KR20110000854A - Composition of food additives of piglets containing igy from egg yolk for preventing of porcine epidemic diarrhea or transmissible gastroenteritis - Google Patents

Composition of food additives of piglets containing igy from egg yolk for preventing of porcine epidemic diarrhea or transmissible gastroenteritis Download PDF

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KR20110000854A
KR20110000854A KR1020090058154A KR20090058154A KR20110000854A KR 20110000854 A KR20110000854 A KR 20110000854A KR 1020090058154 A KR1020090058154 A KR 1020090058154A KR 20090058154 A KR20090058154 A KR 20090058154A KR 20110000854 A KR20110000854 A KR 20110000854A
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박정금
성지현
원미경
김보미
정홍걸
임환
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Abstract

PURPOSE: An additive composition for a piglet feed containing egg yolks is provided to prevent the side effect or the resistance for antibiotics, and to reduce the resistance of consumers by using natural ingredients. CONSTITUTION: An additive composition for a piglet feed containing egg yolks contains the following: 0.05~9.5wt% of egg yolk containing more than one IgY selected from the group consisting of Salmonella Tiphimurium IgY, Transmissible gastroenteritis virus IgY, Porcine epidemic diarrhea IgY, and E.coli IgY; 47.6~71.6wt% of fructo-oligosaccharide; 0.01~0.11wt% of vitamin B6; 0.02~0.22wt% of inositol; 0.05~1.9wt% of microorganism; and 24.2~44.2wt% of purified water.

Description

돼지유행성 설사 또는 돼지전염성 위장염에 대한 항체를 지닌 난황을 함유하는 자돈 사료용 첨가물 조성물{Composition of food additives of Piglets containing IgY from egg yolk for preventing of porcine epidemic diarrhea or transmissible gastroenteritis}Composition of food additives of Piglets containing IgY from egg yolk for preventing of porcine epidemic diarrhea or transmissible gastroenteritis}

본 발명은 자돈 사료용 첨가물 조성물에 관한 것으로, 더욱 상세하게는 돼지유행성 설사(PEDV) 또는 돼지전염성 위장염 바이러스(TGEV)에 대한 특이적인 항체를 함유하는 난황을 포함하는 것을 특징으로 하는 자돈 사료용 첨가물 조성물에 관한 것이다.The present invention relates to an additive composition for piglet feed, and more particularly, to an piglet feed additive composition comprising a yolk containing a specific antibody against swine pancreatic diarrhea (PEDV) or swine infectious gastroenteritis virus (TGEV). It is about.

돼지전염성 위장염 바이러스(transmissible gastroenteritis virus; TGEV)와 돼지유행성 설사 바이러스(porcine epidemic diarrhea virus; PEDV)는 코로나바이러스과에 속하는 바이러스로 모든 일령의 돼지에서 감수성이 있으나 특히 어린 연령일수록 발병률과 치사율이 높다Swine infectious gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) belong to the family of coronaviruses, but are susceptible to all ages of pigs, but the incidence and mortality rate is higher among younger pigs

돼지유행성 설사(PED)는 포유자돈에서 50%이상의 폐사율을 야기하고 급성의 심한 수양성 설사, 구토, 식욕부진, 원기저하 등의 임상증상을 보인다.Swine diarrhea (PED) causes mortality of more than 50% in piglets and has clinical symptoms such as acute severe watery diarrhea, vomiting, anorexia and hypogonadism.

돼지전염성 위장염(TGE)는 흡수불량성 설사와 구토를 일으켜 탈수를 유발하여 2주령 이하의 포유자돈에서 폐사율이 거의 100%에 달하여 양돈산업에 막대한 경제적 손실을 주고 있는데 자돈에서의 빠른 전파와 높은 폐사율을 제외하고는 PED와 매우 유사하다. Porcine infectious gastroenteritis (TGE) causes malabsorption of diarrhea and vomiting, leading to dehydration, resulting in nearly 100% mortality in piglets less than two weeks of age, causing enormous economic losses to the pig industry. It is very similar to the PED except for that.

한편, TGEV는 1954년 경기도의 수입종돈으로부터 국내에 전파된 것으로 추정하고 있고, 이후 겨울철에 꾸준한 발생을 보였으며, PEDV는 1993년 국내에서 처음 발생보고되었다. 근래에는 PED가 TGE 보다 월등히 많은 발생을 보이고 있어 양돈농가에 경제적인 피해를 유발하고 있으며, 2003년 12월까지 한 해 동안 국립수의과학검역원에서 발표한 자료에 의하면 TGE는 1건에 300두에서 발생한 반면, PED의 경우 90건에 40,297두가 발생하였고 2004년도에도 45건에 9,195두에서 발생되었다.On the other hand, TGEV is estimated to have spread domestically from imported sows in Gyeonggi-do in 1954. Since then, TGEV has shown steady occurrence in winter, and PEDV was first reported in Korea in 1993. In recent years, PED has shown much more occurrences than TGE, causing economic damage to pig farms. According to data released by the National Veterinary Research and Quarantine Service during December 2003, TGE occurred in 300 cases per case. On the other hand, in the PED cases, 40,297 heads occurred in 90 cases and 9,195 heads in 45 cases in 2004.

한편, PED가 빈번하게 발생되고 있는 아시아의 양돈국가에서는 PED를 예방하기 위하여 임신돈에 생백신을 접종하여 고역가의 모체이행항체를 포유자돈에 부여하는 방법, 감염자돈의 장유제액으로 모돈에 인공감염시켜 유즙면역을 자극시키는 방법등의 여러 가지 방법을 모색하여 왔다. On the other hand, in pig farming countries in Asia where PED is frequently occurring, a method of inoculating live piglets to pregnant pigs to inoculate live pigs with pregnant vaccines to prevent PEDs, and artificially infecting sows with enteric fluid of infected piglets Various methods, such as the method of stimulating immunity, have been sought.

TGE의 경우에는 강독백신, 약독화 백신, 불활화 백신, 서브유닛(subunit) 백신을 경구, 비강, 근육, 피하 또는 유방에 접종하여 국소면역항체 및 혈중항체를 높여 주는 방법을 많이 이용하고 있다  In the case of TGE, vaccination of oral, nasal, intramuscular, subcutaneous or breast vaccines of attenuated vaccines, attenuated vaccines, inactivated vaccines, and subunit vaccines has been widely used.

그러나 상기의 같은 방법은 국내의 열악한 사양환경이나 양돈 농가의 인식부족으로 인하여 현장에서 손쉽게 사용할 수 없어 PED와 TGE를 예방하는데 어려움이 있다. However, the above method has difficulty in preventing PED and TGE because it is not easily used in the field due to poor domestic environment or lack of recognition of hog farmers.

이에 본 발명은 농가에서 사용하기 편하면서 천연성분으로 구성되어 사람들에게 거부감이 없고, 항생제 내성 및 부작용이 발생하지 않는 자돈 사료용 첨가물 조성물을 제공하는데 그 목적이 있다. Therefore, the present invention is easy to use in farms and is composed of natural ingredients, there is no objection to people, antibiotic resistance and side effects do not occur to provide an additive composition for piglet feed.

상기의 목적을 달성하기 위해 본 발명은 살모넬라 티피뮤리엄(Salmonella Tiphimurium)에 대한 IgY, 살모넬라 콜레라수이스(Salmonella Choleraesuis)에 대한 IgY, TGEV(Transmissible gastroenteritis virus)에 대한 IgY, PEDV(Porcine epidemic diarrhea)에 대한 IgY 또는 대장균(Escherichia coli)에 대한 IgY 중 선택되는 어느 하나 이상의 IgY를 함유하는 난황; 프락토올리고당; 비타민 B6; 이노시톨; 바실러스 서브틸리스(Bacillus subtilis), 비피도박테리움 론굼(Bifidobacterium longum), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 불가리커스(Lactobacillus bulgaricus), 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 애시도필러스(Lactobacillus acidophilus), 락토바실러스 사케이(Lactobacillus sakei), 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 류터리(Lactobacillus reuteri), 락토바실러스 락티스(Lactobacillus lactis), 류코노스톡 메센테로이드(Leuconostoc mesenteroides), 류코노스톡 김치아이( Leuconostoc kimchii), 류코노스톡 시트레 움(Leuconostoc citreum), 류코노스톡 가시코미타툼(Leuconostoc gasicomitatum), 류코노스톡 락티스(Leuconostoc lactis) 또는 스트렙토코커스 써머필러스(Streptococcus thermophilus) 중 선택되는 어느 하나 이상의 미생물; 및 정제수;를 혼합한 혼합물을 포함하는 것을 특징으로 하는 자돈 사료용 첨가물 조성물을 제공한다. The present invention to achieve the above object is Salmonella typhimurium (Salmonella TiphimuriumIgY for Salmonella Cholera suis (Salmonella Choleraesuis), IgY for TGEV (Transmissible gastroenteritis virus), IgY for PEDV (Porcine epidemic diarrhea) or E. coli (Escherichia coliEgg yolk containing any one or more of IgY selected from IgY); Fructooligosaccharides; Vitamin B6; Inositol; Bacillus subtilis (Bacillus subtilis), Bifidobacterium rhomb (Bifidobacterium longum), Lactobacillus permanent (Lactobacillus fermentum), Lactobacillus vulgaris (Lactobacillus bulgaricus), Lactobacillus casei (Lactobacillus casei), Lactobacillus ashdophilus (Lactobacillus acidophilus), Lactobacillus sakei (Lactobacillus sakei), Lactobacillus plantarumLactobacillus plantarum), Lactobacillus lutery (Lactobacillus reuteri), Lactobacillus lactis (Lactobacillus lactis), Leukonostock mesenteroid (Leuconostoc mesenteroides), Ryukonostock Kimchi I (Leuconostoc kimchii), Leukonostock Citrium (Leuconostoc citreum), Ryukonostock Kashikomitatum (Leuconostoc gasicomitatum), Leukonostock lactis (Leuconostoc lactis) Or Streptococcus thermophilus (Streptococcus thermophilusAt least one microorganism selected from; And purified water; provides a piglet feed additive composition comprising a mixture of the mixture.

이하 본 발명의 과제 해결 수단에 대해 상세히 설명하고자 한다.Hereinafter will be described in detail with respect to the problem solving means of the present invention.

돼지유행성 설사(Porcine epidemic diarrhea; PED)는 구토와 수양성 설사를 주증상으로 하는 소화기 질병으로, 소장벽이 얇아지고 황색 수양성 액체가 충만하다. 포유자돈에서 50%이상의 폐사율을 야기하고 구토, 식욕부진, 원기저하 등의 임상증상을 보인다.  Porcine epidemic diarrhea (PED) is a gastrointestinal disease with vomiting and watery diarrhea as the main symptom. It is thin in the intestinal wall and filled with yellow aqueous fluid. It causes more than 50% mortality in piglets and has clinical symptoms such as vomiting, loss of appetite and depression.

돼지전염성 위장염(Transmissible gastroenteritis; TGE)은 고전염성, 급성 전염병으로, 겨울과 봄 사이에 다발하며, 소변으로는 바이러스가 배설되지 않고 변으로 배설된다. 흡수불량성 설사와 구토로 인한 탈수를 유발하여 1주일령 미만의 포유자돈에서 폐사율이 거의 100%에 달하고, 자돈에서 빠른 전파와 높은 폐사율의 특징이 있다. Transmissible gastroenteritis (TGE) is a highly infectious, acute infectious disease that occurs frequently between winter and spring, and excretes into the stool rather than the virus in the urine. It causes dehydration due to malabsorption of diarrhea and vomiting, which leads to almost 100% mortality in piglets less than 1 week old, and has rapid propagation and high mortality in piglets.

본 발명은 상기에서 설명한 어린 연령일수록 발병률과 치사율이 높은 돼지 전염성 위장염 또는 유행성 설사의 발병을 예방하기 위해 반드시 살모넬라 티피뮤리엄(Salmonella Tiphimurium)에 대한 IgY, 살모넬라 콜레라수이스(Salmonella Choleraesuis)에 대한 IgY, TGEV(Transmissible gastroenteritis virus)에 대한 IgY, PEDV(Porcine epidemic diarrhea)에 대한 IgY 또는 대장균(Escherichia coli)에 대한 IgY 중 선택되는 어느 이상의 IgY를 함유하는 난황을 포함한다.The present invention IgY for children ages the more you can IgY, salmonella cholera for sure Salmonella typhimurium (Salmonella Tiphimurium) in order to prevent the onset of incidence and gastroenteritis or the flu diarrhea, high pig infectious mortality database (Salmonella Choleraesuis) described above , IgY for TGEV (Transmissible gastroenteritis virus), IgY for Porcine epidemic diarrhea (PEDV) or Escherichia coli ) and yolk containing any one or more of IgY selected from IgY.

난황항체는 조류의 면역체계를 이용한 수동면역의 한 형태로, 조류 이하의 난생동물의 경우는 어미 닭이 능동면역으로 획득한 면역항체는 난황 중으로 이행되어 자손에게 면역능이 전해진다. 난황 중의 항체 중 가장 많은 비율을 차지하는 것은 혈중의 IgG와 유사한 IgY이다. 이는 분자량이 약 200∼220kDa으로 포유류의 IgG보다 크며 포유류의 항체보다 장점을 가지고 있기 때문에 이용범위가 넓다. Egg yolk antibodies are a form of passive immunization using the bird's immune system. In the case of sub-avian oval animals, the immune antibodies obtained by active chickens from the mother chicken are transferred to egg yolk and the immune function is transmitted to the offspring. The largest proportion of antibodies in egg yolk is IgY, similar to IgG in the blood. Its molecular weight is about 200-220kDa, which is larger than mammalian IgG and has advantages over mammalian antibodies.

한편, 돼지유행성 설사 또는 전염성 위장염을 유발하는 바이러스에 대한 항체 IgY를 함유하는 난황을 생산하기 위해 본 발명은 살모넬라 티피뮤리엄(Salmonella Tiphimurium), 살모넬라 콜레라수이스(Salmonella Choleraesuis), TGEV(Transmissible gastroenteritis virus), PEDV(Porcine epidemic diarrhea) 및 대장균(Escherichia coli)을 배양하여 백신을 제조한 후, 백신을 일정기간 간격으로 산란계에 3차 접종까지 실시하였다. 접종 후, 항체역가를 측정한 결과, 3차 접종 후인 4주부터 가장 높은 역가를 나타내었고, 항체역가가 감소하기 시작한 10주에 추가 접종을 실시한 후, 5~14주까지의 항체역가가 높은 계란의 난황만을 수거하여 수득하였다. Meanwhile, the present invention provides Salmonella typhimurium ( Salmonella) to produce egg yolk containing the antibody IgY against viruses causing swine pandemic diarrhea or infectious gastroenteritis. Tiphimurium , Salmonella Choleraesuis ), TGEV (Transmissible gastroenteritis virus), Porcine epidemic diarrhea (PEDV), and Escherichia coli ) was cultured to prepare a vaccine, and the vaccine was then administered to the laying hens at a time interval up to the third inoculation. After inoculation, the antibody titer was the highest titer from 4 weeks after the 3rd inoculation, and after the inoculation at 10 weeks when the antibody titer began to decrease, the eggs with high antibody titers up to 5-14 weeks Only egg yolk was collected and obtained.

한편, 본 발명의 자돈 사료용 첨가물 조성물은 프락토올리고당을 포함하는데, 프락토올리고당은 비피더스균이나 유산균의 증식효과가 있고, 살모넬라(Salmonella)균과 크로스트리디엄 퍼프리진(Clostridium perfringens)를 억제한다. 특히, 본 발명에서는 감미료로 사용되어 자돈이 사료 섭취시 나타나는 거부감 을 완화시킬 수 있다. On the other hand, piglet feed additive composition of the present invention includes fructooligosaccharide, fructooligosaccharide has a proliferative effect of Bifidobacteria or lactic acid bacteria, inhibit Salmonella (Salmonella) and Crosstridium perfringens (Clostridium perfringens). In particular, in the present invention can be used as a sweetener to alleviate the rejection of piglets appearing when eating feed.

한편, 본 발명의 자돈 사료용 첨가물 조성물은 비타민 B6을 포함하는데, 비타민 B6은 자돈의 생장을 촉진하면서 피부염을 예방할 수 있다. On the other hand, piglet feed additive composition of the present invention includes vitamin B6, vitamin B6 can prevent dermatitis while promoting the growth of piglets.

한편, 본 발명의 자돈 사료용 첨가물 조성물은 이노시톨을 포함하는데, 이노시톨은 당과 같이 결정성이 좋고 감미를 가지기 때문에 자돈이 사료 섭취시 나타나는 거부감을 완화시킬 수 있을 뿐만 아니라, 자돈의 발육을 촉진하며 동맥경화 예방한다. On the other hand, the piglet feed additive composition of the present invention includes inositol, since inositol is crystallized and sweet like sugar, it can not only relieve the rejection of piglets ingesting the feed, but also promote the development of piglets and arteries. Prevent hardening.

한편, 본 발명의 자돈 사료용 첨가물 조성물은 바실러스 서브틸리스(Bacillus subtilis), 비피도박테리움 론굼(Bifidobacterium longum), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 불가리커스(Lactobacillus bulgaricus), 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 애시도필러스(Lactobacillus acidophilus), 락토바실러스 사케이(Lactobacillus sakei), 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 류터리(Lactobacillus reuteri), 락토바실러스 락티스(Lactobacillus lactis), 류코노스톡 메센테로이드(Leuconostoc mesenteroides), 류코노스톡 김치아이(Leuconostoc kimchii), 류코노스톡 시트레움(Leuconostoc citreum), 류코노스톡 가시코미타툼(Leuconostoc gasicomitatum), 류코노스톡 락티스(Leuconostoc lactis) 또는 스트렙토코커스 써머필러스(Streptococcus thermophilus) 중 선택되는 어느 하나 이상의 미생물을 포함하는데, 상기의 미생물은 자돈의 섭취시 자돈에 대해 항생, 항바이러스, 면역 증강 효과가 있다.On the other hand, the piglet feed additive composition of the present invention Bacillus subtilis ( Bacillus subtilis), rongum Bifidobacterium (Bifidobacterium longum ), Lactobacillus fermentum), Lactobacillus Bulgaria carcass (Lactobacillus bulgaricus), Lactobacillus casei (Lactobacillus casei ), Lactobacillus acidophilus ), Lactobacillus sakei), Lactobacillus Planta Room (Lactobacillus plantarum ), Lactobacillus reuteri ), Lactobacillus lactis ), Leuconostoc mesenteroides ), Leuconostoc kimchii , Leuconostoc citreum ), Leuconostoc gasicomitatum ), Leuconostoc lactis ) or Streptococcus thermophilus ) includes any one or more microorganisms selected from the group, the microorganisms have antimicrobial, antiviral, immune enhancing effect on the piglets when ingesting piglets.

한편, 본 발명의 실험예에서는 본 발명에 포함되는 살모넬라 티피뮤리엄(Salmonella Tiphimurium)에 대한 IgY, 살모넬라 콜레라수이스(Salmonella Choleraesuis)에 대한 IgY, TGEV(Transmissible gastroenteritis virus)에 대한 IgY, PEDV(Porcine epidemic diarrhea)에 대한 IgY 및 대장균(Escherichia coli)에 대한 IgY를 함유하는 난황의 돼지유행성 설사(PED)와 돼지전염성 위장염(TGE) 대한 예방효과를 측정하였는데, 그 결과, 대조군에 비해 TGEV(Transmissible gastroenteritis virus), PEDV(Porcine epidemic diarrhea virus) 및 대장균(Escherichia coli)에 대한 항체가 높게 유지되었고, 자돈의 분에서 분리된 대장균 수가 적었으며, 소장과 분에서 검출된 TGEV(Transmissible gastroenteritis virus), PEDV(Porcine epidemic diarrhea)가 감소하였다. On the other hand, in the experimental example of the present invention, Salmonella typhimurium (Salmonella Tiphimurium) IgY, Salmonella choleraesuis could device (Salmonella Choleraesuis) IgY, PEDV (Porcine for IgY, TGEV (Transmissible gastroenteritis virus) for about included in the present invention E. coli IgY and on epidemic diarrhea) (Escherichia coli ) was tested for the prevention of swine pandemic diarrhea (PED) and swine infectious gastroenteritis (TGE) of egg yolk containing IgY. And Escherichia coli ) was maintained high, the number of E. coli isolated from piglets was low, and transmissible gastroenteritis virus (TGEV) and Porcine epidemic diarrhea (PEDV) were detected in small intestine and min.

상기와 같은 특이항체 난황을 포함하는 본 발명의 자돈 사료용 첨가물 조성물을 제조하여 직접 자돈에 섭취시킨 결과, 대조군에 비해 사고두수가 현저히 감소한 것을 확인할 수 있었고, 대조군의 사고율이 28%인 것에 비해 실험군은 4.7%를 나타내 본 발명의 우수한 효과를 확인할 수 있었다. As a result of preparing the piglet feed additive composition of the present invention containing the specific antibody egg yolk as described above and ingesting it directly in piglets, it was confirmed that the accidental head count was significantly reduced compared to the control group. 4.7% was confirmed that the excellent effect of the present invention.

한편, 본 발명의 자돈 사료용 첨가물 조성물은 바람직하게 난황 0.05~9.5 중량%; 프락토올리고당 47.6~71.6 중량%; 비타민 B6 0.01~0.11 중량%; 이노시톨 0.02~0.22 중량%; 바실러스 서브틸리스(Bacillus subtilis), 비피도박테리움 론굼(Bifidobacterium longum), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 불가리커스(Lactobacillus bulgaricus), 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 애시도필러스(Lactobacillus acidophilus), 락토바실러스 사케이(Lactobacillus sakei), 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 류터리(Lactobacillus reuteri), 락토바실러스 락티스(Lactobacillus lactis), 류코노스톡 메센테로이드(Leuconostoc mesenteroides), 류코노스톡 김치아이(Leuconostoc kimchii), 류코노스톡 시트레움(Leuconostoc citreum), 류코노스톡 가시코미타툼(Leuconostoc gasicomitatum), 류코노스톡 락티스(Leuconostoc lactis) 또는 스트렙토코커스 써머필러스(Streptococcus thermophilus) 중 선택되는 어느 하나 이상의 미생물 0.05~1.9 중량% ; 및 정제수 24.2~44.2 중량%로 조성되는 것이 좋다. On the other hand, piglet feed additive composition of the present invention is preferably egg yolk 0.05 ~ 9.5% by weight; Fructooligosaccharide 47.6-71.6 wt%; 0.01 to 0.11 weight percent of vitamin B6; Inositol 0.02-0.22 wt%; Bacillus subtilis subtilis), rongum Bifidobacterium (Bifidobacterium longum ), Lactobacillus fermentum), Lactobacillus Bulgaria carcass (Lactobacillus bulgaricus), Lactobacillus casei (Lactobacillus casei ), Lactobacillus acidophilus ), Lactobacillus sakei), Lactobacillus Planta Room (Lactobacillus plantarum ), Lactobacillus reuteri ), Lactobacillus lactis ), Leuconostoc mesenteroides , Leuconostoc kimchii ), Leuconostoc citreum ), Leuconostoc gasicomitatum ), Leuconostoc lactis ) or Streptococcus thermophilus ) 0.05 to 1.9 wt% of any one or more microorganisms selected from; And 24.2 to 44.2% by weight of purified water.

상기에서 살펴본 바와 같이 본 발명의 자돈 사료용 첨가물 조성물은 50%이상의 폐사율을 야기하는 돼지유행성 설사(PED) 또는 100%의 폐사율을 야기하는 돼지전염성 위장염(TGE)으로 인한 양돈 산업에 막대한 경제적 손실을 예방할 수 있다. As described above, the piglet feed additive composition of the present invention will prevent significant economic losses in the swine industry due to swine pandemic diarrhea (PED) causing mortality of 50% or more or swine infectious gastroenteritis (TGE) causing 100% mortality. Can be.

또한, 천연재료로 구성된 자돈 사료용 첨가물을 사용함으로써 소비자들의 거부감을 감소시킬 수 있다.In addition, the use of natural piglet feed additives can reduce consumer's rejection.

이하, 본 발명의 구성 및 작용에 대해 하기 실시예에서 더욱 상세히 설명하지만, 본 발명의 권리범위가 하기 실시예에만 한정되는 것은 아니고, 이와 등가의 기술적 사상의 변형까지를 포함한다. Hereinafter, the configuration and operation of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples, and includes modifications of equivalent technical spirit.

실시예Example 1: 살모넬라  1: Salmonella 티피뮤리엄(Tippy Museum ( Salmonella TiphimuriumSalmonella Tiphimurium )에)on 대한  About IgYIgY , , 살모Salmo 넬라 Nella 콜레라수이스(Cholera suis ( Salmonella CholeraesuisSalmonella Choleraesuis )에)on 대한  About IgYIgY , TGEV(, TGEV ( TransmissibleTransmissible gastroenteritis  gastroenteritis virusvirus )에 대한 For) IgYIgY , , PEDVPEDV (( PorcinePorcine epidemicepidemic diarrheadiarrhea )에 대한 For) IgYIgY 및 대장균( And E. coli ( EscherichiaEsherichia colicoli )에 대한 For) IgYIgY 을 함유하는 Containing 난황Egg yolk 제조 Produce

실시예 1은 살모넬라 티피뮤리엄(Salmonella Tiphimurium)에 대한 IgY, 살모넬라 콜레라수이스(Salmonella Choleraesuis)에 대한 IgY, TGEV(Transmissible gastroenteritis virus)에 대한 IgY, PEDV(Porcine epidemic diarrhea)에 대한 IgY 및 대장균(Escherichia coli)에 대한 IgY을 함유하는 난황을 제조하였다.Example 1 is IgY for Salmonella Tiphimurium , IgY for Salmonella Choleraesuis , IgY for TGEV (Transmissible gastroenteritis virus), IgY for Escherichia coli (PEDV) and E. coli (E. coli) Esherichia egg yolk containing IgY for coli ) was prepared.

단계 1: 항원생산Stage 1: Antigen Production

살모넬라 티피뮤리엄(Salmonella Tiphimurium), 살모넬라 콜레라수이스(Salmonella Choleraesuis), TGEV(Transmissible gastroenteritis virus), PEDV(Porcine epidemic diarrhea virus) 및 대장균(Escherichia coli)을 블러드아가(blood agar)를 이용하여 37℃ 인큐베이터(incubator)에서 1~2일 혐기배양하였다. 콜로니(colony)를 확인한 후, 준비된 BHI broth에 접종하고 37℃ 쉐이킹인큐베이터(shaking incubator)에서 1~2일 동안 배양하였다. 수득된 배양액을 0.3% 포르말린(formalin)으로 불활화시킨 후, 10000Xg에서 원심분리한 다음 0.1% 포르말린(formalin)으로 다시 불활화를 시켰다. 그리고 스펙트로포토메 터(Spectrophotometer)를 이용하여 1012 cell/ml로 농도를 맞췄다.Salmonella typhimurium (Salmonella Tiphimurium , Salmonella Choleraesuis ), TGEV (Transmissible gastroenteritis virus), Porcine epidemic diarrhea virus (PEDV), and Escherichia coli ) was anaerobicly cultured in blood in a 37 ° C. incubator for 1-2 days. After confirming the colonies (colony), inoculated in the prepared BHI broth and incubated for 1 to 2 days in a 37 ℃ shaking incubator (shaking incubator). The obtained culture was inactivated with 0.3% formalin, then centrifuged at 10000 × g, and then inactivated with 0.1% formalin. The concentration was adjusted to 10 12 cell / ml using a Spectrophotometer.

단계 2: 백신 제조Step 2: Vaccine Preparation

상기 단계 1에서 배양된 균주와 미네랄오일(ISA 70)을 3:7 비율로 혼합한 후, 균질기(Hmogenizer)를 이용하여 유화시킨 후, 무균검사와 불활화 확인시험을 거쳤다.After mixing the strain cultured in step 1 and mineral oil (ISA 70) in a 3: 7 ratio, and then emulsified using a homogenizer (Hmogenizer), the aseptic test and inactivation confirmation test.

단계 3: 살모넬라 Step 3: Salmonella 티피뮤리엄(Tippy Museum ( Salmonella TiphimuriumSalmonella Tiphimurium )에)on 대한  About IgYIgY , , 살모넬Salmonel la 콜레라수이스(Cholera suis ( Salmonella CholeraesuisSalmonella Choleraesuis )에)on 대한  About IgYIgY ,, TGEV(TGEV ( TransmissibleTransmissible gastroenteritis  gastroenteritis virusvirus )에 대한 For) IgYIgY , , PEDVPEDV (( PorcinePorcine epidemicepidemic diarrheadiarrhea )에 대한 For) IgYIgY 및 대장균( And E. coli ( EscherichiaEsherichia colicoli )에 대한 특이항체 Specific antibody to 난황의Egg yolk 생산 production

상기 단계 2에서 제조된 백신을 22주령된 Hy-Line Brown 산란계에 1ml씩 2주 간격으로 가슴에 접종하였으며, 항체 역가의 감소시기인 3차 접종 후, 6주 후에 'Boosting'을 실시하였다.The vaccine prepared in step 2 was inoculated into the chest at 2 weeks intervals by 1 ml in a 22-week-old Hy-Line Brown laying hen, followed by 'Boosting' after 6 weeks after the third inoculation of antibody titers.

난황 중의 특이항체 난황의 역가는 'Indirect ELISA Method'를 이용하였으며, 측정 샘플은 1차 접종 후부터 추가접종 후 2주까지의 난황을 모아 주별로 측정하였다. 난황항체의 활성은 Mine(1997)가 사용한 ELISA 방법을 응용하여 측정하였다.The titer of yolk yolk was measured by 'Indirect ELISA Method', and the measurement samples were measured weekly by collecting yolk from 1 week after inoculation to 2 weeks after booster vaccination. The yolk antibody activity was measured by applying the ELISA method used by Mine (1997).

항원을 200ng/ml의 농도로 100㎕씩 96 웰 폴리스틸렌 프레이트(well poly styrene plate)에 코팅하여 4℃에서 하룻밤 방치하였다. PBS-T(phosphate buffer saline, 0.05% Tween 20, pH 7.4)로 세척 후, 2% BSA가 함유된 PBS 버퍼로 1h/37℃ 블라킹(Blocking)하며 상기와 같은 방법으로 세척하였다. 음성대조군, 양성대조군 및 샘플을 2X씩 희석하여 웰(well)당 100㎕씩 넣고 37℃에서 1시간 동안 정치시켰다. 적절히 희석된 이차 항체를 샘플처리와 같은 방법으로 반응시키고, 10분간 발색시킨 후, 492mm에서 ELISA Reader를 이용하여 흡광도를 측정하였다.Antigen was coated at a concentration of 200 ng / ml in 96 well polystyrene plate (well poly styrene plate) and left overnight at 4 ℃. After washing with PBS-T (phosphate buffer saline, 0.05% Tween 20, pH 7.4), 1h / 37 ℃ blocking with PBS buffer containing 2% BSA (Blocking) and washed in the same manner as described above. The negative control group, the positive control group and the sample were diluted by 2 ×, and 100 µl of each well was added and allowed to stand at 37 ° C. for 1 hour. The appropriately diluted secondary antibody was reacted in the same manner as the sample treatment and developed for 10 minutes, and then the absorbance was measured at 492 mm using an ELISA Reader.

단계 4: Step 4: 난황분말Egg yolk powder 제조 Produce

IgY의 항체 역가가 음성대조군의 160X 이상이 되는 5주부터 14주까지의 계란을 집중적으로 집란하여 할란 후 난황만을 수거하였으며 이를 동결건조하여 난황분말(이하, '특이항체 난황분말'이라고 칭함)을 생산하였다. Eggs from 5 to 14 weeks with IgY antibody titers greater than 160X of the negative control were concentrated and egg yolks were harvested after freezing and egg yolk powder (hereinafter referred to as 'specific antibody yolk powder'). Produced.

하기 실험예에서는 특이항체 난황(IgY)을 경구 투여한 실험군과 투여하지 않은 대조군간의 통계처리는 SAS package(Ver 6.12, USA)를 이용하여 Duncan's new multiple ttest 에 의하여(P<0.05) 수준에서 유의성 검정을 진행하였다.In the following experimental example oral administration of specific antibody yolk (IgY) Between experimental and unadministered controls Statistical analysis was performed on the significance test at the level ( P <0.05) by Duncan's new multiple ttest using SARS (Ver 6.12, USA).

실험예Experimental Example 1: 특이항체  1: specific antibody 난황으로부터From egg yolk 수득된The obtained IgYIgY 를 자돈에 투여Administration to piglets

산차를 고려하여 분만중인 모돈의 초유를 섭취하지 않은 포유자돈 20두를 대상으로 실시하였다. 실험군(Group I)은 10두의 포유자돈을 초유전부터 3일간 2㎖씩 특이항체 난황분말(IgY)을 경구투여하였으며, 대조군(Group Ⅱ)은 10두로써 동일한 조건하에서 특히항체 난황분말을 투여하지 않고 생리식염수를 3일간 2㎖씩 투여하였다. 실험군과 대조군의 포유자돈은 각각의 실험돈방에 넣어 24시간 동안 적응시 킨 후 4일령에 공격접종을 실시 시기별로 채혈하고 체중을 측정하였다(도 1).Taking into account the maternal parity, 20 piglets were fed that did not consume colostrum of sows in labor. The experimental group (Group I) orally administered the specific antibody egg yolk powder (IgY) 2ml each for 10 days from the colostrum from the colostrum, and the control group (Group II) did not administer the antibody yolk powder under the same conditions. 2 mL of saline solution was administered for 3 days. Mammal pigs of the experimental group and the control group were put into each experimental pig room for 24 hours, and then, blood was collected and weight was measured at each day of challenge at 4 days of age (FIG. 1).

실험기간 동안에는 대용유(돈돈밀크, (주)제일제당사료)를 제조사의 권장사항에 따라 급여하였고 음수는 자유롭게 섭취하도록 하였으며 돈방주위는 정기적으로 소독하였다. During the experiment, substitute oil (Dondon Milk, Cheil Jedang Food Co., Ltd.) was fed according to the manufacturer's recommendation, and drinking water was freely consumed.

실험예Experimental Example 2:  2: TGEVTGEV (( TransmissibleTransmissible gastroenteritisgastroenteritis virusvirus ), ), PEDVPEDV (( PorcinePorcine epidemicepidemic diarrheadiarrhea ) 및 대장균() And E. coli ( EscherichiaEsherichia colicoli )에 대한 항체 Antibody to 역가Titer 검사 inspection

상기 실험예 1에서 특이항체 난황분말을 경구투여한 자돈의 수동면역상태를 확인하기 위한 항체역가검사를 위하여 경정맥에서 혈액을 채취하여 응고시킨 후 3,000rpm에서 30분간 원심분리하여 상층액을 이펜도르프 튜브(eppendorf tube)에 옮긴 다음 56℃에서 30분간 비동화시킨 뒤 사용하였다. ELISA로 항체 역가 검사를 실시하였다.In the experiment 1, blood was collected from the jugular vein and coagulated for antibody titer test to check the passive immunity of piglets orally administered with the specific antibody yolk powder. After transfer to (eppendorf tube) was inactivated for 30 minutes at 56 ℃ used. Antibody titer was performed by ELISA.

ELISA를 이용한 항체 역가 검사 결과를 도 2 내지 도 4에 나타내었고, 이를 정리하여 하기 표 1에 나타냈다.   The results of antibody titer test using ELISA are shown in FIGS. 2 to 4, which are summarized in Table 1 below.

처리process PED ELISA 역가PED ELISA Titer 00 77 1010 Group Ⅰ(실험군)Group Ⅰ (Experimental Group) 0.66±0.17a 0.66 ± 0.17 a 0.65±0.10a 0.65 ± 0.10 a 0.72±0.08a 0.72 ± 0.08 a Group Ⅱ(대조군)Group Ⅱ (Control) 0.68±0.15a 0.68 ± 0.15 a 0.59±0.07a 0.59 ± 0.07 a 0.59±0.08b 0.59 ± 0.08 b 처리process TGE ELISA 역가TGE ELISA Titer 00 77 1010 Group Ⅰ(실험군)Group Ⅰ (Experimental Group) 0.75±0.34a 0.75 ± 0.34 a 0.84±0.05a 0.84 ± 0.05 a 0.85±0.07b 0.85 ± 0.07 b Group Ⅱ(대조군)Group Ⅱ (Control) 0.86±0.13a 0.86 ± 0.13 a 0.72±0.33a 0.72 ± 0.33 a 0.72±0.11a 0.72 ± 0.11 a 처리process 대장균 ELISA 역가E. coli ELISA titer 00 77 1010 Group Ⅰ(실험군)Group Ⅰ (Experimental Group) 0.66±0.07a 0.66 ± 0.07 a 0.79±0.07a 0.79 ± 0.07 a 0.79±0.08a 0.79 ± 0.08 a Group Ⅱ(대조군)Group Ⅱ (Control) 0.68±0.15a 0.68 ± 0.15 a 0.74±0.11a 0.74 ± 0.11 a 0.72±0.06b 0.72 ± 0.06 b

항체 역가를 검사한 결과(표 1), PEDV의 경우, 공격접종 전, 공격접종 후 7일 및 10일 실험군과 대조군의 평균 혈청항체가는 각각, 공격접종 전에는 0.66±0.17, 0.68±0.15, 공격접종 후 7일에는 0.65±0.10, 0.59±0.07, 공격접종 후 10일에는 0.72±0.08, 0.59±0.08이었다. TGEV의 경우, 공격접종 전, 공격접종 후 7일 및 10일 실험군과 대조군의 평균 혈청항체가는 각각, 공격접종 전에는 0.75±0.34, 0.86±0.13, 공격접종 후 7일에는 0.84±0.05, 0.72±0.33, 공격접종 후 10일에는 0.85±0.07, 0.72±0.11이었다. 대장균의 경우에는 공격접종 전, 공격접종 후 7일 및 10일 실험군과 대조군의 평균 혈청항체가는 각각, 공격접종 전에는 0.66±0.07, 0.68±0.15, 공격접종 후 7일에는 0.79±0.07, 0.74±0.11, 공격접종 후 10일에는 0.79±0.08, 0.72±0.06이었다.As a result of the antibody titer test (Table 1), the average serum antibody titers of the PEDV test group and the control group 7 days and 10 days before challenge, and 0.66 ± 0.17, 0.68 ± 0.15 before challenge, respectively. After 7 days, it was 0.65 ± 0.10, 0.59 ± 0.07, and on the 10th day after challenge, 0.72 ± 0.08, 0.59 ± 0.08. In the case of TGEV, the mean serum antibody values of the experimental and control groups before and after challenge and on days 7 and 10 were 0.75 ± 0.34, 0.86 ± 0.13 before challenge, and 0.84 ± 0.05 and 0.72 ± 0.33 before challenge, respectively. On the 10th day after challenge, it was 0.85 ± 0.07, 0.72 ± 0.11. In the case of Escherichia coli, the mean serum antibody values of the experimental and control groups before and after challenge and 7 and 10 days of challenge, respectively, were 0.66 ± 0.07, 0.68 ± 0.15 before challenge and 0.79 ± 0.07 and 0.74 ± 0.11 before challenge. On the 10th day after challenge vaccination was 0.79 ± 0.08 and 0.72 ± 0.06.

PEDV는 공격접종한 대조군은 공격접종 후 7일에서 항체가가 다소 감소하였으며 공격접종 후 10일에서는 공격접종 후 7일과 비슷한 상태를 유지하였다. 특이항체 난황 투여 후 PEDV를 공격접종한 실험군은 공격접종 후 7일까지는 별다른 변화를 보이지 않았으며 공격접종 후 10일에서는 다소 증가하였다.The PEDV challenged control group showed a slight decrease in antibody value at 7 days after challenge, and remained similar to 7 days after challenge at 10 days after challenge. The experimental group inoculated with PEDV after the administration of specific antibody yolk showed no change until 7 days after challenge and increased slightly at 10 days after challenge.

TGEV를 공격접종한 대조군은 공격접종 후 7일에서 공격접종 당일에 비해 현저하게 감소하였으며 공격접종 후 10일에서는 공격접종 후 7일과 별다른 변화를 보이지 않았다. 특이항체 난황을 투여 후 TGEV를 공격접종 한 실험군은 공격접종 후 7일에서 현저하게 증가하였으며 공격접종 후 10일에서는 공격접종 후 7일에 비해 약간의 증가만 보였다. The control group treated with TGEV was significantly decreased at 7 days after challenge and compared with the 7 days after challenge at 10 days after challenge. In the experimental group challenged with TGEV after the administration of specific antibody yolk, the increase was significantly increased at 7 days after challenge and only slightly increased at 7 days after challenge.

대장균을 공격접종한 대조군은 공격접종 후 7일과 10일에 약간 증가하였으나, 특이항체난황을 투여한 후, 대장균을 공격접종한 실험군은 공격접종 후 7일과 10일에 대조군보다 증가율이 높았다. The control group inoculated with E. coli increased slightly on days 7 and 10 after challenge vaccination, but the experimental group inoculated with E. coli after challenge with specific antibody yolk increased more than the control group on days 7 and 10 after challenge challenge.

실험예Experimental Example 2:  2: 자돈의Piglets 분에서 분리된 대장균 수 측정 Determination of E. coli Isolated from Minutes

본 실험예 2에서는 상기 실험예 1에서 특이항체 난황분말을 경구투여한 자돈의 분에서 분리된 대장균 수를 측정하였다.In Experimental Example 2, the number of E. coli isolated from the piglets orally administered with the specific antibody yolk powder in Experimental Example 1 was measured.

측정결과(도 5), 대장균을 공격접종한 대조군보다 특이항체 난황분말을 투여한 후, 대장균을 공격접종한 실험군에서 분리된 대장균 수가 적은 것을 확인할 수 있었다. As a result of the measurement (FIG. 5), after the administration of the specific antibody yolk powder than the control group inoculated with E. coli, it was confirmed that the number of E. coli isolated from the experimental group challenged with E. coli.

실험예Experimental Example 3: 특이항체  3: specific antibody 난황을Egg yolk 투여한  Administered 자돈의Piglets 소장과 분에서  In the small intestine and minutes RTRT -- PCRPCR 에 의한 PEDV, By PEDV, TGEVTGEV 검출 detection

상기 실험예 1에서 PEDV, TGEV를 공격접종한 자돈의 분변을 통한 PEDV, TGEV의 배출 여부를 조사하기 위해 매일 소독된 면봉으로 실험자돈의 직장에서 분변을 채취하였다. 실험도중에 폐사한 실험자돈의 소장에서 PEDV를 확인하기 위하여 소장조직을 페스틀(pestle)로 분쇄하여 멸균된 500㎕ PBS(pH 7.2)에 넣어 충분히 혼합시킨 다음 14,000 rpm에서 20분간 원심하여 상층액을 취하여 -70℃에 보존하여 RT-PCR을 실시하였다. In Experimental Example 1, feces were taken from the rectum of experimental pigs with a daily sterilized swab to investigate whether PEDV and TGEV were discharged through feces of piglets challenged with PEDV and TGEV. In order to check the PEDV in the small intestine of the dead piglets, the small intestine tissues were crushed into pestles, mixed in sterile 500 µl PBS (pH 7.2), mixed well, and centrifuged at 14,000 rpm for 20 minutes. RT-PCR was carried out by storage at -70 ° C.

PEDV를 특이적으로 검출하기 위해 'forward primer'는 5'GGGCGCCTGTATAGAGTTTA3', 'reverse primer'는 5'AGACCACCAAGAATGTGTCC3'로 412bp 크기의 프라그먼트(fragment)가 증폭되도록 설계하였다. In order to specifically detect PEDV, 'forward primer' is designed to amplify a fragment of 412bp size with 5'GGGCGCCTGTATAGAGTTTA3 'and' reverse primer 'with 5'AGACCACCAAGAATGTGTCCCC'.

TGEV를 특이적으로 검출할 수 있는 것으로 'forward primer'는 5'GATGGCGACCAGATAGAAGT3', 'reverse primer'는 5'GCAATAGGGTTGCTTGTACC3'로 612bp 크기의 프라그먼트(fragment)가 증폭되도록 설계하였다.  TGEV can be detected specifically 'forward primer' is 5'GATGGCGACCAGATAGAAGT3 ',' reverse primer 'is 5'GCAATAGGGTTGCTTGTACC3' is designed to amplify 612bp fragment.

RNARNA 추출 extraction

분변과 소장조직에서 RNA 추출은 RNeasy Mini kit(QIAGEN, Germany)를 사용하여 실시하였다. RNA extraction from fecal and small intestine tissues was performed using the RNeasy Mini kit (QIAGEN, Germany).

역전사Reverse transcription 반응( reaction( ReverseReverse transcriptiontranscription ; ; RTRT ))

역전사 반응은 RNA 템프레이트(template) 5㎕, 0.2μM의 1쌍의 'reverse primer', 디에틸피로카르보네이트(diethylpyrocarbonate; DEPC) 처리된 증류수 31㎕, 10×RT buffer(MBI, Lithuania), 0.2mM dNTPs (MBI), 40 unit RNasin(Promega, USA), 2.5 mM MgCl2(Promega)를 첨가하여 65℃에서 10분간 반응시킨 뒤 200unit MBI(Molony murine leukemia virus reverse transcriptase)를 첨가하여 50㎕의 반응액으로 42℃에서 1시간, 94℃에서 5분으로 1 사이클(cycle)을 시행하여 cDNA를 합성하였다.Reverse transcription reaction was performed by 5 μl RNA template, 0.2 μM pair of 'reverse primer', 31 μl diethylpyrocarbonate (DEPC) treated distilled water, 10 × RT buffer (MBI, Lithuania), After adding 0.2 mM dNTPs (MBI), 40 unit RNasin (Promega, USA), 2.5 mM MgCl 2 (Promega) and reacting at 65 ° C for 10 minutes, 200 μl MBI (Molony murine leukemia virus reverse transcriptase) was added and 50 μl of CDNA was synthesized by performing one cycle at 42 ° C for 1 hour and 94 ° C for 5 minutes.

중합효소 연쇄반응(Polymerase chain reaction PCRPCR ))

역전사 반응에서 합성된 5㎕ cDNA에 0.2 μM의 프라이머(primer), 10× PCR 버퍼(buffer), 2.5 unit Taq DNA 폴리머라제(polymerase), 0.2mM dNTPs, 2.5mM MgCl2, 멸균된 증류수 32㎕를 첨가한 50㎕의 반응액을 94℃ 5분, 51.5℃ 45초, 72℃ 1분의 반응조건에서 1회 시행한 다음 계속하여 95℃ 45초, 51.5℃ 45초, 72℃ 1분의 반응조건에서 30회 반복하였고 51.5℃ 45초, 72℃5분간 반응조건에서 1회 시행하였다. To 5 μl cDNA synthesized in reverse transcription reaction, 0.2 μM primer, 10 × PCR buffer, 2.5 unit Taq DNA polymerase, 0.2 mM dNTPs, 2.5 mM MgCl 2 , and 32 μl of sterilized distilled water 50 μl of the reaction solution was added once under reaction conditions of 94 ° C. 5 minutes, 51.5 ° C. 45 seconds, and 72 ° C. 1 minute, followed by 95 ° C. 45 seconds, 51.5 ° C. 45 seconds, and 72 ° C. one minute. Repeated 30 times at 51.5 ℃ 45 seconds, was performed once at 72 5 minutes reaction conditions.

증폭된 PCR산물의 확인은 TAE 버퍼(40 mM Trisacetate, 1mM EDTA, pH 8.0)를 전해질로 사용한 1% 아가로스 겔(agarose gel)에서 전기영동을 실시한 후, 40 mM 에티디엄 브로마이드(etidium bromide)용액에서 겔(gel)을 염색하여 'UV transilluminator(Vilberlourmat, France)'로 생성된 밴드(band)를 확인하였으며 DNA 마커(marker)로는 100 bp DNA ladder(Promega, USA)를 사용하였다. Confirmation of the amplified PCR product was performed by electrophoresis on a 1% agarose gel using TAE buffer (40 mM Trisacetate, 1 mM EDTA, pH 8.0), followed by 40 mM etidium bromide solution. In the gel (dye), the band generated by 'UV transilluminator (Vilberlourmat, France)' was identified. As a DNA marker, a 100 bp DNA ladder (Promega, USA) was used.

그룹group 자돈
번호
Piglets
number
분변(Feces)Feces 소장Intestine 누적
검출
(%)
accumulate
detection
(%)
공격접종 후 일(Days)Days after challenge 누적
검출(%)
accumulate
detection(%)
-1-One 00 1One 22 33 44 55 66 77 88 99 1010 Group Ⅰ
(실험군)
Group Ⅰ
(Experimental group)
1One -- -- -- TT -- TT -- -- -- -- -- -- TGEV
26/100
(26)


PEDV
3/100
(3)
TGEV
26/100
(26)


PEDV
3/100
(3)
-- 0/10
(0)
0/10
(0)
22 -- -- TT TT TT TT TT -- -- -- -- -- -- 33 -- -- PP -- PTPT TT PTPT -- -- -- -- -- -- 44 -- -- -- -- TT TT -- -- -- -- -- -- -- 55 -- -- -- -- -- -- -- -- -- -- -- -- -- 66 -- -- TT TT -- -- -- -- -- -- -- -- -- 77 -- -- -- -- TT -- -- -- -- -- -- -- -- 88 -- -- -- TT TT TT TT -- -- -- -- -- -- 99 -- -- -- TT TT TT TT TT TT TT -- -- -- 1010 -- -- -- -- -- -- -- -- -- -- -- -- -- Group Ⅱ
(대조군)
Group Ⅱ
(Control group)
1One -- -- -- PP TT PTPT TT TT TT -- -- --
TGEV
39/91
(42.8)


PEDV
20/91
(21.9)

TGEV
39/91
(42.8)


PEDV
20/91
(21.9)
--

2/10
(20.0)


2/10
(20.0)
22 -- -- TT -- TT TT -- TT TT -- -- -- -- 33 -- -- -- PP PP PP TT TT TT -- -- -- -- 44 -- -- TT TPTP PTPT PTPT TT -- TT -- -- -- -- 55 -- -- -- PP TT TT TT TT TT -- -- -- -- 66 -- -- TT PP TT PP TT -- TT -- -- -- -- 77 -- -- -- PP PP PP PP -- PP PP -- -- -- 88 -- -- TT TT TT TT TT TT -- -- -- -- -- 99 -- -- -- PP PP PP DD ++ 1010 -- -- -- TT TT TT TT TT -- DD ++ a) : - ; negative, + ; positive.
b) : D =died.
a):-; negative, +; positive.
b): D = died.

특이항체 난황분말을 투여한 자돈의 소장과 분에서 RT-PCR에 의한 PEDV, TGEV 검출을 한 결과(표 2), 실험군의 10두에서는 공격접종 후 5일째까지 분변에서 PEDV가 검출되었으며 공격접종 후 8일째까지 분변에서 TGEV가 검출되었다. 공격접종 후 1일째부터 10일째까지 총 100예의 분변재료 중 3예에서 PEDV, 26예에서 TGEV 검출되어 각각 3%, 26%의 검출률을 보였으나 소장에서는 검출되지 않았다.As a result of detection of PEDV and TGEV by RT-PCR in small and small piglets treated with specific antibody yolk powder (Table 2), PEDV was detected in feces until 5 days after challenge challenge. TGEV was detected in feces until day 8. From 1 to 10 days after challenge vaccination, PEDV was detected in 3 cases and TGEV was detected in 26 cases and 3% and 26%, respectively.

한편, 대조군은 공격접종 후 1일째부터 10일째까지 총 91예의 분변재료 중 20예에서 PEDV, 39예에서 TGEV 검출되어 각각 21.9% 42.8%의 검출률을 보였고 폐사한 2두의 소장에서 PEDV와 TGEV가 검출되었다. TGEV와 PEDV의 검출율은 실험군이 대조군에 비해 낮게 검출되었다.In the control group, PEDV was detected in 20 cases and PGEV in 39 cases and TGEV was detected in 21 cases from 21 days to 10 days after challenge vaccination, and 21.9% and 42.8%, respectively. Detected. The detection rate of TGEV and PEDV was lower in the experimental group than in the control group.

실시예Example 2:  2: 자돈Piglets 사료용 첨가물 제조  Preparation of feed additives

실시예 2에서는 상기 실시예 1에서 수득된 특이항체 난황분말을 이용하여 자돈 사료용 첨가물을 제조하였다. In Example 2, an additive for piglets was prepared using the specific antibody yolk powder obtained in Example 1 above.

특이항체 난황분말 20g; 프락토올리고당 240g; 비타민 B6 0.4g; 이노시톨 0.4g; 바실러스 서브틸리스(Bacillus subtilis), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 류코노스톡 메센테로이드(Leuconostoc mesenteroides)를 포함하는 미생물 3.24g; 및 정제수 136ml를 첨가하여 자돈 사료용 첨가물을 제조하였다. Specific antibody yolk powder 20g; 240 g of fructooligosaccharides; 0.4 g of vitamin B6; 0.4 g of inositol; Bacillus subtilis subtilis ), Lactobacillus fermentum ), 3.24 g of microorganisms including Leuconostoc mesenteroides ; And 136 ml purified water was added to prepare an additive for piglets feed.

실험예Experimental Example 4:  4: 자돈Piglets 사료용 첨가물의 효과 측정 Determination of the Effect of Feed Additives

10일령부터 50일령까지만 상기 실시예 2에서 제조된 사료용 첨가물을 섭취시키고 70일령이 되었을 때 체중 및 총 사고두수를 측정하였다. 실시예 2의 사료용 첨가물의 첨가율은 자돈이 10일령~21일령에는 0.6 중량%, 21일령~35일령에는 0.4 중량%, 35일령~50일령에는 0.2 중량% 첨가하였다. 대조군은 상기 실시예 2에서 제제된 사료용 첨가물을 첨가하지 않은 사료를 섭취시킨 자돈이다.Only from 10 days to 50 days of age, the feed additive prepared in Example 2 was ingested, and when it was 70 days of age, body weight and total head count were measured. The feed rate of the feed additive of Example 2 was added to 0.6% by weight in piglets 10 days to 21 days, 0.4% by weight from 21 days to 35 days, 0.2% by weight from 35 days to 50 days of age. The control group is piglets fed the feed without the feed additive prepared in Example 2 above.

먼저, 일반적으로 월간 이유두수 1500두에 대한 발병율, 발병일령, 발병 양상, 폐사율, 폐사일령, 부검소견을 하기 표 3에 나타냈다.First, in general, the incidence rate, onset age, onset pattern, mortality rate, mortality age, autopsy findings for 1500 weaning heads per month are shown in Table 3 below.

월간이유두수Monthly Nipples 1500 두1500 two 발병율Incidence 30~40%30-40% 발병일령Onset age 35일령35 days of age 발병양상Onset 거친피모, 사료섭취량저하, 복식호흡, 잔기침, 설사Rough hair, reduced feed intake, double breathing, residue acupuncture, diarrhea 폐사율Mortality 발병돈 중 80% 폐사80% mortality 폐사일령Dead age 40일령이후(40~50일령 집중)After 40 days of age (40-50 days of age concentrated) 부검소견Autopsy findings 심낭염, 폐 및 복강 내 섬유소성 삼출물, 위궤양, 임파절종대 및 출혈, 폐렴Pericarditis, Fibrous exudate in the lungs and abdominal cavity, Gastric ulcers, Lymphadenopathy and bleeding, Pneumonia

본 실험예 4에서 실시한 자돈 사료용 첨가물을 자돈에 섭취시켰을 때 나타나는 효과를 대조군과 비교하여 하기 표 4에 나타냈다.The effect of ingesting the piglet feed additives carried out in Experimental Example 4 in the piglets is shown in Table 4 in comparison with the control group.

대조군Control 실험군Experimental group 이유평균체중(kg)Average weight (kg) 6.296.29 6.336.33 개시두수Starting head 322322 363363 종료두수End 256256 346346 종료평균체중(kg)End weight (kg) 25.525.5 27.327.3 총 사고두수Total accidents 9696 1717 위축atrophy 2424 44 폐사Our company 5656 99 도태Culling 1616 44 사고율(%)Accident Rate (%) 28.028.0 4.74.7

표 4에 나타난 대조군과 실험군의 종료평균 체중과 사고율을 비교하기 위해 도 6을 작성하였다.FIG. 6 was prepared to compare the end mean body weight and the accident rate of the control group and the experimental group shown in Table 4.

측정결과, 실험군은 위축 및 폐사가 대조군에 비해 현저히 낮게 나타났고, 세균성 및 식이성 하리가 발생되지 않았다. As a result, the experimental group showed significantly lower atrophy and mortality than the control group, and did not cause bacterial and dietary dissociation.

그리고 이를 4개의 농장에서 0~8주의 자돈에 적용한 결과(도 7), 실험군은 대조군에 비해 위축증과 폐사율이 현저히 낮아진 것을 확인할 수 있었다. And as a result of applying this to 0-8 weeks piglets in four farms (Fig. 7), the experimental group was confirmed that significantly reduced atrophy and mortality compared to the control group.

도 1은 실험군과 대조군의 자돈을 각각의 실험 돈방에 넣어 24시간 동안 적응시킨 후 4일령에 공격접종을 실시하고 시기별로 채혈하여 체중을 측정한 실험도이다.1 is an experimental diagram in which the piglets of the experimental group and the control group were put into each experimental pig room for 24 hours, followed by challenge inoculation at 4 days of age, and blood samples were collected at different times.

도 2는 PEDV를 4일령에 공격접종하기 전과 접종 후 7일 10일에 실험군과 대조군 각각 10두씩 시기별로 채혈하여 항체역가를 측정한 도이다. FIG. 2 is a diagram illustrating antibody titers by collecting blood from each of the experimental group and the control group at 10 days before inoculation of PEDV at 4 days of age and at 7 days after inoculation.

도 3은 TGEV를 4일령에 공격접종하기 전과 접종 후 7일 10일에 실험군과 대조군 각각 10두씩 시기별로 채혈하여 항체역가를 측정한 도이다.FIG. 3 is a diagram illustrating antibody titers by collecting blood for 10 heads each of the experimental group and the control group at 7 days and 10 days after inoculation with TGEV at 4 days of age.

도 4는 대장균을 4일령에 공격접종하기 전과 접종 후 7일 10일에 실험군과 대조군 각각 10두씩 시기별로 채혈하여 항체역가를 측정한 도이다.FIG. 4 is a diagram illustrating antibody titers by collecting blood for each of two groups, each of the experimental group and the control group, at 7 days and 10 days after inoculation with E. coli at 4 days of age.

도 5는 대장균을 공격접종 후 실험군과 대조군의 분에서 분리된 대장균의 수를 일별로 나타낸 도이다5 is a diagram showing the number of Escherichia coli separated from the experimental group and the control group daily after inoculation of E. coli.

도 6은 본 발명의 자돈 사료용 첨가물 조성물을 자돈에 섭취시킨 후 체중 및 사고율을 나타낸 도이다.Figure 6 is a diagram showing the weight and accident rate after ingesting the piglet feed additive composition of the present invention in piglets.

도 7은 본 발명의 자돈 사료용 첨가물 조성물을 4개의 농장에서 0~8주의 자돈에 적용한 후, 실험군은 대조군의 위축증과 폐사율을 나타낸 도이다. 7 is a diagram showing the atrophy and mortality of the control group after applying the piglet feed additive composition of the present invention to the piglets 0 to 8 weeks in four farms.

Claims (2)

살모넬라 티피뮤리엄(Salmonella Tiphimurium)에 대한 IgY, 살모넬라 콜레라수이스(Salmonella Choleraesuis)에 대한 IgY, TGEV(Transmissible gastroenteritis virus)에 대한 IgY, PEDV(Porcine epidemic diarrhea)에 대한 IgY 또는 대장균(Escherichia coli)에 대한 IgY 중 선택되는 어느 하나 이상의 IgY를 함유하는 난황; 프락토올리고당; 비타민 B6; 이노시톨; 바실러스 서브틸리스(Bacillus subtilis), 비피도박테리움 론굼(Bifidobacterium longum), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 불가리커스(Lactobacillus bulgaricus), 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 애시도필러스(Lactobacillus acidophilus), 락토바실러스 사케이(Lactobacillus sakei), 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 류터리(Lactobacillus reuteri), 락토바실러스 락티스(Lactobacillus lactis), 류코노스톡 메센테로이드(Leuconostoc mesenteroides), 류코노스톡 김치아이(Leuconostoc kimchii), 류코노스톡 시트레움(Leuconostoc citreum), 류코노스톡 가시코미타툼(Leuconostoc gasicomitatum), 류코노스톡 락티스(Leuconostoc lactis) 또는 스트렙토코커스 써머필러스(Streptococcus thermophilus) 중 선택되는 어느 하나 이상의 미생물; 및 정제수;를 혼합한 혼합물을 포함하는 것을 특징으로 하는 자돈 사료용 첨가물 조성물.IgY for Salmonella Tiphimurium , IgY for Salmonella Choleraesuis , IgY for TGEV (Transmissible gastroenteritis virus), IgY for PEPV (Porcine epidemic diarrhea) or Escherichia egg yolk containing any one or more of IgY selected from IgY for coli ); Fructooligosaccharides; Vitamin B6; Inositol; Bacillus subtilis subtilis), rongum Bifidobacterium (Bifidobacterium longum ), Lactobacillus fermentum), Lactobacillus Bulgaria carcass (Lactobacillus bulgaricus), Lactobacillus casei (Lactobacillus casei ), Lactobacillus acidophilus ), Lactobacillus sakei), Lactobacillus Planta Room (Lactobacillus plantarum ), Lactobacillus reuteri ), Lactobacillus lactis ), Leuconostoc mesenteroides ), Leuconostoc kimchii , Leuconostoc citreum ), Leuconostoc gasicomitatum ), Leuconostoc lactis ) or Streptococcus thermophilus ) any one or more microorganisms selected from; Pigment feed additive composition comprising a mixture of; and purified water; 제1항에 있어서,The method of claim 1, 자돈 사료용 첨가물 조성물은,Pigment feed additive composition, 난황 0.05~9.5 중량%; 프락토올리고당 47.6~71.6 중량%; 비타민 B6 0.01~0.11 중량%; 이노시톨 0.02~0.22 중량%; 바실러스 서브틸리스(Bacillus subtilis), 비피도박테리움 론굼(Bifidobacterium longum), 락토바실러스 퍼멘텀(Lactobacillus fermentum), 락토바실러스 불가리커스(Lactobacillus bulgaricus), 락토바실러스 카제이(Lactobacillus casei), 락토바실러스 애시도필러스(Lactobacillus acidophilus), 락토바실러스 사케이(Lactobacillus sakei), 락토바실러스 플란타룸(Lactobacillus plantarum), 락토바실러스 류터리(Lactobacillus reuteri), 락토바실러스 락티스(Lactobacillus lactis), 류코노스톡 메센테로이드(Leuconostoc mesenteroides), 류코노스톡 김치아이( Leuconostoc kimchii), 류코노스톡 시트레움(Leuconostoc citreum), 류코노스톡 가시코미타툼(Leuconostoc gasicomitatum), 류코노스톡 락티스(Leuconostoc lactis) 또는 스트렙토코커스 써머필러스(Streptococcus thermophilus) 중 선택되는 어느 하나 이상의 미생물 0.05~1.9 중량% ; 및 정제수 24.2~44.2 중량%로 조성되는 것을 특징으로 하는 자돈 사료용 첨가물 조성물.Egg yolk 0.05 to 9.5% by weight; Fructooligosaccharide 47.6-71.6 wt%; 0.01 to 0.11 weight percent of vitamin B6; Inositol 0.02-0.22 wt%; Bacillus subtilis , Bifidobacterium longum ), Lactobacillus fermentum), Lactobacillus Bulgaria carcass (Lactobacillus bulgaricus), Lactobacillus casei (Lactobacillus casei ), Lactobacillus acidophilus ), Lactobacillus sakei), Lactobacillus Planta Room (Lactobacillus plantarum ), Lactobacillus reuteri ), Lactobacillus lactis ), Leuconostoc mesenteroides ), Leuconostoc kimchii , Leuconostoc citreum ), Leuconostoc gasicomitatum ), Leuconostoc lactis ) or Streptococcus thermophilus ) 0.05 to 1.9 wt% of any one or more microorganisms selected from; And purified water 24.2 ~ 44.2 wt% composition for piglet feed, characterized in that the composition.
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