Standard Genotyping Research Articles

Fine-tuning the performance of ddRAD-seq in the peach genome

The advance of Next Generation Sequencing (NGS) technologies allows high-throughput genotyping at a reasonable cost, although, in the case of peach, this technology has been scarcely developed. To date, only a standard Genotyping by Sequencing approach (GBS), based on a single restriction with ApeKI to reduce genome complexity, has been applied in peach. In this work, we assessed the performance of the double-digest RADseq approach (ddRADseq), by testing 6 double restrictions with the restriction profile generated with ApeKI. The enzyme pair PstI/MboI retained the highest number of loci in concordance with the in silico analysis. Under this condition, the analysis of a diverse germplasm collection (191 peach genotypes) yielded 200,759,000 paired-end (2 × 250 bp) reads that allowed the identification of 113,411 SNP, 13,661 InDel and 2133 SSR. We take advantage of a wide sample set to describe technical scope of the platform. The novel platform presented here represents a useful tool for genomic-based breeding for peach.

Genomic epidemiological analysis identifies high relapse among individuals with recurring tuberculosis and provides evidence of recent household-related transmission of tuberculosis in Ghana

ObjectiveTo retrospectively investigate the cause of recurring tuberculosis (rcTB) among participants with pulmonary TB recruited from a prospective population-based study conducted between July 2012 and December 2015. MethodsMycobacterium tuberculosis complex isolates obtained from rcTB cases were characterized by standard mycobacterial genotyping tools, whole-genome sequencing, and phylogenetic analysis carried out to assess strain relatedness. ResultsThe majority (58.3%, 21/36) of study participants with rcTB episodes had TB recurrence within 12 months post treatment. TB strains with isoniazid (INH) resistance were found in 19.4% (7/36) of participants at the primary episode, of which 29% (2/7) were also rifampicin-resistant. On TB recurrence, an INH-resistant strain was found in a larger proportion of participants, 27.8% (10/36), of which 40% (4/10) were MDR-TB strains. rcTB was attributed to relapse (same strain) in 75.0% (27/36) of participants and 25.0% (9/36) to re-infection. ConclusionOur findings indicate that previous unresolved infectiondue to inadequate treatment, may be the major cause of rcTB.

Concordance rate in cattle and sheep between genotypes differing in Illumina GenCall quality score.

Proper quality control of data prior to downstream analyses is fundamental to ensure integrity of results; quality control of genomic data is no exception. While many metrics of quality control of genomic data exist, the objective of the present study was to quantify the genotype and allele concordance rate between called single nucleotide polymorphism (SNP) genotypes differing in GenCall (GC) score; the GC score is a confidence measure assigned to each Illumina genotype call. This objective was achieved using Illumina beadchip genotype data from 771 cattle (12428767 genotypes in total post-editing) and 80 sheep (1557360 SNPs genotypes in total post-editing) each genotyped in duplicate. The called genotype with the lowest associated GC score was compared to the genotype called for the same SNP in the same duplicated animal sample but with a GC score of >0.90 (assumed to represent the true genotype). The mean genotype concordance rate for a GC score of <0.300, 0.300-0.549, and ≥0.550 in the cattle (sheep in parenthesis) was 0.9467 (0.9864), 0.9707 (0.9953), and 0.9994 (0.99997) respectively; the respective allele concordance rate was 0.9730 (0.9930), 0.9849 (0.9976), and 0.9997 (0.99998). Hence, concordance eroded as the GC score of the called genotype reduced, albeit the impact was not dramatic and was not very noticeable until a GC score of <0.55. Moreover, the impact was greater and more consistent in the cattle population than in the sheep population. Furthermore, an impact of GC score on genotype concordance rate existed even for the same SNP GenTrain value; the GenTrain value is a statistical score that depicts the shape of the genotype clusters and the relative distance between the called genotype clusters.

Incidence and impact of allele-specific anti-HLA antibodies and high-resolution HLA genotyping on assessing immunologic compatibility

The ability to identify specific HLA molecules against which a patient has alloantibodies has revolutionized assessment of immunologic compatibility. Anti-HLA antibodies are typically evaluated as reactive against well-defined serologic antigen groups. Thus, donor HLA genotyping is aimed at defining HLA at the serologic split-antigen level to avoid incompatible antigen-antibody combinations. However, anti-HLA antibodies can have reactivities not accurately described by well-defined serologic antigens. While existence of these antibodies is acknowledged, their precise impact on clinical practice is not clear. We performed a single-center review of 2 years of pre-and post-transplant anti-HLA antibody testing data combined with high-resolution HLA genotyping data for living and deceased organ donors to evaluate the clinical impact of anti-HLA antibodies with reactivities outside of commonly defined serologic antigen groups. We find approximately 15% of patients awaiting transplantation have alloantibodies with differential reactivity for HLA proteins encoded by specific alleles within a serologic antigen group. Allele-specific antibodies are associated with positive cellular crossmatches not accurately predicted by standard donor HLA genotyping and can manifest as post-transplant donor-specific antibodies. Our data highlights the importance of evaluating anti-HLA antibodies at the allele-level and provides evidence supporting utility for high-resolution HLA genotyping in solid organ transplantation.

Prevalence of drug resistance mutations in HIV-infected individuals with low-level viraemia under combination antiretroviral therapy: an observational study in a tertiary hospital in Northern Taiwan, 2017-19.

Effective ART is crucial for combating the HIV pandemic. Clinically, plasma viral load monitoring to achieve virological suppression is the guide for an optimal ART. The presence of low-level viraemia (LLV) below the definition level of virological failure is a risk factor for ART failure. However, there is no treatment consensus over LLV yet, mainly due to the limitation of standard HIV-RNA genotyping and the resultant insufficient understanding of LLV characteristics. To better profile drug resistance mutations (DRMs) and the associated factors in cases experiencing LLV. A prospective observational study was conducted from 2017 to 2019. HIV-DNA was used as an alternative to HIV-RNA for HIV genotyping coupled with deep sequencing for ART-naive and ART-failure cases, as well as those with LLV. Eighty-one ART-naive, 18 ART-failure and 16 LLV cases received HIV genotyping in the study. Three-quarters (12/16) of cases experiencing LLV harboured DRMs. Cases with LLV had higher prevalence of DRMs to NNRTIs than the ART-naive group (69% versus 20%, P < 0.001), but lower DRM prevalence to NRTIs than the ART-failure group (25% versus 61%, P < 0.001). Approximately half of the LLV cases had issues of suboptimal ART compliance/ART interruption, and 68.8% (11/16) did not display drug resistance to their ART at the time of LLV. HIV DRM profiles in LLV cases were significantly different to those in ART-naive and ART-failure cases. Approaches to consolidate ART compliance and early exploration of potential ART resistance may be needed for cases experiencing LLV episodes.

Assessment of treatment options for patients with hepatitis C virus recombinant form 2k/1b.

Hepatitis C virus (HCV) intergenotype recombinant form (RF) 2k/1b has been actively circulating in HCV-infected patients, and the prevalence of this RF virus in the Republic of Georgia is one of the highest reported worldwide. The aim of this study was to define the optimal treatment regimen for patients with RF_2k/1b. We analyzed the data of 2735 patients who started treatment at the Medical Center Mrcheveli within Georgia's hepatitis C elimination program from May 2015 through December 2019. The patients were treated with sofosbuvir (SOF)-based regimens. For identification of RF_2k/1b variants, refinement of standard (INNO-LiPA) genotyping results for all patient samples assigned the unspecific HCV genotypes (GT) 2a/2c was carried out by sequencing of core and non-structural protein 5B genes. Overall, 444 patients, representing 66% of GT2 and 16% of the total samples, were RF_2k/1b. Treatment of patients with RF_2k/1b with SOF/ledipasvir and SOF/velpatasvir was highly effective and viral cure rates did not differ among genotypes treated with the same regimen: RF_2k/1b, 99% (343/346); GT1, 99% (876/885); GT2, 96% (156/162); and GT3, 99% (545/552). A separate comparison analysis of sustained virologic response rate, treated with SOF plus ribavirin, showed significantly higher sustained virologic response (96%) in patients with confirmed GT2 (by sequencing) compared to unspecified GT2 (by INNO-LiPA) (79%) (P < 0.05). Sofosbuvir-based regimens are highly effective for treatment of RF 2k/1b patients, and with availability of new pan-genotypic direct-acting antivirals, genotyping to identify RF 2k/1b patients might not be necessary.

Single-step RT-PCR assay for dual genotyping of GI and GII norovirus strains.

Noroviruses are the major cause of acute gastroenteritis (AGE) in people of all ages globally. Standardized genotyping is key for outbreak investigations and surveillance networks. Here we describe the validation of a one-step conventional RT-PCR assay for sequence-based dual typing of GI and GII noroviruses. This polymerase (P) and capsid (C) dual typing assay uses a combination of previously published oligonucleotide primers amplifying a genomic region spanning the 3'-end of ORF1 and 5'end of ORF2 resulting in a 579 bp product for GI and 570 bp product for GII viruses. The limit of detection of the assay ranged from 5 to 50 copies of viral RNA per reaction for GI and GII. To validate the assay, we tested 2,663 noroviruspositive stool samples from outbreaks and sporadic cases of AGE in Bangladesh, Guatemala, Peru, and USA collected between 2010-2019, of which 2,392 (90 %) were genotyped successfully. Most of the known genotypes infecting humans (GI (n = 9) and GII (n = 23)) and P types (GI (n = 15), GII, (n = 20)) could be detected. The remaining 270 samples had low viral load (Ct > 30) by real-time RT-PCR. A panel of 166 samples positive for other enteric viruses (rotavirus, astrovirus, sapovirus, adenovirus type 40/41) tested negative. The use of broadly reactive genotyping assays greatly strengthens exchange of standardized genotype data globally to monitor trends in genotype diversity which is important for both the development of vaccines and to measure their impact.

A novel way to identify specific powdery mildew resistance genes in hybrid barley cultivars

Powdery mildew, a common cereal disease caused by the fungus Blumeria graminis, is a major limiting factor of barley production and genetic resistance is the most appropriate protection against it. To aid the breeding of new cultivars and their marketing, resistance genes can be postulated in homogeneous accessions. Although hybrid cultivars (F1) should be homogeneous, they are often not genetically uniform, especially if more than two genotypes are involved in their seed production or due to undesirable self-pollination, out-crossing and mechanical admixtures. To overcome these problems the accepted method of postulating specific resistance genes based on comparing response type arrays (RTAs) of genetically homogeneous cultivars with RTAs of standard genotypes was substituted by analysing the frequency of response types to clusters of pathogen isolates in segregating F2 generations. This method combines a genetic and phytopathological approach for identifying resistance genes. To assess its applicability six hybrid cultivars were screened and from three to seven with a total of 14 resistance genes were found. Two genes were newly located at the Mla locus and their heritability determined. In addition, three unknown dominant genes were detected. This novel, comprehensive and efficient method to identifying resistance genes in hybrid cultivars can also be applied in other cereals and crops.

Development of Rapid Pharmacogenomic Testing Assay in a Mobile Molecular Biology Laboratory (2MoBiL).

Pharmacogenomics is rapidly assuming an integral part in modern health care. Still, its broad applicability relies on the feasibility of performing pharmacogenomic testing in all clinical settings, including in remote areas or resource-limited settings with budget restrictions. In this study, we describe the development and feasibility of rapid and reliable pharmacogenomics assays using a portable molecular biology laboratory, namely the 2MoBiL (Mobile Molecular Biology Laboratory). More precisely, we demonstrate that the genotyping of rs4149056, located within SLCO1B1, can be efficiently and reliably performed using the 2MoBiL portable laboratory and conventional benchtop laboratory equipment and a gold standard genotyping method (KASP assay) as directly comparable methodologies. Taking into account the compact size of 2MoBiL, which directly and positively impacts on its portability, and the high accuracy achieved, we conclude that the 2MoBiL-based genotyping method is warranted for further studies in clinical practices at remote areas and resource-limited as well as time-constrained planetary health settings. To contextualize the broader and potential future applications of 2MoBiL, we emphasize that genotyping of a limited set of clinically relevant single-nucleotide polymorphisms is often a common endpoint of genomics and pharmacogenomics discovery and translational research pipeline. Hence, rapid genotyping by 2MoBiL can be an essential catalyst for global implementation of pharmacogenomics and personalized medicine in the clinic. The Clinical Trial Registration number is NCT03093818.

Long amplicon sequencing for improved genetic characterization of African swine fever virus

African Swine Fever Virus (ASFV) causes a transmissible and fatal disease in pigs that is currently devastating global swine production. Efficient and economical collection of genetic data from ASFV field isolates is essential for bio-surveillance, to limit and control its spread, and to better understand ASF disease ecology. Standard genotyping and subtyping of ASFV field isolates is currently limited to a few variable regions within the ASFV genome. However, more extensive sequencing is necessary to better understand ASFV molecular evolution and identify regions relevant to genetic diversity. In this study, we developed a method for rapid and efficient next generation sequencing of approximately 40% of the ASFV genome using long PCR amplification of six different genomic regions. The amplified regions contain all segments currently used for genotyping and additional genes predicted to contribute to ASFV diversity. The primers used for amplification are broadly compatible with published ASFV genomes, permitting their use on relevant ASFV isolates. This methodology provides the enhanced depth of coverage of amplicon-based sequencing while mitigating complications associated with ASFV whole-genome sequencing. Implementation of this methodology could substantially increase the scale of ASFV genetic data collection, which is necessary to effectively monitor and combat this critical agricultural disease.

Clinical experience with carrier screening in a general population: support for a comprehensive pan-ethnic approach

PurposeTo present results from a large cohort of individuals receiving expanded carrier screening (CS) in the United States. MethodsSingle-gene disorder carrier status for 381,014 individuals was determined using next-generation sequencing (NGS) based CS for up to 274 genes. Detection rates were compared with literature-reported values derived from disease prevalence and carrier frequencies. Combined theoretical affected pregnancy rates for the 274 screened disorders were calculated. ResultsFor Ashkenazi Jewish (AJ) diseases, 81.6% (4434/5435) of carriers identified did not report AJ ancestry. For cystic fibrosis, 44.0% (6260/14,229)of carriers identified had a variant not on the standard genotyping panel. Individuals at risk of being a silent spinal muscular atrophy carrier, not detectable by standard screening, comprised 1/39 (8763/344,407) individuals. For fragile X syndrome, compared with standard premutation screening, AGG interruption analysis modified risk in 83.2% (1128/1356) premutation carriers. Assuming random pairing across the study population, approximately 1/175 pregnancies would be affected by a disorder in the 274-gene screening panel. ConclusionCompared with standard screening, NGS-based CS provides additional information that may impact reproductive choices. Pan-ethnic CS leads to substantially increased identification of at-risk couples. These data support offering NGS-based CS to all reproductive-aged women.

Sources of resistance to black aphid in cowpea varieties used as green grains

Evaluation of resistance of Vigna unguiculata L. landraces, used as green beans, to Aphis craccivora Koch was tested. Eleven landraces and standard genotypes were used in a free choice protocol: BR 17-Gurguéia, VITA 7 (susceptible), BRS Guariba and TVu 408P2 (resistant). A completely randomized blocks design was used with six replicates. Seedlings were infested with five six-day-old adult insects. The number of alive adults after 48 h and of alive nymphs after 96 h was evaluated. The results evidenced that there are high genetic diversity among the landraces, the antibiosis as the main mechanism of resistance. By means of multivariate analysis we suggest crosses among the commercial cultivar BRS Guariba and the most dissimilar varieties CCE-088, CCE-075, CCE-013 and CCE-062 to obtain superior hybrids.

Sources of resistance to black aphid in cowpea varieties used as green grains

Evaluation of resistance of Vigna unguiculata L. landraces, used as green beans, to Aphis craccivora Koch was tested. Eleven landraces and standard genotypes were used in a free choice protocol: BR 17-Gurguéia, VITA 7 (susceptible), BRS Guariba and TVu 408P2 (resistant). A completely randomized blocks design was used with six replicates. Seedlings were infested with five six-day-old adult insects. The number of alive adults after 48 h and of alive nymphs after 96 h was evaluated. The results evidenced that there are high genetic diversity among the landraces, the antibiosis as the main mechanism of resistance. By means of multivariate analysis we suggest crosses among the commercial cultivar BRS Guariba and the most dissimilar varieties CCE-088, CCE-075, CCE-013 and CCE-062 to obtain superior hybrids.

Molecular Characterization of African Swine Fever Virus Isolates in Estonia in 2014-2019.

After the extensive spread of the African swine fever virus (ASFV) genotype II in Eastern Europe, the first case of African swine fever (ASF) in Estonia was diagnosed in September 2014. By the end of 2019, 3971 ASFV-positive wild boars were found, and 27 domestic pig outbreaks were reported. A selection of ASFV isolates from wild boar and domestic pigs (during the period of September 2014–2019) was molecularly characterized using standardized genotyping procedures. One of the proven markers to characterize this virus is the central variable region (CVR) within the B602L gene. In summer 2015, a new ASFV genotype II CVR variant 2 (GII-CVR2) was confirmed in Estonia. The results suggest that the GII-CVR2 variant was only confirmed in wild boar from a limited area in southern Estonia in 2015 and 2016. In addition to GII-CVR2, a single nucleotide polymorphism (SNP) that resulted in amino acid change was identified within the genotype II CVR variant 1 (GII-CVR1). The GII-CVR1/SNP1 strain was isolated in Estonia in November 2016. Additional GII-CVR1/SNP1 cases were confirmed in two neighbouring counties, as well as in one outbreak farm in June 2017. Based on the available data, no GII-CVR2 and GII-CVR1/SNP1 have been reported by other affected European countries. The spread of variant strains in Estonia has been limited over time, and restricted to a relatively small area.

Evaluation of PCR-based fingerprinting comparatively to the RFLP-PFGE for discrimination of Salmonella sp. isolated from slaughtered pork

Effective epidemiological surveillance and control of Salmonella sp. requires accurate and expeditious genetic typing methods. In the present study, rapid PCR-based methods (ERIC-PCR, M13-PCR and RAPDs) were applied to 73 Salmonella sp. isolates, and the results compared with those previously obtained by RFLP-PFGE (Salmonella gold standard genotyping method), in order to evaluate their discriminatory ability. Results were very diverse among the primers used and, for each primer, the performance level was variable among the different serotypes. ERIC-PCR and RAPD with OPC19 was inefficient for Salmonella sp. discrimination beyond the serotype level. In opposite, M13-PCR, OPC15-RAPD and OPB17-RAPD allowed intraserotype discrimination that, in general, were less discriminative than RFLP-PFGE, indicating that should not be used as a unique typing method in epidemiological studies. Nevertheless, in particular situations, these PCR methods, which are faster and less expensive than RFLP-PFGE, could offers an attractive choice as a preliminary screening of the isolates to reduce the number of suspicious isolates that should be subsequently typed with a more discriminative and accurate methods such as RFLP-PFGE.

Ionizing Radiation Alters the Transition/Transversion Ratio in the Exome of Human Gingiva Fibroblasts.

Little is known about the mutational impact of ionizing radiation (IR) exposure on a genome-wide level in mammalian tissues. Recent advancements in sequencing technology have provided powerful tools to perform exome-wide analyses of genetic variation. This also opened up new avenues for studying and characterizing global genomic IR-induced effects. However, genotypes generated by next generation sequencing (NGS) studies can contain errors, which may significantly impact the power to detect signals in common and rare variant analyses. These genotyping errors are not explicitly detected by the standard Genotype Analysis ToolKit (GATK) and Variant Quality Score Recalibration (VQSR) tool and thus remain a potential source of false-positive variants in whole exome sequencing (WES) datasets. In this context, the transition-transversion ratio (Ti/Tv) is commonly used as an additional quality check. In case of IR experiments, this is problematic when Ti/Tv itself might be influenced by IR treatment. It was the aim of this study to determine a suitable threshold for variant filters for NGS datasets from irradiated cells in order to achieve high data quality using Ti/Tv, while at the same time being able to investigate radiation-specific effects on the Ti/Tv ratio for different radiation doses. By testing a variety of filter settings and comparing the obtained results with publicly available datasets, we observe that a coverage filter setting of depth (DP) 3 and genotype quality (GQ) 20 is sufficient for high quality single nucleotide variants (SNVs) calling in an analysis combining GATK and VSQR and that Ti/Tv values are a consistent and useful indicator for data quality assessment for all tested NGS platforms. Furthermore, we report a reduction in Ti/Tv in IR-induced mutations in primary human gingiva fibroblasts (HGFs), which points to an elevated proportion of transversions among IR-induced SNVs and thus might imply that mismatch repair (MMR) plays a role in the cellular damage response to IR-induced DNA lesions.

SUPERIOR LINES FOR AGRO-TECHNOLOGICAL TRAITS IN SAFFLOWER (Carthamus tinctorius L.)

In safflower (Carthamus tinctorius L.) breeding, determining superior pure lines after crossing for agro-technological traits is the first step to improve new cultivar candidates. Therefore, aim of the study was to determine superior pure lines of sufflower in F9-10 derived three intraspecific crosses evaluating the agronomical and technological (quality) traits. A total of 25 safflower genotypes including 15 pure lines was compared with 10 standard cultivars (Dincer 5-18-1, Centennial, Remzibey-05, Balci, Linas, Olas, Gelendost-2, Gokturk, Montola 2000 and W6 9822) and genotypes along with parents of lines for two years in 2017-2018. GEMO-20 had higher 1000-seed weight, GECE-8 and GEMO-20 had higher oil content, GEMO-19, 28 and GEW6-7 had higher oil yield, GEMO-28 and GEW6-7 had higher seed yield than standard cultivars. According to 1000-seed weight, oil content, seed and oil yield traits, GEMO-19, GEMO-20, GEMO-28 and GEW6-7 lines were identified as superior lines.

Interleukin IL-1B gene polymorphism in Tunisian patients with chronic hepatitis B infection: Association with replication levels.

Approaches based on association studies have proven useful in identifying genetic predictors for many diseases, including susceptibility to chronic hepatitis B. In this study we were interested by the IL-1B genetic variants that have been involved in the immune response and we analyzed their role in the susceptibility to develop chronic hepatitis B in the Tunisian population. IL-1B is a potent proinflammatory cytokine that plays an important role in inflammation of the liver. Polymorphic gene IL-1 (-511, +3954) was analyzed in a total of 476 individuals: 236 patients with chronic hepatitis B from different cities of Tunisia recruited in Pasteur Institute between January 2017 and December 2018 and 240 controls. Genomic DNA was obtained using the standard salting-out method and genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. For -511C>T polymorphism a significant association was found between patients and controls when comparing the genotypic (P = 0.007; χ2 = 9.74 and odds ratio [OR] = 0.60; confidence interval [CI] = 0.41-0.89) and allelic (P = 0.001; χ2 = 10.60) frequencies. When the viral load was taken into account a highly significant difference was found (P = 9 × 10-4 ; χ2 = 10.89). For +3954C>T polymorphism a significant association was found between patients and controls when comparing genotypic (P = 0.0058; χ2 = 7.60 and OR = 1.67; CI = 1.14-2.46) and allelic (P = 0.0029; χ2 = 8.81) frequencies. T allele can be used as a strong marker for hepatitis B virus disease for both polymorphisms.

Effect of Feed Restriction on the Behaviour and Welfare of Broiler Chickens.

Simple SummaryCommercial genotypes of broiler chickens reared for meat purposes in intensive rearing systems are selected for fast growth and high breast yield and kept under conditions that may negatively affect normal behavioural patterns and reduce activity levels. Besides, quantitative feed restriction, used in the field to manipulate chicken growth and improve feed efficiency, may produce hunger and abnormal behaviours. Under these conditions, health and welfare are at risk. Thus, the present study aimed to evaluate the effect of genotype, sex and feed restriction on the welfare of broiler chickens by examining behaviour and corticosterone as a stress indicator during the production cycle. Results showed that behavioural differences between two fast-growing genotypes were limited in quantity and quality, whereas sex and age played a major role. The effect of sex was related especially to size so that heavier males were more active at earlier ages but moved less towards the end of the trial when their walking ability/comfort decreased. Feed restriction stimulated activity during and beyond the restriction phase without relevant effect on stress condition as measured by the plasma and faeces corticosterone.Under intensive rearing conditions, the welfare of broiler chickens may be at risk depending on genotype and sex, due to their different growth rates. The practice of quantitative feed restriction may also impact on welfare. This study aimed to evaluate behaviour and corticosterone content in plasma and faeces at different ages using 896 one-day-old chicks housed in 32 pens, allocated to 8 groups, i.e., 2 genotypes (standard vs. high breast yield) × 2 sex × 2 feeding plans (ad libitum vs. restricted, AL vs. FR). The feeding system affected the percentage of standing (9.84% vs. 11.7% in AL vs. FR; p ≤ 0.001), feeding (7.51% vs. 8.17%; p ≤ 0.01) and sitting/lying (67.0% vs. 64.1%; p ≤ 0.001), and the faeces corticosterone content (12.2 vs. 13.6 ng/g in AL vs. FR; p ≤ 0.10). Sex affected the percentage of pecking other chickens, standing and comfort behaviours. Changes in behaviour were recorded between high and standard breast yield genotypes with faeces corticosterone which tended to be higher in the former (p ≤ 0.10). Significant interactions between the main factors and age were observed. Major changes in behaviour were due to feed restriction, which stimulated activity during restriction.

High discrimination of Mycobacterium bovis isolates in Brazilian herds by spoligotyping

Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis (M. bovis), that leads to economic losses in infected herds and it is also considered an important zoonosis. The molecular typing methods of M. bovis isolates are fundamental for the bovine tuberculosis surveillance system, and spoligotyping is the standard genotyping technique for this species. Thus, the aim of the present study is to analyze the spatial and cluster distribution of M. bovis strains from several regions of Brazil through molecular typing. Spoligotyping technique was applied on 422 isolates identified as M. bovis, and Ripley's K function was used to perform the spatial and cluster analysis of each identified profile. Forty-three (43) different profiles were identified and spoligotype SB0121 was the most frequent and showed a uniform pattern in the spatial distribution while spoligotypes SB0295, SB1380 and SB1050 formed clusters. In addition, three novel spoligotype profiles (SB2361, SB2362, SB2364) were identified in different herds. In this perspective, it is believed that molecular identification and typing can significantly improve the performance of surveillance systems for bovine tuberculosis in Brazil.