Pilar Peral García

Shiga toxin-producing Escherichia coli in beef retail markets from Argentina

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause mild or serious diseases and can lead to people death. This study reports the prevalence and characteristics of STEC O157 and non-O157 in commercial ground beef and environmental samples, including meat table, knife, meat mincing machine, and manipulator hands (n = 450) obtained from 90 retail markets over a nine-month period. The STEC isolates were serotyped and virulence genes as stx (Shiga toxin), rfbO157] (O157 lipopolysaccharide), fliCH7 (H7 flagellin), eae (intimin), ehxA (enterohemolysin) and saa (STEC autoagglutinating adhesin), were determined. STEC O157 were identified in 23 (25.5%) beef samples and 16 (4.4%) environmental samples, while STEC non-O157 were present in 47 (52.2%) and 182 (50.5%), respectively. Among 54 strains isolated, 17 were STEC O157:H7 and 37 were STEC non-O157. The prevalent genotype for O157 was stx2/eae/ehxA/fliCH7 (83.4%), and for STEC non-O157 the most frequent ones were stx1/stx2/saa/ehxA (29.7%); stx2 (29.7%); and stx2/saa/ehxA (27%). None of the STEC non-O157 strains were eae-positive. Besides O157:H7, other 20 different serotypes were identified, being O8:H19, O178:H19, and O174:H28 the prevalent. Strains belonging to the same serotype could be isolated from different sources of the same retail market. Also, the same serotype could be detected in different stores. In conclusion, screening techniques are increasingly sensitive, but the isolation of STEC non-O157 is still a challenge. Moreover, with the results obtained from the present work, although more studies are needed, cross-contamination between meat and the environment could be suspected.

Multiplex–PCR of short amplicons for mtDNA sequencing from ancient DNA

Abstract We here describe a multiplex–PCR method to generate six overlapping short amplicons (100–130 bp) in two separate PCR reactions of non-overlapping fragments for full sequencing of the whole hypervariable region I (HV1). The performance of this multiplex system has been evaluated not only on ancient bone remains (5000–4000 BP) but also on different forensic samples with highly degraded DNA (bone remains, hair shafts, …) that yielded negative PCR results with the mtDNA amplification strategies usually employed in forensic genetics. The multiplex–PCR methodology described in this study also illustrates a potential high throughput strategy to reconstruct the sequence of both coding and non-coding regions of the mtDNA genome from ancient DNA.

Assignment of paternity in a judicial dispute between two neighbor Holstein dairy farmers.

DNA profiling was used as evidence to assign paternity in a dispute between two neighbors in a judicial case of undue appropriation of cattle offspring from five alleged Holstein sires. Five offspring were genotyped using ten genetic markers (nine microsatellites and the BOLA-DRB3 locus). The computer program CERVUS was used to estimate the LOD score values and the confidence of paternity assignments. The results presented here show that three out of five paternity cases were assigned at 95% of confidence to a single sire with a LOD score ranging from 2.53 to 3.55. A fourth male was assigned using its delta value. Finally, all alleged sires were excluded from the paternity of the fifth offspring, probably due to the existence of an non-sampled male in the studied population. We concluded that the likelihood-based approach, included into CERVUS program, was a powerful tool in cattle kinship analysis when dealing with judicial dispute particularly when the dams genotype was absent, allowing the assignments of paternity at 95% level of confidence in situations usually used by dairy and beef cattle producers in Argentine (e.g., multi-sire pasture mating).

Analysis of apoptosis and DNA damage in bovine cumulus cells after exposure in vitro to different zinc concentrations.

The purpose of this study was to investigate the effect of Zn (zinc) concentration on CCs (cumulus cells) during in vitro maturation. For this purpose, DNA integrity of CCs by addition of different Zn concentrations [0 (control); 0.7 μg/ml (Zn1); 1.1 μg/ml (Zn2) and 1.5 μg/ml (Zn3)] to the culture medium was evaluated by comet assay. In addition, early apoptosis was analysed by annexin staining assay. CCs treated with Zn showed a significant decrease in the DNA damage in a dose‐dependent manner. Comet assay analysed for TM (tail moment) was significantly higher in cells cultured without Zn (control, P<0.01) with respect to cells treated with Zn (control: 5.24±16.05; Zn1: 1.13±5.31; Zn2: 0.10±0.36; Zn3: 0.017±0.06). All treatments were statistically different from the control (P=0.014 for Zn1; P<0.01 for Zn2 and Zn3). The frequency of apoptotic cells was higher in the control group (control: 0.142±0.07; Zn1: 0.109±0.0328; Zn2:0.102±0.013; Zn3: 0.0577±0.019). Statistical differences were found between control and Zn1 (P=0.0308), control and Zn2 (P=0.0077), control and Zn3 (P<0.0001), Zn1 and Zn3 (P<0.001) and Zn2 and Zn3 (P=0.0004). No differences were found between Zn1 and Zn2. In conclusion, low Zn concentrations increase DNA damage and apoptosis in CCs cultured in vitro. However, adequate Zn concentrations ‘protect’ the integrity of DNA molecule and diminish the percentage of apoptotic CC.

Prediction of Non-Genotoxic Carcinogenicity Based on Genetic Profiles of Short Term Exposure Assays

Non-genotoxic carcinogens are substances that induce tumorigenesis by non-mutagenic mechanisms and long term rodent bioassays are required to identify them. Recent studies have shown that transcription profiling can be applied to develop early identifiers for long term phenotypes. In this study, we used rat liver expression profiles from the NTP (National Toxicology Program, Research Triangle Park, USA) DrugMatrix Database to construct a gene classifier that can distinguish between non-genotoxic carcinogens and other chemicals. The model was based on short term exposure assays (3 days) and the training was limited to oxidative stressors, peroxisome proliferators and hormone modulators. Validation of the predictor was performed on independent toxicogenomic data (TG-GATEs, Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System, Osaka, Japan). To build our model we performed Random Forests together with a recursive elimination algorithm (VarSelRF). Gene set enrichment analysis was employed for functional interpretation. A total of 770 microarrays comprising 96 different compounds were analyzed and a predictor of 54 genes was built. Prediction accuracy was 0.85 in the training set, 0.87 in the test set and increased with increasing concentration in the validation set: 0.6 at low dose, 0.7 at medium doses and 0.81 at high doses. Pathway analysis revealed gene prominence of cellular respiration, energy production and lipoprotein metabolism. The biggest target of toxicogenomics is accurately predict the toxicity of unknown drugs. In this analysis, we presented a classifier that can predict non-genotoxic carcinogenicity by using short term exposure assays. In this approach, dose level is critical when evaluating chemicals at early time points.

Comprehensive Evaluation and Implementation of Improvement Actions in Butcher Shops.

Foodborne pathogens can cause acute and chronic diseases and produce a wide range of symptoms. Since the consumption of ground beef is a risk factor for infections with some bacterial pathogens, we performed a comprehensive evaluation of butcher shops, implemented improvement actions for both butcher shops and consumers, and verified the impact of those actions implemented. A comprehensive evaluation was made and risk was quantified on a 1–100 scale as high-risk (1–40), moderate-risk (41–70) or low-risk (71–100). A total of 172 raw ground beef and 672 environmental samples were collected from 86 butcher shops during the evaluation (2010–2011) and verification (2013) stages of the study. Ground beef samples were analyzed for mesophilic aerobic organisms, Escherichia coli and coagulase-positive Staphylococcus aureus enumeration. Salmonella spp., E. coli O157:H7, non-O157 Shiga toxin-producing E. coli (STEC), and Listeria monocytogenes were detected and isolated from all samples. Risk quantification resulted in 43 (50.0%) high-risk, 34 (39.5%) moderate-risk, and nine (10.5%) low-risk butcher shops. Training sessions for 498 handlers and 4,506 consumers were held. Re-evaluation by risk quantification and microbiological analyses resulted in 19 (22.1%) high-risk, 42 (48.8%) moderate-risk and 25 (29.1%) low-risk butcher shops. The count of indicator microorganisms decreased with respect to the 2010–2011 period. After the implementation of improvement actions, the presence of L. monocytogenes, E. coli O157:H7 and stx genes in ground beef decreased. Salmonella spp. was isolated from 10 (11.6%) ground beef samples, without detecting statistically significant differences between both study periods (evaluation and verification). The percentage of pathogens in environmental samples was reduced in the verification period (Salmonella spp., 1.5%; L. monocytogenes, 10.7%; E. coli O157:H7, 0.6%; non-O157 STEC, 6.8%). Risk quantification was useful to identify those relevant facts in butcher shops. The reduction of contamination in ground beef and the environment was possible after training handlers based on the problems identified in their own butcher shops. Our results confirm the feasibility of implementing a comprehensive risk management program in butcher shops, and the importance of information campaigns targeting consumers. Further collaborative efforts would be necessary to improve foodstuffs safety at retail level and at home.

A Case of Multiple Assignments (Paternity/Maternity) in an Equine‐Out Breeding System*

Abstract:  Recently, the use of DNA markers has provided a more accurate method of identifying individuals and verifying parentage. In this report, we describe foal assignment in a farm bred jumping horses (Silla argentino). Ten mares were freely served by two stallions, resulting in nine foals. Weaning occurred without registration of the mare of each offspring, resulting in a failure to identify either the mare or the sire of each foal. Animals were typed using 12 microsatellite systems and four biochemical polymorphisms in order to determine the paternity/maternity of each foal. We used the CERVUS program to evaluate the parentage of each offspring. It was possible to determine maternity in eight cases, and paternity in all of them. We concluded that this set of codominant markers analyzed following a likelihood‐based approach included in the CERVUS package, are useful tools to solve parentage assignments in domestic horses.

First report of cerebellar abiotrophy in an Arabian foal from Argentina

Evidence of cerebellar abiotrophy (CA) was found in a six-month-old Arabian filly with signs of incoordination, head tremor, wobbling, loss of balance and falling over, consistent with a cerebellar lesion. Normal hematology profile blood test and cerebrospinal fluid analysis excluded infectious encephalitis, and serological testing for Sarcocystis neurona was negative. The filly was euthanized. Postmortem X-ray radiography of the cervical cephalic region identified not abnormalities, discounting spinal trauma. The histopathological analysis of serial transverse cerebellar sections by electron microscopy revealed morphological characteristics of apoptotic cells with pyknotic nuclei and degenerate mitochondria, cytoplasmic condensation and areas with absence of Purkinje cells, matching with CA histopathological characteristics. The indirect DNA test for CA was positive in the filly, and DNA test confirmed the CA carrier state in the parents and the recessive inheritance of the disease. To our knowledge this is the first report of a CA case in Argentina.

Forensic validation studies on the Y-Plex™ 6 Kit

Abstract The results of some forensic validation studies on the Y-PLEX™ 6 Kit (Reliagene Technologies) are presented. The evaluation of specificity (including imbalanced male/female mixtures, DNA from microorganisms) and sensitivity (28–32 cycles) allowed us to conclude that the Y-Plex 6 Kit system is a sensitive and reproducible Y-STR multiplex system. However, some nonspecific PCR products amplified from female DNA were observed for the yellow channel.

Analysis of mitochondrial 12S rRNA gene sequence variation in four ethnically defined populations

Abstract In this study, we have analysed the nucleotide sequence variation of the 12S rRNA mitochondrial gene (648–1601 bp) from five different populations (Spanish Caucasian, Autochthonous from the Basque Country, Chinese, New Guinea Highlander and Africans of Benin) by full sequencing of two overlapping PCR fragments using d-rhodamine cycle sequencing coupled with an ABI377 sequencer. Preliminary data indicate different patterns of sequence variation between Africans and non-Africans. Africans are much more polymorphic than non-African populations, which have only a very restricted subset of haplotypes. Furthermore, the greater part of Africans analysed showed two specific nucleotide substitutions (769G→A and 1018G→A) that were not observed in non-African individuals. In conclusion, the mtDNA 12SRNA gene in combination with other systems could be an interesting ethnic marker that could help to differentiate between African and non-African maternal lineages.