Evolution and diversity of the EMA families of the divergent equid parasites, Theileria equi and T. haneyi.

The equine parasite Theilera equi continues to curtail global equine commerce due primarily to its ability to persist indefinitely in the immunocompetent horse. Details regarding the parasite life cycle, pathogenesis and mechanism of persistence remain unclear. The recently discovered T. haneyi is also capable of persistence in the horse, creating a potential reservoir for additional infections. These two divergent parasites share a unique gene family that expresses surface merozoite antigens, or equi merozoite antigens (EMAs). The EMA family was maintained in number and size in both parasites despite a species divergence of over 30 million years ago. This family is unique amongst Theilerias in number, structure and biochemical properties. In silico analysis revealed no evidence of selection for diversity within this family, indicating a role in host adaptation and persistence rather than antigenic variation and immune escape. Biochemical analysis revealed the presence of a conserved domain, homologous to the hemolysin toxin found in cobra venom. This finding combined with data from protein interaction prediction models may indicate interaction with the structural components of the host erythrocyte and a role in merozoite entry or escape. Additional predicted protein interactions focus on disruption of the enzymatic functions of the host cell, potentially resulting in enhanced parasite survival.

Verification of post-chemotherapeutic clearance of Theileria equi through concordance of nested PCR and immunoblot.

Certain countries including the United States remain non-endemic for particular infectious diseases such as equine piroplasmosis through import restrictions and surveillance. Endemic regions often employ premunition as the primary method to control disease, however in non-endemic countries, chemosterilization combined with methods to confirm parasite elimination are required to maintain disease-free status. The ability of imidocarb diproprionate (ID) to clear persistent Theileria equi infection from infected horses has been shown through the inability of treated horses to transmit via blood transfer. However, the common lengthy persistence of anti-T. equi antibody causes regulatory tests such as cELISA or IFA to remain positive for extended periods. Persistence of positive testing creates challenges for regulatory veterinary medicine and international trade. Concordance between nested polymerase chain reaction (nPCR) targeting the ema1 gene and immunoblotting (IB) measuring declination in anti-EMA1 and anti-EMA2 antibody were used to verify clearance of T. equi from 179 ID-treated horses. These data support the use of IB to demonstrate declining anti-EMA1 and EMA2 titers in T. equi-infected horses subsequent to successful ID treatment. Such data provide concordant support to a negative nPCR and allow for a more timely determination of effective ID clearance of T. equi. The post ID treatment results indicate that while nPCR was consistently negative by 14 days and cELISA generally remained positive after 1 year, immunoblot was on average negative after 4 months and 100% in agreement with nPCR.

Deletion variant near ZNF389 is associated with control of ovine lentivirus in multiple sheep flocks.

Ovine lentivirus (OvLV) is a macrophage-tropic lentivirus found in many countries that causes interstitial pneumonia, mastitis, arthritis and cachexia in sheep. There is no preventive vaccine and no cure, but breed differences suggest marker-assisted selective breeding might improve odds of infection and control of OvLV post-infection. Although variants in TMEM154 have consistent association with odds of infection, no variant in any gene has been associated with host control of OvLV post-infection in multiple animal sets. Proviral concentration is a live-animal diagnostic measure of OvLV control post-infection related to severity of OvLV-induced lesions. A recent genome-wide association study identified a region including four zinc finger genes associated with proviral concentration in one Rambouillet flock. To refine this region, we tested additional variants and identified a small insertion/deletion variant near ZNF389 that showed consistent association with proviral concentration in three animal sets (P < 0.05). These animal sets contained Rambouillet, Polypay and crossbred sheep from multiple locations and management conditions. Strikingly, one flock had exceptionally high prevalence (>87%, including yearlings) and mean proviral concentration (>950 copies/µg), possibly due to needle sharing. The best estimate of proviral concentration by genotype, obtained from all 1310 OvLV-positive animals tested, showed insertion homozygotes had less than half the proviral concentration of other genotypes (P < 0.0001). Future work will test additional breeds, management conditions and viral subtypes, and identify functional properties of the haplotype this deletion variant tracks. To our knowledge, this is the first genetic variant consistently associated with host control of OvLV post-infection in multiple sheep flocks.

Review of equine piroplasmosis.

Equine piroplasmosis is caused by one of 2 erythrocytic parasites Babesia caballi or Theileria equi. Although the genus of the latter remains controversial, the most recent designation, Theileria, is utilized in this review. Shared pathogenesis includes tick-borne transmission and erythrolysis leading to anemia as the primary clinical outcome. Although both parasites are able to persist indefinitely in their equid hosts, thus far, only B. caballi transmits across tick generations. Pathogenesis further diverges after transmission to equids in that B. caballi immediately infects erythrocytes, whereas T.equi infects peripheral blood mononuclear cells. The recent re-emergence of T.equi in the United States has increased awareness of these tick-borne pathogens, especially in terms of diagnosis and control. This review focuses in part on factors leading to the re-emergence of infection and disease of these globally important pathogens.

Evaluation of mass spectrometric methods for detection of the anti-protozoal drug imidocarb.

Imidocarb [N,N'-bis[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]urea, C(19)H(20)N(6)O(1), m.w. 348.41] is a symmetrical carbanilide derivative used to treat disease caused by protozoans of the Babesia genus. Imidocarb, however, is also considered capable of suppressing Babesia-specific immune responses, allowing Babesia-positive horses to pass a complement fixation test (CFT) without eliminating the infection. This scenario could enable Babesia-infected horses to pass CFT-based importation tests. It is imperative to unequivocally identify and quantify equine tissue residues of imidocarb by mass spectrometry to address this issue. As a pretext to development of sensitive tissue assays, we have investigated possibilities of mass spectrometric (MS) detection of imidocarb. Our analyses disclosed that an unequivocal mass spectral analysis of imidocarb is challenging because of its rapid fragmentation under standard gas chromatography (GC)-MS conditions. In contrast, solution chemistry of imidocarb is more stable but involves distribution into mono- and dicationic species, m/z 349 and 175, respectively, in acid owing to the compound's inherent symmetrical nature. Dicationic imidocarb was the preferred complex as viewed by either direct infusion-electrospray-MS or by liquid chromatography (LC)-MS. Dicationic imidocarb multiple reaction monitoring (MRM: m/z 175 → 162, 145, and 188) therefore offer the greatest opportunities for sensitive detection and LC-MS is more likely than GC-MS to yield a useful quantitative forensic analytical method for detecting imidocarb in horses.

Dynamics of bovine spleen cell populations during the acute response to Babesia bovis infection: an immunohistological study.

The spleen is a critical organ in defence against haemoparasitic diseases like babesiosis. Many in vitro and ex vivo studies have identified splenic cells working in concert to activate mechanisms required for successful resolution of infection. The techniques used in those studies, however, remove cells from the anatomical context in which cell interaction and trafficking take place. In this study, an immunohistological approach was used to monitor the splenic distribution of defined cells during the acute response of naïve calves to Babesia bovis infection. Splenomegaly was characterized by disproportionate hyperplasia of large versus small leucocytes and altered distribution of several cell types thought to be important in mounting an effective immune response. In particular, the results suggest that the initial crosstalk between NK cells and immature dendritic cells occurs within the marginal zone and that immature dendritic cells are first redirected to encounter pathogens as they enter the spleen and then mature as they process antigen and migrate to T-cell-rich areas. The results of this study are remarkably similar to those observed in a mouse model of malarial infection, suggesting these dynamic events may be central to the acute response of naïve animals to haemoparasitic infection.

Ovine progressive pneumonia virus capsid antigen as found in CD163- and CD172a-positive alveolar macrophages of persistently infected sheep.

In situ detection of ovine progressive pneumonia virus (OPPV) and the phenotypic identification of the cells that harbor OPPV have not been described for the OPPV-affected tissues, which include lung, mammary gland, synovial membranes of the carpal joint, and choroid plexus of the brain. In this study, the authors first developed a single enzyme-based automated immunohistochemical (IHC) analysis for detection of OPPV capsid antigen (CA) on OPPV-affected tissues, using 2 anti-CAEV CA monoclonal antibodies, 5A1 and 10A1, and 2 enzyme-based IHC systems. Out of 10 naturally and persistently OPPV-infected ewes, OPPV CA was detected in intercellular regions of the carpal synovial membrane of 1 ewe, in cells resembling alveolar macrophages and pulmonary interstitial macrophages in lung tissue of 3 ewes, and in mammary alveolar cells of 1 ewe. Furthermore, dual enzyme-based automated IHC analyses revealed that OPPV CA was predominantly detected in CD172a- or CD163-positive alveolar macrophages of the lungs and mammary gland. That anti-inflammatory (CD163) and downregulatory (CD172a) types of alveolar macrophage harbor OPPV CA leads to the possibility that during persistent infection with OPPV, the host alveolar macrophage might serve to limit inflammation while OPPV persists undetected by the host adaptive immune response in the lung and mammary gland.

Scrapie resistance in ARQ sheep.

Variation in the ovine prion protein amino acid sequence influences scrapie progression, with sheep homozygous for A(136)R(154)Q(171) considered susceptible. This study examined the association of survival time of scrapie-exposed ARQ sheep with variation elsewhere in the ovine prion gene. Four single nucleotide polymorphism alleles were associated with prolonged survival. One nonsynonymous allele (T112) was associated with an additional 687 days of survival for scrapie-exposed sheep compared to M112 sheep (odds ratio, 42.5; P = 0.00014). The only two sheep homozygous for T112 (TARQ) did not develop scrapie, suggesting that the allelic effect may be additive. These results provide evidence that TARQ sheep are genetically resistant to development of classical scrapie.

Characterization of Anaplasma phagocytophilum major surface protein 5 and the extent of its cross-reactivity with A. marginale.

Major surface protein 5 (Msp5) of Anaplasma marginale is highly conserved in the genus Anaplasma and the antigen used in a commercially available competitive enzyme-linked immunosorbent assay (cELISA) for serologic identification of cattle with anaplasmosis. This study analyzes the degrees of conservation of Msp5 among various isolates of Anaplasma phagocytophilum and the extent of serologic cross-reactivity between recombinant Msp5 (rMsp5) of Anaplasma marginale and A. phagocytophilum. The msp5 genes from various isolates of A. phagocytophilum were sequenced and compared. rMsp5 proteins of A. phagocytophilum and A. marginale were used separately in an indirect ELISA to detect cross-reactivity in serum samples from humans and dogs infected with A. phagocytophilum and cattle infected with A. marginale. Serum samples were also tested with a commercially available competitive ELISA that uses monoclonal antibody ANAF16C1. There were 100% sequence identities in the msp5 genes among all of the A. phagocytophilum isolates from the United States and a horse isolate from Sweden. Sheep isolates from Norway and dog isolates from Sweden were 99% identical to one another but differed in 17 base pairs from the United States isolates and the horse isolate. Serologic cross-reactivity was identified when serum samples from cattle infected with A. marginale were reacted with rMsp5 of A. phagocytophilum and when serum samples from humans and dogs infected with A. phagocytophilum were reacted with rMsp5 of A. marginale in an indirect-ELISA format. Serum samples from dogs or humans infected with A. phagocytophilum did not cross-react with rMsp5 of A. marginale when tested with the commercially available cELISA. These results suggest that rMsp5 of A. phagocytophilum is highly conserved among United States and European isolates and that serologic distinction between A. phagocytophilum and A. marginale infections cannot be accomplished if rMsp5 from either organism is used in an indirect ELISA.

The innate immune response in calves to Boophilus microplus tick transmitted Babesia bovis involves type-1 cytokine induction and NK-like cells in the spleen.

The innate immune response to Babesia bovis infection in cattle is age-related, spleen-dependent and, in stabilate inoculated calves, has type-1 characteristics, including the early induction of IL-12 and IFN-gamma. In this study with three calves, parameters of innate immunity were followed for 2 weeks after tick transmission of B. bovis. Each calf survived the acute disease episode without drug intervention, and responded with increased levels of plasma interferon-gamma and type-1 cytokine expression, monocyte/macrophage activation, and CD8+ cellular proliferation in the spleen. The proliferating CD8+ population consisted primarily of NK-like cells, and the expansion occurred in parallel with an increase in IL-15 mRNA expression in the spleen.

Prime-boost vaccination with plasmid DNA encoding caprine-arthritis encephalitis lentivirus env and viral SU suppresses challenge virus and development of arthritis.

This study evaluated the efficacy of prime-boost vaccination for immune control of caprine arthritis-encephalitis virus (CAEV), a macrophage tropic lentivirus that causes progressive arthritis in the natural host. Vaccination of Saanen goats with pUC-based plasmid DNA expressing CAEV env induces T helper type 1 (Th1) biased immune responses to vector-encoded surface envelope (SU), and the plasmid-primed Th1 response is expanded following boost with purified SU in Freund's incomplete adjuvant (SU-FIA) (J. C. Beyer et al., 2001, Vaccine 19, 1643-1651). Four goats vaccinated with env expression plasmids and boosted with SU-FIA were challenged intravenously with 1 x 10(4) TCID(50) of CAEV at 428 days after SU-FIA boost and evaluated by immunological, virological, and disease criteria. Controls included two goats primed with pUC18 and eight unvaccinated goats. Goats receiving prime-boost vaccination with CAEV env plasmids and SU-FIA became infected but suppressed postchallenge virus replication, provirus loads in lymph node, and development of arthritis for at least 84 weeks.

Validation of a competitive enzyme-linked immunosorbent assay for diagnosing Babesia equi infections of Moroccan origin and its use in determining the seroprevalence of B. equi in Morocco.

A highly specific and sensitive competitive enzyme-linked immunosorbent assay for detection of specific antibody to Babesia equi in serum from equids was validated for use in Morocco. The assay is based on the specific inhibition of binding of a monoclonal antibody to a conserved epitope within a recombinant parasite peptide by serum from infected animals. The assay was compared to an established indirect immunofluorescence assay, with a concordance of 91%. The assay was used to determine seroprevalence for B. equi infections in donkeys and horses throughout Morocco. A total of 578 sera (163 horses and 415 donkeys) from 6 locations representing different bioclimatic regions were assayed. An analysis of variance, indicated no significant effect of location; however, donkeys were significantly more likely than horses to be seropositive. Management conditions contribute to greater tick infestations and thus Babesia exposure in donkeys than in horses.

Immunostimulatory CpG-modified plasmid DNA enhances IL-12, TNF-alpha, and NO production by bovine macrophages.

The immunogenicity of DNA vaccines is partially attributable to the adjuvant properties of bacterial plasmid DNA (pDNA) for B lymphocytes and professional antigen-presenting cells. In mice, modification of immunostimulatory sequences (ISSs), including CpG motifs, in pDNA vectors or oligodeoxynucleotides can increase or decrease their adjuvant properties. ISSs that stimulate optimal responses reportedly differ for murine and human leukocytes. We have previously characterized the mitogenic properties of oligodeoxynucleotides containing one AACGTT motif for bovine B lymphocytes. We now define cytokine responses by macrophages stimulated with pDNA engineered to contain an ISS comprising two AACGTT motifs. Macrophages activated with CpG-modified pDNA secreted significantly more interleukin-12, tumor necrosis factor-alpha, and nitric oxide than macrophages stimulated with unmodified pDNA or modified pDNA that contained nucleotides scrambled to remove CpG motifs. Engineered CpG-pDNA or CpG-oligodeoxynucleotides should be useful as vaccines or adjuvants to promote the enhanced type 1 responses important for protection against intracellular pathogens.

Long-term in vivo depletion of functional CD4+ T lymphocytes from calves requires both thymectomy and anti-CD4 monoclonal antibody treatment.

In vivo depletion of lymphocyte subsets is a direct approach used for dissection of the mechanisms of protective immunity. Long-term in vivo depletion of bovine T lymphocyte subpopulations with monoclonal antibody (mAb) treatment alone has been difficult to achieve. The objective of this study was to determine whether both thymectomy and anti-CD4 mAb treatment would optimize long-term in vivo depletion of functional bovine CD4+ T lymphocytes. Calves were thymectomized and treated with high doses of anti-CD4 mAb (approximately 5 mg/kg) over 4 days followed by subsequent lower doses (approximately 0.3 mg/kg) administered twice weekly for an additional 7 weeks. Depletion of CD4+ T lymphocytes from blood, spleen and peripheral lymph nodes was significantly improved in thymectomized calves compared to thymus-intact anti-CD4 mAb-treated calves. Significant differences in percentages of CD4+ T lymphocytes between thymectomized and thymus-intact calves were sustained for the duration of the 8-week study. Depletion of CD4+ T lymphocytes from thymectomized calves resulted in complete abrogation of lymphoproliferative responses to ovalbumin. In addition, thymectomized calves treated with anti-CD4 mAb had significantly reduced immunoglobulin G1 and no detectable immunoglobulin G2 ovalbumin-specific antibody responses compared to thymus-intact anti-CD4 mAb-treated calves. The results of this study demonstrate that both thymectomy and treatment with anti-CD4 mAb are required for long-term in vivo depletion of functional bovine CD4+ T lymphocytes.

Use of repetitive DNA elements to define genetic relationships among Anaplasma marginale isolates.

Anaplasma marginale genomic DNA was tested for the presence of repetitive extragenic palindromic (REP) and enterobacterial repetitive intergenic consensus (ERIC)-like sequences in order to evaluate the genetic diversity of multiple A. marginale isolates. A. marginale isolates were obtained from cattle of six different states of Brazil, from the US and an Anaplasma centrale strain was obtained from Uruguay. Patterns obtained from A. marginale isolates varied from 14 to 17 fragments by REP-polymerase chain reaction (PCR) and 6 to 14 fragments by ERIC-PCR. All A. marginale isolates presented a 0.75-kb fragment by REP and two common fragments (0.38 and 1.0 kb) by ERIC-PCR. These two fragments were not detectable in A. centrale. Both methods produced similar patterns (80%) among A. marginale isolates obtained from the same region, although some isolates within regions shared less similarity. Isolates from Parana and Pernambuco, were differentiated by these methods. The study demonstrates the presence of ERIC and REP-like elements in A. marginale isolates and shows that A. marginale isolates and strains can be differentiated by these methods.

Prp-c and Prp-Sc at the fetal-maternal interface.

Scrapie is a naturally occurring prion (PrP) disease causing a fatal neurodegenerative disorder in sheep and goats. Previous studies suggest that scrapie is transmitted naturally through exposure to the scrapie agent in wasted placentas of infected ewes. This study determined the distribution and biochemical properties of PrP cellular (PrP-C) and the distribution of PrP scrapie (PrP-Sc) in reproductive, placental, and selected fetal tissues and fetal fluids in sheep. Glycosylated, N-terminally truncated, proteinase K-sensitive PrP-C with apparent molecular masses of 23-37 kDa was present in reproductive, placental, and fetal tissues and fetal fluids. PrP-C was low or undetectable in intercotyledonary chorioallantois, amnion, urachus, amniotic fluid, and fetal urine. In pregnant ewes, cotyledonary chorioallantois, allantoic fluid, and caruncular endometrium contained higher levels of PrP-C than did intercaruncular endometrium, myometrium, oviduct, ovary, fetal bladder, or fetal kidney. Caruncular endometrial PrP-C was up-regulated during pregnancy. Despite the wide distribution of PrP-C in reproductive, placental, and selected fetal tissues and fetal fluid, PrP-Sc was detected only in caruncular endometrium and cotyledonary chorioallantois of pregnant scrapie-infected ewes. The embryo/fetus may not be exposed to scrapie in utero because it is separated physically from PrP-positive allantois and chorioallantois by PrP-negative amnion.

Efficient use of a small genome to generate antigenic diversity in tick-borne ehrlichial pathogens.

Ehrlichiae are responsible for important tick-transmitted diseases, including anaplasmosis, the most prevalent tick-borne infection of livestock worldwide, and the emerging human diseases monocytic and granulocytic ehrlichiosis. Antigenic variation of major surface proteins is a key feature of these pathogens that allows persistence in the mammalian host, a requisite for subsequent tick transmission. In Anaplasma marginale pseudogenes for two antigenically variable gene families, msp2 and msp3, appear in concert. These pseudogenes can be recombined into the functional expression site to generate new antigenic variants. Coordinated control of the recombination of these genes would allow these two gene families to act synergistically to evade the host immune response.

Sensitivity and specificity of the complement fixation test for detection of cattle persistently infected with Anaplasma marginale.

The complement fixation (CF) test commonly is used to identify cattle infected with Anaplasma marginale prior to interstate or international movement. Estimates of the accuracy of the CF test in detecting animals persistently infected with A. marginale vary widely. In this study, the sensitivity and specificity of the CF test for detection of carrier animals was determined using serum from 232 cattle previously defined as A. marginale positive or negative by nested polymerase chain reaction methods and hybridization. Considering results from 2 independent laboratories and interpreting a 1:5 suspect reaction as positive, the best estimate of CF test sensitivity was 20%, with a specificity of 98%. Using a 1:10 cutoff, sensitivity decreased to 14% and specificity increased to 99%. Results of this study indicate that the CF test is ineffective for identifying cattle persistently infected with A. marginale and thus is inadequate for anaplasmosis regulatory and surveillance programs.

Cellular prion protein is expressed on peripheral blood mononuclear cells but not platelets of normal and scrapie-infected sheep.

BACKGROUND AND OBJECTIVES: Transmissible spongiform encephalopathies (TSEs) including sheep scrapie are characterized by the conversion of a normal, cellular prion protein (PrPc) to an abnormal protease-resistant form (PrPSc). Like human peripheral blood, the peripheral blood of scrapie-infected sheep remains one possible source of disease transmission. As a first step in understanding the disease requirements in the natural scrapie host, the presence of PrPc was evaluated in peripheral blood cells from five normal and five scrapie-infected Suffolk sheep. DESIGN AND METHODS: Live peripheral blood cells from normal and scrapie-infected sheep were analyzed for the presence of PrP using flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: PrP mRNA was detected in peripheral blood mononuclear cells (PBMC) but not in platelets or granulocytes. Consistent with PrP mRNA expression, cell-surface expressed PrP was detected on PBMC, but was not detected on granulocytes, platelets, or erythrocytes. Two-color flow cytometric analysis of PBMC specific phenotypes revealed that regardless of scrapie-status, expression of PrP was significantly higher on B2 positive B-lymphocytes than on CD4, CD8, WC1 positive T-lymphocytes or CD14 positive monocytes. In addition, PrP expressed on PBMC from normal and scrapie-infected sheep was sensitive to proteinase K (PK)and phosphatidylinositol-specific phospholipase C (PIPLC). INTERPRETATION AND CONCLUSIONS: Regardless of the scrapie-status of the sheep, resting PBMC transcribe PrPc and express PrPc as a cell-surface protein sensitive to both PK and PIPLC. Because of the abundance of PrPc on PBMC, future diagnostic tests using PK and PIPLC to discriminate between protease sensitive and resistant PrP must be carefully evaluated.