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.     

The significance of bredavirus as a diarrhea agent in calf herds in Lower Saxony]

The objective of this investigation was to determine the distribution of Bredavirus in cattle herds in Lower Saxony and to evaluate its significance as potential cause of diarrhea in calves. Fecal samples and paired blood samples of 119 diarrheic and 46 healthy calves up to two months of age were collected from herds where diarrhea of calves was a problem. Fecal samples were examined for Breda-, rota- and coronavirus by solid phase immune electron microscopy and by ELISA, for K99-positive E. coli and salmonella by microbiological methods, and for cryptosporidia in smears. Antibody titers against Bredavirus, total serum protein and serum gamma globulin content were evaluated in the blood samples. Bredavirus was found in fecal samples from 5% (n = 6) of diarrheic calves which came from four different herds, but not in healthy calves. Rotavirus (31.9%), coronavirus (18.5%) and cryptosporidia (29.9%) were detected more frequently in fecal samples than Bredavirus. In this investigation rotavirus, coronavirus and cryptosporidia were present in addition in all herds where Bredavirus was found. In contrast to the low percentage of fecal samples containing Bredavirus, antibody titers in 75% of calves confirmed the high prevalence of Bredavirus infection in the cattle population of Lower Saxony.     

Molecular epidemiology of bovine noroviruses in South Korea

Since the prevalence of bovine norovirus (BNoV) and their genetic diversity have only been reported in the USA, England, Germany and The Netherlands, this study examined the prevalence and genetic diversity of BNoVs in diarrheic calves in South Korea using 645 diarrheic fecal specimens from calves by RT-PCR and nested PCR assays. Overall, 9.3% of the diarrheic fecal samples tested positive for BNoVs by either RT-PCR or nested PCR, of which 5.9% samples also tested positive for other enteric pathogens including the bovine coronavirus, bovine viral diarrhea virus, bovine torovirus, bovine groups A, B and C rotaviruses, bovine enteric Nebraska-like calicivirus and Escherichia coli. The genetic diversity was determined by direct sequencing of the partial RdRp region of 12 BNoVs detected from the fecal samples by nested PCR. Among the BNoVs examined, one Korean BNoV strain had the highest nucleotide (86.8%) and amino acid (99.1%) identity with the genotype 1 BNoV (GIII-1) strain, while the remaining 11 Korean BNoVs shared a higher nucleotide (88.0-90.5%) and amino acid (93.5-99.1%) identity with the genotype 2 BNoV (GIII-2) strains. The phylogenetic data for the nucleotide and amino acid sequences also demonstrated that one Korean BNoV strain clustered with GIII-1 but the remaining eleven strains clustered with GIII-2. In conclusion, BNoV infections are endemic and there are two distinct genotypes with GIII-2 being the main genotype circulating in the calf population in South Korea.     

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.     

Infection with Cryptosporidium spp. in humans and cattle in Manitoba.

Between October 1, 1983 and October 31, 1984, fecal specimens from 3656 persons with enteritis and 182 calves, representing 148 herds having a neonatal diarrhea problem, were examined for oocysts of Cryptosporidium spp. Oocysts were found in 1% of human and 25% of bovine specimens. All infected persons were immunocompetent. Children under five years of age had an infection rate of 25/100,000 compared to 1.4/100,000 in older people (p less than 0.005). Rates in northern communities were four to seven times as high as those in southern Manitoba. Human infections occurred most commonly in late summer and fall. In beef calves infection occurred in winter and spring, the calving season in Manitoba. Epidemiological association between the infection in people and in cattle could not be established.     

Prevalence of enteric pathogens in dogs of north-central Colorado

To evaluate the prevalence of enteric pathogens in dogs of north-central Colorado, fecal samples were obtained from client-owned dogs presented to the Veterinary Teaching Hospital at Colorado State University for evaluation of acute small-bowel, large-bowel, or mixed-bowel diarrhea (n=71) and from age-matched, client-owned, healthy dogs (n=59). Infectious agents potentially associated with gastrointestinal disease were detected in 34 of 130 (26.1%) fecal samples. Agents with zoonotic potential were detected in feces from 21 (16.2%) of 130 dogs and included Giardia spp. (5.4%), Cryptosporidium parvum (3.8%), Toxocara canis (3.1%), Salmonella spp. (2.3%), Ancylostoma caninum (0.8%), and Campylobacter jejuni (0.8%). Positive test results occurred in dogs with or without gastrointestinal signs of disease. Dogs, particularly those in homes of immunocompromised humans, should be evaluated for enteric zoonotic agents.     

Sporadic cryptosporidiosis in a rural population is asymptomatic and associated with contact to cattle

Fecal samples each followed by a completed questionnaire were obtained from 233 persons representing 80 households. Using the formalinether concentration method combined with the acid-fast staining oocysts of Cryptosporidium were found in 7 (3 %) persons. 63 persons had contact to cattle, and asymptomatic cryptosporidiosis was diagnosed in 6 of them. Cryptosporidiosis was not found in any of the 51 persons with contact to some other animal but not cattle, and none of the 7 Cryptosporidium positive persons had been traveling abroad. We also obtained fecal samples from 15 calves from 7 households. Six of the calves (40 %) harbored Cryptosporidia and manifest enteritis was detected in those under age of 1 month. Five out of 14 farms had problems of neonatal calf diarrhea. Human cryptosporidiosis was more frequent on such problem farms. The results indicate that in rural population, human cryptosporidiosis is mainly asymptomatic and occurs mainly in residents of cattle-rearing farms.     

Experimentally induced coronavirus infections in calves: viral replication in the respiratory and intestinal tracts

Eleven 3- to 50-day-old colostrum-deprived gnotobiotic calves and seven 25- to 63-day-old colostrum-deprived conventional calves were allotted into 3 groups. Each group was inoculated with a fecal isolate of bovine coronavirus via different routes: orally/intranasally OR/IN, No. 1 through 8, group 1 calves; OR, No. 9 through 13, group 2 calves; IN, No. 14 through 18, group 3 calves. Nasal swab specimens and fecal specimens were collected daily and were examined for coronavirus antigen by use of direct immunofluorescent staining (nasal epithelial cells) or by use of immune electron microscopy (fecal specimens). All but 4 calves (No. 11, 13, 17, and 18) were euthanatized on postinoculation days (PID) 3 to 7. Calves 11 and 17 became severely dehydrated and died at PID 5. Calves 13 and 18 were evaluated for nasal and fecal shedding of coronavirus through PID 14. Distribution of coronavirus antigen in the respiratory and intestinal tracts of the 14 euthanatized calves was evaluated by use of direct immunofluorescent staining. All calves developed profuse diarrhea by PID 2 to 4; however, calves did not develop clinical signs of respiratory tract disease before euthanasia or death. Inoculated calves shed coronavirus in their feces as detected by use of immune electron microscopy. Infected nasal epithelial cells were detected in all but 2 orally inoculated calves (No. 9 and 10). Route of inoculation influenced the sequence of initial detection of coronavirus antigen from fecal specimens or nasal swab specimens.(ABSTRACT TRUNCATED AT 250 WORDS)     

An overview of calf diarrhea - infectious etiology,diagnosis, and intervention

Calf diarrhea is a commonly reported disease in young animals, and still a major cause of productivity and economic loss to cattle producers worldwide. In the report of the 2007 National Animal Health Monitoring System for U.S. dairy, half of the deaths among unweaned calves was attributed to diarrhea. Multiple pathogens are known or postulated to cause or contribute to calf diarrhea development. Other factors including both the environment and management practices influence disease severity or outcomes. The multifactorial nature of calf diarrhea makes this disease hard to control effectively in modern cow-calf operations. The purpose of this review is to provide a better understanding of a) the ecology and pathogenesis of well-known and potential bovine enteric pathogens implicated in calf diarrhea, b) describe diagnostic tests used to detect various enteric pathogens along with their pros and cons, and c) propose improved intervention strategies for treating calf diarrhea.     

Comparison of fecal samples collected per rectum and off the ground for estimation of environmental contamination attributable to beef cattle

OBJECTIVES: To determine whether sampling feces off the ground replicates prevalence estimates for specific pathogens obtained from fecal samples collected per rectum of adult cows, and to determine characteristics of feces on the ground (fecal pats) that are associated with subsequent identification of Campylobacter spp, Cryptosporidium parvum, and Giardia duodenalis. ANIMALS: A random sample of adult beef cattle from 25 herds located throughout California. PROCEDURE: 1,115 rectal and ground fecal samples were obtained. Samples were submitted for culture of Campylobacter spp and examined, using a direct fluorescent antibody assay, to detect C parvum oocysts and G duodenalis cysts. Characteristics of fecal pats, such as volume and consistency, were recorded. RESULTS: Prevalence of Campylobacter spp was 5.0% (20/401) for rectal fecal samples, which was significantly greater than prevalence determined for ground fecal samples (2/402; 0.5%). Most isolates were C jejuni subsp jejuni. Prevalence of C parvum was higher in rectal fecal samples (6/557; 1.1%) than in ground fecal samples (1/558; 0.2%), but this difference was not significant. Prevalence of G duodenalis did not differ for rectal (36/557; 6.5%) versus ground (26/558; 4.7%) fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Evaluation of ground fecal samples may not accurately indicate the prevalence of Campylobacter spp or C parvum in cattle but may reflect prevalence of G duodenalis. Differences in prevalence estimates between the 2 methods suggest inactivation of pathogens in feces after cattle have defecated. Prevalence estimates generated by evaluation of ground fecal samples, however, may more accurately estimate environmental pathogen burden.     

Involvement of microbial respiratory pathogens in acute interstitial pneumonia in feedlot cattle

OBJECTIVE: To evaluate viral and bacterial respiratory pathogens and Mycoplasma spp isolated from lung tissues of cattle with acute interstitial pneumonia (AIP) and cattle that had died as a result of other causes. SAMPLE POPULATION: 186 samples of lung tissues collected from cattle housed in 14 feedlots in the western United States. PROCEDURE: Lung tissues were collected during routine postmortem examination and submitted for histologic, microbiologic, and toxicologic examinations. Histologic diagnoses were categorized for AIP, bronchopneumonia (BP), control samples (no evidence of disease), and other disorders. RESULTS: Cattle affected with AIP had been in feedlots for a mean of 1272 days before death, which was longer than cattle with BP and control cattle. Detection of a viral respiratory pathogen (eg, bovine respiratory syncytial virus [BRSV], bovine viral diarrhea virus, bovine herpesvirus 1, or parainfluenza virus 3) was not associated with histologic category of lung tissues. Bovine respiratory syncytial virus was detected in 8.3% of AIP samples and 24.0% of control samples. Histologic category was associated with isolation of an aerobic bacterial agent and Mycoplasma spp. Cattle with BP were at greatest risk for isolation of an aerobic bacterial agent and Mycoplasma spp. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these results suggests that AIP in feedlot cattle is not a consequence of infection with BRSV. The increased, risk of isolation of an aerobic bacterial agent from cattle with AIP, compared with control cattle, may indicate a causal role or an opportunistic infection that follows development of AIP.     

Natural and experimental infection of neonatal calves with Clostridium difficile

Clostridium difficile toxins were associated with calf diarrhea in a recent retrospective study; however, no causal relationship has been prospectively investigated. This infection study tested whether the oral inoculation of neonatal calves with a toxigenic strain of C. difficile (PCR-ribotype 077) results in enteric disease. Fourteen 6-24 h old male colostrums-fed Holstein calves, received either three doses of C. difficile (1.4 x 10(8) +/- 3.5 x 10(8) cfu) (n = 8) or sterile culture broth (n = 6). Calves were euthanized on day 6 or after the onset of diarrhea, whichever came first. Fecal and intestinal samples were blindly cultured for C. difficile, and tested for its toxin A/B (C. difficile TOX A/B II ELISA, Techlab). PCR-ribotyping was used to compare inoculated and recovered isolates. Diarrhea was observed in all control calves and 3/8 of inoculated calves (p = 0.03), but it did not occur in calves that tested positive for C. difficile toxins. Fecal toxins were identified only from two controls. PCR-ribotyping confirmed the presence of C. difficile PCR-ribotype 077 in samples of all inoculated calves, but not from controls. The identification of five other PCR-ribotypes in 3/8 (37.5%) and 2/6 (33.3%) of inoculated and control calves, respectively, indicated early natural infection (< or = 24h of age). Five of 14 cecal samples had C. difficile (p = 0.01). In conclusion, the oral administration of C. difficile PCR-ribotype 077 to neonatal calves resulted in fecal/intestinal colonization but not in detection of toxins, or signs of enteric disease. Further studies are required to investigate the clinical relevance of C. difficile in calves.     

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。     

Evaluation of concurrent shedding of bovine coronavirus via the respiratory tract and enteric route in feedlot cattle

OBJECTIVE: To assess the relationship between shedding of bovine coronavirus (BCV) via the respiratory tract and enteric routes and the association with weight gain in feedlot cattle. ANIMALS: 56 crossbred steers. PROCEDURES: Paired fecal samples and nasal swab specimens were obtained and were tested for BCV, using antigen-capture ELISA. Paired serum samples obtained were tested for antibodies to BCV, using antibody-detection ELISA. Information was collected on weight gain, clinical signs, and treatments for enteric and respiratory tract disease during the study period. RESULTS: Number of samples positive for bovine respiratory coronavirus (BRCV) or bovine enteric coro navirus (BECV) was 37/224 (17%) and 48/223 (22%), respectively. Some cattle (25/46, 45%) shed BECV and BRCV. There were 25/29 (86%) cattle positive for BECV that shed BRCV, but only 1/27 (4%) cattle negative to BECV shed BRCV. Twenty-seven of 48 (56%) paired nasal swab specimens and fecal samples positive for BECV were positive for BRCV. In contrast, only 10/175 (6%) paired nasal swab specimens and fecal samples negative for BECV were positive for BRCV. Only shedding of BECV was associated with significantly reduced weight gain. Seroconversion to BCV during the 21 days after arrival was detected in 95% of the cattle tested. CONCLUSIONS AND CLINICAL IMPLICATIONS: Feedlot cattle infected with BCV after transport shed BCV from the respiratory tract and in the feces. Fecal shedding of BCV was associated with significantly reduced weight gain. Developing appropriate control measures for BCV infections could help reduce the decreased weight gain observed among infected feedlot cattle.