Contamination, error, and nonspecific molecular tools

ABSTRACT Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) are widely used in studies of genetic variation. Although it is recognized that contamination should be avoided in DNA samples, little is known about the potential hazards of low level bacterial contamination of samples from which DNA is extracted for RAPD or AFLP analyses. We found that contamination of Aphanomyces cochlioides cultures with a prokaryote at visibly undetectable levels markedly altered the results of RAPD and AFLP analyses. The contamination resulted in seven contaminant-specific RAPD products and in the suppression of eight products characteristic of uncontaminated A. cochlioides cultures. Prokaryote contamination resulted in 39 contaminant-specific AFLP products, but did not cause suppression of AFLP products. Comparing A. cochlioides samples with outgroup A. euteiches did not clearly indicate the presence of contaminant DNA, because uneven product suppression in RAPD analysis increased the apparent similarity between contaminated samples and A. euteiches and because a high proportion of the contaminant-specific amplified products comigrated with products from A. euteiches in both RAPD and AFLP analyses. Work with organisms that are prone to contamination should employ techniques such as restriction fragment length polymorphism or DNA sequence comparisons rather than relying solely on RAPD or AFLP analyses.     

Serologic survey of Neospora caninum infection in a closed dairy cattle herd in Maryland: risk of serologic reactivity by production groups

Prevalence of antibodies to Neospora caninum was determined in a cross-sectional consensus survey of 1029 bovines in a dairy herd with endemic Neospora-induced abortion. Sera were screened by indirect fluorescent antibody test (IFAT). The prevalence of N. caninum antibody in the IFAT was 17.9% in 107 neonates, 26.2% in 233 yearling heifers and steers, 39.07% in 218 mature heifers, and 26.9% in 465 milking cows. Serologic reactivity was associated with production grouping on the farm with the greatest risk of serologic reactivity appearing in the yearling and mature heifers. There was an increasing risk of serologic reactivity with increasing age only in the parity one and greater animals in the herd. Castrated males were at half the risk of similarly aged females of possessing antibodies to N. caninum. There was no clear relationship between the serologic status of dams and offspring.     

Nonsuppurative meningoencephalitis associated with Aleutian mink disease parvovirus infection in ranch mink.

Severe nonsuppurative meningoencephalitis associated with Aleutian mink disease parvovirus (ADV) infection was observed in adult ranch mink. Brain lesions included severe, locally extensive to coalescing lymphoplasmacytic meningoencephalitis with accompanying gliosis, satellitosis, and mild extension of inflammation into the leptomeninges. ADV was identified in mesenteric lymph node, spleen, brain, and liver of affected mink by polymerase chain reaction techniques. Sequences of the ADV isolate (TH5) revealed 2 unique residues in the region of the viral genome that determines pathogenicity. These findings suggest that certain strains of ADV may preferentially cause disease in the nervous system. ADV infection should be considered in the differential diagnosis of neurologic disorders in mink.     

Implications of carbon forestry for local livelihoods and leakage

? Context

An inequitable distribution of the costs and benefits of carbon forestry could undermine its role in tackling climate change, but safeguarding local livelihoods could undercut its effectiveness.

? Aims

We simulate a reforestation program in a densely populated locality in central Mexico to analyze indirect land-use change, or leakage, associated with the program and its implications for local livelihoods.

? Methods

An agent-based, general equilibrium model simulates scenarios that deconstruct the sources of leakage and livelihood outcomes.

? Results

Simulations reveal how conditions linking land, labor, and food markets determine the costs and benefits of reforestation and simultaneously the potential for leakage. Leakage is lowest in remote and poorly integrated localities where declining wages foster local food production while discouraging consumption. Since leakage is tied to consumption, there is a trade-off between the program’s effectiveness and an equitable outcome.

? Conclusion

An ideal strategy could target those localities with few remaining forests, where a program might lead to agricultural intensification rather than expanding the agricultural frontier. Alternatively, the scheme could incorporate remaining forests to avoid deforestation while encouraging reforestation. An uneven distribution of costs and benefits, where some stakeholders may draw benefits from others’ losses, could nevertheless set the stage for conflict. Acknowledging these trade-offs should help design a politically feasible program that is effective, efficient, and equitable.     

Identification of Apoptotic Bodies in Equine Semen

Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37°C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin‐V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer‐assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.