A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections

Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5′ non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89 TCID₅₀. The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine.     

First report of porcine circovirus type 2 infections in Cuba

To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba and the probable association of PCV2 with other porcine viruses, tissue samples collected from ill pigs were evaluated using polymerase chain reaction (PCR). The PCR analysis showed that 67.7% of the samples (23/34) from seven swine herds of six different geographic regions were detected to be positive for PCV2. Ten of the 23 PCV2 positive samples (43.5%) shown a concurrent infection with porcine parvovirus (PPV) and 17 of 23 PCV2 positive samples (73.9%) exhibited a concomitant infection with classical swine fever virus (CSFV). This study is the first report of PCV2 infecting pigs with different clinical conditions in Cuban swine herds and provides evidence of PCV2 co-infection with PPV and CSFV in the field.     

Differing nitrogen use strategies of two tropical rainforest late successional tree species in French Guiana: Evidence from 15N natural abundance and microbial activities

Previous studies in lowland tropical rainforests of French Guiana showed that, among non-N₂-fixing trees, two groups of late successional species contrasting in their leaf ¹⁵N natural abundance coexist, suggesting two different main ways of nitrogen acquisition. Two abundant late-successional species typically co-occurring in rainforests in French Guiana, namely Eperua falcata and Dicorynia guianensis, were chosen as representative of each group. Stable isotope techniques and measurements of potentials of microbial N transformation were performed to assess to what extent leaf ¹⁵N natural abundance of these species could be related to (i) δ¹⁵N signatures of soil mineral N sources and (ii) the capacity of soil to express nitrification and denitrification (both processes being directly involved in the balance between NH₄⁺ and NO₃^–). Soil δ¹⁵N-NH₄⁺ was roughly similar to leaf δ¹⁵N of D. guianensis (around 3.5‰), suggesting a preferential use of NH₄⁺, whereas in E. falcata, leaf δ¹⁵N values were closer to root δ¹⁵N-NO₃^? values (0.2 and ?2.0‰, respectively), suggesting a preferential use of NO₃^?. These differences in N source utilization were not accompanied by differences in availability in soil NO₃^? or in intensity of microbial functions responsible for soil N mineral evolution. However, (i) under both tree species, these functions showed clear spatial partitioning, with denitrification occurring potentially in soil and nitrification in the litter layer, and (ii) E. falcata fine roots colonized the litter layer much more strongly than D. guianensis fine roots. This strongly suggests that (i) the contrasted leaf δ¹⁵N values found in the two late-successional species reveal distinct N acquisition strategies and (ii) the ability of roots to predominantly exploit the litter layer (E. falcata) or the soil (D. guianensis) may constitute an important explanation of the observed differences. A complementarity between tree species, based on mineral N resource partitioning (itself resulting from a spatially structured location of the microbial functions responsible for the balance between NH₄⁺ and NO₃^–), can thus be supposed.     

First evidence of the emergence of novel putative infectious bronchitis virus genotypes in Cuba

The emergence of new infectious bronchitis virus (IBV) genotypes or serotypes along with the poor cross-protection observed among IBV serotypes have complicated the avian infectious bronchitis (IB) control programs in different geographic regions. In Cuba, the lack of genetic information regarding IBV and the increasing epidemiological importance of this virus in Cuban chicken flocks demand further characterization of IBV isolates. In the present work, studies of genetic diversity and phylogenetic relationships among recent IBV isolates from Cuban chicken flocks showing respiratory disorders were performed. Two putative genotypes genetically different to the Massachusetts genotype H120 strain used in the Cuban vaccination program were found in the flocks assessed. In addition, a potential nephropathogenic IBV isolate was found by first time in Cuba.     

Molecular detection of Torque teno sus virus in lymphoid tissues in concomitant infections with other porcine viral pathogens

In this study, 40 pigs with respiratory and wasting disorders from Cuban swine herds were screened by PCR for the presence of TTSuV1, TTSuV2, PCV-2, PPV and CSFV in spleen samples. The variability of the porcine TTSuV sequences obtained was investigated by phylogenetic analysis. This study showed for the first time that TTSuV1 and TTSuV2 were present in Cuban swine herds. The investigation revealed the following infection rates: TTSuV1 40%, TTSuV2 37.5%, PCV-2 70%, PPV 37.5% and CSFV in 52.5%. The presence of two or more of these viruses at different rates in the same spleen samples was revealed. Also, a higher genetic diversity of TTSuV2 sequences was observed regarding TTSuV1 sequences.     

Melengestrol acetate as a tool for inducing early ovulation in transitional mares

The efficacy of melengestrol acetate (MGA) to shorten the vernal transition of mares by synchronising and accelerating the first ovulation of the year after 60 days of phototherapy was determined by ultrasonographic monitoring. Sixteen mares in late transition were fed two doses of MGA (150 mg/mare/day and 100 mg/mare/day, respectively) for 10 days. A luteolytic dose of prostaglandin was administered to each mare one day after the end of MGA treatment. The presence and duration of oestrus, follicular growth, uterine oedema and presence of ovulation were monitored by ultrasonography and the cervical tone was evaluated by rectal palpation. Ovulation was detected in 87.5% of the mares treated with 150 mg MGA/mare/day for 10 days, and in 62.5% of the mares receiving 100 mg MGA/mare/day for 10 days. This was statistically different (P = 0.03) from the untreated control mares having an ovulation rate of 20%. Mares that received 150 mg MGA/day for 10 days had a mean treatment to ovulation interval of 13.1 +/- 5.97 days after the end of treatment, while mares that received 100 mg MGA/day for 10 days had a mean of 25.6 +/- 10.50 days (P = 0.01) to ovulation. These results suggest that MGA can be used for synchronising and hastening the first ovulation of the year in mares.     

Phylogenetic networks to study the origin and evolution of porcine circovirus type 2 (PCV2) in Cuba

Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of postweaning multisystemic wasting syndrome (PMWS), which is considered one of the most economically important swine diseases worldwide. In this study, a comparison between methodologies based on classical phylogenetic trees and networks to infer the origin of PCV2 in Cuba was performed. In addition, the mechanisms supporting the genetic variability of Cuban PCV2 populations were investigated. A retrospective study, using pig sera collected in Cuba from 1993 to 2004, to evaluate the presence of PCV2 genome and PCV2-specific antibodies was also conducted and revealed a lack of evidence of PCV2 infection in Cuban swine from years 1993 to 2004. A total of 24 complete Cuban PCV2 sequences collected between 2005 and 2009 from different regions of the country were analyzed. Three classical methods of phylogenetic analysis, namely Neighbour-Joining, Maximum Parsimony and Bayesian Inference, as well as haplotype network construction, were used. Whereas the classical phylogenetic trees suggested different origins for the Cuban PCV2 strains, the haplotype network revealed a direct connection between all the Cuban sequences in agreement with the obtained epidemiological and viral sequence data. Moreover, the importation of pigs carried out in 2005 from the Quebec-Ontario region, Canada, seems to be the most likely origin of PCV2 in Cuba. Likewise, the genetic variability of Cuban PCV2 sequences was supported by geographic segregation and positive selection pressure with estimated rates of nucleotide substitution on the order of 3.12×10(-3) and 6.57×10(-3) substitutions/site/year, which are closer to those reported for RNA viruses.     

Does variability in litter quality determine soil microbial respiration in an Amazonian rainforest?

Tree species-rich tropical rainforests are characterized by a highly variable quality of leaf litter input to the soil at small spatial scales. This diverse plant litter is a major source of energy and nutrients for soil microorganisms, particularly in rainforests developed on old and nutrient-impoverished soils. Here we tested the hypothesis that the variability in leaf litter quality produced by a highly diverse tree community determines the spatial variability of the microbial respiration process in the underlying soil. We analyzed a total of 225 litter-soil pairs from an undisturbed Amazonian rainforest in French Guiana using a hierarchical sampling design. The microbial respiration process was assessed using substrate-induced respiration (SIR) and compared to a wide range of quality parameters of the associated litter layer (litter nutrients, carbon forms, stoichiometry, litter mass and pH). The results show that the variability of both litter quality and SIR rates was more important at large than at small scales. SIR rates varied between 1.1 and 4.0 μg g⁻¹ h⁻¹ and were significantly correlated with litter layer quality (up to 50% of the variability explained by the best mixed linear model). Total litter P content was the individual most important factor explaining the observed spatial variation in soil SIR, with higher rates associated to high litter P. SIR rates also correlated positively with total litter N content and with increasing proportions of labile C compounds. However, contrary to our expectation, SIR rates were not related to litter stoichiometry. These data suggest that in the studied Amazonian rainforest, tree canopy composition is an important driver of the microbial respiration process via leaf litter fall, resulting in potentially strong plant-soil feedbacks.