Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction

The potential role of Boophilus microplus as a natural tick vector of Babesia equi and Babesia caballi in Brazilian horses was assessed using nested polymerase chain reaction (PCR)-based marker assay. B. equi merozoite-specific 218bp gene fragment was detected in almost 96% of horse blood samples, and 45.3-62.5% of females, eggs, larvae, and nymphs of B. microplus collected from 47 horses at Campo Grande in the State of Matto Grosso, Brazil. Except for the partially-fed female ticks, the B. caballi-specific 430bp gene fragment was amplified from horse blood samples, and all developmental stages. Parasite DNA from both species was detected in horse blood samples and B. microplus, with the preponderance of B. equi DNA. No DNA samples were positive solely for B. caballi parasite. Only 32% of the Giemsa-stained thin blood smears were positive for Babesia parasites, as against detection of B. equi parasite DNA in 95.7% of the blood samples by nested PCR. We have obtained molecular evidence that strengthens earlier experimental and ultrastructural studies in Brazil incriminating B. microplus as a natural vector of B. equi, and possibly of B. caballi. The detection of B. equi and B. caballi DNA in eggs and larvae of B. microplus is likewise suggestive of the possibility of both transovarial and transstadial parasite transmission in this tick vector.     

Farm forestry in the black forest southern nature park from a socio-political point of view

In this study the interactions between forest management and the preservation of rural structures, as well as the economic implications should there be a shut-down of silvicultural activities in the small-scale private forestry of the Southern Nature Park of the Black Forest in Germany, are examined. The jobs, which are connected to the production of wood and the quality of the landscape, are characterized and projected on the region as far as possible. The creation of value by owners managing forests for production is demonstrated for the area of investigation. In combination with estimated average monetary values for the infrastructural benefits of the forests, scenarios point out the consequences of management as well as of a shut-down of the smallscale private forests. Finally, the paper explores whether it is it is appropriate to extend the meaning of the term ‘Environmentally Sound Forest Management’ beyond the conventional sense of forest functions, and to an holistic approach which integrates the people as well as forests and other landscape features. If an holistic approach is adopted, a redefined model of the mixed farm forest enterprise, which pursues many targets in the same area, must be developed. In this way, it seems possible to solve conflicts regarding land-use in rural areas and to achieve desirable socio-political development.     

Impact of Soaking and Drying Conditions on Rice Chalkiness as Revealed by Scanning Electron Microscopy

Chalkiness is one of the most influential factors on head rice yield. Parboiling is known to be an effective way to remove chalkiness and improve head rice yield. However, the steps involved in the removal of chalkiness are still not completely resolved. This study investigated the effects of soaking temperature, soaking duration, and drying conditions on the removal of rice chalkiness. Chalky brown rice kernels were selected and soaked at 25, 65, 70, or 75°C for 3 h. After 1, 2, or 3 h, the rice samples were frozen before drying or immediately dried. Soaking at 25°C did not remove chalkiness and caused no morphological change in starch granules. When the soaking temperature increased from 25 to 65, 70, and 75°C, the chalkiness decreased from 100% to 34.1, 29.7, and 15.9%, respectively. Soaking rice at temperatures above the starch glass transition temperature but below the gelatinization temperature reduced chalkiness owing to rearrangement of starch granules and protein denaturation to fill the void spaces in the chalky area. During soaking, the morphology of starch granules also changed from round to angular in shape. Drying at temperatures above the starch glass transition temperature also facilitated rearrangement of starch granules to further reduce rice chalkiness.     

Concurrent infection in chickens with fowlpox virus and infectious laryngotracheitis virus as detected by immunohistochemistry and a multiplex polymerase chain reaction technique

A concurrent infection of chickens with infectious laryngotracheitis virus (ILTV), a herpesvirus, and fowlpox virus (FWPV), an avipoxvirus, is described. Two techniques, an immunohistochemistry (IHC) technique and a multiplex polymerase chain reaction (PCR), were used to examine 11 tissue samples from chickens clinically diagnosed as FWPV-infected, but only IHC was used to examine six tissue-paraffin blocks prepared from turkeys suspected of having FWPV infection. By multiplex PCR, both FWPV and ILTV were detected from three chicken samples (FI-90, FI-93, and FI-94); both FWPV and ILTV were detected from only two samples (FI-93 and FI-94) by IHC. All chicken samples were positive for FWPV by both PCR and IHC. Viral DNA from these samples was further confirmed by restriction enzyme analysis. When turkey samples were analyzed by the double-stain IHC, all six samples showed the presence of FWPV antigens, but no ILTV antigens. The double IHC technique, using monoclonal antibodies against FWPV and ILTV, was successful in simultaneous demonstration of specific FWPV and ILTV antigens colocalized in infected tissue samples as well as within individual cells. This paper emphasizes the importance of reliable tests that detect specifically the presence of ILTV and FWPV in infected tissue samples. The multiplex PCR assay holds potential to be versatile, rapid, and more sensitive (100%) than IHC (67%) for the simultaneous detection of two different avian viruses. Furthermore, the presence of mixed infection should always be kept in mind in the virologic analysis of respiratory sickness of poultry.     

Development of loop-mediated isothermal amplification (LAMP) method for diagnosis of equine piroplasmosis

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid method whereby DNA is amplified with high specificity, efficiency, and rapidity under isothermal conditions using a set of four specifically designed primers and a DNA polymerase with strand displacement activity. In this study, we used LAMP primer sets designed from EMA-1 and Bc 48 genes for detection of Theileria equi and Babesia caballi infections, respectively. These primer sets specifically amplified DNA of the respective parasites. Both primer sets amplified T. equi and B. caballi up to 10(-6) dilution of 10-fold serially diluted samples. Furthermore, DNA extracted from blood collected from a horse experimentally infected with T. equi was amplified by a T. equi LAMP primer set from days 2 to 35 post-infection, demonstrating the high sensitivity of these primers. Of 55 samples collected from China, 81.8% and 56.3% were positively detected by LAMP for T. equi and B. caballi infections, respectively. In contrast, 91.8% and 45.9% of the 37 samples collected from South Africa were LAMP positive for T. equi and B. caballi, respectively. These results suggest that LAMP could be a potential diagnostic tool for epidemiological studies of equine piroplasmosis.     

Development of a single-round and multiplex PCR method for the simultaneous detection of Babesia caballi and Babesia equi in horse blood

With the aim of developing more simple diagnostic alternatives, a differential single-round and multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of Babesia caballi and Babesia equi, by targeting 18S ribosomal RNA genes. The multiplex PCR amplified DNA fragments of 540 and 392 bp from B. caballi and B. equi, respectively, in one reaction. The PCR method evaluated on 39 blood samples collected from domestic horses in Mongolia yielded similar results to those obtained from confirmative PCR methods that had been established earlier. Thus, the single-round and multiplex PCR method offers a simple tool for the differential diagnosis of B. caballi and B. equi infections in routine diagnostic laboratory settings as well as in epidemiological studies.     

Gametogenic asynchrony of mussels Mytilus in a mixed-species area: Implications for management

Marine mussels Mytilus edulis, Mytilus galloprovincialis, Mytilus trossulus and their hybrids have recently been reported to occur sympatrically in both wild and farmed populations in Scotland. The presence of M. trossulus has led to significant economic losses at some aquaculture sites. Interest in understanding the reproductive cycles of these mussels in a mixed-species aquaculture area, and the potential to use this information to favour cultivation of the preferred species M. edulis, has motivated a seasonal study of the maturation stages of M. edulis, M. trossulus and M. trossulus × M. edulis hybrids, the most frequent genotypes at one site in Loch Etive, Scotland. To investigate larval abundance of these genotypes, plankton samples were collected simultaneously at the same site over the year and analysed using a real-time PCR assay for the identification of Mytilus species-specific alleles. The main spawning events of M. trossulus and M. trossulus × M. edulis hybrids occurred slightly later, but also more frequently and over a longer period than those of M. edulis. M. trossulus alleles were detected in plankton samples throughout the year, always in larger numbers than M. edulis alleles, except during February. Overall, results suggest that the settlement of M. trossulus and/or M. trossulus × M. edulis hybrids may occur during most of the year. This implies that management of the timing of the deployment of settlement ropes to significantly favour the settlement and production of M. edulis is unlikely to be successful. The real-time PCR methodology has provided valuable information on the temporal patterns of abundance of alleles of each species at the planktotrophic stage of development, and demonstrated the potential to identify and quantify Mytilus larvae in plankton samples.     

Operational Effectiveness of Centralized Nursery for Small-Scale Forestry in San Carlos City, Negros Occidental, the Philippines: Lessons Learned in Seedling Production for Commercial Energy Crop Plantations

One way to maximize potential income of smallholder tree farmers is to ensure high quality of planting material. In San Carlos City, Negros Occidental, the Philippines, great importance is placed on seedling quality because Energy Crop Plantations (ECPs) managed under a Short-Rotation Coppice (SRC) system will involve a single planting for several three-year coppices. High seedling quality requires superior seed provenances and seedling propagation methods. Throughout the establishment of the nursery facility, and the propagation of planting stock, data collection, reporting and database systems have been designed to capture relevant costs and technical information. Training has been provided to nursery personnel and labourers to enable them to operate the nursery and produce high quality seedlings. This paper reports a practical operational evaluation of all materials and procedures followed in nursery establishment and seedling production. Economies of scale in nursery operation are achieved at a production level of at least 90,000 seedlings per planting season of three months in the year. The production cost is estimated at PhP 3.57/seedling, excluding the cost of the nursery infrastructure and overheads. Cost comparison with decentralized or contracted nurseries shows little variation at a total cost of PhP 4.12/seedling. However, with these alternative nursery arrangements there is high risk of producing low quality seedlings by possible reduction of inputs and skipping quality enhancement procedures to reduce production cost.