牛病毒性腹泻的鉴定以及治疗

为了鉴定甘肃武威地区某牛场中牛病毒性腹泻病的发病情况,分别采集了13只病牛的血液样品、粪便样品配合诊断,通过血清学诊断方法和PCR鉴定的方法对样本进行了检测,结果显示血清学方法中的13份血液样本有8份为阳性,粪便样本提取RNA,进行RT-PCR同样可以检测到8份样本中扩增得到片段大小为267bp的条带,检测的13份样本有8份样本的血清学检测和粪便PCR检测均为阳性,本次检测的阳性率为61.54%,结果表明本次检测的甘肃武威地区某养殖场的牛病毒性腹泻阳性率相对较高,需要加强牛病毒性腹泻的防控,采取科学的手段治理该类疾病降低其对养牛产业的损失。     

Primary isolation of cytopathic bovine rotaviruses on fetal rhesus monkey kidney cells

Primary isolation of bovine rotaviruses was successfully performed on rolling cultures of MA104 cells following trypsin treatment of fecal samples and cells. Fifty-one fecal samples were obtained from 22 herds affected with naturally-occurring acute diarrhea in calves during a period of over two years. Rotavirus particles were demonstrated in only 10 fecal samples by electron microscopy. Fourteen cytopathic bovine rotaviruses were isolated from positive samples and could be serially cultivated on MA104 cells. The presence of virus was identified by specific immunofluorescence in infected cells. These data indicated that approximately 30% of the herds affected with acute diarrhea in their calves were associated with rotavirus infection.     

Isolation of picornavirus from feces and semen from an infertile bull.

Virus was isolated from semen and fecal samples from a bull with orchitis, testicular degeneration, aspermatogenesis, and loss of libido. Both isolates were classified as picornavirus, bovine enterovirus serotype I, on the basis of physical, chemical, and serologic characteristics. Veterinary practitioners that may suspect viral infection as a cause of bovine infertility should submit both semen and fecal samples for virus isolation and identification.     

规模化奶牛场犊牛腹泻的病原检测与分析报告

为了调查某规模化奶牛场犊牛腹泻的发病原因,试验采用血清抗体检测、细菌分离培养、寄生虫卵镜检和病毒核酸检测方法对腹泻犊牛的血样和粪便进行了检查。结果表明,该奶牛场牛病毒性腹泻/黏膜病病毒抗体阳性率为65.00%,牛轮状病毒抗体阳性率25.00%,牛冠状病毒抗体阳性率15.00%;分离到1 株结肠弯曲杆菌和1 株空肠弯曲杆菌;所检腹泻犊牛粪便和血液样品中,4 份粪便样品和1 份血液样品检出牛冠状病毒,2 份粪便样品检出轮状病毒,1 份粪便样品和1 份血液样品检出牛病毒性腹泻/黏膜病病毒,同一份粪便样品中同时检出牛病毒性腹泻/黏膜病病毒和牛冠状病毒;通过镜检,球虫卵囊检出率77.78%,在1 份粪样中发现蛔虫卵,2 份粪样中发现其他线虫卵。该牛场流行性腹泻的原因可能为夏季高温潮湿引起犊牛免疫力下降导致的多病原体感染。     

Technical note: a procedure for the preparation and quantitative analysis of samples for titanium dioxide

A procedure was developed for the rapid analysis of titanium dioxide (TiO2) concentrations in feed and fecal samples. Samples were digested in concentrated H2SO4 for 2 h, followed by addition of 30% H2O2, and absorbance was measured at 410 nm. Standards were prepared by spiking blanks with increasing amounts of TiO2, resulting in a linear standard curve. Complete analysis using this procedure can typically be accomplished within 4.5 h. This procedure was compared to a previously published dry-ash procedure for the analysis of TiO2 in bovine fecal samples. Three sources of OM devoid of TiO2 (a forage sample, a bovine fecal sample without Cr2O3, and a bovine fecal sample containing Cr2O3) were spiked with graded amounts (0, 2, 4, 6, 8, or 10 mg) of TiO2. With our procedure, TiO2 recoveries averaged 96.7, 97.5, and 98.5%, for the three OM sources, respectively, vs. 74.3, 83.8, and 53.1% for the same samples analyzed using the dry-ash method. These results suggest that our procedure is a rapid and accurate alternative to dry-ash procedures for the determination of TiO2.     

The vaginal and fecal microbiomes are related to pregnancy status in beef heifers

Background: The greatest impact on profitability of a commercial beef operation is reproduction. However, in beef heifers, little is known about the vaginal and fecal microbiota with respect to their relationship with fertility. To this end, we followed heifers through gestation to examine the dynamics of vaginal and fecal microbial composition throughout pregnancy.Results: Heifers were exposed to an estrus synchronization protocol, observed over a 12-day period, artificially inseminated 12 h to 18 h after observed estrus, and subsequently exposed to bulls for a 50-day breeding season.Vaginal samples were taken at pre-breeding(n = 72), during the first(n = 72), and second trimester(n = 72) for all individuals, and third trimester for individuals with confirmed pregnancies(n = 56). Fecal samples were taken at prebreeding(n = 32) and during the first trimester(n = 32), including bred and open individuals. Next generation sequencing of the V4 region of the 16 S rRNA gene via the Illumina Mi Seq platform was applied to all samples.Shannon indices and the number of observed bacterial features were the same in fecal samples. However,significant differences in vaginal microbiome diversity between gestation stages were observed. No differences in beta-diversity were detected in vaginal or fecal samples regarding pregnancy status, but such differences were seen with fecal microbiome over time. Random Forest was developed to identify predictors of pregnancy status in vaginal(e.g., Histophilus, Clostridiaceae, Campylobacter) and fecal(e.g., Bacteroidales, Dorea) samples.Conclusions: Our study shows that bovine vaginal and fecal microbiome could be used as biomarkers of bovine reproduction. Further experiments are needed to validate these biomarkers and to examine their roles in a female's ability to establish pregnancy.     

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.     

Rapid and sensitive detection of bovine coronavirus and group a bovine rotavirus from fecal samples by using one-step duplex RT-PCR assay

Bovine coronavirus (BCoV) and group A bovine rotavirus (BRV) are two of major causes for neonatal calf diarrhea. In the present study, a one-step duplex RT-PCR was established to detect and differentiate BCoV and group A BRV from fecal samples. The sensitivity of this method for BCoV and group A BRV was 10 PFU/100 μl and 1 PFU/100 μl, respectively. Twenty-eight diarrhea fecal samples were detected with this method, the result showed that 2 samples were identified as co-infected with BCoV and group A BRV, 26 samples were group A BRV positive, and 2 samples were negative. It proved that this method is sensitive for clinical fecal samples and is worth applying to laboratory diagnosis for BCoV and group A BRV.     

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.     

Paratuberculosis in cattle and free-living exotic deer.

Paratuberculosis was studied among dairy cows and exotic deer that shared grazing areas at Point Reyes National Seashore, California. Of the 10 dairy herds tested, 5 (50%) were infected with Mycobacterium paratuberculosis (based on results of fecal culture). Mycobacterium paratuberculosis was cultured from 9 (8.7%) of the 103 bovine fecal samples and from 4 (3.9%) of the 103 bovine rectal mucosa scapings tested. Of 89 fecal samples from 52 axis deer (Axis axis) and 37 fallow deer (Dama dama), 5 (9.6%) and 3 (8.1%), respectively, contained M paratuberculosis. Culture of intestinal necropsy samples from the same deer indicated that 3 (5.8%) of the axis deer and 2 (5.4%) of the fallow deer were infected with M paratuberculosis. The cows were tested for serum antibodies by the complement-fixation test and by radioimmunoassay. Of 95 sera tested by complement fixation, 15 (15.8%) were positive, as were 15 (14.7%) of 102 sera tested by radioimmunoassay. Culture results and serologic test results were compared on a herd basis.     

黑龙江省大庆市部分地区牛轮状病毒腹泻的分子流行病学调查

为了对黑龙江省大庆市部分地区犊牛轮状病毒腹泻的流行情况进行调查,应用RT-PCR技术对随机采取的6份犊牛腹泻粪便样品的轮状病毒VP7基因进行扩增,采用多重半套式PCR方法对VP7基因阳性样本进行分型鉴定。结果显示,6份犊牛腹泻粪便样品中牛轮状病毒VP7基因均为阳性;VP7基因阳性样本经RT-PCR分型鉴定,均属于G10型。该研究结果表明大庆地区引起犊牛轮状病毒腹泻的轮状病毒主要为G型,因此需要针对G型轮状病毒加以防控。     

Ribotyping of Streptococcus uberis from a dairy's environment, bovine feces and milk

Streptococcus uberis is a major cause of bovine mastitis and infections commonly result from environmental exposure to the pathogen. To identify specific sources of mastitis-causing S. uberis strains, samples were collected monthly from the environment and feces of dry cows in a grazing herd. Environmental and fecal strains of S. uberis were compared to those found in milk. S. uberis was detected in 63% of 94 environmental samples, including water, soil, plant matter, bedding material, flies, and hay, in 23% of 107 fecal samples, and in 4% of 787 milk samples. Automated PvuII ribotyping revealed 48 ribotypes among 266 isolates. Per sample, up to five ribotypes were detected. The distribution of ribotypes did not differ significantly among environmental, fecal and milk samples. Specific environmental sources or strains of udder-pathogenic S. uberis were not identified. Fecal shedding was not persistent and did not differ between dry-off and calving. The proportion of fecal samples containing S. uberis was highest during the summer grazing season. S. uberis was common in farm soil (31 of 35 samples) but not in non-farm soil (0 of 11 samples). We hypothesize that fecal shedding of S. uberis may play a role in maintenance of S. uberis populations in the dairy ecosystem.     

Detection and molecular characterization of porcine group C rotaviruses in South Korea

Group C rotaviruses (GCRVs) cause acute diarrhea in humans and animals worldwide and the evidence for a possible zoonotic role of GCRVs has been recently provided. However, there is little evidence of porcine GCRV infections or of their genetic diversity in South Korea. We examined 137 diarrheic fecal specimens from 55 farms collected from six provinces. RT-PCR utilizing primer pairs specific for the GCRV VP6 gene detected GCRV-positive reactions in 36 (26.2%) diarrheic fecal samples. Of these, 17 samples (12.4%) tested positive for porcine GCRVs alone and 19 samples (13.8%) were also positive for other pathogens. Other enteric pathogens except for GCRV were detected in 64 feces samples (46.7%) and no enteric pathogens were evident in 37 feces samples (27.0%). Phylogenetic and sequence homology analyses of GCRV partial VP6 gene between 23 Korean and other known porcine GCRVs demonstrated that Korean strains belonged to the porcine lineage. Furthermore, one Korean porcine strain shared the highest nucleotide (89.7–89.0%) and deduced amino acid sequence (92.9–93.9%) identities with bovine GCRV strains and was placed in the bovine GCRV lineage indicative of bovine origin. In conclusion, porcine GCRV infections are widespread in piglets with diarrhea in South Korea. The infecting porcine GCRVs mostly belong to the porcine lineage with the exception of one bovine-like GCRV, which possibly originated from bovine GCRV due to interspecies transmission.     

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.     

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.     

Evaluation of bacteriologic culture of pooled fecal samples for detection of Mycobacterium paratuberculosis

OBJECTIVES: To compare sensitivity of several methods of bacteriologic culture of pooled bovine fecal samples for detection of Mycobacterium paratuberculosis and evaluate homogeneity in number of M paratuberculosis in pooled fecal samples. SAMPLE POPULATION: Feces from 10 dairy cows that shed M paratuberculosis at various concentrations and 1 dairy cow known to be free of infection with M paratuberculosis. PROCEDURE: 5 fecal pooling methods, 2 culture methods, and 2 pool sizes were evaluated. Each pooled sample contained 1 infected sample and 4 or 9 uninfected samples. RESULTS: Sensitivity of detection of M paratuberculosis was greater with smaller pool size (5 vs 10 samples/pool). Detection sensitivity was also associated with concentration of bacteria in the infected sample. Results indicated that, compared with concurrent bacterial culture of individual infected samples, 37 to 44% of pooled samples with low bacterial concentrations yielded positive culture results and 94% of pooled samples with high bacterial concentrations yielded positive results. CONCLUSIONS AND CLINICAL RELEVANCE: Bacteriologic culture of pooled fecal samples may provide a valid and cost-effective method of detecting M paratuberculosis infection in cattle herds.     

Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and fecal-PCR for the diagnosis of bovine Johne's disease (BJD) in India

Country lacks indigenous diagnostic kits against Johne's disease in animals. Indigenous ELISA and IS 900 PCR kits, originally developed for goats and sheep, have been adapted for screening of lactating cows. Multiple diagnostic tests were used to screen 26 lactating dairy cows against Johne's disease. Milk ELISA was evaluated with fecal culture, milk culture and fecal PCR. Of the 26 samples from lactating cows, 84.6, 96.1, 88.4 and 23.0% were positive in fecal culture, milk culture, m-ELISA and m-PCR, respectively. Comparatively milk sediment and milk fat culture detected 84.6 and 76.9% cows positive, respectively. Comparatively fecal culture and milk culture detected 84.6 and 96.1% cows positive, respectively. M-ELISA detected 11.5, 0.0, 11.5, 61.0 and 15.3%, cows as negative, suspected, low positive, positive and strong positive, respectively. There was good correlation between milk and fecal culture with m-ELISA. Three negative cows in m-ELISA were also detected in milk and fecal culture. Of the 26 decontaminated fecal samples, 23.0% cows were positive using specific IS 900 f-PCR. Comparative evaluation of m-ELISA with fecal and milk culture showed agreement in 80.7 and 84.6% cows, respectively. Sensitivity of m-ELISA with respect to fecal and culture was 90.9 and 95.6%, respectively. Comparative evaluation of four tests (milk culture, fecal culture, m-ELISA and f-PCR) showed that only 15.3% cows were detected in all the four tests. In three tests (fecal and milk culture and m-ELISA), 57.6% cows were detected positive. None of the cow was exclusively detected in f-PCR. Of the four diagnostic tests used milk culture was most sensitive (96.15%), followed by fecal culture (86.6%), m-ELISA (76.9%) and IS 900 PCR (23.0%) for the diagnosis of bovine Johne's disease (BJD). Milk ELISA detected only one cow extra, which was negative in milk culture. In view of the simplicity, rapidity and efficacy present milk ELISA kit employing soluble protoplasmic antigen from native Map 'Bison type' genotype of goat origin can be reliable for screening of bovine population against Johne's disease in India.     

吉林省肉牛冠状病毒感染状况调查及分析

旨在全面了解牛冠状病毒(BCoV)在吉林省肉牛群的流行情况,选择吉林省东中西部地区的12个县市的规模化、养殖合作社、家庭型养殖户等牛场在不同季节采集血液、鼻拭子、粪拭子及临床病死牛组织脏器,采用血清学及分子生物学诊断检测技术,对BCoV进行流行病学调查,了解BCoV在该吉林省部分地区的流行情况。共采集临床血清样品1 298份,粪便样品、肝、肺、脾、气管等组织样品462份,应用商品化BCoV抗体检测试剂盒检测血清抗体和纳米PCR新型检测技术对临床样品进行PCR检测,并对核酸检测出的阳性结果测序分析。结果显示BCoV抗体血清阳性率为1.08%,粪便、肝等临床样品阳性率21.10%。经测序分析调查地区BCoV流行株与我国四川流行株相似性达99%以上。本研究对吉林省中部地区BCoV流行情况进行了全面调查,丰富了牛冠状病毒的流行病学调查数据,为指导牛冠状病毒的防控工作奠定了基础。     

新疆北疆某规模化牛场奶牛副结核病的检测与分析

为查明导致新疆北疆某规模化牛场奶牛腹泻、消瘦的主要病原菌,本研究采用微生物学与分子生物学方法对采集的患牛粪便进行检测与鉴定。结果显示:2份粪样抗酸染色阳性,IS900基因及亚型分型基因检测阳性,IS900基因与GenBank中多个副结核分枝杆菌序列同源性在99%以上,亚型分型基因与Ⅱ型同源性为99.81%,从而确定2份样本均被Ⅱ型(牛型)副结核分枝杆菌感染。