Detection of ruminant meat and bone meals in animal feed by real-time polymerase chain reaction: result of an interlaboratory study

The commercialization of animal feeds infected by prions proved to be the main cause of transmission of bovine spongiform encephalopathy (BSE). Therefore, feed bans were enforced, initially for ruminant feeds, and later for all feeds for farmed animals. The development and validation of analytical methods for the species-specific detection of animal proteins in animal feed has been indicated in the TSE (Transmissible Spongiform Encephalopathies) Roadmap (European Commission. The TSE (Transmissible Spongiform Encephalopathy) roadmap. URL: http://europa.eu.int/comm/food/food/biosafety/bse/roadmap_en.pdf, 2005) as the main condition for lifting the extended feed ban. Methods based on polymerase chain reaction (PCR) seem to be a promising solution for this aim. The main objective of this study was to determine the applicability of four different real-time PCR methods, developed by three National expert laboratories from the European Union (EU), for the detection and identification of cattle or ruminant species in typical compound feeds, fortified with meat and bone meals (MBM) from different animal species at different concentration levels. The MBM samples utilized in this study have been treated using the sterilization condition mandatory within the European Union (steam pressure sterilization at 133 degrees C, 3 bar, and 20 min), which is an additional challenge to the PCR methods evaluated in this study. The results indicate that the three labs applying their PCR methods were able to detect 0.1% of cattle MBM, either alone or in mixtures with different materials such as fishmeal, which demonstrates the improvement made by this technique, especially when compared with results from former interlaboratory studies.     

Prevalence and risk factors associated with goat gastrointestinal helminthiasis in the Sertão region of Paraíba State,Brazil

Gastrointestinal helminthiasis represents an obstacle to goat raising, causing severe damage to herds such as growth retardation, weight loss, and even death. In this study, we aimed to determine the prevalence and risk factors associated to goat gastrointestinal helminthiasis in the Sertão region of Paraíba State, Brazil. A total of 256 goats from 54 farms were systematically sampled. Blood and fecal samples were collected from each animal for egg per gram (EPG), larval culture, and packed cell volume (PCV) analyses. We found that 79.3 % of the goats investigated were parasitized with gastrointestinal helminths. Significant correlation (p?=?0.004) was observed between the EPG and PCV of the animals studied, and it was observed that the EPG increases as the PCV decreases. In the larval culture, the most prevalent helminth was Haemonchus sp. (83.2 %). Age and sex were significant variables (p?≤?0.20) for the development of gastrointestinal helminths: 86.8 % of animals over 36 months of age and 81.7 % of females were infected. The variable type of animal exploitation was also significant, with 90.3 % (p?≤?0.20) of the animals presenting double suitability (milk and meat). The Sertão region of Paraíba State presents high prevalence of gastrointestinal helminthiasis in goats, and age and type of animal exploitation are the most relevant risk factors to the development of these parasites.     

Identification and quantification of benzimidazole resistance polymorphisms in Haemonchus contortus isolated in Northeastern Brazil

Haemonchus contortus is the most prevalent nematode in Brazil. The objective of this study was to select 6 populations of H. contortus of known or suspected benzimidazole resistance status and characterize these using quantitative real-time polymerase chain reaction (qPCR) for single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A in the β-tubulin isotype 1 gene. qPCR was performed using DNA from a pool of 10 adult male H. contortus from a single animal per farm. Faecal egg count reduction test (FECRT) and egg hatch test (EHT) were used to determine the resistance status. Samples were obtained from 6 farms located in 5 counties in the Ceará State: Tauá, Boa Viagem, Quixadá, Santa Quitéria and Solonópole. The inbred-susceptible-Edinburgh (ISE) isolate was used as reference for comparative purposes in the qPCR. Benzimidazole resistance was detected by FECRT on all farms with efficacy values ranging from 0 to 51%. EC50 values as determined by EHT were all above 1.49 μg/ml. High frequencies of the resistant SNPs F200Y and F167Y alleles were detected but no resistance was detected at SNP E198A. Our results suggest that the SNPs F167Y and F200Y are both important for benzimidazole resistance in the studied populations.     

Prion protein genotypes of sheep as determined from 3343 samples submitted from Ontario and other provinces of Canada from 2005 to 2012

This study analyzed sheep prion protein (PrP) genotypes of samples submitted from Ontario and other provinces of Canada to the Animal Health Laboratory at the University of Guelph, Guelph, Ontario, between 2005 and 2012. In Ontario, the proportion of scrapie-resistant sheep increased from 2005 to 2012 as evidenced by an increase in the ARR haplotype. When Canadian provinces (Alberta, Ontario, Quebec, and Nova Scotia) were compared from 2008 to 2012, a high proportion of scrapie-resistant sheep was found in all the provinces. The proportions of resistant sheep were lower in Alberta and Quebec than in Ontario and Nova Scotia. Alberta had higher proportions of susceptible sheep and a higher frequency of VRQ alleles, and Quebec had a higher frequency of the ARQ allele.     

Panagrellus redivivus mass produced on solid media as live food for Litopenaeus vannamei larvae

The free‐living soil nematode Panagrellus redivivus is well known to be an excellent food source for first feeding fish larvae. It represents an alternative to the highly expensive Artemia, which is commonly used. The lack of a proper method for mass production of P. redivivus has prevented its wider use in commercial hatcheries. A new cultivation method allows the production of a sufficient quantity of nematodes to deliver a standardized and permanently available live food of high quality, throughout the larval rearing period. In two experiments – carried out at the Centro de Investigación en Alimentación y Desarrollo, Mexico – several feeding regimes were established to prove the quality of the mass produced P. redivivus for larvae of Litopenaeus vannamei, the Pacific white shrimp. Two different nematode treatments were compared with a no‐feed group and a control group that was fed with Artemia. All treatments had an additional algal co‐feed and were run in five replicates. Panagrellus redivivus was cultured on two different media (wheat/corn flour and oat flour) to compare these for their suitability as high‐quality live food for the larvae. Shrimp fed nematodes grown on wheat/corn medium reached the postlarval stage earlier than those from other treatments. The nematode treatments showed promising results; however, further research is needed on the development of improved culture media or enrichment methods to further increase the nutritional value of P. redivivus.     

The genetic structure of Mytilus chilensis (Hupé 1854) populations along the Chilean coast based on RAPDs analysis

The Chilean blue mussel Mytilus chilensis is an important commercial species. However, little has been published on the population genetics of this species, despite the need to implement management and conservation policies. Randomly amplified polymorphic DNA‐polymerase chain reaction analysis was used to estimate genetic variation within and between eight natural populations along the whole range of its Chilean natural distribution (ca. 1900 km from Arauco (VIII Region) to Punta Arenas (XII Region)). The values of Nei's unbiased genetic distance, D (0.030–0.107), among populations were small, despite the large geographic separation. A mantel test using 50 000 randomizations showed evidence for a significant correlation (r=0.74, P<0.05) between genetic and geographic (coastal) distance. Punta Arenas population was the most genetically differentiated from the others, although the scale of differentiation was not large (D=0.076–0.107). The levels of gene flow (Nm=1.55) found in this study prevent differentiation among populations by genetic drift. This is the result of the long‐lived planktotrophic larvae of M. chilensis, which provides this species with considerable dispersal ability throughout its range, which is favoured by the ocean currents along the Chilean coast. A restricted larval dispersal towards the north due to the Cape Horn Current derived from the West Wind Drift could be the cause of the higher genetic differentiation of Punta Arenas population from the northern populations. For management purposes of the M. chilensis fishery, the results provide no evidence for discrete stocks, with the possible exception of the Punta Arenas population. The present study provides the baseline data in order to continue further characterization of these mussel populations, considering the great increase in aquaculture of this species.     

Assessing habitat fragmentation on marine epifaunal macroinvertebrate communities: an experimental approach

Habitat fragmentation is considered a major cause of biodiversity loss, both on terrestrial and marine environments. Understanding the effects of habitat fragmentation on the structure and dynamics of natural communities is extremely important to support management actions for biodiversity conservation. However, the effects of habitat fragmentation on marine communities are still poorly understood. Here we evaluated whether habitat fragmentation affects the structure of epifaunal communities in the sublittoral zone, in the northern coast of São Paulo state, Brazil. Five experimental landscapes were constructed, each one forming a large continuous patch. After 4 weeks, each landscape was cut on three patches of different sizes. Epifaunal macroinvertebrate communities were sampled at the edge and interior of experimental landscapes before manipulation to evaluate edge effects. After four more weeks, communities from the three patch sizes were also sampled to evaluate patch size effects. We compared the diversity of communities at different levels of fragmentation by total abundance, rarefied taxon richness, Shannon–Wiener diversity index, Simpson’s dominance index, and abundance of dominant taxa. Higher taxon richness and gastropod abundance were recorded in the patch edges, but no significant differences were found among patch sizes. We found a significant effect of habitat fragmentation, with lower abundances of Gammaridea (the dominant taxon), Ophyuroidea, and Pycnogonida after the experimental fragmentation. Lower abundances of dominant taxa resulted in higher diversity and lower dominance in fragmented landscapes when compared to integral, pre-manipulation landscapes. Our results suggest that fragmentation of landscapes in the system studied can reduce dominance, and that even small patch sizes can be important for the conservation of macroinvertebrate diversity.     

Epitheliocystis disease in the cultured amberjack, Seriola dumerili Risso (Carangidae)

Epitheliocystis infection affecting the gill and the pseudobranch of the cultured amberjack, Seriola dumerili Risso, is described. In hyperinfected fish, proliferative cell response around the epitheliocystis capsule resulted in gill and pseudobranch lamellar fusion, which led to mass mortalities in the 0+age class. Histopathological and scanning electron microscope observations showed that the target cell was the chloride cell since (1) epitheliocystis organisms were first found within the chloride cell; (2) chloride cells underwent degeneration in the filament epithelium, proliferated along the lamellae, and hypertrophied; and (3) cysts were found only in the trailing edge of the gill filament and in the interlamellar spaces where, in healthy fish, chloride cells are mainly located. In some cases, the pseudobranch of infected fish was much more severely affected than the gill. It is therefore suggested that, for diagnostic purposes, the pseudobranch should also be sampled when epitheliocystis disease is suspected.     

Quantitative genetic parameter estimates for size and growth rate traits in Pacific white shrimp, Penaeus vannamei (Boone 1931) when reared indoors

Heritabilities of size traits and growth rate traits, as well as genetic, phenotypic and environmental correlations were estimated at three ages for a captive population of Pacific white shrimp (Penaeus vannamei) grown indoors. A covariate, mean size or mean growth rate during early growth in individual tanks before tagging and mixing, was introduced in the analyses to reduce the error caused by unique previous growth conditions on variance components. Heritabilities of size traits increased with age, with the h² for TL, AL, TWt and Wi1AS being 0.15, 0.20, 0.20 and 0.22, respectively, at 17 weeks, increasing to 0.28, 0.33, 0.34 and 0.35 at 29 weeks of age. Heritabilities of growth rate traits estimated between consecutive growth periods decreased from the first (h² for ΔTL 0.65, ΔAL 0.71, ΔTWt 0.63 and ΔWi1AS 0.84) to the second period (h² for ΔTL 0.34, ΔAL 0.50, ΔTWt 0.54 and ΔWi1AS 0.52). Phenotypic correlations were always larger than genetic correlations for both, size and growth rate traits. Genetic correlations between size traits within age were high (r_G >0.95), but those between the same size trait at different ages decreased as the age difference increased in spite of a consistently high environmental correlation (r_E 0.80–0.85) between the same trait at different ages. Phenotypic and genetic correlation's between the same growth rate trait at the two different growth periods evaluated were negative or zero (r_G TL –0.26, AL –0.24, Wi1AS 0.00) with the exception of total weight (r_G TW 0.35) and the environmental correlations between growth periods were also low (r_E 0.13–0.32).     

Characterization of Amaranthus cruentus L. seed proteins by 2-DE and LC/MS–MS: Identification and cloning of a novel late embryogenesis-abundant protein

Amaranth is taking great attention as an important cereal crop that could fulfill food requirements for the growing population, especially in developing countries. However, the protein composition of these seeds is not well known yet. We have used the proteomics tools to characterize amaranth seed proteome. About 400 proteins spots were resolved on two-dimensional gel electrophoresis (2-DE) and protein spots were analyzed by LC-MS/MS (liquid chromatography tandem mass spectrometry). Identified proteins were related to stress and defense responses, metabolic, respiratory, and oxide-reduction processes. One abundant spot was identified as a Late Embryogenesis Abundant (LEA) protein and the gene was cloned and characterized. The AcLEA cDNA contains a 418 bp open reading frame (ORF) encoding a polypeptide of 139 amino acids. Bioinformatics analysis showed that AcLEA belongs to LEAs Group 5. Proteomics is a powerful technique that could be used even in non-sequenced organisms such as amaranth. The obtained information reveals that amaranth seed, beyond the classical seed storage proteins, contains proteins related to protection against stress. The identification of these proteins opens the door to the application of new strategies to improve the quality of amaranth production.