Genealogy of an in-vivo passaged isolate of western Canadian bovine respiratory syncytial virus.

Bovine respiratory syncytial virus (BRSV) is a primary respiratory pathogen in calves. Clinical infection with this pathogen has been experimentally modelled to assess vaccine efficacy using a field isolate (Asquith) of BRSV that has been sequentially passaged in vivo in neonatal calves to maintain virulence. The objective of this retrospective cumulative analysis of passages over approximately 20 years was to determine if there have been any changes in the viral genome of this isolate because of this process. Sequence analyses indicated that the Asquith isolate placed genetically in a clade comprising US and some European isolates and a recently described Chinese BRSV isolate (DQ). Furthermore, there were rare changes in bases over time in the N, G, and F gene segments examined when comparing among different passages ranging from 1996 to 2019. These results indicated the absence of significant mutations in the absence of significant adaptive immunological pressure.

Le virus respiratoire syncitial bovin (BRSV) est un agent pathogène respiratoire primaire chez les veaux. Une infection clinique avec cet agent pathogène a été expérimentalement modélisée pour évaluer l'efficacité vaccinale en utilisant un isolat de champ (Asquith) de BRSV qui a été passé séquentiellement in vivo chez des veaux nouveau-nés pour maintenir sa virulence. L'objectif de cette analyse rétrospective cumulative des passages sur une période d'approximativement 20 ans était de déterminer s'il y avait eu des changements dans le génome viral de cet isolat à cause de ce processus. L'analyse des séquences indiquaient que l'isolat Asquith se positionnait génétiquement dans un clade comprenant des isolats américains et quelques isolats européens et un isolat chinois de BRSV récemment décrit (DQ). Également, il y avait de rares changements de bases dans le temps dans les segments de gènes N, G et F examinés lors de la comparaison parmi les différents passages allant de 1996 à 2019. Ces résultats indiquent l'absence de mutation significative en absence de pression immunologique adaptative significative.(Traduit par Docteur Serge Messier).

Antibody recognition of cathepsin L1-derived peptides in Fasciola hepatica-infected and/or vaccinated cattle and identification of protective linear B-cell epitopes.

Fasciola hepatica infection causes important economic losses in livestock and food industries around the world. In the Republic of Ireland F. hepatica infection has an 76% prevalence in cattle. Due to the increase of anti-helminthic resistance, a vaccine-based approach to control of Fasciolosis is urgently needed. A recombinant version of the cysteine protease cathepsin L1 (rmFhCL1) from F. hepatica has been a vaccine candidate for many years. We have found that vaccination of cattle with this immunodominant antigen has provided protection against infection in some experimental trials, but not in others. Differential epitope recognition between animals could be a source of variable levels of vaccine protection. Therefore, we have characterised for first time linear B-cell epitopes recognised within the FhCL1 protein using sera from F. hepatica-infected and/or vaccinated cattle from two independent trials. Results showed that all F. hepatica infected animals recognised the region 19-31 of FhCL1, which is situated in the N-terminal part of the pro-peptide. Vaccinated animals that showed fluke burden reduction elicited antibodies that bound to the regions 120-137, 145-155, 161-171 of FhCL1, which were not recognised by non-protected animals. This data, together with the high production of specific IgG2 in animals showing vaccine efficacy, suggest important targets for vaccine development.

Efficacy of intranasal vaccination with a multivalent vaccine containing temperature-sensitive modified-live bovine herpesvirus type 1 for protection of seronegative and seropositive calves against respiratory disease.

OBJECTIVE To evaluate efficacy and duration of immunity of the bovine herpesvirus type 1 (BHV-1) fraction of a trivalent vaccine also containing parainfluenza virus-3 and bovine respiratory syncytial virus fractions administered intranasally (IN) for protection of calves against infectious bovine rhinotracheitis (IBR). DESIGN Controlled challenge study. ANIMALS 120 dairy calves (3 to 8 days old) seronegative for antibody against BHV-1 (experiments 1 and 2) or seropositive for maternally derived antibody against BHV-1 (experiment 3). PROCEDURES In 3 separate experiments, calves were vaccinated IN via 2 nostrils (experiment 1) or 1 nostril (experiments 2 and 3) with a vaccine containing or not containing a BHV-1 fraction. For seronegative calves, the test vaccine contained a minimum immunizing dose of BHV-1; for seropositive calves, it contained a commercial dose of BHV-1. Calves were challenged IN with virulent BHV-1 on day 28 or 193 (seronegative calves) or day 105 (seropositive calves) after vaccination to evaluate vaccine efficacy. Frequency and duration of clinical signs, rectal temperatures, virus shedding, and serologic responses were compared between treatment groups within experiments. RESULTS In all experiments, BHV-1 vaccinated calves had lower frequencies or shorter durations of clinical signs of IBR than did control calves. Following viral challenge, peak rectal temperatures and degrees of virus shedding were lower and serologic responses were higher in vaccinated versus control calves. CONCLUSIONS AND CLINICAL RELEVANCE IN vaccination against BHV-1 protected all calves against clinical IBR disease, regardless of serologic status at the time of vaccination, and suppressed virus shedding. A single dose of this IN vaccine has the potential to protect seronegative calves for at least 193 days and override maternally derived antibody to protect seropositive calves for at least 105 days.

Differential endometrial cell sensitivity to a cholesterol-dependent cytolysin links Trueperella pyogenes to uterine disease in cattle.

Purulent disease of the uterus develops in 40% of dairy cows after parturition, when the epithelium of the endometrium is disrupted to expose the underlying stroma to bacteria. The severity of endometrial pathology is associated with isolation of Trueperella pyogenes. In the present study, T. pyogenes alone caused uterine disease when infused into the uterus of cattle where the endometrial epithelium was disrupted. The bacterium secretes a cholesterol-dependent cytolysin, pyolysin (PLO), and the plo gene was identical and the plo gene promoter was highly similar amongst 12 clinical isolates of T. pyogenes. Bacteria-free filtrates of the T. pyogenes cultures caused hemolysis and endometrial cytolysis, and PLO was the main cytolytic agent, because addition of anti-PLO antibody prevented cytolysis. Similarly, a plo-deletion T. pyogenes mutant did not cause hemolysis or endometrial cytolysis. Endometrial stromal cells were notably more sensitive to PLO-mediated cytolysis than epithelial or immune cells. Stromal cells also contained more cholesterol than epithelial cells, and reducing stromal cell cholesterol content using cyclodextrins protected against PLO. Although T. pyogenes or plo-deletion T. pyogenes stimulated accumulation of inflammatory mediators, such as IL-1beta, IL-6, and IL-8, from endometrium, PLO did not stimulate inflammatory responses by endometrial or hematopoietic cells, or in vitro organ cultures of endometrium. The marked sensitivity of stromal cells to PLO-mediated cytolysis provides an explanation for how T. pyogenes acts as an opportunistic pathogen to cause pathology of the endometrium once the protective epithelium is lost after parturition.

Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum.

BACKGROUND: Anaplasma phagocytophilum is an intracellular organism in the Order Rickettsiales that infects diverse animal species and is causing an emerging disease in humans, dogs and horses. Different strains have very different cell tropisms and virulence. For example, in the U.S., strains have been described that infect ruminants but not dogs or rodents. An intriguing question is how the strains of A. phagocytophilum differ and what different genome loci are involved in cell tropisms and/or virulence. Type IV secretion systems (T4SS) are responsible for translocation of substrates across the cell membrane by mechanisms that require contact with the recipient cell. They are especially important in organisms such as the Rickettsiales which require T4SS to aid colonization and survival within both mammalian and tick vector cells. We determined the structure of the T4SS in 7 strains from the U.S. and Europe and revised the sequence of the repetitive virB6 locus of the human HZ strain. RESULTS: Although in all strains the T4SS conforms to the previously described split loci for vir genes, there is great diversity within these loci among strains. This is particularly evident in the virB2 and virB6 which are postulated to encode the secretion channel and proteins exposed on the bacterial surface. VirB6-4 has an unusual highly repetitive structure and can have a molecular weight greater than 500,000. For many of the virs, phylogenetic trees position A. phagocytophilum strains infecting ruminants in the U.S. and Europe distant from strains infecting humans and dogs in the U.S. CONCLUSIONS: Our study reveals evidence of gene duplication and considerable diversity of T4SS components in strains infecting different animals. The diversity in virB2 is in both the total number of copies, which varied from 8 to 15 in the herein characterized strains, and in the sequence of each copy. The diversity in virB6 is in the sequence of each of the 4 copies in the single locus and the presence of varying numbers of repetitive units in virB6-3 and virB6-4. These data suggest that the T4SS should be investigated further for a potential role in strain virulence of A. phagocytophilum.

Diversity of Ehrlichia ruminantium major antigenic protein 1-2 in field isolates and infected sheep.

Proteins expressed from the map1 multigene family of Ehrlichia ruminantium are strongly recognized by immune T and B cells from infected animals or from animals that were infected and have recovered from heartwater disease (although still remaining infected carriers). Analogous multigene clusters also encode the immunodominant outer membrane proteins (OMPs) in other ehrlichial species. Recombinant protein analogs of the expressed genes and DNA vaccines based on the multigene clusters have been shown to induce protective immunity, although this was less effective in heterologous challenge situations, where the challenge strain major antigenic protein 1 (MAP1) sequence differed from the vaccine strain MAP1. Recent data for several ehrlichial species show differential expression of the OMPs in mammalian versus tick cell cultures and dominant expression of individual family members in each type of culture system. However, many genes in the clusters appear to be complete and functional and to generate mRNA transcripts. Recent data also suggest that there may be a low level of protein expression from many members of the multigene family, despite primary high-level expression from an individual member. A continuing puzzle, therefore, is the biological roles of the different members of these OMP multigene families. Complete genome sequences are now available for two geographically divergent strains of E. ruminantium (Caribbean and South Africa strains). Comparison of these sequences revealed amino acid sequence diversity in MAP1 (89% identity), which is known to confer protection in a mouse model and to be the multigene family member primarily expressed in mammalian cells. Surprisingly, however, the greatest sequence diversity (79% identity) was in the less-studied map1-2 gene. We investigated here whether this map1-2 diversity was a general feature of E. ruminantium in different cultured African strains and in organisms from infected sheep. Comparison of MAP1-2s revealed amino acid identities of 75 to 100% (mean of 86%), compared to 84 to 100% (mean of 89%) for MAP1s. Interestingly, MAP1-2s varied independently of MAP1s such that E. ruminantium strains with similar MAP1s had diverse MAP1-2s and vice versa. Different MAP1-2s were found in individual infected sheep. Different regions of a protein may be subjected to different evolutionary forces because of recombination and/or selection, which results in those regions not agreeing with a phylogeny deduced from the whole molecule. This appears to be true for both MAP1 and MAP1-2, where statistical likelihood methods detect heterogeneous evolutionary rates for segments of both molecules. Sera from infected cattle recognized a MAP1-2 variable-region peptide in enzyme-linked immunosorbent assay, but less strongly and consistently than a MAP1 peptide (MAP1B). Heterologous protective immunity may depend on recognition of a complex set of varying OMP epitopes.

Evaluation of E. ruminantium genes in DBA/2 mice as potential DNA vaccine candidates for control of heartwater.

Heartwater caused by the rickettsia Ehrlichia ruminantium (E. ruminantium) is an acute and fatal tick-borne disease of domestic and some wild ruminants. A user-friendly vaccine does not exist. We selected and tested nine genes of E. ruminantium for protection against challenge in a DBA/2 mouse model, in order to identify candidate genes for incorporation into a recombinant vaccine. Of the nine DNA vaccine constructs tested, four DNA constructs 14HWORF1/VR1012, 14HWORF2/VR1012, 27HWORF1/VR1012, and HSP58/VR1012 were not protective and were excluded from the study. The remaining five DNA constructs-MAP2/ VR1012, 1HWORF3/ VR1012, 4HWORF1/ VR1012, 18HWORF1/ VR1012, and 3GDORF3/ VR1012-offered partial protection against lethal challenge demonstrated by reduced mortalities compared to control groups. Protection was augmented when DNA primed mice were boosted with a respective homologous recombinant protein. Protection in these five groups was associated with the induction of cell-mediated or T helper 1 (Th1) type of immune responses characterized by the production of large amounts of interferon-gamma and interleukin-2 in in vitro proliferation assays using E. ruminantium antigens for stimulation. These responses were enhanced when the DNA-vaccinated DBA/2 mice were boosted with specific homologous recombinant protein vaccination. In a preliminary follow-up study, protection conferred by DNA vaccination with individual gene constructs was not enhanced when the protective constructs were administered in combination (including the map-1 gene of E. ruminantium). Further evaluation of these and other untested DNA constructs is necessary to optimize their expression in vivo in the presence of molecular adjuvants, such as the IFN-gamma gene, GM-CSF gene, IL-12 gene, and CpG motifs to fully evaluate their protective value.

Infection of a goat with a tick-transmitted Ehrlichia from Georgia, U.S.A., that is closely related to Ehrlichia ruminantium.

We detected a novel tick-transmitted Ehrlichia in a goat following exposure to lone star ticks (Amblyomma americanum) from a park in the metropolitan area of Atlanta, GA, U.S.A. Nineteen days after infestation with field-collected adult ticks, the goat developed a fever of two days duration, which coincided with mild clinical pathologic changes and the presence of DNA from a novel Ehrlichia in peripheral blood. The goat transmitted ehrlichiae to uninfected nymphal A. americanum that fed upon the goat, and the ticks maintained the pathogen transstadially. Five months after exposure, immunosuppression of the goat resulted in transient ehrlichemia with transmission of ehrlichiae to feeding ticks. Sequencing and phylogenetic reconstructions of the 16S rRNA, gltA, map1, map2, and ribonuclease III genes suggest the agent might be a divergent strain of Ehrlichia ruminantium, the agent of heartwater, or a new, closely related species. Convalescent serum from the goat reacted with the MAP-1 protein of E. ruminantium and with whole-cell Ehrlichia chaffeensis antigen. DNA from the novel Ehrlichia was detected in 5/302 field-collected adult A. americanum from the park. Our data suggest that A. americanum is a natural vector and reservoir of this Ehrlichia and that domestic goats can be reservoirs. The geographic range of the agent and its pathogenicity to humans and livestock needs to be evaluated.

Ticks and tick-borne disease in Guatemalan cattle and horses.

Blood samples and ticks were collected from 48 cattle and 74 horses from seven sites in the Peten region of Guatemala. Data on body condition, mucous membrane capillary refill time and tick infestation levels were recorded for each animal in the study. Horses had significantly higher levels of tick infestation than cattle, as well as poorer body condition scores. Seroprevalence of Babesia spp. was 95.8% for B. bovis in cattle, 89.6% for B. bigemina in cattle, and 92.7% for B. equi in horses. Seroprevalence of Anaplasma marginale in cattle was 87.5%, similar to reports in animals from other regions of Central America. This is the first time that A. phagocytophilum has been reported in animals from this region, with overall PCR-prevalence of 27.6% in cattle and horses, and seroprevalence of 28.4% (52% in cattle and 13% in horses). An agent was identified with serological cross-reactivity and close genetic relatedness to Ehrlichia ruminantium, but further testing confirmed that the agent in Guatemalan cows was not the agent of heartwater. Ticks were identified to species with the predominant species identified on cattle as Boophilus microplus and Amblyomma cajennense, while Anocentor nitens and A. cajennense were most commonly found on horses. Prevalence of infection, tick infestation levels, host factors and environmental data were analyzed for association; A. nitens was significantly associated with A. phagocytophilum prevalence by village.

Increasing risks of introduction of heartwater onto the American mainland associated with animal movements.

Opportunities to introduce heartwater onto the American mainland through animal movements include importation from Africa of tick-infested reptiles and of subclinically infected wild ungulates and importation of livestock from islands in the Caribbean infested with Amblyomma variegatum ticks. Measures to control importation of heartwater vectors on reptiles include importation bans of infested species, treatment of imported reptiles, and eradication of established infestations on the American mainland. Measures to control importation of infected wildlife must focus on improved methods, such as the PCR assay, of screening animals to prevent the entry of carriers of Cowdria ruminantium. Measures to control importation of infected animals from the Caribbean must be based on knowledge of the islands that are infected with C. ruminantium so that the risk of dissemination of heartwater can be established.

Control and eradication of chelonian tick infestations, with particular reference to vectors of heartwater.

Studies using the African tortoise tick (Amblyomma marmoreum) and leopard tortoises (Geochelone pardalis) demonstrated that cyfluthrin and permethrin were safe and efficacious acaricides for control of Amblyomma ticks on tortoises. A protocol was developed that successfully eradicated an A. sparsum infestation from a tortoise breeding facility in Florida. It involved treatment of all tortoises with a permethrin formulation, followed by treatment of the premises with a cyfluthrin formulation. Sentinel tortoises were later placed on the treated premises to establish successful tick eradication.

Evaluation of safety and efficacy of acaricides for control of the African tortoise tick (Amblyomma marmoreum) on leopard tortoises (Geochelone pardalis).

Nine acaricides (amitraz, carbaryl, chlorpyrifos, cyfluthrin, fipronil, lindane, permethrin, phenothrin, and pyrethrins) were studied for their efficacy in killing the African tortoise tick (Amblyomma marmoreum). Only four of the acaricides (chlorpyrifos, cyfluthrin, lindane, and permethrin) produced 100% mortality within 24 hr of application, and only two (cyfluthrin and permethrin) continued to cause 100% mortality when diluted to as low as 1:10,000. Five of the acaricides (amitraz, carbaryl, chlorpyrifos, cyfluthrin, and permethrin) were studied for toxicity to the leopard tortoise (Geochelone pardalis), the most common host of A. marmoreum. The results indicate that cyfluthrin and permethrin, which were herein found to be the two most effective acaricides for control of A. marmoreum, are also the safest acaricides for use on leopard tortoises.

Ehrlichia ruminantium infection (heartwater) in wild animals.

Several wild animal species have been implicated as hosts of Ehrlichia ruminantium (formerly Cowdria ruminantium), the rickettsial agent causing heartwater, a fatal disease of domestic ruminants in sub-Saharan Africa and eastern Caribbean. However, evidence for infection in most wild species is inconclusive because of inadequate diagnostic techniques. Infection has been proven only in 12 African ruminants, three non-African ruminants and two African rodents. A subclinical carrier state occurs in eight of the African ruminant species. Further studies on E. ruminantium infection in wild animal species are needed in order to determine the host range of this pathogen accurately. The host range of Ehrlichia ruminantium in wildlife is reviewed here and the role played by these species in the epidemiology and spread of heartwater is discussed.

Comparison of three enzyme-linked immunosorbant assays with the indirect immunofluorescent antibody test for the diagnosis of canine infection with Ehrlichia canis.

The aim of this study was to compare three different enzyme-linked immunosorbant assays (recombinant major antigenic protein 2 (rMAP2)-ELISA, the Immunocomb (Biogal, Israel) and the Snap 3Dx assay (IDEXX Laboratories Inc., USA)) with the indirect immunofluorescent antibody test in detecting anti-Ehrlichia canis immunoglobulin-G (IgG) antibodies. Samples tested were collected from dogs suspected to be naturally infected with E. canis and from experimentally infected dogs. When qualitative results (positive/negative) were compared, there was an overall agreement of 81% (54/67) between the indirect immunofluorescence antibody (IFA) test and the rMAP2-ELISA. An overall agreement of 94% (63/67) was found between the IFA test and the Immunocomb, and an overall agreement of 91% (61/67) was found between the IFA test and the Snap 3Dx assay. In 50 of 67 (74.6%) samples tested, complete agreement in the qualitative results was found in all four tests. Sixteen of 17 samples with disagreement in the qualitative results were found to have IFA titers of 1:320 or less. The sensitivities and specificities of the tests were found to be 0.71 and 0.85 for the rMAP2-ELISA, 0.86 and 0.98 for the Immunocomb, and 0.71 and 1.00 for the Snap 3Dx assay. The tests performed in this study were found to be highly specific in detecting E. canis antibodies. Their sensitivity was found to be low with sera having IFA titers of < or =1:320, while high with sera having titers greater than 1:320. Repeating the serological tests 1-2 weeks after the first antibody assay may overcome the sensitivity problem with titers of < or =1:320.

Immunisation of cattle against heartwater by infection with Cowdria ruminantium elicits T lymphocytes that recognise major antigenic proteins 1 and 2 of the agent.

There is growing evidence that immunity of cattle to Cowdria ruminantium infection is mediated by T lymphocytes. C. ruminantium antigens that stimulate these responses are therefore of considerable importance to the development of a sub-unit vaccine against the disease. We have examined T cell responses against recombinant analogues of the surface-exposed C. ruminantium major antigen 1 (MAP1) a 28.8 kDa protein and MAP2 (21 kDa) antigen in cattle immunised by infection and treatment. Vigorous and sustained proliferative responses to both antigens were observed in peripheral blood mononuclear cells from immune cattle. MAP1-specific responses were predominantly restricted to cluster of differentiation four antigen positive T cells (CD4+ T cells). Reverse transcription polymerase chain reaction (RT-PCR) analysis of cytokine expression by T cell lines derived from this population revealed strong expression of interferon gamma (IFN-gamma), interferon alpha (IFN-alpha), tumour necrosis factor alpha (TNF-alpha), tumour necrosis factor beta (TNF-beta), interleukin-2 receptor alpha (IL-2Ralpha) transcripts, and weak expression of IL-2 and IL-4. Supernatants from these T cell cultures contained IFN-gamma protein. CD4+ T cell clones specific for MAP1 were generated. Two of these clones proliferated in the presence of autologous infected endothelial cells. In contrast, the response to MAP2 was characterised largely by proliferation of gamma delta (gammadelta) T cells. RT-PCR analysis of cytokine expression by T cell lines which were dominated by gammadelta T cells revealed expression of IFN-gamma, TNF-alpha, TNF-beta, IL-2Ralpha transcripts. Supernatants of these T cell cultures also contained IFN-gamma protein. Our findings indicate that immunisation of cattle by infection with C. ruminantium results in generation of MAP1- and MAP2-specific T cell responses that may play a role in protection against the pathogen.

DNA vaccination with map1 gene followed by protein boost augments protection against challenge with Cowdria ruminantium, the agent of heartwater.

A DNA vaccine encoding the immunodominant MAP1 protein of Cowdria ruminantium (Crystal Springs (CS) strain) was shown to partially protect DBA/2 mice against homologous lethal challenge. To enhance the protective capacity of this DNA vaccine, the effects of length of interval between vaccinations and of prime-boost regimes were investigated. Increasing the interval between vaccinations from 2 to 12 weeks did not result in better protection (P=0.900). However, boosting DNA vaccine-primed mice with recombinant MAP1 protein significantly augmented protection on homologous challenge in various trials from 13-27 to 53-67% (P<0.050). The augmented protection by the prime-boost regimen correlated with augmented T(H1) type immune responses that were induced by the DNA vaccine. These responses were characterized by production of IFN-gamma, IL-2 and anti-MAP1 antibodies of predominantly IgG2a isotype, and were critical for protection against C. ruminantium infection. Cytokine analyses were done at 48h after in vitro stimulation of splenocytes with C. ruminantium or control antigens. In contrast, splenocytes of DNA vector control mice produced no cytokines and these mice were fully susceptible to challenge. In addition, DBA/2 mice immunized with the recombinant MAP1 protein without DNA vaccine priming produced non-protective T(H2) type immune responses which were characterized by production of IL-4, IL-5, IL-10 and IgG1 anti-MAP1 antibodies. A second DNA vaccine containing map1 gene from the Mbizi strain of C. ruminantium also delivered by a prime-boost regime, conferred less protection against heterologous challenge. Hence, in developing DNA vaccines against heartwater that contain map1 gene, a prime-boost regimen should be adopted and gene sequence heterogeneity of field isolates should also be considered.