Supplementation of honey bee production colonies with a native beneficial microbe mixture.

Honey bee colonies form a complex superorganism, with individual and social immune defences that control overall colony health. Sometimes these defences are not enough to overcome infections by parasites and pathogens. For that reason, several studies have been conducted to evaluate different strategies to improve honey bee health. A novel alternative that is being studied is the use of beneficial microbes. In a previous study, we isolated and characterised bacterial strains from the native gut microbiota of honey bees. Four Apilactobacillus kunkeei strains were mixed and administered in laboratory models to evaluate their potential beneficial effect on larvae and adult bees. This beneficial microbe mixture was safe; it did not affect the expression of immune-related genes, and it was able to decrease the mortality caused by Paenibacillus larvae infection in larvae and reduced the Nosema ceranae spore number in infected adult honey bees. In the present study, we aimed to delve into the impact of the administration of this beneficial microbe mixture on honey bee colonies, under field conditions. The mixture was administered in sugar syrup using lyophilised bacterial cells or fresh cultures, by aspersion or sprayed and feeder, once a week for three consecutive weeks, in autumn or spring 2015, 2017 and 2019. Colony strength parameters were estimated before the administration, and one and three months later. Simultaneously different samples were collected to evaluate the infection levels of parasites and pathogens. The results showed that administering the beneficial microbe mixture decreased or stabilised the infection by N. ceranae or Varroa destructor in some trials but not in others. However, it failed to improve the colony's strength parameters or honey production. Therefore, field studies can be a game-changer when beneficial microbes for honey bees are tested, and meticulous studies should be performed to test their effectiveness.

Impact of Nutritional Stress on Honeybee Gut Microbiota, Immunity, and Nosema ceranae Infection.

Honeybees are important pollinators, having an essential role in the ecology of natural and agricultural environments. Honeybee colony losses episodes reported worldwide and have been associated with different pests and pathogens, pesticide exposure, and nutritional stress. This nutritional stress is related to the increase in monoculture areas which leads to a reduction of pollen availability and diversity. In this study, we examined whether nutritional stress affects honeybee gut microbiota, bee immunity, and infection by Nosema ceranae, under laboratory conditions. Consumption of Eucalyptus grandis pollen was used as a nutritionally poor-quality diet to study nutritional stress, in contraposition to the consumption of polyfloral pollen. Honeybees feed with Eucalyptus grandis pollen showed a lower abundance of Lactobacillus mellifer and Lactobacillus apis (Firm-4 and Firm-5, respectively) and Bifidobacterium spp. and a higher abundance of Bartonella apis, than honeybees fed with polyfloral pollen. Besides the impact of nutritional stress on honeybee microbiota, it also decreased the expression levels of vitellogenin and genes associated to immunity (glucose oxidase, hymenoptaecin and lysozyme). Finally, Eucalyptus grandis pollen favored the multiplication of Nosema ceranae. These results show that nutritional stress impacts the honeybee gut microbiota, having consequences on honeybee immunity and pathogen development. Those results may be useful to understand the influence of modern agriculture on honeybee health.

Impact of nutritional stress on the honeybee colony health.

Honeybees Apis mellifera are important pollinators of wild plants and commercial crops. For more than a decade, high percentages of honeybee colony losses have been reported worldwide. Nutritional stress due to habitat depletion, infection by different pests and pathogens and pesticide exposure has been proposed as the major causes. In this study we analyzed how nutritional stress affects colony strength and health. Two groups of colonies were set in a Eucalyptus grandis plantation at the beginning of the flowering period (autumn), replicating a natural scenario with a nutritionally poor food source. While both groups of colonies had access to the pollen available in this plantation, one was supplemented with a polyfloral pollen patty during the entire flowering period. In the short-term, colonies under nutritional stress (which consumed mainly E. grandis pollen) showed higher infection level with Nosema spp. and lower brood and adult bee population, compared to supplemented colonies. On the other hand, these supplemented colonies showed higher infection level with RNA viruses although infection levels were low compared to countries were viral infections have negative impacts. Nutritional stress also had long-term colony effects, because bee population did not recover in spring, as in supplemented colonies did. In conclusion, nutritional stress and Nosema spp. infection had a severe impact on colony strength with consequences in both short and long-term.

Lactobacillus kunkeei strains decreased the infection by honey bee pathogens Paenibacillus larvae and Nosema ceranae.

Due to their social behaviour, honey bees can be infected by a wide range of pathogens including the microsporidia Nosema ceranae and the bacteria Paenibacillus larvae. The use of probiotics as food additives for the control or prevention of infectious diseases is a widely used approach to improve human and animal health. In this work, we generated a mixture of four Lactobacillus kunkeei strains isolated from the gut microbial community of bees, and evaluated its potential beneficial effect on larvae and adult bees. Its administration in controlled laboratory models was safe for larvae and bees; it did not affect the expression of immune-related genes and it was able to decrease the mortality associated to P. larvae infection in larvae and the counts of N. ceranae spores from adult honey bees. These promising results suggest that this beneficial microorganism's mixture may be an attractive strategy to improve bee health. Field studies are being carried out to evaluate its effect in naturally infected colonies.

Milk fatty acid profile from grazing buffaloes fed a blend of soybean and linseed oils / Perfil de ácidos graxos do leite de búfalas a pasto recebendo uma mistura de óleo de soja e linhaça na dieta

The aim of the study was to examine the changes in milk fatty acid (FA) profile of grazing buffaloes fed either low (L, 276g/d) or high (H, 572g/d) doses of a blend (70:30, wt/wt) of soybean and linseed oils. Fourteen multiparous Mediterranean buffaloes grazing on a native pasture were fed 4 kg/day of a commercial concentrate containing no supplemental oil over a pre-experimental period of ten days. The baseline milk production and composition and milk FA profile were measured over the last three days. After this pre-experimental period the animals received the same concentrate added with either the L or H oil doses for 26 additional days. Milk yield (g/animal/day) did not differ at the start (1776 ± 522 and 1662 ± 291 for L and H, respectively, P<0.622) or at the end of the trial (4590 ± 991 and 4847 ± 447 in L and H, respectively, P<0.543). Baseline milk fat content (g/kg) averaged 77.1 (±20.5) in L and 74.3 (±9.9) in H (P<0.10) and was reduced (P<0.031) to 60.7 (±23.6) and 49.4 (±11.2) (P<0.0031) respectively after L and H with no differences between treatments (P<0.277). Baseline milk protein content (L=43.2 ± 3.4 and H= 44.3 ± 6.9g/kg) increased after oil supplementation (P<0.0001) in both L (73.2 ± 6.0g/kg) and H (68.4 ± 4.9g/kg) without differences between oil doses (P<0.123). Milk fat content of 14:0 decreased after oil supplementation only in the H treatment (5.29 to 4.03, P<0.007) whereas that of 16:0 was reduced (P<0.001) at both L (24.49 to 19.75g/100g FA) and H (25.92 to 19.17g/100g FA) doses. The reduction of total content of 12:0 to 16:0 was higher (P<0.052) in H (32.02 to 23.93g/100g FA) than L (30.17 to 25.45g/100g FA). Vaccenic acid content increased (P<0.001) from 5.70 to 13.24g/100g FA in L and from 5.25 to 16.77 in H, with higher results in the in H treatment (P<0.001). Baseline rumenic acid was sharply increased (P<0.001) in L (1.80 to 4.09g/100g FA, +127%) and H (1.60 to 4.61g/100g FA, +187%) with no differences between [...](AU)

O objetivo do presente estudo foi avaliar as mudanças no perfil de ácidos graxos do leite de búfalas leiteiras recebendo baixas (B, 276g/d) ou altas (A, 572g/d) doses de uma mistura de óleos de soja e linhaça (70:30, peso/peso) na dieta. Quatorze búfalas multíparas da raça Mediterrânea, mantidas em pastagens nativas, receberam 4kg/dia de um concentrado comercial sem adição de óleo (pré-tratamento) ao longo de umperíodopré-experimental de 10 dias. A produção de leiteindividual e amostras de leite foram coletadas individualmente para determinação dos valores basais de composição e perfil de ácidos graxos do leite nos últimos trêsdias. Após este período, os animais receberam o mesmo concentrado adicionado deBou Apor 26 dias. A produção de leite (g/animal/dia) não diferiu no início (1776 ± 522 e 1662 ± 291para B e A, respectivamente (P<0,622) e no final do período experimental(4590 ±991e4847 ± 447 para LeH, respectivamente, P<0,543). O teor de gordura do leite (g/100g) apresentou valores médios de 77,1(±20,5)paraBe74,3 (±9,9)paraA(P<0,10) durante o período pré-tratamento,mas foi reduzido (P<0,03) após o fornecimento das dietas com óleo para 60,7 (± 23,6) e 49,4 (± 11,2), respectivamente para B e A, não havendo diferenças entre tratamentos (P<0,277). Os teores basais de proteína do leite (B=43,2 ± 3,4 e A=44,3 ± 6,9g/kg) aumentaram após a suplementação com óleo (P<0,0001) em ambos B (73,2 ± 6,0g/kg) e A (68,4 ± 4,9g/kg), não ocorrendo diferenças entre tratamentos (P<0,123). O teor médio basal de 14:0 na gordura do leite (4,76g/100g AG) foi reduzido após a suplementação da dieta com óleo somente no tratamento A (5,29 para 4,03, P<0,007). O teor de 16:0 na gordura do leite foi reduzido (P<0,001) nos tratamentos B (24,49 para 19,75g/100g AG) e A (25,92 para 19,17g/100g AG). A redução nos teores de 12:0+14:0+16:0 na gordura do leite foi maior (P<0,052) em A (32,02 para 23,93g/100g AG) do que em B (30,17 para 25,45g/100g AG). O teor de ácido vacênico (AV) [...](AU)

Milk fatty acid profile from grazing buffaloes fed a blend of soybean and linseed oils / Perfil de ácidos graxos do leite de búfalas a pasto recebendo uma mistura de óleo de soja e linhaça na dieta

The aim of the study was to examine the changes in milk fatty acid (FA) profile of grazing buffaloes fed either low (L, 276g/d) or high (H, 572g/d) doses of a blend (70:30, wt/wt) of soybean and linseed oils. Fourteen multiparous Mediterranean buffaloes grazing on a native pasture were fed 4 kg/day of a commercial concentrate containing no supplemental oil over a pre-experimental period of ten days. The baseline milk production and composition and milk FA profile were measured over the last three days. After this pre-experimental period the animals received the same concentrate added with either the L or H oil doses for 26 additional days. Milk yield (g/animal/day) did not differ at the start (1776 ± 522 and 1662 ± 291 for L and H, respectively, P<0.622) or at the end of the trial (4590 ± 991 and 4847 ± 447 in L and H, respectively, P<0.543). Baseline milk fat content (g/kg) averaged 77.1 (±20.5) in L and 74.3 (±9.9) in H (P<0.10) and was reduced (P<0.031) to 60.7 (±23.6) and 49.4 (±11.2) (P<0.0031) respectively after L and H with no differences between treatments (P<0.277). Baseline milk protein content (L=43.2 ± 3.4 and H= 44.3 ± 6.9g/kg) increased after oil supplementation (P<0.0001) in both L (73.2 ± 6.0g/kg) and H (68.4 ± 4.9g/kg) without differences between oil doses (P<0.123). Milk fat content of 14:0 decreased after oil supplementation only in the H treatment (5.29 to 4.03, P<0.007) whereas that of 16:0 was reduced (P<0.001) at both L (24.49 to 19.75g/100g FA) and H (25.92 to 19.17g/100g FA) doses. The reduction of total content of 12:0 to 16:0 was higher (P<0.052) in H (32.02 to 23.93g/100g FA) than L (30.17 to 25.45g/100g FA). Vaccenic acid content increased (P<0.001) from 5.70 to 13.24g/100g FA in L and from 5.25 to 16.77 in H, with higher results in the in H treatment (P<0.001). Baseline rumenic acid was sharply increased (P<0.001) in L (1.80 to 4.09g/100g FA, +127%) and H (1.60 to 4.61g/100g FA, +187%) with no differences between...

O objetivo do presente estudo foi avaliar as mudanças no perfil de ácidos graxos do leite de búfalas leiteiras recebendo baixas (B, 276g/d) ou altas (A, 572g/d) doses de uma mistura de óleos de soja e linhaça (70:30, peso/peso) na dieta. Quatorze búfalas multíparas da raça Mediterrânea, mantidas em pastagens nativas, receberam 4kg/dia de um concentrado comercial sem adição de óleo (pré-tratamento) ao longo de umperíodopré-experimental de 10 dias. A produção de leiteindividual e amostras de leite foram coletadas individualmente para determinação dos valores basais de composição e perfil de ácidos graxos do leite nos últimos trêsdias. Após este período, os animais receberam o mesmo concentrado adicionado deBou Apor 26 dias. A produção de leite (g/animal/dia) não diferiu no início (1776 ± 522 e 1662 ± 291para B e A, respectivamente (P<0,622) e no final do período experimental(4590 ±991e4847 ± 447 para LeH, respectivamente, P<0,543). O teor de gordura do leite (g/100g) apresentou valores médios de 77,1(±20,5)paraBe74,3 (±9,9)paraA(P<0,10) durante o período pré-tratamento,mas foi reduzido (P<0,03) após o fornecimento das dietas com óleo para 60,7 (± 23,6) e 49,4 (± 11,2), respectivamente para B e A, não havendo diferenças entre tratamentos (P<0,277). Os teores basais de proteína do leite (B=43,2 ± 3,4 e A=44,3 ± 6,9g/kg) aumentaram após a suplementação com óleo (P<0,0001) em ambos B (73,2 ± 6,0g/kg) e A (68,4 ± 4,9g/kg), não ocorrendo diferenças entre tratamentos (P<0,123). O teor médio basal de 14:0 na gordura do leite (4,76g/100g AG) foi reduzido após a suplementação da dieta com óleo somente no tratamento A (5,29 para 4,03, P<0,007). O teor de 16:0 na gordura do leite foi reduzido (P<0,001) nos tratamentos B (24,49 para 19,75g/100g AG) e A (25,92 para 19,17g/100g AG). A redução nos teores de 12:0+14:0+16:0 na gordura do leite foi maior (P<0,052) em A (32,02 para 23,93g/100g AG) do que em B (30,17 para 25,45g/100g AG). O teor de ácido vacênico (AV)...

Worldwide HLA-E nucleotide and haplotype variability reveals a conserved gene for coding and 3' untranslated regions.

The human leukocyte antigen-E (HLA-E) locus is a human major histocompatibility complex (MHC) gene associated with immune-modulation and suppression of the immune response by the interaction with specific natural killer (NK) and T cell receptors (TCRs). It is considered one of the most conserved genes of the human MHC; however, this low nucleotide variability seems to be a consequence of the scarce number of studies focusing on this subject. In this manuscript we assessed the nucleotide variability at the HLA-E coding and 3' untranslated regions (3'UTRs) in Brazil and in the populations from the 1000Genomes Consortium. Twenty-eight variable sites arranged into 33 haplotypes were detected and most of these haplotypes (98.2%) are encoding one of the two HLA-E molecules found worldwide, E*01:01 and E*01:03. Moreover, three worldwide spread haplotypes, associated with the coding alleles E*01:01:01, E*01:03:01 and E*01:03:02, account for 85% of all HLA-E haplotypes, suggesting that they arose early before human speciation. In addition, the low nucleotide diversity found for the HLA-E coding and 3'UTR in worldwide populations suggests that the HLA-E gene is in fact a conserved gene, which might be a consequence of its key role in the modulation of the immune system.

A nonsynonymous mutation at HLA-E defines the new E*01:06 allele in Brazilian individuals.

We report a novel nonclassical class I HLA-E*01:06 allele observed in Brazilian individuals.

A novel HLA-E allele, E*01:03:05, identified in two Brazilian individuals.

Here, we report a novel non-classical class I HLA-E allele found in the Brazilian population.

Identification of two new HLA-G alleles, G*01:01:03:03 and G*01:01:21, in Brazilian individuals.

Here we report two new non-classical class I HLA-G alleles found in the Brazilian population.

Non-classical HLA-E gene variability in Brazilians: a nearly invariable locus surrounded by the most variable genes in the human genome.

The non-classical human leukocyte antigen (HLA) class I genes present a very low rate of variation. So far, only 10 HLA-E alleles encoding three proteins have been described, but only two are frequently found in worldwide populations. Because of its historical background, Brazilians are very suitable for population genetic studies. Therefore, 104 bone marrow donors from Brazil were evaluated for HLA-E exons 1-4. Seven variation sites were found, including two known single nucleotide polymorphisms (SNPs) at positions +424 and +756 and five new SNPs at positions +170 (intron 1), +1294 (intron 3), +1625, +1645 and +1857 (exon 4). Haplotyping analysis did show eight haplotypes, three of them known as E*01:01:01, E*01:03:01 and E*01:03:02:01 and five HLA-E new alleles that carry the new variation sites. The HLA-E*01:01:01 allele was the predominant haplotype (62.50%), followed by E*01:03:02:01 (24.52%). Selective neutrality tests have disclosed an interesting pattern of selective pressures in which balancing selection is probably shaping allele frequency distributions at an SNP at exon 3 (codon 107), sequence diversity at exon 4 and the non-coding regions is facing significant purifying pressure. Even in an admixed population such as the Brazilian one, the HLA-E locus is very conserved, presenting few polymorphic SNPs in the coding region.

Genomic alterations detected by comparative genomic hybridization in ovarian endometriomas.

Endometriosis is a complex and multifactorial disease. Chromosomal imbalance screening in endometriotic tissue can be used to detect hot-spot regions in the search for a possible genetic marker for endometriosis. The objective of the present study was to detect chromosomal imbalances by comparative genomic hybridization (CGH) in ectopic tissue samples from ovarian endometriomas and eutopic tissue from the same patients. We evaluated 10 ovarian endometriotic tissues and 10 eutopic endometrial tissues by metaphase CGH. CGH was prepared with normal and test DNA enzymatically digested, ligated to adaptors and amplified by PCR. A second PCR was performed for DNA labeling. Equal amounts of both normal and test-labeled DNA were hybridized in human normal metaphases. The Isis FISH Imaging System V 5.0 software was used for chromosome analysis. In both eutopic and ectopic groups, 4/10 samples presented chromosomal alterations, mainly chromosomal gains. CGH identified 11q12.3-q13.1, 17p11.1-p12, 17q25.3-qter, and 19p as critical regions. Genomic imbalances in 11q, 17p, 17q, and 19p were detected in normal eutopic and/or ectopic endometrium from women with ovarian endometriosis. These regions contain genes such as POLR2G, MXRA7 and UBA52 involved in biological processes that may lead to the establishment and maintenance of endometriotic implants. This genomic imbalance may affect genes in which dysregulation impacts both eutopic and ectopic endometrium.

Genomic alterations detected by comparative genomic hybridization in ovarian endometriomas

Endometriosis is a complex and multifactorial disease. Chromosomal imbalance screening in endometriotic tissue can be used to detect hot-spot regions in the search for a possible genetic marker for endometriosis. The objective of the present study was to detect chromosomal imbalances by comparative genomic hybridization (CGH) in ectopic tissue samples from ovarian endometriomas and eutopic tissue from the same patients. We evaluated 10 ovarian endometriotic tissues and 10 eutopic endometrial tissues by metaphase CGH. CGH was prepared with normal and test DNA enzymatically digested, ligated to adaptors and amplified by PCR. A second PCR was performed for DNA labeling. Equal amounts of both normal and test-labeled DNA were hybridized in human normal metaphases. The Isis FISH Imaging System V 5.0 software was used for chromosome analysis. In both eutopic and ectopic groups, 4/10 samples presented chromosomal alterations, mainly chromosomal gains. CGH identified 11q12.3-q13.1, 17p11.1-p12, 17q25.3-qter, and 19p as critical regions. Genomic imbalances in 11q, 17p, 17q, and 19p were detected in normal eutopic and/or ectopic endometrium from women with ovarian endometriosis. These regions contain genes such as POLR2G, MXRA7 and UBA52 involved in biological processes that may lead to the establishment and maintenance of endometriotic implants. This genomic imbalance may affect genes in which dysregulation impacts both eutopic and ectopic endometrium.

Evaluation of computational methods for the reconstruction of HLA haplotypes.

Human leukocyte antigen (HLA) haplotypes are frequently evaluated for population history inferences and association studies. However, the available typing techniques for the main HLA loci usually do not allow the determination of the allele phase and the constitution of a haplotype, which may be obtained by a very time-consuming and expensive family-based segregation study. Without the family-based study, computational inference by probabilistic models is necessary to obtain haplotypes. Several authors have used the expectation-maximization (EM) algorithm to determine HLA haplotypes, but high levels of erroneous inferences are expected because of the genetic distance among the main HLA loci and the presence of several recombination hotspots. In order to evaluate the efficiency of computational inference methods, 763 unrelated individuals stratified into three different datasets had their haplotypes manually defined in a family-based study of HLA-A, -B, -DRB1 and -DQB1 segregation, and these haplotypes were compared with the data obtained by the following three methods: the Expectation-Maximization (EM) and Excoffier-Laval-Balding (ELB) algorithms using the arlequin 3.11 software, and the PHASE method. When comparing the methods, we observed that all algorithms showed a poor performance for haplotype reconstruction with distant loci, estimating incorrect haplotypes for 38%-57% of the samples considering all algorithms and datasets. We suggest that computational haplotype inferences involving low-resolution HLA-A, HLA-B, HLA-DRB1 and HLA-DQB1 haplotypes should be considered with caution.