Danofloxacin (Advocin) reduces the spread of contagious bovine pleuropneumonia to healthy in-contact cattle

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides SC (MmmSC), is one of the most important diseases of cattle in Sub-Saharan Africa. The live T1/44 vaccine is normally used for its control but produces only transient protection and gives rise to adverse reactions. The present study evaluated the efficacy of danofloxacin (2.5% Advocintrade mark, Pfizer Ltd.) for the treatment of naturally infected cattle and in the prevention of CBPP transmission to in-contact cattle. Adult cattle, taken from a natural outbreak, were placed into two groups of 10 animals and kept on a research farm in paddocks 50m apart. One group was treated with 2.5mg/kg danofloxacin on days 0, 1 and 2; the other group were saline treated. On day 2, 10 CBPP-free, seronegative cattle were placed in contact with each of the two groups. All cattle were monitored for 3.5 months. No differences were seen in clinical improvement of the CBPP-affected cattle treated with danofloxacin compared with the untreated CBPP-affected cattle with approximately half of each group being withdrawn because of CBPP or showing CBPP lesions at post mortem examination. Clinical scores of the two groups were also similar. However cattle kept in contact with the danofloxacin-treated CBPP-affected animals showed significantly fewer lesions, less mortality and fewer animals were seropositive (P<0.02) and had reduced clinical scores (P<0.001) compared to cattle kept in contact with untreated CBPP-affected cattle. MmmSC was also isolated from fewer contact controls kept with the treated group. These findings could have important implications for the control of CBPP in Africa.     

Development of an indirect fluorescent antibody test, using microfluorometry as a diagnostic test for bovine anaplasmosis

The indirect fluorescent antibody test for the diagnosis of Anaplasma marginale infection in cattle was modified for use with microfluorometry. The test was standardized by use of a fluorometer that measures intensity of fluorescence. Standardization included A marginale-infected blood smears on microscope slides as antigen, serum from an inoculated calf as a positive control containing specific antibody, and an affinity-purified fluorescein-conjugated anti-bovine immunoglobulin as 2nd antibody. The modified test and microfluorometry allowed for titration of sera from A marginale (Florida isolate)-inoculated cattle with a degree of accuracy exceeding visual determinations. In addition, the fluorometric test was more sensitive than the complement fixation or card agglutination tests in identifying cattle that had previous Anaplasma infections.     

Development of a diagnostic blood test for tuberculosis in alpacas (Lama pacos)

Vaccination of alpacas (Lama pacos) with heat-killed Mycobacterium bovis (BCG) in oil adjuvant produces an immune response that is able to be measured in vitro. Lymphocyte transformation was present 2 weeks after boosting while antibody as measured by the enzyme-linked immunosorbent assay was present 4-6 weeks after primary vaccination. Two vaccinated animals were positive to the intradermal skin test at the conclusion of the experiment and showed signs of systemic inflammation 72 hours after the skin test, while the controls remained negative for all tests. The BCG vaccine studies showed that these laboratory tests can also identify M. bovis-specific reactivity, so the technique has potential for the diagnosis of tuberculosis in alpacas. Apart from diagnosing M. bovis-specific reactivity, the blood test for tuberculosis may also be used to diagnose non-specific mycobacterial sensitisation in farmed New Zealand alpacas.     

Prevalence study on bovine tuberculosis and molecular characterization of its causative agents in cattle slaughtered at Addis Ababa municipal abattoir, Central Ethiopia

A cross-sectional study was conducted on 500 cattle slaughtered at Addis Ababa abattoir to determine the prevalence of bovine tuberculosis (BTB) and characterize its causative agents. Postmortem examination, mycobacteriological culturing, region of difference-4 (RD4)-based PCR and spoligotyping were applied. The prevalence of BTB was 5 % on the basis of postmortem inspection alone but 1.2 % based on molecular confirmation. Factors including age, sex, and breed showed statistically significant association with BTB (p?<?0.05). Gross lesions were observed most frequently (68 %) in the lungs and lung-associated lymph nodes compared to other organs and lymph nodes. Of the 25 grossly suspicious TB lesions processed and cultured, only six (24 %) were culture-positive, yielding Mycobacterium bovis confirmed by RD4 deletion typing. Further characterization of the six M. bovis isolates at the strain level by using spoligotyping revealed that one did not belong to any previously known type, while the others belonged to types SB1176 (two), SB1477 (two), and SB0133 (one). The new strain was submitted to the international M. Bovis.org database for international code designation. The study confirms the considerable prevalence of bovine tuberculosis in cattle slaughtered at Addis Ababa abattoir and highlights the need for control of bovine tuberculosis in the country.     

Evaluation of PetrifilmsTM as a diagnostic test to detect bovine mastitis organisms in Kenya

The study purpose was to validate Petrifilms^TM (3M Microbiology, 2005) against standard culture methods in the diagnosis of bovine mastitis organisms in Kenya. On 128 smallholder dairy cattle farms in Kenya, between June 21, 2010 and August 31, 2010, milk samples from 269 cows that were positive on California Mastitis Test (CMT) were cultured using standard laboratory culture methods and Petrifilms^TM (Aerobic Count and Coliform Count –3M Microbiology, 2005), and results were compared. Staphylococcus aureus was the most common bacterium isolated (73 % of samples). Clinical mastitis was found in only three cows, and there were only two Gram-negative isolates, making it impossible to examine the agreement between the two tests for Gram-negative- or clinical mastitis samples. The observed agreement between the standard culture and Petrifilm^TM (3M Microbiology, 2005) results for Gram-positive isolates was 85 %, and there was fair agreement beyond that expected due to chance alone, with a kappa (κ) of 0.38. Using culture results as a gold standard, the Petrifilms^TM had a sensitivity of 90 % for Gram-positive samples and specificity of 51 %. With 87 % of CMT-positive samples resulting in Gram-positive pathogens cultured, there was a positive predictive value of 93 % and a negative predictive value of 43 %. Petrifilms^TM should be considered for culture of mastitis organisms in developing countries, especially when Gram-positive bacteria are expected.     

Development of a novel diagnostic test for detection of bovine viral diarrhea persistently infected animals using hair

The purpose of this study was to determine whether manually plucked hairs might serve as an alternative sample for a quantitative real time polymerase chain reaction (qRT-PCR) testing. Twenty three, 1~3 week old, non-bovine viral diarrhea virus (BVDV) vaccinated calves, found to be positive for BVDV by immunohistochemical staining, were selected and hairs were manually plucked from the ear. qRT-PCR was performed on samples consisting of more than 30 hairs (30~100) and whole blood. All 23 animals were positive for the virus by qRT-PCR performed on the whole blood and when samples of more than 30 hairs were assayed. Additionally, qRT-PCR was performed on groups of 10 and 20 hairs harvested from 7 out of 23 immunohistochemical staining-positive calves. When groups of 20 and 10 hairs were tested, 6 and 4 animals, respectively, were positive for the virus.     

牛结核病巢式PCR快速检测方法的建立

根据分枝杆菌（mycobacteria）保守的插入序列IS1081设计4条特异性引物，建立了快速检测牛结核病的巢式PCR方法。该方法一次扩增的敏感性是1．35pg，二次扩增的敏感性是1．35fg。在对95份PPD阳性牛临床病料组织和23份血液样本的PCR检测中，用引物TB—Q1和TB-Q2做一次扩增，阳性检出率分别为36／95（37．5％）和5／23（21．7％）；用引物TB—B1和TB—B2做二次扩增，阳性检出率分别为81／95（85．3％）和14／23（60．9％）。该方法作为辅助PPD试验的快速检测方法用于牛结核病的流行病学调查，具有重要的实用价值和应用前景。     

Evaluation of the sensitivity and specificity of bovine tuberculosis diagnostic tests in naturally infected cattle herds using a Bayesian approach

Test-and-slaughter strategies have been the basis of bovine tuberculosis (BT) eradication programs worldwide; however, eradication efforts have not succeeded in certain regions, and imperfect sensitivity and specificity of applied diagnostic techniques have been deemed as one of the possible causes for such failure. Evaluation of tuberculosis diagnostic tools has been impaired by the lack of an adequate gold standard to define positive and negative individuals. Here, a Bayesian approach was formulated to estimate for the first time sensitivity (Se) and specificity (Sp) of the tests [single intradermal tuberculin (SIT) test, and interferon-gamma (IFN-γ) assay] currently used in Spain. Field data from the first implementation of IFN-γ assay (used in parallel with SIT test 2-6months after a first disclosure SIT test) in infected beef, dairy and bullfighting cattle herds from the region of Castilla and Leon were used for the analysis. Model results suggested that in the described situation: (i) Se of SIT test was highly variable (40.1-92.2% for severe interpretation, median=66-69%), and its Sp was high (>99%) regardless interpretation criteria; (ii) IFN-γ assay showed a high Se (median=89-90% and 83.5% for 0.05 and 0.1 cut-off points respectively) and an acceptable Sp (85.7% and 90.3% for 0.05 and 0.1 thresholds) and (iii) parallel application of both tests maximized the combined Se (95.6% using severe SIT and 0.05 cut-off point in the IFN-γ assay). These results support the potential use of the IFN-γ assay as an ancillary technique for routine BT diagnosis.     

检测牛结核抗体新型ELISA方法的建立及应用

利用分子克隆和Splicing by overlapping extension(SOE)技术,表达了重组蛋白rM70-83-E6,Western blot分析rM70-83-E6具有很好的特异性。以蛋白rM70-83-E6作为诊断抗原建立了间接ELISA方法,ELISA诊断方法的判定标准,即S/P≥0.5为阳性,S/P<0.4为阴性,0.5>S/P≥0.4为可疑。ELISA方法的特异性为96.0%、敏感性为69.4%。对67份PPD皮试阳性和50份PPD皮试阴性(117份)血清样品进行了检测,并与PPD皮试比较。67份PPD皮试阳性血清样品中有46份为ELISA检测阳性,阳性符合率为68.7%,50份PPD皮试阴性牛血清都为阴性,阴性符合率为100%,本ELISA方法与PPD皮试诊断方法总复合率为82.1%。研究表明,ELISA方法具有很好的开发和应用前景。     

Development of a simple, rapid in vitro cellular assay for bovine tuberculosis based on the production of gamma interferon

Whereas in vitro assays of antibody responses to infectious agents are commonly used for routine diagnostic purposes, diagnostic tests for cellular responsiveness are confined to in vivo intradermal tests. This paper describes a simple and rapid in vitro cellular assay for bovine tuberculosis. The assay system is based on the detection of gamma interferon (gamma IFN), which is released in response to specific antigen. This assay can be carried out with whole blood samples thus avoiding the time-consuming task of isolating lymphocytes. A simple bioassay has been developed to quantitate the amount of gamma IFN produced, but this will eventually be replaced with an enzyme-linked immunoassay using monoclonal antibodies specific for bovine gamma IFN. The application of this system to bovine tuberculosis and other infectious diseases may provide a convenient in vitro cellular assay for routine diagnostic purposes.     

Evaluation of enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in Korea

An enzyme-linked immunosorbent assay (ELISA) using bulk tank milk samples was evaluated as a screening test for bovine tuberculosis (TB), a contagious chronic disease of cattle. An ELISA with MPB70, a major antigen of Mycobacterium bovis was performed using paired sets of milk and sera samples from 33 tuberculin-positive and 43 tuberculin-negative cattle. Anti-MPB70 antibodies were detected in milk samples and there was a significant correlation between seroreactivities of milk and sera samples (R² = 0.83). Using the tuberculin skin test as the reference test, the sensitivities of ELISA using milk and sera samples were 87.8% and 81.8%, respectively, and the specificities were 97.7% and 100%, respectively.In the screening test using bulk tank milk samples from 931 dairy herds in Whasung, Gyeonggi-do, Korea, the positive rate for anti-MPB70 antibody was 4.5% (42/931) and the tuberculin-positive rate was 2.8% (26/931). Individual milk samples (n = 253) were collected from randomly selected 8 problematic and 3 negative herds (positive and negative in the screening test by MPB70 ELISA using bulk tank milk samples, respectively) and tested by MPB70 milk ELISA. In the problematic herds, positive rates were 10.5% (20/190) for anti-MPB70 antibodies in milk ELISA and 2.1% (4/190) in the tuberculin skin test. More than one dairy cows were positive by milk ELISA among the problematic herds, and all tuberculin-positive dairy cows were positive in the milk ELISA. Further, no positive cows were detected in negative herds both by milk ELISA and tuberculin skin test. These results suggest that an ELISA, using bulk tank milk samples, might be a potential efficient screening test for bovine TB of dairy cows.     

致奶牛乳腺炎金黄色葡萄球菌快速鉴别及药敏试验选择培养液的培养特性

本研究在Baird-parker培养基基础上,研制出致奶牛乳腺炎金黄色葡萄球菌(SA)快速鉴别培养及药敏试验用选择mBP培养液.在此培养液中,除SA生长良好外,链球菌等4种革兰氏阳性茵和大肠杆茵等6种革兰氏阴性菌均不生长;将10μL奶样接种入mBP培养液,37℃培养24 h后即可根据培养液是否浑浊和产生黑色产物判断奶样中有无SA,检测灵敏度为60个细菌:经与标准纸片法药敏试验对比证明,mBP培养液对常用于奶牛乳腺炎治疗的7类8种抗生素的抑茵活性无明显影响:将10μL奶样或选择培养物接种入含标准抑茵浓度抗生素的mBP培养液中,35℃培养24 h后即能根据培养液是否浑浊和产生黑色产物判断奶样中SA对所试抗生素的敏感性.以mBP培养液为基础建立的致奶牛乳腺炎金黄色葡萄球茵快速鏊剐及药敏试验方法具有简单、快速、价廉和不需专门设备等优点,特别适合我国中、小型奶牛场和基层兽医站使用.     

蜜蜂属生物的物种分子诊断标签:动物DNA条形码

蜜蜂属生物构成了膜翅目昆虫一个代表属,蜜蜂属生物的物种分子诊断标签普遍选用动物DNA条形码;蜜蜂属生物的物种DNA条形码经过生物信息学数据分析,成为动物DNA条形码数据库一组不可或缺的生物样本分类学资源。本文从如下三个方面综述了蜜蜂属生物的物种DNA条形码研究进展,即蜜蜂属生物的物种DNA条形码含义,蜜蜂属生物的物种DNA条形码生物信息学数据分析方法,以及蜜蜂属生物的物种DNA条形码分类学地位。     

Molecular epidemiology of bovine tuberculosis in wild animals in Spain: a first approach to risk factor analysis

In human tuberculosis (Mycobacterium tuberculosis), molecular epidemiology has accurately indicated the risk factors involved in active transmission of the disease, by comparing individuals whose isolates belong to a cluster with patients whose strains are considered unique. Nevertheless, this application has not been used in bovine tuberculosis (Mycobacterium bovis). Our study describes the integration of epidemiological data into molecular classification data on M. bovis isolates. These were isolated from wild ungulates in Extremadura (western Spain) with the objective of detecting the risk factors linked to the association of strains in clades, which are indicators of the active spread of the disease. The molecular markers used were spoligotyping + VNTR typing (loci: VNTR 2165, VNTR 2461, VNTR 0577, VNTR 0580, VNTR 3192 VNTR 2163a and VNTR 2163b) on a population of 59 M. bovis strains isolated from deer (Cervus elaphus), 112 from wild boar (Sus scrofa), six from bovines, 28 from pigs and 2 from goats (n = 207). Epidemiological variables included the animal species from which the strain was isolated, pathological condition of the host (incipient lesion, early and late generalisation), date of sampling (during or after the reproductive period) and hunting season. Bivariant analysis was used to establish the risk factors connected to the association of strains and later, the variables were evaluated by means of logistic regression. Molecular typing grouped a total of 131 strains (64.21%) in 28 clusters and 76 isolates shows unique profiles. The association of strains was connected to the appearance of macroscopic lesions during the reproductive period (O.R. 4.80; 95% CI 1.09–22.99, P < 0.005), showing a possible higher transmission during the courting period. This happened mainly during the last hunting season analysed (2002–2003, O.R. 3.69; 95% CI 1.27–11.9, P < 0.05), clashing with the time of higher prevalence of the disease in wild ungulates. Active spread was not connected to any species in particular, or to any concrete pathological condition.     

Development of a specific latex agglutination test based on a recombinant hemagglutinin protein to detect antibodies to H5 avian influenza viruses

A latex agglutination test (LAT) was developed for rapid detection of antibodies to H5 avian influenza viruses (AIVs). The hemagglutinin protein of H5 AIV was covalently linked to carboxylated latex by ethyl-dimethyl-amino-propyl carbodiimide to prepare the sensitized latex beads. The LAT was evaluated with the hemagglutination inhibition (HI) assay as the reference test. The H5-LAT showed a sensitivity of 87.0% and specificity of 88.9% in detecting 126 serum samples from experimentally infected chickens and a sensitivity of 82.5% and specificity of 86% in detecting 587 field chicken serum samples from mostly vaccinated chickens. The agreement ratio between H5-LAT and HI was found to be 87.3% and 83.1% for the two groups of samples, respectively. Difficulty with background agglutination in stored chicken sera was overcome by serum pretreatment with either dried chicken liver powder or dilution buffer containing detergent Tween-20. The H5-LAT has advantages over a previously reported whole-virus LAT in terms of biosafety in preparation, chemical stability, and higher specificity. It is a rapid and simple test suitable for field monitoring of antibodies to H5-type AIV.     

Bayesian estimation of diagnostic accuracy of a new bead-based antibody detection test to reveal Toxoplasma gondii infections in pig populations

The success of a Toxoplasma gondii surveillance program in European pig production systems depends partly on the quality of the test to detect infection in the population. The test accuracy of a recently developed serological bead-based assay (BBA) was investigated earlier using sera from experimentally infected animals. In this study, the accuracy of the BBA was determined by the use of sera from animals from two field subpopulations. As no T. gondii infection information of these animals was available, test accuracy was determined through a Bayesian approach allowing for conditional dependency between BBA and an ELISA test. The priors for prevalence were based on available information from literature, whereas for specificity vague non-informative priors were used. Priors for sensitivity were based either on available information or specified as non-informative. Posterior estimates for BBA sensitivity and specificity were (mode) 0.855 (Bayesian 95% credibility interval (bCI) 0.702–0.960) and 0.913 (bCI 0.893–0.931), respectively. Comparing the results of BBA and ELISA, sensitivity was higher for the BBA while specificity was higher for ELISA. Alternative priors for the sensitivity affected posterior estimates for sensitivity of both BBA and ELISA, but not for specificity. Because the difference in prevalence between the two subpopulations is small, and the number of infected animals is small as well, the precision of the posterior estimates for sensitivity may be less accurate in comparison to the estimates for specificity. The estimated value for specificity of BBA is at least optimally defined for testing pigs from conventional and organic Dutch farms.     

A longitudinal study to evaluate the diagnostic potential of a direct faecal quantitative PCR test for Johne's disease in sheep

A longitudinal study was carried out to evaluate the diagnostic potential of the previously developed direct faecal real-time quantitative PCR (QPCR) assay (Kawaji et al., 2007) for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) infected sheep. Of the 58 sheep, 38 were orally inoculated with MAP, while 20 controls were maintained separately from the infected group throughout the trial. All animals were tested by QPCR, faecal culture and serum ELISA pre-inoculation and at 4, 8 and 13 months post-inoculation, and were necropsied at 13 months post-inoculation. Eighteen out of 38 inoculated sheep were detected by QPCR to be shedding MAP in faeces at 4 months post-inoculation, while only one sheep was positive in faecal culture at this time point. At 8 months post-inoculation, MAP DNA was detected in faeces of all inoculated sheep by QPCR, while MAP organisms were isolated from only 34% of the inoculated animals by faecal culture. The QPCR results for faecal samples that were collected at necropsy demonstrated that faecal QPCR was more sensitive than culture of intestinal tissues for MAP. The QPCR assay was confirmed to be a sensitive and specific ante-mortem diagnostic test for MAP in sheep, circumventing faecal culture which is a less sensitive and highly time consuming test. Quantification of MAP DNA in faeces by QPCR may provide immediate information to estimate the stage of the infection as well as the risk of transmission from infected animals.     

A field study evaluation of Petrifilm™ plates as a 24-h rapid diagnostic test for clinical mastitis on a dairy farm

Clinical mastitis is one of the most common and expensive diseases of dairy cattle. To make an informed treatment decision, it is important to know the causative pathogen. However, no detection of bacterial growth can be made in approximately 30% of all clinical cases of mastitis. Before selecting the treatment regimen, it is important to know whether the mastitis-causing pathogen (MCP) is Gram-positive or Gram-negative. The aim of this field study was to investigate whether using two 3M Petrifilm™ products on-farm (which conveys a higher degree of sample freshness but also bears a higher risk for contamination than working in a lab) as 24-h rapid diagnostic of clinical mastitis achieved results that were comparable to the conventional microbiological diagnostic method.