Evaluation of the performance of the IFN-γ release assay in bovine tuberculosis free herds from five European countries.

The diagnostic methods for granting and maintenance of the official tuberculosis-free (OTF) status and for intra-Community movement of cattle are the tuberculin skin tests (single or comparative) and the interferon-γ (IFN-γ) release assay (IGRA). However, until now, IGRAs have been primarily applied in infected farms in parallel to the skin test to maximize the number of infected animals detected. Therefore, an evaluation of the performance of IGRAs in OTF herds to assess whether if their specificity is equal to or higher than that of the skin tests is needed. For this, a panel of 4365 plasma samples coming from 84 OTF herds in six European regions (five countries) was assembled and analysed using two IGRA kits, the ID Screen® Ruminant IFN-g (IDvet) and the Bovigam™ TB Kit (Bovigam). Results were evaluated using different cut-offs, and the impact of herd and animal-level factors on the probability of positivity was assessed using hierarchical Bayesian multivariable logistic regression models. The percentage of reactors ranged from 1.7 to 21.0% (IDvet: S/P ≥ 35%), and 2.1-26.3% (Bovigam: ODbovis-ODPBS ≥ 0.1 and ODbovis-ODavium ≥ 0.1) depending on the region, with Bovigam disclosing more reactors in all regions. The results suggest that specificity of IGRAs can be influenced by the production type, age and region of origin of the animals. Changes in the cut-offs could lead to specificity values above 98-99% in certain OTF populations, but no single cut-off yielding a sufficiently high specificity (equal or higher than that of skin tests) in all populations was identified. Therefore, an exploratory analysis of the baseline IFN-γ reactivity in OTF populations could help to assess the usefulness of this technique when applied for the purpose of maintaining OTF status.

Molecular detection of Brucella spp. in ruminant herds in Greece.

Brucellosis is a worldwide distributed infectious disease. Ruminants and other animal species (swine, dogs, equids, etc.), as well as wild mammals, can be affected. The disease can be transmitted to humans through the food chain or by direct contact with infected animals. Because of the relatively high economic burden due to abortions within a herd, significant efforts have been employed and hence the disease in most European countries has been eradicated. Accordingly, Greece applies both control and eradication programs concerning small ruminants (sheep and goats) and bovines depending on the geographical area. Current challenges in the standard antibody-based laboratory methods used for Brucella detection are the failure to differentiate antibodies against the wild strain from the ones against the vaccine strain Rev1 and antibodies against B. melitensis from those against B. abortus. The aim of the study was to reexamine and combine previously published protocols based on PCR analysis and to generate a rapid, not expensive, and easy to perform diagnostic tool able to confirm the doubtful results delivered from serology. For this reason, 264 samples derived from 191 ruminants of the farm and divided in 2 groups (male/female) were examined with a modified DNA extraction and PCR protocol. Molecular examination revealed the presence of Brucella spp. in 39 out of 264 samples (derived from 30 animals). In addition, Brucella spp. was detected in infected tissues such as testicles, inguinal lymph nodes, fetal liver, and fetal stomach content.

Validation of a competitive ELISA for diagnosis of Brucella melitensis infection in sheep and goats.

A competitive enzyme-linked immunosorbent assay (cELISA) was validated for the serodiagnosis of Brucella melitensis infection in small ruminants using 2108 positive and 2154 negative reference sera from sheep and goats. The optimum cut-off values, offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp), determined by receiver operating characteristic analysis, were at 23.6%, 21.8% and 25.0% inhibition of the conjugate control for sheep, goats and both species, respectively. The DSns of the cELISA for sheep, goats and both species at these cut-off values were 89.2% (95% confidence interval 87.1-91.1%), 74.0% (95% CI 71.4-76.5%) and 77.9% (95% CI 76.1-79.7%), whereas DSps were 96.4% (95% CI 95.2-97.4%), 92.9% (95% CI 91.1-94.3%) and 97.2% (95% CI 96.4-97.8%), respectively. Compared to cELISA, indirect ELISA and fluorescence polarisation assay have higher DSns and DSps. However, the results obtained with the cELISA were in good agreement with those of the complement fixation test (CFT) under field conditions using 5735 sheep and goat sera. The cELISA can be used as an alternative to the CFT for diagnosing B. melitensis infection in small ruminants.

Epidemiological and clinical aspects of human brucellosis in Central Greece.

The confirmed cases of human brucellosis in the area of Larissa in Central Greece from 2003 to 2005 were analyzed to assess the features of the affected population and to determine the factors influencing the acquisition of infection. Data of patients infected by Brucella spp. concerning age, gender, occupation, date of diagnosis and the observed symptoms were collected from regional hospitals, health centres and private practitioners. The incidence of human brucellosis in the area was 32.49 cases/100,000 inhabitants. Males, due to their professions, were affected more often by brucellosis than females. The majority of the cases were attributed to direct contact with animals or their products. Only in 8.49% of the cases was the infection attributed to the consumption of dairy products. The urban population is not at potential risk for acquiring brucellosis because all commercialized dairy products in Greece are produced from pasteurized milk. The occurrence of human brucellosis shows seasonality, with the majority of the cases diagnosed from December to May. Direct contact with animals, the season of the year and gender were the risk factors influencing the acquisition of infection. Fever, arthralgia, profuse sweating and anorexia were the symptoms most often observed.

Liver injury after ischemia and reperfusion: the role of oxygen free radicals.

OBJECTIVE: This article describes conditions of ischemia and reperfusion in dog's liver. The aim of the study was to observe hepatic injury from oxygen free radicals and to evaluate mannitol and ascorbic acid as scavengers. METHODS: The criteria for evaluation were histopathological and biochemical. Twenty-one dogs were divided into three equal groups: group A with 90 minutes of ischemia and 10 minutes of reperfusion, and groups B and C with 120 minutes of ischemia and 10 minutes of reperfusion. In treatment group C, before the ischemia, mannitol and ascorbic acid were injected. RESULTS: Differences were significant for malondialdehyde in A and B group postischemia (p < 0.001). In group C, a significant difference (p < 0.091) was not observed. The histopathological examination revealed fatty degeneration, alterations of mitochondrial structure, and membranic cysts. In group C, the alterations were considerably milder. CONCLUSIONS: We can suggest the use of ascorbic acid and mannitol at the clinical level for scavenging and inhibition of oxygen free radicals in ischemic liver.