Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.
Small ruminant lentiviruses include members that infect sheep (ovine lentivirus [OvLV]; also known as ovine progressive pneumonia virus/maedi-visna virus) and goats (caprine arthritis encephalitis virus [CAEV]). Breed differences in seroprevalence and proviral concentration of OvLV had suggested a strong genetic component in susceptibility to infection by OvLV in sheep. A genetic marker test for susceptibility to OvLV has been developed recently based on the TMEM154 gene with validation data from over 2,800 sheep representing nine cohorts. While no single genotype has been shown to have complete resistance to OvLV, consistent association in thousands of sheep from multiple breeds and management conditions highlight a new strategy for intervention by selective breeding. This genetic marker-assisted selection (MAS) has the potential to be a useful addition to existing viral control measures. Further, the discovery of multiple additional genomic regions associated with susceptibility to or control of OvLV suggests that additional genetic marker tests may be developed to extend the reach of MAS in the future. This review will cover the strengths and limitations of existing data from host genetics as an intervention and outline additional questions for future genetic research in sheep, goats, small ruminant lentiviruses, and their host-pathogen interactions.
small ruminantlentivirus; susceptibility; marker-assisted selection; sheep; goats; TMEM154
Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. haemolytica has been isolated by culture less frequently than the other bacteria. We hypothesized that the growth of M. haemolytica is inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whether P. multocida inhibits the growth of M. haemolytica. Although in monoculture both bacteria exhibited similar growth characteristics, in coculture with P. multocida there was a clear inhibition of growth of M. haemolytica. The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth of M. haemolytica was inhibited when both bacteria were separated by a membrane that allowed contact (pore size, 8.0 μm) but not when they were separated by a membrane that limited contact (pore size, 0.4 μm). Lytic bacteriophages or bactericidal compounds could not be detected in the culture supernatant fluid from monocultures of P. multocida or from P. multocida-M. haemolytica cocultures. These results indicate that P. multocida inhibits the growth of M. haemolytica by a contact- or proximity-dependent mechanism. If the inhibition of growth of M. haemolytica by P. multocida occurs in vivo as well, it could explain the inconsistent isolation of M. haemolytica from the lungs of pneumonic BHS.
Theileria equi immune plasma was infused into young horses (foals) with severe combined immunodeficiency. Although all foals became infected following intravenous challenge with homologous T. equi merozoite stabilate, delayed time to peak parasitemia occurred. Protective effects were associated with a predominance of passively transferred merozoite-specific IgG3.
Bighorn sheep (BHS) are more susceptible than domestic sheep (DS) to Mannheimia haemolytica pneumonia. Although both species carry M. haemolytica as a commensal bacterium in the nasopharynx, DS carry mostly leukotoxin (Lkt)-positive strains while BHS carry Lkt-negative strains. Consequently, antibodies to surface antigens and Lkt are present at much higher titers in DS than in BHS. The objective of this study was to determine whether repeated immunization of BHS with multivalent Mannheimia-Bibersteinia vaccine will protect them upon M. haemolytica challenge. Four BHS were vaccinated with a culture supernatant vaccine prepared from M. haemolytica serotypes A1 and A2 and Bibersteinia trehalosi serotype T10 on days 0, 21, 35, 49, and 77. Four other BHS were used as nonvaccinated controls. On the day of challenge, 12 days after the last immunization, the mean serum titers of Lkt-neutralizing antibodies and antibodies to surface antigens against M. haemolytica were 1:160 and 1:4,000, respectively. Following intranasal challenge with M. haemolytica A2 (1 × 105 CFU), all four control BHS died within 48 h. Necropsy revealed acute fibrinonecrotic pneumonia characteristic of M. haemolytica infection. None of the vaccinated BHS died during the 8 weeks postchallenge observation period. Radiography at 3 weeks postchallenge revealed no lung lesions in two vaccinated BHS and mild lesions in the other two, which resolved by 8 weeks postchallenge. These results indicate that if BHS can be induced to develop high titers of Lkt-neutralizing antibodies and antibodies to surface antigens, they are likely to survive M. haemolytica challenge which is likely to reduce the BHS population decline due to pneumonia.
Mannheimia haemolytica consistently causes fatal bronchopneumonia in bighorn sheep (BHS; Ovis canadensis) under natural and experimental conditions. Leukotoxin is the primary virulence factor of this organism. BHS are more susceptible to developing fatal pneumonia than the related species Ovis aries (domestic sheep [DS]). In BHS herds affected by pneumonia, lamb recruitment is severely impaired for years subsequent to an outbreak. We hypothesized that a lack of maternally derived antibodies (Abs) against M. haemolytica provides an immunologic basis for enhanced susceptibility of BH lambs to population-limiting pneumonia. Therefore, the objective of this study was to determine the titers of Abs directed against M. haemolytica in the sera of BH and domestic lambs at birth through 12 weeks of age. Results revealed that BH lambs had approximately 18-fold lower titers of Ab against surface antigens of M. haemolytica and approximately 20-fold lower titers of leukotoxin-neutralizing Abs than domestic lambs. The titers of leukotoxin-neutralizing Abs in the serum and colostrum samples of BH ewes were approximately 157- and 50-fold lower than those for domestic ewes, respectively. Comparatively, the higher titers of parainfluenza 3 virus-neutralizing Abs in the BH lambs ruled out the possibility that these BHS had an impaired ability to passively transfer Abs to their lambs. These results suggest that lower levels of leukotoxin-neutralizing Abs in the sera of BH ewes, and resultant low Ab titers in their lambs, may be a critical factor in the poor lamb recruitment in herds affected by pneumonia.
We report an outbreak of equine piroplasmosis in southern Texas, USA, in 2009. Infection prevalence reached 100% in some areas (292 infected horses). Amblyomma cajennense was the predominant tick and experimentally transmitted Theileria equi to an uninfected horse. We suggest that transmission by this tick species played a role in this outbreak.
equine piroplasmosis; tick-borne diseases; vector-borne infections; Theileria equi; Amblyomma cajennense; equids; equine diseases; horses; parasites; ticks; Texas; expedited; dispatch
Rhipicephalus (Boophilus) microplus is an economically important tick of cattle involved in the transmission of Babesia bovis, the etiological agent of bovine babesiosis. Commercial anti-tick vaccines based on the R. microplus Bm86 glycoprotein have shown some effect in controlling tick infestation; however their efficacy as a stand-alone solution for tick control has been questioned. Understanding the role of the Bm86 gene product in tick biology is critical to identifying additional methods to utilize Bm86 to reduce R. microplus infestation and babesia transmission. Additionally, the role played by Bm86 in R. microplus fitness during B. bovis infection is unknown.
Here we describe in two independent experiments that RNA interference-mediated silencing of Bm86 decreased the fitness of R. microplus females fed on cattle during acute B. bovis infection. Notably, Bm86 silencing decreased the number and survival of engorged females, and decreased the weight of egg masses. However, gene silencing had no significant effect on the efficiency of transovarial transmission of B. bovis from surviving female ticks to their larval offspring. The results also show that Bm86 is expressed, in addition to gut cells, in larvae, nymphs, adult males and ovaries of partially engorged adult R. microplus females, and its expression was significantly down-regulated in ovaries of ticks fed on B. bovis-infected cattle.
The R. microplus Bm86 gene plays a critical role during tick feeding and after repletion during blood digestion in ticks fed on cattle during acute B. bovis infection. Therefore, the data indirectly support the rationale for using Bm86-based vaccines, perhaps in combination with acaricides, to control tick infestation particularly in B. bovis endemic areas.
The conversion of normal cellular prion protein to disease-associated prion protein (PrPSc) is a fundamental component of prion disease pathogenesis. The molecular mechanisms contributing to prion conversion and the impact of PrPSc accumulation on cellular biology are not fully understood. To further define the molecular changes associated with PrPSc accumulation in cultured cells, the transcriptional profile of PrPSc-accumulating primary ovine microglia was compared to the profile of PrPSc-lacking microglia using the Affymetrix Bovine Genome Array. The experimental design included three biological replicates, each with three technical replicates, and samples that were collected at the point of near maximal PrPSc accumulation levels as measured by ELISA. The array analysis revealed only 19 upregulated genes and 30 downregulated genes in PrPSc-accumulating microglia. The results support the hypothesis that chronic PrPSc accumulation in cultured microglia results in a limited transcriptional response.
ovine; microglia; prion; scrapie; microarray
This report describes the genome sequences of Mannheimia haemolytica serotype A2 isolated from pneumonic lungs of two different ruminant species, one from Ovis aries, designated ovine (O), and the other from Bos taurus, designated bovine (B).
A caprine arthritis-encephalitis virus (CAEV)/maedi-visna virus (MVV) indirect enzyme-linked immunosorbent assay (iELISA) was validated with samples from U.S. sheep and by the use of radioimmunoprecipitation as the standard for comparison. The sensitivity and the specificity were 86.0% (±5.8%) and 95.9% (±2.9%), respectively. The iELISA format and phylogenetic differences based on the MVV gag sequence contribute to the reduced sensitivity.
Mannheimia (Pasteurella) haemolytica is the only pathogen that consistently causes severe bronchopneumonia and rapid death of bighorn sheep (BHS; Ovis canadensis) under experimental conditions. Paradoxically, Bibersteinia (Pasteurella) trehalosi and Pasteurella multocida have been isolated from BHS pneumonic lungs much more frequently than M. haemolytica. These observations suggest that there may be an interaction between these bacteria, and we hypothesized that B. trehalosi overgrows or otherwise inhibits the growth of M. haemolytica. Growth curves (monoculture) demonstrated that B. trehalosi has a shorter doubling time (∼10 min versus ∼27 min) and consistently achieves 3-log higher cell density (CFU/ml) compared to M. haemolytica. During coculture M. haemolytica growth was inhibited when B. trehalosi entered stationary phase (6 h) resulting in a final cell density for M. haemolytica that was 6 to 9 logs lower than expected with growth in the absence of B. trehalosi. Coculture supernatant failed to inhibit M. haemolytica growth on agar or in broth, indicating no obvious involvement of lytic phages, bacteriocins, or quorum-sensing systems. This observation was confirmed by limited growth inhibition of M. haemolytica when both pathogens were cultured in the same media but separated by a filter (0.4-μm pore size) that limited contact between the two bacterial populations. There was significant growth inhibition of M. haemolytica when the populations were separated by membranes with a pore size of 8 μm that allowed free contact. These observations demonstrate that B. trehalosi can both outgrow and inhibit M. haemolytica growth with the latter related to a proximity- or contact-dependent mechanism.
Anaplasma marginale subsp. centrale is a naturally attenuated subtype that has been used as a vaccine for a century. We sequenced the genome of this organism and compared it to those of virulent senso stricto A. marginale strains. The comparison markedly narrows the number of outer membrane protein candidates for development of a safer inactivated vaccine and provides insight into the diversity among strains of senso lato A. marginale.
Babesia are emerging health threats to humans and animals in the United States. A collaborative effort of multiple disciplines to attain optimal health for people, animals and our environment, otherwise known as the One Health concept, was taken during a research workshop held in April 2009 to identify gaps in scientific knowledge regarding babesioses. The impetus for this analysis was the increased risk for outbreaks of bovine babesiosis, also known as Texas cattle fever, associated with the re-infestation of the U.S. by cattle fever ticks.
The involvement of wildlife in the ecology of cattle fever ticks jeopardizes the ability of state and federal agencies to keep the national herd free of Texas cattle fever. Similarly, there has been a progressive increase in the number of cases of human babesiosis over the past 25 years due to an increase in the white-tailed deer population. Human babesiosis due to cattle-associated Babesia divergens and Babesia divergens-like organisms have begun to appear in residents of the United States. Research needs for human and bovine babesioses were identified and are presented herein.
The translation of this research is expected to provide veterinary and public health systems with the tools to mitigate the impact of bovine and human babesioses. However, economic, political, and social commitments are urgently required, including increased national funding for animal and human Babesia research, to prevent the re-establishment of cattle fever ticks and the increasing problem of human babesiosis in the United States.
Antimicrobial treatment of persistent infection to eliminate transmission risk represents a specific challenge requiring compelling evidence of complete pathogen clearance. The limited repertoire of antimicrobial agents targeted at protozoal parasites magnifies this challenge. Using Babesia caballi as both a model and a specific apicomplexan pathogen for which evidence of the elimination of transmission risk is required for international animal movement, we tested whether a high-dose regimen of imidocarb dipropionate cleared infection from persistently infected asymptomatic horses and/or eliminated transmission risk. Clearance with elimination of transmission risk was supported by the following four specific lines of evidence: (i) inability to detect parasites by quantitative PCR and nested PCR amplification, (ii) conversion from seropositive to seronegative status, (iii) inability to transmit infection by direct inoculation of blood into susceptible recipient horses, and (iv) inability to transmit infection by ticks acquisition fed on the treated horses and subsequently transmission fed on susceptible horses. In contrast, untreated horses remained infected and capable of transmitting B. caballi using the same criteria. These findings establish that imidocarb dipropionate treatment clears B. caballi infection with confirmation of lack of transmission risk either by direct blood transfer or a high tick burden. Importantly, the treated horses revert to seronegative status according to the international standard for serologic testing and would be permitted to move between countries where the pathogen is endemic and countries that are free of the pathogen.
A multiplex real-time PCR was developed using a single pair of primers and fluorescent probes specific for five malignant catarrhal fever viruses and an internal positive control. The assay was able to simultaneously detect and differentiate the viruses in clinical samples with high sensitivity (97.2%) and specificity (100%).
The cattle tick Rhipicephalus (Boophilus) microplus is involved in the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Interactions between ticks and protozoa are poorly understood and the investigation of tick genes that affect tick fitness and protozoan infection can set the stage for dissecting the molecular interactions between the two species.
In this study, RNA interference was used to silence R. microplus genes that had been previously shown to be up-regulated in response to B. bovis infection. The silencing of a putative immunophilin gene (Imnp) in female ticks fed on a calf acutely infected with B. bovis decreased the hatching rate and survival of larval progeny. Interestingly, Imnp was up-regulated significantly in ovaries of R. microplus in response to B. bovis infection and its silencing in female ticks significantly increased the infection rate of the protozoan in larval progeny. The results also showed that the silencing of a putative Kunitz-type serine protease inhibitor (Spi) gene and a putative lipocalin (Lpc) gene decreased the fitness of R. microplus females, but had no significant effect on the infection rate of B. bovis in larval progeny.
The silencing of the Imnp, Spi or Lpc genes decreased the fitness of R. microplus females fed on a calf during acute B. bovis infection. The Imnp gene data suggest that this putative immunophilin gene is involved in the defense system of R. microplus against B. bovis and may play a role in controlling the protozoan infection in tick ovaries and larval progeny.
Studies were undertaken to determine whether anti-ovine progressive pneumonia virus (OPPV) antibody responses in serum or OPP provirus levels in peripheral blood associate with the degree of histologically measured tissue lesions in naturally OPPV-infected sheep. Sections of formalin-fixed, paraffin-embedded, and hematoxylin- and eosin-stained lung, mammary gland, carpal synovial membrane, and brain tissues from 11 OPPV-infected ewes (mean age of 8.6 years) and 5 OPPV-uninfected ewes (mean age of 6 years) were evaluated for lesion severity. Ovine progressive pneumonia (OPP) provirus levels and anti-OPPV antibody titers in peripheral blood and serum samples, respectively, were measured upon euthanasia and 3 years prior to euthanasia. Both mean peripheral OPP provirus levels and mean serum anti-surface envelope glycoprotein (anti-SU) antibody titers at the time of euthanasia were significantly higher in ewes with moderate to severe histological lesions than in ewes with no to mild histological lesions. However, although mean peripheral blood OPP provirus levels at euthanasia and 3 years prior to euthanasia significantly correlated with the highest histological lesion score for any affected tissue (two-tailed P values, 0.03 and 0.02), mean serum anti-SU antibody titers, anti-capsid antibody titers, and anti-transmembrane 90 antibody titers at euthanasia did not show a significant correlation with the highest histological lesion score for any tissue (two-tailed P values, 0.32, 0.97, and 0.18, respectively). These data are the first to show that OPP provirus levels predict and correlate with the extent of OPPV-related histological lesions in various OPPV-affected tissues. These findings suggest that peripheral OPP provirus levels quantitatively contribute more to the development of histological lesions than the systemic anti-SU antibody host immune response.
Strain superinfection occurs when a second pathogen strain infects a host already carrying a primary strain. Anaplasma marginale superinfection occurs when the second strain carries a variant repertoire different from that of the primary strain, and the epidemiologic consequences depend on the relative efficiencies of tick-borne transmission of the two strains. Following strain superinfection in the reservoir host, we tested whether the presence of two A. marginale (sensu lato) strains that differed in transmission efficiency altered the transmission phenotypes in comparison to those for single-strain infections. Dermacentor andersoni ticks were fed on animals superinfected with the Anaplasma marginale subsp. centrale vaccine strain (low transmission efficiency) and the A. marginale St. Maries strain (high transmission efficiency). Within ticks that acquired both strains, the St. Maries strain had a competitive advantage and replicated to significantly higher levels than the vaccine strain. The St. Maries strain was subsequently transmitted to naïve hosts by ticks previously fed either on superinfected animals or on animals singly infected with the St. Maries strain, consistent with the predicted transmission phenotype of this strain and the lack of interference due to the presence of a competing low-efficiency strain. The vaccine strain was not transmitted by either singly infected or coinfected ticks, consistent with the predicted transmission phenotype and the lack of enhancement due to the presence of a high-efficiency strain. These results support the idea that the strain predominance in regions of endemicity is mediated by the intrinsic transmission efficiency of specific strains regardless of occurrence of superinfection.
The relative fitness of arthropod-borne pathogens within the vector can be a major determinant of pathogen prevalence within the mammalian host population. Strains of the tick-borne rickettsia Anaplasma marginale differ markedly in transmission efficiency, with a consequent impact on pathogen strain structure. We have identified two A. marginale strains with significant differences in the transmission phenotype that is effected following infection of the salivary gland. We have proposed competing hypotheses to explain the phenotypes: (i) both strains are secreted equally, but there is an intrinsic difference in infectivity for the mammalian host, or (ii) one strain is secreted at a significantly higher level and thus represents delivery of a greater pathogen dose. Quantitative analysis of pathogen replication and secretion revealed that the high-efficiency St. Maries strain replicated to a 10-fold-higher titer and that a significantly greater percentage of infected ticks secreted A. marginale into the saliva and did so at a significantly higher level than for the low-efficiency Israel vaccine strain. Furthermore, the transmission phenotype of the vaccine strain could be restored to that of the St. Maries strain simply by increasing the delivered pathogen dose, either by direct inoculation of salivary gland organisms or by increasing the number of ticks during transmission feeding. We identified morphological differences in the colonization of each strain within the salivary glands and propose that these reflect strain-specific differences in replication and secretion pathways linked to the vector-pathogen interaction.
Sheep scrapie is the prototypical transmissible spongiform encephalopathy (prion disease), which has a fundamental pathogenesis involving conversion of normal cellular prion protein (PrPC [C superscript stands for cellular]) to disease-associated prion protein (PrPSc [Sc superscript stands for sheep scrapie]). Sheep microglial cell cultures, derived from a prnp 136VV/171QQ near-term fetal brain, were developed to study sheep scrapie in the natural host and to investigate potential cofactors in the prion conversion process. Two culture systems, a primary cell culture and a cell line transformed with the large T antigen of simian virus 40, were developed, and both were identified as microglial in origin as indicated by expression of several microglial phenotype markers. Following exposure to PrPSc, sheep microglial cells demonstrated relatively low levels (transformed cell line) to high levels (primary cell line) of PrPSc accumulation over time. The accumulated PrPSc demonstrated protease resistance, an inferred beta-sheet conformation (as determined by a commercial enzyme-linked immunosorbent assay), specific inhibition by anti-PrP antibodies, and was transmissible in a dose-dependent manner. Primary microglia coinfected with a small-ruminant lentivirus (caprine arthritis encephalitis virus-Cork strain) and PrPSc demonstrated an approximately twofold increase in PrPSc accumulation compared to that of primary microglia infected with PrPSc alone. The results demonstrate the in vitro utility of PrPSc-permissive sheep microglial cells in investigating the biology of natural prion diseases and show that small-ruminant lentiviruses enhance prion conversion in cultured sheep microglia.
Strain superinfection affects the dynamics of epidemiological spread of pathogens through a host population. Superinfection has recently been shown to occur for two genetically distinct strains of the tick-borne pathogen Anaplasma marginale that encode distinctly different surface protein variants. Superinfected animals could serve as a reservoir for onward transmission of both strains if the tick vector is capable of acquiring and transmitting both strains. Whether competition among strains during development within the tick vector, which requires sequential invasion and replication events, limits colonization and subsequent transmission to a single strain is unknown. We tested this possibility by acquisition feeding Dermacentor andersoni ticks on a reservoir host superinfected with the genetically distinct St. Maries and EMΦ strains. Although the St. Maries strain consistently maintained higher bacteremia levels in the mammalian host and the EMΦ strain had an early advantage in colonization of the tick salivary glands, individual ticks were coinfected, and there was successful transmission of both strains. These results indicate that a genetically distinct A. marginale strain capable of superinfecting the mammalian host can subsequently be cotransmitted and become established within the host population despite the presence of an existing established strain.
A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.