J. Anim. Sci. 2003. 81:2139-2144
© 2003 American Society of Animal Science
Effect of dietary Echinacea purpurea on viremia and performance in porcine reproductive and respiratory syndrome virus-infected nursery pigs1
J. R. Hermann*,
M. S. Honeyman*,2,
J. J. Zimmerman
,
B. J. Thacker,
P. J. Holden* and
C. C. Chang
* Department of Animal Science and
and
Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames 50011
 |
Abstract
|
|---|
The effect of dietary Echinacea purpurea on performance, viremia, and ontogeny of the humoral antibody response against porcine reproductive and respiratory syndrome virus (PRRSV) infection was evaluated in weaned pigs. In three replicates, 120 weaned pigs (25 ± 1 d of age; 8.46 ± 0.48 kg of BW) from a PRRSV-naíve herd were allotted randomly to one of eight pens (diets) in two separate rooms (four pens/room), with each pen containing five pigs. Pigs began one of four dietary treatments (as-fed basis) 1 wk before inoculation with PRRSV: 1) basal diet composed of corn, soybean meal, whey, and essential vitamins and minerals; 2) basal diet plus carbadox (0.055 g/kg of diet; as-fed basis); 3) basal diet plus Echinacea 2% (2% of the total diet); 4) basal diet plus Echinacea 4% (4% of the total diet). The diets were formulated to be isocaloric and isolysinic. Echinacea purpurea was purchased in powder form and determined by chemical analysis to contain 1.35% cichoric acid (as-fed basis). Seven days after starting the diets, all pigs in one room were intranasally inoculated with PRRSV isolate ATCC VR-2332 at a concentration of 104 tissue culture infectious dose50/mL. To monitor the effects of Echinacea and PRRSV challenge, BW and blood samples were obtained from all pigs at 7-d intervals. Serum samples were analyzed for the presence of PRRSV and PRRSV-specific antibodies. All challenged pigs became infected with PRRSV, and all unchallenged pigs remained free of infection. No differences (P > 0.10) in ADG, ADFI, or gain:feed (G:F) were observed in PRRSV-challenged compared with unchallenged animals. For PRRSV-challenged animals receiving diets supplemented with Echinacea at 2 or 4%, no differences (P > 0.10) were observed in ADG, ADFI, or G:F ratio. Among PRRSV-challenged pigs, dietary Echinacea did not affect (P > 0.10) the rate or level of the ELISA-detectable antibody response from d 7 to 42 or the level and duration of PRRSV in serum. For PRRSV-unchallenged animals receiving diets supplemented with Echinacea at 2 or 4%, no differences (P > 0.10) were observed in ADG, ADFI, and G:F ratio. Under the conditions of this study, dietary Echinacea did not enhance growth, exhibit antiviral effects to PRRSV, or show any evidence of immune enhancing properties.
Key Words: Echinacea • Piglets • Porcine Reproductive and Respiratory Syndrome • Viremia
|
Introduction
|
|---|
Antimicrobial agents are used in food animals as therapy for an infection or, in the absence of disease, for the subtherapeutic purpose of growth promotion as measured by increased rate of gain and improved feed efficiency (Stahly et al., 1980
; Gorbach, 2001). Increased interest in curbing antibiotic use to reduce antimicrobial resistance has led to a growing interest in alternative growth promoters. The use of herbal remedies or botanicals in swine diets has been proposed because of their natural stimulation of the immune system and/or enhanced growth performance. Extracts from Echinacea have been shown to have nonspecific immunostimulatory properties in vitro (Bauer and Wagner, 1991), including increased phagocytosis (Stotzem et al., 1992), increased cytokine production (Burger et al., 1997), and natural killer cell activity (See et al., 1997). Rehman et al. (1999) showed an increase in primary and secondary immunoglobin G response in rats treated with Echinacea. Hypothetically, these immune enhancing properties of Echinacea could provide resistance to viral swine diseases, such as porcine reproductive and respiratory syndrome virus (PRRSV), and assist in the control of secondary bacterial infections. A few scientific studies have assessed the efficacy of Echinacea in vivo with varying results (Melchart et al., 1998; Grimm and Müller, 1999; Turner et al., 2000). We know of no studies that have involved pigs as an animal model in assessing the efficacy of Echinacea as an immunostimulant. Work evaluating Echinacea as a possible growth promotant for pigs is also limited (Holden and McKean, 2000).
Our objective was to determine the effects of dietary Echinacea purpurea, with a defined chemical profile, on growth performance, level of viremia, and ontogeny of antibody response in PRRSV-challenged nursery-age pigs.
|
Materials and Methods
|
|---|
Animals
Three replicate, successive trials involving a total of 120 pigs (average initial BW = 8.46 ± 0.48 kg; 25 ± 1 d of age) were conducted at the Iowa State University Livestock Infectious Disease Isolation Facility (LIDIF), Ames. Imposition of treatments began 1 wk after a 7-d acclimation period. All pigs were farrowed and reared at the Iowa State University Lauren Christian Swine Research and Demonstration Farm, Atlantic. Sows were vaccinated 2 wk before their expected farrowing date with Clostridium perfringens type C/Escherichia coli bacterin-toxoid (Littergard-LTC, Pfizer, Inc., NY) and farrowed in crates. In each trial, 40 crossbred piglets (Duroc terminal sires crossed on predominantly Yorkshire x Landrace sows) from this PRRSV-naïve herd were weaned between 17 to 19 d of age and allotted to one of eight pens in two separate rooms (four pens per room) at the LIDIF. Pigs were allotted to balance for pig weight and litter source. Animal housing and care was conducted under supervision of the Iowa State University Committee on Animal Care (Log No. 6-1-4861-S) and in accordance with FASS (1999). At birth, pigs were tail docked, ear notched, teeth clipped, males castrated, and injected subcutaneously with 2 mL of iron dextran (iron hydrogenated dextran injection, Durvet Inc., Blue Springs, MO) and 0.5 mL of ceftiofur sodium (Naxel, Pfizer, New York). At 7 d of age, all pigs received a second injection of 1 mL of ceftiofur sodium. At weaning, all pigs received injections of 0.5 mL of ivermectin (Ivomec, Merial, Duluth, GA) and 1 mL of penicillin (Han-Pen B, Hanford Pharmaceuticals, Syracuse, NY), were weighed and allotted, and then moved to their assigned treatment. One pig was removed from the experiment due to a locomotor injury, and one pig died prior to PRRSV inoculation due to intestinal torsion. The experimental design included three replications of two rooms (PRRSV-positive and PRRSV-negative), four pens per room, five pigs per pen, and four dietary treatments.
Body/Feed Weights and Blood Collection
Pigs and feeders were weighed initially and at 7-d intervals (including a 7-d pretrial period) until completion of each 42-d trial. Feed was weighed and added as needed. For each pen, ADG and ADFI (as-fed basis) were calculated. Blood was collected from the anterior vena cava of each pig at 7-d intervals (including the 7-d pretrial period). Blood sampling methods used were described by Straw et al. (1999). The blood was centrifuged at 2,000 x g for 10 min at 4°C. The serum was stored in Falcon 5-mL polystyrene round-bottom tubes (Becton Dickinson Labware, Franklin Lakes, NJ) at -20°C until tested.
Housing
The LIDIF is a biosafety Level 2 building. The environment in each room is strictly controlled (humidity 70%, temperature 26.6°C). The pigs were housed in an infected room or a noninfected control room. Five pigs were penned on nursery decks (1.22 x 2.43 m) with plastic-slatted floors. Each pen had one nipple waterer and a four-hole Kane polyethylene nursery feeder (60 x 20 x 60 cm). Heat lamps were used for the 7-d pretrial period.
Experimental Diets
Pigs (pens) began one of four dietary treatments 1 wk (d 0) before inoculation with PRRSV. The treatment diets (as-fed basis) were: 1) basal diet composed of corn, soybean meal, whey, and essential vitamins and minerals (no additive); 2) basal diet plus carbadox (0.055 g/kg); 3) basal diet plus Echinacea 2% (2% of the total diet); and 4) basal diet plus Echinacea 4% (4% of the total diet). Four phases of each diet were fed to coincide with different stages of growth. For d -7 to 0, 0 to 7, 7 to 26, and 26 to 42, the pigs were fed Phases A, B, C, and D diets, respectively (Table 1). The pigs were moved to the rooms and fed Phase A for the 7-d acclimation period. Phase B was fed beginning on d 0, which was the beginning of the trial. All diets were in meal form, and the pigs were given ad libitum access to feed. Diets were formulated to meet or exceed nutritional requirements (NRC, 1998).
The gross energy of Echinacea was determined to be 3,344 kcal/kg (adiabatic bomb calorimeter, Parr Instrument Co., Inc., Moline, IL). Using the gross energy value of Echinacea and values from the NRC (1998) and Ewan (1996), the diet was adjusted to compensate for the lower energy content of Echinacea. Soy oil was added with the Echinacea, and equal amounts of corn were removed. The diets contained nutrient concentrations that met or exceeded the estimated nutrient requirements of nursery pigs (NRC, 1998). Feeding mats (0.42 x 0.77 m) were placed in front of the feeders for the pretrial period. All pigs were fed Phase A in the self-feeders with additional feed on the nursery mats. Decreasing amounts of feed were placed on the mats until they were removed on d 0. Wasted feed was minimal and not recorded.
Certified organic Echinacea purpurea root was used for the presumed immunostimulatory activity of the caffeic acid derivative, cichoric acid (Bauer et al., 1989). Echinacea was fed before inoculation with PRRSV until the end of the 42-d trial to allow for possible effects on immune function to be observed. Three-year-old plants were harvested in September, and the root was dried with forced air to a moisture content of 9% (Nature’s Cathedral, Inc., Blairstown, IA). The root was ground and sifted to a powder, and then mixed with basal diets and fed. The 2 and 4% Echinacea treatment levels were chosen based on preliminary work at Iowa State University (Holden and McKean, 2000). Determination of phenolics in Echinacea was performed by Alpha Laboratories Division (Petaluma, CA). High-pressure liquid chromatography was used to determine the content of caftaric acid, chlorogenic acid, cichoric acid, and echinacoside in the dried, powdered Echinacea purpurea root. The final results are expressed as a percentage of the total components in the material analyzed. The Echinacea contained (as-fed basis) 0.39% caftaric acid, 0.01% chlorogenic acid, less than 0.01% echinacoside, and 1.35% cichoric acid.
Virus Preparation
A North American prototype PRRSV isolate ATCC VR-2332 (American Type Culture Collection, Manassas, VA; Benfield et al., 1992; Collins et al., 1992) that had one pig passage (Chang et al., 2002) was used. The virus was propagated on MARC-145 cells, a clone of the African monkey kidney cell line MA-104 considered highly permissive to PRRSV (Kim et al., 1993). The concentration of 104 tissue culture infectious dose (TCID)50/mL of the virus was adjusted for the challenge dose. Pigs were challenged on d 7 by the oral nasal route with 2 mL (1 mL/naris) of clarified virus supernatant 104 TCID50/mL. Pigs were inoculated with PRRSV after d 21 of age to guarantee that the immune system was developed (Varley, 1995).
Virus Titration
A microtitration infectivity assay was performed to estimate the concentration of PRRSV in serum samples collected over time from inoculated pigs. Samples were serially 10-fold diluted (10-0 to 10-6) in culture medium. One hundred microliters of each dilution was added to three wells of a 96-well microtitration plate (Corning, Inc., Corning, NY) containing 24-h-old confluent MARC-145 cell monolayers. Inoculated cells were incubated at 37°C in a 5% CO2 humidified incubator. Each sample was run in duplicate. The cells were monitored daily for cytopathic effect for up to 7 d. If cytopathic effect was not evident, the cells were fixed with 80% acetone, dried, stained with fluoroisothiocyanate-conjugated monoclonal antibody (Mab) specific for the N protein of PRRSV (Mab SDOW17, Rural Technologies, Brookings, SD), and visualized with fluorescence microscopy. The presence of PRRSV was based on the visualization of virus-specific cytopathic effect and/or fluorescence reaction. Virus titers were determined using the method described by Reed and Muench (1938) and expressed as TCID50/mL. Serum antibody titers confirmed that the pigs were PRRSV naïve at weaning and before virus challenge.
Enzyme-linked Immunosorbent Assay
A commercial ELISA kit (Herdchek Porcine Reproductive and Respiratory Syndrome Virus Antibody Kit, IDEXX Laboratories, Westbrook, ME) was used to detect PRRSV-specific antibody in serum samples by following the procedures recommended by the manufacturer. A sample was classified as positive for PRRSV antibody if the sample:positive ratio was equal to or greater than 0.4.
Data Analysis
Data were analyzed with fixed effect models with week as a repeated measure using the GLM procedure of SAS (SAS Inst. Inc., Cary, NC). The dependent variables were growth performance and viremia. The independent variables were PRRSV, diet, and time. The interactions of PRRS x time, PRRS x diet, diet x time, and PRRS x diet x time were examined for statistical significance. If higher-level interactions were not statistically significant, then lower-level terms were tested. The pen was considered the experimental unit. Data were reported as least squares means. A significance level of less than 0.05 was required as the minimum acceptable P-value.
|
Results
|
|---|
Pig Performance
No dietary treatment x PRRSV interactions (P > 0.80) were observed for performance data (Table 2). A dietary treatment x time (P = 0.015) interaction was observed for ADG; however, no other interactions between diet or PRRSV and time (P > 0.10) were observed for ADG, ADFI, or gain:feed ratio (Table 2). Notwithstanding the dietary treatment x time interaction, no differences were observed on individual sampling day (data not shown), and averaged across sampling day, no differences on ADG were observed between Echinacea at either 2 or 4% compared with controls. Likewise, diets supplemented with carbadox had no effect on ADG (P = 0.499). Pigs fed diets supplemented with Echinacea grew at a comparable rate to pigs fed diets supplemented with carbadox. Feeding Echinacea at either 2 or 4% and supplementing diets with carbadox did not affect ADFI (P = 0.340) or gain:feed (P = 0.298) compared to control (Table 2). Porcine reproductive and respiratory syndrome virus status had no effect on ADG (P = 0.192), ADFI (P = 0.393), or gain:feed (P = 0.630; Table 2).
View this table:
[in this window]
[in a new window]
|
Table 2. Effects of feeding Echinacea purpurea on performance in porcine reproductive and respiratory syndrome virus (PRRSV) unchallenged and challenged nursery pigsa
|
|
Viremia and Serum Antibody Response
For pigs inoculated with PRRSV, no dietary treatment x time interactions (P > 0.40) were observed for serum titers or serum to positive ratios (Table 3). Averaged across time, no differences were detected in pigs among the dietary treatments in level of ELISA-detectable antibody response or number of pigs positive for PRRSV antibody (P = 0.186). Likewise, no differences were detected in level or duration of viremia (P = 0.947) with dietary treatment.
View this table:
[in this window]
[in a new window]
|
Table 3. Effects of feeding dietary Echinacea purpurea on serum porcine reproductive and respiratory syndrome virus (PRRSV) titers and ELISA serum:positive ratiosab
|
|
|
Discussion
|
|---|
Porcine reproductive and respiratory syndrome virus infection became pandemic in the domestic swine population during the 1990s (Zimmerman, 2003). Clinical signs vary widely within and between herds, but infection is uniformly persistent in individual pigs (Horter et al., 2001; Thacker, 2003). Porcine reproductive and respiratory syndrome virus was selected for our study because of its economic importance to pork producers and our laboratory’s extensive experience with the virus.
Although PRRSV infection is reported to inhibit growth in pigs (Spurlock et al., 1997; Greiner et al., 2001), differences in performance between PRRSV-challenged and -unchallenged animals were not observed in this experiment. The absence of growth differences may have been the result of the high health status of the pigs, and the low potential for infection by secondary pathogens. Additionally, the possible growth effects of dietary Echinacea were not observed, perhaps for similar reasons.
Extracts from Echinacea have been shown to have nonspecific immunostimulatory properties in vitro (Bauer and Wagner, 1991), including increased phagocytosis (Stotzem et al., 1992), increased cytokine production (Burger et al., 1997), and natural killer cell activity (See et al., 1997). Echinacea has been shown to increase immunoglobulin G levels in rats (Rehman et al., 1999). Current information suggests that the immunostimulatory activity of Echinacea species depends on the combined action of caffeic acid derivatives and alkylamides (Bauer et al., 1989; Bauer and Wagner, 1991), although the mode of action of these compounds on immune function has not been documented. Echinacea products vary widely in chemical composition, making standardization difficult (Osowski et al., 2000; Perry et al., 2001). The species of Echinacea, the soil type in which the plant is grown, the phase of plant development at harvest, the parts of the plant selected, and processing procedures influence the chemical composition and activity (Letchamo et al., 1999; Perry et al., 2001). The absence of antiviral effects in this study could be attributed to variable chemical activity. The efficacy of dietary Echinacea may vary physiologically and metabolically across various animal species. Work comparing Echinacea in various animal species has not been done.
|
Implications
|
|---|
The need to find alternative dietary growth promotants and immune modulators has led to interest in Echinacea. Echinacea purpurea fed in this study (0.39% caftaric acid, 0.01% chlorogenic acid, less than 0.01% echinacoside, and 1.35% cichoric acid) as 2% or 4% of the basal diet did not augment the antigen-specific antibody response to porcine reproductive respiratory syndrome virus, inhibit virus replication, improve elimination of virus, or promote growth in nursery-age pigs. The role of Echinacea as a possible antiviral, immune-enhancing, or growth-promoting compound was not supported by this study. Additional work examining the possible efficacy of Echinacea in relation to other common swine pathogens or combinations of pathogens could be conducted.
|
Footnotes
|
|---|
1 This project was supported by the Leopold Center for Sustainable Agriculture, Hatch Act funds, and State of Iowa funds. Mention of company or product names is for presentation clarity and does not imply endorsement by the authors or Iowa State University, nor exclusion of any other products that may also be suitable for application. The authors gratefully acknowledge B. Fowles for her work in serology, D. McDonald at LIDIF, and employees at the swine nutrition farm, S. Medford, P. Horton, L. Mosman, T. Lutz, and R. Evans. Also, thanks to B. Wheeler, J. Gilbert, A. Penner, N. Will, D. Horter, K. Krumm, C. Johnson, and C. Kulchaiyawat for their help in data and sample collection. 
2 Correspondence— phone: 515-294-4621; fax: 515-294-6210; E-mail: honeyman{at}iastate.edu.
Received for publication October 7, 2002.
Accepted for publication June 2, 2003.
|
Literature Cited
|
|---|
Bauer, R., P. Remiger, K. Juric, and H. Wagner. 1989. Influence of Echinacea extracts on phagocytotic activity. Z. Phytother. 10:43–48.
Bauer, R., and H. Wagner. 1991. Echinacea species as potential immunostimulatory drugs. Pages 253–321 in Economic and Medicinal Plant Research. N. R. Farnsworth and H. Wagner, ed. Academic Press Limited, London, U.K.
Benfield, D. A., E. Nelson, J. E. Collins, L. Harris, S. M. Goyal, D. Robison, W. T. Christianson, R. B. Morrison, D. Gorcyca, and D. Chladek. 1992. Characterization of swine infertility and respiratory syndrome (SIRS) virus (isolate ATCC VR-2332). J. Vet. Diagn. Invest. 4:127–133.[Abstract/Free Full Text]
Burger, R. A., A. R. Torres, R. P. Warren, V. D. Caldwell, and B. G. Hughes. 1997. Echinacea-induced cytokine production by human macrophages. Int. J. Immunopharmacol. 19:371–379.[Medline]
Chang, C. C., K. J. Yoon, J. J. Zimmerman, K. M. Harmon, P. M. Dixon, C. M. Dvorak, and M. P. Murtaugh. 2002. Evolution of porcine reproductive and respiratory syndrome virus during sequential passages in pigs. J. Virol. 76:4750–4763.[Abstract/Free Full Text]
Collins, J. E., D. A. Benfield, W. T. Christianson, L. Harris, J. C. Hennings, D. P. Shaw, S. M. Goyal, S. McCullough, R. B. Morrison, H. S. Joo, D. Gorcyca, and D. Chladek. 1992. Isolation of swine infertility and respiratory syndrome virus (isolate TCC VR-2332) in North America and experimental reproduction of the disease in gnotobiotic pigs. J. Vet. Diagn. Invest. 4:117–126.[Abstract/Free Full Text]
Ewan, R. C. 1996. Energy Values of Feed Ingredients. 5th rev. ed. Iowa State Univ., Ames.
FASS. 1999. Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching. 1st ed. Fed. Anim. Sci. Soc., Savoy, IL.
Gorbach, S. L. 2001. Antimicrobial use in animal feed-time to stop. N. Engl. J. Med. 16:1202–1203.
Greiner, L. L., T. S. Stahly, and T. J. Stabel. 2001. The effect of dietary soy genistein on pig growth and viral replication during a viral challenge. J. Anim. Sci. 79:1272–1279.[Abstract/Free Full Text]
Grimm, W., and H. H. Müller. 1999. A randomized controlled trial of the effect of fluid extract of Echinacea purpurea on the incidence and severity of colds and respiratory infections. Am. J. Med. 106:138–143.[Medline]
Holden, P. J., and J. D. McKean. 2000. Botanicals for Pigs—Echinacea II. ASL-R647. 2000 ISU Swine Research Report, AS-644. Iowa State Univ., Ames.
Horter, D., C. C. Chang, R. Pogranichnyy, J. Zimmerman, and K. J. Yoon. 2001. Persistence of porcine reproductive and respiratory syndrome in pigs. Adv. Exp. Med. Biol. 494:91–94.[Medline]
Kim, H. S., J. Kwang, I. J. Yoon, H. S. Joo, and M. L. Frey. 1993. Enhanced replication of Porcine Reproductive and Respiratory Syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line. Arch. Virol. 133:477–483.[Medline]
Letchamo, W., J. Livesey, T. J. Arnason, C. Bergeron, and V. S. Krutilina. 1999. Cichoric acid and isobutylamide content in Echinacea purpurea as influenced by flower developmental stages. Pages 494–498 in Perspectives on New Crops and New Uses. J. Janick, ed. ASHS Press, Alexandria, VA.
Melchart, D., E. Walther, K. Linde, R. Brandmaier, and C. Lersch. 1998. Echinacea root extracts for the prevention of upper respiratory tract infections: A double-blind, placebo-controlled randomized trial. Arch. Fam. Med. 7:541–545.[Abstract/Free Full Text]
NRC. 1998. Nutrient Requirements of Swine. 10th ed. Natl. Acad. Sci., Washington, DC.
Osowski, S., M. Rostock, H. H. Bartsch, and U. Massing. 2000. Pharmaceutical comparability of different therapeutic Echinacea preparations. Forsch. Komplementarmed Klass Naturheilkd. 7:294–300.[Medline]
Perry, N. B., E. J. Burgess, and V. L. Glennie. 2001. Echinacea standardization: Analytical methods for phenolic compounds and typical levels in medicinal species. J. Agric. Food Chem. 49:1702–1706.[Medline]
Reed, L. J., and H. Muench. 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27:493–497.
Rehman, J., J. M. Dillow, S. M. Carter, J. Chou, B. Lee, and A. S. Maisel. 1999. Increased production of antign-specific immunoglobulins G and M following in vivo treatment with medicinal plants Echinacea angustifolia and Hydrastis canadensis. Immunol. Lett. 68:391–395.[Medline]
See, D. M., N. Broumand. L. Sahl, and L. G. Tilles. 1997. In vitro effects of Echinacea and ginseng on natural killer and antibody-dependent cell toxicity in healthy subjects and chronic fatigue syndrome or acquired immunodeficiency syndrome patients. Immunopharmacology 35: 229–235.[Medline]
Spurlock M. E., G. R. Frank, G. M. Willis, J. L. Kuske, and S. G. Cornelius. 1997. Effect of dietary energy source and immunological challenge on growth performance and immunological variables in growing pigs. J. Anim. Sci. 75:720–726.[Abstract/Free Full Text]
Stahly, T. S., G. L. Cromwell, and H. J. Monegue. 1980. Effects of the dietary inclusion of copper and (or) antibiotics on the performance of weanling pigs. J. Anim. Sci. 51:1347–1351.
Stotzem, C. D., U. Hungerland, and U. Mengs. 1992. Influence of Echinacea purpurea on phagocytosis of human granulocytes. Med. Sci. Res. 20:719–720.
Straw, B. E., S. D’Allaire, W. L. Mengeling, and D. J. Taylor. 1999. Diseases of Swine. 8th ed. Iowa State Univ. Press, Ames.
Thacker, B. 2003. Infection and disease. In The Porcine Reproductive and Respiratory Syndrome Compendium: A Comprehensive Reference on PRRS for Pork Producers, Veterinary Practitioners, and Researchers. 2nd ed. J. J. Zimmerman, K. J. Yoon, ed. Natl. Pork Board, Des Moines, Iowa (In press).
Turner, R. B., D. K. Riker, and J. D. Gangemi. 2000. Ineffectiveness of Echinacea for prevention of experimental Rhinovirus colds. Antimicrob. Agents Chemother. 44:1708–1709.[Abstract/Free Full Text]
Varley, M. A. 1995. The Neonatal Pig, Development and Survival. CAB International, Oxon, U.K.
Zimmerman, J. 2003. Historical overview. In The Porcine Reproductive and Respiratory Syndrome Compendium: A Comprehensive Reference on PRRS for Pork Producers, Veterinary Practitioners, and Researchers (2nd ed.). J. J. Zimmerman and K. J. Yoon, ed. Natl. Pork Board, Des Moines, Iowa (In press).
Top
Abstract
Introduction
