【病毒外文文献】1997 Passive protection of neonatal calves against bovine coronavirus-induced diarrhea by administration of egg yolk or

ELSEVIER Veterinary MicrobioLogy 58 1997 105 111 veterinary microbiology Passive protection of neonatal calves against bovine coronavirus induced diarrhea by administration of egg yolk or colostrum antibody powder Yutaka Ikemori Masashi Ohta Kouji Umeda Faustino C Icatlo Jr Masahiko Kuroki Hideaki Yokoyama Yoshikatsu Kodama Ghen Corporation Immunology Research Institute 839 1 Sano Gifu City 501 11 Japan Received 12 December 1996 accepted 14 May 1997 Abstract The protective effect of egg yolk and colostrum powders prepared from hens and cows vaccinated with inactivated bovine coronavirus BCV antigen was evaluated in a challenge model with a virulent BCV strain Twenty three calves from BCV free herds were randomly divided into control and several treatment groups All calves were orally challenged with 1 x 10 9 TCIDso of the virulent Kakegawa strain of BCV at 24 to 36 h after birth Calves in treatment groups received either egg yolk powder or cow colostrum containing BCV specific antibodies Daily treatment with these antibody preparations started 6 h until 7 days post challenge Control calves which received no antibody had severe diarrhea and all died within 6 days after infection In contrast calves fed milk containing egg yolk or colostrum with neutralization titers of 1 2560 or 1 10 240 respectively all survived and had positive weight gain unlike the other treatment groups These results indicate that the orally administered egg yolk and colostrum powders protected against BCV induced diarrhea in neonatal calves and that the egg yolk used provided a higher degree of protection compared to colostrum powder on a titer basis Treatment with whole egg yolk from immunized hens therefore provides a more efficacious alternative to the existing methods of specific passive protection against BCV 1997 Elsevier Science B V Keywords Cattle viruses Bovine coronavirus Passive immunization ImmunoLogy Egg yolk Colostrum Corresponding author Tel 81 58 235 7303 fax 81 58 235 7505 0378 1135 97 17 00 1997 Elsevier Science B V All rights reserved PII S0378 1 1 35 97 00144 2 106 E lkemori et al Veterinary Microbiolog3 58 1997 105 111 1 Introduction Bovine coronavirus BCV is an important agent of neonatal calf diarrhea and is associated with acute diarrhea of adult cattle referred to as winter dysentery This virus is known to cause a more severe disease and higher mortalities than those caused by the bovine rotavirus because it multiplies in both the small intestine and the large intestine whereas the rotavirus infects only the small intestine Kapil et al 1990 To determine protective immune responses against BCV there have been reports analyzing the titers of passive and active antibody isotypes to BCV in serum and mucosal surface Heckert et al 1991a b Coronavirus vaccines are available to control BCV diarrhea but have not been found to be efficacious in protecting against infection Heckert et al 1991c Kapil et al 1990 In the case of passive immunization there were few reports on oral administration of antibodies derived from serum or colostrum against BCV Recently chicken egg yolk antibodies have been used for prophylaxis and treatment of infectious intestinal diseases We have been investigating the efficacy of the chicken egg yolk antibodies in calves against enterotoxigenic Escherichia coli ETEC Ikemori et al 1992 and bovine rotavirus Kuroki et al 1993 The objectives of the study reported here were l to evaluate the efficacy of chicken egg yolk antibodies and cow colostrum antibodies against BCV induced diarrhea in calves where BCV causes a more severe disease than bovine rotavirus and 2 to compare the therapeutic value of chicken egg yolk and cow colostrum antibody 2 Material and methods 2 1 Virus strains The strain NCDC of BCV was kindly provided by Dr Murakami the National Institute of Animal Health Tsukuba Japan This strain was originally isolated by Mebus et al Mebus et al 1973 in the U S A The Kakegawa strain of BCV was pathogenic to calves and was obtained from the Japanese Association of Veterinary Biologics Both viruses were certified free from adventitious agents and grown in confluent human rectal tumour cell line HRT 18 with Eagle s MEM as previously described Tsunemitsu et al 1991 The viruses were used for challenge exposure of calves and for production of antibody in chickens and cows 2 2 Titration for infectiL e L iruses and neutralization test Virus titration and neutralization test were performed in HRT 18 cell cultures grown in microdilution plates and conducted by the method previously described Tsunemitsu et al 1991 Briefly for the titration serial 10 fold dilutions of viruses were made and inoculated to 4 wells of HRT 18 cells with 0 1 ml of each dilution The plates were incubated for 7 days at 37 C and the infectivity was expressed by median tissue culture infective dose TCIDs0 as determined by the appearance of CPE For the neutralization test egg yolk and colostrum powders were prepared as 10 suspension in PBS Serial 2 fold dilutions of these samples were mixed with the same Y Ikemori et al Veterinary Microbiology 58 1997 105 111 107 volume of virus suspensions containing 200 TCIDs0 0 1 ml and incubated for 1 h at 37 C Two wells of HRT 18 cells were inoculated with 0 I ml of each mixture and incubated for 7 days at 37 C The neutralizing antibody titers were expressed as the reciprocal of the highest dilution that inhibited 50 of CPE multiplied by the antibody powder dilution factor 2 3 Preparation of specific antibody against BCV Two kinds of specific antibody against BCV were prepared egg yolk powder and bovine colostrum powder NCDC antigen containing about 108 5 TCIDs0 ml was added with 0 3 formalin and inactivated for 1 week at 4 C The antigen was mixed with an equal volume of oil adjuvant with 5 mannide monooleate Maine Biological Laborato ries Maine and 1 0 ml of the mixture was injected intramuscularly into 10 white Leghorn hens Six weeks after the initial injection booster inoculation were adminis tered in a similar manner and eggs were harvested 2 weeks later The yolks were carefully separated from the egg white and was then applied to a spray dry machine operated at air inlet temperature of 140 C The dried egg powder was stored in a desiccator at room temperature until use The cows used were 3 to 5 years old and had no history of disease or vaccination against BCV One ml formalin inactivated NCDC vaccine was administered intramuscu larly in two cows 6 weeks before the anticipated calving followed by a similar dose spaced 3 weeks apart Early colostrum was collected from cows after parturition and applied to the spray dry machine operated under the same conditions The dried colostrum was stored in a desiccator at room temperature until use 2 4 Challenge exposure and clinical observations Twenty three colostrum deprived newborn Holstein calves from farms that were free from neonatal diarrhea due to bovine coronavirus rotavirus viral diarrhea virus and ETEC were used in this study and were fed a commercially available milk formula Calves were randomly distributed as non treated control group group 1 egg powder treatment groups groups 2 and 3 and colostrum powder treatment groups groups 4 5 and 6 At 24 to 36 h after birth calves were orally challenge exposed with 1 109 0 TCIDs0 of the virulent Kakegawa strain Fig l At 6 h after BCV challenge the treatment groups were given between 1 5 and 2 1 of milk containing the egg yolk or colostrum powder Subsequently this was given 2 times per day for 7 days after challenge exposure Particularly the egg powder treatment groups 2 and 3 were given milk containing antibody titers of 1 1280 and 1 2560 0 25 and 0 5 g of egg powder respectively On the other hand colostrum powder treatment groups 4 5 and 6 were given milk containing antibody titers of 1 2560 1 5120 and 1 10 240 0 5 1 0 and 2 0 g of colostrum powder respectively The control group received no antibody The clinical response of each calf was recorded throughout the experiment and evaluated in terms of fecal consistency score weight gain and mortality Fecal scoring was done by persons who did not know the treatment condition for each calf Scoring was done 2 times per day with numerical scores as follows 0 normal 1 soft consistency 2 mild 108 K lkemorietal Veterinao Microbiology 58 1997 105 111 Challenge Antibody treatment Clinical observation Fecal sampling Serology Necropsy I I i I I I I I I O 1 2 3 4 5 6 7 days AAAAAAAAAAAAAA 0 0 0 0 0 0 0 0 O Fig I Summary of the experimental protocol O Q diarrhea 3 severe watery diarrhea and death The cumulative fecal score of each calf was expressed as total score for 7 days Examination of feces for infectious BCV was done daily for 7 days by culturing the homogenates of the specimens in HRT 18 cells On day 7 post challenge samples of the middle part of jejunum and colon were tested for infectious BCV by the same method Body weight gain of calves was expressed as percentage weight gain at day 7 over initial body weight on day 0 The serum of calves collected at the start and at the end of the experiment were checked for anti BCV antibodies by neutralization test 2 5 Statistical analysis The Student s t test was used to assess the statistical significance of differences in fecal consistency scores total positive days of virus detection percentage of body weight change and virus titers in intestines whereas the Fischer exact test was used to assess differences in mortality between treated and control calves 3 Results 3 1 Production of antibody powders from immunized egg yolk and colostrums The in vitro neutralizing antibody titers of egg yolk powder from chickens and colostrum powder from cows immunized with the strain NCDC were almost the same 1 5120 against the homologous strain Apparently there was not much difference in the magnitude of antibody response between chickens and cows The antibody titers 1 5120 against the strain Kakegawa were the same as those against the homologous strain in both powders A cross reactivity between the two strains in the neutralizing antibody test revealed a close serological relationship between the NCDC and Kakegawa strains titer of 1 5120 using egg yolk or colostrum against each strain Y lkemori et al Veterinary Microbiology 58 1997 105 111 109 3 2 Clinical response of calves after challenge exposure All calves of the control group group 1 when exposed to coronavirus developed severe watery diarrhea including mucus and blood from postchallenge exposure PE day 1 to day 2 and died on PE day 6 Table 1 Severe diarrhea was observed with a mean change in body weight of 7 4 2 3 at the time of death Calves of group 2 which was treated with 1 1600 antibody titer of the egg yolk antibody were not protected against severe diarrhea and mortality Three calves in this group developed severe diarrhea of which two calves died on PE days 4 and 7 respectively Although the other calves of group 2 survived the infection cumulative fecal score was 20 0 17 4 and dehydration resulted in weight loss of 5 1 All calves of group 3 recovered from the disease Table 1 Diarrhea was temporary and was not accompanied by weight loss in these calves The difference in cumulative fecal score and weight gain between groups 1 and 3 was significant P 0 01 Based on isolation of coronavirus from feces and intestinal specimens the total number of days of positive virus detection in the feces in group 3 was 1 8 1 5 days which was of shorter duration than that of group 1 difference not significant The titers of coron avirus in the intestines of this group were very low The virus was not detected in the small intestine of group 3 calves On the other hand all calves of the colostrum antibody 1 2560 treated group 4 developed severe watery diarrhea and later died on PE day 4 Table 1 Two calves in group 5 1 5120 also died with severe diarrhea and the cumulative fecal score and weight loss in this group were as high as those in the control group In group 6 all calves recovered from disease but 3 calves in this group developed watery diarrhea which continued for 4 to 5 days The cumulative fecal score in this group was a high 14 8 11 3 together with a low weight gain The mean total positive days of virus detection in feces among groups 4 5 and 6 were shorter than that of group 1 but this was not statistically significant Table 2 Titers of coronavirus in the intestine of groups 4 5 and 6 decreased with increasing colostrum dose and virus titers in the small intestine were generally higher not statistically significant than in the large intestine in these 3 groups The neutralizing antibody titers of the serum of all calves were 10 against Table 1 Clinical response of calves to challenge exposure with BCV and subsequent treatment with egg yolk or colostrum antibody powder of different titers Group Antibody powder Antibody titer No diarrhea No dead Cumulative Weight gain total no total no fecal score 1 4 4 100 4 4 100 30 5 6 1 7 4 2 3 2 Egg 1280 3 4 75 2 4 50 20 0 7 4 5 1 10 4 3 Egg 2560 2 4 50 0 4 0 3 8 3 8 6 3 2 4 4 Colostrum 2560 3 3 100 3 3 100 32 3 0 6 9 6 1 5 5 Colostrum 5120 4 4 100 2 4 50 28 8 3 4 8 2 5 3 6 Colostrum 10 240 3 4 75 0 4 0 14 8 11 3 1 2 3 2 P O 05 P O Ol 110 E lkemori et al Veterinao Microbiology 58 1997 105 111 Table 2 Detection of BCV in feces from daily fecal samples after challenge exposure and from intestines at the time of necropsy among calves treated with egg yolk or colostrum powder of different titers Group Total positive days of virus detection mean S D Virus titers TCIDs0 g Small intestine mean S D Large intestine mean S D 1 6 0 0 8 4 63 0 14 5 00 t 00 2 5 5 1 9 2 38 2 74 4 00 1 70 3 1 8 1 5 0 0 00 1 25 1 44 4 6 3 0 6 5 41 0 14 5 08 0 72 5 5 5 1 0 2 81 2 08 2 75 2 02 6 3 5 2 4 1 25 1 40 0 63 1 25 All mean values had n 4 except for group 4 where n 3 P 0 05 BCV before challenge Similar titers were obtained 7 days later indicating that antibod ies from administered egg yolk and colostrum did not enter the systemic circulation of calves 4 Discussion In this study we evaluated the efficacy of the antibody preparations derived from egg yolks of immunized laying hens and colostrums of immunized cows against experimen tally induced bovine coronavirus calf diarrhea This is to fill the gap in the literature concerning passive protection against BCV using specific antibody Our data showed that significant protection was achieved in calves treated with high titers of the egg yolk and colostrum antibodies against the challenge strain Table 1 Survival against coronavirus induced mortality was 100 when 1 2560 and 1 10 240 antibody titers were used for treatment of calves with the egg yolk or colostrum powder respectively Particularly the calves treated with the egg yolk antibody did not have severe diarrhea and had higher weight gains About 8 g of egg yolk powder were obtained from one chicken egg and only one egg yolk was needed to protect one calf from diarrhea over a 7 day course of treatment The present data corroborate an earlier protection trial using egg antibody specific for ETEC in ETEC infected calves Ikemori et al 1992 The minimal antibody titers against fatal diarrhea were found to be 1 2560 for the egg yolk and 1 10 240 for the colostrum Although diarrhea was observed in calves treated with these antibody powders the cumulative fecal scores were higher in the calves treated with colostrum than those treated with egg yolk It appears that greater amount of colostrum powder than egg yolk powder is necessary to prevent diarrhea The virus titers in the intestines of groups 4 to 6 calves were high irrespective of the dose of antibody given virus titers in the small intestines tended to be higher than in the large intestines in the above groups Table 2 It was speculated that continued viral proliferation in the small intestine was the reason for the persistent diarrhea observed in these groups of calves The difference in the minimal protective titers of the antibody powders between the egg yolk and the colostrum may have two possible explanations Firstly the avidity of Y lkemori et al Veterinary Microbiology 58 1997 105 111 111 antibodies derived from colostrum is lower than that of antibodies obtained from egg yolk Ikemori et al 1993 Compared to colostral antibodies the BCV specific antibody from egg yolk may have reacted more strongly and stably with coronavirus epitopes in vivo in the neutralization reaction Secondly less chicken antibody may have been digested and inactivated by the gastric juice The antibodies that escaped digestion in the stomach are still functional in the small intestine The antibody of the egg yolk is thought to be of almost the same stability or slightly more susceptible to gastric juice than mammalian antibody Shimizu et al 1992 However the present data demon strated that the antibody from egg yolk was more effective than colostral antibody suggesting that yolk components in the egg yolk powder such as proteins and fats may have protected the immunoglobulin fraction from digestive enzymes and allowed safe passage of yolk immunoglobulins through the stomach enough to confer protection in the target areas of the small intestine of calves On the other hand colostral proteins and fats may be more susceptible to digestion by gastric juice or quantitatively less than those of the egg yolk In conclusion the egg yolk powder from chickens immunized with bovine coron avirus has a greater therapeutic potential against BCV induced diarrhea than colostrum powder from immunized cows It took about four times more colostral antibody than egg yolk antibody to prevent mortality in calves Oral egg yolk powder containing specific antibodies may therefore provide an alternative approach to passive prevention of BCV infections in the field References Heckert R A Sail L J Mengel J P Myers G W 1991a Mucosal and systemic antibody responses to bovine coronavirus structural proteins in experimentally challenge exposed calves fed low or high amounts of colostral antibodies Am J Vet Res 52 700 708 Heckert R A Sail L J Myers G W 1991b Mucosal and systemic isotype specific antibody responses to bovine coronavirus structural proteins in naturally infected dairy calves Am J Vet Res 52 852 857 Heckert R A Saif L J Myers G W Agnes A G 1991c Epidemiologic factors and isotype specific antibody responses in serum and mucosal secretions of dairy calves with bovine coronavirus respiratory tract and enteric tract infections Am J Vet Res 52 845 851 Ikemori Y Kuroki M Peralta R C Yokoyama H Kodama Y 1992 Protection of neonatal calves against fatal enteric colibacillosis by administration of egg yolk powder from hens immunized with K99 piliated enterotoxigenic Escherichia coli Am J Vet Res 53 2005 2008 lkemori Y Peralta R C Kuroki M Yokoyama H Kodama Y 1993 Research note avidity of chicken yolk antibodies to enterotoxigenic Escherichia coli fimbriae Poultry Sci 72 2361 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