DNA sequence analysis of glycoprotein G gene of bovine ephemeral fever virus and development of a double oil emulsion vaccine against bovine ephemeral fever

The surface glycoprotein G is considered as the major neutralizing and protective antigen of bovine ephemeral fever virus (BEFV). Comparison of the deduced amino acid sequence of G protein of BEFV isolates during the period 1984-2004 outbreaks in Taiwan showed amino acid substitutions in the neutralizing epitopes. All the isolates differ markedly in the neutralizing epitope at the same amino acid positions compared to the currently available killed vaccine strain (Tn73). Tn88128 strain isolated in 1999 showed the maximum variability of 12 amino acids, 5 amino acid in the neutralization epitope and 7 apart from, respectively. Combinations of both Tn88128 (1999) and commercially available vaccine strain (Tn73) were developed and its safety was evaluated in mice, guinea pigs, calves, and pregnant cows. None of the animals showed any adverse effect or clinical signs. Calves were immunized with commercial vaccine (Tn73) and, combined vaccine (Tn73 and Tn88128), respectively, with adjuvants such as Al-gel and water-in-oil-in-water (w/o/w) oil and PBS alone and challenged with Tn88128 strains. Except PBS administered animals, all the vaccinated animals showed protective immune response. However, animals immunized with combined vaccine plus w/o/w adjuvant elicited stronger neutralization antibodies and long lasting immunity compared to other vaccines.     

Production and characterization of monoclonal antibodies against bovine leukaemia virus using various crude antigen preparations: a comparative study

A total of 59 monoclonal antibodies (mAbs) specific against the bovine leukaemia virus (BLV) using different antigen preparations was produced. The five antigen preparations for immunizing BALB/c mice were: live cells (CEL), sonicated and ultracentrifuged cells (SOC), cell lysates (LYS), semi-purified BLV (PV), and formalin-treated cells (FOR) from two cell lines permanently infected with BLV (FLK-BLV and BLV-bat2). These viral component presentations were selected to obtain mAbs against specific BLV proteins: located on the cell surface (FOR and CEL), in free virus particles (PV) and intracellular viral proteins (SOC and LYS). Two antigen preparations (SOC and LYS) were lethal to the mice following the intravenous and intrasplenic routes. Six fusions were performed in this study that rendered specific antibodies against BLV. The highest number of hybridomas was produced with SOC; however, the majority of the hybridomas produced (> 90%) were against cellular proteins. Even though immunization with PV gave the lowest number of hybridomas, the majority of them were specific against BLV. Based on the reactivity of the mAbs in Western blot (WB), we classified the mAbs into five groups, namely anti-gp51SU (39 mAbs), anti-gp30TM (six mAbs), anti-Pr72env (nine mAbs), anti-Pr66gag-pro (one mAb) and anti-Prgag (four mAbs). A very high percentage of the mAbs produced (48 of 59) reacted with gp51SU, suggesting that this is the most immunogenic and accessible BLV protein presented in the different antigen preparations. The majority of our mAbs recognized more than one band in WB, suggesting that, aside from reacting with mature proteins, the mAbs also recognized viral precursors.     

A comparative proteomic study of plasma in feline pancreatitis and pancreatic carcinoma using 2-dimensional gel electrophoresis to identify diagnostic biomarkers: A pilot study

While pancreatitis is now recognized as a common ailment in cats, the diagnosis remains challenging due to discordant results and suboptimal sensitivity of ultrasound and specific feline pancreatic lipase (Spec fPL) assay. Pancreatitis also shares similar clinical features with pancreatic carcinoma, a rare but aggressive disease with a grave prognosis. The objective of this pilot study was to compare the plasma proteomes of normal healthy cats (n = 6), cats with pancreatitis (n = 6), and cats with pancreatic carcinoma (n = 6) in order to identify potential new biomarkers of feline pancreatic disease. After plasma protein separation by 2-dimensional gel electrophoresis, protein spots were detected by Coomassie Brilliant Blue G-250 staining and identified by mass spectrometry. Alpha-1-acid glycoprotein (AGP), apolipoprotein-A1 (Apo-A1), and apolipoprotein-A1 precursor (Pre Apo-A1) appeared to be differentially expressed, which suggests the presence of a systemic acute-phase response and alteration of lipid metabolism in cats with pancreatic disease. Future studies involving greater case numbers are needed in order to assess the utility of these proteins as potential biomarkers. More sensitive proteomic techniques may also be helpful in detecting significant but low-abundance proteins.     

A comparative study on the immune response of sheep to foot and mouth disease virus vaccine type Asia-1 prepared with different inactivants and adjuvants.

Foot and mouth disease virus type Asia-1 was inactivated either with formaldehyde or binaryethylenimine (BEI). Inactivated vaccines were prepared incorporating aluminium hydroxide gel or mineral oil as an adjuvant. The antibody response to the adult sheep was studied by ELISA and SN test for a period of 6 months. There was no difference in the antibody response between vaccines inactivated with formaldehyde or BEI. Whereas significant difference in the antibody response was observed between gel and oil vaccines. The high titres of antibody stimulated by oil vaccines persisted longer than those of gel vaccines within the period of study.     

ESAT-6及PPD ELISA检测疫区和非疫区牛结核病的比较研究

本实验克隆、表达了牛结核早期分泌靶抗原蛋白6（ESAT-61抗原,建立了ESAT-6酶联免疫吸附检测方法（ELISA）。同时应用牛结核提纯菌素（PPD）ELISA和ESAT-6-ELISA对采自疫区的641头牛和非疫区的324头牛进行检测,其中PPD-ELISA检测中,疫区有581头阳性牛,阳性率为90．64％,非疫区有2头阳性牛,阳性率为0、62％;ESAT-6-ELISA检测中,疫区有567头阳性牛,阳性率为88．45％,非疫区没有阳性牛。对采自疫区的23例变态反应阳性牛和非疫区的7例变态反应阳性牛进行检测,其中PPD-ELISA检测中,疫区有20头为阳性。与变态反应的符合率为86．90％,非疫区有1头为阳性,与变态反应的符合率为14％;在ESAT-6-ELISA检测中,疫区有18头为阳性,与变态反应的符合率为78．30％,非疫区没有阳性。这些结果表明：ESAT-6-ELISA的敏感性要低于PPD-ELISA;牛结核ELISA在非疫区应用效果较差,在疫区更具有应用价值。     

A comparative study on the clinical, parasitological and molecular diagnosis of bovine trypanosomosis in Uganda

The clinical, parasitological and molecular diagnosis of bovine trypanosomosis were compared using samples from 250 zebu cattle exposed to natural trypanosome challenge in Uganda. Clinical examination, molecular and parasitological diagnoses detected 184 (73.6%), 96 (38.4%) and 36 (14.4%) as diseased, respectively. The sensitivity and specificity of clinical examination were 87.5% and 35%, and 78 % and 27 % based on molecular and parasitological diagnoses, as gold standards, respectively. Of the 33, 3, 13 and 12 parasitological-positive cattle that had Trypanosoma brucei, Trypanosoma congolense, Trypanosoma vivax or mixed infections, 78 %, 33 %, 84 % and 100 % respectively manifested clinical signs. Of the 24, 89, 12, 3, 6 and 27 cattle detected by molecular diagnosis to have mixed infections, T. brucei, T. vivax, T. congolense forest-, Savannah- and Tsavo-type, 100%, 83%, 91%, 100%, 67% and 81 % had clinical signs, respectively. In conclusion, treatment of cattle based on clinical examination may clear up to 87.5 % or 78 % of the cases that would be positive by either molecular or parasitological diagnosis, respectively. Under field conditions, in the absence of simple and portable diagnostic tools or access to laboratory facilities, veterinarians could rely on clinical diagnosis to screen and treat cases of bovine trypanosomosis presented by farmers before confirmatory diagnosis in diagnostic centres for few unclear cases is sought.     

Assessment of immunogenicity and protective efficacy of Microsporum canis secreted components coupled to monophosphoryl lipid-A adjuvant in a vaccine study using guinea pigs

Microsporum canis is the most common dermatophyte in pets and is of zoonotic importance but currently there is no effective vaccine available to prevent dermatophytosis. The aim of this work was to assess the immunogenicity and protective efficacy of secreted components (SC) from M. canis adjuvanted with the monophosphoryl lipid-A (MPLA), in a vaccine study using the guinea pig as an experimental model. Animals were vaccinated with either the SC adjuvanted with the MPLA, the MPLA adjuvant alone or PBS three times at two-week intervals, until 42 days prior to M. canis infection. A blind evaluation of dermatophytosis symptoms development and fungal persistence in skin was monitored weekly. The antibody response towards the SC and the levels of Interferon (IFN)γ and Interleukin-4 expressed in peripheral blood mononuclear cells were assessed along or at the end of the study period respectively. The animals that received MPLA had a significantly lower clinical score than those inoculated with PBS. However, no significant difference was observed between the guinea pigs vaccinated with the SC adjuvanted with the MPLA and those having received MPLA alone. The results also showed that vaccination induced a strong antibody response towards the SC and an increase in IFNγ mRNA level. Our results show that the MPLA adjuvant used in this vaccine study can induce per se a partial protection against a M. canis infection. Although they induce a delayed-type hypersensitivity reaction in guinea pigs, the SC do not confer a protection under the present experimental conditions.     

A comparative longitudinal study of bovine trypanosomiasis in tsetse-free and tsetse-infested zones of the Amhara Region, northwest Ethiopia

A study was conducted to determine the incidence of trypanosome infections in cattle in tsetse-free and tsetse-infested zones of the Amhara Region of northwest Ethiopia. A total of six sentinel herds were established and the cattle observed during a period of 8 consecutive months. The prevalence of seropositive cattle was high in both the tsetse-free and tsetse-infested zones. The average monthly incidence of trypanosome infection, determined using molecular diagnostic tools, was 20.9% and 25.7% in the tsetse-free and the tsetse-infested zones, respectively. In the tsetse-free, Trypanosoma vivax was responsible for 90.9% of the cattle trypanosome infections. In the tsetse-infested zone, Trypanosoma congolense and T. vivax contributed almost equally to the trypanosome infections in cattle. Trypanosome infection, regardless of species, resulted in anaemia as evidenced by a significant decrease in the packed cell volume of the infected animal. The outcome of this longitudinal study suggests that control of trypanosomiasis in the Amhara Region cannot be achieved by tsetse control alone. Supplemental measures to include drug therapy and biting fly control are discussed.     

Feline melanoma: a comparative study of ocular, oral, and dermal neoplasms

Melanomas diagnosed in 29 cats over an 11 year period included 19 ocular (16 intraocular, three palpebral), five oral, and five dermal melanomas. Intraocular melanomas involved the ciliary body and iris in 12; the whole eye was involved in four. The average age of cats with intraocular melanomas was 11 years; the female : male ratio was 9 : 7. Histologically, eight intraocular tumors were mixed, six were epithelioid, and two were spindle cell. Ten of 16 cats (62.5%) with intraocular melanomas were killed because of the tumor at a mean of 156 days; four are living with no evidence of disease (average, 255 days). The mean time of death in cats with palpebral melanoma was 409 days. Metastasis occurred in 63% of cats with intraocular melanoma and all cats with palpebral melanoma. Four cats with oral melanoma were killed at a mean of 61 days; all had metastasis. Of five cats with cutaneous melanoma, one was killed with metastasis at 90 days; three cats were alive without evidence of recurrence or metastasis greater than 365 days after surgery. Results of this study indicate that in the cat, ocular melanomas are more common than oral and dermal melanomas, and ocular and oral melanomas are more malignant than dermal melanomas, with higher rates of mortality and metastasis.     

A comparative study of Sephadex, glass wool and Percoll separation techniques on sperm quality and IVF results for cryopreserved bovine semen

The aim of this study was to compare the effects of spermatozoa separation techniques on sperm quality and in-vitro fertilization (IVF) results for cryopreserved bovine semen. Sephadex, glass wool and Percoll gradient separation techniques were used for sperm separation and sperm motility, morphology and membrane integrity were evaluated before and after separation. Also, cleavage and blastocyst developmental rate were investigated after IVF with sperm recovered by each separation technique. The motility of samples obtained by the three separation techniques were greater compared to the control samples (p < 0.05). The percentage of spermatozoa with intact plasma-membrane integrity, identified by 6-carboxyfluoresceindiacetate/propidium iodide fluorescent staining and the hypo-osmotic swelling test, was highest in the glass wool filtration samples (p < 0.05). The cleavage and blastocyst rate of total oocytes produced from glass wool filtration samples were also higher than the control and Sephadex filtration samples (p < 0.05), but were not significantly different from Percoll separation samples. However, a significantly greater number of cleaved embryos produced by glass wool filtration developed to blastocyst stage than those produced by Percoll separation (p < 0.05). These results indicate that spermatozoa with good quality can be achieved by these three separation techniques and can be used for bovine IVF. In particular, it suggests that glass wool filtration would be the most effective method of the three for improving sperm quality and embryo production for cryopreserved bovine spermatozoa.     

Kinematic characteristics of Andalusian, Arabian and Anglo-Arabian horses: a comparative study

The aim of this study was to compare the kinematic trot characteristics of three different breeds of horse: Andalusian (AN, n = 15), Arabian (AR, n = 7) and Anglo-Arabian (AA, n = 5) using standard computer-assisted videography (25 Hz). Linear, temporal and angular parameters in fore- and hind limbs were analysed in six randomly selected strides per horse. Normalised angle-time diagrams along the complete stride were obtained for all joints angles in each breed and specific kinematic characteristics were detected graphically. AA horses displayed longer swing durations in both limbs ans a shorter angular range of motion (ARM) in scapula and pelvis inclination and in shoulder, hip and forelimb retraction-protraction angles. At lift off, stifle and tarsal joint angles were more flexed. In general, only small differences were observed in AR horse kinematics when compared with the other 2 breeds. AN horses presented negative overtracking length, which was positive in AR and AA. In AN horses the elbow and carpal joints were more flexed at the moment of maximal elevation, elbow and fore-fetlock joints also exhibited a larger ARM due to a smaller angle at maximal flexion. In the hind limbs, tarsal, hind fetlock and retraction-protraction angles presented a larger ARM in AN horses due to greater maximal flexion in the tarsal and hind fetlock joints. Fore- and hind fetlocks were also more flexed in horses from this breed. In conclusion, differences between kinematic variables at the trot were observed in the three breeds studied here, mainly in forelimb joints. The most outstanding feature was the greater forelimb flexion recorded in AN horses than in the other breeds which is consistent with the elevated movements in this breed. In AA horses, the ARM of proximal joints involved in retraction protraction in both fore- and hind limbs was smaller. All the differences observed highlighted the idiosyncratic nature of the trot in each breed; this may influence the functional capacity of each breed.     

Cervical vertebral interbody fusion in the horse: a comparative study of bovine xenografts and autografts supported by stainless steel baskets

A modified form of the Cloward technique for anterior cervical fusion in human beings was used in the application of different grafts for evaluation of their effectiveness in stabilizing equine cervical vertebrae. Results of bovine xenograft implants in 8 horses were compared with results of stainless steel baskets (SSB) packed with cancellous autogenous bone in 8 horses. Graft material was incorporated in all cases. Evidence of graft rejection was not present. Both forms of implants decreased the mobility of the intervertebral space in which they were implanted. A fibrous connective tissue union was prevalent after xenograft implantation. Osseous union was prevalent after implantation of the SSB. Reduction in range of motion was greatest at those sites with the SSB.     

Rat, caprine, equine and bovine erythrocyte ghosts exposed to t-butyl hydroperoxide as a model to study lipid peroxidation using a chemiluminescence assay

The aim of the present study was to analyze the time-course of t-butyl hydroperoxide-induced changes in lipid peroxidation, fatty acid composition and chemiluminescence intensity in rat, caprine, equine and bovine erythrocyte ghosts. A relatively high content of arachidonic acid (C20:4 n6) and docosahexaenoic acid (C22:6 n3) was characteristic of the rat erythrocyte ghosts. The fatty acid composition of native erythrocyte ghosts obtained from caprine, equine and bovine was characterized by a high content of oleic acid (C18:1 n9) and a low content of the peroxidable polyunsaturated fatty acids (C20:4 n6 and C22:6 n3). The proportion of linoleic acid (C18:2 n6) was higher in equine and bovine compared to rat and caprine. Increase in lipid peroxidation in rat erythrocyte ghosts was maximal within 12 min of incubation, t-butyl hydroperoxide concentration dependent and was paralleled by a decrease in C18:2 n6, C20:4 n6 and C22:6 n3 and an increase in chemiluminescence formation. Polyunsaturated fatty acids (PUFAs) present in rat erythrocyte ghosts exhibit the highest sensitivity to oxidative damaged and their sensitivity increases as a power function of the number of double bonds per fatty acid molecule. Light emission in caprine, equine and bovine erythrocyte ghosts was very low, t-butyl hydroperoxide concentration-dependent but changes in fatty acid composition were not observed. The main conclusion of this work is that a low unsaturation degree of fatty acids in erythrocyte ghosts of caprine, equine and bovine prevent the lipid peroxidation on those membranes when they are incubated with t-butyl hydroperoxide.     

Detection of antibodies against hog cholera virus and bovine viral diarrhea virus in porcine serum. A comparative examination using CF, PLA and NPLA assays

The antibodies in serum samples from an outbreak of low-virulent hog cholera in Spielbach, West Germany, 1966, as well as serum samples from pigs inoculated with hog cholera (HG) virus and bovine viral diarrhea (BVD) virus, respectively, were examined by means of 3 different methods:

1. A modified direct complement fixation (GF) test,
2. A peroxidase-linked antibody (PLA) assay based on microplates with fixed, viral-antigen containing cells,
3. A neutralization assay carried out in microplates using the “chessboard” principle and read by means of the peroxidaselinked antibody (NPLA) assay.

A good correlation was found in their ability to detect the antibodies. Generally neutralizing antibodies could be found 2 weeks after inoculation. By CF and PLA antibodies could be detected at the same time or up to 2 weeks later. All sera were tested by the 3 methods against both HG viral antigen and BVD viral antigen. HC-antibodies could not be distinguished from BVD-antibodies by CF but to a certain degree by PLA. BVD-antibodies could to a certain degree be distinguished from HG-antibodies by CF but not by PLA. This means that CF and PLA together provide a good possibility for differentiation between the two types of antibodies. NPLA could to a high degree of reliability distinguish between HG-antibodies and BVD-antibodies.