Dental pathology in conventionally fed and pasture managed dairy cattle.

Healthy teeth are important in the first stages of digestion for dairy cattle, yet little is known about bovine dental disease. This study aimed to investigate dental pathology of dairy cattle in two parts. First dairy cattle cadaver heads (n=11) were examined at the time of culling. Second, the authors performed oral exams in cattle fed a total mixed ration (TMR) (n=200) and pasture-based (n=71) grazing cattle. Cadaver heads were imaged using radiography and computed tomography before gross dissection to study dental anatomy and pathology. The most prevalent dental abnormalities were excessive transverse ridging of the occlusal surface, the presence of diastemas and third molar dental overgrowths (M3DO) in cadaver heads. Average thickness of subocclusal dentine ranged from 3.5 mm to 5.8 mm in cheek teeth but was >10 mm in maxillary teeth with M3DO. Radiographic findings were compared with oral examinations in live cattle. Prevalence of M3DO upon oral examination was 19 per cent and 28 per cent in herds of cattle fed a TMR diet and 0 per cent in a herd of grazing cattle. Dental abnormalities are prevalent in dairy cattle but due to thin subocclusal dentine in the cheek teeth, established equine dental treatment methodology is not appropriate for bovine cheek teeth with the exception of those that have developed M3DO.

S-adenosylmethionine synthetase in cultured normal and oncogenically-transformed human and rat cells.

We have investigated the enzymatic formation of S-adenosylmethionine in extracts of a variety of normal and oncogenically-transformed human and rat cell lines which differ in their ability to grow in medium in which methionine is replaced by its immediate precursor homocysteine. We have localized the bulk of the S-adenosylmethionine synthetase activity to the post-mitochondrial supernatant. We show that in all cell lines a single kinetic species exists in a dialyzed extract with a Km for methionine of about 3-12 microM. In selected lines we have demonstrated a requirement for Mg2+ in addition to that needed to form the Mg X ATP complex for enzyme activity and have shown that the enzyme can be regulated by product feedback inhibition. Because we detect no differences in the enzymatic ability of these cell extracts to utilize methionine for S-adenosylmethionine formation in vitro, we suggest that the failure of oncogenically-transformed cell lines to grow in homocysteine medium may result from the decreased methionine pools in these cells or from the loss of ability of these cells to properly metabolize homocysteine, adenosine, or their cellular product S-adenosylhomocysteine.

Identification of an amino acid on VP2 that affects neutralization of bluetongue virus serotype 10.

Genome segment 2, coding for the VP2 protein, of a neutralization resistant variant was compared to segment 2 of the bluetongue virus (BTV) serotype 10 parent from which the variant was derived. Full-length double-stranded cDNA of BTV segment 2 RNA, which was prepared by reverse transcription, was used as template to prepare overlapping subgenomic cDNA products by PCR. Purified PCR cDNA fragments were sequenced by the dideoxy chain termination reaction. Each base was determined an average of 3.7 times. Comparison of the sequence of segment 2 of the neutralization resistant variant with segment 2 of the parental virus showed two base changes, one of which resulted in a changed amino acid. This change was in a different region of VP2 than those previously reported in other neutralization resistant variants of BTV. In addition to this change, both the parental virus and the variant virus differed in two amino acids from the previously published sequence of VP2 of BTV serotype 10.

Verification of bluetongue virus S9 segment nucleotide sequences.

During the course of our bluetongue virus (BTV) nucleic acid sequence investigations, conflicts among United States (US) prototype BTV S9 genome segment sequences deposited in GenBank were noted. In order to rectify these inter-laboratory discrepancies, the S9 segments of Arthropod-borne Animal Diseases Research Laboratory (ABADRL)-stored US prototype BTV 2, BTV 10, BTV 11, BTV 13, and BTV 17 isolates were resequenced. Our S9 sequences, determined by direct sequencing of full-length reverse transcriptase-polymerase chain reaction (RT-PCR) generated amplicons, shared 99% or greater nucleotide identity with one or more respective S9 sequences previously reported. Possible sources of remaining unsupported US prototype BTV S9 sequences were evaluated by amplifying and sequencing the S9 segments of BTV 2 Ona A strain, South African (SA) prototype BTV 1, BTV 2, and BTV 4 strains, and the North American (NA) prototype epizootic hemorrhagic disease virus (EHDV) serotype 2 (Alberta) strain. Comparative analysis using these S9 sequences, as well as sequences of US BTV 2 field isolates, identified potential contributors to inter-laboratory sequence disagreements.

Geographical genetic variation in the gene encoding VP3 from the Alberta isolate of epizootic hemorrhagic disease virus.

The complete nucleic acid and deduced amino acid sequences of gene segment 3 and the encoded VP3 from the North American, Alberta isolate of epizootic hemorrhagic disease virus serotype 2 (EHDV-2) are reported. Complementary DNA corresponding to segment 3 was 2768 nucleotides in length with an open reading frame of 2697 base pairs which encoded a VP3 polypeptide of 899 amino acid residues. Sequence comparison with genome segment 3 and VP3 from the Australian strain of EHDV-2 indicated genotypic and phenotypic homologies of 79% and 94%, respectively. Two North American field isolates of EHDV-2, as well as EHDV-1 (New Jersey isolate), had virtually identical homology to the Alberta isolate. Sequence analysis delineated North American EHDV strains as members of a genetically homologous and geographically distinct group of orbiviruses (topotype). The data support the hypothesis that geographic isolation between North American and Australian orbiviruses has permitted the viral topotypes to maintain their genetic distinctness.

The smallest gene of the orbivirus, epizootic hemorrhagic disease, is expressed in virus-infected cells as two proteins and the expression differs from that of the cognate gene of bluetongue virus.

The smallest gene (S10) of the virus of epizootic hemorrhagic disease of deer (EHD, serotype 2) is expressed as two proteins in virus-infected cells. By contrast, the non-structural proteins (NS3 and NS3A) encoded in the smallest gene of bluetongue (BT) viruses are difficult to detect in virus-infected cells. The nucleotide sequence of S10 of EHDV-2 contains two in-frame initiation codons which allow for translation of proteins of mol. wt. 25503 and 23921 analogous to NS3 and NS3A of BT viruses. The S10 genes of BT viruses are highly conserved (82%-99%); the nucleotide sequence similarity of S10 of EHDV-2 and BT viruses is about 64%. Some structural features of NS3 and NS3A are conserved in the two viruses, despite the divergence in the amino acid sequences of the proteins. The hydrophobic domains of the proteins and the putative transmembrane sequences are conserved, as are potential glycosylation sites in the proteins. A cluster of proline residues, which is conserved at residues 36-50 in all of the published sequences of NS3 of BT viruses, is conserved exactly in the alignment of the sequence of NS3 of EHDV-2 with that of the BT viruses. An explanation for the differences in expression of NS3/NS3A in EHD and BT viruses was not evident in comparing the nucleotide sequences of S10 of the viruses.

Phylogenetic relationships of bluetongue viruses based on gene S7.

Previous phylogenetic analyses based on bluetongue virus (BTV) gene segment L3, which encodes the inner core protein, VP3, indicated a geographical distribution of different genotypes. The inner core protein, VP7, of BTV has been identified as a viral attachment protein for insect cell infection. Because the inner core proteins are involved with infectivity of insect cells, we hypothesized that certain VP7 protein sequences are preferred by the insect vector species present in specific geographic locations. We compared the gene segment S7, which encodes VP7, from 39 strains of BTV isolated from Central America, the Caribbean Basin, the United States, South Africa and Australia. For comparison, the S7 sequences from strains of the related orbiviruses, epizootic hemorrhagic disease virus (EHDV) and African horse sickness virus (AHSV) were included. The S7 gene was highly conserved among BTV strains and fairly conserved among the other orbiviruses examined. VP7 sequence alignment suggests that the BTV receptor-binding site in the insect is also conserved. Phylogenetic analyses revealed that the BTV S7 nucleotide sequences do not unequivocally display geographic distribution. The BTV strains can be separated into five clades based on the deduced VP7 amino acid sequence alignment and phylogeny but evidence for preferential selection by available gnat species for a particular VP7 clade is inconclusive. Differences between clades indicate allowable variation of the VP7 binding protein.

Monoclonal antibodies to bluetongue virus define two neutralizing epitopes and a hemagglutinating epitope.

Monoclonal antibodies were used to characterize neutralizing epitopes on VP2 of bluetongue virus serotype 10 (BTV-10). Six neutralizing monoclonal antibodies that immune precipitated VP2 demonstrated two distinct patterns of reactivity in the competitive enzyme-linked immune absorbent assay (ELISA). These results suggest that there are at least two distinct domains of neutralization on VP2 of BTV. Monoclonal antibodies defining the two domains were serotype-restricted in plaque neutralization, immune precipitation or ELISA. One of the two neutralizing domains also demonstrated significant hemagglutinating activity. Both cattle and sheep infected with BTV-10 produce antibodies to the two neutralizing epitopes.

In vitro neutralization of antigenic variants of bluetongue virus is related to in vivo protection.

Neutralization resistant variants of bluetongue virus serotype 10 were selected with a neutralizing monoclonal antibody. Three variants were selected for further characterization. One of these variants was completely resistant to neutralization, while the other two variants showed intermediate resistance to neutralization as compared to the parent virus. The completely resistant variant failed to bind the selecting monoclonal antibody as determined by immune precipitation and enzyme linked immunosorbent assay; whereas, the other two variants bound antibody to a similar extent as the parent virus as determined by these tests. The ability of the variants to bind monoclonal antibody correlated with passive protection in the newborn mouse model. These results indicate that the ability of the virus to bind antibody is directly related to in vivo protection and that in vitro neutralization and in vivo protection are also related.

Immunodetection of bluetongue virus and epizoötic hemorrhagic disease virus in Culicoides variipennis (Diptera: Ceratopogonidae).

The avidin-biotin complex immunoperoxidase system has been used with nitrocellulose membranes to detect bluetongue virus serotypes 10 (BTV-10) and 11 (BTV-11) and epizootic hemorrhagic disease virus (EHDV) serotype 1 (EHDV-1) in individual specimens of Culicoides variipennis (Coquillett). The cross reactivity between BTV-10 and BTV-11 indicates that polyclonal antisera cannot be used to distinguish between flies infected with BTV-10 and BTV-11; however, the procedure can be used to discriminate flies infected with BTV-10 or BTV-11 from flies infected with EHDV-1. Because the test is rapid and sensitive and does not require expensive equipment, it could be used in the field to determine the relative numbers of flies infected with BTV or EHDV.

Sources of variation in an enzyme-linked immunoassay of bluetongue virus in Culicoides variipennis (Diptera: Ceratopogonidae).

An enzyme-linked immunoassay for detecting bluetongue virus in infected Culicoides variipennis was evaluated using a nested analysis of variance to determine sources of experimental error in the procedure. The major source of variation was differences among individual insects (84% of the total variance). Storing insects at -70 degrees C for two months contributed to experimental variation in the ELISA reading (14% of the total variance) and should be avoided. Replicate assays of individual insects were shown to be unnecessary, since variation among replicate wells and plates was minor (2% of the total variance).

Complex interactions between vectors and pathogens: Culicoides variipennis sonorensis (Diptera: Ceratopogonidae) infection rates with bluetongue viruses.

Two laboratory colonies of Culicoides variipennis sonorensis Wirth & Jones were allowed to take blood meals containing the five U.S. serotypes (2, 10, 11, 13, and 17) of bluetongue (BLU) virus. After 14 d of extrinsic incubation, the flies were assayed for the presence of virus using an antigen capture enzyme-linked immunosorbent assay (ELISA). There was a significant effect of the serotype on infection of C. v. sonorensis with BLU virus. There was no significant difference in infection of the two colonies when averaged across the five BLU virus treatments. However, there was a statistically significant interaction between the colonies and the virus serotypes, which was demonstrated by a higher rate of infection of the AA colony with BLU virus serotype 13 and a higher rate of infection of the AK colony with BLU virus serotype 11.

Bluetongue virus in laboratory-reared Culicoides variipennis sonorensis: applications of dot-blot, ELISA, and immunoelectron microscopy.

An avidin-biotin complex (ABC) dot-blot, an antigen capture enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy (IEM) were used to detect bluetongue (BLU) virus or viral antigen or both in adult Culicoides variipennis sonorensis Wirth & Jones. The dot-blot and ELISA procedures detected viral antigen in 10-22% (depending on serotype) of the biting midges infected with BLU-2, BLU-10, BLU-13, and BLU-17 and approximately 68% of the midges infected with BLU-11. IEM analyses revealed BLU virus in salivary glands, fat body, and thoracic muscle tissue from infected insects. There appeared to be selective growth of the virus in salivary gland tissue.

Applications of dot-blot, ELISA, and immunoelectron microscopy to field detection of bluetongue virus in Culicoides variipennis sonorensis: an ecological perspective.

An avidin-biotin complex (ABC) dot-blot, an antigen capture enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy (IEM) were used to detect bluetongue (BLU) virus and viral antigen in field-collected C. varriipennis sonorensis Wirth & Jones from an enzootic BLU area in northeastern Colorado. This is the 1st attempt to apply these immunodiagnostic methods to an epidemiologically relevant, large-scale ecological system. One of the 1,800 midges (0.0005%) was positive by the dot-blot procedure, 2 (0.0011%) were positive by the ELISA, and BLU virus was identified in 8 midges (0.0044%) by IEM. These data are interpreted in context of the "whole system" of the disease to provide a framework for determining the knowledge gaps in our understanding and directing future studies in these areas. Our basic model of BLU ecology suggests that the infection rates found by the diagnostic methods are within expected ranges, thus strongly supporting the proposed ecological model and the work used to parameterize the model. This integration of immunodiagnostic methods and ecology makes it evident that further investigations of daily mortality during the extrinsic incubation period are vital to a better understanding of BLU virus occurrence in Culicoides vector and vertebrate host populations.

Detection of bluetongue virus in Culicoides variipennis (Diptera: Ceratopogonidae) by an antigen capture enzyme-linked immunosorbent assay.

An antigen capture enzyme-linked immunosorbent assay (ELISA) was developed to detect bluetongue virus (BTV) in the vector Culicoides variipennis (Coquillett). This ELISA used polyclonal and monoclonal antibodies in a biotin-avidin immunoperoxidase system to detect BTV in individual adult female flies that had taken an infectious blood meal. Detection of BTV with the ELISA correlated with detection of BTV from the same flies by plaque assay in cell culture. The ELISA was group-specific for the five U.S. serotypes of BTV and did not cross react with closely related epizootic hemorrhagic disease virus.

Seasonal transmission of bluetongue virus by Culicoides sonorensis (Diptera: Ceratopogonidae) at a southern California dairy and evaluation of vectorial capacity as a predictor of bluetongue virus transmission.

Vectorial capacity of Culicoides sonorensis Wirth & Jones for the transmission of bluetongue (BLU) virus was examined at a southern California dairy from January 1995 to December 1997. Insects were collected one to two times per week in five CDC-type suction traps (without light) baited with CO2 at a constant release rate of 1,000 ml/min. BLU virus was detected in midges collected from May through December with an estimated overall infection rate of 0.08%. The BLU virus infection rate of field-captured midges was not correlated with sentinel calf seroconversions to BLU virus. Sentinel calf seroconversions were highly seasonal, occurring from August through November with most calves seroconverting during September and October. Vector competence of field-collected nulliparous flies fed a locally acquired serotype of BLU virus in the laboratory was stable among years (17-23%). Vectorial capacity was strongly correlated with BLU virus transmission (measured by sentinel calf seroconversions) during 1995 and 1996, but not during 1997. Host biting rate estimated for traps nearest to the sentinel calves was the index best correlated with BLU virus transmission for all study years and was most highly correlated with sentinel seroconversions 4 wk later. The utility of vectorial capacity and its component variables is discussed for this system.

T lymphocyte subset alterations following bluetongue virus infection in sheep and cattle.

To determine potential mechanisms of differential disease expression in ruminants infected with bluetongue virus (BTV), clinically normal, BTV-seronegative, yearling sheep and cattle were infected subcutaneously with a standardized insect-source inoculum of BTV serotype 17 (BTV-17) (three infected and one contact control each) or animal adapted BTV serotype 10 (BTV-10) (three sheep only). BTV was isolated from peripheral blood cell components of infected sheep and cattle and all infected animals showed evidence of seroconversion by 14 days post infection (PI). Sheep infected with both serotypes of BTV developed pyrexia, oral lesions, and leukopenia which were most severe on days 7-8 PI. Analysis of peripheral blood mononuclear leukocytes with specific monoclonal antibodies and flow cytometry revealed panlymphocytopenia on day 7 PI. This response was further characterized by an increase in the CD4/CD8 ratio (greater than 3) resultant from a greater decrease in absolute numbers of circulating SBU-T8(CD8+) ("cytotoxic/suppressor") lymphocytes compared to SBU-T4 (CD4)+ ("helper") lymphocytes. SBU-T19+ lymphocytes were also decreased below baseline values on days 5-14 post infection. On day 14 PI there were increased CD8+ lymphocytes and decreased CD4/CD8 ratios (approximately 0.6) in these sheep. Clinical and hematologic changes in cattle infected with BTV-17 were minimal and consisted of mild pyrexia (rectal temperature 103 degrees F) on day 9 PI in two of three infected animals and mild leukopenia on several days PI in one animal. This leukopenia was the result of a pan T lymphocytopenia with CD4/CD8 ratios in the expected range (1-2). Similar to infected sheep, infected cattle did have a shift (decrease, approximately 0.8) in the peripheral CD4/CD8 ratio associated with an increase in circulating BoT8 (CD8)+ lymphocytes on day 14 post infection. Lymphocytes in the peripheral blood of all sheep and cattle infected with BTV-17 proliferated in vitro in response to purified BTV-17. These results confirm and extend those of previous studies that indicate species differences in the hematologic response to an equivalent BTV infection in domestic ruminants.

Development of an enzyme-linked immunosorbent assay for the detection of antibody to epizootic hemorrhagic disease of deer virus.

An enzyme-linked immunosorbent assay has been developed to detect antibodies to epizootic hemorrhagic disease of deer virus (EHDV). The assay incorporates a monoclonal antibody to EHDV serotype 2 (EHDV-2) that demonstrates specificity for the viral structural protein, VP7. The assay was evaluated with sequential sera collected from cattle experimentally infected with EHDV serotype 1 (EHDV-1) and EHDV-2, as well as the four serotypes of bluetongue virus (BTV), BTV-10, BTV-11, BTV-13, and BTV-17, that currently circulate in the US. A competitive and a blocking format as well as the use of antigen produced from both EHDV-1- and EHDV-2-infected cells were evaluated. The assay was able to detect specific antibody as early as 7 days after infection and could differentiate animals experimentally infected with EHDV from those experimentally infected with BTV. The diagnostic potential of this assay was demonstrated with field-collected serum samples from cattle, deer, and buffalo.

Bluetongue virus detection: a safer reverse-transcriptase polymerase chain reaction for prediction of viremia in sheep.

A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the use of the highly toxic methyl mercury hydroxide denaturant.

Detection of bluetongue virus from blood of infected sheep by use of an antigen-capture enzyme-linked immunosorbent assay after amplification of the virus in cell culture.

An antigen-capture ELISA was used to detect bluetongue virus (BTV) from blood of infected sheep. A rabbit-origin capture antibody and a mouse-origin detection antibody combined with biotin-avidin amplification were used for the assay. The antigen-capture ELISA could not detect virus directly from the blood of infected sheep because of low virus titer. To enhance detection, virus from infected blood was amplified in cell culture. Virus could then be detected from cell culture supernatant fluids, using the ELISA. This amplification step increased the sensitivity of the assay comparable to that of assays performed in cell culture measuring cytopathic effects. The ELISA procedure was specific for BTV and did not mistakenly identify the antigenically related epizootic hemorrhagic disease virus. The antigen-capture ELISA permitted indirect quantitation and identification of BTV from the blood of infected sheep.