Specificity of assays used by regulatory agencies to detect antibiotic residues in tissues of culled dairy cows

OBJECTIVE: To determine percentage of false-positive test results for assays used by regulatory agencies to detect antibiotic residues in tissues. DESIGN: Prospective study. ANIMALS: 426 dairy cows. PROCEDURE: Dairy cows scheduled for culling that were identified as being unlikely to have antibiotic residues in tissues on the basis of strict inclusion criteria were used. A sample of kidney obtained from each cow at slaughter was tested on-site, using the swab test on premises (STOP; 97 samples) or the fast antibiotic screening test (FAST; 329 samples). Frozen samples (n = 1,278) of liver, muscle, and kidney were thawed and retested at a federal laboratory, using the same screening assays. Kidney and liver samples (n = 852) were also tested using the 7-plate bioassay confirmation test used for confirmation and identification of antibiotic residues. RESULTS: Results of screening assays performed onsite were negative. When frozen samples were retested, 20 (12 liver, 7 kidney, and 1 muscle) had positive FAST results, but none had positive STOP results. Of the samples tested with the 7-plate bioassay confirmation test, 4 liver samples had results indicating a tetracycline (n = 3) or an unidentified microbial inhibitor (1) as a residue. CLINICAL IMPLICATIONS: Results suggest it is unlikely that regulatory action will be taken against producers sending untreated cattle to market. However, because results of the FAST and 7-plate bioassay confirmation test were positive when applied to frozen tissue, use of assays based on microbial inhibition may not be valid for confirmation of residues.     

Elimination kinetics of ceftiofur hydrochloride after intramammary administration in lactating dairy cows

OBJECTIVE: To determine the elimination kinetics of ceftiofur hydrochloride in milk after intramammary administration in lactating dairy cows. DESIGN: Prospective study. ANIMALS: 5 lactating dairy cows. PROCEDURE: After collection of baseline milk samples, 300 mg (6 mL) of ceftiofur was infused into the left front and right rear mammary gland quarters of each cow. Approximately 12 hours later, an additional 300 mg of ceftiofur was administered into the same mammary gland quarters after milking. Milk samples were collected from each mammary gland quarter every 12 hours for 10 days. Concentrations of ceftiofur and its metabolites in each milk sample were determined to assess the rate of ceftiofur elimination. RESULTS: Although there were considerable variations among mammary gland quarters and individual cows, ceftiofur concentrations in milk from all treated mammary gland quarters were less than the tolerance (0.1 microg/mL) set by the FDA by 168 hours (7 days) after the last intramammary administration of ceftiofur. No drug concentrations were detected in milk samples beyond this period. Ceftiofur was not detected in any milk samples from nontreated mammary gland quarters throughout the study. CONCLUSIONS AND CLINICAL RELEVANCE: Ceftiofur administered by the intramammary route as an extra-label treatment for mastitis in dairy cows reaches concentrations in milk greater than the tolerance set by the FDA. Results indicated that milk from treated mammary gland quarters should be discarded for a minimum of 7 days after intramammary administration of ceftiofur. Elimination of ceftiofur may be correlated with milk production, and cows producing smaller volumes of milk may have prolonged withdrawal times.     

盐酸头孢噻呋注射液对奶牛的安全性研究

试验旨在研究盐酸头孢噻呋注射液对奶牛的安全性。试验选择20头健康泌乳奶牛,随机分成4组,即1×(2.2 mg/kg bw)、3×(6.6 mg/kg bw)、5×(11.0 mg/kg bw)推荐剂量组和0.9%生理盐水对照组,观察用药后奶牛的直肠温度、精神状态以及乳房局部症状等临床症状,并进行血液生理和血清生化指标的测定。结果显示,盐酸头孢噻呋注射液1×、3×、5×推荐剂量组所有试验动物用药后均无明显的不良反应,血液生理和血清生化指标均在正常范围内,表明盐酸头孢噻呋注射液在临床上用于靶动物奶牛是安全的。     

A comprehensive review of ceftiofur sodium and hydrochloride formulations for treatment of acute bovine foot rot

Seven well-controlled studies conducted under multiple management conditions demonstrated that ceftiofur, a late-generation veterinary parenteral cephalosporin, is effective for the treatment of bovine foot rot in beef and dairy cattle. Two preliminary dosage titration studies using a challenge model compared the efficacy of ceftiofur (1.1 mg or 2.2 mg ceftiofur equivalents [CE]/kg administered once daily for 3 days) with placebo. One preliminary clinical study evaluated the efficacy of ceftiofur sodium (1.0 mg CE/kg once daily for 3 days) in lactating dairy cows. Two clinical trials evaluated the efficacy of ceftiofur sodium versus placebo for naturally occurring foot rot, and two trials compared the efficacy of ceftiofur sodium or hydrochloride (1.0 mg CE/kg) with oxytetracycline (6.6 or 10 mg/kg), each administered once daily for 3 days, for treatment of acute foot rot in beef cattle. All trials demonstrated the efficacy of ceftiofur for treatment of acute bovine foot rot. Ceftiofur and oxytetracycline were comparable in efficacy, with ceftiofur having excellent injection-site tolerance and short or no milk discard or preslaughter withdrawal.     

Association between ceftiofur use and isolation of Escherichia coli with reduced susceptibility to ceftriaxone from fecal samples of dairy cows

OBJECTIVE: To estimate the association between ceftiofur use and the isolation of Escherichia coli with reduced ceftriaxone susceptibility from fecal samples of dairy cow populations. ANIMALS: 1,266 dairy cows on 18 farms in Ohio. PROCEDURES: Individual fecal samples from all cows in the study herds were tested for Escherichia coli with reduced ceftriaxone susceptibility. Herd antimicrobial use policy and antimicrobial treatment records were also obtained. Plasmid DNA from these isolates was tested for the presence of the AmpC beta-lactamase gene (blaCMY-2). Minimum inhibitory concentrations to a standard panel of 16 antimicrobial drugs were determined by use of a broth microdilution system. RESULTS: Herds for which ceftiofur use was reported were more likely to have cows from which reduced susceptibility E coli was isolated than herds that did not report ceftiofur use (odds ratio, 25.0). However, at the individual cow level, no association was found between recent ceftiofur treatment and isolation of reduced-susceptibility E coli (adjusted odds ratio, 1.01). No observed linear relationship was found between the percentage of cows from which E coli with reduced ceftriaxone susceptibility was isolated and the percentage of cows in the herd recently treated with ceftiofur. CONCLUSIONS AND CLINICAL RELEVANCE: Our observation of a herd-level but not an individual cow-level association between ceftiofur use and isolation of E coli with reduced ceftriaxone susceptibility from fecal samples suggests that interventions to reduce the spread of antimicrobial resistance genes in agricultural animals will be most effective at the herd level.     

Efficacy of ceftiofur hydrochloride sterile suspension administered parenterally for the treatment of acute postpartum metritis in dairy cows

OBJECTIVE: To evaluate the efficacy of ceftiofur hydrochloride sterile suspension administered parenterally for treatment of acute postpartum metritis (APM) in dairy cows. DESIGN: Multilocation, randomized block, field trial. ANIMALS: 406 cows in the first 14 days postpartum. PROCEDURE: Cows with rectal temperatures > or = 39.5 degrees C (103.1 degrees F) without clinical signs of respiratory or gastrointestinal tract disease and with a fetid vaginal discharge were allocated randomly in blocks of 3 to 3 treatment groups: sterile saline (0.9% NaCl) solution administered at a dosage of 2 mL/45.4 kg (2 mL/100 lb), SC or IM, once daily for 5 days (control); or ceftiofur hydrochloride administered at a dosage of 1.1 or 2.2 mg of ceftiofur equivalents (CE)/kg (0.5 or 1 mg/lb, respectively), SC or IM, once daily for 5 days. Cows were evaluated on days 6, 10, and 14, and clinical cure or failure to cure was determined. Clinical cure was defined as no additional antimicrobial treatment administered, rectal temperature < 39.5 degrees C, and absence of a fetid vaginal discharge. RESULTS: On day 14, clinical cure rates were 77%, 65%, and 62% for the 2.2 mg of CE/kg, 1.1 mg of CE/kg, and control groups, respectively. No significant differences were detected in clinical cure rates between control and treatment groups on day 10 or 6. CONCLUSIONS AND CLINICAL RELEVANCE: Ceftiofur hydrochloride administered at a dosage of 2.2 mg of CE/kg, SC or IM, once daily for 5 days was efficacious for treatment of APM in dairy cows.     

The effects on the pharmacokinetics of intravenous ceftiofur sodium in dairy cattle of simultaneous intravenous acetyl salicylate (aspirin) or probenecid

Ceftiofur sodium is a third-generation cephalosporin antibiotic. It is possible that non-steroidal anti-inflammatory drugs such as acetyl salicylate (aspirin) may be used concomitantly with ceftiofur sodium in dairy cattle. Therefore this study evaluated potential pharmacokinetic interactions between ceftiofur sodium and aspirin. In addition, this study evaluated the potential for interaction between ceftiofur and its active metabolites and the organic anion transporter. The organic anion transporter substrate used in this evaluation was probenecid. Ten healthy, non-pregnant, non-lactating dairy cows were used in a randomized complete three-way crossover design. In repeated experiments all cows were administered: (1) 2 mg of ceftiofur sodium per kg body weight by intravenous bolus or (2) 10 mg of probenecid per kg body weight by intravenous bolus, followed immediately by 2 mg of ceftiofur sodium per kg body weight by intravenous bolus or (3) 26 mg of aspirin per kg body weight by intravenous bolus, followed immediately by 2 mg of ceftiofur sodium per kg body weight by intravenous bolus. For treatment with ceftiofur sodium alone, the mean volume of distribution at steady-state V_d(33) was 0.2 ± 0.06 L/kg, the mean volume of distribution by the area method V_d(area) was 0.38 ± 0.22 L/kg, mean residence time (MRT) was 6.5 ± 1.8 h, mean residence time in peripheral tissues (MRT_p) was 2.6 ± 1.0 h, total body clearance (Cf) was 0.032 ± 0.013 L/kg/h and elimination rate constant (P) was 0.097 ± 0.044 h^-1(mean ± standard deviation). No statistically significant changes were detected as a result of preceding treatment with aspirin. Preceding treatment with probenecid resulted in a decrease in both Cl (0.007 ± 0.005 L/kg/h) and MRT_p (0.89 ± 0.45 h). These results suggest that ceftiofur or its metabolites may interact with the organic anion transporter, but that consideration of alterations to dose and dose interval may not be necessary when ceftiofur sodium is administered to the cow concomitantly with a single dose of aspirin.     

屡配不孕奶牛血清抗精子抗体水平和孕酮含量的测定

应用精子凝集试验测定五个牧场共33头屡配不孕奶牛和86头正常牛的血清抗精子抗体水平,发现30头屡配不孕牛的血清稀释到1∶8时,仍可见明显的凝集反应,而85头正常牛的血清稀释到1∶8时,凝集反应阴性。因此,将1∶8的血清稀释倍数作为诊断母牛是否不孕的指标,阳性和阴性准确率分别可达90.91％和98.84％。此外,用微滴板酶免疫法测定8头屡配不孕牛和30头正常牛的血清孕酮含量分别为9.83±1.85和1.95±0.74n g/ml,两者统计差异显著(P<0.01)。     

Pharmacokinetics of ceftiofur in plasma and uterine secretions and tissues after subcutaneous postpartum administration in lactating dairy cows

A study was conducted to measure concentrations of potentially active ceftiofur derivatives, in plasma, in uterine tissues (endometrium and caruncles) and in uterine secretions at different time points after a single subcutaneous administration of ceftiofur hydrochloride (Excenel RTU Sterile Suspension) at the dose of 1 mg/kg body weight in Holstein-Friesian dairy cows. The animals (n=4) were injected within 24 h of calving, after expulsion of the foetal membranes. Plasma, lochial fluid, caruncles and endometrium were collected before ceftiofur hydrochloride administration and at 1, 2, 4, 8, 12 and 24 h after treatment. For each cow the concentrations of ceftiofur in the biological matrices were quantified using an high-performance liquid chromatography (HPLC) assay. The limit of quantification of the method was 0.1 microg/mL for plasma and 0.1 microg/g for lochial fluid, caruncles and endometrium. The concentrations of potentially active ceftiofur derivatives detected in plasma reached a maximum of 2.85 +/- 1.11 microg/mL at 2 h and decreased to 0.64 +/- 0.14 microg/mL at 24 h after administration. In lochial fluid, these concentrations reached a maximum of 0.97 +/- 0.25 microg/g at 4 h and decreased to 0.22 +/- 0.21 microg/g at 24 h after administration. In endometrium, these concentrations reached a maximum of 2.23 +/- 0.82 microg/g at 4 h and decreased to 0.56 +/- 0.14 microg/g at 24 h following the injection, whereas these levels in caruncles were 0.96 +/- 0.45 and 0.60 +/- 0.39 microg/g obtained at 8 and 24 h, respectively. At the dose of 1 mg/kg body weight in healthy dairy cows, subcutaneous administration of ceftiofur (as ceftiofur hydrochloride) after parturition results in concentrations of ceftiofur derivatives in uterine tissues and in lochial fluid that exceed the reported minimal inhibitory concentrations (MICs) for the common pathogens (Escherichia coli, Fusobacterium necrophorum, Bacteroides spp., and Arcanobacterium pyogenes) associated with acute puerperal metritis.     

黑龙江省鸡西地区奶牛子宫内膜炎病原菌分离鉴定及耐药性研究

从黑龙江省鸡西地区部分奶牛场选取25头临床诊断为子宫内膜炎的患牛,并从这25头患牛体内分离得到39株细菌,其中大肠杆菌17株、葡萄球菌13株、链球菌5株、其他菌4株。耐药性试验表明,大肠杆菌、葡萄球菌和链球菌都对头孢曲松钠和头孢噻呋钠敏感,对庆大霉素比较敏感,对其他几种常用抗生素存在不同程度耐药性。     

Ceftiofur distribution in plasma and joint fluid following regional limb injection in cattle

The objective of this study was to evaluate the efficacy of regional intravenous (i.v.) injection of ceftiofur in delivery of this drug to joint fluid and plasma in a limb distal to a tourniquet in five, healthy, adult, mixed breed beef cattle. A tourniquet was positioned in the mid-metacarpal region, and 500 mg of ceftiofur was administered through a catheter in the dorsal common digital vein (DCDV). Plasma samples were collected from the catheter at 15, 30 and 45 min postinjection, and from the abaxial proper palmar vein (APPV) at 15 min postinjection. Synovial fluid was collected from the metacarpal phalangeal joint at 45 min postinjection. Ceftiofur concentrations were estimated in plasma and synovial fluid using high-pressure liquid chromatography (HPLC) and a microbiological assay utilizing Pasteurella haemolytica as the test organism. Both assays indicated highest plasma concentrations of ceftiofur at 15 min, with the concentrations declining with time. Concentrations of ceftiofur in plasma obtained from the DCDV were not significantly different from APPV levels, indicating rapid distribution of ceftiofur within the limb. Microbiological assay always demonstrated higher concentrations of ceftiofur compared with HPLC assay, because the former probably also detected the active metabolites of ceftiofur as well as the parent compound. At 45 min, ceftiofur concentrations determined by HPLC were 251+/-97 and 15+/-5 microg/mL in plasma and synovial fluid, respectively. Regional intravenous injection appears to be a feasible technique to produce rapid distribution of ceftiofur within the limb well above therapeutic concentrations.     

Multilocation trial of ceftiofur for treatment of postpartum cows with fever

OBJECTIVE: To evaluate the effect of ceftiofur for treatment of postpartum cows with fever. DESIGN: Multilocation randomized complete block design trial. ANIMALS: 330 cows. PROCEDURE: Cows with rectal temperature > or = 39.5 C (103.1 F) during the first 10 postpartum days were randomly assigned to a treatment (ceftiofur; 1 mg/kg [0.45 mg/lb] of body weight daily for 3 days) or untreated control group. Cure (no additional or alternative antimicrobial treatment used, rectal temperature < 39.5 C, and no other concurrent clinical signs of disease when evaluated at 9 or 10 days after enrollment), milk production, and rectal temperature were evaluated. RESULTS: Ceftiofur-treated cows were significantly more likely to be cured than control cows (56.0 vs 28.9%, respectively), with an odds ratio of 3.14 when vaginal discharge (a factor with moderate interaction with treatment) was present at enrollment. Among cows that had an abnormal calving (a significant interaction factor), treated cows had first milking yield 2.27 kg (5 lb) greater than control cows. Treated cows had a significantly greater reduction in rectal temperature (1.19 C [2.14 Fl), compared with control cows (1.04 C [1.87 F]). CONCLUSIONS AND CLINICAL RELEVANCE: Parenteral administration of ceftiofur significantly improved cure rate, milk yield, and rectal temperature in postpartum cows with fever and vaginal discharge or dystocia. These findings provide information to determine appropriate treatment for postpartum cows, which for years has been debated in the dairy industry.     

盐酸吡利霉素治疗奶牛乳房炎临床药效学及对乳品质影响的研究

本研究依据对黑龙江省部分牧场奶牛乳房炎致病菌分离鉴定以及药敏试验结果,选取盐酸吡利霉素、头孢噻呋钠和复方阿莫西林为试验药物,对患有隐性乳房炎的奶牛进行治疗并对乳品质进行跟踪监测。结果表明盐酸吡利霉素对隐性乳房炎的治愈率最高,对临床型乳房炎的治疗效果略差于复方阿莫西林,盐酸吡利雷素与复方阿莫西林对两种类型乳房炎治疗的总有效率相当,但盐酸吡利霉素对乳品质（尤其是降低体细胞数方面）的改善作用显著好于复方阿莫西林治疗组。     

Penetration of ceftiofur into sterile vs. Mannheimia haemolytica-infected tissue chambers in beef calves after subcutaneous administration of ceftiofur crystalline free acid sterile suspension in the ear pinna

The effect of Mannheimia haemolytica infection on the penetration of ceftiofur and desfuroylceftiofur metabolites into tissue chambers was studied in cattle after subcutaneous administration of ceftiofur crystalline free acid sterile suspension (CCFA-SS). Four tissue chambers were implanted subcutaneously in each of 12 calves. Approximately 45 days after implantation, two chambers were inoculated with M. haemolytica (10(6) colony-forming units per chamber) while the remaining two chambers were inoculated with sterile phosphate-buffered saline. Twenty-four hours after inoculation, CCFA-SS was administered subcutaneously in the middle third of the caudal ear pinna of each calf. Chamber fluid and blood samples were collected at predetermined times for 10 days following dosing and analyzed for ceftiofur and desfuroylceftiofur metabolites by high-performance liquid chromatography. Concentrations of ceftiofur and desfuroylceftiofur metabolites in plasma and tissue chamber fluid remained above a threshold of 0.2 microg/mL for at least 8 days. Infected tissue chamber fluid concentrations of ceftiofur and desfuroylceftiofur metabolites were significantly higher than those in non-infected tissue chamber fluid, which correlated with significantly higher total protein concentration in infected tissue chambers. These results indicate that single subcutaneous administration of CCFA-SS at 6.6 mg/kg can be expected to provide effective therapy of susceptible bacterial infections for a period of at least 1 week.     

一株牛源乡间布丘菌的分离鉴定及耐药性研究

为了解奶牛乳房炎病原微生物的生物学特性及其耐药性,本研究从奶牛乳房炎乳中分离到的一株革兰氏阴性菌,命名为HLJ-36,对其进行形态特征、生化鉴定、分子生物学鉴定、小鼠致病性试验及药敏试验。生化试验结果显示,木糖、ONPG、柠檬酸盐等反应呈阳性,氧化酶、吲哚、尿素酶等反应呈阴性。理化特性与分子生物学试验结果均显示,分离株与乡间布丘菌特征相符,与人源乡间布丘菌肠道分离株（GenBank登录号：NR_041968.1）同源性最高,达99%。小鼠致病性试验结果显示,试验组小鼠72 h内未发生死亡,表明其致病性较弱。药敏试验结果显示,该菌对头孢噻呋、阿奇霉素、环丙沙星等耐药,对头孢西丁、美罗培南、氯霉素、阿莫西林-克拉维酸、庆大霉素敏感。结果表明,乡间布丘菌可能是引起奶牛乳房炎的一种病原菌,并有一定程度的耐药性,由于该菌与人源乡间布丘菌肠道分离株（GenBank登录号：NR_041968.1）同源性最高,有通过乳及乳制品传播给人类的可能,本试验具有一定的公共卫生学意义。     

Efficacy of extended intramammary ceftiofur therapy against mild to moderate clinical mastitis in Holstein dairy cows: A randomized clinical trial

Few studies have investigated the efficacy of extended ceftiofur therapy and none have focused on extended therapy for naturally occurring clinical mastitis. The objective of this study was to compare the efficacy of extended intramammary ceftiofur therapy of 8 d duration with a standard 2-day regimen for the treatment of naturally occurring mild to moderate clinical mastitis in lactating dairy cows. Holstein cows from 22 dairy herds (n = 241) were randomly allocated to the 2 treatment groups. For each case of mastitis, 125 mg of ceftiofur hydrochloride was administered intramammary once a day for 2 or 8 d. Clinical cure, 21 d after the last treatment, was 89% (98/110) in each group. Bacteriological cure 21 d after the last treatment for the 2- and 8-day regimens were 32% (15/47) and 61% (25/41), respectively, for all bacteria (P = 0.007), 64% (9/14) and 82% (9/11), respectively, for streptococci (P = 0.50), and 0% (0/20) and 47% (9/19), respectively, for Staphylococcus aureus (P = 0.0004). There were no statistical differences between groups for new intramammary infections. Overall, ceftiofur extended therapy increased cure when compared to a 2-day regimen for the treatment of naturally occurring mild to moderate clinical mastitis in lactating dairy cows.     

Evaluation of the agar gel immunodiffusion test for diagnosis of subclinical paratuberculosis in cattle

Concurrent bacteriologic culture of feces and agar gel immunodiffusion (AGID) testing was performed on all cows and bred heifers over 14 months old in 10 dairy herds during a 32-month period to determine the effectiveness of the AGID test for the detection of subclinical paratuberculosis. Herds were sampled 5 times and, when possible, culled animals were tested again at slaughter. During 5 herd-wide samplings, Mycobacterium paratuberculosis was isolated from 139 fecal specimens obtained from 109 cattle. Results of the AGID test were simultaneously positive 40 of 139 times (28.8%). Thirty-six of the 109 cattle (33.0%) determined to be infected had a positive AGID test result at some point during the 5 herd-wide samplings. When results of tests performed at time of slaughter were included, 117 cattle were identified as infected by culture methods; 55 of these (47.0%) were AGID test-positive at some point during the study. The upper limit of the maximal false-positive rate for the AGID test was 2.1%. On the basis of colony counts from cultures, subclinically infected cows shedding higher numbers of M paratuberculosis in their feces were more likely to have positive AGID test results (P less than 0.0001). In known infected cattle, neither the culture nor AGID test results were consistently positive on repeated testing. Of 48 official calfhood paratuberculosis vaccinates tested as adults, 3 had positive AGID test results and in 1 of these, M paratuberculosis was also isolated from the feces, indicating that the rate of false-positive AGID test results in calfhood vaccinates is low.     

The use of beef bull semen reduced the risk of abortion in Neospora-seropositive dairy cows

There is an evidence that the epidemiology of neosporosis differs in dairy and beef cattle, such that beef cattle carry a lower risk of abortion. The aim of the present study was to establish whether artificial insemination using semen from beef bulls could reduce the risk of abortion in dairy cows seropositive for the Neospora caninum parasite. Our study was based on yearly serological screening for neosporosis and on the confirmation of Neospora infection in aborted fetuses in two high-producing dairy herds with a mean 28% seroprevalence of N. caninum antibodies. The study population comprised of 273 pregnancies in seropositive animals: 156 pregnancies monitored after insemination using Holstein-Friesian semen and 117 after insemination using beef bull semen. Abortion rates for these animals were 28.2% (77 of 273), 34.6% (54 of 156) and 19.7% (23 of 117). Logistic regression analysis indicated no significant effects of lactation number and previous abortion on the abortion rate. Based on the odds ratio, a 1-unit increase in the Neospora antibody titre yielded a 1.01-fold increase in the abortion rate. The likelihood of abortion was two times higher for cows in one of the two herds and 2.8 times lower (one of 0.36) for pregnant cows inseminated with beef bull semen rather than Holstein-Friesian semen. Our results indicate that the use of beef bull semen can reduce the risk of abortion in dairy cows, and suggest that annual screening for neosporosis, specifically the antibody titre to the protozoan, could be an useful predictor of abortion risk in reproductive health programmes.     

Predictive values for clinical signs of abomasal ulcer disease in adult dairy cattle

A 12-month prospective study was performed to determine the predictive values for clinical signs of abomasal ulcer disease in a hospital population of 296 adult dairy cows which were believed to have gastrointestinal dysfunction. There were 26 ulcer-positive cows (prevalence 8.8%). The occult blood test was a more reliable indicator of abomasal ulcer disease than were the abdominal pain or anemia tests. Only three ulcer-positive cows were negative for all three tests.

Forty-five cows were positive for at least one of the three clinical tests. The principal diseases present in the 22 cows that had false-positive results were traumatic reticuloperitonitis, abomasal displacement, liver disease, cecal volvulus and pneumonia or pleuritis.     

中国荷斯坦奶牛CCR5基因克隆、生物信息学及表达分析

试验旨在克隆中国荷斯坦奶牛CC趋化因子受体5(C-C chemokine receptor type 5,CCR5)基因,对其进行生物信息学分析,并探究CCR5基因在奶牛炎性和健康组织中的表达水平。采用PCR技术扩增并克隆荷斯坦奶牛CCR5基因CDS区全长序列,连接pMD18-T载体并转化大肠杆菌DH5α感受态细胞,通过蓝白斑方法筛选阳性克隆并测序,对序列进行相似性比对及系统进化树构建;应用多种在线生物信息学软件对其编码蛋白进行分析,并利用实时荧光定量PCR方法检测CCR5基因在健康和炎症奶牛乳腺组织中的表达情况。结果显示,中国荷斯坦奶牛CCR5基因CDS区全长1 059 bp,编码352个氨基酸。相似性和遗传进化分析结果显示,奶牛CCR5基因与绵羊的遗传距离最近,高达96.0%,与鸡遗传关系最远,为61.0%,且在不同物种之间CCR5基因高度保守。中国荷斯坦奶牛CCR5蛋白分子质量为40.235 ku,理论等电点(pI)为9.30,为疏水性蛋白但不是分泌蛋白,主要存在于细胞质内;在CCR5蛋白二级结构中α-螺旋和无规则卷曲分别占51.14%和32.95%,三级结构模型预测结果与二级结构一致。实时荧光定量PCR结果显示,CCR5基因在健康奶牛乳腺组织中的表达量极显著低于炎性奶牛乳腺组织(P<0.01),提示其可能参与奶牛乳腺炎的发生过程。本试验结果为进一步研究奶牛CCR5蛋白的功能提供了理论依据,对探究奶牛CCR5基因在奶牛乳腺炎中的调控功能等具有重要意义。