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The use of cheap and thermoresistant vaccines in poor tropical countries for the control of animal diseases is a key issue. Our work aimed at designing and validating a process for the large-scale production of a ready-to-use inactivated vaccine for ruminants. Our model was heartwater caused by the obligate intracellular bacterium Ehrlichia ruminantium (ER). The conventional inactivated vaccine against heartwater (based on whole bacteria inactivated with sodium azide) is prepared immediately before injection, using a syringe-extrusion method with Montanide ISA50. This is a fastidious time-consuming process and it limits the number of vaccine doses available. To overcome these issues, we tested three different techniques (syringe, vortex and homogenizer) and three Montanide ISA adjuvants (50, 70 and 70M). High-speed homogenizer was the optimal method to emulsify ER antigens with both ISA70 and 70M adjuvants. The emulsions displayed a good homogeneity (particle size below 1 μm and low phase separation), conductivity below 10 μS/cm and low antigen degradation at 4 °C for up to 1 year. The efficacy of the different formulations was then evaluated during vaccination trials on goats. The inactivated ER antigens emulsified with ISA70 and ISA70M in a homogenizer resulted in 80% and 100% survival rates, respectively. A cold-chain rupture assay using ISA70M+ER was performed to mimic possible field conditions exposing the vaccine at 37 °C for 4 days before delivery. Surprisingly, the animal survival rate was still high (80%). We also observed that the MAP-1B antibody response was very similar between animals vaccinated with ISA70+ER and ISA70M+ER emulsions, suggesting a more homogenous antigen distribution and presentation in these emulsions. Our work demonstrated that the combination of ISA70 or ISA70M and homogenizer is optimal for the production of an effective ready-to-use inactivated vaccine against heartwater, which could easily be produced on an industrial scale. [ABSTRACT FROM AUTHOR]

Abstract: A statistical model of the population of first-order olfactory receptor neurons (ORNs) is proposed and analysed. It describes the relationship between stimulus intensity (odour concentration) and coding variables such as rate and latency of the population of several thousand sex-pheromone sensitive ORNs in male moths. Although these neurons likely express the same olfactory receptor, they exhibit, at any concentration, a relatively large heterogeneity of responses in both peak firing frequency and latency of the first action potential fired after stimulus onset. The stochastic model is defined by a multivariate distribution of six model parameters that describe the dependence of the peak firing rate and the latency on the stimulus dose. These six parameters and their mutual linear correlations were estimated from experiments in single ORNs and included in the multidimensional model distribution. The model is utilized to reconstruct the peak firing rate and latency of the message sent to the brain by the whole ORN population at different stimulus intensities and to establish their main qualitative and quantitative properties. Finally, these properties are shown to be in agreement with those found previously in a vertebrate ORN population. This article is part of a Special Issue entitled: Neural Coding. [Copyright &y& Elsevier]

The antennal lobe (AL) of the Noctuid moth Agrotis ipsilon has emerged as an excellent model for studying olfactory processing and its plasticity in the central nervous system. Odor-evoked responses of AL neurons and input-to-output transformations involved in pheromone processing are well characterized in this species. However, the intrinsic electrical properties responsible of the firing of AL neurons are poorly known. To this end, patch-clamp recordings in current- and voltage-clamp mode from neurons located in the two main clusters of cell bodies in the ALs were combined with intracellular staining on A. ipsilon males. Staining indicated that the lateral cluster (LC) is composed of 85% of local neurons (LNs) and 15% of projection neurons (PNs). The medial cluster (MC) contains only PNs. Action potentials were readily recorded from the soma in LNs and PNs located in the LC but not from PNs in the MC where recordings showed small or no action potentials. In the LC, the spontaneous activity of about 20% of the LNs presented irregular bursts while being more regular in PNs. We also identified a small population of LNs lacking voltage-gated Na + currents and generating spikelets. We focused on the firing properties of LNs since in about 60% of LNs, but not in PNs, action potentials were followed by depolarizing afterpotentials (DAPs). These DAPs could generate a second action potential, so that the activity was composed of action potential doublets. DAPs depended on voltage, Ca 2+ -channels and possibly on Ca 2+ -activated non-specific cationic channels. During steady state current injection, DAPs occurred after each action potential and did not require high-frequency firing. The amplitude of DAPs increased when the interspike interval was small, typically within bursts, likely arising from a Ca 2+ build up. DAPs were more often found in bursting than in non-bursting LNs but do not support bursting activity. DAPs and spike doublets also occurred during odor-evoked activity suggesting that they can mediate olfactory integration in the AL. [ABSTRACT FROM AUTHOR]

Although there is evidence that family violence increased in the United States during the COVID-19 pandemic, few studies have characterized longitudinal trends in family violence across the course of initial stay-at-home orders. The purpose of the present study is to investigate patterns and predictors of family violence, such as child maltreatment and harsh punishment, during the first eight weeks of the pandemic after initial stay-at-home orders in North Carolina. Participants included 120 families with children ages 4–11 (53% non-White, 49% female) and a primary caregiver (98% female) living in rural and suburban areas in North Carolina. Participants were recruited based on high risk of pre-pandemic family violence exposure. Caregivers completed weekly surveys during the pandemic assessing family violence, caregiver employment status, and caregiver emotion reactivity. In addition, all caregivers completed pre-pandemic surveys on family violence. Mixed-effects models revealed that family violence was highest following initial stay-at-home orders and decreased linearly over time. Higher pre-pandemic child violence exposure and caregiver unemployment were associated with higher initial family violence. Higher caregiver emotion reactivity was associated with changes in family violence across time. We observed high levels of family violence following stay-at-home orders, especially in families with higher baseline violence, higher caregiver emotion reactivity, and caregiver unemployment or underemployment. These associations suggest that vulnerable families may respond to the additional stressor of stay-at-home orders with increased violence and thus need additional support in moments of crisis. [ABSTRACT FROM AUTHOR]

Abstract: Ehrlichia ruminantium (ER) is a member of the order Rickettsiales transmitted by Amblyomma ticks. This obligatory intracellular bacterium is the causative agent of a fatal disease in ruminants, named heartwater. It represents a constraint on breeding development in sub-Saharan Africa and in the Caribbean. The genetic diversity of the strains of ER, which could be a limiting factor to obtain effective vaccines, needs to be better characterized. For this purpose, we developed a molecular typing technique based on the polymorphism of variable number tandem repeat (VNTR) sequences, MLVA (multiple locus VNTR analysis). Eight (out of 21) VNTR candidates were validated using 17 samples representing a panel of ER strains from different geographical origins from West, South Africa, and Caribbean areas and in ER infected ticks and goat tissues. This result demonstrated the ability of these VNTRs to type a wide range of strains. The stability of the selected VNTR markers was very good, at the time scale needed for epidemiological purposes: in particular, no difference in the VNTR profiles was observed between virulent and attenuated strains (for Gardel and Senegal strains) and between strains (Gardel and Blonde strains) isolated in the same area 19years apart. We validated the strong discriminatory power of MLVA for ER and found a high level of polymorphism between the available strains, with 10 different profiles out of 13 ER strains. The MLVA scheme described in this study is a rapid and efficient molecular typing tool for ER, which allows rapid and direct typing of this intracellular pathogen without preliminary culture and gives reliable results that can be used for further epidemiological studies. [Copyright &y& Elsevier]

Abstract: Cowdriosis or heartwater is a major tick-borne disease on ruminants in Africa and the Caribbean. The causative agent is Ehrlichia ruminantium, an intracellular bacterium. Development of vaccines against heartwater has been hampered the limited efficiency of vaccine in the field, thought to be a consequence of the high genetic diversity of strains circulating in a same area. A sampling scheme was set to collect ticks over 2 years in a delimited area and well identified flock. Prevalence was low at about 3%. A set of 37 strains was considered for MLST analysis along with two reference strains, i.e. ERGA and ERWO, for which full-length genome was available, using a previously described scheme based on the genes gltA, groEL, lepA, lipA, lipB, secY, sodB and sucA. Two populations were identified both with limited genetic variability but with differing evolutionary patterns. Population 1 is in genomic stasis, in agreement with the paradigm for intracellular bacteria. The two reference strains, one from the Caribbean separated from West African strains three centuries ago and another one isolated in South Africa, belong to Population 1. Population 2 is on expansion following a recent clonal emergence from Population 1. The founder strain was identified as strain 395. Strain 623 displays a particularly high rate of mutations in groEL. Owing to the chaperone function of GroEL, this might indicate another clonal emergence under way. This work brings further insight in the genomic plasticity of E. ruminantium and its impact on vaccine strategy. [Copyright &y& Elsevier]

Abstract: In order to identify the appropriate strains to use in vaccination trials against heartwater in Burkina Faso, the protective effect of Gardel and Welgevonden strains was assessed against local strains on sheep vaccinated by infection-and-treatment method: Gardel protected significantly against Burkina Faso strains tested (survival rate 59% for immunised sheep vs 13% for control sheep) while Welgevonden did not (survival rate 45% for immunised sheep vs 25% for control sheep). The efficacy of the ISA50 inactivated vaccine, produced under industrial process, was evaluated in sheep during field challenges in two successive years. During year 1, there was a limited protective effect of the Gardel vaccine with 65% of survival rate for the vaccinated group compared to 49% for the control group (N =153, p =0.053). During year 2, the vaccine containing Gardel and a local strain gave an increased protective effect compared to the first trial: 72% of the vaccinated animals survived compared to 47% of the naïve animals (N =173, p <0.001). There was an important genetic diversity of strains in the field with detection of 11 different map1 genotypes in brains from control and vaccinated sheep post mortem. Map1 genotyping of strains detected in brains from control sheep showed that genotype distribution varied according to time and study areas, which could explain the difference in efficacy of the vaccine. [Copyright &y& Elsevier]

Abstract: Understanding bacterial genetic diversity is crucial to comprehend pathogenesis. Ehrlichia ruminantium (E. ruminantium), a tick-transmitted intracellular bacterial pathogen, causes heartwater disease in ruminants. This model rickettsia, whose genome has been recently sequenced, is restricted to neutrophils and reticulo-endothelial cells of its mammalian host and to the midgut and salivary glands of its vector tick. E. ruminantium harbors a multigene family encoding for 16 outer membrane proteins including MAP1, a major antigenic protein. All the 16 map paralogs are expressed in bovine endothelial cells and some are specifically translated in the tick or in the mammalian host. In this study, we carried out phylogenetic analyses of E. ruminantium using sequences of 6 MAP proteins, MAP1, MAP1-2, MAP1-6, MAP1-5, MAP1+1 and MAP1-14, localized either in the center or at the borders of the map genes cluster. We show that (i) map1 gene is a good tool to characterize the genetic diversity among Africa, Caribbean islands and Madagascar strains including new emerging isolates of E. ruminantium; (ii) the different map paralogs define different genotypes showing divergent evolution; (iii) there is no correlation between all MAP genotypes and the geographic origins of the strains; (iv) The genetic diversity revealed by MAP proteins is conserved whatever is the scale of strains sampling (village, region, continent) and thus was not related to the different timing of strains introduction, i.e. continuous introduction of strains versus punctual introduction (Africa versus Caribbean islands). These results provide therefore a significant advance towards the management of E. ruminantium diversity. The differential evolution of these paralogs suggests specific roles of these proteins in host–vector–pathogen interactions that could be crucial for developing broad-spectrum vaccines. [Copyright &y& Elsevier]

Abstract: Heartwater, caused by the intracellular bacterium Ehrlichia ruminantium, is a major tick-borne disease of livestock in Africa also introduced in the Caribbean. The main problem encountered with the control of this disease is the lack of efficient vaccine in the field. This is thought to be related to the high genetic diversity of strains circulating in a same area. A set of eight circulating strains was isolated from a herd of cows in a small locality in Burkina-Faso and analyzed along with two reference strains, i.e. ERGA and ERWO, for which full-length genome was available. A MLST analysis was developed based on the genes gltA, groEL, lepA, lipA, lipB, secY, sodB and sucA. Phylogeny analysis was conducted both on concatenated MLST loci and on each individual locus. This showed differing phylogenies for each individual target gene. Most of the recorded polymorphism was borne by three strains: 331, 469 and 623. The neutrality hypothesis could not be rejected. Recombination and linkage disequilibrium were shown to have occurred. A core of seven strains displayed little polymorphism and signs of most likely ancient recombination events. The two reference strains, one from the Caribbean separated from west African strains three centuries ago and another one isolated in South Africa, were very closely related to the core strains whereas the three differing strains displayed recombination and most of the parcimony informative sites. These data suggest that some strains are in genomic stasis, as expected for intracellular parasites, while others emerge in the same area with DNA polymorphism. This work also shows that the MLST scheme developed can discriminate between these two kinds of strains. [Copyright &y& Elsevier]

Abstract: Ehrlichia ruminantium is the causative agent of heartwater, a major tick-borne disease of livestock in Africa introduced in the Caribbean and threatening to emerge and spread in the American mainland. Complete genome sequencing was done for two isolates of E. ruminantium of differing phenotype, isolates Gardel (Erga) from Guadeloupe Island and Welgevonden (Erwe) originating from South Africa and maintained in Guadeloupe. The type strain of E. ruminantium (Erwo), previously isolated and sequenced in South Africa; is identical to Erwe with respect to target genes. They make the Erwe/Erwo complex. Comparative analysis of the genomes shows the presence of 49 unique CDS and 28 truncated CDS differentiating Erga from Erwe/Erwo. Three regions of accumulated differences (RAD) acting as mutational hot spots were identified in E. ruminantium. Ten CDS, six unique CDS and four truncated CDS corresponding to major genomic changes (deletions or extensive mutations) were considered as targets for differential diagnosis on four isolates of E. ruminantium: Erga, Erwe/Erwo, Senegal and Umpala. Pairs of PCR primers were developed for each target gene. PCR analysis of the target genes generated strain-specific patterns on Erga and Erwe/Erwo as predicted by comparative genomics, but also for isolates Senegal and Umpala. The target genes identified by bacterial comparative genomics are shown to be highly efficient for strain-specific PCR diagnosis of E. ruminantium and further vaccine management tools. [Copyright &y& Elsevier]

Abstract: We report Marie Galante as one of the Caribbean islands most heavily infested by the tropical bont tick (TBT) Amblyomma variegatum which is associated with two major diseases of ruminants: heartwater and dermatophilosis. In 2005, a survey was undertaken to assess the prevalence of TBT infestation in cattle, the prevalence of Ehrlichia ruminantium infection in TBTs, and the tick control measures implemented by livestock owners. A random sample of 195 cattle herds out of 1885 recorded on the island was investigated by thoroughly counting adult ticks on each animal and filling a questionnaire. A randomly collected sample of 136 TBTs was tested for infection by E. ruminantium by pCS20 nested PCR. Cattle herd prevalence (hp) was 73.8% for infestation by at least one TBT, 17.9% for infestation by at least one engorged female TBT, and 8.2% for clinical dermatophilosis. Cattle individual prevalence was 42.3% for infestation by at least one TBT, 6.6% for infestation by at least one engorged female TBT, and 2.2% for clinical dermatophilosis. The minimum, maximum and average numbers of TBTs per infested animal were, respectively 1, 108 and 11.5. Prevalence of TBT infection by E. ruminantium was 19.1%. No significant difference in herd prevalence was found among parishes or among ecological zones. For cattle owners treating against ticks (97.9% of all owners), all used aspersion of amitraz and herd prevalence was significantly different among those treating every 1–2-week (hp=69.6%, n =148), and less often than every 2-week (hp=88.6%, n =35) (P =0.031). Of the 42 herd subunits treated less than 4 days before the survey, 27 (64%) were infested with at least one TBT, and 6 (14%) with at least one engorged female TBT. These results indicate a high level of TBT infestation in Marie Galante, the inefficacy of tick treatments currently performed, and the need for an improved tick control strategy. Persisting high levels of infestation in Marie Galante threaten the success of on-going TBT eradication programs in the Caribbean because TBT can spread through migrating birds and trade of animals or of animal hides to other islands and potentially the American continent. [Copyright &y& Elsevier]

Ehrlichia ruminantium is the causative agent of heartwater, an important tick-borne disease of livestock in Africa and the Caribbean that threatens the American mainland. The genome sequences of three strains of E. ruminantium have recently been published, revealing the presence of specific features related to genomic plasticity. E. ruminantium strains have traces of active genomic modifications, such as high substitution rates, truncated genes and the presence of pseudogenes and many tandem repeats. The most specific feature is the presence in all Ehrlichia of independent long-period tandem repeats, which are associated with expansion or contraction of intergenic regions. [Copyright &y& Elsevier]

Abstract: This work evaluates the effect of purification process and storage conditions (buffer formulation and temperature) on the efficacy of Ehrlichia ruminantium (ER) elementary bodies to be used as an inactivated vaccine candidate against heartwater. In vitro assays revealed that, to avoid major losses in ER integrity and corresponding antigenic properties, a buffer with pH between 5.6 and 8 and an osmolality above 100mOsmol/kg H2O is recommended. Amongst the tested formulations, both PBS and NaCl have shown to stabilize ER antigens at −20°C. To assess the protective properties of the different vaccine formulations, in vivo experiments were performed using a goat model. The results obtained showed that the preparation of ER antigens using a novel membrane-based purification strategy and a simple vaccine formulation (NaCl, −20°C) induced equivalent protection to the conventional vaccine based on ER antigens prepared by a multistep centrifugation methodology and stored at −20°C in PBS buffer. [Copyright &y& Elsevier]

Abstract: The aim of this work was to evaluate the minimal protective dose of the inactivated vaccine against heartwater. In order to conduct a reliable vaccination trial, an in vitro quantification method of Ehrlichia ruminantium (ER) challenge doses was developed. In experimental conditions, homologous Gardel challenges with 2.1×104 to 9.4×104 live ER elementary bodies were reproducible and mimicked a natural challenge. Similar results were obtained when animals were challenged with 3×104 live elementary bodies from five different ER strains. A 28-fold reduction of the vaccine dose did not decrease protection when compared to the conventional dose of inactivated vaccine. Two injections of 35μg of ER antigen induced good protection against heartwater. Moreover, we found that the amount of IFNγ secreted in blood from vaccinated animals after in vitro stimulation with ER antigen was not a reliable predictor of survival and could not be used to test the potency of the inactivated vaccine. [Copyright &y& Elsevier]

Interferon gamma (IFN-γ) is considered as a key mediator of protective cell-mediated immunity against intracellular pathogens in general, and against Ehrlichia ruminantium, the causative agent of tick-borne heartwater disease of ruminants, in particular. However, the source of this important cytokine in animals immunized against E. ruminantium remains largely unknown. We have analyzed in goats protected by vaccination with a killed E. ruminantium vaccine, the potential of individual, genuine (i.e., non-cloned), T cell subsets to produce IFN-γ after antigenic recall in vitro. In all vaccinated but none control animals, E. ruminantium-induced IFN-γ secretion was observed in 24 h stimulated blood. Flow cytometric analysis of stimulated peripheral blood mononuclear cells (PBMCs) collected after each vaccine inoculation indicated that immune CD4+ and CD8+ T cells contribute to the same extent to the production of IFN-γ, while WC1+ T cells are less important. This was confirmed by blocking the secretion of IFN-γ with anti-classes I and II major histocompatibility complex antibodies. Blocking experiments also suggest that CD8+ need the help of CD4+ T cells in order to produce IFN-γ. Thus, this work underlines the key role of CD4+ T cells in the production of IFN-γ by immune goat PBMC. It also describes, for the first time in ruminants, E. ruminantium-specific CD8+ effector T cells. Since CD4+ and CD8+ T cells collectively contribute to the production of IFN-γ in most vaccinated animals, and since these responses are associated with protection, it may be that a recombinant vaccine will need to incorporate E. ruminantium antigens capable of driving both responses. [Copyright &y& Elsevier]

The major antigenic protein 1 (MAP1) of the tick-borne rickettsial pathogen Cowdria ruminantium is encoded by a multigene family containing conserved and variable genes. The part of a locus containing the map1 multigene family that was characterized contained three homologous, but non-identical map1 genes, designated map1-2, map1-1, and map1. Reverse transcriptase-polymerase chain reaction was used to study the transcriptional activity of these genes in isolates of C. ruminantium grown in bovine endothelial cells, in two different tick cell lines, and in Amblyomma variegatum ticks. The map1 gene was always transcribed, whereas transcription of map1-2 was not detected under any of the tested conditions. The map1-1 gene transcript was detected in A. variegatum ticks, but was not found in virulent C. ruminantium Senegal grown in bovine endothelial cells at 30 or 37°C. Interestingly, transcripts of map1-1 were also found in different passages of the in vitro attenuated Senegal isolate grown in bovine endothelial cells, as well as in the Gardel isolate grown in two tick cell lines. When transcribed, map1-1 was present on a polycistronic messenger together with map1. [Copyright &y& Elsevier]