ANTIVIRAL EFFECTS OF NARASIN IN SWINE FEED

Abstract

The present invention relates to a composition for ameliorating viral infections in nursery pigs. The composition contains the polyether ionophore narasin, and is supplied to the nursery pigs in an orally-acceptable form. The composition is effective in reducing viral shedding and the severity of diarrhea after challenge of nursery pigs with Porcine Epidemic Diarrhea Virus (PEDV).

Claims

1. A method of treating porcine epidemic diarrhea virus (PEDV) infection, comprising administering to a nursery pig narasin with an orally-acceptable carrier.

2. The method of claim 1, wherein the orally-acceptable carrier is selected from the group comprising an animal feed, a liquid composition other than an animal feed, and a solid composition other than an animal feed.

3. The method of claim 1, wherein the concentration of narasin is selected from the group of about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, and about 60 mg/kg of the composition.

4. A method of treating porcine epidemic diarrhea virus (PEDV) infection, comprising administering to a nursery pig narasin with an animal feed, wherein narasin is present at a concentration of about 60 mg/kg of the animal feed.

5. (canceled)

6. (canceled)

7. (canceled)

8. A composition comprising a concentration of narasin and an orally-acceptable carrier; wherein the composition provides an antiviral effect to a nursery pie.

9. The composition of claim 8, wherein the orally-acceptable carrier is selected from the group comprising an animal feed, a liquid composition other than an animal feed, and a solid composition other than an animal feed.

10. The composition of claim 8, wherein the concentration of narasin is selected from the group of about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, and about 60 mg/kg of the composition.

11. The method of claim 2, wherein the concentration of narasin is selected from the group of about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, and about 60 mg/kg of the composition.

Description

EXAMPLE 1

Experimental Design

[0020] Eighty-one weaning stage (21.8+/−0.6 days) commercial crossbred piglets (Wilson's Prairie View Farms, Burlington, Wis.) free of PEDV infection arrived at the site and six were used as potential replacement animals during the pre-challenge period (Day −7 to Day 0). Six piglets were removed at Day 0 immediately prior to PEDV challenge, resulting in fifteen pens containing five piglets/pen (n=75) animals. There were five pens per treatment. For balance of sentinel piglets within a block, a total of thirty-nine piglets of one gender and thirty-six of the other gender were used. The use of five pens (twenty-five piglets) per treatment group was estimated to detect significant differences (P<0.10) between treatment groups in growth performance and incidence of viral shedding.

[0021] The seventy-five piglets were randomly assigned to one of three treatment groups: control (no ionophore), narasin (30 mg/kg) and narasin (60 mg/kg). They acclimatized to their environment and were fed daily for seven days prior to exposure with PEDV on Day 0. Treatment continued for 14 days following challenge. Pigs from the three treatment groups were administered orally with 4×10.sup.4 TCID.sub.50/mL of the PEDV isolate PEDV/USA/NC/2013/49469 (College of Veterinary Medicine, Iowa State University). The piglets were monitored daily for changes in clinical parameters, clinical score for diarrhea, depression and gauntness, rate of food consumption, and rate of body weight change and viral swabs were taken daily to determine viral shedding.

Statistical Methods

[0022] Viral shedding values was evaluated by RMANOVA and the PROC MIXED procedure of SAS (SAS, Cary, N.C.). Other variables for analysis included growth performance data (average daily gain, or ADG; average daily feed intake, or ADFI; and feed efficiency (unit of weight gain per unit of feed consumed), or G:F), incidence and severity of diarrhea, depression, and gauntness scores, and intestinal histology score and immunohistochemistry. The analysis of the growth performance outcomes were conducted using a generalized linear mixed model and the PROC MIXED procedure of SAS.

[0023] Incidence and severity of the score data (diarrhea, depression, and gauntness) were summarized by frequencies and mean scores on a daily basis.

Diet Formulations

[0024] Diet formulations were manufactured at Provimi (Lewisburg, Ohio) and fed in meal form. Composition of diets were analyzed by Minnesota Valley Testing Laboratories (New Ulm, Minn.), and nutrient analysis values were found to be similar to formulated levels. Narasin levels were analyzed by Covance Laboratories (Greenfield, Ind.) and were found to be 0, 29.8, and 64.1 mg/kg, which were similar to formulated levels of 0, 30, and 60 mg/kg, respectively.

[0025] No ractopamine (last feed drug) was detected in the experimental diets.

Growth Performance

[0026] The effects of narasin inclusion level on mean body weights (BW), average daily weight gain (ADG), average daily feed intake (ADFI), feed efficiency (G:F), and feed conversion ratio (F:G) are shown in Table 1.

TABLE-US-00001 TABLE 1 Least-squares means for the effects of narasin inclusion level on the growth performance of nursery pigs challenged with PEDV Narasin inclusion level, mg/kg Item 0 30 60 SEM P-value No. of pens 5 5 5 — — BW, lb Day −7.sup.1 14.3 14.9* 14.9* 0.72 0.04 Day 0.sup.2 16.6 16.9 16.7 0.68 0.72 Day 5.sup.3 18.1 18.7 19.2 0.78 0.12 Day 14.sup.4 26.5 26.9 26.9 1.01 0.92 ADG, lb Day −7 to 0 0.32 0.29 0.26* 0.017 0.08 Day 0 to 5 0.24 0.38* 0.48* 0.056 0.03 Day 5 to 14 0.94 0.92 0.85 0.066 0.61 Day 0 to 14 0.65 0.70 0.70 0.047 0.74 ADFI, lb Day −7 to 0 0.36 0.37 0.36 0.032 0.67 Day 0 to 5 0.65 0.68 0.71 0.035 0.51 Day 5 to 14 1.23 1.23 1.22 0.102 1.00 Day 0 to 14 0.99 1.00 1.01 0.069 0.98 G:F, lb:lb Day −7 to 0 0.882 0.802 0.742 0.0755 0.40 Day 0 to 5 0.375 0.551 0.670* 0.0767 0.05 Day 5 to 14 0.772 0.772 0.708 0.0609 0.59 Day 0 to 14 0.659 0.711 0.698 0.0486 0.73 F:G, lb:lb Day −7 to 0 1.14 1.33 1.38 0.112 0.22 Day 0 to 5 3.80 1.93 1.56 0.744 0.12 Day 5 to 14 1.30 1.38 1.42 0.114 0.64 Day 0 to 14 1.52 1.48 1.44 0.106 0.85 *Means are significantly different than control treatment (P < 0.10). .sup.1Day −7: Animal arrival to test facility; allotment to study. Includes extra 6 pigs. .sup.2Day 0: Pigs challenged with PEDV. .sup.3Day 5: Sentinel animal (1 pig/pen) removed from pen, euthanized, and necropsied. .sup.4Day 14: End of study.

[0027] Feeding 30 and 60 mg/kg narasin numerically increased overall ADG (7.7% and 7.7%, respectively) and G:F (7.9% and 5.9%, respectively) compared to the control (0 mg/kg).

[0028] Feeding 30 and 60 mg/kg narasin increased (P<0.10) ADG (58.3% and 100%, respectively) in the Day 0 to 5 period (Table 1) compared to the control, although feeding 60 mg/kg narasin resulted in a decreased (P<0.10) ADG (18.8%) in the Day −7 to 0 period. In addition, feeding 60, but not 30 mg/kg narasin, increased (P<0.10) G:F in the Day 0 to 5 period (78.7%) compared to 0 mg/kg narasin. There were no statistical differences for ADG, ADFI, G:F, or F:G in the Day 5 to 14 period. There was no effect (P>0.10) of narasin level on overall (Day 0 to 14) growth performance.

Clinical Scoring

[0029] Table 2 indicates the scoring system used to measure diarrhea, depression, and gauntness. Table 3 compares the mean scores for diarrhea, depression and gauntness in nursery pigs whose diet included 0, 30 or 60 mg/kg narasin.

TABLE-US-00002 TABLE 2 Enteric Clinical Observation Scoring System Clinical Evaluation Clinical Signs Diarrhea 1 = Normal (no diarrhea present) 2 = Pasty (semi-solid; cow-pie consistency) 3 = Semi-liquid (loose with some solid material; oatmeal consistency) 4 = Liquid (watery feces with little or no solid material) Depression 0 = Normal (bright, alert, and responsive) 1 = Mild (may stand isolated but will quickly respond to stimulation) 2 = Moderate (may stand isolated with head down and possible signs of muscle weakness; delayed response to stimulation) 3 = Severe (severely depressed; recumbent and reluctant to rise) Gauntness 0 = Normal abdominal fill; flank is full and round 1 = Decreased gut fill; flank is flat 2 = Severely gaunt; flank is hollow

[0030] The mean of diarrhea scores measured over the 14-d period decreased numerically with increasing narasin level (1.49, 1.45, and 1.37 for 0, 30, and 60 mg/kg, respectively). Severity of diarrhea decreased with increasing narasin level (3.16, 2.72, and 2.48 for 0, 30, and 60 mg/kg, respectively; Table 3).

[0031] The mean of depression scores measured over the 14-d period was not affected by narasin level, however, a low incidence of depression observed in animals is noted (5, 4, and 3 animals for 0, 30, and 60 mg/kg narasin, respectively). Severity of depression decreased with increasing narasin level, although this was based on relatively few animals exhibiting the condition (Table 3).

[0032] There was little effect of narasin level on mean or severity of gauntness scores measured over the 14-d period (Table 3).

TABLE-US-00003 TABLE 3 Means for the effects of narasin inclusion level on diarrhea scores of nursery pigs challenged with PEDV Narasin inclusion level, mg/kg Item 0 30 60 No. of pens 5   5   5   Diarrhea score Mean of Day 0 to 14 1.49 1.45 1.37 Severity.sup.1 3.16 2.72 2.48 Incidence 22/25 19/25 19/25 Depression score Mean of Day 0 to 14 0.02 0.03 0.02 Severity.sup.1 0.20 0.16 0.12 Incidence  5/25  4/25  3/25 Gauntness score.sup.1 Mean of Day 0 to 14 0.19 0.14 0.14 Severity.sup.1 0.64 0.68 0.52 Incidence 14/25 14/25 11/25 .sup.1Severity: Average of worst score observed for each pig over 14-d period.

Viral Shedding

[0033] The effects of narasin levels (0, 30, 60 mg/kg) on within-day viral shedding of nursery pigs (Table 4) and percentage of nursery pigs shedding PEDV (Table 5) are compared. Fecal swabs were collected from all pigs on test daily from Day 0 to 14 of study. Each sample was labeled with pig identification, pen number, and sample day. All samples were frozen at the time of collection and stored until required for analysis. Only fecal swab samples collected from days 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 14 were sent for analysis. Fecal swab samples were sent to the Iowa State Veterinary Diagnostic Laboratory (ISVDL) and were analyzed using real-time RT-PCR to determine viral shedding of PEDV.

TABLE-US-00004 TABLE 4 Least-squares means for the effects of narasin inclusion level within day on viral shedding of nursery pigs challenged with PEDV P-values Item Narasin Day Narasin × Day Viral shedding 0.01 <0.0001 <0.0001 Narasin inclusion level, mg/kg Item 0 30 60 No. of pens 5      5     5 Viral shedding, cells/mL.sup.1,2 Day 0 0      0     0 Day 1 1      1     0 Day 2 802,918     1,005*      1* Day 3 46,321,178    152,889*     4,085* Day 4 299,600,217  7,543,717    18,980* Day 5 2,577,353,893 108,303,640  .sup. 593,842* Day 6 2,546,739,463 879,972,048  5,551,647* Day 7 2,642,072,514 1,422,120,182   171,586,154  Day 8 83,710,799 268,967,479 58,813,444 Day 9 5,615,075  63,817,042 16,189,481 Day 14 3,665     2,256    9,262 *Within day, means are significantly different than control treatment (P < 0.10). .sup.1Values were ln(count + 1) transformed prior to the statistical analysis. .sup.2Values presented are only from pigs testing positive for shedding. Values for pigs testing negative (i.e., 0) are not included in the data set.

[0034] When compared to the control, pigs fed 30 mg/kg narasin had lower (P<0.10) viral shedding on Day 2 and Day 3 of study, and consistently had numerically lower viral shedding through Day 7 of study. Pigs fed 60 mg/kg narasin had lower (P<0.10) viral shedding on Day 2, 3, 4, 5, and 6 of study, and had numerically lower viral shedding on Day 7 and 8 of study (Table 4).

[0035] There was no effect (P>0.10) of narasin level on the percentage of pigs shedding PEDV, but the duration of shedding generally decreased as narasin level increased.

Histology

[0036] The effects of narasin inclusion level on the intestinal histology score and immunohistochemistry were analyzed. As narasin level increased from 0, 30, 60 mg/kg), histology scores decreased from 2 to 0 while the immunohistochemistry score decreased only at the 60 mg/kg level (from 3 to 2) although differences were not significant (P>0.05).