Endotoxemia in neonatal calves given antiserum to a mutant Escherichia coli (J-5)

Endotoxemia was characterized in neonatal calves given a small amount of colostrum and smooth Escherichia coli endotoxin by small-dosage (0.5 microgram/kg of body weight), slow (5-hour) IV infusion to mimic natural conditions. Responses were compared among 22 calves freely allotted to groups treated with saline solution (group I), preimmunization plasma (PP, group II), or antiserum to the rough mutant of E coli O111:B4 (J-5, group III) before endotoxin was infused. Bovine J-5 antiserum was produced by immunization of 4 cattle with J-5 boiled cell bacterin. The antiserum titers of immunoglobulin (Ig) M, IgG1, and IgG2 to the J-5 boiled cells, as determined by enzyme-linked immunosorbent assay, were 240, 7,680, and 960, respectively. The PP had enzyme-linked immunosorbent assay titers to J-5 of 240, 480, and 60 of IgM, IgG1, and IgG2, respectively. Endotoxemia in the 3 groups was characterized by significant (P less than 0.05) time-related changes in rectal temperature, heart rate, respiratory rate, capillary refill time, oral mucous membranes, nose moistness, scleral injection, attitude, PCV, total plasma protein concentration, WBC count and differential, plasma glucose, and lactate concentrations. The only significant treatment effects on clinical or laboratory values were higher mean total plasma protein concentrations in groups II and III 10 to 30 hours after endotoxin infusion was started than that in group I and increasing mean most-severe attitude abnormality score in groups I, III, and II (P less than 0.05). The administration of bovine J-5 antiserum to neonatal calves resulted in significantly higher serum IgG1 and IgG2 titers to J-5 boiled cells (P less than 0.05), and cross-reactive IgG2 to the challenge endotoxin (P less than 0.01) than did treatment with PP or saline solution; however, this antiserum did not mitigate the effects of sublethal endotoxemia. There was a significant negative correlation between IgG2 to J-5 at base line and the mean attitude abnormality score at 4.5 hours after infusion was started (P less than 0.05).     

Occurrence of enterotoxigenic Escherichia coli in calves with acute neonatal diarrhoea

Enterotoxigenic Escherichia coli (ETEC) were isolated from 16.4% of diarrheic calves of up to 30 days old. Of 1-2-day-old calves nearly one half (45.0%) harboured ETEC, while this was the case with only 4.9% of 8-day-old calves. Among 206 9-30-day-old calves just three were found to harbour ETEC.     

Availability of oral carbohydrates to neonatal calves.

Four experiments were conducted to determine the availability of 3 carbohydrate sources given orally to 1- to 3-week-old clinically normal calves. In terms of effect on plasma glucose concentration, commercial fruit pectin was equivalent to glucose, corn syrup was less effective (P less than 0.05) than glucose, and sucrose was totally unavailable. The addition of 80 mM NaCl to a 10% glucose solution enhanced glucose absorption (P less than 0.01). Increasing the glucose concentration of a commercial product from 1% to 3% resulted in a significant (P less than 0.01) increase in plasma glucose concentration. Lactose availability from suckled fluids was greater (P less than 0.01) than that from fluids given by stomach tube.     

Tolerance of a rice-based oral rehydration solution given to normal calves

The objective of the study was to test the tolerance of a rice-based oral rehydration formula when fed to calves. Six healthy Holstein calves, 1 week of age, were fed the formula instead of milk replacer for 3 days. Pre- and posttreatment results of clinical examination and laboratory parameters were compared. Vital signs, attitude, appetite, clinical hydration status, urine specific gravity, and most routine serum biochemistry test results did not vary and remained within the normal range. Five of the 6 calves developed diarrhea when fed the rice-based formula, which was accompanied by a reduction in fecal pH and presence of reducing sugars in the feces. This effect was reversed when calves were returned to the milk replacer diet at the end of the study. Diarrhea was accompanied by increased water consumption, which allowed the calves to maintain normal hydration status. These results suggest that calves are unable to properly digest the rice-derived carbohydrate, and this type of formula is not recommended for oral rehydration of calves.     

Effect of combined rotavirus and Escherichia coli in neonatal gnotobiotic calves

Gnotobiotic calves (24 hours old) were monoinfected with calf rotavirus (CRV) strain NCDV, an enterotoxigenic Escherichia coli (ETEC) strain B44 (K99+), or a nonenterotoxigenic E coli (NETEC) strain 123 (K99-). Calves also were dually infected with CRV and either ETEC or NETEC. Eighteen calves equally allotted between 6 treatment groups were used in these studies: Noninfected controls--group A; CRV--group B; ETEC--group C; NETEC--group D; CRV + ETEC--group E; and CRV + NETEC--group F. Severe diarrhea and villous atrophy were observed in calves of treatment groups B, C, E, and F. Mortality was present only in treatment groups C and E as result of ETEC infection. There were no significant differences in the clinical responses or enteric lesions between treatments B and F, although a significant increase in the concentrations of NETEC was demonstrated in calves dually infected with CRV + NETEC (group F) as compared with calves monoinfected with NETEC (group D). Calves inoculated with ETEC (group C) had severe villous atrophy, neutrophilic infiltration of intestinal lumen, and moderate enterocyte necrosis. Calves dually inoculated with CRV + ETEC (group E) had the most extensive and severe lesions, similar to those in group C, plus a pronounced necrotic fibrino-hemorrhagic enteritis. Infection of enterocytes by CRV did not affect in any way the adherence of ETEC to the intestinal mucosa. Dual viral and bacterial infections of the same enterocytes were evident.     

Passive transfer of antibodies to Shiga toxin-producing Escherichia coli O26, O111 and O157 antigens in neonatal calves by feeding colostrum

To study whether or not passive immunity of neonatal calves against Shiga toxin-producing Escherichia coli (STEC) O26, O111, and O157 was obtained by colostrum administration, serum antibodies in calves after the feeding were determined by enzyme-linked immunosorbent assay (ELISA) in comparison with antibodies in colostrum and sera from donor dams. The highest antibody titers to STEC in colostrum from dams were detected soon after parturition. The antibody titers were found to be elevated in sera of neonatal calves (4-9 hr after birth) orally administered with colostrum with high antibody titers, suggesting that passive immunity of neonatal calves to STEC infection may be obtained by feeding colostrum. These results suggest that colostrum administration to neonatal calves may play an important role in elevating serum antibodies against STEC in neonatal calves.     

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsa-stained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a co-agglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.     

Weaned pig responses to Escherichia coli K88 oral challenge when receiving a lysozyme supplement

Lysozyme is a low-molecular-weight protein with antimicrobial properties. An experiment was conducted to investigate the response of piglets receiving a water-soluble lysozyme supplement [Entegard (EG), Neova Technologies Inc., Abbotsford, British Columbia, Canada; 4,000 lysozyme units/mg] after oral challenge with enterotoxigenic Escherichia coli (ETEC). A total of 36 individually housed weanling pigs were randomly allotted to 1 of the 4 treatments, with 9 replicates per treatment. Treatments were a control (CONT, no additive), antibiotic (AB; 2.5 g/kg of feed of antibiotic with chlortetracycline, sulfamethazine, and penicillin), and EG delivered in the drinking water at concentrations of 0.1% (EG1) and 0.2% (EG2). All pigs received a basal diet similar in composition and nutrients, except for pigs receiving the AB diet, which had an added antibiotic. Pigs were acclimated to treatments for a 7-d period to monitor growth performance. On d 8, blood samples were collected from each pig to obtain serum, and each pig was gavaged with 6 mL (2 × 10(9) cfu/mL) of ETEC solution. Pigs were monitored for another 7 d to assess incidences of diarrhea and growth performance, and then all pigs were killed to obtain intestinal tissue and digesta samples. Treatments did not influence growth performance throughout the study. Greater ETEC counts were observed in the ileal mucosal scrapings (P = 0.001) and colonic digesta (P = 0.025) of pigs in the CONT group compared with pigs in the AB and EG1 groups. Pigs receiving AB and EG1 had greater (P < 0.05) small intestinal weights and ileal villus heights than pigs receiving CONT; however, the ileal villus height-to-crypt depth ratio was greater in pigs fed the AB diet (1.69) compared with those fed the CONT diet (1.34), whereas pigs receiving EG1 were intermediate. Pigs in the EG1 group showed greater (P < 0.001) serum tumor necrosis factor α and IL-6 concentrations before ETEC challenge; however, at 7 d postchallenge, pigs receiving EG2 showed the least (P < 0.05) circulating tumor necrosis factor α and IL-6 concentrations. Overall, better intestinal growth and development, as well as decreased ETEC counts on the intestinal mucosa and serum proinflammatory cytokines, suggest that EG can maintain gut health and function in piglets commensurate with antibiotics. However, it is noteworthy that at the largest dose tested, EG seemed to have a dramatic effect on proinflammatory cytokines but had a minimal or no effect on the other response criteria.     

Pathotyping and antimicrobial susceptibility testing of Escherichia coli isolates from neonatal calves

Neonatal calf mortality is a major concern to livestock sector worldwide. Neonatal calf diarrhoea (NCD), an acute severe condition causes morbidity and mortality in calves. Amongst various pathogens involved in NCD, E. coli is considered as one of the major causes. The study was targeted to characterize E. coli isolates from neonatal calves for diarrhoeagenic Escherichia coli (DEC) types (pathotyping), antimicrobial resistance (AMR) profiling and to correlate with epidemiological parameters. From neonates, a total of 113 faecal samples were collected, out of that 308, lactose fermenting colonies were confirmed as E. coli. Pathotypable isolates (12.3%) were represented by STEC (6.1%), EPEC (2.9%), ETEC (1.9%), EAEC (0.9%) and EHEC (0.3%). Occurrence of STEC was more in non-diarrhoeic calves, whereas ETEC was observed more in diarrhoeic calves. EPEC occurrence was observed in both diarrhoeic and non-diarrhoeic calves. Fishers extract test showed no significant association for occurrence of DEC types to type of dairies, health status, species, breed, age and sex of neonatal calves. Two hundred and eighty isolates were tested for antimicrobial susceptibility. The isolates showed maximum resistance towards ampicillin (55.4%) followed by tetracycline (54.3%), while minimum resistance was observed towards meropenem (2.5%). Multidrug resistant E. coli isolates were found to be 139 (49.6%), and Extended-spectrum beta-lactamase (ESBL) producers were 120 (42.9%). DEC pathotypes like STEC, ETEC, EHEC and EAEC that are also multidrug resistant present in neonatal calves have zoonotic potential and hence are of public health significance.

     

Vaccination of pregnant cows with K99 antigen of enterotoxigenic Escherichia coli and protection by colostrum in newborn calves

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.     

Effect of maternal cells transferred with colostrum on cellular responses to pathogen antigens in neonatal calves

OBJECTIVE: To assess the effect of maternal cells or cellular components on neonatal immune responses to intracellular pathogens in calves. ANIMALS: 15 Holstein calves. PROCEDURES: Calves were fed whole colostrum, frozen colostrum, or cell-free colostrum within 4 hours after birth. Leukocytes were obtained from calves before feeding colostrum and 1, 2, 7, 14, 21, and 28 days after ingestion. Proliferative responses against bovine viral diarrhea virus (BVDV) and mycobacterial purified protein derivatives were evaluated. Dams received a vaccine containing inactivated BVDV, but were not vaccinated against mycobacterial antigens. RESULTS: All calves had essentially no IgG in circulation at birth, but comparable and substantial concentrations by day 1. Calves that received whole colostrum had enhanced responses to BVDV antigen 1 and 2 days after ingestion of colostrum. In contrast, calves that received frozen colostrum or cell-free colostrum did not respond to BVDV. No differences were identified among the 3 groups in response to mycobacterial antigens. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that transfer of live maternal cells from colostrum to neonatal calves enhanced responses to antigens against which the dams had previously responded (BVDV), but not to antigens to which the dams were na?ve (mycobacterial purified protein derivatives). Results suggested that cell-mediated immune transfer to neonates can be enhanced by maternal vaccination.     

Virulence factors in Escherichia coli isolated from the blood of bacteremic neonatal calves

Twenty-five Escherichia coli isolates from the blood of critically ill bacteremic calves sampled in two separate studies on a calf-rearing farm housing over 15,000 calves, in the San Joaquin Valley, California were studied.Isolates were characterized for O serogroups and for pathotypes as determined by the presence of specific virulence factors including heat-labile enterotoxin (LT), heat-stable enterotoxins a and b (STa, STb), verotoxins 1 and 2 (VT1, VT2), cytotoxic necrotizing factor (CNF), aerobactin, intimin Eae and P, F17 and CS31A fimbrial adhesins, and resistance to bactericidal effects of serum.These isolates constituted a heterogeneous group. However, isolates were mostly aerobactin positive and often resistant to the bactericidal effects of serum. Isolates of pathotypes O78 (n=6), O119:CS31a (n=3), and P positive but O non-typeable (n=3) were associated with a high mortality rate. The remaining isolates belonged to diverse pathotypes, often possessing the adhesins P, F17, CS31A and Eae but belonging to O serogroups other than O78 and O119, and were less frequently associated with mortality.Although no virulence factor common to all isolates was identified, the capacity to use iron by the presence of aerobactin which is important to the capture of iron was a predominant factor. Moreover, certain pathotypes appear to be associated with primary colisepticemia whereas other pathotypes may cause a bacteremia without necessarily leading to septicemia.     

The role of oral immunisation in stimulating Escherichia coli antibody of the IgM class in porcine colostrum.

Oral immunisation with Escherichia coli polysaccharide antigens provided a primary antigenic stimulus which facilitated the production of humoral IgM antibody following a single parenteral antigen dose. The peak antibody response of preparturient sows was manipulated to coincide with the period of colostrum formation so that high levels of IgM antibody were made available for neonatal defence. The characteristics of the immune response remained unchanged on reintroduction of the immunisation schedule for a second gestation period.     

Protection of neonatal chicks against a lethal challenge of Escherichia coli using DNA containing cytosine-phosphodiester-guanine motifs

Oligodeoxynucleotides (ODN) containing cytosine-phosphodiester-guanine (CpG) motifs have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. We recently have shown that CpG ODN protects against extracellular bacterial infections in mature chickens. The objective of this study was to investigate the effect of CpG ODN on Escherichia coli septicemia in neonatal broiler chicks. Two-day-old chicks, or embryonated eggs that had been incubated for 18 or 19 days, received 50 microg CpG ODN. Three days after exposure to CpG ODN, a virulent isolate of E. coli was inoculated subcutaneously in the neck of each bird. Birds were examined for 7 days post-E. coli challenge and dinical, pathologic, and bacteriologic assessments were conducted. The control group of birds that received no CpG ODN had a survival rate of 0% to 20%. In contrast, groups that received CpG ODN, either by intramuscular or in ovo routes, had significantly higher survival rates (P < 0.0001). Bacterial counts in air sacs were significantly lower when birds or embryos were treated with CpG ODN as compared with controls. A dose as low as 10 microg of CpG ODN, administered intramuscularly, was able to protect birds significantly against E. coli challenge. Formulation of CpG ODN with 30% Emulsigen did not enhance the protection. This study demonstrates that CpG ODN has systemic protective effects in broiler chicks against E. coli infections. This is the first time that CpG ODN has been demonstrated to have an immunoprotective effect against a bacterial infection in chicks following in ovo delivery.