Evaluation of three enzyme-linked immunosorbent assays (ELISAs) for the detection of serum antibodies in sheep infected with Echinococcus granulosus

The aim of this study was to develop an immunological method for the identification of sheep infected with Echinococcus granulosus which would allow the monitoring of animals imported into countries free from hydatidosis and as an aid to countries where control schemes for the disease are in operation. Three enzyme-linked immunosorbent assays (ELISAs) were developed and validated, using as antigen either a purified 8 kDa hydatid cyst fluid protein (8kDaELISA), a recombinant EG95 oncosphere protein (OncELISA) or a crude protoscolex preparation (ProtELISA). Sera used for the assay validations were obtained from 249 sheep infected either naturally or experimentally with E. granulosus and from 1012 non-infected sheep. The highest diagnostic sensitivity was obtained using the ProtELISA at 62.7 and 51.4%, depending on the cut-off. Assay sensitivities were lower for the 8kDaELISA and the OncELISA. Diagnostic specificities were high, ranging from 95.8 to 99.5%, depending on the ELISA type and cut-off level chosen. A few sera from 39 sheep infected with T. hydatigena and from 19 sheep infected with T. ovis were recorded as positive. Western immunoblot analysis revealed that the dominant antigenic components in the crude protoscolex antigen preparation were macromolecules of about 70-150 kDa, most likely representing polysaccharides. This study demonstrated that the ProtELISA was the most effective immunological method of those assessed for detection of infection with E. granulosus in sheep. Because of its limited diagnostic sensitivity of about 50-60%, it should be useful for the detection of the presence of infected sheep on a flock basis and cannot be used for reliable identification of individual animals infected with E. granulosus.     

Immunosuppression in specific-pathogen-free broilers administered infectious bursal disease virus vaccines by in ovo route

The effect of two infectious bursal disease virus (IBDV) vaccines (IBDV-immune complex [Icx] and IBDV-2512), administered in ovo, on the cell-mediated immunity of specific-pathogen-free (SPF) broilers was examined. A decrease (P < 0.05) in the T-cell mitogenic response occurred in birds vaccinated with both vaccines on days 9 and 21 post in ovo vaccination (PIOV), but an increase (P < 0.05) occurred on day 15 PIOV. The T cells from birds given the IBDV-2512 were less responsive. There were no significant differences in proportions of lymphocytes expressing CT4+CT8 and CT8+CT4- except on day 21 PIOV, where an increase (P < 0.05) in IBDV-2512-vaccinated birds and a decrease (P < 0.05) in percentage of CT4+CT8- in IBDV-Icx-vaccinated birds was observed. There was an increase (P < 0.05) in percentage of CT8+CT4- T cells on day 21 PIOV in both vaccinated groups. A decrease (P < 0.05) in B-cell percentage was observed on day 21 PIOV in birds given both vaccines. Results indicated that although humoral immunosuppression is associated with destruction of B cells (bursal atrophy), cell-mediated immunosuppression induced by these two IBDV vaccines in SPF birds was not associated with altered helper (CT4+CT8-) or cytotoxic (CT8+CT4-) subpopulations of T lymphocytes.     

甜瓜蔓枯病菌子实体法分离及A型菌株产孢条件研究

为解决甜瓜蔓枯病菌(Didymellabryoniae)组织分离法中枯萎病菌污染和单孢分离法操作复杂的问题,采用挑取单个子实体分离纯化获得甜瓜蔓枯病菌A型菌株。该菌株在12h荧光/12h黑暗的常规光照条件下,不能形成分生孢子器,但经过一定时间的暗处理和间歇紫外灯(12h紫外灯/12h黑暗)照射后则能够产生大量分生孢子。研究了光照、温度、pH值、培养基、碳源、氮源对产孢量的影响。综合各种因素,A型菌株产生分生孢子的最佳条件为:7d暗培养和4d间歇紫外灯处理的光照条件下,只含有磷酸二氢氨的马铃薯平板培养基,25℃时产生的分生孢子量最多。     

Estimation of receiver-operating characteristic curves to determine accuracy of a competitive enzyme-linked immunosorbent assay for the serodiagnosis of Brucella infection in domestic water buffalo (Bubalus bubalis) and cattle

OBJECTIVE: To estimate receiver-operating characteristic (ROC) curves for a competitive ELISA (c-ELISA) that is used in serodiagnosis of brucellosis in water buffalo and cattle, to determine the most appropriate positive cutoff value for the c-ELISA in confirmation of infection, and to evaluate species differences in c-ELISA function. SAMPLE POPULATION: Sera from 4 herds of cattle (n = 391) and 4 herds of water buffalo (381). PROCEDURE: Serum samples were evaluated for Brucella-specific antibodies by use of a c-ELISA. On the basis of previous serologic test results, iterative simulation modeling was used to classify animals as positive or negative for Brucella infection without the use of a gold standard. Accuracy of c-ELISA for diagnosis of infection was compared between cattle and water buffalo by comparison of areas under ROC curves. RESULTS: A positive cutoff value of 30% inhibition for c-ELISA yielded sensitivity and specificity estimates, respectively, of 83.9 and 92.6% for cattle and 91.4 and 95.4% for water buffalo. A positive cutoff value of 35% inhibition yielded sensitivity and specificity estimates, respectively, of 83.9 and 96.2% for cattle and 88.0 and 974% for water buffalo. Areas under ROC curves were 0.94 and 0.98 for cattle and water buffalo, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: ROC curves can be estimated by use of iterative simulation methods to determine optimal cutoff values for diagnostic tests with quantitative outcomes. A cutoff value of 35% inhibition for the c-ELISA was found to be most appropriate for confirmation of Brucella infection in cattle and water buffalo.     

Multi-scale Mexican spotted owl (<Emphasis Type="Italic">Strix occidentalis lucida</Emphasis>) nest/roost habitat selection in Arizona and a comparison with single-scale modeling results

Context

Organisms commonly respond to their environment across a range of scales, however many habitat selection studies still conduct selection analyses using a single-scale framework. The adoption of multi-scale modeling frameworks in habitat selection studies can improve the effectiveness of these studies and provide greater insights into scale-dependent relationships between species and specific habitat components.

Objectives

Our study assessed multi-scale nest/roost habitat selection of the federally “Threatened” Mexican spotted owl (Strix occidentalis lucida) in northern Arizona, USA in an effort to provide improved conservation and management strategies for this subspecies.

Methods

We conducted multi-scale habitat modeling to assess habitat selection by Mexican spotted owls using survey data collected by the USFS. Each selected covariate was included in multi-scale models at their “characteristic scale” and we used an all-subsets approach and model selection framework to assess habitat selection.

Results

The “characteristic scale” identified for each covariate varied considerably among covariates and results from multi-scale models indicated that percent canopy cover and slope were the most important covariates with respect to habitat selection by Mexican spotted owls. Multi-scale models consistently outperformed their analogous single-scale counterparts with respect to the proportion of deviance explained and model predictive performance.

Conclusions

Efficacy of future habitat selection studies will benefit by taking a multi-scale approach. In addition to potentially providing increased explanatory power and predictive capacity, multi-scale habitat models enhance our understanding of the scales at which species respond to their environment, which is critical knowledge required to implement effective conservation and management strategies.

     

Characterization of a wild-type strain of Francisella tularensis isolated from a cat.

Francisella tularensis type A is the primary cause of tularemia in animals and humans in North America. The majority of research on F. tularensis has been done with the attenuated live vaccine strain (LVS), which is a type B, but very few wild-type F. tularensis strains have been characterized. A gram-negative coccobacillus that was isolated in pure culture from the lungs of a cat that died after being lost for 5 days was received for identification at the Virginia-Maryland Regional College of Veterinary Medicine Teaching hospital. The isolate (strain TI0902) was not identified (or was misidentified) by commercial identification systems; however, it was identified as F. tularensis subspecies tularensis (type A) by sequencing a portion of the 16S ribosomal RNA gene. Furthermore, repetitive extragenic palindromic sequences-polymerase chain reaction amplified a 4-kb DNA fragment from TI0902 that was characteristic of F. tularensis type A but not type B. The electrophoretic profile of the lipopolysaccharide of strain TI0902 was identical to that of the LVS by Western blotting with antiserum to LVS. The protein-enriched outer membrane of strain TI0902 contained 6-8 proteins, which were similar in molecular size to those from the LVS. Electron microscopy of negatively stained and alcian blue-stained LVS and TI0902 cells showed that both strains were coccobacillary in shape and may be encapsulated. However, after mouse challenge, the TI0902 strain was clearly more virulent than the LVS strain. Results of this study indicate that the genotype and phenotype of wild-type F. tularensis type A strain TI0902 is similar, but not identical, to that of the LVS strain. Further studies will help determine whether pathogenesis and host-pathogen interactions are also similar between the 2 strains.     

Tetanus in a Camel (Camelus dromedarius) – A Case Report

Twenty days after an open castration, a 5-year-old dromedary was presented to the Dubai Camel Hospital with severe central nervous symptoms. The dromedary showed the following signs: off feed, stiff gait with extended neck, external swelling of the preputial sheath and groin region, and foamy saliva drooling from the mouth. The dromedary was unable to swallow. Three days after admission, the camel developed lockjaw, and on the fifth day it was unable to stand owing to paralysis of the hindquarters. Because of the severity of the disease and because it did not respond to treatment, the camel was euthanized 26 days after the operation and submitted to the Central Veterinary Research Laboratory for further investigation. Both castration wounds were closed and spermiducts were filled with necrotic masses from which Clostridium tetani was isolated. Two mice, which were injected with the filtrate of the thioglycolate broth, developed typical signs of tetanic spasm of the hind leg. Faecal samples from camel and horse paddocks that were only 50 metres apart were negative for C. tetani. However, C. tetani was isolated from two soil samples of the horse paddock. It is recommended that camels should be vaccinated against tetanus prior to castration.     

Rapid clonal propagation of rhubarb by in vitro culture of shoot-tips

In vitro culture of shoot-tips of rhubarb (Rheum rhaponticum L.) on the medium of Murashige and Skoog (1962), containing 1 mg/l of 6 benzylaminopurine and 1 mg/l of 3-indolebutyric acid, induced the development of axillary buds. Rooting was easily attained in 4–5 days by omitting the cytokinin from the basal medium. The temperature was 25 ± 1° C during the 16-h photoperiod and 20 ± 1° C during the 8-h dark period. The rooted plantlets were transplanted to soil medium with 70% success. The method described gives a multiplication rate of 5 per month.