Expression of 4 truncated fragments of Pasteurella multocida toxin and their immunogenicity

Pasteurella multocida toxin (PMT) is a poor antigen that becomes more immunogenic after its native structure has been destroyed. In contrast, partially truncated PMT proteins, which are predicted to be good antigens when used as a vaccine, might be used to improve the control of atrophic rhinitis in pigs. In this study, 4 truncated PMT fragments were expressed in Escherichia coli, and those 4 fragments were inoculated into mice to produce the polyclonal antibodies. The results of an enzyme-linked immunosorbent assay (ELISA) revealed that #1 and #4 fragments were the most immunogenic. Immunized mice were subsequently challenged intraperitoneally with P. multocida type D. Five of the eight #1 fragment-immunized mice showed some protection against death and bacterial clearance. Pigs immunized with #1 fragment produced no or mild atrophic rhinitis (turbinate conchal score) after challenge, suggesting that this #1 fragment could be a good candidate for a subunit recombinant-type vaccine.     

Stable carbon isotopes accurately predict diet selection by sheep fed mixtures of C3 annual pastures and saltbush or C4 perennial grasses

The stable carbon isotope technique has been widely used to infer the dietary ecology of a range of animal species; however calibration of the technique with animals fed known diets is essential for accurate back-calculation of dietary preferences. The aim of this study was to identify suitable samples and back-calculation methods to predict short-term (2 to 3 week) dietary selection by sheep among plants with C₃ and C₄ photosynthetic pathways. Variation in integration time of dietary carbon into plasma and faeces; diet-tissue discrimination of carbon isotopes (fractionation) and the importance of accounting for the digestible or indigestible components of the diet was investigated. The results indicate that faecal and rumen samples provided the most accurate prediction of short term dietary changes in sheep selecting between C₃ and C₄ plants. The most accurate back-calculation method for these samples used δ¹³C of the C₃ and C₄ plants and accounted for both diet-tissue discrimination and differences in the indigestibility between the C₃ and C₄ forage. For faecal samples, the organic matter content of the diet originating from C₄ plants could be predicted with a mean error as low as 2.7%. Wool and plasma samples were not conducive to predicting proportion of C4 forage in the diet within 18 days after a change in diet; however plasma could be used to discriminate between animals fed 100% C₃ and C₄ diets after 3 days. The δ¹³C technique provides a valuable tool for researchers when designing pastures for dual environmental and production purposes. An understanding of what sheep select allows for development of appropriate grazing management strategies to optimise productivity and/or persistence of target species.     

B亚群禽白血病病毒SDAU09C2株的TCID50与p27抗原之间的相关性研究

为了研究B亚群禽白血病病毒(ALV-B)的半数细胞培养物感染量(TCID₅₀)与p27抗原的酶联免疫吸附试验(ELISA)的S/P值之间的相关性,本试验将ALV-B SDAU09C2 株接种鸡胚成纤维细胞(CEF细胞),换维持液后连续10 d取样,检测10 d的TCID₅₀值与p27抗原的S/P值之间的相关性;同时,将该毒株在DF-1细胞系上传代至20代,取其中的第1、5、10、15和20代分别进行TCID₅₀滴度的测定和p27抗原检测。结果表明,在CEF细胞上接种的ALV-B SDAU09C2株连续10 d的TCID₅₀值与p27抗原之间存在显著的正相关(r=0.94002;P<0.0001);在DF-1细胞系上传的不同代数之间也呈显著正相关(r=0.96449;P=0.0080)。由此可推测ALV-B的TCID₅₀与p27抗原呈显著正相关,可以用ELISA法测得的p27抗原的S/P值来估测病毒的TCID₅₀值。     

Infection of specific-pathogen-free lambs with parainfluenza virus type 3, Pasteurella haemolytica and Mycoplasma ovipneumoniae

Groups of specific-pathogen-free lambs were inoculated with combinations of parainfluenza virus type 3 (PI₃), Pasteurella haemolytica (P.h.) and Mycoplasma ovipneumoniae ( (M.o.). Acute, necrotising bronchopneumonia developed in 8/9 lambs inoculated with PI₃ followed by P.h. whereas only 1/5 lambs inoculated with PI₃ followed by a combination of M.o. and P.h. developed a pneumonic lesion. When M.o. was inoculated 29 days before PI₃ and P.h., pneumonia developed in 3/4 lambs but M.o. was not reisolated from any of the lungs. Pneumonia was observed in 1/5 lambs inoculated with P.h. alone and in 1/5 inoculated with M.o. plus P.h. In addition, one lamb in the latter group died of acute septicaemic pasteurellosis. None of the lambs inoculated with M.o. alone, PI₃ alone or PI₃ followed by M.o. had any gross or microscopic evidence of pneumonia although the virus alone, or in combination, did produce minor pulmonary lesions.These data suggest that M.o. is not an important primary or secondary lung pathogen.     

牛病毒性腹泻病毒E2蛋白的亚细胞定位研究

为深入研究牛病毒性腹泻病毒E_2蛋白的生物学功能,了解其在哺乳动物细胞中的亚细胞定位情况,采用RT-PCR扩增牛病毒性腹泻病毒Changchun184株E_2基因,将其克隆至真核表达载体pc DNA3.1/V5His A中,并进行酶切鉴定和测序分析,将构建的重组质粒pc DNA3.1/V5His A-E_2经脂质体介导转染至MDBK细胞。继续培养48h后,在激光共聚焦显微镜下观察E_2蛋白在细胞中的亚细胞定位情况。结果表明,E_2蛋白在细胞核与细胞质中均有表达,且绿色荧光呈点状均匀分布。该研究为进一步研究牛病毒性腹泻病毒E_2蛋白的相关功能奠定了基础。     

Conditioning Horses at v10 3 Times per Week Does Not Enhance v4

This study examined the effect of exercising horses 3 times per week with two bouts of 5-minutes' duration at their v₁₀. Six Thoroughbreds were treadmill-conditioned for 6 weeks. A standardized exercise test (SET) was performed at the beginning of the conditioning period to determine the blood lactate–running speed (BLRS) relation, and the SET was repeated every 2 weeks. After each SET, the BLRS relation was used to calculate the horse's speed, which produced a blood lactate (LA) concentration of 10 mmol/L (v₁₀) and 4 mmol/L (v₄). Each horse was then conditioned for the next 2 weeks (3 times/week) at its individual v₁₀ for two 5-minute bouts with a 5-minute walking phase in between. Exercise speed was individually adapted to the new v₁₀ every 2 weeks. The v₄ of horses decreased after the first 2 weeks (from 6.23 ± 0.41 m/s to 5.95 ± 0.33 m/s, mean ± SD; P < .05), increased in the following 2 weeks (6.33 ± 0.58 m/s; P < .01), and stayed constant thereafter (P > .05). The conclusion drawn was that exercising horses 3 times per week at their v₁₀ for two 5-minute bouts did not improve v₄.     

Studies of maternally-acquired antibodies in the foal to equine influenza A1 and A2, and equine rhinopneumonitis

Serum and colostrum were collected from 50 mares at parturition. Pre- and post-nursing serum samples were obtained from their foals. Bi-weekly serum samples were obtained from 25 of the foals for eight weeks. Hemagglutination-inhibiting (HAI) antibody titers to equine influenza viruses A₁ and A₂ (EIVA₁ and EIVA₂) and serum-neutralizing antibody titers to equine herpes virus 1 (EHV₁) were measured in serum and colostrum samples. IgG levels in serum and colostrum were determined.No antibody was detected in any foal's pre-nursing serum sample. Foal post-nursing antibody and IgG levels were equivalent to those measured in their dam's sera (EHVA₁ p=0.86; EHVA₂ p=0.54; EHV₁ p=0.91; IgG p=0.58). The half-life of maternally-acquired serum antibody in the foals was determined to be: EIVA₁=28.88 days (26.4 to 31.7 days); EIVA₂=29.1 days (26.7 to 32.1 days); EHV₁=31.0 days (28.1 to 34.8 days). Colostrum contained antibody and IgG at levels ranging from 2 to 8 times higher (4.3 average) than those detected in the mare's serum.     

Immunological properties of the SLLTEVET epitope of Influenza A virus in multiple display on filamentous M13 phage

Small peptides require large carriers to stimulate the humoral immune system. The filamentous phages, such as M13, have been proposed as vectors for expressing and carrying these peptides on their capsid surface. M2e 2-9 (SLLTEVET) residues of the transmembrane protein M2 of Influenza A virus are conserved and considered as a suitable target for immunization against a wide range of Influenza A virus strains. Here, M2e (2-9) sequence of Influenza A virus was fused to the N-terminus of the major coat protein gpVIII of M13 phage and was used to immunize broiler chickens. The results showed that the SLLTEVET peptide on the surface of M13 phage was expressed, the hybrid phage was immunogenic and produced specific antibodies against M2e (2-9) in broiler chickens.     

重组犬瘟热病毒F-H融合基因乳酸菌的构建及表达

为了构建重组犬瘟热病毒(CDV)F-H融合基因工程乳酸菌,采用重叠延伸PCR(SOE-PCR)技术扩增CDV F-H融合基因。将F-H融合基因亚克隆至穿梭载体p SIP409多克隆位点上,构建p SIP-F-H表达重组子,并电转化至植物乳杆菌NC8感受态细胞中。利用SDS-PAGE和Western blotting检测F-H融合蛋白的表达情况。结果表明,成功地扩增了CDV F-H融合基因,构建了p SIP-F-H重组表达质粒,并电转化至乳酸杆菌感受态细胞中。SDS-PAGE和Western blotting检测表明,重组乳酸杆菌表达了分子量约为62.96 ku的F-H融合蛋白,且该蛋白能与CDV阳性抗体反应,具有反应原性。     

Characteristics of Gas Generation (NH3, CH4, N2O, CO2, H2O) From Horse Manure Added to Different Bedding Materials Used in Deep Litter Bedding Systems

The aim of this study was to analyze the influence of horse manure added to different bedding materials on the generation of gases (ammonia (NH₃), nitrous oxide, carbon dioxide, methane, water vapor) from deep litter bedding under standardized laboratory conditions. Two different types of straw (wheat and rye) and wood shavings were analyzed. The deep litter (substrate) was made of 25 kg of the respective bedding material, 60 kg horse feces, and 60 L ammonium chloride solution (urea), and spread out in identical chambers over 19 days (n = 3). On days 1, 8, 15, and 19, total nitrogen, total carbon, and dry matter content of the substrate, as well as the pH in 500-g samples, were measured along with. At the end of each test period, the nitrite nitrogen, nitrate nitrogen, and ammonium nitrogen contents of the leachate were analyzed. The wheat straw substrate emitted the highest concentration of NH₃ (4.31 mg/m³; P < .0001) and the wood shavings substrate emitted the lowest (1.73 mg/m³; P < .0001); the rye straw substrate generated 3.05 mg/m³. In addition, significant differences occurred during days 1 to 3 with respect to the generation of the gases NH₃, methane, nitrous oxide, carbon dioxide, and water vapor, and after the opening of the chamber on day 15. The nitrogen losses through the leachate occurred mainly in the form of nitrate, where the leachate from the wheat straw substrate had a significantly higher amount of nitrate nitrogen (44.56 mg) as compared with the leachates of the rye straw (14.49 mg; P ≤ .0001) and the wood shaving substrates (22.62 mg; P = .0010).     

表达新城疫病毒融合蛋白基因禽痘病毒重组体的构建

通过RT-PCR扩增新城疫病毒F基因,利用禽痘病毒复合启动子、SV40 PolyA终止信号序列构建F基因表达盒。将F基因表达盒与loxp-GFP-loxP序列串联插入禽痘病毒重组臂,构建了禽痘病毒转移质粒载体。在脂质体介导下转染CEF,获得了带有绿色荧光信号的重组病毒rFPVFGFP。通过二次转染,利用Cre-loxP位点特异性重组将重组病毒基因组的GFP自动去除,结果获得了只含新城疫病毒F基因表达盒的重组禽痘病毒rFPVF。试验结果表明,rFPVF复制稳定,表达的F蛋白具有免疫原性,能够刺激鸡只产生抗新城疫病毒的中和抗体。     

Non-epidermidis coagulase-negative Staphylococcus isolated from farm animals can inhibit the hemagglutinating activity of Newcastle disease virus and bovine parainfluenza virus type 3

The Embp protein of Staphylococcus epidermidis inhibits the hemagglutination of the H1N1 influenza virus and protects birds from a viral respiratory infection. Several species of Coagulase-negative Staphylococcus (CoNS) are present in the respiratory cavity, particularly in nostrils. We hypothesize that non-epidermidis CoNS found in animals can have the same function as observed in S. epidermidis. Thirty Non-epidermidis CoNS isolates were obtained from poultry, sheep, goat, pig, and dairy cow nostrils. Haemagglutination inhibition (HI) activity was assayed in bacteria-free supernatants from non-epidermidis CoNS against Newcastle disease virus (NDV) and bovine parainfluenza virus type 3 (BPIV). In 13 of the 30 strains (43.3 %), bacteria-free supernatants showed HI activity for NDV and BPIV-3. Staphylococcus xylosus supernatants from poultry (one isolate), sheep (two isolates), goat (one isolate), and dairy cow (three isolates) had the highest frequency of HI activity on NDV and BPIV-3, followed by Staphylococcus sp. supernatants from goat (one isolate), dairy cow (two isolates), and finally Staphylococcus equorum, Staphylococcus chromogens and Staphylococcus gallinarum supernatants with single isolation from poultry, pig and poultry, respectively. Nine isolates had the homologous gene to the embp gene of S. epidermidis, and it was associated with HI activity in the studied viruses. By Pulsed‐field gel electrophoresis, S. xylosus isolates showed to be different clones and related to the origin of isolation and HI activity. These results demonstrate that non-epidermidis CoNS supernatants from different animals and origins have the ability of HI on NDV and BPIV-3, indicating that not only S. epidermidis has the same function.     

Recombinant pseudorabies virus expressing P12A and 3C of FMDV can partially protect piglets against FMDV challenge

One of the crucial factors for evaluation of an effective genetically engineered vaccine is whether susceptible animals are protected from virus challenge after vaccination. In this study, a recombinant pseudorabies virus (PRV-P12A3C) that expressed capsid precursor polypeptide P12A and nonstructural protein 3C of foot-and-mouth disease virus (FMDV) was used as a vaccine. The expression of P12A3C and its immunogenicity and protective efficacy against FMDV challenge were measured. Humoral and cellular immune responses were evaluated after each immunization. Subsequently, each piglet was challenged with 1000 ID₅₀ (50% infection dose) FMDV serotype O, named OR/80, which is used to produce vaccine in China. PRV-P12A3C induced a high level of neutralizing antibody and FMDV-specific lymphocytes. Inactivated vaccine provided 100% protection, and the vector strain (TK⁻/gE⁻/gI⁻) showed no protection. PRV-P12A3C induced 60% protection, compared with piglets that were vaccinated with TK⁻/gE⁻/gI⁻. The severity of clinical signs for the remaining two piglets was lighter and the appearance of vesicles was delayed.     

The pathogenesis of sequential infection with parainfluenza virus type 3 and Pasteurella haemolytica in sheep

Conventionally-delivered colostrum-deprived lambs were inoculated with either parainfluenza virus type 3 (PI₃) alone or PI₃ followed, 6 days later, by Pasteurella haemolytica (P.h.). Six out of 20 lambs died or were killed in extremis within 3 days of the inoculation of P.h.; the remainder were selected at random and killed from 1 to 28 days after the inoculation of P.h.Extensive pneumonic lesions developed in a large proportion of lambs inoculated with both agents but in none of those inoculated with the virus alone. Histologically, the pneumonic lesions fell into two categories: necrotic lesions, demarcated by a zone of either oat-cell or neutrophil infiltration, and a milder, purulent bronchopneumonia. Bacterial numbers tended to be higher in necrotic lesions than they were in purulent lesions. Virus titres in nasal secretions, on the day of inoculation of P.h. (day 6), also were higher in animals that developed necrotic lesions than they were in those that developed milder lesions. Nevertheless, titres were similar in both groups on day 4.Necrotic lesions persisted for at least 21 days as residual encapsulated abscesses which still contained viable P.h. whereas the milder, purulent bronchopneumonia was not detected later than 3 days after the inoculation of P.h..     

Genome-wide linkage and QTL mapping in porcine F2 families generated from Pietrain, Meishan and Wild Boar crosses

Three informative pig F₂ families based on European Wild Boar (W), Meishan (M) and Pietrain (P) crosses have been used for genome‐wide linkage and quantitative trait loci (QTL) analysis. Altogether 129 microsatellites, 56 type I loci and 46 trait definitions (specific to growth, fattening, fat deposition, muscling, meat quality, stress resistance and body conformation) were included in the study. In the linkage maps of M × P, W × P and W × M families, average spacing of markers were 18.4, 19.7 and 18.8 cM, the numbers of informative meioses were 582, 534 and 625, and the total lengths of autosomes measured were 27.3, 26.0 and 26.2 Morgan units, respectively. Maternal maps were on average 1.3 times longer than paternal maps. QTLs contributing more than 3% of F₂ phenotypic variance could be identified at p < 0.05 chromosome‐wide level. Differences in the numbers and positions of QTLs were observed between families. Genome‐wide significant QTL effects were mapped for growth and fattening traits on eight chromosomes (1, 2, 4, 13, 14, 17, 18 and X), for fat deposition traits on seven chromosomes (1, 2, 3, 4, 6, 7 and X), for muscling traits on 11 chromosomes (1, 2, 3, 4, 6, 7, 8, 12, 14, 15 and X), for meat quality and stress resistance traits on seven chromosomes (2, 3, 6, 13, 16, 18 and X), and QTLs for body‐conformation traits were detected on 14 chromosomes. Closely correlated traits showed similar QTL profiles within families. Major QTL effects for meat quality and stress resistance traits were found on SSC6 in the interval RYR1‐A1BG in the W × P and M × P families, and could be attributed to segregation of the RYR1 allele T derived from Pietrain, whereas no effect in the corresponding SSC6 interval was found in family W × M, where Wild Boar and Meishan both contributed the RYR1 allele C. QTL positions were mostly similar in two of the three families for body conformation traits and for growth, fattening, fat deposition and muscling traits, especially on SSC4 (interval SW1073‐NGFB). QTLs with large effects were also mapped on SSC7 in the major histocompatibility complex (MHC) (interval CYP21A2‐S0102) and affected body length, weight of head and many other traits. The identification of DNA variants in genes causative for the QTLs requires further fine mapping of QTL intervals and a positional cloning. However, for these subsequent steps, the genome‐wide QTL mapping in F₂ families represents an essential starting point and is therefore significant for animal breeding.     

Specific sequence amplification of bovine virus diarrhea virus (BVDV) and hog cholera virus and sequencing of BVDV nucleic acid.

The pestiviruses are small enveloped RNA viruses and are causative agents of economically important animal diseases in cattle, swine, sheep and goats worldwide. We used the polymerase chain reaction to amplify one common fragment of several different strains of both hog cholera virus and bovine virus diarrhea virus (BVDV). The fragment is located at the 5'-end of the genome immediately upstream of the open reading frame. This is a highly conserved region among the different published pestivirus sequences. An internal restriction digest of the amplified fragment with XhoI and PstI was performed in order to confirm specificity of the amplified fragment. The fragment was sequenced for a number of different BVDV strains, and the sequences obtained were compared to those published and used to deduce genetic relationships between strains. Apart from this common fragment we have amplified several other fragments of the Danish BVDV strain Ug59 and obtained specific amplification fragments of the expected size.     

Studies with Bovine Parainfluenza — 3 Virus in U.A.R. (Egypt)

A bovine strain of myxovirus parainfluenza-3 (MP3) virus, designated S virus, was isolated from lung tissue collected from cattle with respiratory illness in 1963. The virus agglutinates mammalian and avian erythrocytes, and is sensitive to ether, sodium desoxycholate and trypsin. It grows in primary calf kidney, buffalo kidney, dog kidney, camel kidney and MS cell cultures. The S virus forms well-defined plaques in buffalo and calf kidney cells on the 5th or 6th day after inoculation. Examination of cell cultures following inoculation with S virus revealed giant cell formation, and introcytoplasmic and intranuclear inclusions. At 37°C the virus titer dropped from 10^10.4 to 10^2.6 in 3 days. Virus was completely inactivated at 56°C within 15 minutes. Growth-curve studies in tissue culture monolayer cells revealed a latent period of 10 hours. The intracellular virus titer was slightly lower than that of extracellular virus. The isolate was identified as MP3 virus by serum neutralization and hemagglutination-inhibition tests. Antibodies (HI) to S virus were shown to be present in a significant proportion of Egyptian cattle. The epidemiological significance of MP3 (bovine strain) virus in U.A.R. is discussed.     

Effects of dietary T3 on growth parameters and hormone levels in normal and sex-linked dwarf chickens

Tri-iodothyronine (T₃) has been administered in the diet, from day of hatch until 8 weeks of age, to sex-linked dwarf and normal chicks of both sexes from a brown-egg slow-growing strain. Feed was supplemented with either 0.1 ppm or 0.5 ppm T₃. A significant genotype by treatment interaction was observed on body weight: the effect of T₃ in males was significantly positive for dwarfs and null for normals, the effect in females was null for dwarfs and significantly negative for normals. Feed efficiency was rather decreased by the treatment in both genotypes. Abdominal fatness was decreased in a dose-dependent manner in both genotypes, while rectal temperature was raised in dwarf chicks only. Plasma T₃ was raised to normal levels in dwarfs receiving 0.1 ppm exogenous T₃, while the 0.5 ppm dose yielded hyperthyroid levels. Plasma GH levels were decreased in a dose-dependent manner by the T₃ treatment, the effect being larger in dwarfs. Surprisingly, plasma IGF-I was unchanged in spite of the GH decrease, whatever the genotype or the sex. It was concluded that exogenous T₃ alone can have a stimulatory effect on growth in dwarfs but can not fully restore a normal growth rate. Both T₃ and IGF-I are important for a normal growth and the relationships between T₃ and IGF-I production should be further investigated in order to better understand the physiological modifications due to the sex-linked dwarf gene.     

VITAMIN B12 AND COPPER SUPPLEMENTATION IN BEEF CALVES

SUMMARY Bodyweight responses to subcutaneous injections of vitamin B₁₂ and copper were investigated using Hereford calves in the southeast of South Australia, an area known to produce cobalt and copper deficient sheep. Calves were allocated to one of four groups: control; copper; vitamin B₁₂; copper plus vitamin B₁₂. Responses in bodyweight gain to vitamin B₁₂, and to copper were obtained during the trial of one year. The results of biochemical analysis of blood, hair and faeces from calves and of dam's milk are reported. It is concluded that calves raised on cobalt-deficient pastures will require cobalt or vitamin B₁₂ supplementation prior to weaning.