Stable isotope analyses—A method to distinguish intensively farmed from wild frogs

Consumption of frog legs is increasing worldwide, with potentially dramatic effects for ecosystems. More and more functioning frog farms are reported to exist. However, due to the lack of reliable methods to distinguish farmed from wild‐caught individuals, the origin of frogs in the international trade is often uncertain. Here, we present a new methodological approach to this problem. We investigated the isotopic composition of legally traded frog legs from suppliers in Vietnam and Indonesia. Muscle and bone tissue samples were examined for δ¹⁵N, δ¹³C, and δ¹⁸O stable isotope compositions, to elucidate the conditions under which the frogs grew up. We used DNA barcoding (16S rRNA) to verify species identities. We identified three traded species (Hoplobatrachus rugulosus, Fejervarya cancrivora and Limnonectes macrodon); species identities were partly deviating from package labeling. Isotopic values of δ¹⁵N and δ¹⁸O showed significant differences between species and country of origin. Based on low δ¹⁵N composition and generally little variation in stable isotope values, our results imply that frogs from Vietnam were indeed farmed. In contrast, the frogs from the Indonesian supplier likely grew up under natural conditions, indicated by higher δ¹⁵N values and stronger variability in the stable isotope composition. Our results indicate that stable isotope analyses seem to be a useful tool to distinguish between naturally growing and intensively farmed frogs. We believe that this method can be used to improve the control in the international trade of frog legs, as well as for other biological products, thus supporting farming activities and decreasing pressure on wild populations. However, we examined different species from different countries and had no access to samples of individuals with confirmed origin and living conditions. Therefore, we suggest improving this method further with individuals of known origin and history, preferably including samples of the respective nutritive bases.     

Population genetic structure of wild boars in the West Balkan region

We investigated the population genetic structure of wild boars from Vojvodina (Serbia), Slavonija (Croatia) and Bosnia using four microsatellite markers. All loci presented a high degree of polymorphism and a total of 76 alleles (mean 19 alleles per locus) were detected. Average observed heterozygosity (H _o) value was 0.60. Deviation from Hardy-Weinberg equilibrium was found due to significant heterozygote deficiency detected for three of the four analyzed loci and for all populations. F _IS value over all loci and all populations was 0.29, and the effective number of migrants based on private alleles was 1.64. Sufficient levels of gene flow were found between all populations and the spatial structure showed slightly closer nuclear gene pool affinity of Vojvodina and Slavonija populations in relation of Bosnia population.     

Serological survey of influenza A virus infection in Japanese wild boars (Sus scrofa leucomystax)

We conducted a serological survey to detect antibodies against influenza A virus (IAV) in Japanese wild boars in Kagoshima prefecture, Japan, between 2014 and 2017. Seroprevalence against a pandemic‐like swine H1N1 (H1N1pdm) virus was identified in 27.1% of specimens, and 1.7% were positive for both swine H1N2 and H3N2 viruses, indicating that wild boars could play an important role in the dynamics of H1N1pdm viral dispersion in the wild. The high frequency of positive results for sera against the H1N1pdm virus suggests that cross‐species IAV transmission between wild boars, livestock, and humans is a threat to veterinary and public health.     

Antibodies to selected viral disease agents in wild boars from the Czech Republic

Blood samples were collected from wild boar (Sus scrofa) shot during the hunting season from 1999 to 2005 in the Czech Republic. Sera were tested by enzyme-linked immunosorbent assay for the presence of antibodies against classical swine fever virus (CSFV), swine vesicular disease virus (SVDV), Aujeszky's disease virus (ADV), and bovine viral diarrhea virus (BVDV). Indirect fluorescence antibody test was used for detection of antibodies against porcine circovirus type 2 (PCV-2) and transmissible gastroenteritis virus (TGEV). Antibodies against ADV, BVDV, PCV-2, and TGEV were detected in 30% (101 of 338), 1% (2 of 352), 43% (57 of 134), and 1% (1 of 134) of wild boars, respectively. Sera of 6,471 and 362 tested wild boars were negative for the presence of antibodies against CSFV and SVDV, respectively. This is the first survey of TGEV antibodies in wild boars and the first serologic survey of viral diseases in wild boars in the Czech Republic. Wild boars in the Czech Republic may act as a potential reservoir of ADV and thus have a role in the epidemiology of this disease.     

A signature-based method to distinguish time-of-flight secondary-ion mass spectra from biological samples

Time-Of-Flight Mass Spectrometry (TOF-SIMS) was used to determine elemental and biomolecular ions from isolated protein samples. We identified a set of 23 mass-to-charge ratio (m/z) peaks that represent signatures for distinguishing biological samples. The 23 peaks were identified by Singular Value Decomposition (SVD) and Canonical Analysis (CA) to find the underlying structure in the complex mass-spectra data sets. From this modified data, SVD was used to identify sets of m/z peaks, and we used these patterns from the TOF-SIMS data to predict the biological source from which individual mass spectra were generated. The signatures were validated using an additional data set different from the initial training set used to identify the signatures. We present a simple method to identify multiple variables required for sample classification based on mass spectra that avoids overfit. This is important in a variety of studies using mass spectrometry, including the ability to identify proteins in complex mixtures and for the identification of new biomarkers.     

Antibodies to selected viral and bacterial pathogens in European wild boars from southcentral Spain

Serum samples from 78 European wild boars (Sus scrofa) harvested during the 1999-2000 hunting season were tested for antibodies to Brucella spp., classical swine fever virus, Erysipelothrix rhusiopathiae, Haemophilus parasuis, Leptospira interrogans serovar pomona, Mycoplasma hyopneumoniae, pseudorabies virus (PRV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus, Salmonella serogroups B, C, and D, Streptococcus suis, and swine influenza virus (SIV) serotypes H1N1 and H3N2. Samples were collected from Sierra Morena and Montes de Toledo in southcentral Spain. Antibodies were detected to PRV (36%), L. interrogans serovar pomona (12%), PPV (10%), E. rhusiopathiae (5%), SIV serotype H1N1 (4%), Salmonella serogroup B (4%), and Salmonella serogroup C (3%). Our results suggest that more research is needed to describe the epidemiology of infectious diseases of Spanish wild boars.     

A serological study on brucellosis in wild boars in Germany

A recent outbreak of brucellosis in an outdoor pig herd, where wild boars were identified as the most probable source of infection, prompted us to conduct a serological study on wild boars from five federal states of Germany. A total of 885 sera were examined using a combination of screening and confirmatory testing, i.e. indirect enzyme-linked immunosorbent assay (ELISA) followed by complement fixation and slow agglutination tests. Seroprevalences of Brucella suis antibodies in the various regions were between 0 and 28.5% in the ELISA. After confirmatory testing, the amount of positive sera was lower and reached only up to 12.1%, dependent on the method. Although wild boars usually harbour B. suis biovar 2, which is less virulent for humans, a zoonotic risk for persons dealing with these animals and their carcasses cannot be ruled out.     

A method to distinguish microbial acclimation from reduced growth rates in perturbed ecosystems

Abstract Predicting variations in microbial growth rates (μ) in Nature requires a method that will detect departures from optimal μ, determine the time scale at sub-optimal μ, and measure the restoration or reversibility toward optimal μ. Isotopic flux measurements are equivocal because of uncertainties in their relationship to μ, particularly in non-steady state condictions. After perturbing a natural microbial community and a clone of the phytoplankter Selenastrum capricornutum with two different toxicants, we examined the kinetics of the adenylate energy charge, and followed changes in biomass over time with the total adenylate and chlorophyll a concentrations. We determined that exposure to cadmium resulted in a reduction in μ; reversible within a short time, whereas exposure to the industrial effluent caused an exponential loss of adenylates and complete inhibition of growth. Upon perturbing a microbiol community, the study of adenylate energy charge kinetics detects departures from optimal μ, measures their duration and reversibility.     

Prevalence of antibody to six Leptospira servovars in Swedish wild boars

Zoonotic Leptospira bacteria are pathogens that may increase in importance with climate change. We investigated the prevalence of antibody to six Leptospira serovars (sv) in the Swedish wild boar (Sus scrofa) population, which is increasing in number and geographic distribution. The serovars we selected cause disease in pigs or may be of use as sentinel serovars to measure the potential spread in Swedish fauna. In total, 386 serum samples from wild boars collected between 2005 and 2007 were investigated using a microscopic agglutination test for Leptospira interrogans sv Bratislava strain Jez Bratislava, sv Icterohaemorrhagiae strain Kantorowicz, sv Pomona strain Pomona, Leptospira kirschneri sv Grippotyphosa strain Duyster, and Leptospira borgpetersenii sv Tarassovi strain Perepelitsin, and a domestic strain closely related to sv Sejroe. Twelve (3.1%) of the analyzed samples were antibody-positive. Of those, nine (2.3%) were positive for sv Bratislava and 0.8% for sv Icterohaemorrhagiae. All antibody-positive samples originated from areas where wild boars are reported to be common. We conclude that Leptospira infection is less common in Swedish wild boar than in continental Europe. However, we recommend continuous surveillance to follow the effects of climate change and an increasing wild boar population.     

Identification of mitochondrial markers for genetic traceability of European wild boars and Iberian and Duroc pigs

Iberian pigs and wild boars are the source of highly priced meat and dry-cured products. Iberian maternal origin is mandatory for labeled Iberian products, making necessary the authentication of their maternal breed origin. Discrimination between wild and domestic pig maternal origin may be useful to distinguish labeled wild boar meat obtained from hunting or farming. In order to detect useful polymorphisms to trace Iberian, Duroc and wild boar maternal lineages, we herein investigated the complete porcine mitochondrial DNA (mtDNA) using three complementary approaches. Near-complete mtDNA sequences (16989 bp), excluding the minisatellite present in the displacement loop region (D-loop), were successfully determined in six Iberian pigs, two Duroc and six European wild boars. To complete the mtDNA analysis, the D-loop minisatellite region was also analyzed in the same set of samples by amplification and capillary electrophoresis detection. Finally, the frequencies of Asian and European Cytochrome B (Cyt B) haplotypes were estimated in Iberian (n = 96) and Duroc (n = 125) breeds. Comparison of near-complete mtDNA sequences revealed a total of 57 substitutions and two Indels. Out of them, 32 polymorphisms were potential Iberian markers, 10 potential Duroc markers and 16 potential wild boar markers. Fourteen potential markers (five Iberian and nine Duroc), were selected to be genotyped in 96 Iberian and 91 Duroc samples. Five wild boar potential markers were selected and tested in samples of wild boars (73) and domestic pigs including: 96 Iberian, 16 Duroc, 16 Large White and 16 Landrace. Genotyping results showed three linked markers (m.7998C>T, m.9111T>C, m.14719A>G) absent in Duroc and present in Iberian pigs with a frequency 0.72. Six markers (m.8158C>T, m.8297T>C, m.9230G>A, m.11859A>G, m.13955T>C, m.16933T>C), three of them linked, were absent in Iberian pigs and present in Duroc with a joint frequency of almost 0.50. Finally three linked markers (m.7188G>A, m.9224T>C, m.15823A>G) were solely detected in wild boars with a frequency 0.22. The D-loop minisatellite results showed overlapping ranges of fragment sizes and suggested heteroplasmy, a result that nullify the use of this region for the development of breed diagnostic markers. The Cyt B haplotype results showed the presence of European haplotypes in Iberian while one of the Asian haplotypes was detected in Duroc with a frequency 0.22, linked to the Duroc marker m.9230G>A. Our results are valuable to resolve the problems of Iberian and wild boar maternal origin determination but additional markers are required to achieve totally useful genetic tests.     

A fluorescent antibody method to distinguish vegetative cells from spheroplasts of anEscherichia coli strain

An immunological method for distinguishing bacteria from spheroplasts of anE. coli strain is presented. Antisera prepared against fractions of a lysate of spheroplasts are able, after absorption of the O antibodies, to label spheroplasts but not normal bacteria by the fluorescent antibody method. The immunologic bases of the specificity are studied. By diffusion precipitation tests it was shown that spheroplasts contain at least 7 antigens, 2 of which are part of the O antigen complex of the bacterium.This investigation was supported by the Fonds National de la Recherche Scientifique. The author wishes to thand Doctor J. Salmon for useful suggestions and help in immunological methods. The technical assistance of Miss E. Ligot is gratefully acknowledged.     

Grouping-patterns and inter-group relationships of Japanese wild boars (Sus scrofa leucomystax) in the Rokko mountain area

Grouping-patterns and inter-group relationships of Japanese wild boars (Sus scrofa leucomystax) were studied at a provisioning site in the Rokko mountain area, Hyogo Prefecture, Japan, from 1982 to 1983. The observations were divided into four study periods. Almost all wild boars were individually identified and their kin-relationships were followed from the beginning of the study. To examine grouping-patterns of Japanese wild boars, the degree of association between any two individuals, based on coexisting time at the provisioning site, was analyzed for each study period. Mother and her litter group (MLG), female littermate group (FLG) and solitary individual were recognized grouping-patterns. Groups did not associate with each other. Male offspring separated from their mother before 1 year of age and become solitary. Separation of female offspring occurred before the subsequent parturition of their mother. The proximate cause of separation was increased aggression by the mother. After separation, female offspring formed a FLG. However, the FLG seemed to be generally a temporary one, maintaining its stability only until parturition of it's members. Thereafter adult females had no close association with any other individuals, except for their own infants. Male offspring disappeared from their natal area, while female offspring could remain there.     

Estimation of the fertility rates of Japanese wild boars (Sus scrofa leucomystax) using fetuses and corpora albicans

Effective population control of Japanese wild boar (Sus scrofa leucomystax) requires reliable information about population dynamics. Fertility rate is the fundamental component of reproduction to evaluate population dynamics. However, little is known regarding the fertility rate of Japanese wild boar. The traditional hunting practices make it difficult to obtain pregnant females and calculate the fertility rate by checking fetuses as is performed in other countries. Therefore, we focused on the corpora albicans (CA) as the CA remains in the ovaries of postpartum females after pregnancy. This study aimed to evaluate the utility of CA and estimate the fertility rate of Japanese wild boars using CA. Histological analysis of ovaries enabled us to discriminate type 1 CA, which remains for 1 year after breeding. Type 1 CA is a superior indicator compared with lactation in the non-pregnancy season because it allows verification of postpartum females over a long period. The fertility rate was calculated by the combination of pregnant and postpartum females using fetuses and type 1 CA from April to November. The fertility rate of the females captured after the second pregnancy season was 90.3 % during the pregnancy period and 100 % during the non-pregnancy period. The high fertility rate of adult females suggests that intensive adult female harvesting is needed. Our new method to determine fertility rates contributes to developing a monitoring system to adequately control Japanese wild boar population.     

A genetic approach to the species problem in wild potato

Wild potatoes are native to the Americas, where they present very wide geographical and ecological distribution. Most are diploid, obligate out-crossers due to a multiallelic gametophytic self-incompatibility (S) locus that prevents self-fertilisation and crossing between individuals carrying identical S-alleles. They have two alternative modes of reproduction: sexual (by seeds) and asexual (by stolons and tubers), which provide, respectively, for genetic flexibility in changing environments and high fitness of adapted genotypes under stable conditions. Since the early twentieth century, their taxonomic classification has been mostly based on morphological phenotypes (Taxonomic Species Concept). More recently, attempts have been made to establish phylogenetic relationships, applying molecular tools in samples of populations (accessions) with a previously assigned specific category. However, neither the reproductive biology and breeding relations among spontaneous populations nor the morphological and genetic variability expected in obligate allogamous populations are considered when the taxonomic species concept is applied. In nature, wild potato populations are isolated through external and internal hybridisation barriers; the latter, which are genetically determined, can be either pre-zygotic (pollen-pistil incompatibility) or post-zygotic (abortion of embryo, endosperm or both tissues, sterility, and hybrid weakness and breakdown in segregating generations). The internal barriers, however, can be incomplete, providing opportunities for hybridisation and introgression within and between populations and ploidy levels in areas of overlap. The widespread occurrence of spontaneous hybrids in nature was recognised in the mid-twentieth century. Using genetic approaches, results have been obtained that provide strong support to the assertion that populations are at different stages of genetic divergence and are not at the end of the evolutionary process, as presupposed by the Taxonomic Species Concept. Furthermore, since wild potatoes have uniparental and biparental overlapping generations, the Biological Species Concept - developed for sexually reproducing biparental organisms - cannot be applied to them. In this paper, morphological, genetic, molecular and taxonomic studies in wild potato are reviewed, considering the genetic consequences of their reproductive biology, in an attempt to shed light on the species problem, because of its relevance in germplasm conservation and breeding.     

划分土壤类型的一种新方法--以功能生态学理论为基础

借助功能生态学的理论 ,提出了运用土壤的特征物质 (CM)和特征时间 (CT)区分土壤类型的新方法。通过数学模拟 ,分别计算了灰色森林土和黑钙土的特征物质和特征时间 :当灰色森林土和黑钙土的年凋落物量分别为 5 .5t·hm-2 和 11.2t·hm-2 时 ,它们的CM和CT分别为 133t·hm-2 、80年和 82 0t·hm-2 、6 5 0年。CM和CT的值随着输入土壤的物质量和土壤中分解速度的变化而变化 ,而且受外界环境因素 (包括自然因素和人为因素 )的影响。CT随土壤深度的增加而增大     

A rapid PCR based method to distinguish between Lactococcus and Enterococcus

Phenotypic characterisation of Lactococcus and Enterococcus species remains unreliable as strains of both genera have been isolated which do not conform to the traditional criteria for separation of these genera. A bank of 131 isolates was phenotypically characterised by three methods: (a) traditional broth tests, (b) API Rapid ID 32 Strep and (c) BBL Crystal ID kits. Differences in genus designation between commercial kits were evident for 12 strains (9%), while 7 strains (5%) remained unidentified by either kit. Published 16S rRNA sequences were aligned and used to design genus-specific primers which, when used in separate PCR reactions, were capable of distinguishing all type strains of Lactococcus and Enterococcus. These primers did not react with known species of Streptococcus, Pediococcus, Lactobacillus, Leuconostoc or Tetragenococcus. Isolates which could not be identified by phenotype were assigned to either genus on the basis of the gene primers.     

The response of Mediterranean herbaceous community to soil disturbance by native wild boars

Native wild boar (Sus scrofa lybicus) populations in Israel increased in the last decades, and their rooting activity presents an apparent severe disturbance that may affect herbaceous plant communities. We studied the extent of wild boar rooting and its effect on herbaceous vegetation in an east Mediterranean garrigue. We compared the effects of rooting soil disturbance (RSD) between summer, winter, and RSD-free control plots on herbaceous plants. RSD affected four percent of the area covered by herbaceous vegetation in winter and spring, and only 2 % in summer. Summer RSD increased herbaceous species diversity in the following spring by decreasing the abundance of the dominant species, but it did not affect total herbaceous biomass. Winter RSD lowered herbaceous biomass but did not affect herbaceous plant abundance or diversity. We conclude that Mediterranean herbaceous plant community appears to be resilient to RSD, causing only minor changes in species composition and small decrease of their biomass. The contrast between the relatively large extent of wild boars RSD, and its limited impact on the herbaceous plant community, can be attributed to the long mutual evolutionary history of wild boars and these vegetation communities.