Molecular typing of Brucella melitensis endemic strains and differentiation from the vaccine strain Rev-1

In the present study forty-four Greek endemic strains of Br. melitensis and three reference strains were genotyped by Multi locus Variable Number Tandem Repeat (ML-VNTR) analysis based on an eight-base pair tandem repeat sequence that was revealed in eight loci of Br. melitensis genome. The forty-four strains were discriminated from the vaccine strain Rev-1 by Restriction Fragment Length Polymorphism (RFLP) and Denaturant Gradient Gel Electrophoresis (DGGE). The ML-VNTR analysis revealed that endemic, reference and vaccine strains are genetically closely related, while most of the loci tested (1, 2, 4, 5 and 7) are highly polymorphic with Hunter-Gaston Genetic Diversity Index (HGDI) values in the range of 0.939 to 0.775. Analysis of ML-VNTRs loci stability through in vitro passages proved that loci 1 and 5 are non stable. Therefore, vaccine strain can be discriminated from endemic strains by allele’s clusters of loci 2, 4, 6 and 7. RFLP and DGGE were also employed to analyse omp2 gene and reveled different patterns among Rev-1 and endemic strains. In RFLP, Rev-1 revealed three fragments (282, 238 and 44 bp), while endemic strains two fragments (238 and 44 bp). As for DGGE, the electrophoretic mobility of Rev-1 is different from the endemic strains due to heterologous binding of DNA chains of omp2a and omp2b gene. Overall, our data show clearly that it is feasible to genotype endemic strains of Br. melitensis and differentiate them from vaccine strain Rev-1 with ML-VNTR, RFLP and DGGE techniques. These tools can be used for conventional investigations in brucellosis outbreaks.     

Leukocyte counts in bronchoalveolar lavage fluid obtained from normal and Maedi–Visna‐infected sheep

Background: Bronchoalveolar lavage has proven helpful for the diagnosis of certain ovine diseases of the lungs. There is insufficient data concerning the leukocyte profile of bronchoalveolar lavage fluid (BALF) from MaediVisna infected sheep. Objective: The objective of this study was to assess the differential leukocyte profile of BALF associated with Maedi virus infection in sheep and to determine whether cytologic examination of BALF is an effective way to diagnose Maedi or determine the severity of lung lesions. Methods: BALF and serum samples were analyzed from 400 sheep. Sediment smears of bronchoalveolar lavage were stained with Diff‐Quik and examined microscopically to obtain a 200‐cell differential cell count. Serum was tested using a commercial kit for Maedi–Visna virus antibodies. Lung samples obtained at the time of slaughter were weighed and examined histologically. Results: Maedi‐infected sheep (n=267; seropositive with lung lesions) had a significantly higher percentage of lymphocytes and lower percentage of macrophages in BALF than normal sheep (n=133; seronegative and no lung lesions). These differences were significantly more severe in animals with advanced vs moderate lung lesions. Using classification trees, a cut‐off of 13.5% lymphocytes was predictive of Maedi infection and a cut‐off of 24.5% lymphocytes was predictive of advanced lung lesions. Conclusions: Cytologic examination of BALF is useful for the clinical diagnosis of Maedi in sheep and provides important information about the severity of the lung lesions.     

The "effects" of Rev-1 vaccination of sheep and goats on human brucellosis in Greece

Vaccination of young animals (3-6-month-old sheep and goats) with Rev-1 vaccine for 15 years in Greece, importantly decreased the abortions in sheep and goats as well as the incidence of brucellosis in humans. After the stop of vaccination in 1994, all over Greece, the prevalence of brucellosis in animals and the incidence in humans quickly increased. It was a positive rank correlation (0.90) among these variables. Once an emergency mass-vaccination programme of young and adult animals with Rev-1 vaccine was started in 1998, the human incidence again decreased. The association of the vaccination coverage of animals and incidence of brucellosis in humans was not linear; the decrease in human brucellosis incidence was observed when the vaccination coverage of animals was >30%.     

Validation of fluorescence polarization assay (FPA) and comparison with other tests used for diagnosis of B. melitensis infection in sheep

Fluorescence polarization assay (FPA) is a new test for the serological diagnosis of Brucella spp. infection in animals. The FPA is validated for the diagnosis of B. melitensis infection in sheep. For this purpose, 166 sera originated from natural infected sheep (verified by culture) and 851 sera originated from healthy animals (reared in areas where B. melitensis was never been isolated) were tested. The optimum cut-off value that offers the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp) was determined at 87mP with the use of ROC analysis. The DSn and DSp of FPA using this cut-off value are calculated at 97.6 and 98.9% with a 95% confidence interval (CI) of 93.9-99.3% and 98.0-99.5%, respectively. The DSn and DSp of FPA have been assessed also using as positive reference (n=587), sera that gave positive results at least in two tests used for diagnosis of B. melitensis in sheep as Rose Bengal Test (RBT), modified Rose Bengal Test (m-RBT), complement fixation test (CFT), indirect Elisa (i-Elisa) and competition Elisa (c-Elisa) originated from animals reared in flocks infected by B. melitensis. The optimum cut-off value using the above panel of positive reference sera was the same offering a DSn of 95.9% with a 95% CI, 94.0-97.4%, since the DSp remains the same. The DSn and DSp as well as performance, accuracy and agreement of FPA's result were compared with those of other tests used. The accuracy of FPA is very high, similar with that of i-Elisa. FPA is a promising assay, which offers a DSn and accuracy better that of those of the tests currently approved for the diagnosis of B. melitensis in sheep and goats. Due to its simplicity, the sort time that results can be obtained and its accuracy it can be used and improve the laboratory testing capacity as well as the efficacy of the eradication program based on test-and-slaughter policy.     

Evaluation of a portable glucose meter for use in cattle and sheep

Background: In farm animal practice, determination of blood glucose concentration under field conditions is often necessary. Objective: As there is no portable glucose meter device developed for use in farm animals, the analytical accuracy of a portable glucometer designed for people was evaluated for its use in cattle and sheep. Methods: Blood samples from 90 cattle and 101 sheep were used in the study. Glucose concentration was determined in whole blood immediately after blood collection from the jugular vein with the One Touch Vita portable glucometer and in serum with an enzymatic colorimetric method. The agreement between methods was assessed by Passing and Bablok regression analysis. The precision and the accuracy of the measurements were determined using the concordance correlation coefficient. Results: There was a strong linear relationship between the glucose values obtained using the portable glucometer and those obtained by the bench method in both cattle and sheep. Precision was 95% for cattle and 88% for sheep, whereas accuracy was 92% and 99%, respectively. The mean glucose values obtained using the portable glucometer were significantly lower by 8.3% in cattle and 3.2% in sheep than those determined by the bench method. Conclusion: The One Touch Vita portable glucometer can be used in clinical practice to determine blood glucose concentrations in cattle and sheep, but reference intervals (RI) must be corrected to allow for negative bias. Based on these equations the RI for blood glucose in cattle and sheep using the portable glucometer were corrected to 1.84–4.17 and 2.41–4.35 mmol/L, respectively.     

Adjuvant regulation of cytokine profile and antibody isotype of immune responses to Mycoplasma agalactiae in mice

Traditionally, adjuvants have been administered with antigens to enhance immunity. We studied the effect of several adjuvants such as Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), lipopolysaccharide (LPS), homopolymers of polyinosinic-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U), lithium chloride (LiCl), saponin Quil A and calcium phosphate gel (CaHPO(4)) on the immune response of mice to formalin-inactivated Mycoplasma agalactiae. The specific antibody or cytokine producing splenocytes were detected by ELISAspot and immunocytochemistry, respectively. Depending on the adjuvant given, the number of M. agalactiae-specific antibody producing cells was increased 2.5-6-fold. IgG was the major class of M. agalactiae-specific antibodies followed by IgM, IgA and IgE. Among IgG isotypes, FCA, FIA, Quil A and CaHPO(4) induced an IgG1 response with substantial increase of the IgG2a, IgG2b and IgG3 isotypes while poly I:C shifted the response toward an IgG2a/IgG3 production. Finally, poly A:U induced an IgG2b response while LPS and LiCl augmented the IgG3/IgG1/IgG2a secretion. FCA augmented IL-4, IL-5 and IL-10 production suggesting a strong Th2 response, while IFN-gamma and IL-12 remained low; poly I:C enhanced IFN-gamma, IL-12 and TNF-alpha eliciting a Th1 response; poly A:U resulted in a IL-10, IL-5, IL-6 and IL-12 secretion; and LPS enhanced the IL-10, IL-6 and TNF-alpha production. Our data show that adjuvants augment M. agalactiae-specific antibody production and lead to B cell isotype-switching via the appropriate cytokine milieu. Certain adjuvants, such as poly I:C, therefore, appear as promising immune enhancers for vaccination against M. agalactiae infections.     

Perspectives of a scrapie resistance breeding scheme targeting Q211, S146 and K222 caprine PRNP alleles in Greek goats

The present study investigates the potential use of the scrapie-protective Q211 S146 and K222 caprine PRNP alleles as targets for selective breeding in Greek goats. Genotyping data from a high number of healthy goats with special emphasis on bucks, revealed high frequencies of these alleles, while the estimated probabilities of disease occurrence in animals carrying these alleles were low, suggesting that they can be used for selection. Greek goats represent one of the largest populations in Europe. Thus, the considerations presented here are an example of the expected effect of such a scheme on scrapie occurrence and on stakeholders.     

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.     

Liver flukes promote cholelithiasis in sheep

The main objective of this study was to investigate whether cholelithiasis in sheep is related to parasitism or other commonly observed disorders such as liver abscesses. Additionally, the features of the observed biliary calculi are described. The livers of 254 randomly selected clinically healthy adult dairy sheep were used. All visible concretions in the bile were considered as stones. Based on the macroscopical examination, 60 livers were normal, 40 were parasitized with Fasciola hepatica, 42 were parasitized with Dicrocoelium dendriticum, 28 were parasitized with both D. dendriticum and F. hepatica, 40 livers had abscesses and 44 had hydatid cysts. Biliary calculi were detected in 40 livers. Twenty livers had pigment stones and 20 livers had cholesterol stones. The percentage of cholelithiasis was significantly higher in livers parasitized with flukes compared with the others (P<0.05) and the parasitoses with F. hepatica and D. dendriticum were proven to be significant risk factors (P<0.05) for the presence of cholelithiasis in sheep.     

Pelargonium net vein agent and pelargonium petal streak as beneficial infections of commercial pelargoniums

Pelargonium Net Vein Agent (PNVA) and Pelargonium Petal Streak Agent (PPSA) have been shown to be graft-transmissable, singly and together, to both ivy-leaf and zonal pelargoniums. In zonal pelargoniums, PNVA symptoms are seasonal; PPSA results in petal curling in some cultivars and in most petal streaking is absent or only on the lower petal surface. PNVA symptoms were stable in the ivy-leaf cultivars studied, as were those of PPSA. There was no interaction between PPSA and PNVA, doubly-infected ivy-leaf cultivars showing both symptom types.Symptoms induced by PNVA and PPSA were eliminated by meristem culture.The commercial potential for the exploitation of PPSA and PNVA is discussed.