the injection of SPF roosters between 18 and 24 woa with killed bacteria (E. coli) was shown by Iseri and Klasing (2013) to increase plasma AGP at 18, 24, and 48 h after injection. A similar response to the inoculation of E. coli-derived lipopolysaccharide in SPF chickens at 2 woa was observed by Nakamura et al. (1998). The postinoculation serum AGP levels in those birds increased between 6 h and 4 d, with a peak at 2 d, after which levels returned to normal. The injection of E. coli-derived lipopolysaccharide in broiler chicks at 26 d of age was also shown to result in increases in their plasma AGP concentrations when

5 1400 Peebles et al. blood was collected 16 h after injection (Takahashi et al., 1997). At 45 woa, there was also a significant (P ) day treatment interaction for AGP (Table 2). Similar to that at 10 woa, there were no significant differences in the serum AGP concentrations of the birds before the various vaccine treatments were administered on d 0 at 45 woa. However, by d 1 (24 h) after the birds were treated, serum AGP levels were higher in the ts11mg & MGBac and HpFMG treatment group in comparison with that in the ts11mg and HpFMG, MGBac and HpFMG, and double control groups. The AGP levels of the single control group were also higher than that in the MGBac and HpFMG group, as well as the double control group, and AGP in the ts11mg and HpFMG birds was higher than that in the double controls. On d 3, 7, and 14, serum AGP in the single control group and the MGBac, ts11mg, and ts11mg & MGBac treatment groups that were later vaccinated with HpFMG at 45 woa were not different, but all were significantly higher than that in the double control group. Conversely, on d 28, circulating AGP concentrations were higher in the MGBac and HpFMG treatment group in comparison to the ts11mg & MGBac and HpFMG group as well as the double control group. In addition, the ts11mg and HpFMG, and the single control treatment birds had higher AGP levels than those in the double control birds (Table 2). Holt and Gast (2002) have reported that elevated serum AGP levels were observed in SPF Single Comb White Leghorn hens at 85 and 88 woa in response to the injection of live Salmonella enteritidis, with elevated levels being recorded on d 3 and 9 postchallenge. Similarly, in the current study, except for the MGBac treatment on d 1 and the ts11mg & MGBac treatment on d 28, the wk 45 HpFMG vaccination overlay significantly elevated AGP concentrations in all the treatment groups in comparison with the double control group on d 1, 3, 7, 14, and 28 postvaccination. Furthermore, at 45 woa, the results indicated overall that the previous treatment of birds with the MGBac, ts11mg, or ts11mg & MGBac vaccines did not exacerbate the stimulatory effect of the wk 45 HpFMG overlay vaccine on circulating AGP concentrations on d 1, 3, 7, 14, and 28 postvaccination. Finally, a review of the 9 and 43 woa baseline means for AGP showed that by 43 woa, AGP concentrations returned to those observed at 9 woa (before any vaccinations were administered) regardless of treatment at 10 woa. Serum AGP concentrations in vaccinated birds at 10 and 45 woa on d 1, 3, 7, 14, and 28 fell in the range of those that were reported by Iseri and Klasing (2013) at 18, 24, and 48 h after SPF White Leghorn roosters, aged 18 to 24 woa, were injected intravenously with dead E. coli. The results of this current study also showed that circulating AGP concentrations in commercial laying hens increase in response to an infection by HpFMG at 45 woa in association with depressed performance. However, results also showed that AGP concentrations were increased in response to subclinical infections by MGBac and ts11mg that were initiated at 10 woa. More specifically, elevated AGP levels in these birds were sustained up to 28 d after the inclusion of MGBac, ts11mg, or their combination in vaccines that were delivered at 10 woa. Because MGBac is a killed bacterium, it is noteworthy that whether live or dead, M. gallisepticum was able to similarly elicit a significant elevation of AGP in the blood. Nevertheless, it is of further interest that the combination of ts11mg and MGBac led to a greater response on d 1 and 3 postvaccination than did the use of ts11mg or MGBac given individually at 10 woa. Elevated AGP levels in these birds were also sustained up to 28 d after the inclusion of HpFMG in an overlay vaccine that was delivered at 45 woa. This occurred after AGP concentrations had returned to baseline levels by 43 woa. In conclusion, the observation of elevated circulating AGP concentrations may be a useful tool in confirming subclinical infections as well as those that coincide with depressed performance in layers up to 28 d after their exposure to M. gallisepticum. More specifically, elevated circulation AGP concentrations may be used Table 2. Mean serum α 1 -acid glycoprotein concentrations (mg/ml) in layer pullets on d 0, 1, 3, 7, 14, and 28 in the no 10 or 45 wk of age vaccination treatment group (double control), and in the 10 wk of age no vaccination (single control), Mycoplasma gallisepticum bacterin (MGBac), ts11 strain M. gallisepticum (ts11mg), and ts11mg and M. gallisepticum bacterin (ts11mg & MGBac) treatment groups that were later vaccinated with high passage F-strain Mycoplasma gallisepticum at 45 wk of age 1,2 Day Double control Single control MGBac ts11mg ts11mg & MGBac d ab cd bc a b a a a a b a a a a b a a a a c ab a ab bc a d Means within a row with no common superscript differ significantly (P 0.05). 1 Two duplicate samples from each of 3 birds in each of 2 replicate units were used to calculate each mean. 2 Pooled SEM =

6 to detect and confirm subclinical infections in pullets up to 28 d after having been vaccinated with ts11mg, MGBac, or their combination. Furthermore, in association with depressed performance, elevated serum AGP concentrations in layers may be used to confirm Hp- FMG infections up to 28 d after its use as a vaccine. REFERENCES Abd-el-Motelib, T. Y., and S. H. Kleven A comparative study of Mycoplasma gallisepticum vaccines in young chickens. Avian Dis. 37: Association of Official Analytical Chemists (AOAC) Official Methods of Analysis. 9 CFR (b), AOAC Intl., Vol. 1, 15th ed. AOAC, Arlington, VA. Barbour, E. K., S. K. Hamadeh, and A. Eid Infection and immunity in broiler chicken breeders vaccinated with a temperature-sensitive mutant of Mycoplasma gallisepticum and impact on performance of offspring. Poult. Sci. 79: Branton, S. L., H. Gerlach, and S. H. Kleven Mycoplasma gallisepticum isolation in layers. Poult. Sci. 63: Branton, S. L., B. D. Lott, J. W. Deaton, J. M. Hardin, and W. 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