Validation of a Field Based Chromatin Dispersion Assay to Assess Sperm DNA Fragmentation in the Bottlenose Dolphin (Tursiops truncatus)

Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non‐fragmented DNA displayed small compact haloes surrounded by a dense core of non‐dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two‐tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field.     

Light-scattering study of the structure of aggregates and gels formed by heat-denatured whey protein isolate and beta-lactoglobulin at neutral pH

The structure of aggregates and gels formed by heat-denatured whey protein isolate (WPI) has been studied at pH 7 and different ionic strengths using light scattering and turbidimetry. The results were compared with those obtained for pure beta-lactoglobulin (beta-Lg). WPI aggregates were found to have the same self-similar structure as pure beta-Lg aggregates. WPI formed gels above a critical concentration that varied from close to 100 g/L in the absence of added salt to about 10 g/L at 0.2 M NaCl. At low ionic strength (<0.05 M NaCl) homogeneous transparent gels were formed, while at higher ionic strength the gels became turbid but had the same self-similar structure as reported earlier for pure beta-Lg. The length scale characterizing the heterogeneity of the gels increased exponentially with increasing NaCl concentration for both WPI and pure beta-Lg, but the increase was steeper for the former.     

A comparison of the interferon gamma assay with the absorbed ELISA for the diagnosis of Johne''s disease in cattle

Two interferon gamma (IFN-gamma) assays, the IFN-gamma enzyme immunoassay (EIA) and the IFN-gamma bioassay and an absorbed ELISA were used to screen 6 cattle herds for Johne's disease. Each herd had a history of Johne's disease but the majority of infected animals did not show clinical signs. The disease status of the cattle, which were removed from the herds, was confirmed by bacteriological culture of faeces or histopathological examination and culture of tissues collected at necropsy. The sensitivities of the IFN-gamma assays and the absorbed ELISA were determined using test results from infected animals. The sensitivity of the IFN-gamma EIA in detecting subclinical (71.8 to 93.3%) and clinical animals (100%) was not significantly different. However, the IFN-gamma bioassay and the absorbed ELISA were more sensitive in detecting cattle with advanced infections (80%) than those that were subclinically affected (16.7 to 33.3%).     

Gamma Interferon Production and Plasma Concentrations of Pregnancy‐Associated Glycoproteins 1 and 2 in Gestating Dairy Cows Naturally Infected With Neospora caninum

Gamma interferon (IFN‐γ) production and cross‐breed pregnancy have been attributed a role in protecting dairy cows infected with Neospora caninum against abortion. Plasma levels of pregnancy‐associated glycoproteins‐1 (PAG‐1) are a marker of placental/foetal well‐being and of PAG‐2 is an abortion risk indicator in chronically N. caninum‐infected animals. The present study examines, in cross‐breed pregnancies, interactions between IFN‐γ production and levels of PAG‐1 and PAG‐2 in non‐aborting naturally Neospora‐infected dairy cows. Data were obtained from 60 pregnant Holstein‐Friesian cows: 44 Neospora‐seropositive and 16 Neospora‐seronegative; 12 became pregnant using Holstein‐Friesian semen and 48 using Limousin semen. Blood samples were collected on Days 40, 90, 120, 150, 180 and 210 of gestation. Gamma interferon was only detected in the plasma of nine of the 44 Neospora‐seropositive cows, all of them became pregnant using Limousin semen. Through GLM procedures, in cows inseminated with Limousin semen and Neospora‐seropositive cows showing no IFN‐γ production, PAG‐1 concentrations were high and increased throughout gestation compared to the levels detected in cows inseminated with Holstein‐Friesian semen and Neospora‐seropositive cows producing IFN‐γ, respectively. In Neospora‐seronegative cows and in Neospora‐seropositive cows showing no IFN‐γ production, significantly increased PAG‐2 concentrations were observed on gestation Day 120. Our findings indicate that IFN‐γ production correlates negatively and the production of antibodies against N. caninum is uncorrelated with plasma PAG concentrations during gestation in Neospora‐infected dairy cows. Accordingly, IFN‐γ production could be linked to the transplacental migration of tachyzoites, which may cause a reduction in PAG levels.     

Molecular differentiation of Mycobacterium bovis isolates. Review of main techniques and applications

Until recently, none of the Mycobacterium bovis typing techniques permitted a satisfactory differentiation of isolates. During the last 10 years, the genome of pathogenic mycobacteria has been extensively studied, and phylogenetic analyses have shown that all (except Mycobacterium avium) belong to a single genetic species: the Mycobacterium tuberculosis complex. This increase in knowledge about the genome of these bacteria has lead to the discovery of molecular markers that allow us to differentiate isolates. Because of the phylogenetic proximity of the strains, even if most of these markers have been discovered in M. tuberculosis, they could be successfully adapted to the other bacteria of the M. tuberculosis complex, especially M. bovis. The most common markers in use today are the IS6110 insertion sequence, the direct repeat (DR) region, the poly(GC) rich (PGRS) sequences and the variable number tandem repeats (VNTR) sequences. The corresponding typing techniques are briefly described, and current knowledge of polymorphism and marker stability is detailed. If molecular markers are to offer wide perspectives for field studies, these two characteristics (polymorphism and stability) must be taken into account when choosing the marker(s) used in a study. In this context, examples of the application of molecular typing techniques for M. bovis are reviewed, on the one hand with epidemiological studies for which the major problem is the comparison between isolates and, on the other, with more general studies about the population genetics of M. bovis in a given country, and about its history and its phylogeny.     

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 10⁴ to 9.0 × 10¹⁰ WSSV copies μg^–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 10⁹ WSSV copies μg^–1 of DNA were detected which is equivalent to 5.7 × 10¹⁰ WSSV copies g^–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).