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.     

Pathogenicity characterization of a bovine triple reassortant rotavirus in calves and piglets

Rotaviruses are important human and animal pathogens with high impact on public health and livestock industry. There is little evidence about the cross-species pathogenicity and extra-intestinal infections of animal and human reassortant rotaviruses, particularly based on all 11 genotyping data. In this study, the bovine triple reassortant KJ56-1 strain harboring two bovine-like genome segments, eight porcine-like genome segments, and one human-like genome segment was used to evaluate the cross-species pathogenicity in its parent species, calves and piglets, and to determine its abilities of causing viremia and extra-intestinal tropisms in piglets. The KJ56-1 strain isolated from a calf diarrhea fecal sample replicated without causing diarrhea and severe intestinal pathology in calves. However, piglets inoculated with this strain showed persistent severe diarrhea and marked intestinal pathology. By SYBR Green real-time RT-PCR, viral RNA was detected in the sera, mesenteric lymph node, lung, liver, choroid plexus, and cerebrospinal fluid in the experimental piglets. An immunofluorescence assay confirmed viral replication in these extra-intestinal organs and tissues. These results indicated that the bovine triple reassortant KJ56-1 strain was virulent to piglets but not to calves. Our data also demonstrated that the reassortant rotaviruses had the ability to spread to the bloodstream from the gut, enter and amplify in the mesenteric lymph node, and disseminate to the extra-intestinal organs and tissues.     

Acute undifferentiated neonatal diarrhea in beef calves. I. Occurence and distribution of infectious agents.

Beef calves in a 48-cow herd were studied during one calving season from birth to ten days of age to determine the presence or absence of potentially enteropathogenic bacteria, viruses, and/or chlamydia in both normal and diarrheic calves. Calves were born and raised outside in large pens unless the ambient temperature was below minus 10 degrees F when calving was done inside. Fecal swabs, fecal aliquots, and nasal swabs were taken from each calf at 32, 128 plus or minus 3, and 248 plus or minus 3 hours of age and as soon after the onset of diarrhea as possible. Diarrhea was defined as that condition in which the feces contained less than 10% dry matter. Enteropathogenic Escherichia coli in feces were identified using the ligated gut loop procedure in calves and by feeding broth cultures to colostrum fed lambs seven to 16 hours old. Potentially enteropathogenic viruses were detected using a variety of methods which included tissue culture, fluorescent antibody, hemadsorption, and electron microscope techniques. Of the 40 calves studied, 32 (80%) developed diarrhea before ten days of age. Twenty-two strains of Escherichia coli which caused dilation of calf ligated intestinal loops were isolated from 11 scouring calves and from one normal calf. Nine out of ten strains of Escherichia coli which dilated ligated loops also caused diarrhea when fed to colostrum-fed lambs seven to 16 hours old. Using antibody technique a Reo-like virus was detected in the feces of 15 calves before, during, and after the onset of diarrhea. Four calves excreted both loop dilating strains of E. coli and Reo-like virus in the feces before ten days of age; in all cases the loop dilating E. coli were isolated from the feces prior to the demonstration of Reo-like virus. A Corona-like virus was also demonstrated in three of the 15 calves infected with Reo-like virus and a noncytopathogenic strain of bovine virus diarrhea virus was isolated from two of the 15 calves infected with Reo-like virus. A loop dilating strain of Citrobacter was isolated from one diarrheic calf. There was no consistent pattern of onset or duration of diarrhea in calves which excreted different infectious agents. Salmonella species, infectious bovine rhinotracheitis virus, parvovirus, adenoviruses, parainfluenza-3 virus, and Chlamydia species could not be demonstrated in any of the calves or their dams. No potentially enteropathogenic agents could be demonstrated in 11 of the 32 calves which scoured. These findings emphasize the complexity of the infectious aspect of the neonatal diarrhea syndrome and illustrate the difficulty in making an etiological diagnosis in field outbreaks of the calf scours complex.     

Coronavirus as an agent of neonatal calf diarrhea in a Chinese dairy cattle farm.

Neonatal calf diarrhea is a significant health problem in dairy industry in the whole world. Although the aetiological agents of neonatal diarrhea are diverse, it is obvious that viruses play an important role. One of them is bovine coronavirus (BCV). Since BCV was discovered (MEBUS et al., 1969), there have been many published reports describing the virus and its importance in calves. In China, we first investigated the seroepidemiological status of BCV infections in cattle (YAO et al., 1990). The results showed that the incidence of infections with BCV in cattle in different chinese regions are high. Further work on direct diagnosis of BCV as a causative agent of diarrhea was therefore necessary. In the last years we have developed diagnostic methods including a monoclonal antibody-based ELISA to detect BCV and other diarrheal viruses. In this study, the diagnosis of an outbreak of BCV in chinese dairy cattle is described.     

Porcine group C rotavirus as a cause of neonatal diarrhea in a Quebec swine herd.

A porcine group C rotavirus was found to be the unique cause of a problem of enzootic neonatal diarrhea in a minimal disease herd composed of 190 sows on a continuous farrowing program. During the outbreaks of diarrhea, 10 to 80% of the litters were affected with a morbidity rate of 100% and case fatality rates of 5 to 10%. Clinical signs began 24 to 48 h after birth and were characterized by a profuse yellow diarrhea lasting a few days. Piglets from different outbreaks of diarrhea were necropsied. They had multifocal villous atrophy in the small intestine, especially in the ileum. Group C rotavirus was demonstrated by direct immunofluorescent staining of frozen intestinal sections and by polyacrylamide gel electrophoresis of viral RNA extracted from the intestinal contents of diarrheic piglets. The infection with clinical illness and lesions was reproduced experimentally in newborn piglets by oral inoculation of a suspension prepared from a pool of intestinal contents from diarrheic piglets.     

Diagnostic investigation of bovine viral diarrhea infection in a Minnesota dairy herd

Bovine viral diarrhea (BVD) virus infection was diagnosed in neonatal calves with enteritis. Successful diagnostic procedures included direct immunofluorescence of frozen tissue sections, histopathology, and virus isolation. Virus isolation from buffy coats and serum was successful in detecting infected animals, whereas direct immunofluorescence of buffy coat samples was found to be less reliable. Virus was not isolated from any fecal samples. Booster vaccinations and the culling of animals shedding virus resulted in improved calf viability in this herd. It is suggested that procedures for the diagnosis of BVD virus infection should always be included in the diagnosis of neonatal calf enteritis.     

Neonatal calf diarrhea caused by a virus that induces villous epithelial cell syncytia.

Intestinal lesions caused by a virus serologically unrelated to the calf diarrheal rotavirus or coronavirus were studied in gnotobiotic calves. The virion purified from feces from infected calves was a fringed particle with a diameter of about 100 nm. The incubation period from time of inoculation per orum to onset of diarrhea in calves was as short as 8 hours. The viral infection in bacteria-free calves or calves not contaminated with pathogenic bacteria caused severe illness for only 24 hours. When bacteria such as the K99 antigen Escherichia coli were present, the combined infection caused mortality. Lesions occurred only in the small intestinal villous epithelium. Calves euthanatized shortly before or after the onset of diarrhea had developed villous epithelial cell syncytia that contained numberous virions in the cytoplasm. Within 2 to 3 hours after onset of diarrhea, the infected cells were shed and the villi had denuded tips or had cuboidal to squamous epithelial cells.     

Effect of infection with bovine viral diarrhea virus alone,bovine rotavirus alone,or concurrent infection with both on enteric disease in gnotobiotic neonatal calves

OBJECTIVE: To compare experimentally induced concurrent infection with bovine viral diarrhea virus (BVDV) and bovine rotavirus (BRV) with infection of either virus alone in calves. ANIMALS: Seventeen 1-day-old gnotobiotic calves. PROCEDURE: Calves were allotted to 8 treatments as follows: group 1, mock-infected control calves (n = 2); group 2, inoculated with BVDV on day 1 (2); groups 3, 5, and 7, inoculated with BRV on days 1 (2), 4 (1), or 7 (2), respectively; and groups 4, 6, and 8, inoculated with BVDV on day 1 and with BRV on days 1 (2), 4 (2), or 7 (4), respectively. Concentrations of BVDV in serum and ileal tissues were measured, and BRV shedding in feces was determined. Histologic examination and immunohistochemical analysis were conducted to detect lesions and viral antigens. RESULTS: Neonatal calves inoculated with BVDV alone or with BVDV on day 1 and BRV on day 7 developed villus atrophy and submucosal inflammation of the intestines. Concurrent BVDV and BRV infections acted synergistically in the intestinal tract, causing more severe enteric disease than infection with either virus alone. Severe lymphoid depletion was associated with BVDV infection in calves regardlesss of concurrent BRV infection. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with BVDV played direct and indirect roles in enteritis in neonatal calves, causing villus atrophy in the duodenum and submucosal inflammation of the intestines. Also, BVDV potentiated effects of BRV. Concurrent infection with BVDV and BRV resulted in more severe enteric disease in neonatal calves than infection with BRV or BVDV alone.     

Coronaviruses associated with outbreaks of transmissible enteritis of turkeys in Quebec: hemagglutination properties and cell cultivation

Coronaviruses were observed by electron microscopy in the intestinal contents of turkeys in Quebec flocks where repeated outbreaks of enteritis occurred. Three isolates could be serially propagated in turkey embryos inoculated by the amniotic route with clarified intestinal contents. Purification and concentration of viral particles contained in intestinal contents of infected embryos were achieved by precipitation with polyethylene glycol and ultracentrifugation on sucrose density gradients. Three particle types were demonstrated: intact virions with a density of 1.18 to 1.20 g/ml and incomplete particles with densities of 1.14 and 1.24 g/ml. Hemagglutination of rabbit and guinea pig erythrocytes was demonstrated with the intact viral particles; the hemagglutinin was not dependent on incubation temperature. All the isolates were antigenically related, as shown by hemagglutination-inhibition. The turkey coronaviruses did not cross-react with antisera against coronaviruses of avian infectious bronchitis, porcine transmissible enteritis, bovine neonatal calf diarrhea, or mouse hepatitis. One of the Quebec isolates was shown to induce syncytia formation on its third passage in primary chicken-embryo kidney cell cultures. Electron-microscopic examination of infected cell-culture fluids revealed characteristics coronavirus particles identical to those found in intestinal contents of infected turkeys.     

Identification of a bovine enteric syncytial virus as a nongroup A rotavirus

An atypical or nongroup A rotavirus was identified in feces obtained from gnotobiotic calves in which fecal preparations originally derived during an epizootic of neonatal calf diarrhea had been serially passaged. The epizootic was previously reported to be caused by a noncharacterized viral agent that induced the formation of epithelial syncytia on small intestinal villi of experimentally infected calves. This bovine, nongroup A rotavirus was found to be antigenically related to a described atypical rotavirus of rats by immunofluorescence and by enzyme immunoassay. Complementary DNA derived from the atypical rat rotavirus cross hybridized with RNA obtained from the bovine virus, but not with RNA extracted from group A rotaviruses. Complementary DNA derived from SA-11 group A rotavirus cross hybridized with other group A rotavirus RNA, but not with RNA obtained from either the rat or bovine nongroup A isolates. Additionally, another similar, if not identical, bovine atypical rotavirus was identified in a second epizootic of neonatal calf diarrhea that occurred several hundred kilometers and 8 months apart from the original epizootic.     

中西医结合治疗犊牛消化不良体会

犊牛消化不良是犊牛胃肠道发生机能紊乱,引起胃肠道粘膜卡他性炎症及消化功能障碍的一种以腹泻或排粪迟滞为特征的犊牛疾病。本病多发于10—20日龄犊牛,常因犊牛初生后管护不到位而引发。通过采用中西医结合的治疗方法,有效提高了治愈率,获得了满意的疗效。     

Viruses and virus-like particles detected during examination of feces from calves and piglets with diarrhea

A total of 763 fecal or intestinal samples from diarrheic calves and piglets were examined for viral content by immunofluorescence, electron microscopy or cell culture. Routine fluorescent antibody and cultural tests detected rotavirus (n=126), coronavirus (n=80) and bovine viral diarrhea virus (n=13). Electron microscopy detected rotaviruses (n=24) and coronaviruses (n=17) not identified by standard fluorescent antibody tests. Other viruses detected by electron microscopy included Breda virus-like particles (n=49), astroviruses (n=1), caliciviruses (n=1), rhabdoviruses (n=1), parvoviruses (n=2), enteroviruses (n=3), togavirus-like particles (n=2), and “chained” particles (n=5). Mixtures of several of the viruses were detected in a number of fecal samples.

The survey emphasized the value of electron microscopy as a broad-spectrum diagnostic tool.

     

Lactobacillus GG does not affect D-lactic acidosis in diarrheic calves, in a clinical setting

D-lactate, produced by gastrointestinal fermentation, is a major contributor to metabolic acidosis in diarrheic calves. Lactobacillus rhamnosus GG survives gastrointestinal transit in the neonatal calf and does not produce D-lactate. To determine whether this probiotic reduces gastrointestinal D-lactate production or severity of diarrhea or both, 48 calves (mean, 11 days old; range, 2-30 days) admitted to the clinic for treatment of diarrhea were randomly allocated to 2 groups. The experimental group was given Lactobacillus rhamnosus GG (1 x 10(11) cfu/d) PO, dissolved in milk or oral electrolyte solution, in addition to clinic treatment protocols; the other group served as a control. Serum and fecal samples were obtained at admission and at 24 and 48 hours after initial administration of Lactobacillus rhamnosus GG. All samples were analyzed for D- and L-lactate by using high-pressure liquid chromatography. Feces were also analyzed for pathogens, Lactobacillus rhamnosus GG recovery, and dry matter. D-lactic acidemia (>3 mmol/L) was present in 37/48 calves at admission. Lactobacillus rhamnosus GG was recovered in the feces of 13 experimental calves and 0 control calves 24 hours after administration. No difference in serum or fecal D- or L-lactate between the groups was detected at any time point. After therapy, D-lactic acidosis was absent at 48 hours in all but 1 calf. No relation between fecal pathogen (viral, bacterial, or protozoal) and degree of D-lactic acidosis was observed. The reduction in mortality and greater fecal dry matter in Lactobacillus rhamnosus GG-treated calves was not statistically significant.     

Scanning electron, light, and immunofluorescent microscopy of intestine of gnotobiotic calf infected with calf diarrheal coronavirus.

Intestinal lesions in 2 gnotobiotic calves given (oral inoculation) calf diarrheal coronavirus were studied by scanning electron, light, and immunofluorescent microscopy. The calves were euthanatized at 34 and 73 hours after the onset of diarrhea. Lesions in the small intestine were similar to those reported in animals affected with transmissible gastroenteritis of swine. Small intestinal villi were shortened, some adjacent villi were fused, and villous epithelium was composed of low cuboidal to squamous cells. In the ansa spiralis coli, there were atrophy of the colonic ridges and marked differences in length and spacing of the microvilli on individual epithelial cells.     

低聚果糖对早期断奶犊牛生长性能和血液理化指标及肠黏膜形态的影响

选用同一牛场的荷斯坦公犊牛25头,饲喂添加不同浓度低聚果糖(FOS)的日粮,研究其对早期断奶犊牛生长性能、血液理化指标及肠黏膜形态的影响。试验结果表明:日粮中添加FOS极显著提高犊牛的体重及日增重(P<0.01);各FOS组犊牛牛血清IgG、IgA含量肠绒毛高度、固有膜厚度、血清总蛋白、血清白蛋白显著高于对照组(P<0.05);但总胆固醇含量显著降低。说明FOS能提高犊牛的生长性能,能防止早期断奶犊牛肠道萎缩、维持肠道的正常形态结构,能影响犊牛代谢,提高犊牛免疫性能;缓解断奶对犊牛的应激。     

Malabsorption due to selected oral antibiotics

This article represents an overview of recent research conducted on antibiotic-induced malabsorption in calves. The authors feel strongly that this work identifies a serious and ill-defined problem in the management of neonatal calves. Too often the solution utilized by veterinarian and stockmen for controlling neonatal diarrhea has been to administer oral antibiotics. In many cases, this has been done on the basis of antibiotic sensitivity testing, an approach that seems appropriate. Unfortunately, little consideration has been given to the relative sensitivity of the neonatal intestinal mucosa, with its very rapid turnover, to the potentially detrimental effects of oral antimicrobial therapy. The data that we have collected over the past 3 years conclusively demonstrate that high levels of four commonly used oral antibiotics, especially neomycin and chloramphenicol but also tetracycline and ampicillin, can cause a malabsorption diarrhea in normal calves. This action is not due to viral agents or overgrowth of resistant microbes but is the result of direct modification of the intestinal mucosa. Extrapolation of these data to different dose levels may not be accurate. Oral antibiotics may be of value in treating neonatal enteritis. Conversely, there can be too much of a good thing, and many cases of chronic diarrhea following use of oral antibiotics may be the result of an overenthusiastic and prolonged dosage regimen. Be cautious!     

Plasma corticoid changes during diarrhea in neonatal calves.

An acute, and usually fatal, diarrhea was induced in 11 healthy neonatal dairy calves by oral administration of an enteropathogenic viral inoculum. Fluid and electrolyte therapy was administered to several affected calves. Plasma corticoid values were determined by radio-immunoassay and competitive protein-binding assay, after purification of the plasma by paper chromatography. Statistical comparisons between control and diarrheic groups of calves were made on 5 corresponding experimental days. Plasma aldosterone values of 2 groups of diarrheic calves which died with and without treatment were greater (P less than 0.01) just before death than those of controls and diarrheic calves which recovered. Plasma hydrocortisone and progesterone values in the calves not treated were higher (P less than 0.05) on the day preceding death than in the controls. Plasma corticosterone concentration was increased (P less than 0.05) throughout the disease in both groups of calves that recoverd. These results indicate that the adrenal cortex of the neonatal calf can respond to the severe stressor influences and fluid-electrolyte losses of diarrhea.     

A Case of Viral Neonatal Calf Diarrhea in a Québec Dairy Herd

This report is concerned with a consistent problem of neonatal calf diarrhea (NCD) in a dairy herd in which, for nearly two years, the morbidity had approached 100% and the mortality had varied from 20% to 45%. Generally, diarrhea appeared at three days of age. By the fluorescent antibody tissue section technique the two Nebraska NCD viruses (reo-like and corona-like) were detected in the cytoplasm of many absorptive cells of the small intestine from a calf submitted for necropsy. Reo-like virus antigen was not detected in the absorptive and crypt cells of the colon but coronavirus-like antigen was. An adenovirus was also isolated from the small intestine of this calf. The disease was reproduced experimentally in a two day old colostrum deprived calf with a bacteria free intestinal homogenate obtained from the naturally infected calf. Both Nebraska NCD viruses were demonstrated in this experimental animal. However, the adenovirus was not re-isolated. Histological lesions observed in the small and large intestines of the naturally and experimentally infected calves were similar and because of their good correlation with the immunofluorescent findings, a combination of the two Nebraska NCD viruses was thought to be a major cause of the neonatal calf diarrhea problem afflicting this dairy herd.     

Negative contrast electron microscopic diagnosis of viruses of neonatal calf diarrhea.

Ninety-one cases of neonatal calf diarrhea were examined for viruses with negative contrast electron microscopy. Viruses were demonstrated in 41% of the cases. Reo-like viruses and corona-like viruses, and mixed virus populations were observed in 12%, 20% and 9%, respectively. Twenty-six of the cases were examined by negative contrast electron microscopy, and by virus isolation or by fluorescent antibody technique. There was an 81% agreement in obtained results. The disagreements resulted from the demonstration of a viral agent by negative contrast electron microscopy while the other techniques did not indicate a virus. The results suggest that negative contrast electron microscopy is a more sensitive diagnostic tool for demonstration of viruses associated with neonatal calf diarrhea than are viral isolation or the fluorescent antibody technique.     

Bovine Viral Diarrhea Virus and Escherichia Coli in Neonatal Calf Enteritis

This study was initiated to determine the etiologic and pathogenic significance of an American strain of bovine viral diarrhea (BVD) virus (strain NADL-MD) in enteritis of neonatal calves (calf scours).

Three colostrum-fed calves from dams exposed intravenously to BVD virus at 6, 16 and 25 days prepartum, respectively, had moderate diarrhea persisting until the eighth day of life. The BVD virus was isolated from all 3 calves and persisted up to 93 days in 1 calf, indicating either that BVD was transmitted in utero or via the dam's milk.

Three specific pathogen free (SPF) calves permitted dams' colostrum for the first 4 feedings and then given milk replacer were exposed orally on the day of birth to BVD virus. One calf died of neonatal enteritis 28 hours post-exposure and at necropsy the BVD virus was isolated from several of its organs. The remaining 2 calves had a mild diarrhea persisting to the eighth day of age.

Two calves permitted dams' colostrum ad lib. for 72 hours, and then weaned, were exposed orally to BVD virus. Both calves had a mild persistent diarrhea and BVD virus was isolated from their blood for 56 days post-exposure.

Of 13 SPF, colostrum-deprived calves exposed orally or intranasally at birth to the BVD virus, 4 had severe diarrhea and died of neonatal enteritis from 38 hours to 13 days postexposure. Isolations of BVD virus were made from several of the organs of the calves at necropsy. All of the 9 surviving calves had a moderate to severe diarrhea frequently persisting for 7 to 10 days, and BVD virus was isolated from the survivors up to 103 days postexposure.

Several strains of Escherichia coli were isolated from calves after the second day of life, but were neither pathogenic for mice, nor serologically related to strains of E. coli usually associated with outbreaks of calf scours. Four colostrum-deprived SPF calves were exposed orally at birth to a strain of E. coli isolated from the intestine of the calf with the most acute symptoms and fatal neonatal enteritis. None of the four calves receiving the E. coli had diarrhea. One calf, however, had respiratory distress and died on day 5.

Two SPF colostrum-deprived control calves had neither diarrhea nor respiratory distress.

The above findings support the conclusion that BVD virus should not be overlooked as a primary cause of the neonatal calf enteritis complex.