key: cord-0008925-wwqwa6im authors: Valente, C.; Fruganti, G.; Tesei, B.; Ciorba, A.; Cardaras, P.; Floris, A.; Bordoni, E. title: Vaccination of pregnant cows with K99 antigen of enterotoxigenic Escherichia coli and protection by colostrum in newborn calves date: 2002-11-05 journal: Comp Immunol Microbiol Infect Dis DOI: 10.1016/0147-9571(88)90037-9 sha: 8556784ee620c0c0061e5dc4f58eda2802ad93a2 doc_id: 8925 cord_uid: wwqwa6im The immune response to the K99 was tested in 45 pregnant cows, subcutaneously vaccinated, for protecting the newborn calves. Serological tests were performed in the blood sera of all animals and in the milk and colostrum sera; hemogram, inhibition of the adhesion to the brush border and histological tests were performed. The calves from vaccinated cows survived the experimental infection after the suction of colostrum in spite of the fact that the calves from control dams died with diarrhea. The enterotoxigenic strains of Escherichia coli are able to induce enteric syndromes in newborn calves producing enterotoxins and colonizing at the enterocytes by K99 antigen or other Pili [1] [2] [3] [4] [5] [6] . Epidemiological surveys in different countries show however that the incidence of enterotoxigenic strains can reach a high percentage [7] [8] [9] [10] . The disease usually occurs in calves less than three days old it can be reproduced experimentally by administering the bacterial suspension per os to calves, lambs or pigs early after birth either to the conventional or gnotobiotic animals [11] [12] [13] . The resistance to the infection develops rapidly with age and the enterocytes of calves one week old are resistant probably because E.T.E.C. is not able to colonize [14] . The first defensive action of the newborn comes from the colostrum. The secretion of many cows lacks the antibodies capable of inhibiting the bacterial colonization. The antigen K99 could constitute a logical basis for the preparation of a vaccine for administration to pregnant females. The aim of this study was to test the immunological capacity of K99 by vaccination of pregnant cows and also to verify the degree of colostral protection in newborn calves. The enterotoxigenic strains ofEscherichia coli B41 (O101 :K-:H , K99, F41) were used for the preparation of the antigen and Escherichia coli B44 (O9:K30:H , K99, F41) for the experimental infection of calves [15] . The colonizing antigen K99 was obtained from the enterotoxigenic Escherichia coli B41 by means of chemical detachment of the Pili according to a process of partial purification [16] [17] [18] and was injected at a protein concentration of 15 mg in 10 ml of saline solution [19] with the somatic residual antigens associated with the incomplete Freund adjuvant [20] . Forty-nine pregnant cows were used, 6-8 years old, clinically healthy, belonging to a 200 head herd and fed silage corn, grain and concentrates. Forty-five pregnant cows were vaccinated subcutaneous 6 and 4 weeks before calving; 4 pregnant cows were maintained as controls, they received a broth culture lacking bacteria [21, 22] . Escherichia coli B44, grown for 18 h at 37°C in Minca-Is [23] was administered to calves 5-10 h old after the first suckling of colostrum. The infective dose of 4 x l0 ~° colonies forming units/ml, was given orally to calves born. A rectal swab was obtained from each calf before exposure to the challenge [13] . The blood samples were obtained from cows at the time of vaccination and then 15 and 25 days later, within 24 h of calving and 7 days post partum. The colostrum was collected before the suction by the calf and 7 days after calving. The blood samples were obtained from calves after the suction of colostrum at 12-24 h after birth and at 7 days old. All the subjects underwent a direct clinical control, when samples of blood and colostrum were taken from the cows and the calves daily. The blood samples were examined for the hemogram according to the usual hematological techniques. Agglutination tests on the colostrum and milk serum, obtained by microbiological digestion and centrifugation, and on the blood serum samples were performed in microtiter plates using as antigen both Escherichia coli B41 and its K99 mutant [18, 24] . The test, employing epitelial cells from the small intestine of a newborn calf, was performed with Escherichia coli B41 in the presence of milk and colostrum serum. An immune serum anti K99 was utilized as a positive control [18, 25, 26] . Twenty enterocytes were examined for every test. The inhibition index was expressed in percent and the value was 100 only if none of the bacteria were attached to the epithelial cells. The immune serum anti K99 gave a value of 70%. Intestinal fragments were collected from the small intestine, immersed in formalin, dehydrated, enclosed in paraffin and stained for histological control [13, 27] . The results were prepared for the analysis of the variant in relation to a single criterium of classification [28] . The vaccinated cows, including the controls, did not have local reactions or general changes. The gestation and parturition were normal, followed by birth at the end of term and the milk production was within the average of the herd. Of the 45 calves born alive and viable from vaccinated mothers only 3 presented slight depression and water diarrhea in the second and third day of age. The 4 control calves died, one at 24 h and the others at the third-fourth day with profuse diarrhea, serious dehydration with insufficient peripheral circulation (Table 1 ). In the vaccinated and the control cows the hematomorphological examination showed hypochromic anemia and neutrophilic granulocytosis during the whole period of observation, let alone a decrease, statistically significant, of the eosinophils in the third and fourth samples (Tables 2 and 3 ). In the calves from vaccinated cows the hemogram performed within 24 h from birth revealed anemia and neutrophilia; further checks showed the persistence of the anemia and the inversion of the granulocyte/lymphocyte relationship ( Table 4 ). The results of the serological tests with K99 antigen and the acapsular mutant are shown in Tables 5 and 6 , Figs 1 and 2. No variation was observed in the antibody level with the acapsular mutant. The colostrum antibody levels were higher than in milk. However the highest titers, 7 days from calving, found the level in milk coincided with the lower levels of the colostrum. The calves, 12 24 h, from vaccinated cows had high antibody titers, higher than those registered in the control subjects (Table 7 and Fig. 3) . The capacity of milk and colostrum to inhibit, in vitro, the colonization of K99 antigen to enterocytes furnished the results expressed in percentages, inferior to 30 ( Table 8) . The histological checks carried out on the control calves demonstrated marked exfoliation and shortening of the villi. Furthermore from the feces and intestinal content of those animals was isolated Escherichia coli B44, which was irregularly present in the rectal swabs of calves from vaccinated cows. The analysis of the results, in the cows vaccinated with K99 antigen, shows that the antibodies, significantly increased from the 25th day from the first inoculation until 24 h after calving. This was statistically significant and its drop is equally significant after 7 days. The serological values of the colostrum and milk of the cows and blood serum of the calves show interesting and significant decreases of the antibody levels against K99 antigen, in the milk colostrum relation of the vaccinated cows, and in the sera of their calves checked when 7 days old. The antibody levels for K99 antigen were determined using a heterologous serotype Escherichia coli B41, having in common with Escherichia coli B44 only the K99 antigen ensuring in this way the antibody specificity. As a further verification the acapsular mutant was also used. The results of the colonization inhibition test at the enterocytes, even if low percentage, showed significant differences between colostrum and milk, and larger concentrations of specific antibodies in the colostrum according to the serologic test. The fact that the calves from vaccinated cows resisted the challenge after suckling the colostrum and those of the controls did not, confirms the validity of the immunization. A further confirmation comes from the isolation of enterotoxigenic bacteria from the feces and intestinal content of the control calves and dysepithelization with shortening of the intestinal villi. Therefore this result supports the validity of the vaccine, obtained from Pili, against the enterotoxigenic strains of Escherichia coli administered to pregnant females at 5 weeks before calving [21, 29, 30] . In similar conditions piglets and lambs suckling colostrum from vaccinated mothers resisted to the challenge [31] [32] [33] . In the cows the enterotoxigenic bacteria were injected differently, either as whole bacteria or as colonizing purified antigen or partially purified or associated to Rotavirus, each with different results [20-22, 29, 30, 34, 35] . It is important to note that there was a seroconversion against the acapsular mutant in all of the subjects of the experiment. This was presumably due to the high diffusion of Escherichia coli in the environment and due to the incomplete purification of K99 antigen in our vaccine that also contained the somatic antigen. The seroconversion against antigen K99, also in non vaccinated cows with higher levels 24 h after calving and in control calves, which is spite of this immunity did not resist to the infection, justifies the existence of other colonizing antigens. It also confirms the hypothesis that not always the degree of protection corresponds to the level of specific antibody values present in the colostrum [1, 3, 25, [36] [37] [38] . Considering the results of this paper it is necessary in the future to establish the different antibodies of the colostrum and to find the lowest serological titer able to protect newborn calves. Fr6quence des Pili FY et K99 parmi des souches de Escherichia coli isol6es de veaux diarrh6iques en France Differences in the response of rabbit small intestine to heat labile and heat stable enterotoxins of Escherichia coli Adhesion in vitro and in vivo associated with an adhesive antigen (F41) produced by a K99 mutant of the reference strain Escherichia coli B4 I Episome carried surface antigen K88 of Escherichia coli. I Transmission of determinant of the K88 antigen and influence on the transfer of chromosomal markers Further observations on Escherichia coli enterotoxins with particular regard to those produced by atypical piglet strains and by calf and lamb strains: the transmissible nature of these enterotoxins and of a K antigen possessed by calf and lamb strains Acute indifferentiated neonatal diarrhea of beef calves: the prevalence of enterotoxigenic E. coli, reo-like (rota) virus and other enteropathogenes in cow calf herds Etiologic diagnosis of diarrheal diseases of calves: frequency and methods for detecting enterotoxin and K99 antigen production by Escherichia coli Occurrence and characteristics of enterotoxigenic Escherichia coli isolated from calves with diarrhea Characterization of enterotoxigenic bovine Escherichia coli isolated from newborn calves Int6r6t de l'agneau axenique pour de l'ent6ropathog6nicit6 d'Escherichia coli d'origine bovine. Observations cliniques, microbiologiques et histologiques The experimental production of diarrhoea in colostrum deprived axenic and gnotaxenic calves with enteropathogenic Escherichia coli, rotavirus, coronavirus and in a combined infection of rotavirus and Escherichia coli Diarrhea in lambs: experimental infection with enterotoxigenic Escherichia coli Pathogenesis of enteric disease caused by Escherichia coli Haemagglutinating and adhesive properties associated with the K99 antigen of bovine strains of Escherichia coli Purification, characterization and partial covalent structure of Escheriehia coli adhesive antigen K99 New method for isolation of immunologically pure pili from Escherichia coli Purificazione e parziale caratterizzazione dell'antigene K99 di Escherichia coli Protein measurement with the folin phenol reagent Passive immunisation of neonatal lambs against infection with enteropathogenic Escherichia coli via colostrum of ewes immunised with crude and purified K99 pili Vaccination of cows with purified K99 antigen anucleated live E. coli, and whole cell bacterins containing enterotoxigenic E. coli for prevention of enterotoxigenic colibacillosis of calves Passive immunity in calf diarrhea: vaccination with K99 antigen of enterotoxigenic Escherichia coli and rotavirus Improved Minca medium for the detection of K99 antigen in calf enterotoxigenic strains of Escherichia coli Escherichia coli isolated from calves with diarrhoea: mannose resistant haemagglutination and colonisation factor Attachment des Escherichia coli ent6ropathog6nes la muquese intestinale. Gastro-ent6rites n6onatales du veau Adhesion of enteropathogenic Escherichia coli to pig intestinal Brush Border: the existence of two pig phenotypes Quantitation of small intestinal structure and function in unthrifty piglets Specific protection by colostrum from cows vaccinated with the K99 antigen in newborn calves experimentally infected with E. coli ENT + K99 + Vaccination of cows with a K99 extract to protect newborn calves against experimental enterotoxic colibacillosis Immunization of suckling pigs against enterotoxigenic Escherichia coli induced diarrheal disease by vaccinating dams with purified 987 or K99 pili; protection correlates with pilus homology of vaccine and challenge Protection of lambs against enteric colibacillosis by vaccination of ewes Passive protection of lambs against experimental enteric colibacillosis by colostrum transfer of antibodies from K99 vaccinated ewes Antibody response of cows to Escherichia coli pilus antigen K99 oral vaccination with live or dead bacteria Enteric colibacillosis in the calves: immunogenicity and antigenicity of Escherichia coli antigens Serological comparison of the Escherichia eoli prototype strains for the F (Y) and Art 25 adhesins implicated in neonatal diarrhoea in calves Colibacilles ent6rotoxinog6nes du veau poss6dant un antigone d'attachement diff6rent de l'antig6ne K99 Antibacterial activity in colostrum and milk associated with protection of piglets against enteric disease caused by K88 positive Escherichia coli