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Animal African trypanosomosis, caused by blood protozoan parasites transmitted mainly by tsetse flies, represents a major constraint for millions of cattle in sub-Saharan Africa. Exposed cattle include trypanosusceptible indicine breeds, severely affected by the disease, and West African taurine breeds called trypanotolerant owing to their ability to control parasite development, survive and grow in enzootic areas. Until now the genetic basis of trypanotolerance remains unclear. Here, to improve knowledge of the biological processes involved in trypanotolerance versus trypanosusceptibility, we identified bovine genes differentially expressed in five West African cattle breeds during an experimental infection by Trypanosoma congolense and their biological functions. To this end, whole blood genome-wide transcriptome of three trypanotolerant taurine breeds (N'Dama, Lagune and Baoulé), one susceptible zebu (Zebu Fulani) and one African taurine x zebu admixed breed (Borgou) were profiled by RNA sequencing at four time points, one before and three during infection. As expected, infection had a major impact on cattle blood transcriptome regardless of the breed. The functional analysis of differentially expressed genes over time in each breed confirmed an early activation of the innate immune response, followed by an activation of the humoral response and an inhibition of T cell functions at the chronic stage of infection. More importantly, we highlighted overlooked features, such as a strong disturbance in host metabolism and cellular energy production that differentiates trypanotolerant and trypanosusceptible breeds. N'Dama breed showed the earliest regulation of immune response, associated with a strong activation of cellular energy production, also observed in Lagune, and to a lesser extent in Baoulé. Susceptible Zebu Fulani breed differed from other breeds by the strongest modification in lipid metabolism regulation. Overall, this study provides a better understanding

Macrophages are major cells of the innate immunity. Macrophages derived from monocyte precursors undergo specific differentiation depending on the local tissue environment. Similar to the T helper type 1 and T helper type 2 polarization, two distinct states of polarized activation for macrophages have been defined in mouse and humans: the classically activated (M1) macrophage and the alternatively activated (M2) macrophage phenotypes. On the other hand, these different patterns of macrophage differentiation drive adaptive responses during the stages of infection, hence restraining inflammation and favoring tissue repair. In vitro generation and characterization of these subpopulations are essential to perform relevant studies understanding the host-pathogen interactions. Currently, several in vitro differentiation and polarization protocols are used to induce M1 or M2 mouse and human macrophages but none have been developed for the bovine species. We developed a method for in vitro differentiation and polarization of bovine macrophages using GM-CSF and IFNg to induce M1, and IL-4 to induce M2 phenotype. We characterized M1/M2 macrophages by specific morphology, production of cytokines (IL-10, IL-12, TNF-a), NO production, and phenotypic markers such as CD206 and TLR2.

Macrophages are major cells of the innate immunity. Macrophages derived from monocyte precursors undergo specific differentiation depending on the local tissue environment. Similar to the T helper type 1 and T helper type 2 polarization, two distinct states of polarized activation for macrophages have been defined in mouse and humans: the classically activated (M1) macrophage and the alternatively activated (M2) macrophage phenotypes. On the other hand, these different patterns of macrophage differentiation drive adaptive responses during the stages of infection, hence restraining inflammation and favoring tissue repair. In vitro generation and characterization of these subpopulations are essential to perform relevant studies understanding the host-pathogen interactions. Currently, several in vitro differentiation and polarization protocols are used to induce M1 or M2 mouse and human macrophages but none have been developed for the bovine species. We developed a method for in vitro differentiation and polarization of bovine macrophages using GM-CSF and IFNg to induce M1, and IL-4 to induce M2 phenotype. We characterized M1/M2 macrophages by specific morphology, production of cytokines (IL-10, IL-12, TNF-a), NO production, and phenotypic markers such as CD206 and TLR2.

Background Today, when more than 60% of animal diseases are zoonotic, understanding their origin and development and identifying protective immune responses in ruminants are major challenges. Robust, efficient and cost-effective tools are preconditions to solve these challenges. Cytokines play a key role in the main mechanisms by which the immune system is balanced in response to infectious pathogens. The cytokine balance has thus become the focus of research to characterize immune response in ruminants. Currently, SYBR Green reverse transcriptase quantitative PCR (RT-qPCR) is the most widely method used to investigate cytokine gene expression in ruminants, but the conditions in which the many assays are carried out vary considerably and need to be properly evaluated. Accordingly, the quantification of gene expression by RT-qPCR requires normalization by multiple reference genes. The objective of the present study was thus to develop an RT-qPCR assay to simultaneously quantify the expression of several cytokines and reference genes in three ruminant species. In this paper, we detail each stage of the experimental protocol, check validation parameters and report assay performances, following MIQE guidelines. Results Ten novel primer sets were designed to quantify five cytokine genes (IL-4, IL-10, IL-12B, IFN-? and TNF-?) and five reference genes (ACTB, GAPDH, H3F3A, PPIA and YWHAZ) in cattle, sheep, and goats. All the primer sets were designed to span exon-exon boundaries and use the same hybridization temperature. Each stage of the RT-qPCR method was detailed; their specificity and efficiency checked, proved and are reported here, demonstrating the reproducibility of our method, which is capable of detecting low levels of cytokine mRNA up to one copy whatever the species. Finally, we checked the stability of candidate reference gene expression, performed absolute quantification of cytokine and reference gene mRNA in whole blood samples and relative expression of cyt

Background Animal African Trypanosomosis particularly affects cattle and dramatically impairs livestock development in sub-Saharan Africa. African Zebu (AFZ) or European taurine breeds usually die of the disease in the absence of treatment, whereas West African taurine breeds (AFT), considered trypanotolerant, are able to control the pathogenic effects of trypanosomosis. Up to now, only one AFT breed, the longhorn N'Dama (NDA), has been largely studied and is considered as the reference trypanotolerant breed. Shorthorn taurine trypanotolerance has never been properly assessed and compared to NDA and AFZ breeds. Methodology/Principal Findings This study compared the trypanotolerant/susceptible phenotype of five West African local breeds that differ in their demographic history. Thirty-six individuals belonging to the longhorn taurine NDA breed, two shorthorn taurine Lagune (LAG) and Baoulé (BAO) breeds, the Zebu Fulani (ZFU) and the Borgou (BOR), an admixed breed between AFT and AFZ, were infected by Trypanosoma congolense IL1180. All the cattle were genetically characterized using dense SNP markers, and parameters linked to parasitaemia, anaemia and leukocytes were analysed using synthetic variables and mixed models. We showed that LAG, followed by NDA and BAO, displayed the best control of anaemia. ZFU showed the greatest anaemia and the BOR breed had an intermediate value, as expected from its admixed origin. Large differences in leukocyte counts were also observed, with higher leukocytosis for AFT. Nevertheless, no differences in parasitaemia were found, except a tendency to take longer to display detectable parasites in ZFU. Conclusions We demonstrated that LAG and BAO are as trypanotolerant as NDA. This study highlights the value of shorthorn taurine breeds, which display strong local adaptation to trypanosomosis. Thanks to further analyses based on comparisons of the genome or transcriptome of the breeds, these results open up the way for better knowledge of h

Background Animal African Trypanosomosis particularly affects cattle and dramatically impairs livestock development in sub-Saharan Africa. African Zebu (AFZ) or European taurine breeds usually die of the disease in the absence of treatment, whereas West African taurine breeds (AFT), considered trypanotolerant, are able to control the pathogenic effects of trypanosomosis. Up to now, only one AFT breed, the longhorn N'Dama (NDA), has been largely studied and is considered as the reference trypanotolerant breed. Shorthorn taurine trypanotolerance has never been properly assessed and compared to NDA and AFZ breeds. Methodology/Principal Findings This study compared the trypanotolerant/susceptible phenotype of five West African local breeds that differ in their demographic history. Thirty-six individuals belonging to the longhorn taurine NDA breed, two shorthorn taurine Lagune (LAG) and Baoulé (BAO) breeds, the Zebu Fulani (ZFU) and the Borgou (BOR), an admixed breed between AFT and AFZ, were infected by Trypanosoma congolense IL1180. All the cattle were genetically characterized using dense SNP markers, and parameters linked to parasitaemia, anaemia and leukocytes were analysed using synthetic variables and mixed models. We showed that LAG, followed by NDA and BAO, displayed the best control of anaemia. ZFU showed the greatest anaemia and the BOR breed had an intermediate value, as expected from its admixed origin. Large differences in leukocyte counts were also observed, with higher leukocytosis for AFT. Nevertheless, no differences in parasitaemia were found, except a tendency to take longer to display detectable parasites in ZFU. Conclusions We demonstrated that LAG and BAO are as trypanotolerant as NDA. This study highlights the value of shorthorn taurine breeds, which display strong local adaptation to trypanosomosis. Thanks to further analyses based on comparisons of the genome or transcriptome of the breeds, these results open up the way for better knowledge of h

Background Today, when more than 60% of animal diseases are zoonotic, understanding their origin and development and identifying protective immune responses in ruminants are major challenges. Robust, efficient and cost-effective tools are preconditions to solve these challenges. Cytokines play a key role in the main mechanisms by which the immune system is balanced in response to infectious pathogens. The cytokine balance has thus become the focus of research to characterize immune response in ruminants. Currently, SYBR Green reverse transcriptase quantitative PCR (RT-qPCR) is the most widely method used to investigate cytokine gene expression in ruminants, but the conditions in which the many assays are carried out vary considerably and need to be properly evaluated. Accordingly, the quantification of gene expression by RT-qPCR requires normalization by multiple reference genes. The objective of the present study was thus to develop an RT-qPCR assay to simultaneously quantify the expression of several cytokines and reference genes in three ruminant species. In this paper, we detail each stage of the experimental protocol, check validation parameters and report assay performances, following MIQE guidelines. Results Ten novel primer sets were designed to quantify five cytokine genes (IL-4, IL-10, IL-12B, IFN-? and TNF-?) and five reference genes (ACTB, GAPDH, H3F3A, PPIA and YWHAZ) in cattle, sheep, and goats. All the primer sets were designed to span exon-exon boundaries and use the same hybridization temperature. Each stage of the RT-qPCR method was detailed; their specificity and efficiency checked, proved and are reported here, demonstrating the reproducibility of our method, which is capable of detecting low levels of cytokine mRNA up to one copy whatever the species. Finally, we checked the stability of candidate reference gene expression, performed absolute quantification of cytokine and reference gene mRNA in whole blood samples and relative expression of cyt

La présente invention concerne l'utilisation de l'aprotinine dans le traitement des maladies ou infections parasitaires animales qui affectent les mammifères domestiqués, en particulier le bétail, notamment les bovins. En particulier, l'aprotinine trouve application dans la lutte contre les effets pathogènes induits par les parasites de la famille des Trypanosomatidae, tels que les protozoaires appartenant au genre Trypanosoma ou au genre Leishmania. L'invention concerne également l'utilisation de l'aprotinine pour le pronostic de la tolérance bovine aux infections trypanosomiennes.

Background: The Senepol cattle breed (SEN) was created in the early XXth century from a presumed cross between a European (EUT) breed (Red Poll) and a West African taurine (AFT) breed (N'Dama). Well adapted to tropical conditions, it is also believed trypanotolerant according to its putative AFT ancestry. However, such origins needed to be verified to define relevant husbandry practices and the genetic background underlying such adaptation needed to be characterized. Methodology/Principal Findings: We genotyped 153 SEN individuals on 47,365 SNPs and combined the resulting data with those available on 18 other populations representative of EUT, AFT and Zebu (ZEB) cattle. We found on average 89% EUT, 10.4% ZEB and 0.6% AFT ancestries in the SEN genome. We further looked for footprints of recent selection using standard tests based on the extent of haplotype homozygosity. We underlined i) three footprints on chromosome (BTA) 01, two of which are within or close to the polled locus underlying the absence of horns and ii) one footprint on BTA20 within the slick hair coat locus, involved in thermotolerance. Annotation of these regions allowed us to propose three candidate genes to explain the observed signals (TIAM1, GRIK1 and RAI14). Conclusions/Significance: Our results do not support the accepted concept about the AFT origin of SEN breed. Initial AFT ancestry (if any) might have been counter-selected in early generations due to breeding objectives oriented in particular toward meat production and hornless phenotype. Therefore, SEN animals are likely susceptible to African trypanosomes which questions the importation of SEN within the West African tsetse belt, as promoted by some breeding societies. Besides, our results revealed that SEN breed is predominantly a EUT breed well adapted to tropical conditions and confirmed the importance in thermotolerance of the slick locus.

La présente invention concerne l'utilisation de l'aprotinine dans le traitement des maladies ou infections parasitaires animales qui affectent les mammifères domestiqués, en particulier le bétail, notamment les bovins. En particulier, l'aprotinine trouve application dans la lutte contre les effets pathogènes induits par les parasites de la famille des Trypanosomatidae, tels que les protozoaires appartenant au genre Trypanosoma ou au genre Leishmania. L'invention concerne également l'utilisation de l'aprotinine pour le pronostic de la tolérance bovine aux infections trypanosomiennes.

Background: The Senepol cattle breed (SEN) was created in the early XXth century from a presumed cross between a European (EUT) breed (Red Poll) and a West African taurine (AFT) breed (N'Dama). Well adapted to tropical conditions, it is also believed trypanotolerant according to its putative AFT ancestry. However, such origins needed to be verified to define relevant husbandry practices and the genetic background underlying such adaptation needed to be characterized. Methodology/Principal Findings: We genotyped 153 SEN individuals on 47,365 SNPs and combined the resulting data with those available on 18 other populations representative of EUT, AFT and Zebu (ZEB) cattle. We found on average 89% EUT, 10.4% ZEB and 0.6% AFT ancestries in the SEN genome. We further looked for footprints of recent selection using standard tests based on the extent of haplotype homozygosity. We underlined i) three footprints on chromosome (BTA) 01, two of which are within or close to the polled locus underlying the absence of horns and ii) one footprint on BTA20 within the slick hair coat locus, involved in thermotolerance. Annotation of these regions allowed us to propose three candidate genes to explain the observed signals (TIAM1, GRIK1 and RAI14). Conclusions/Significance: Our results do not support the accepted concept about the AFT origin of SEN breed. Initial AFT ancestry (if any) might have been counter-selected in early generations due to breeding objectives oriented in particular toward meat production and hornless phenotype. Therefore, SEN animals are likely susceptible to African trypanosomes which questions the importation of SEN within the West African tsetse belt, as promoted by some breeding societies. Besides, our results revealed that SEN breed is predominantly a EUT breed well adapted to tropical conditions and confirmed the importance in thermotolerance of the slick locus.

Background: Many tools used to analyze microarrays in different conditions have been described. However, the integration of deregulated genes within coherent metabolic pathways is lacking. Currently no objective selection criterion based on biological functions exists to determine a threshold demonstrating that a gene is indeed differentially expressed. Methodology/Principal Findings: To improve transcriptomic analysis of microarrays, we propose a new statistical approach that takes into account biological parameters. We present an iterative method to optimise the selection of differentially expressed genes in two experimental conditions. The stringency level of gene selection was associated simultaneously with the p-value of expression variation and the occurrence rate parameter associated with the percentage of donors whose transcriptomic profile is similar. Our method intertwines stringency level settings, biological data and a knowledge database to highlight molecular interactions using networks and pathways. Analysis performed during iterations helped us to select the optimal threshold required for the most pertinent selection of differentially expressed genes. Conclusions/Significance: We have applied this approach to the well documented mechanism of human macrophage response to lipopolysaccharide stimulation. We thus verified that our method was able to determine with the highest degree of accuracy the best threshold for selecting genes that are truly differentially expressed.

Background: Many tools used to analyze microarrays in different conditions have been described. However, the integration of deregulated genes within coherent metabolic pathways is lacking. Currently no objective selection criterion based on biological functions exists to determine a threshold demonstrating that a gene is indeed differentially expressed. Methodology/Principal Findings: To improve transcriptomic analysis of microarrays, we propose a new statistical approach that takes into account biological parameters. We present an iterative method to optimise the selection of differentially expressed genes in two experimental conditions. The stringency level of gene selection was associated simultaneously with the p-value of expression variation and the occurrence rate parameter associated with the percentage of donors whose transcriptomic profile is similar. Our method intertwines stringency level settings, biological data and a knowledge database to highlight molecular interactions using networks and pathways. Analysis performed during iterations helped us to select the optimal threshold required for the most pertinent selection of differentially expressed genes. Conclusions/Significance: We have applied this approach to the well documented mechanism of human macrophage response to lipopolysaccharide stimulation. We thus verified that our method was able to determine with the highest degree of accuracy the best threshold for selecting genes that are truly differentially expressed.