The possible role of bovine herpesvirus type‐4 infection in cow infertility

Experimental and field studies have proven that the bovine herpesvirus type‐4 (BHV‐4) infection leads to various reproductive system problems. In this study, the role of BHV‐4 infection in repeat breeding was investigated serologically. Eighty‐four samples were obtained from repeat‐breeding diagnosed cows in two organized dairy herds; an equal number of healthy cows were sampled from the same farms. The rest of the samples (105) were obtained from reproductively normal cows that were breeding in 18 small enterprises as a control group. The seropositivity proportion in repeat‐breeding diagnosed cows was found to be significantly higher (69% (58/84)) than other cows (44% (37/84)) on the same farms. The lowest antibody positivity value for BHV‐4 was detected as 24.7% (26/105) in the samples from family‐type small farms. The odds ratio (OR) value was calculated as 2.834 in repeat‐breeding diagnosed and healthy cows on the same farms, while 6.778 was determined in cows with and without reproductive problems on organized farms compared to small farms. As a result, the BHV‐4 infection can be considered one of the reasons for repeat breeding besides other reproductive disorders.     

Bovine herpesvirus 4-associated postpartum metritis in a Spanish dairy herd

In more than 10 Spanish dairy cows, a bovine herpesvirus 4 (BHV4) associated postpartum metritis was confirmed by virus isolation, BHV4-glycoprotein B (gB) PCR and/or serology. In this study, 12 cows with, and, at the time of sampling, 3 cows without clinical signs of acute postpartum metritis from one large dairy herd in Spain were examined for bacterial and viral infections. Blood, placenta/caruncles and uterine contents were collected between day 1 and day 20 post-calving, and examined for the presence of bacteria and for viruses by virus isolation, BHV4 DNA by BHV4-gB PCR and/or BHV4 antibody titres. Bovine herpesvirus 4 was detected in 83% of the cases with clinical signs of acute postpartum metritis by virus isolation and/or BHV4-gB PCR. An increase of BHV4 antibodies was detected in all examined postpartum metritis cows and in the 3 cows without clinical metritis. Two of these 3 cows developed severe metritis a few dayss after collecting the first blood sample. A concurrent infections of BHV4 and bacteria, mainly Arcanobacterium pyogenes and Streptococcus sp., were detected in 73% of the examined uterine contents collected from postpartum metritis affected cows. This case-report study showed a clear association between BHV4 infections and acute postpartum metritis in dairy cows. In addition, the BHV4-associated postpartum metritis appeared to be an emerging syndrome in this Spanish herd.     

牛疱疹病毒I型二重LUX荧光PCR鉴别检测方法的建立

采用LUX新型荧光PCR技术原理，建立了快速检测牛疱疹病毒I型（BHV-1）以及鉴别野毒感染和基因缺失疫苗免疫动物的二重实时荧光PCR方法。结果显示，该方法对多株BHV-1病毒均呈典型gE、gC双基因阳性反应，对其他常见动物疱疹病毒以及健康牛基因组DNA等均呈阴性反应；对细胞增殖病毒液的gE、gC双基因鉴别的检测敏感性可达0．4～O．04TCID50比常规PCR方法高100倍以上；对带毒牛血清、抗凝全血、牛新鲜精液和冷冻精液基因鉴别的检测敏感性分别达0．04TCID50、0．4TCID50、0．4TCID50和4TCID50。采用该方法从临床牛血样、鼻拭子样品中检出IBRV阳性样品，检测全程仅需约2h。对单基因克隆质粒的检测进一步证实该方法能特异地鉴定gE、gC基因，检测灵敏度分别达90、30拷贝。结果表明，该方法可应用于临床快速诊断BHV-1病毒感染，鉴别BHV-1病毒感染与对应的基因缺失疫苗免疫动物。     

Molecular detection of BHV-1 in artificially inoculated semen and in the semen of a latently infected bull treated with dexamethasone

Two polymerase chain reaction (PCR) assays specific for glycoprotein B (gB) and glycoprotein E (gE) gene detection, respectively, were adopted for the detection of bovine herpesvirus-1 (BHV-1) in naturally infected bulls. The methods were tested on bovine semen artificially inoculated with BHV-1 and were compared with an optimised virus isolation method. Raw and extended semen samples were diluted in minimal essential medium (MEM) and spiked with equal dose of BHV-1. The extended semen was found to be more toxic for the cells than the raw semen, while the viral DNA could be detected by the PCR method in all tested dilutions of raw and extended semen samples. The sensitivity of both methods was compared also for BHV-1 detection in semen, nasal swabs and leucocytes of a seropositive bull in a different time period after virus reactivation with dexamethasone treatment. The sensitivity of virus detection by the PCR method was equivalent to that of virus isolation in cell culture. However, PCR was shown to be faster and easier to perform and may be a good alternative to virus isolation especially when bovine semen has to be screened for BHV-1 prior to artificial insemination.     

Detection of bovine herpesvirus type 4 antibodies and bovine lymphotropic herpesvirus in New Zealand dairy cows

AIM: To detect the presence of bovine herpesvirus (BoHV) type 4 in New Zealand dairy cows with clinical metritis.

METHODS: Serum samples taken from 92 dairy cows with clinical metritis, each from a different farm, were tested for the presence of antibodies against BoHV-4 using a commercially available, indirect ELISA. Peripheral blood mononuclear cells (PBMC) were collected from 10 BoHV-4 seropositive cows, and PBMC were examined by a pan-herpesvirus nested PCR to detect herpesvirus. PCR products were sequenced directly and a proportion of the PCR products were cloned and sequenced to identify the virus present.

RESULTS: Antibodies to BoHV-4 were detected in 23/92 (25%) serum samples. The pan-herpesvirus PCR was positive in 8/10 PBMC samples. Cloning and sequencing identified that all of the eight PCR-positive PBMC contained bovine lymphotropic herpesvirus (BLHV); no BoHV-4 DNA was detected.

CONCLUSIONS: This study reports the finding of the presence of apparent antibodies to BoHV-4, and BLHV DNA in New Zealand dairy cows affected by metritis.

CLINICAL RELEVANCE: Bovine herpesvirus type 4 and BLHV are reported to have the potential to cause reproduction failure in cows. This is the first report of apparent BoHV-4 antibodies, and BLHV in New Zealand. The importance and epidemiology of these viruses in cattle in New Zealand requires further investigation.     

An investigation of the aetiological role of bovine herpesvirus 4 in bovine endometritis

Uteri from 31 infertile cattle were examined for the presence of bovine herpesvirus 4 (BoHV-4) by nested polymerase chain reaction (PCR). Samples were also tested for bacteria, including chlamydiae and Mycoplasma bovis. BoHV-4 was detected by PCR in 27/31 (87.1%) samples, but the presence and amount of viral DNA was not correlated with histological and bacteriological findings. Arcanobacterium pyogenes, Histophilus somni and Pasteurella multocida were isolated from five cows with endometritis. Chlamydiae were detected in four cases (12.9%), but only two of these had endometritis. The study does not support a role for BoHV-4 as primary agent in bovine endometritis.     

Feeding Saccharomyces cerevisiae fermentation products lessens the severity of a viral–bacterial coinfection in preweaned calves

We have previously reported that supplementation with Saccharomyces cerevisiae fermentation products (SCFP) ameliorates clinical signs and lung pathology following experimental bovine respiratory syncytial virus (BRSV) infection in preweaned dairy calves. The objectives of this study were to determine the effect of SCFP supplementation on the metabolic and endocrine responses, and disease outcome of a viral–bacterial coinfection in preweaned calves. Twenty-seven, 1- to 2-d-old Holstein-Angus cross calves were enrolled in the study; one SCFP calf was removed from the trial during the pre-challenge phase due to complications from nephritis. Calves were assigned to two treatment groups: control or SCFP-treated, base milk replacer with 1 g/d SCFP (Smartcare, soluble formula) and calf starter top dressed with 5 g/d SCFP (NutriTek, insoluble formula). Calves were infected with BRSV on day 21, followed 6 d later by intratracheal inoculation with Pasteurella multocida (PM). Calves were euthanized on day 10 post-viral infection. Calves receiving SCFP had reduced thoracic ultrasonography scores on day 7 post-viral infection (P = 0.03) and a tendency toward reduced scores on day 10 post-viral infection (P = 0.09). Calves receiving SCFP also had less severe lung pathology scores at necropsy (P = 0.06). No differences between treatments were observed in lung viral loads (P = 0.48) or bacterial lung recovery (P = 0.34); however, there was a distinction in the lung location for PM recovery, with PM isolated more frequently from the cranial lobes in SCFP-treated calves, but more frequently from the caudal lobes of control calves. Calves treated with SCFP tended (P = 0.07) to have higher serum IL-6 concentrations following the coinfection. Calves treated with SCFP had lower concentrations of serum nonesterified fatty acids and beta-hydroxybutyric acid compared with controls following experimental challenge (P = 0.03 and P = 0.08, respectively), suggesting metabolic changes favoring growth and development. There were no differences between groups in gene expression of insulin receptor, insulin-like growth factor 1 (IGF-1), IGF-1 receptor (IGF-1R), growth hormone receptor, or haptoglobin in the liver. Results from this study suggest that supplementing with SCFP may moderate the impact of a respiratory viral–bacterial coinfection on preweaned calves through metabolic and immune modifications.     

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID₅₀ tissue culture infective dose, 50%     

Oral administration of Lactobacillus plantarum and Bacillus subtilis on rumen fermentation and the bacterial community in calves

The objective of this study was to assess the effect of dietary probiotics on rumen fermentation and the bacterial community in dairy calves. Twelve Holstein calves were randomly allocated to three treatments: a basal diet, the basal diet supplemented with Lactobacillus plantarum GF103 (LB) or basal diet supplemented with a mixture of Lactobacillus plantarum GF103 and Bacillus subtilis B27 (LBS). A milk replacer was fed to calves from 8 days of age. A starter and alfalfa hay was offered ad libitum from 21 and 28 days of age, respectively, and the orts were weighted daily. The ruminal fluid was sampled at 56 and 83 days of age to determine the rumen fermentation characteristics. The bacterial community was analyzed by denaturing gradient gel electrophoresis (DGGE) and the number of certain bacteria was quantified by real‐time polymerase chain reaction. The ratio of total dry matter intake to average body wieght was higher in the control (P < 0.05). The DGGE fingerprint of the 16S ribosomal RNA gene was affected by the blended probiotics at 83 days of age. The number of Ruminococcus albus was lower in the LB and LBS treatment (P < 0.05). Oral administration of the probiotics affected the rumen bacterial community and the numbers of cellulolytic bacteria decreased.     

猪圆环病毒3型荧光定量PCR检测方法的建立及其在猪体中的组织分布研究

为了解猪圆环病毒3型(PCV3)在猪体中的组织分布情况,针对PCV3 Cap蛋白基因设计筛选出一对特异性引物和探针,通过对荧光PCR引物、探针浓度进行优化,建立了基于TaqMan探针实时荧光定量PCR技术的PCV3检测方法,并应用该方法对人工感染PCV3猪的多种组织器官进行病毒含量检测。结果显示,建立的PCV3实时荧光定量PCR检测方法特异性较好且灵敏度高,可检测低至1 copy/μL的PCV3病毒核酸量,而对其他几种常见猪病毒病原的检测结果均为阴性。PCV3主要存在于肺脏、淋巴结,在扁桃体和脾脏中有少量存在。试验表明,所建立的荧光定量PCR检测方法可用于PCV3的快速定量检测,对PCV3在猪体中的组织分布情况的研究为进一步揭示PCV3的组织嗜性和致病机理提供理论依据。     

Effects of two different rearing protocols for Holstein bull calves in the first 3 weeks of life on health status,metabolism and subsequent performance

The aim of this study was to investigate the impact of weight gain of calves within the first 3 weeks of life on health status and subsequent performance. Holstein bull calves were reared either intensively (IR; individual hutches and ad libitum milk feeding for the first 3 weeks of life; n = 24), or according to the established protocol [ER; 4 l milk/day in hutches during week 1 and 720 g/day milk replacer (MR) from day 8 to 21 in a group pen; n = 24]. Water, hay and concentrates were freely available to all calves. From week 4, calves of both groups were housed together in a group pen and fed 720 g MR/day; step‐down weaning was performed between week 5 and 10. Key metabolic blood parameters were analysed on day 2, 12, 21 and 70 of life. After weaning, all animals were fed concentrates and corn silage until slaughter at an age of 8 months. Within the first 3 weeks, average daily weight gain was threefold higher in IR calves in relation to ER calves (1.28 vs. 0.38 kg/day, p < 0.001). Neither incidence nor duration of scouring differed significantly between groups. Starter intake (week 4–10) was higher in IR calves in relation to ER calves (49.7 vs. 38.0 kg/calf, p = 0.006). Serum glucose, urea, albumin and insulin were higher at an age of 21 days in IR calves in relation to ER calves; no differences were obvious at an age of 70 days. Plasma GH and IGF‐I concentrations revealed an uncoupling of the somatotropic axis in ER calves within the first 3 weeks of life. At slaughter, body weight of IR calves tended to be higher than that of the ER calves (320 vs. 309 kg, p = 0.07). In conclusion, intensive feeding and individual housing during the first 3 weeks of life had positive long‐term effects on subsequent performance.     

The studies on the aetiology of diarrhoea in neonatal calves and determination of virulence gene markers of Escherichia coli strains by multiplex PCR

The purpose of this study was to determine aetiological agents of diarrhoea in neonatal calves and to investigate virulence gene markers of Escherichia coli strains isolated from calves by multiplex polymerase chain reaction (PCR). Eighty-two diarrhoeic calves and 18 healthy calves were used as subjects. Faeces were taken from the rectums of all the calves and were subjected to bacterial culture. Antigen enzyme-linked immunosorbent assay (ELISA) was performed to detect rotavirus, coronavirus and E. coli K99 in faeces of all the calves. A multiplex PCR was used to characterize E. coli strains in all the calves. Escherichia coli was isolated from 37 faeces samples, Enterococcus ssp. was isolated from 22 faeces samples and Salmonella was isolated from one faeces sample in diarrhoeic calves. Furthermore, only E. coli was isolated from all 18 faeces samples of healthy calves. Of the 37 E. coli isolated from diarrhoeic calves, K99 (18.9%), F41 (18.9%), heat-stable enterotoxin a (STa) (18.9%), Shiga toxin 1 (Stx1; 13.5%) and Shiga toxin 2 (Stx2; 5.4%) and intimin (8.1%) genes were identified by multiplex PCR. Of the 18 E. coli isolated from healthy calves, K99 (16.6%) and intimin (55.5%) genes were identified by PCR. A total of 15 rotavirus, 11 coronavirus and 11 E. coli K99 were detected in diarrhoeic calves by the antigen ELISA. As a result, this study shows that rotavirus, coronavirus, E. coli and Enterococcus ssp. were determined to play a role in the aetiology of diarrhoea in the neonatal calves. K99, F41, STa, Stx1 and Stx2 were found as the most common virulence gene markers of E. coli strains isolated from calves with diarrhoea. Multiplex PCR may be useful for characterization of E. coli isolated from calves.     

禽腺病毒血清4型感染鸡组织中NLRP3基因转录水平的实时荧光定量PCR分析

旨在分析禽腺病毒血清4型(FAdV-4)感染鸡组织中NLRP3基因的转录水平,本研究设计鸡NLRP3特异性引物,利用RT-PCR扩增NLRP3基因180 bp片段并克隆至pMD-18T载体,制备重组质粒pMD-18T-NL-RP3.以pMD-18T-NLRP3质粒作为标准品进行荧光定量PCR并建立标准曲线.通过反应条件...     

Extension of the culture period for the in vitro growth of bovine oocytes in the presence of bone morphogenetic protein‐4 increases oocyte diameter,but impairs subsequent developmental competence

Bone morphogenetic protein‐4 (BMP‐4) inhibits luteinization of granulosa cells during in vitro growth (IVG) culture of bovine oocytes; however, oocytes derived from a 12 day IVG were less competent for development than in vivo‐grown oocytes. We herein investigated whether an extended IVG culture with BMP‐4 improves oocyte growth and development to blastocysts after in vitro fertilization. Oocyte‐granulosa cell complexes (OGCs) were cultured for 14 or 16 days with BMP‐4 (10 ng/mL), while a 12 day culture with BMP‐4 served as the in vitro control. OGC viability was maintained for the 16 day culture with BMP‐4 (83.2%), but was significantly lower without BMP‐4 (58.9%) than the control (83.0%). Prolong‐cultured oocytes at 16 days had statistically greater diameter (114.6 μm) than the control (111.7 μm). IVG oocytes with BMP‐4 for the 16 day culture had a similar nuclear maturation rate to the control (approximately 67%); however, blastocyst rates in BMP‐4 treated oocytes of 14 (1.8%) and 16 day (0%) IVG were statistically lower than that of 12 day IVG (9.0%). In conclusion, BMP‐4 maintained OGC viability and promoted oocyte growth in a prolonged culture, but impaired the developmental competence of oocytes. Prolonged culture may not be an appropriate strategy for enhancing the developmental competence of IVG oocytes.     

家禽骨髓内红细胞、白细胞发生的显微和亚显微结构特点

利用光镜和电镜对鹅、鸭和鸡骨髓内红细胞和白细胞发生的显微和亚显微结构进行了研究。结果显示 :鹅骨髓内红细胞系的体积比鸭的略大。光镜下鸭、鹅异嗜性粒细胞 ,在早幼阶段胞质内出现少量圆形颗粒 ;在晚幼阶段出现较多暗红色的圆形、杆状、梭形颗粒。嗜酸性粒细胞在早幼阶段胞质内出现少量着桔红色的圆形颗粒 ;晚幼阶段颗粒多呈杆状 ,胞核轮廓清楚。嗜碱性粒细胞在各阶段胞质内散布紫红色的细小颗粒。电镜下鸡原始红细胞多附着在窦壁上 ,核周隙窄 ,核孔数量较多 ;而成熟红细胞多分布在近血窦中央处 ,核周隙宽 ,核孔数量较少。异嗜性颗粒在早幼阶段可分 A型和 B型。嗜碱性颗粒分为 型和 型 ,它们分别在中幼和晚幼阶段出现。嗜酸性颗粒呈均质的圆形     

Production of bioactive bovine fibroblast growth factor 4 in E. coli based on the common nucleotide sequence of its structural gene in three breeds

Fibroblast growth factor 4 (FGF4) is considered a crucial gene in the proper development of bovine embryos. We recently determined the FGF4 gene sequence in eight cattle derived from three breeds and revealed a common nucleotide sequence of the structural gene encoding FGF4, which leads to the deletion and mutation of amino acid sequences in the mature FGF4 (Pro³²‐Leu²⁰⁶) compared with the sequence previously reported. In the present study, HisbFGF4, a 6× histidine‐tagged bovine FGF4 (Pro³²‐Leu²⁰⁶), was produced in Escherichia coli based on the validated nucleotide sequence and purified by heparin column chromatography. In primary bovine fibroblasts, HisbFGF4 showed significant mitogenic activity, whereas, intriguingly, the activity of a commercially available recombinant human FGF4 (Gly²⁵‐Leu²⁰⁶) produced in E. coli was weaker than that of HisbFGF4. In conclusion, the present study provides a simple method for the production of a bioactive bovine FGF4 derivative in E. coli utilizing its structural gene elucidated by us.     

凉山州腹泻仔猪猪圆环病毒2型感染情况调查

为了解凉山州腹泻仔猪猪圆环病毒2型(PCV2)的感染情况,采用PCR方法对采自四川省凉山州西昌市和喜德县7个猪场85份腹泻仔猪粪便、病变组织等样品进行PCV2检测,并将扩增到的PCV2 ORF2基因片段进行测序和序列分析。结果显示,PCV2阳性样品有22份,阳性率为25.9%,阳性猪场占71.4%(5/7)。22个ORF2基因片段长度均为459 bp,核苷酸同源性为92.8%～100%,氨基酸同源性为93.5%～100%。构建的系统进化树显示22个测序序列分别处于2个分支:PCV2b和PCV2d,但未形成明显的地理分支。结果表明凉山州猪群中PCV2感染较为普遍,且以PCV2d亚型为主,并存在一定程度的遗传变异。     

家兔BMP7成熟肽编码区克隆及BMP7、BMP4基因组织表达谱分析

用RT-PCR方法克隆家兔BMP 7基因成熟肽编码区cDNA序列,并对2月龄家兔BMP 7和BMP 4基因的组织表达谱进行半定量分析。结果表明,家兔BMP 7成熟肽编码序列长414 bp,与人、小鼠BMP 7的同源性分别为91.89%、89.32%,三者的同源性为94.98%。在家兔心、肝、脾、肺、肾、脑、脊髓、小肠8种组织中检测到BMP 7和BMP 4基因表达。BMP 7基因在心、脾组织以低丰度表达,在肺、脊髓、十二指肠以中等丰度表达,在肝、肾、脑中以高丰度表达;BMP 4基因在脊髓以低丰度表达,在脑、心、脾、小肠等器官组织中呈中等丰度表达,在肝、肺、肾中以高丰度表达。因此,家兔BMP 7基因成熟肽编码区在进化过程中高度保守。BMP 7和BMP 4具有广泛的组织表达谱和组织表达特异性。     

黄淮山羊骨形态发生蛋白4结构与功能分析

基于克隆得到的黄淮山羊骨形态发生蛋白(BMP4)全长cDNA序列,推导氨基酸序列,运用生物信息学方法从蛋白质理化性质、信号肽、结构域、Motif、二级和三级结构预测等方面对BMP4结构和功能进行分析。结果表明,BMP4具有信号肽、TGFβ超家族7个保守半胱氨酸结构特征和潜在的糖基化等位点,无跨膜区,成熟肽与模板2h62A有91.176%的氨基酸序列一致。因此,山羊BMP4具有BMPs家族的典型结构特征,这些结构特征构成了该蛋白在山羊软骨和骨组织形成、原始卵泡发育及早期胚胎发育中发挥作用的基础。