A serological survey of Akabane virus infection in cattle and sheep in northwest China

Purpose

Akabane disease characterized mainly by fetal damage is a ruminant disease caused by insect-transmitted Akabane virus infection.

Methods

We investigated Akabane disease using serum neutralization tests in 446 blood samples collected from 187 cattle and 259 sheep of Xinjiang province, northwest China.

Results

(1) The overall prevalence rate of neutralizing antibody was 19.06?% (85/446), (2) the prevalence rates of Akabane disease in cattle and sheep were 20.32?% (38/187) and 18.15?% (47/259), respectively, (3) the disease prevalence rates were not significantly different between cattle and sheep, but significantly different among samples collected from different sampling months, (4) the disease was most prevalent in July when mosquitoes and culicoides were most active, and (5) the disease prevalence rates were significantly different between individuals with abortion experience and without abortion experience (P?<?0.05), suggesting that Akabane virus infection may significantly increase abortion risk in cattle and sheep.

Conclusions

To our knowledge, this is the first report confirming that Akabane virus infection is common in cattle and sheep of Xinjiang province, northwest China and providing useful epidemiological information for cattle and sheep abortion prevention and control.     

赤羽病间接ELISA检测方法的建立和标化

应用赤羽病病毒(AKV)OBE-1株和牛标准阴阳性血清，以蔗糖密度梯度离心纯化细胞毒为包被抗原，在国内首次建立了检测AKV抗体的间接ELISA方法。该方法与中和试验相关系数0．7614，特异性和重复性良好。应用此方法对南京、上海附近奶牛抽样检测，阳性率分别为26．7％和32．7％。对澳大利亚、加拿大、美国进口牛13600头份进行检测，全部阴性。     

Genetic analysis of Akabane virus isolates from cattle in Korea

Bayesian Inference (BI) and Neighbor Joining (NJ) analyses of the phylogenetic relationships between the nucleotide sequences of the N gene of Akabane virus revealed an unclear topology among genogroups I–III, which was probably caused by genetic reassortment or recombination between these genogroups. In contrast, nucleotide and amino acid phylogenetic tree analyses of the M RNA segment agreed with the topologies obtained by using the BI and NJ methods. Therefore, distinct genogrouping of Akabane virus isolates should be performed using the M RNA segment. Four Korea isolates were classified into genogroup II together with Akabane virus strains isolated from all areas of Japan, including Okinawa Island. However, more nationwide isolates and more clinical data from Korean cattle farms will be required in the future to confirm the precise relationships between genotypes and pathogenicity.     

Improvement of the reproducibility of the Elisa test for detecting anti-Trypanosoma congolese antibodies in cattle

Simplicity and the potential automatization make the ELISA test a universal tool for the detection of antibodies, and, more recently, of antigens. But the reproducibility of results is not very good, due to many varying factors. We tried to improve the reproducibility of the ELISA test for the detection of anti-Trypanosoma congolense antibodies in cattle. For that, buffers are always used at room temperature to avoid temperature gradients in the plates. All volumes are increased to 200 microliters per well. Non-activated rabbit serum is used to block nonspecific sites and to reduce background signal. Better results were obtained with a homologous antigen (T. congolense) than with a heterologous one (T. evansi). Titration of the antigen concentration to be used must be checked for each new batch. A distribution is proposed which would permit a testing of each serum four times per plate and thus to calculate the mean and standard deviation for each serum. Finally, we propose that the reading should no longer be a function of time after contact between enzyme and substrat, but a function of the progress of the reaction measured in a target serum. Results show a very good reproducibility and in addition we propose a computerized monitoring system.     

Determination of an optimized cut-off value for the Neospora agglutination test for serodiagnosis in cattle

A definitive diagnosis of neosporosis in cattle implies the examination of the aborted fetus. However, in many instances fetal material is not available. Therefore, most diagnosis are based on serological tests. At the moment, there are no consensuses about the cut-off for serodiagnosis of neosporosis in cattle, for any test. The objective of the present study was to estimate the best cut-off for the Neospora agglutination test (NAT) for serodiagnosis in cattle. For that purpose, 246 serum samples from 4 groups of dairy cows (aborted Neospora positive; not aborted healthy; aborted other diseases and herds endemic neosporosis) were collected and antibodies anti-N. caninum were determined by NAT. Additionally, immunoblot (IB) was performed with sera from all cows of the endemic neosporosis group and the patterns of seroreactivity were contrasted with the NAT titers for this group of cows. Evaluation of the optimized sensitivity and specificity was calculated using Youden's J-statistics. The best Youden's J-statistic was obtained at 1:40 titer, presenting 100% of sensitivity and 90.4% of specificity with negative and positive predictive values of 100 and 75.0%, respectively. The comparison between NAT titers and the IB banding pattern support the results of the statistical analysis, i.e. titers of 1:40 and higher showed a complex pattern of bands, while titers lower than 1:40 did not precipitate any bands. These results indicate that 1:40 was an optimized NAT cut-off for serodiagnosis in cattle.     

Modified indirect fluorescent antibody test for the serodiagnosis of Anaplasma marginale infections in cattle

A modified indirect fluorescence antibody technique was used for the serodiagnosis of Anaplasma marginale infections in cattle. Nonspecific antibodies adherent to infected erythrocytes were removed, using acidic glycine buffer. Evans blue was used as a counterstain.     

Appearance of slow-reacting and complement-requiring neutralizing antibody in cattle infected with Akabane virus

Slow-reacting complement-requiring neutralizing (NT) antibody was detected in sera from cattle 2 weeks after infection with Akabane virus. Bovine sera obtained 3 or 4 weeks after infection contained slow-reacting noncomplement-requiring NT antibody. The slow-reacting complement-requiring NT antibody was sensitive to 2-mercaptoethanol (2-ME), whereas the slow-reacting noncomplement-requiring NT antibody was resistant to 2-ME. The initial phase may represent the IgM response and the later phase a change to IgG. A NT test was developed in which virus-serum mixtures were incubated at 4 degrees C for 48 h and then with complement at 37 degrees C for 60 min; this gave an improved sensitivity over the previous incubation at 37 degrees C for 60 min.     

Use of the latex test in the serodiagnosis of enzootic leukosis of cattle]

Two antigenic extracts, prepared by different methods, were tried on 317 serum samples in the microscope-slide agglutination, sedimentation and tube agglutination tests. In comparison with the haematological findings, both antigens and all the tests gave a proportion of non-specific positive results, which made it impossible to obtain specific results. Various ways of reducing the proportion of non-specific reactions were tried, without success.     

Akabane virus infection in the pregnant ewe. 2. Pathology of the foetus

A strain of Akabane virus (CSIRO 16) isolated from Culicoides brevitarsis was given three different passage treatments in the laboratory and then inoculated into ewes that were 32 to 36 days pregnant. The foetuses from these ewes were examined between the 69th and 106th days of gestation. The 39 infected ewes produced 55 foetuses of which 44 (80%) had severe developmental defects. Arthrogryposis and agenesis of the brain or hydranencephaly, were present in 43 of the foetuses. Other gross defects affecting variable proportions of foetuses were porencephaly, brachygnathism, scoliosis, hypoplasia of the lungs and agenesis or hypoplasia of the spinal cord. Histopathological findings covered a wide spectrum of defects that have previously been considered to occur over an extended range of foetal ages. These defects included skeletal muscle atrophy and degeneration, and in the brain, particularly in the cerebrum, cystic areas and malacia, general oedema, subependymal gliosis, perivascular cuffing and mineralised plaques. Similar lesions were seen in the pons and cerebellum. Extensive lesions, with and without inflammation were seen in the spinal cord.     

Hemolytic activity of Akabane virus

Akabane virus was shown to lyse as well as to agglutinate pigeon erythrocytes. The hemolytic activity of the virus was markedly enhanced by repeated freeze-thawing, but its hemagglutinating activity was not affected. Hemolysis (HL) with the virus, like its hemagglutination, was affected by the NaCl concentration as well as by the pH of the diluent. HL was markedly affected by the incubation temperature, but hemagglutination (HA) was not; HL activity was highest at 37°C, somewhat lower at 25°C, very low at 4°C, and did not occur at 0°C. While pigeon erythrocytes were positive for both HL and HA, goose erythrocytes were positive for HA but negative for HL. Erythrocytes from cattle, sheep, rabbits, guinea pigs, mice and day-old chickens were tested for HA as well as for HL activity with negative results. A linear relationship was shown, in a wide range of the virus concentrations, between the percent HL and the virus concentration, as expressed on a logarithmic scale. Based on these findings we developed an assay method for Akabane virus hemolysin. Analysis by CsCl equilibrium density gradient centrifugation indicated the hemolytic as well as the hemagglutinating activity to be structurally associated with the virion. Scanning electron microscopy of pigeon erythrocytes undergoing HL with the virus revealed the appearance of a depressed area with a hole on the cell surface. The hemolytic activity of the virus was specifically inhibited by antisera to the virus and an HL-inhibition test was developed.     

赤羽病病毒RT-PCR检测方法的建立

利用BHK 2 1细胞繁殖赤羽病病毒 (AKV)美国株 ,以 30 %蔗糖垫底超速离心浓缩病毒。根据病毒SRNA序列设计 3对引物 ,初步建立了检测AKV的RT PCR方法 ,850bp扩增产物的序列与已知序列同源性 99%以上。人工攻毒鼠检测特异灵敏。     

牛羊赤羽病

赤羽病是由吸血昆虫进行传播的一种虫媒性病毒病 ,常导致牛羊繁殖障碍及新生胎儿发生关节弯曲和积水性无脑症 ,因而对牛羊危害较大。该病毒于 1 959年在日本群马县赤羽村首次被分离到。我国 1 998年首次分离并鉴定了该病毒 ,目前已证实我国至少有 1 3个省市地区有本病的流行。本文从病原、流行病学、致病机理、诊断与防治等方面对该病进行了综述 ,以期为该病的防制和研究提供可参考资料。     

赤羽病病毒核衣壳蛋白抗体间接ELISA检测方法的建立

以纯化的重组赤羽病病毒核衣壳蛋白作为诊断抗原，建立了检测牛血清特异性核衣壳蛋白抗体的间接ELISA方法，初步组装成便于现地使用的试剂盒。经对试验条件进行优化，确定最佳抗原包被量为每孔1μg（100μL），样品稀释度为1：100，兔抗牛IgG辣根过氧化物酶标记抗体稀释度为1：8000。经特异性试验和重复性试验证明该方法特异性高、重复性好。应用初步研制的间接ELISA试剂盒和微量中和试验法分别对云南省的89份、内蒙古的100份牛血清样本进行了检测，以中和试验为参照，经统计学处理，得出检测临界值分别为0．411和0．303，2种方法的符合率分别为72．7％（56／77）和91．4％（85／93）。试剂盒在37℃保存3d，对敏感性无明显影响。     

Encephalitis of cattle caused by Iriki isolate, a new strain belonging to Akabane virus

A disease characterized by nervous signs was found in 10 calves in two districts in Kagoshima Prefecture, Japan, from October to November, 1984. Histopathological changes of nonpurulent encephalitis were found in every case. An agent, named Iriki isolate, was isolated from the cerebellum of a calf in HmLu-1 cell cultures. All of the affected calves possessed neutralizing antibody to the virus. A high seropositive rate to the virus in cohabiting cattle and cattle kept in the epizootic area, and seroconversion to the virus in 1984, were disclosed. Experimental infection of calves with Iriki isolate produced severe nervous signs and histopathological changes similar to those of the natural infection. These seroepidemiological findings and animal experiments established that Iriki isolate is the causative agent of the disease. Iriki isolate was considered as a variant of Akabane virus since the virus showed cross reaction with Akabane virus in virus neutralization tests.     

The development of Akabane virus-induced congenital abnormalities in cattle

A prospective study of the incidence and severity of congenital deformities of calves, attributable to maternal infection by Akabane virus, was carried out on a population of 174 susceptible animals that were between one and nine months pregnant at the time of infection. The study was carried out in the Hunter Valley of New South Wales during 1983, after an epidemic of Akabane virus infection in late February to early March 1983. The incidence of virus-induced abnormalities in calves and fetuses was 17.8 per cent (31/174). The highest incidence of abnormalities occurred during the third and sixth months of gestation (27 to 29 per cent). The earliest abnormality was observed after infection at 76 days of gestation, and the last after infection at 249 days. The development of the pathological entities of hydranencephaly/porencephaly and arthrogryposis were found to be quite distinct. Cases of hydranencephaly and porencephaly developed after infection between 76 and 104 days of gestation whereas arthrogryposis developed after infection between 103 and 174 days of infection. It was concluded that the type of congenital deformity produced by maternal infection with Akabane virus was dependent on the stage of fetal development at the time of infection. The data suggest that the infection was transplacental and that fetuses of less than two months of age were protected from infection.     

Evaluating the protective efficacy of a trivalent vaccine containing Akabane virus,Aino virus and Chuzan virus against Schmallenberg virus infection

Schmallenberg virus (SBV), an arthropod borne pathogen, spread rapidly throughout the majority of Europe since 2011. It can cause a febrile disease, milk drop, diarrhea, and fetal malformation in ruminants. SBV, a member of the Simbu serogroup within the genus Orthobunyavirus, is closely related to Akabane virus (AKAV) and Aino virus (AINOV) among others. In the present study, 4 Holstein-Friesian calves were immunized twice four weeks apart with a multivalent, inactivated vaccine against AKAV and AINOV. Another 4 calves were kept as unvaccinated controls. All animals were clinically, serologically and virologically examined before and after challenge infection with SBV. AKAV- and AINOV-specific neutralizing antibodies were detected one week before challenge infection, while SBV-specific antibodies were detectable only thereafter. SBV genome was detected in all vaccinated animals and 3 out of 4 controls in serum samples taken after challenge infection. In conclusion, the investigated vaccine was not able to prevent an SBV-infection. Thus, vaccines for other related Simbu serogroup viruses can not substitute SBV-specific vaccines as an instrument for disease control.     

Diagnostic performance of the equine IgM capture ELISA for serodiagnosis of West Nile virus infection

The objectives of these studies were to assess the diagnostic performance (sensitivity and specificity) of the IgM capture enzyme-linked immunosorbent assay (ELISA; MAC) for diagnosis of West Nile (WN) virus in horses and to examine the performance of this test by using different criteria for seropositivity. A total of 36 horses classified as WN virus infected (group 1) and 383 horses from 4 subpopulations of hoses classified as noninfected (groups 2, 3, 4, and 5) were used in the study. The sensitivity (proportion of infected horses that tested positive for WN virus IgM antibodies) and specificity (proportion of noninfected horses that tested negative) were calculated at different cutoff points by using receiver operating curve (ROC) analysis. Using a selected cutoff point = 2.0, the sensitivity and specificity of the MAC were 91.7 and 99.2%, respectively. The area under the ROC curve = 0.95 (95% confidence interval [CI], 0.89 to 1.0), suggesting that the MAC is a useful tool for diagnosis of recent WN virus exposure in horses. In fulfillment of the 2nd objective, 2 other indices were developed and these indices approached 1.0 for the AUC with smaller 95% CIs. These indices were then used to test 602 additional diagnostic samples submitted from suspect horses between 2002 and 2004. Using the standard cutoff, 194 (32%) of the horses were interpreted as positive. Utilizing newly predicted cutoff criteria from each index, additional horses were identified as positive. In conclusion, the MAC as used for identification of WN virus-diseased horses undergoing recent exposure performs reliably at the standard cutoff for seropositivity. A negative test might not completely rule out WN virus disease, but horses that test negative were most likely not exposed to WNV. Performance of the test can be further improved by investigation of other indexes of seropositivity.     

Evidence for bovine immunodeficiency virus infection in cattle in Zambia

We report herein on the first evidence for the presence of bovine immunodeficiency virus (BIV) in Zambia. Serological surveillance of BIV and bovine leukemia virus (BLV) was conducted in traditional cattle herds in Zambia. Out of a total of 262 sera analyzed, 11.4% were found positive for anti-BIV p26 antibodies as determined by Western blot analysis, while 5.0% were positive for anti-BLV gp51 antibodies as detected by immunodiffusion test. Peripheral blood mononuclear cells from BIV seropositive cattle were found to have BIV-provirus DNA, as detected by nested polymerase chain reaction. A nucleotide sequence corresponding to a 298 bp fragment of the BIV pol gene was also analyzed. Amino acid sequences of these Zambian pol gene products showed 98.0 to 100% homology to the American strain BIV R29, 97.0 to 99.0% to Japanese BIV isolates, and divergence ranged from 0.0 to 2.0% among Zambian BIV isolates.     

Experimental placental transfer of Akabane virus in the hamster

Transplacental infection of hamster fetuses was produced by inoculation of pregnant hamsters with 10(6.3) plaque-forming units (PFU) of Akabane virus by either the intraperitoneal or the subcutaneous route. Virus with titers as high as 10(7.5) PFU/g of tissue was detected first in the placenta and later in the fetus. Virus could also be readily isolated from blood, lung, spleen, and liver of both pregnant and nonpregnant hamsters, but it reached higher titers and persisted longer in the placenta and fetus. Young dying at birth had Akabane virus titers as high as 10(7.3) PFU/g of brain tissue. Litter size was reduced by inoculation of the pregnant hamster at gestational day 11 or earlier, and survival of the newborn to 1 week of age was decreased by inoculation at gestational day 9 or later.