Search

Early studies in transgenic mouse lines have shown that the coexpression of endogenous murine prion protein (PrP C ) and transgenic PrP C from another species either inhibits or allows the propagation of prions, depending on the infecting prion strain and interacting protein species. The way whereby this phenomenon, so-called "interference," is modulated remains to be determined. In this study, different transgenic mouse lines were crossbred to produce mice coexpressing bovine and porcine PrP C , bovine and murine PrP C , or murine and porcine PrP C These animals and their respective hemizygous controls were inoculated with several prion strains from different sources (cattle, mice, and pigs) to examine the effects of the simultaneous presence of PrP C from two different species. Our results indicate interference with the infection process, manifested as extended survival times and reduced attack rates. The interference with the infectious process was reduced or absent when the potentiality interfering PrP C species was efficiently converted by the inoculated agent. However, the propagation of the endogenous murine PrP Sc was favored, allowing us to speculate that host-specific factors may disturb the interference caused by the coexpression of an exogenous second PrP C IMPORTANCE Prion propagation can be interfered with by the expression of a second prion protein in the host. In the present study, we investigated prion propagation in a host expressing two different prion protein genes. Our findings indicate that the ability of the second prion protein to interfere with prion propagation is related to the transmissibility of the prion in the host expressing only the interfering prion protein. The interference detected occurs in a prion strain-dependent manner. Interestingly, a bias favoring the propagation of the murine PrP allele has been observed. These results open the door to future studies in order to determine the role of host factors other than the PrP amino acid sequence in the interference in prion propagation., (Copyright © 2021 Espinosa et al.)

Breeding towards genetic resistance to prion disease is effective in eliminating scrapie. In sheep, classical forms of scrapie have been eradicated almost completely in several countries by breeding programs using a prion protein (PrP) gene (PRNP) amino acid polymorphism. For goats, field and experimental studies have provided evidence for several amino acid polymorphisms that are associated with resistance to scrapie, but only limited data are available concerning the susceptibility of caprine PRNP genotypes to BSE. In this study, goat kids representing five PRNP genotypes based on three polymorphisms (M142, Q211 and K222 and the wild type I142, R211 and Q222) were orally challenged with bovine or goat BSE. Wild type goats were killed with clinical signs between 24-28 months post inoculation (mpi) to both challenges, and goats with genotype R/Q211 succumbed between 29-36 mpi. I/M142 goats developed clinical signs at 44-45 mpi and M/M142 goats remained healthy until euthanasia at 48 mpi. None of the Q/K222 goats showed definite clinical signs. Taken together the highest attack ratios were seen in wild type and R/Q211 goats, and the lowest in I/M142, M/M142 and Q/K222. In all genotype groups, one or more goats remained healthy within the incubation period in both challenges and without detectable PrP deposition in the tissues. Our data show that both the K222 and M142 polymorphisms lengthen the incubation period significantly compared to wild type animals, but only K222 was associated with a significant increase in resistance to BSE infection after oral exposure to both BSE sources.

Epsilon toxin is one of the four major toxins of Clostridium perfringens. It is the third most potent clostridial toxin after botulinum and tetanus toxins and is thus considered as a potential biological weapon classified as category B by the Centers for Disease Control and Prevention (CDC). In the case of a bioterrorist attack, there will be a need for a rapid, sensitive and specific detection method to monitor food and water contamination by this toxin, and for a simple human diagnostic test. We have produced and characterized five monoclonal antibodies against common epitopes of epsilon toxin and prototoxin. Three of them neutralize the cytotoxic effects of epsilon toxin in vitro. With these antibodies, we have developed highly sensitive tests, overnight and 4-h sandwich enzyme immunoassays and an immunochromatographic test performed in 20 min, reaching detection limits of at least 5 pg/mL (0.15 pM), 30 pg/mL (0.9 pM) and 100 pg/mL (3.5 pM) in buffer, respectively. These tests were also evaluated for detection of epsilon toxin in different matrices: milk and tap water for biological threat detection, serum, stool and intestinal content for human or veterinary diagnostic purposes. Detection limits in these complex matrices were at least 5-fold better than those described in the literature (around 1 to 5 ng/mL), reaching 10 to 300 pg/mL using the enzyme immunoassay and 100 to 2000 pg/mL using the immunochromatographic test.

Dairy ruminants experiencing a severe postpartum negative energy balance (NEB) are considered to be more susceptible to mastitis. Although the genetic variability of mastitis resistance is well established, the biological basis of the link between energy metabolism and resistance is mostly unknown. The aim of this study was to characterize the effect of NEB on metabolism and immune response according to the genetic background for mastitis resistance or susceptibility. Forty-eight ewes from high and low somatic cell score (SCS) genetic lines were allocated to 2 homogeneous subgroups 2 wk after lambing: one group (NEB) received an energy-restricted diet to cover 60% of their energy requirements, and the other group received a control (positive energy balance: PEB) diet. Both diets met the protein requirements. After 10 d on either the NEB or PEB diet, all ewes were injected with a Pam3CSK4/MDP solution in one half-udder to induce an inflammatory response. The ewes were monitored for milk production, somatic cell count (SCC), body weight (BW), body condition score (BCS), and blood metabolites. Differential milk cell counts were determined by flow cytometry. Plasma concentrations of glucose, insulin, nonesterified fatty acids (NEFA), β-hydroxybutyrate (BHB), and triiodothyronine were determined. Energy restriction resulted in an increased fat:protein ratio in milk and decreased milk yield, BW, and BCS. The NEB ewes had significantly higher NEFA and BHB and lower plasma glucose concentrations than PEB ewes, reflecting a mobilization of body reserves and ketone body synthesis. High-SCS ewes had a higher SCS than low-SCS throughout the experiment, except after the inflammatory challenge, which resulted in similar SCS in all 4 groups. A noteworthy interaction between genetic background and diet was evidenced on metabolic parameters and BW. Indeed, high-SCS ewes subjected to NEB showed greater decrease in BW and increased NEFA and BHB concentrations compared with low-SCS ewes. Thus, NEB in early lactation led to extensive mobilization of body reserves and intense ketone body synthesis in mastitis-susceptible sheep. These results reinforce the hypothesis of a genetic association between mastitis susceptibility and energy metabolism and open the way to further studies on the biological basis for this association., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Mastitis is an infectious disease mainly caused by bacteria invading the mammary gland. Genetic control of susceptibility to mastitis has been widely evidenced in dairy ruminants, but the genetic basis and underlying mechanisms are still largely unknown. We describe the discovery, fine mapping and functional characterization of a genetic variant associated with elevated milk leukocytes count, or SCC, as a proxy for mastitis. After implementing genome-wide association studies, we identified a major QTL associated with SCC on ovine chromosome 3. Fine mapping of the region, using full sequencing with 12X coverage in three animals, provided one strong candidate SNP that mapped to the coding sequence of a highly conserved gene, suppressor of cytokine signalling 2 (Socs2). The frequency of the SNP associated with increased SCC was 21.7% and the Socs2 genotype explained 12% of the variance of the trait. The point mutation induces the p.R96C substitution in the SH2 functional domain of SOCS2 i.e. the binding site of the protein to various ligands, as well-established for the growth hormone receptor GHR. Using surface plasmon resonance we showed that the p.R96C point mutation completely abrogates SOCS2 binding affinity for the phosphopeptide of GHR. Additionally, the size, weight and milk production in p.R96C homozygote sheep, were significantly increased by 24%, 18%, and 4.4%, respectively, when compared to wild type sheep, supporting the view that the point mutation causes a loss of SOCS2 functional activity. Altogether these results provide strong evidence for a causal mutation controlling SCC in sheep and highlight the major role of SOCS2 as a tradeoff between the host's inflammatory response to mammary infections, and body growth and milk production, which are all mediated by the JAK/STAT signaling pathway.

Transmissible spongiform encephalopaties (TSEs) are fatal neurodegenerative diseases characterized by the aggregation and accumulation of the misfolded prion protein in the brain. Other proteins such as β-amyloid, tau or Serum Amyloid-A (SAA) seem to share with prions some aspects of their pathogenic mechanism; causing a variety of so called prion-like diseases in humans and/or animals such as Alzheimer's, Parkinson's, Huntington's, Type II diabetes mellitus or amyloidosis. The question remains whether these misfolding proteins have the ability to self-propagate and transmit in a similar manner to prions. In this review, we describe the prion and prion-like diseases affecting animals as well as the recent findings suggesting the prion-like transmissibility of certain non-prion proteins., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Mice overexpressing the prion protein (PrP) sequence from various host species are widely used for measuring infectious titers in prion disease. However, the impact that the transgene expression level might have on the susceptibility to infection raises some concerns about the final biological relevance of these models. Here we report that endpoint titration of a sheep scrapie isolate in sheep and in mice overexpressing the ovine PrP results in similar estimates of the infectious titer.

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M142, N/D146, S/D146, R/Q211, and Q/K222) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I142R154R211Q222) goats and homozygous and/or heterozygous I/M142, R/H154, R/Q211, and Q/K222 goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M142 PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H154, Q211, and K222 PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H154 and Q211 allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K222 and a small proportion of homozygous K222 goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K222 goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie.

The PrP gene polymorphisms at codons 142 (I/M), 154 (R/H), 211 (R/Q), 222 (Q/K) and 240 (S/P) and their association with susceptibility to classical scrapie infection were investigated in five French goat herds displaying a high disease prevalence (>10%). On the basis of PrP(Sc) detection in the central nervous system and in various lymphoid tissues, 301 of 1343 goats were found to be scrapie infected. The statistical analyses indicated that while P(240) mutation had no direct impact on scrapie infection risk, the H(154), Q(211) and K(222) mutations were associated with high resistance to scrapie. The M(142) mutated allele was associated with a limited protection level against the disease. These results further reinforce the view that, like in sheep, the control and eradication of classical scrapie through the selection of certain PrP alleles could be envisaged in commercial goat population.

The association between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health and raises the possibility that other ruminant TSEs may be transmissible to humans. In recent years, several novel TSEs in sheep, cattle and deer have been described and the risk posed to humans by these agents is currently unknown. In this study, we inoculated two forms of atypical BSE (BASE and H-type BSE), a chronic wasting disease (CWD) isolate and seven isolates of atypical scrapie into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP). Upon challenge with these ruminant TSEs, gene-targeted Tg mice expressing human PrP did not show any signs of disease pathology. These data strongly suggest the presence of a substantial transmission barrier between these recently identified ruminant TSEs and humans.

Staphylococcus aureus is a prevalent pathogen for mastitis in dairy ruminants and is responsible for both clinical and subclinical mastitis. Mammary epithelial cells (MEC) represent not only a physical barrier against bacterial invasion but are also active players of the innate immune response permitting infection clearance. To decipher their functions in general and in animals showing different levels of genetic predisposition to Staphylococcus in particular, MEC from ewes undergoing a divergent selection on milk somatic cell count were stimulated by S. aureus. MEC response was also studied according to the stimulation condition with live bacteria or culture supernatant. The early MEC response was studied during a 5 h time course by microarray to identify differentially expressed genes with regard to the host genetic background and as a function of the conditions of stimulation. In both conditions of stimulation, metabolic processes were altered, the apoptosis-associated pathways were considerably modified, and inflammatory and immune responses were enhanced with the upregulation of il1a, il1b, and tnfa and several chemokines known to enhance neutrophil (cxcl8) or mononuclear leukocyte (ccl20) recruitment. Genes associated with oxidative stress were increased after live bacteria stimulation, whereas immune response-related genes were higher after supernatant stimulation in the early phase. Only 20 genes were differentially expressed between Staphylococcus spp-mastitis resistant and susceptible animals without any clearly defined role on the control of infection. To conclude, this suggests that MEC may not represent the cell type at the origin of the difference of mastitis susceptibility, at least as demonstrated in our genetic model. Supernatant or heat-killed S. aureus produce biological effects that are essentially different from those induced by live bacteria.

Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor) and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-kappaB in the nucleus of TNFalpha-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein.

Background: While the genomic era is accumulating a tremendous amount of data, the question of how genomics can describe a bacterial species remains to be fully addressed. The recent sequencing of the genome of the Mycoplasma agalactiae type strain has challenged our general view on mycoplasmas by suggesting that these simple bacteria are able to exchange significant amount of genetic material via horizontal gene transfer. Yet, events that are shaping mycoplasma genomes and that are underlining diversity within this species have to be fully evaluated. For this purpose, we compared two strains that are representative of the genetic spectrum encountered in this species: the type strain PG2 which genome is already available and a field strain, 5632, which was fully sequenced and annotated in this study., Results: The two genomes differ by ca. 130 kbp with that of 5632 being the largest (1006 kbp). The make up of this additional genetic material mainly corresponds (i) to mobile genetic elements and (ii) to expanded repertoire of gene families that encode putative surface proteins and display features of highly-variable systems. More specifically, three entire copies of a previously described integrative conjugative element are found in 5632 that accounts for ca. 80 kbp. Other mobile genetic elements, found in 5632 but not in PG2, are the more classical insertion sequences which are related to those found in two other ruminant pathogens, M. bovis and M. mycoides subsp. mycoides SC. In 5632, repertoires of gene families encoding surface proteins are larger due to gene duplication. Comparative proteomic analyses of the two strains indicate that the additional coding capacity of 5632 affects the overall architecture of the surface and suggests the occurrence of new phase variable systems based on single nucleotide polymorphisms., Conclusion: Overall, comparative analyses of two M. agalactiae strains revealed a very dynamic genome which structure has been shaped by gene flow among ruminant mycoplasmas and expansion-reduction of gene repertoires encoding surface proteins, the expression of which is driven by localized genetic micro-events.

Inducible promoter systems are powerful tools for studying gene function in prokaryotes but have never been shown to function in mollicutes. In this study we evaluated the efficacy of the tetracycline-inducible promoter Pxyl/tetO(2) from Bacillus subtilis in controlling gene expression in two mollicutes, the plant pathogen Spiroplasma citri and the animal pathogen Mycoplasma agalactiae. An S. citri plasmid carrying the spiralin gene under the control of the xyl/tetO(2) tetracycline-inducible promoter and the TetR repressor gene under the control of a constitutive spiroplasmal promoter was introduced into the spiralin-less S. citri mutant GII3-9a3. In the absence of tetracycline, expression of TetR almost completely abolished expression of spiralin from the xyl/tetO(2) promoter. Adding tetracycline (>50 ng ml(-1)) to the medium induced high-level expression of spiralin. Interestingly, inducible expression of spiralin was also detected in vivo: in S. citri-infected leafhoppers fed on tetracycline-containing medium and in S. citri-infected plants watered with tetracycline. A similar construct was introduced into the M. agalactiae chromosome through transposition. Tetracycline-induced expression of spiralin proved the TetR-Pxyl/tetO(2) system to be functional in the ruminant pathogen, suggesting that this tetracycline-inducible promoter system might be of general use in mollicutes.

In sheep, susceptibility to scrapie is mainly influenced by polymorphisms of the PrP gene. In goats, there are to date few data related to scrapie susceptibility association with PrP gene polymorphisms. In this study, we first investigated PrP gene polymorphisms of the French Alpine and Saanen breeds. Based on PrP gene open reading frame sequencing of artificial insemination bucks (n=404), six encoding mutations were identified at codons 127, 142, 154, 211, 222 and 240. However, only seven haplotypes could be detected: four (GIH(154)RQS, GIRQ(211)QS, GIRRK(222)S and GIRRQP(240)) derived from the wild-type allele (G(127)I(142)R(154)R(211)Q(222)S(240)) by a single-codon mutation, and two (S(127)IRRQP(240) and GM(142)RRQP(240)) by a double-codon mutation. A case-control study was then implemented in a highly affected Alpine and Saanen breed herd (90 cases/164 controls). Mutations at codon 142 (I/M), 154 (R/H), 211 (R/Q) and 222 (Q/K) were found to induce a significant degree of protection towards natural scrapie infection. Compared with the baseline homozygote wild-type genotype I(142)R(154)R(211)Q(222)/IRRQ goats, the odds of scrapie cases in IRQ(211)Q/IRRQ and IRRK(222)/IRRQ heterozygous animals were significantly lower [odds ratio (OR)=0.133, P<0.0001; and OR=0.048, P<0.0001, respectively]. The heterozygote M(142)RRQ/IRRQ genotype was only protective (OR=0.243, P=0.0186) in goats also PP(240) homozygous at codon 240. However, mutated allele frequencies in French Alpine and Saanen breeds were low (0.5-18.5 %), which prevent us from assessing the influence of all the possible genotypes in natural exposure conditions.

Poxviruses are among the largest and most complex viruses known. Vaccinia virus, the prototype of the family Poxviridae, has been studied much more than myxoma virus. The aim of this work was to have a better knowledge about myxoma virus morphogenesis. The characterization of the main stages of MV morphogenesis was achieved by ultrastructural and immunological analysis. Specific antibodies were raised against M022L and M071L, two envelope proteins of extracellular enveloped virus and intracellular mature virus, respectively. The main stages of assembly were similar to those seen with other poxviruses, and the duration of the whole replication cycle was estimated to be around 16 h, longer than what was described for vaccinia virus. Morphological changes of infected cells were associated with the development of long cellular projections and enlarged microvilli. Intracellular enveloped viruses are associated with the cytoskeleton to move through the cell. Unlike earlier studies, as many cell-associated enveloped viruses as intracellular enveloped viruses were observed in relation with specialized microvilli, although these structures were rarely noticed. Finally, an unusual spreading process was observed, which uses cytoplasmic corridors.

Downregulation of MHC class I molecules is a strategy developed by some viruses to escape cellular immune responses. Myxoma virus (MV), a poxvirus causing rabbit myxomatosis, encodes MV-LAP that is known to increase MHC-I endocytosis and degradation through a C(4)HC(3) motif critical for an E3 ubiquitin ligase activity. Here, we performed a functional mapping of MV-LAP and showed that not only the C(4)HC(3) motif is necessary for a marked downregulation of MHC-I but also a conserved region in the C-terminal part of the protein. We also showed that the putative transmembrane domains are responsible for a specific subcellular localization of the protein: they retain MV-LAP in the ER in transfected cells and in the endolysosomal compartments in infected cells. We observed that a specific MV infection context is necessary for a fully efficient downregulation of MHC-I. Our data suggest that the functionality of viral LAP factors, inherited by herpes- and poxviruses from mammalian cells, is more complex than anticipated.

Microbial pathogens have developed a variety of strategies to manipulate host-cell functions, presumably for their own benefit. We propose the term "cyclomodulins" to describe the growing family of bacterial toxins and effectors that interfere with the eukaryotic cell cycle. Inhibitory cyclomodulins, such as cytolethal distending toxins (CDTs) and the cycle inhibiting factor (Cif), block mitosis and might constitute powerful weapons for immune evasion by inhibiting clonal expansion of lymphocytes. Cell-cycle inhibitors might also impair epithelial-barrier integrity, allowing the entry of pathogenic bacteria into the body or prolonging their local existence by blocking the shedding of epithelia. Conversely, cyclomodulins that promote cellular proliferation, such as the cytotoxic necrotizing factor (CNF), exemplify another subversion mechanism by interfering with pathways of cell differentiation and development. The role of these cyclomodulins in bacterial virulence and carcinogenesis awaits further study and will delineate new perspectives in basic research and therapeutic applications.

NF-kappaB is one of the most important elements that coordinate stress-induced, immune, and inflammatory responses. Myxoma virus, a member of the Poxviridae family responsible for rabbit myxomatosis, codes for several factors that help its survival in the host. In this study, we focused on the product of the M150R gene. We show that the protein has nine ankyrin repeats (ANKs), with the eighth having a close similarity with the nuclear localization signal-containing ANK of I-kappaBalpha, which regulates NF-kappaB activity by sequestering it in the cytosol. Because the viral protein is targeted to the nucleus, it was named MNF, for myxoma nuclear factor. This localization was lost when the eighth ANK was removed. In tumor necrosis factor alpha-treated cells, MNF and NF-kappaB colocalized as dotted spots in the nucleus. In vivo experiments with a knockout virus showed that MNF is a critical virulence factor, with its deletion generating an almost apathogenic virus. Detailed histological examinations revealed an increase in the inflammatory process in the absence of MNF, consistent with the interference of MNF with the NF-kappaB-induced proinflammatory pathway. Because MNF has homologs in other poxviruses, such as vaccinia, cowpox, and variola viruses, this protein is probably part of a key mechanism that contributes to the immunogenic and pathogenic properties of these viruses.