Association of BoLA-DRB3 alleles identified by a sequence-based typing method with mastitis pathogens in Japanese Holstein cows

The association of the polymorphism of bovine leukocyte antigen ( BoLA-DRB3 ) genes identified by the polymerase chain reaction sequence-based typing (PCR-SBT) method with resistance and susceptibility to mastitis caused by pathogenic bacteria was investigated. Blood samples for DNA extraction were collected from 194 Holstein cows (41 healthy cows and 153 mastitis cows including 24 mixed-infection cows infected with 2 or 3 species of pathogens) from 5 districts of Chiba prefecture, Japan. Sixteen BoLA-DRB3 alleles were detected. The 4 main alleles of DRB3*0101 , *1501 , *1201 , and *1101 constituted 56.8% of the total number of alleles detected. Mastitis cows were divided into 2 groups: group 1 with single-infection cows and group 2 with all mastitis cows including 24 mixed-infection cows. The differences in the frequencies of BoLA-DRB3 alleles and the number of cows homozygous or heterozygous for each BoLA-DRB3 allele between healthy cows and the 2 groups of mastitis cows were evaluated. Furthermore, similar comparisons were performed between healthy cows and the 2 groups of mastitis cows for each mastitis pathogen. It was considered that the 4 alleles, namely, DRB3*0101 , *1501 , *1201 , and *1101 had specific resistance and susceptibility to 4 different mastitis pathogens. Thus, DRB3*0101 might be associated with susceptibility to coagulase-negative Staphylococci and Escherichia coli , and DRB3*1501 might be associated with susceptibility to Escherichia coli . However, DRB3*1101 might be associated with resistance to Streptococci and coagulase-negative Staphylococci , and DRB3*1201 , with resistance to Streptococci , Escherichia coli , and Staphylococcus aureus .     

Presence of glutamine at position 74 of pocket 4 in the BoLA-DR antigen binding groove is associated with occurrence of clinical mastitis caused by Staphylococcus species

Potential relationships between amino acid motifs in the antigen binding groove of various alleles of the bovine major histocompatibility complex DR (BoLA-DR) molecule and occurrence of clinical mastitis caused by Staphylococcus species (non-Staphylococcus aureus) were investigated in a case-control study. A significant association (P< or =0.05) was detected between the presence of glutamic acid at position beta 74 and occurrence of mastitis caused by Staphylococcus spp. with a relative risk of 11. This motif is present in BoLA-DRB3.2*22, *23 and *24 alleles. Presence of a positively charged residue (arginine or lysine) at position 13 also showed a tendency (P< o r=0.1) towards an association with a higher risk of clinical mastitis caused by the same bacteria. This motif is present in BoLA-DRB3.2*23 and *8 alleles. Similarly, presence of arginine at position beta 71 (present in alleles *23 and *22) was associated with occurrence of this disease. These positions (beta 13, beta 71 and beta 74) form pocket 4 of the antigen binding groove, which plays an instrumental role in antigen binding and recognition by T-lymphocytes. Thus, it can be concluded that pocket 4 of the BoLA-DR molecule is involved in conferring susceptibility to clinical mastitis caused by Staphylococcus spp.     

Detection of bovine leukocyte antigen DRB3 alleles as candidate markers for clinical mastitis resistance in Holstein x Zebu

Bovine leukocyte antigen DRB3 alleles from Holstein x Zebu crossbred dairy cows (n = 409) were analyzed using the PCR-RFLP technique. Exon II of DRB3 was amplified using locus-specific primers (HLO30/HLO32), followed by digestion with 3 restriction enzymes (RsaI, BstyI, and HaeIII). Forty alleles were found with frequency ranging from 0.005 to 0.139. The most frequently detected alleles of Holstein x Zebu were DRB3*16, *51, *23, *11, *8, and *1, accounting for 61.12% of the alleles in the population. Detection of candidate alleles for clinical mastitis occurrence was performed by logistic regression. It was found that percentage of Holstein fraction in crossbred cows had a nonsignificant effect (P > 0.05). However, parity had a significant effect on mastitis occurrence. In addition, DRB3*1 and *52 were the most associated with the occurrence of clinical mastitis, whereas *15, *51, and *22 were associated with resistance in crossbred populations. This is the first report of association of DRB3*15 and *51 with mastitis resistance. The association was validated by examining the candidate alleles in another commercial population. Highly susceptible (n = 43) and resistant (n = 42) groups of Holstein x Zebu cows were investigated. The result confirmed that DRB3*1 and *52 could be considered as susceptibility alleles, whereas *15, *51, and *22 could be considered as resistant alleles in Holstein x Zebu raised under tropical conditions. In addition, allele effects on 305-d milk production were estimated by BLUP. It was shown that most alleles associated with high clinical mastitis occurrence were related to increased milk yield. This study revealed that allele DRB3*10 had the greatest effect on increasing milk yield with moderate resistance to clinical mastitis, which could be used as a potential marker for selection in dairy genetic evaluation.     

Study on the association of BoLA-DRB3.2 alleles with clinical mastitis in Norwegian Red cows

Genotyping of bovine leucocyte antigen DRB3.2 (BoLA-DRB3.2) in a total of 523 Norwegian Red (NR) cows from two groups selected for high protein yield and low clinical mastitis, respectively, identified 27 previously reported BoLA-DRB3.2 alleles across the groups. Significant differences in BoLA-DRB3.2 allele frequencies were found between the selection groups. Alleles *13, *18, *22 and *27 had a significantly higher frequency in cows selected for low clinical mastitis, while alleles *3, *9, *11 and *26 had a higher frequency in cows selected for high protein yield. Associations between BoLA-DRB3.2 alleles and clinical mastitis were analysed based on mastitis data from 741,072 first-lactation NR cows, of which 452 were genotyped. Alleles *22 and *26 were found to be associated with increased clinical mastitis, while alleles *7, *11, *18 and *24 had a favourable effect on mastitis resistance. Contradictory results from different studies investigating associations between BoLA-DRB3.2 alleles and mastitis indicate that future studies should focus on associations of mastitis with BoLA haplotypes rather than with single BoLA genes.     

Allelic frequency of PCR-RFLP type of the BoLA-DRB3 in Japanese Holstein herds and the relation to mastitis

Holstein Cows ( n  = 702) from 26 dairy herds in the Tama area of Tokyo, Japan were examined for polymorphisms of the BoLA-DRB3 allele using a PCR-RFLP method. Twenty alleles were observed and allelic frequencies ranged from < 1% to 20.3%. Nine alleles ( DRB3.2 * 24, * 16, * 8, * 23, * 22, * 3, * 11 , * 10 and * 7 in order) constituted 90.0% of all alleles. Somatic cell counts (SCC) were used to classify healthy (group 1), mastitis (group 2) and suspected (group 3) cows. Frequencies of DRB3.2 * 11 and DRB3.2 * 23 were slightly higher in group 1 than in group 2, whereas, frequencies of DRB3.2 * 8 and DRB3.2 * 16 were slightly higher in group 2 than in group 1. However, none of the differences in frequencies between the two groups were statistically significant. For combinations of alleles, frequencies of DRB3.2 * 8/ * 23 ( P  < 0.1) and DRB3.2 * 16/ * 24 ( P  < 0.05) were significantly higher in group 2 than in group 1, and their odds ratios were 2.1, 2.5, respectively. However, there were no significant differences between genotypes in their effects for SCC. On the other hand, frequency of DRB3.2 * 23/ * 23 including combinations of DRB3.2 * 23 with minor alleles was significantly higher in group 1 than in group 2 ( P  < 0.01), and the odds ratio was 0.3. Therefore, it was considered that mastitis resistance or susceptibility of cows may vary with the combination of BoLA-DRB3 alleles.     

BoLA-DQA、DRB3* exon2多态性及其与奶牛乳房炎的关联分析

采用PCR-SSCP技术，检测到9种中国荷斯坦牛BoLA-DQA^＊ exon2基因基因型、15种BoLA-DRB3^＊ exon2基因的基因型和3种单体型，分析比较了各基因型、单体型与奶牛乳房炎的关联性。结果在中国荷斯坦牛中，没有发现与乳房炎易感性或抗性有关联的基因和基因型，但发现DQA-B／DRB3-C、DQA-G／DRB3-F两种单体型可能与奶牛乳房炎易感性有关，而DQBF／DRB3-E单体型可能与奶牛乳房炎抗性有关。     

The Distribution of Staphylococcus intermedius and Coagulase-negative Staphylococci on the Hair, Skin Surface, within the Hair Follicles and on the Mucous Membranes of Dogs

Abstract— The distribution of Staphylococcus intermedius and coagulase-negative staphylococci on the hair, skin surface, within the hair follicles and on the mucous membranes of a group of dogs is reported. Staphylococcus intermedius was recovered in higher numbers from the distal hair (mean 0.5 log₁₀ colony forming units plus one [C.F.U. g^-1]) rather than the proximal hair (mean 0.13 log₁₀ C.F.U. g^-1), P = 0.01. Staphylococcus intermedius was recovered in higher numbers from the hair follicle (mean 0.11 log₁₀ C.F.U. g^-1) rather than the skin surface (mean 0.02 log₁₀ C.F.U. g^-1), P = 0.02. No such difference was found for coagulase-negative staphylococci. Differences were not found in the distribution of either Staphylococcus intermedius or coagulase-negative staphylococci on the mucous membranes.     

A new variant in exon VII of bovine β-casein gene (CSN2) and its distribution among European cattle breeds

An undescribed bovine CSN2 variant was detected and characterized. It is based on a mutation in exon VII, which leads to a substitution of the aminoacid 93 (Met→Leu) in comparison to allele CSN * A² . The new allele was named CSN2 * I . A PCR-SSCP based DNA-test for the differentiation of the common alleles A¹, A², A³, B, C, and of the new variant I was developed. Genotyping of 318 unrelated animals belonging to seven European breeds (Italian Holstein Friesian, Italian Red Pied, Piemontese, Angler, Maremmana, Polish Red and White, Polish Red) showed a wide distribution of the new variant with a frequency up to 0.14 in Italian Red Pied.     

Association of BoLA‐DRB3 alleles with mastitis resistance and susceptibility in Japanese Holstein cows

In this study, 714 cows from 26 dairy herds were reclassified as healthy or mastitic cows on the basis of long‐term somatic cell count (SCC) in milk. Cows with more than three consecutive lactation records of SCC from the first or second to fifth lactation, were selected, and their BoLA‐DRB3 (DRB3) alleles were identified using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method. Cows with an SCC of < 200 000 cells/mL in all monthly records were classified as healthy (n = 91). Cows with an SCC of > 300 000 cells/mL in two consecutive tests or four non‐consecutive tests or cows with an SCC of > 500 000 cells/mL in any one test during lactation, regardless of parity, were classified as mastitic (n = 201). Mastitic cows (n = 153) from another 40 herds were considered to be infected if bacteriological testing revealed mastitis pathogens in milk. Their DRB3 alleles were identified using PCR‐sequence‐based typing (PCR‐SBT). The differences in DRB3 allelic frequencies between healthy cows and cows with various degrees of mastitis were re‐investigated. Moreover, the associations of various amino acid motifs in DRB3 alleles with resistance or susceptibility to mastitis pathogens were re‐examined. DRB3.2*8(DRB3*1201) and DRB3.2*16(DRB3*1501) alleles were found to be associated with susceptibility, while DRB3.2*22(DRB3*1101), DRB3.2*23(DRB3*2703), and DRB3.2*24(DRB3*0101) alleles were found to be associated with resistance.     

Effect of nonessential amino acids on nitrogen retention in growing pigs fed on a protein-free diet supplemented with sulphur amino acids, threonine and tryptophan

Two N balance experiments were conducted on growing pigs to study the effect of essential and nonessential amino acids added to a protein-free diet on N retention. In Expt. 1, the addition of sulphur amino acids, threonine and tryptophan to a protein-free diet at levels two times the maintenance requirements reduced (p > 0.1) daily N loss from –131 to –108 mg/kg^0.75. A further addition of nonessential amino acids equivalent to 250 mg N/kg^0.75/d resulted in a marked increase (p < 0.01) in daily N retention to 28 mg/kg^0.75. In Expt. 2, nonessential amino acids were added to a protein-free diet supplemented with sulphur amino acids, threonine and tryptophan at levels corresponding to 100, 200 and 300 mg N/kg^0.75/d. N retention increased linearly as dietary nonessential N increased. The slope of the best-fit regression line indicated that the marginal efficiency of nonessential N utilization for protein accretion was 0.26. The results suggest that nonessential amino acids may be a limiting factor for the re-utilization of amino acids released by body protein breakdown or that they may serve as precursors for de novo synthesis of amino acids by gut microorganisms, thus contributing to the amino acid requirements of the pig.     

Post-prandial serum bile acid concentrations and ammonia tolerance in Maltese dogs with and without hepatic vascular anomalies

SUMMARY: Post-prandial serum bile acid concentrations were measured in 200 Maltese dogs in an attempt to identify those with subclinical portosystemic shunts. Five of these were later shown to have hepatic pathology or abnormal liver function. In the other 195 Maltese bile acid concentrations ranged from 1 to 362 μmol.L^-1 (mean ± SD, 70 ±50 μmol.L^-1; median, 65.0 μmol.L^-1). Of these, 79% were above the reference range (0 to 31 μmol.L^-1) established from 23 mixed-breed control dogs. It was therefore not possible to determine the prevalence of subclinical portosystemic shunts on the basis of bile acid determinations.
Further investigation of liver function was performed to investigate why bile acid concentrations were increased in these dogs. Rectal ammonia tolerance tests were normal in 102 of 106 Maltese tested and liver samples (11 dogs) and plasma biochemistry profiles (9 dogs) demonstrated no significant hepatic disease or dysfunction.
Of 2 Maltese with hyperammonaemia after administration of ammonium chloride, one had a large congenital portosystemic shunt that was confirmed at surgery. In the other there were no macroscopic portosystemic communications, but a liver biopsy showed histological changes consistent with microscopic portovascular dysplasia.
Total serum bile acid concentrations were consistently lower when assessed by highperformance liquid chromatography than by an enzymatic spectrophotometric method. This discrepancy was substantially larger in Maltese than in control dogs, suggesting the presence of an additional reacting substance in the serum of Maltese dogs.     

Evaluation of wraps covering the distal aspect of pelvic limbs for prevention of bacterial strike-through in an ex vivo canine model

Objective— To determine differences in bacterial strike-through for materials commonly used to cover the distal aspect of the pelvic limb during operative site preparation.
Study Design— Randomized block design; ex vivo model.
Animals— Canine cadaveric pelvic limbs (n=40).
Methods— Pelvic limbs (n=40) were randomly assigned to 4 treatment groups: Group 1=Vetrap^™+sterile Coban^™; Group 2=latex glove+Vetrap^™+sterile Coban^™; Group 3=latex glove+Vetrap^™+sterile Coban^™+sterile latex glove+sterile Coban^™; and Group 4=latex glove+Vetrap^™+sterile disposable drape+sterile Coban^™. Limbs were contaminated with a standardized bacterial solution and routinely prepared using the assigned distal leg wrap. Bandages were fluid challenged with a saline (0.9% NaCl) solution-soaked laparotomy sponge for 30 seconds. The wrap surface was sampled for microbial culture before surgical preparation, immediately after, and 60 minutes after applying a sterile leg wrap.
Results— Bacterial growth occurred in all Group 1 cultures, 90% of Group 2 cultures, and none of the Group 3 and 4 cultures, 60 minutes after applying the sterile wrap.
Conclusion— A distal leg wrap of Vetrap^™+sterile Coban^™ is not effective in preventing bacterial strike-through.
Clinical Relevance— If similar results occur in the live animal, then a sterile impermeable barrier must be incorporated into the distal leg wrap to prevent bacterial strike-through.     

北京黑猪MYH3和MYH13基因多态性与肉质性状的关联分析

【目的】 试验旨在研究肌球蛋白重链3（myosin heavy chain 3，MYH3）和肌球蛋白重链13（myosin heavy chain 13，MYH13）基因遗传变异对猪肉质性状的影响。【方法】 利用北京黑猪背最长肌样本进行肉质性状（肌内脂肪（intramuscular fat，IMF）、水分、滴水损失、酸碱度（pH）、肉色L^*值、肉色a^*值和肉色b^*值）表型数据的测定。利用PCR和Sanger测序技术对MYH3和MYH13基因启动子区和CDS区进行基因分型。将性别、场、日龄作为协变量，采用协方差分析，确定与猪肉质性状相关的SNPs，利用实时荧光定量PCR检测基因表达水平。【结果】 试验共筛选到9个SNPs，其中MYH3基因CDS区有1个错义突变（c.5782 G>C）与IMF显著相关（P<0.05），且MYH3基因表达水平与IMF呈正相关；MYH13基因CDS区有4个SNPs，其中2个（c.1923 G>A、c.1308 G>A）与肉色a^*值显著相关，1个（c.963 G>A）与滴水损失显著相关，1个（c.237 G>T）与肉色L^*值显著相关（P<0.05）；MYH13基因启动子区有4个SNPs，其中2个（rs699287502、rs318639161）与肉色L^*值显著相关，2个（rs321315318、rs330770991）与滴水损失显著相关（P<0.05），且MYH13基因表达水平与滴水损失呈负相关。【结论】 MYH3基因有1个SNP与IMF显著相关，且其基因表达水平与IMF呈正相关。MYH13基因中存在3个SNPs与滴水损失显著相关，且该基因启动子区2个SNPs基因表达水平与滴水损失呈负相关；3个SNPs与肉色L^*值显著相关；2个SNPs与肉色a^*值显著相关。以上SNPs均可作为影响北京黑猪肉质性状的候选基因功能位点。     

A comparison of the various routes of administration of erythromycin in cattle

A comparison of i.v., i.m. and s.c. administration erythromycin base in polyethylene glycol at 15 mg/kg and 30 mg/kg body weight was carried out in beef-type calves of approximately 200 kg body weight. Additional evaluations were carried out with oral administration of erythromycin phosphate and erythromycin stearate. Absorption of erythromycin was very slow by both the i.m. and s.c. routes of administration with a K_ab of 0.0135 min^-1 and 0.0185 min^-1 for i.m. and 0.0032 min^-1 and 0.0074 min^-1 for s.c. at 15 mg/kg and 30 mg/kg, respectively. The bioavailability (32–42%) and peak serum concentrations were much lower with s.c. than with i.m. (60–65%) administration. The disposition of erythromycin administered i.v. appeared to be representative of dose-dependent kinetics rather than dose-independent first-order kinetics inasmuch as the elimination half-time ( t _1/2B) increased from 174.5 ± 13 min for the 15 mg/kg dosage to 239 ± 10.8 min with 30 mg/kg dosage. An acute apparent cardiovascular effect accompanied i.v. administration of erythromycin at 30 mg/kg dosage but not at 15 mg/kg. Severe diarrhea followed oral administration of either erythromycin phosphate or erythromycin stearate.     

Cutaneous presentation of canine intravascular lymphoma (malignant angioendotheliomatosis)

A 9-year-old female spayed Boxer dog presented with variably sized, firm, black, raised, exudative subcutaneous masses on her head, neck and trunk, that tended to fluctuate in size and frequently ulcerate. Skin biopsy showed that the dermis was expanded by a densely cellular mass of proliferative capillaries distended with large pleomorphic neoplastic round cells mixed with fibrin and erythrocytes. Intravascular lymphoma was diagnosed and immunostains were compatible with a CD8⁺ T lymphocyte histogenesis (CD3⁺/CD79a⁻/TCRαβ⁺/CD8α⁺). Post-mortem examination, four months after diagnosis, revealed neoplastic T-cells within meningeal arteries. We are unaware of other reports of a cutaneous presentation and ante-mortem diagnosis of intravascular lymphoma in the dog. Additionally, this vasoproliferative form of intravascular lymphoma has not been previously described in dogs.     

High-Frequency Jet Ventilation in Anesthetized, Paralyzed Dogs and Cats Via Transtracheal and and Endotracheal Tube Routes

The ability of the SAV 6 high-frequency jet ventilator to effectively ventilate three anesthetized, paralyzed cats (3.2–4.2 kg), two small dogs (7.2 and 10.0 kg), six medium-sized dogs (20.5–25.0 kg), and three large dogs (36.0–43.0 kg) via a 14-gauge (dogs) or a 16-gauge (cats) catheter placed percutaneously into the trachea via the cricothyroid membrane or into a preplaced endotracheal tube was evaluated. The lowest driving pressure within the range of 0.25 to 2.0 kg/cm² (1 kg/cm²= 14.2 psi) and the highest cycle rate within the range of 60 to 240 per minute that would generate a PaCO₂ of 30 ± 3 mm Hg were determined.
All animals could be ventilated to a PaC0₂ of 30 ± 3 mm Hg by the endotracheal tube and transtracheal route, except the largest dogs, which couid be ventilated to an average PaC0₂ of 36 mm Hg by the transtracheal route. The transtracheal route consistently required higher driving pressures and lower cycle rates than did the endotracheal tube route. Cats could be ventilated with a driving pressure of 0.25 kg/cm²; small dogs could be ventilated with 0.5 to 1.0 kg/cm₂; medium-sized dogs with 1.0 to 1.5 kg/cm²; and large dogs with 1.5 to 2.0 kg/cm².
The SAV 6 high-frequency jet ventilator can effectively ventilate cats and dogs (7.2–43.0 kg) via a transtracheal catheter and an endotracheal tube.     

4株NDV流行株的分离鉴定、生物学特性及遗传学特性的研究

为研究近年国内的新城疫病毒（Newcastle disease virus,NDV）流行株与传统疫苗株（Mukteswar、La Sota及Clone 30）的亲缘关系和遗传演化情况,试验从广东、广西和海南地区患病的免疫鸡群病料中分离鉴定得到4株NDV流行株。通过RT-PCR技术对各毒株的F和HN基因进行扩增、克隆与序列分析,然后与GenBank上发表的NDV序列进行同源性比对、遗传进化树分析,并测定各毒株致病指数。结果显示,本试验中HN-08株的鸡胚半数感染量（EID₅₀）、平均死亡时间（MDT）、脑内致病指数（ICPI）和静脉致病指数（IVPI）分别为10^-8.37/0.2 mL、58.5 h、1.78和2.45,XX-08株分别为10^-6.50/0.2 mL、75.0 h、1.61和2.41,YS-09株分别为10^-7.75/0.2 mL、64.5 h、1.71和2.38,LF-09株分别为10^-7.60/0.2 mL、63.8 h、1.84和2.38。4株鸡源NDV流行株彼此间F和HN基因推导的氨基酸序列同源性在95.8%以上,与鸡源流行株GX11/03、GM株的同源性为96.8%~98.6%,而疫苗株Mukteswar、La Sota及Clone 30株的同源性为86.7%~90.6%,与标准强毒株F₄₈E₉的同源性为88.4%~91.3%。表明4株NDV流行株均为强毒株,与近年来国内流行的GX11/03、GM株有较近的亲缘关系,而与传统疫苗株的关系相对较远。     

Haematological findings in captive dolphins and whales

Objective To examine haematological features in five species of healthy, captive marine mammals.
Animals Twenty bottlenose dolphins ( Tursips truncatus ), seven Pacific white-sided dolphins ( Lagenorhynchus obliquidens ), five Risso dolphins ( Grampus griseus ) and five false killer whales ( Pseudorca crassidens ).
Results and conclusion The red blood cell count was 4.21 × 10¹²/L in bottlenose dolphins, 5.32 × 10¹²/L in Pacific white-sided dolphins, 4.35 × 10¹²/L in Risso dolphins and 4.43 × 10¹²/L in false killer whales. The haemoglobin concentration was 1.51 g/L and packed cell volume 44.7% in bottlenose dolphins; the corresponding values were 1.71 g/L and 48.9% in Pacific white-sided dolphins, 1.72 g/L and 49.4% in Risso dolphins, and 1.52 g/L and 47.8% in false killer whales. The white blood cell count was 7.097 × 10⁹/L in bottlenose dolphins, 5.928 × 10⁹/L in Pacific white-sided dolphins, 5.001 × 10⁹/L in Risso dolphins and 7.921 × 10⁹/L in false killer whales. There were no significant differences in these values among bottlenose dolphins and Pacific white-sided dolphins. The proportion of eosinophils in the differential leukocyte count ranged from 10.3% to 11.5% in bottlenose dolphins, Pacific white-sided dolphins and false killer whales, but was only 0.4% in Risso dolphins. The eosinophilic granules were larger in Risso dolphins and false killer whales than in bottlenose and Pacific white-sided dolphins.     

Metabolites of Monascus ruber in silages

A total of 233 silages were examined and found that Monascus ruber was present in 43 samples with counts between 1 × 10³ and 9 × 10⁶ colony-forming units (CFU)/g (mean: 2 × 10⁵ CFU/g). Monacolin K_L and the hydroxy acid monacolin K_A were detected by liquid chromatography-mass spectrometry in 45 and 50 of 233 samples at levels ranging from 25–15 600 and 28–65 400  μ g/kg, respectively. Citrinin was found with high-performance liquid chromatography-fluorescence detection (FLD) in 14 (6%) samples, the concentrations varied between 2.4 and 64.2  μ g/kg. The concentrations of citrinin were low and toxic effects are not anticipated. Monacolin K_A and monacolin K_L occur frequently and in considerable amounts in silages. These metabolites are believed to influence the metabolic activity of rumen anaerobic fungi resulting in a poorer digestion of crude fibre.     

Isolation,Identification of Four Newcastle Disease Virus Strains and Study on Their Biological Characteristics and Genetic Characteristics

In order to study the relationship and evolution between the latest Newcastle disease virus (NDV) epidemic strain and the vaccine strain (Mukteswar,La Sota and Clone 30 strains),four NDV epidemic strains were initially isolated and identified from immunized chicken groups in Guangdong,Guangxi and Hainan provinces. Fusion (F) gene and hemaglutinin neuraminidase (HN) gene of four isolated stains were amplified,cloned and sequenced by RT-PCR method,and the homology analysis of amino acid sequences deduced by F and HN genes of NDV and other strains published in GenBank were studied, the phylogenetic tree were build,and the pathogenicity index of each strain (EID₅₀,MDT, ICPI and IVPI) were determined. The results showed that the EID₅₀,MDT,ICPI and IVPI of the HN-08 strain were 10^-8.37/0.2 mL,58.5 h,1.78 and 2.45,that of the XX-08 strain were 10^-6.50/0.2 mL,75.0 h,1.61 and 2.41,that of the YS-09 strain were 10^-7.75/0.2 mL,64.5 h,1.71 and 2.38,and of the LF-09 strain were 10^-7.60/0.2 mL,63.8 h,1.84 and 2.38.In this experiment,the similarity of amino acid sequences of F and HN genes was over 95.8% among four strains of NDV isolated from chicken,the similarity of four NDV strains with chicken epidemic strains (GX11/03 and GM strains),vaccine strains (Mukteswar,La Sota and Clone 30 strains) and standard virulent strain (F₄₈E₉ strain) were from 96.8% to 98.6%,86.7% to 90.6% and 88.4% to 91.3%,respectively. The results showed that four NDV epidemic strains were virulent strains,which were closely related to the GX11/03 and GM strains that were popular in China in recent years,but relatively far from the traditional vaccine strains.