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.

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.

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.

PrP(c) mRNA, but not PrP(Sc) is found in the salivary glands of scrapie-infected sheep.

Transmission studies in transmissible spongiform encephalopathies (TSEs) have become increasingly important due to the possible transmission of bovine spongiform encephalopathy to humans resulting in new variant Creutzfeldt-Jacob disease. The horizontal transmission of scrapie, a TSE of sheep, is poorly understood. Possible sources of horizontal transmission are the submandibular and parotid salivary glands. TSEs like natural sheep scrapie are characterized by the conversion of a normal protease sensitive prion protein, PrP(c), to an abnormal protease resistant prion protein, PrP(Sc). Since the presence of PrP(Sc) is an indicator of disease, the salivary glands of scrapie-infected sheep were examined for the presence of PrP(Sc). Although PrP(c) mRNA was detected in the salivary glands, PrP(Sc) was not found in the salivary glands of scrapie-infected sheep. These data suggest that the salivary glands are unlikely sources of horizontal transmission of natural sheep scrapie.

Association of GP51 expression and persistent CD5+ B-lymphocyte expansion with lymphomagenesis in bovine leukemia virus infected sheep.

Alterations in the circulating CD5+ B-lymphocyte population, in vitro GP51 expression, and in vivo tax/rex expression that may precede lymphomagenesis were characterized prospectively in ten experimentally BLV-infected sheep. Infection with pathogenetic BLV resulted in a significant expansion of the circulating CD5+ B-lymphocyte population in six infected sheep. Of the remaining four infected sheep that did not have persistently elevated CD5+ B-lymphocyte counts, three developed lymphoid neoplasia within 14 months post-inoculation. Neoplastic cells from two of these three sheep were CD5- B-lymphocytes, while cells from the third were CD5+ B-lymphocytes. In vitro GP51 expression was a consistent feature of circulating lymphocytes from all three sheep developing tumors, but high level tax/rex gene transcription was not detected in circulating lymphocytes prior to lymphomagenesis. Neither in vitro GP51 expression nor high level tax/rex gene transcription was associated with expansion of the CD5+ B-lymphocyte population in sheep with significantly elevated CD5+ B-lymphocyte counts. These observations indicate that BLV infection in sheep results in expansion of the circulating CD5+ B-lymphocyte population, and that this expansion is not required for the subsequent development of BLV-associated lymphoid neoplasia.

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.

Genetic and biochemical analysis of erythrocyte-stage surface antigens belonging to a family of highly conserved proteins of Babesia equi and Theileria species.

Erythrocyte-stage Babesia equi expresses a 34-kDa immunodominant antigen recognized by antibody from persistently infected horses worldwide. This erythrocyte-stage surface protein, equi merozoite antigen-1 (EMA-1) is encoded by a single copy gene, and was previously shown to share 33% amino acid identity with similar sized proteins of Theileria sergenti and T. buffeli. A mean homology of 31% amino acid identity extends to similar sized proteins of T. parva, T. annulata and T. mutans. Genomic and cDNA copies of a second B. equi gene, ema2 were cloned. The single copy ema2 gene encodes a 30-kDa protein (EMA-2) that shares 52% amino acid identity with EMA-1. EMA-2 also shares a mean amino acid identity of 31% with proteins of similar molecular mass from Theileria species. EMA-1 and EMA-2 each contain a glycosylphosphatidylinositol anchor. These unique erythrocyte-stage surface proteins of B. equi and Theileria species lack antigenic repeats, and excluding the signal peptide, contain one or no cysteines. Consistent with the hypothesis that this family of proteins interacts with the erythrocyte surface, the T. species proteins possess a basic isoelectric point. The B. equi proteins have acidic isoelectric points, but 24-mer peptides within them have strongly basic net charges.

An in vivo model to investigate lymphocyte-mediated immunity during acute hemoparasitic infections. Use of a monoclonal antibody to selectively deplete CD4+ T lymphocytes from thymectomized calves.

Thymectomized calves were selectively depleted of CD4+ T lymphocytes with a monoclonal antibody (mAb) specific for the bovine CD4 monomer (ILA-11). Calves were treated with high loading doses of ILA-11 during the first week of the study then treated with subsequent lower maintenance doses. Depletion of CD4+ T lymphocytes was assessed weekly by flow cytometric analysis of PBMC and mononuclear cells from lymph node and spleen biopsies. Treatment with high doses of ILA-11 resulted in rapid and marked depletion of CD4+ T lymphocytes from the peripheral blood, peripheral lymph nodes, and spleen. Although CD4+ T lymphocytes slowly returned to the peripheral blood, peripheral lymph nodes, and spleen by day 21 post-treatment, the numbers of CD4+ T lymphocytes in depleted calves remained below pre-depletion levels for the duration of the study. CD4+ T lymphocytes failed to be effectively depleted from a non-thymectomized calf treated with the mAb ILA-11. Development of a T lymphocyte depletion model in thymectomized calves will permit testing of the hypothesis that CD4+ T lymphocytes and IFN-gamma are required in cattle for control of acute anaplasmosis. In subsequent planned studies, thymectomized calves depleted of CD4+ T lymphocytes will be experimentally infected with A. marginale and parameters of disease compared between depleted and non-depleted calves.

Malignant catarrhal fever virus. Characterization of a United States isolate and development of diagnostic assays.

Malignant catarrhal fever (MCF) is a severe lymphoproliferative disease of certain domestic and wild ruminants. Two distinct but closely related viruses cause clinically indistinguishable syndromes in susceptible ruminant species: wildebeest-associated MCF virus (WA-MCFV) and sheep-associated MCF virus (SA-MCFV). Neither the pathogenesis nor the epidemiology of SA-MCF is understood, primarily because of a lack of adequate detection methods for the etiologic agent or antibody against that agent. Work designed to develop these tests has been under way in our laboratory. To obtain basic information about the virus, the in vitro growth properties of a US isolate of MCF virus were studied and its major viral proteins identified and characterized by a panel of monoclonal antibodies generated against the isolate. A monoclonal antibody to a broadly conserved epitope of MCF virus was identified, and a competitive-inhibition ELISA (CI-ELISA) was developed for detection of anti-MCF antibody in sheep and other ruminants. The monoclonal antibody (15-A) reacted with an epitope located on a glycoprotein complex, which was present in all isolates of MCF virus examined. Antibody from a wide variety of ruminants infected with MCF virus of both sheep and wildebeest origin competed with the monoclonal antibody 15-A for the epitope, which was not present on 14 other common ruminant viruses. The assay detected antibody in inapparently infected sheep, and in cattle, deer, and bison with clinical MCF. A PCR assay for DNA of the sheep-associated virus was developed, based on previously reported primers. Comparative studies demonstrated that the CI-ELISA was specific for MCFV antibody and that the PCR was more reliable for diagnosis of clinical MCF.

Prospective study for the detection of Anaplasma marginale Theiler, 1911 (Rickettsiales: Anaplasmataceae) in Costa Rica.

A prospective study was conducted to assess the dynamics of the infection and host response to Anaplasma marginale in one closed herd in the dry tropical forest of Costa Rica. The study subjects were the dams and their calves born during 1 breeding season (1995-1996). All cows were sampled at 3 month intervals for antibody detection using a competitive ELISA (cELISA) and for antigen detection using PCR/nonradioactive probe assay. All 24 calves born during the study were individually identified at birth and subsequently sampled each month for PCR and cELISA. Ticks were identified from all animals throughout the entire study period. The results from this study confirmed that the cELISA is a reliable assay for identifying new and carrier infections and that carrier infections can exist at levels below that detectable by PCR. In addition, it was demonstrated that calves born in this region will most likely be exposed to Anaplasma within the first 6 months of age.

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.

Caprine arthritis-encephalitis lentivirus (CAEV) challenge of goats immunized with recombinant vaccinia virus expressing CAEV surface and transmembrane envelope glycoproteins.

This study evaluated infection and disease following caprine arthritis-encephalitis lentivirus (CAEV) challenge of goats with existent immune response to CAEV surface and transmembrane envelope glycoproteins. Six Saanen goats were vaccinated three times with recombinant vaccinia virus rWR63 expressing glycoproteins encoded by the CAEV-63 envelope gene. Two goats were immunized with rWRSC11, a control vaccinia virus derived from the pSC11 vaccinia expression plasmid without the CAEV envelope gene. One pair of rWR63 vaccinated goats received a booster immunization with recombinant surface glycoprotein in Freund's complete adjuvant, a second pair was boosted by intravenous inoculation with rWR63, and the third pair was boosted by immunization with HPLC purified native CAEV surface glycoprotein in Freund's complete adjuvant. All six goats vaccinated with rWR63 developed antibody responses to CAEV envelope glycoproteins; however, CAEV-63 neutralizing antibody was not detected. Neither of the rWRSC11-vaccinated goats developed CAEV reactive antibody. All goats were challenged by intravenous inoculation with 10(6) TCID50 CAEV-63. All goats became infected following challenge infection, shown by detection of serum antibody to CAEV core proteins and virus isolation. Existent CAEV-63 immune responses did not detectably alter the severity of inflammatory joint lesions at 24 weeks postchallenge.

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.

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.

Conserved recombinant antigens of Anaplasma marginale and Babesia equi for serologic diagnosis.

The competitive inhibition ELISA (CI-ELISA) format overcomes problems associated with antigen purity since the specificity of the CI-ELISA depends solely on the monoclonal antibody (mAb) used. Therefore, the CI-ELISA format is well suited for use with recombinant antigens. Molecular clones expressing a conserved 19 kDa protein of Anaplasma marginale and a 34 kDa protein of Babesia equi were derived and characterized. The 19 kDa A. marginale protein, conserved in all recognized Anaplasma species, and present in the infected tick salivary gland, was reactive with all bovine immune sera tested. The 34 kDa B. equi protein contains a protein epitope bound by antibody in equine immune sera from 19 countries. Monoclonal antibodies reactive with these proteins were derived and applied with recombinant copies of the 19 kDa A. marginale and 34 kDa B. equi proteins in a CI-ELISA format.

Experimental infection of cattle with the agents of transmissible mink encephalopathy and scrapie.

Cattle are susceptible to experimental infection with the Stetsonville isolate of the transmissible mink encephalopathy (TME) agent. To determine if they are susceptible to other TME isolates, two groups of calves were inoculated intracerebrally with homogenate of mink brain containing the Hayward isolate or the Blackfoot isolate. For comparison, a third group was inoculated with a brain homogenate from a steer infected with the Stetsonville isolate in its primary cattle passage and a fourth group was inoculated with a pool of brain homogenate from three cattle experimentally infected with a sheep and goat scrapie agent in its primary cattle passage. Clinical signs of neurological disease appeared in each steer of every group between 15 and 25 months after inoculation. An encephalopathy characterized by severe spongiform change and pronounced astrocytosis occurred in the three groups inoculated with the TME agent. In contrast, the neurohistological changes in the steers inoculated with the cattle-passaged scrapie agent were slight and subtle. Analysis of the octapeptide repeat region of the bovine protease-resistant protein (PrP) gene showed that variations in incubation period, clinical signs, and neurohistological changes were unrelated to the homozygous or heterozygous condition of six or six/five octapeptide repeats.