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  • Author or Editor: Cecelia A. Whetstone x
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SUMMARY

A nested polymerase chain reaction (pcr) test was developed to examine infection with the bovine lentivirus, bovine immunodeficiency-like virus (biv), in cattle. Primers were designed to amplify 2 separate regions of the pol and env segments of the biv genome. Two calves were experimentally infected with an isolate derived from the original strain of biv, R29, or with a recent field isolate, FL491. Serial blood samples were collected and examined by virus isolation, protein immunoblot, and nested pcr. The nested pcr test detected biv infection by 3 days after inoculation, earlier than the other 2 methods, and continued to identify infected cattle 9 to 15.5 months after inoculation, even when results from virus isolation and serology became negative. Nested pcr also detected multiple-size env products in samples obtained later in the infection from the calf that received FL491, giving evidence that viral quasispecies were selected during in vivo replication of the virus. Results indicated that the nested pcr test is more sensitive than virus isolation or serology for the detection of biv infection in cattle.

Free access
in American Journal of Veterinary Research

Summary

The Cooper isolate of bovine herpesvirus-1, which causes abortion in cattle, was used to construct a thymidine kinase- negative (tk- ) deletion mutant virus. Twelve heifers were inoculated iv at 25 to 29 weeks of pregnancy with either tk- or thymidine kinase-positive (tk+ ) Cooper virus. All heifers developed fevers of 1 to 2 C during the first week after inoculation. Temperatures of tk+ inoculates were slightly higher and remained above normal a few days longer than in tk- inoculates. Viremia was detected in 5 of 6 tk+ inoculates and in all 6 tk- inoculates. More virus isolations were made from nasal and vaginal swab specimens of tk+ inoculates than from swab specimens of tk- inoculates. All heifers developed virus neutralizing antibody within 14 days after inoculation and antibody titers were similar between the 2 groups. None of the tk- inoculated heifers aborted and their calves did not have neutralizing antibody at birth. Abortion occurred in 5 of 6 heifers given tk+ virus. All aborted fetuses were infected with bovine herpesvirus-1, as demonstrated by virus isolation or detection of viral antigen in fetal tissues. These results indicate that inactivation of the tk gene reduces abortifacient activity of bovine herpesvirus-1.

Free access
in American Journal of Veterinary Research

SUMMARY

The Cooper isolate of bovine herpesvirus 1 (bhv-1) was used to produce a thymidine kinase-negative ( tk ) recombinant by insertion of a β-galactosidase (bgal) expression cassette into the tk coding region. The recombinant virus (tk bgal+) was tested for abortifacient activity in cattle by inoculation of 5 pregnant heifers at 25 to 29 weeks gestation. Five additional heifers were inoculated with the Cooper tk positive ( tk +) virus to serve as controls. After inoculation, both groups of heifers developed similar febrile responses and neutralizing antibody titers. Virus was isolated from blood of all heifers during the first postinoculation (pi) week, and isolation frequencies were similar for both groups. In contrast, whereas virus was isolated from many of the nasal and vaginal swab specimens of heifers inoculated with tk + virus, only rare virus isolations were made from the heifers given tk bgal+ virus. All heifers inoculated with tk + virus aborted between pidays 19 and 35. The finding of characteristic microscopic lesions and viral antigen in fetal tissues indicated that the abortions were caused by bhv-1 infection. Virus was isolated from 3 fetuses, and all isolates were tk +. Two heifers inoculated with tk bgal+ virus aborted at PI days 25 and 39. Fetal tissues had typical bhv-1 microscopic lesions and viral antigen. Virus was isolated from blood of both fetuses, and the isolates were tk bgal+. Results of this study indicate that inactivation of the tk gene reduces, but does not eliminate, the abortifacient activity of bhv-1.

Free access
in American Journal of Veterinary Research

SUMMARY

Leukocytosis (34,600 wbc/μl of blood) was detected in an apparently healthy 7-day-old Holstein heifer. Analysis of blood samples obtained over the next 41 days revealed chronic progressive neutrophilia, which peaked at > 85% neutrophils and exceeded 100,000 wbc/μl. In vitro assessment of isolated blood neutrophils obtained from the heifer at 38 and 45 days of age revealed selected functional abnormalities. Endocytosis of immunoglobulin-opsonized Staphylococcus aureus and killing of this test organism by the calf’s neutrophils were significantly diminished, as were phagocytosis-associated superoxide generation, chemiluminescence activity, and myeloperoxidase-catalyzed iodination. Diminished H2O2 elaboration by the calf’s neutrophils was evident during ingestion of opsonized zymosan or on exposure to phorbol myristate acetate. Extracellular release (secretion) of elastase during ingestion of zymosan was also diminished, although total cell content of elastase was normal, compared with that of neutrophils from age-matched calves, and granular or other morphologic abnormalities of the calf’s neutrophils were not evident by ultrastructural examination. Abnormalities of random migration were inconsistently detected, and normal or high degree of antibody-dependent cytotoxicity or natural killing by the calf’s neutrophils was observed. Similar in vitro assessment of neutrophils obtained from the calf’s dam revealed no functional abnormalities. The calf died at 48 days of age, with persistent fever and chronic diarrhea, despite administration of antibiotics. Histologic examination at necropsy revealed large numbers of intravascular neutrophils in most tissues, including massive neutrophil sequestration in spleen. However, a striking lack of extravascular neutrophils was evident in inflamed submucosa adjacent to intestinal ulcers heavily contaminated with enteric microorganisms. Bone marrow examination revealed diffuse myeloid hyperplasia, but no other abnormalities.

The clinical and pathologic features in this calf were similar to those in previously reported human patients or Irish Setters with genetic deficiency of the CD11/CD18 leukocyte glycoprotein complex, thus prompting further postmortem evaluations. Results of immunoblot analyses of the neutrophil lysates of the heifer calf (isolated and stored prior to death) documented severe deficiency of Mac-1 (CD11b/CD18). Results of immunofluorescent analyses indicated substantially diminished (intermediate) amounts ofthe Mac-1 β subunit (CD18) on blood neutrophils of the calf's dam and sire and on neutrophils of 8 of 15 paternal half-siblings; findings were consistent with an autosomal recessive trait in the proband's kindred. Findings also indicate that genetic abnormalities of CD11/CD18 proteins may underlie the molecular pathogenesis of disease in this calf as well as other previously described examples of the granulocytopathy syndrome in Holstein cattle.

Free access
in American Journal of Veterinary Research