Isolation of bovine coronavirus from feces and nasal swabs of calves with diarrhea.

Fecal and nasal samples were collected from 180 calves with diarrhea and 36 clinically normal co-habitants, and tested for virus using HRT-18 cell cultures derived from human rectal adenocarcinoma. A cytopathic virus was isolated from 5 fecal and 56 nasal samples obtained from diarrheic calves. All calves in which the virus was isolated from diarrheic feces were positive for virus isolation from nasal swabs. The virus was also isolated from the nasal swabs of 10 clinically normal calves that were co-habitants with diarrheic calves. Because they were morphologically similar to coronavirus, agglutinated mouse erythrocytes and serologically identical with the Nebraska calf diarrhea coronavirus, new isolates were identified as bovine coronavirus. The demonstration of viral antigens in nasal epithelial cells by a direct immunofluorescence was in close agreement with the virus isolation in HRT-18 cell cultures. This is the first report on the isolation of bovine coronavirus from newborn calves with diarrhea in Japan. The evidence that the virus was frequently isolated from nasal swabs is of great interest for understanding the pathogenesis of bovine coronavirus infection.     

Bovine Cryptosporidiosis: Clinical and Pathological Findings in Forty-two Scouring Neonatal Calves

Cryptosporidia organisms were identified in 42 of 161 (26%) neonatal, diarrheic calves, over a 32 month period commencing July 1979. Forty of the 161 calves were submitted alive and cryptosporidiosis was diagnosed in 63% (25 of 40) of them. The cryptosporidia infected calves were usually one to two weeks old and came from 26 herds where the typical history was profuse, watery diarrhea in nearly all neonatal calves. The diarrhea usually started around one week of age, was unresponsive to all conventional antidiarrhea therapies, lasted for two or more weeks and was usually fatal. Twenty-nine (69%) of the cryptosporidia infected calves were submitted between December and February. These calves were often hutch reared.

Histopatholoical examination revealed large numbers of the coccidial parasite Cryptosporidium sp embedded in the microvilli of jejunal and ileal absorptive enterocytes of all affected calves. The organisms were identified as trophozoites and schizonts (asexual stages) and macrogametes (female sexual stages) with the electron microscope. Microgametes (male sexual stages) were not identified. Occasionally a merozoite (asexual stage) was also seen apparently burrowing into or about to be enveloped by a host microvillus. Observation of the organisms was much easier when diarrheic calves were submitted alive. Enterotoxigenic Escherichia coli were often cultured from intestines of dead calves and occasionally from calves submitted alive. Coronavirus particles were seen in one calf. In the last year of this study, oocysts were identified in fecal smears stained with May-Grünwald-Giemsa stain and fecal samples using a dichromate solution flotation technique.

     

Certain enteropathogens in calves of Finnish dairy herds with recurrent outbreaks of diarrhea

The occurrence and distribution of certain enteropathogenic organisms and the manifestation of diarrhea, especially in relation to colostral management, were studied in neonatal calves of eight southern Finnish dairy herds with recurrent outbreaks of enteric disturbances. The fecal samples were obtained from the calves and were provided by the farmers. Potentially pathogenic agents were demonstrated in 52 of the 68 calves studied. Cryptosporidium was found in 36 of them while rotavirus was found in 29 and Escherichia coli in 15 of them, either singly or in combination. Salmonella, coronavirus or parvovirus were not detected in any of the calves. Twenty-six of the 36 calves contracted their Cryptosporidium infection and 18 of the 29 calves their rotavirus infection within the first week of life.

Eighteen of the 52 infected calves developed diarrhea of microbial origin within 20 days of age; the most frequent finding was mixed infection with Cryptosporidium and rotavirus. In addition there were six diarrheic calves where no infection could be demonstrated. Only 10 of the 68 calves had neither infections nor diarrhea. There was a highly significant difference between the mean times of the first colostral feeding of the uninfected non-diarrheic and diarrheic calves and a significant difference between the mean times of the first colostral feeding of the subclinically infected and diarrheic calves. The importance of sucking of colostrum at the first feeding was observed. The smallest amount protecting the calves from infections was 0.75 1 given within 1 h of delivery.     

Bovine cryptosporidiosis in Denmark. 2. Cryptosporidia associated with neonatal calf diarrhea

In recent years cryptosporidia have often been identified in diarrheic calves of under one month old, either as the sole pathogenic agent or in combination with other diarrhea-causing infectious agents. In 203 cases of bovine cryptosporidiosis, recorded over a period of two years, cryptosporidia were present in such great numbers that they would seem to be of causal significance. The calves were from three days to five weeks old. In 111 cases (55%) a monoinfection with cryptosporidia occurred, while in the rest of the cases mixed infections with other agents associated with calf diarrhea were demonstrated, especially rotavirus (61 cases) and coronavirus (32 cases); in addition, mixed infections with ETEC (four cases), septicaemic E. coli (two cases), salmonellosis (11 cases), and BVD and coccidiosis (one case each) were seen. In 87 (10%) of 849 4-21-day-old dead calves a massive occurrence of cryptosporidia was demonstrated. Extreme emaciation was noted in 36 of 122 autopsied calves. Most of these calves were more than 14 days old, and mono-infection with cryptosporidia was demonstrated in two thirds of them. In younger calves, more acute cases of cryptosporidiosis were commoner, often in connection with concomitant infections with rota- or coronavirus.     

D-lactate production and excretion in diarrheic calves

The origin of D-lactate, the most important acid contributing to metabolic acidosis in the diarrheic calf, is unknown. We hypothesized that because D-lactate is produced only by microbes, gastrointestinal fermentation is the source. The objective of this study was to determine whether D-lactate production occurs in the rumen, colon, or both, and to measure D- and L-lactate concentrations in urine. Fecal, rumen, blood, and urine samples were obtained from 16 diarrheic and 11 healthy calves. Serum electrolyte concentrations were measured in both groups, and blood gas analyses were performed for diarrheic calves. All samples were analyzed for D- and L-lactate by high performance liquid chromatography (HPLC). Diarrheic calves were generally hyperkalemic with high serum anion gap, depressed serum bicarbonate, and low blood pH. L-lactate was markedly higher in rumen contents (22.7 mmol/ L [median]) and feces (8.6 mmol/L) of diarrheic calves than healthy calves (0.5 mmol/L and 5.1 mmol/L, respectively), but not different in serum or urine. Rumen, fecal, serum, and urine D-lactate concentrations were all significantly higher (P < .05) in diarrheic calves (17.0, 25.4, 13.9, and 19.2 mmol/L, respectively) than in healthy calves (0.5, 9.1, 1.4, and 0.5 mmol/L, respectively). Higher D-lactate concentrations in the rumen and feces of diarrheic calves suggests these sites as the source of D-lactate in blood and urine.     

A seroepidemiological study of the importance in cow-calf pairs of respiratory and enteric viruses in beef operations from northwestern Quebec.

Serum antibody analyses for bovine herpesvirus type 1 (BHV-1), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCV), and bovine rotavirus (BRV) were performed on 527 randomly selected cows, before calving, and on 407 three-week-old calves. In cows and calves, BCV and BRV were the most seroprevalent viruses (80% to 100% according to virus and vaccination status). Bovine respiratory syncytial virus was the least seroprevalent in the cows, independent of the vaccination status. In nonvaccinated cows the seroprevalence to BRSV was 36.7%, and 53.5% in cows vaccinated less than two weeks prior to collecting blood, and 67.6% in cows vaccinated two weeks or more prior to blood collection. In their calves, BHV-1 was the least seroprevalent, independent of the vaccination status. The serological status and antibody titers in calves were generally associated with those of the dam. The occurrence of respiratory diseases in the calves was associated with cow and calf serological profiles (BHV-1, BRSV and BCV in the nonvaccinated group, BHV-1, BVDV and BCV in the vaccinated group). The occurrence of diarrhea was not associated with cow and calf serological profiles but was negatively associated with high level calf serum IgG in the nonvaccinated group (odds ratio = 0.73). Bovine coronavirus and BRV were shed by 1.4% and 4.9% of calves in the nonvaccinated group, and by 0% and 9.9% of calves in the vaccinated group, respectively. Bovine rotavirus shedding was associated with fecal diarrheic consistency at the moment of fecal sampling but not with previous occurrence of diarrhea.     

Molecular detection and characterization of unclassified bovine enteric caliciviruses in South Korea

The unclassified bovine enteric calicivirus (BEC) is a new bovine enteric calicivirus that is different from bovine norovirus, and causes diarrhea and pathologies in the small intestine of calves. This virus includes Nebraska (NB)- and Newbury agent 1 (NA1)-like strains. The prevalence of this BEC and its genetic characterization has only been reported in the UK and the USA. This study examined the prevalence and genetic diversity of these BECs in diarrheic calves in South Korea. Among a total of 645 diarrheic fecal specimens obtained from 629 cattle herds, these unclassified BECs were detected in 59 (9.1%) diarrheic fecal samples from 57 herds (9.3%) by either RT-PCR or nested PCR. Sequence and phylogenetic analyses of the partial RdRp gene showed that all the Korean BECs clustered together and were closely related to the NB-like viruses (80.9-88.1% nucleotide and 84.5-98.4% amino acid) but not to the NA1-like viruses (75.8-78.4% nucleotide and 79.7-82.8% amino acid). Although these viruses could not be classified into NA1- and NB-like viruses from the sequence and phylogenetic data of the entire capsid gene, all the Korean BECs clustered together on a branch separate from the other known BECs. These results show that these BEC infections are endemic in diarrheic calves in South Korea. The infecting strains are genetically closer to the NB-like viruses but have a distinct evolutionary pathway.     

A comparison of dichromate solution floatation and fecal smears for diagnosis of cryptosporidiosis in calves

Fecal samples from eleven calves experimentally infected with cryptosporidia were examined by two methods to evaluate their sensitivity and ease of use as diagnostic techniques. Comparison of a dichromate solution floatation and fecal smear techniques indicated that the former method was more sensitive. Oocysts were detected in feces earlier and for a longer period following exposure, and were easier to visualize using the dichromate solution floatation procedure. In addition, the dichromate solution floatation technique eliminated Candida albicans which can sometimes be confused with cryptosporidial oocysts in fecal smears. Another advantage was that oocysts could readily be detected in fecal samples stored as long as 120 days in dichromate solution. Using the dichromate solution floatation technique, it was shown that shedding of cryptosporidial oocysts in feces occurred from two to 20 days after oral challenge but was not always accompanied by diarrhea. Infection with cryptosporidia was confirmed in two calves at necropsy. Calves which were not sacrificed for portmortem examination recovered without treatment. The dichromate solution floatation technique is simple, rapid, inexpensive and should facilitate detection of cryptosporidia by diagnostic laboratories and some veterinary practitioners.     

Detection of rotavirus and coronavirus shedding in two beef cow herds in Idaho

Fecal samples were taken at the time of pregnancy examinations and at parturition from two beef herds. They were also taken from sick calves at the onset of disease, and from 25% of the healthy calves at 15 days of age. All fecal samples were examined by electron microscopy for viruses.

Four cows in herd A were detected excreting coronavirus, one at the time of the pregnancy examinations and three at parturition. The first cow was removed from the herd and the others calved at the end of the season. There were no sick calves.

No cows in herd B were detected excreting virus at the time of pregnancy checks, but fourteen coronavirus and two rotavirus carrier cows were found at parturition. All but two calves sampled had large numbers of virus particles in their feces. Clinical illness was associated with dams shedding virus and with nightly low temperatures.

     

Virus-like particles, 45 to 65 nm, in intestinal contents of neonatal calves.

Enveloped virus particles 45 to 65 nm in diameter, tentatively called minicorona virus, were detected by electron microscopy in the intestinal contents of one normal and seven diarrheic calves in Quebec dairy herds. The agent was shown to be antigenically unrelated to the Nebraska calf diarrhea coronavirus and to the bovine viral diarrhea virus by counterimmunoelectrophoresis and fluorescent-antibody techniques. Antibodies against these particles were demonstrated in the serum of affected calves using immunoelectron microscopy. The agent could not be isolated in cell cultures and its possible role as etiological agent in calf diarrhea is still to be determined.     

A case-control study of selected pathogens including verocytotoxigenic Escherichia coli in calf diarrhea on an Ontario veal farm.

A case-control study of diarrheal disease in veal calves was conducted over a three month period on a single large veal farm in southern Ontario. One hundred diarrheic calves (cases) were identified by visual examination of their feces. Each case was matched to two nondiarrhetic controls from the same room on the same day, and a fecal sample was obtained from each animal. Fecal consistency of cases and controls was observed daily for one week following sample collection. Control calves which developed diarrhea during that period were excluded from the study. Breed, sex and the date and nature of antimicrobial drugs administered to each calf were recorded. Moisture content of fecal samples was measured by weighing samples before and after oven drying. Samples were screened for verocytotoxigenic Escherichia coli (VTEC) using a Vero cell assay, for enterotoxigenic E. coli (ETEC) using an immunoblot procedure with anti-K99 monoclonal antibodies, and for Salmonella species using modified semi-solid Rappaport-Vassiliadis medium. A latex agglutination test was used to detect rotaviruses, and samples were examined for cryptosporidia using sucrose wet mounts. No VTEC were identified in cases or controls. One calf was positive for Salmonella and three were positive for ETEC. Rotaviruses were detected in four cases and four controls. A significant positive association was found between diarrhea and infection with Cryptosporidium. This study thus provided no evidence of an association between diarrhea and infection with either VTEC, ETEC, Salmonella spp. or rotaviruses in the population examined. On the other hand our results do suggest that Cryptosporidium infection may promote transient diarrheal disease in veal calves in Ontario.     

Development of nasal, fecal and serum isotype-specific antibodies in calves challenged with bovine coronavirus or rotavirus

Unsuckled specific pathogen free calves were inoculated at 3-4 weeks of age, either intranasally (IN) or orally (O) with bovine coronavirus or O plus IN (O/IN) or O with bovine rotavirus. Shedding of virus in nasal or fecal samples, and virus-infected nasal epithelial cells were detected using immunofluorescent staining (IF), ELISA or immune electron microscopy (IEM). Isotype-specific antibody titers in sera, nasal and fecal samples were determined by ELISA. Calves inoculated with coronavirus shed virus in feces and virus was detected in nasal epithelial cells. Nasal shedding persisted longer in IN-inoculated calves than in O-inoculated calves and longer than fecal shedding in both IN and O-inoculated calves. Diarrhea occurred in all calves, but there were no signs of respiratory disease. Calves inoculated with rotavirus had similar patterns of diarrhea and fecal shedding, but generally of shorter duration than in coronavirus-inoculated calves. No nasal shedding of rotavirus was detected. Peak IgM antibody responses, in most calves, were detected in fecal and nasal speciments at 7-10 days post-exposure (DPE), preceeding peak IgA responses which occurred at 10-14 DPE. The nasal antibody responses occurred in all virus-inoculated calves even in the absence of nasal shedding of virus in rotavirus-inoculated calves. Calves inoculated with coronavirus had higher titers of IgM and IgA antibodies in fecal and nasal samples than rotavirus-inoculated calves. In most inoculated calves, maximal titers of IgM or IgA antibodies correlated with the cessation of fecal or nasal virus shedding. A similar sequence of appearance of IgM and IgA antibodies occurred in serum, but IgA antibodies persisted for a shorter period than in fecal or nasal samples. Serum IgG1 antibody responses generally preceeded IgG2 responses and were predominant in most calves after 14-21 DPE.     

Genetically diverse group C rotaviruses cause sporadic infection in Korean calves

This study examined the prevalence and genetic diversity of the bovine group C rotaviruses (GCRVs) in a total of 127 diarrhea fecal samples of calves from 52 Korean native beef calf herds using RT-PCR and nested PCR. Overall, seven of the 127 fecal samples (5.5%) from seven of the 52 herds (13.5%) tested positive for bovine GCRVs only by nested PCR. Sequence and phylogenetic analyses of a partial VP6 gene showed that Korean bovine GCRVs had marked genetic diversity; two Korean strains belonged to the bovine lineage, whereas five Korean strains belonged to the porcine lineage. These results suggest that the genetically diverse bovine GCRVs cause sporadic infections in diarrheic calves in South Korea.     

Association of diarrhea in cattle with torovirus infections on farms.

An epidemiologic survey was performed to determine the incidence of torovirus infections in 2 disease entities of cattle: diarrhea of replacement calves up to 2 months old, and winter dysentery of adult cattle. Samples were obtained from 187 diarrheal and 115 healthy calves from 15 farms, as well as 149 diarrheal and 67 healthy cows from 27 farms with or without winter dysentery. Enzyme-linked immunosorbent assays for detection of torovirus, rotavirus, and coronavirus antigen in feces, and of torovirus and coronavirus antibodies in serum were used to monitor infections in these groups. Torovirus was detected in 9 of the 15 farms in the study, and in 6% of calves with diarrhea, which was significantly higher than in healthy calves (2%). Seroconversion to torovirus was found significantly more often after winter dysentery episodes than on farms without a disease history; coronavirus seroconversion was less common.     

河南省犊牛腹泻主要病原调查研究

[目的]为了解河南省犊牛腹泻主要病原流行情况和腹泻病因,有效防控犊牛腹泻,加快养牛业健康发展。[方法]用ELISA方法对从河南省12个地区采集1～6月龄有腹泻临床症状和无腹泻临床症状犊牛粪便样品187份,血液样品305份进行检测。[结果]在305份血液样品中,共检测到病毒性腹泻病毒(BVDV)阳性血样17份;副结核分支杆菌阳性血样13份。187份粪便样品中,检测到微小隐孢子虫阳性样品47份;轮状病毒阳性样品3份;大肠杆菌K99⁺阳性样品8份;冠状病毒阳性样品5份。[结论]发现犊牛腹泻主要是由病原感染引起的,其中微小隐孢子虫的感染率为较高,同时以单一病原感染为主。     

Fecal D- and L-lactate, succinate and volatile fatty acid levels, and relationships with fecal acidity and diarrhea in neonatal calves

To clarify the significance of fecal organic anions in neonatal diarrhea, a total of 252 fecal samples (91 diarrheic, 161 normal) were collected from 136 dairy calves (including three crossbreds) less than 4 weeks old. Fecal pH, D‐ and L‐lactate, succinate and volatile fatty acid (VFA) were analyzed. In normal feces, lactate was highest and VFA was lowest at week 1 of age, and lactate progressively decreased and VFA progressively increased with advancing age. In diarrheic samples, although higher pH and lower lactate levels were confirmed at week 1, samples at weeks 3–4 showed lower pH and VFA accompanied by higher lactate of D and L‐isomers. In diarrhea, fecal butyrate was significantly lower at all stages, but succinate levels did not differ significantly. The proportion of lactate to organic anions (sum of lactate, succinate and VFA) in diarrheic feces was lower at week 1, and higher in weeks 2–4, while that of VFA to organic anions showed the opposite pattern. Strong relationships were observed between fecal pH and lactate, and VFA proportions in organic anions, though the relationship was weak in diarrhea. Most of the elevated lactate was observed in fecal samples with lower VFA. However, succinate had no relationship with VFA or lactate levels.     

牛病毒性腹泻病毒、大肠杆菌和奇异变形杆菌混合感染致犊牛腹泻的研究

为确定甘肃省临夏州某奶牛场犊牛腹泻的病因,并提供合适的治疗方案和防控措施,试验采集该牛场13头腹泻犊牛的粪便和血清,通过胶体金技术、ELISA方法、细菌分离鉴定、Kirby-Bauer法分别进行病毒病原学检测、病毒血清学抗体检测、病原菌鉴定和药物敏感性试验。病毒学检测结果显示,13份粪样中未检测出牛轮状病毒（BRV）、牛冠状病毒（BCV）的抗原,牛病毒性腹泻病毒（BVDV）抗原阳性率为23.08%（3/13）;未检出BRV和BCV的抗体,BVDV血清学抗体阳性率为38.46%（5/13）。病原菌检测结果显示,13份粪便样品中,分离出13株大肠杆菌和7株奇异变形杆菌。药敏试验表明,分离的大肠杆菌和奇异变形杆菌对20种常规药物均产生了不同程度的耐药,且无对两种细菌均有效的药物。此次犊牛腹泻是由BVDV、大肠杆菌、奇异变形杆菌混合感染引起的,且大肠杆菌和奇异变形杆菌的耐药现象严重,本试验结果为该牛场进一步治疗此次的犊牛腹泻病提供了合理有效的依据。     

Prevalence and genotypes of Giardia duodenalis in dairy and beef cattle in farms around Charlottetown, Prince Edward Island, Canada

Prevalence of Giardia duodenalis in dairy and beef cattle on farms around Charlottetown, Prince Edward Island (Canada) was determined by analyzing feces using direct immunofluorescence antibody microscopy. Genotypes were determined by 16S-rRNA sequencing. Fecal samples (n = 892) were collected from adult cattle in dairy tie-stall, dairy free-stall, and beef herds (10 herds each), and from calves (n = 183) from 11 dairy farms. Prevalence rates were 38% and 51% in cows and calves, respectively. Giardia duodenalis was present in all dairy herds, in 9/10 beef herds and in calves from 10/11 herds examined. Prevalence rates were 40% and 41% for cows in tie- and free-stall herds, respectively, and 27% for beef cows. Zoonotic Assemblage A was found in 12.2% of calves concomitantly infected with Assemblage E. All successfully sequenced samples (114/128) from cows corresponded to Assemblage E. Giardia duodenalis is highly prevalent in cattle herds in Prince Edward Island and Assemblage A in calves is a potential public health concern.