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Objective: To determine microscopic features and involvement of viruses in teat-end lesions (TEL) of dairy cows during winter., Sample Population: Teats with TEL on lactating Holstein cows and from udders of carcasses., Procedure: Tissues obtained from TEL of 10 teats from 7 cows on 2 research farms during the winter of 1994 to 1995 and 13 teats with TEL excised from udders of carcasses at an abattoir during February 1995 were submitted for virus isolation. During the winter of 1995 to 1996, an increased prevalence of TEL was observed in a research herd. After a decrease in ambient temperature, TEL were identified, and a full-thickness section of epidermis was removed from skin surrounding teat orifices. Tissues were examined by use of light and electron transmission microscopy., Results: Viruses were not isolated from TEL tissues. Lesions ranged from mild elevations of the epidermis to thickened oval regions that encircled the teat orifice. The most severe lesions were dark and had thick crusts. Histologically, TEL were composed of thickened regions of epidermis most notably caused by hyperplasia of cells within the stratum spinosum. Excess production of keratinocytes was also evident, and the keratinocyte layer often contained bacteria. Ultrastructurally, squamous cells contained large amounts of keratin, but virions were not detected. Evidence of a viral etiologic agent for TEL was not detected., Clinical Implications: Development of TEL may be associated with decreases in ambient temperature. Numerous bacteria were evident in the keratin of TEL. Lesions and associated bacteria may predispose cows to mastitis.

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.

New Zealand white rabbits, which had been prepared for inoculation by intraperitoneal treatment with thioglycollate, were inoculated intraperitoneally with bovine immunodeficiency-like virus (BIV). Infected materials from various sources were used including cultured cells and culture fluids, peripheral blood leukocytes from infected cattle and spleen tissue from previously infected rabbits. Virus isolations and serological responses detected by western blotting provided clear evidence that infections had been established in inoculated rabbits and that the spleen was an important site of BIV infectivity. These results indicate that rabbits may be a useful species when testing for BIV infectivity in materials too toxic or highly contaminated to be inoculated directly into cell cultures. Furthermore, rabbits may also be useful in testing effects of coinfections with other bovine viruses on progression of BIV infection and for the initial evaluation of therapeutic regimens designed to suppress or eliminate BIV infections.

Bovine herpesvirus type 1 (BHV-1) isolates are classified into 3 subtypes by use of restriction endonuclease analysis. Isolates from aborted fetuses have been either subtype 1 or 2a, whereas subtype 2b viruses have not been associated with abortion. We assessed the abortifacient property of isolates representing each of the 3 BHV-1 subtypes by IV inoculation of heifers with the virus 25 to 27 weeks after breeding. Three heifers were given Cooper (subtype 1) isolate, 3 heifers were given FI (subtype 2a) isolate, and 5 heifers were given K22 (subtype 2b) isolate. All heifers developed fever and viremia 2 to 5 days after inoculation. Heifers given Cooper or FI isolate aborted between 17 and 85 days after inoculation. The 5 heifers given K22 isolate delivered full-term calves. Placenta was obtained from 4 of the 5 heifers, and K22 virus was isolated from each placenta. Four calves had BHV-1 neutralizing antibody in precolostral serum, with titer ranging from 1:4 to 1:512.

Leukocytosis (34,600 WBC/microliter 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 greater than 85% neutrophils and exceeded 100,000 WBC/microliter. 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.

Levamisole, a compound that has been used widely as an anthelmintic in man and domestic animals, has also been found to be an immunomodulator. It was, thus, of interest to determine whether treatment with levamisole would affect bovine leukemia virus infections in cattle and sheep or the results of serological and virological tests routinely used to identify infected animals. Studies of cattle and sheep given either the recommended anthelmintic dose of levamisole or repeated larger doses of the drug failed to provide evidence of significant changes in antibody titer or virus replication. It is, therefore, concluded that levamisole neither potentiated nor repressed bovine leukemia virus replication or the associated immunological responses.

Fluoresceinated, heat-aggregated bovine immunoglobulins (B-IgG) and human immunoglobulins (H-IgG) were used to detect a receptor for the crystallizable fragment (Fc) of the immunoglobulin molecule on peripheral blood lymphocytes (PBL) of cattle. The aggregated and B-IgG and H-IgG bound to the bovine PBL, but aggregated H-IgG was found to be more sensitive for the detection of Fc receptors. The specificity of aggregated H-IgG binding to the Fc receptors was established by demonstrating that antigen-antibody complexes inhibited this binding, and unaggregated H-IgG did not bind significantly to PBL. Double-labeling experiments suggested that all Fc+ cells have surface immunoglobulins (SIg), a marker for B lymphocytes. The percentage of Fc+ and SIg+ cells in normal animals was 9.5% (range 4-15%) and 16.2% (range 4.5-30.2%), respectively. Persistent lymphocytotic cows had 2.71 times more Fc+ and 3.85 times more SIg+ lymphocytes than did normal cows. Cows with lymphosarcoma had a lower percentage of Fc+ and SIg+ cells than did cows with persistent lymphocytosis. Cases with thymic lymphosarcoma and those with the skin form of leukemia had normal percentages of Fc+ and SIg+ cells.

A bovine herpesvirus-1 (BHV-1) isolate (FI) from an aborted fetus was used to infect 9 heifers at various stages of gestation. Two heifers were inoculated IV on postbreeding day (PBD) 1, 7, or 14, and 3 heifers were inoculated in the sixth month of pregnancy. Plasma progesterone assays were used to monitor corpus luteum function in heifers inoculated during early pregnancy. Low progesterone values and infertility were seen in the 2 heifers inoculated on PBD 1. Luteal function remained normal in heifers inoculated on PBD 7 or 14. These 4 heifers inoculated on PBD 7 or 14 carried their fetuses to term, and their calves were free of BHV-1 infection at birth. Three heifers inoculated during the sixth month of pregnancy also carried their fetuses to term. Two calves were born alive, and BHV-1 was not isolated from nasal swab samples of either calf; the third calf was stillborn. Virus was not isolated from the stillborn calf's tissues, but BHV-1 was isolated from the placenta. Lesions were not detected in several tissues examined by light microscopy, and BHV-1 antigen was not detected by immunohistochemical examination of paraffin sections. Restriction endonuclease analysis of viral DNA was used to compare the FI virus to other BHV-1 isolates (Colorado-1, Iowa, and K22). On the basis of restriction endonuclease analysis, the FI isolate should be classified as a type-2 (infectious pustular vulvovaginitis) virus, specifically subtype a.

Leukocytes from 1 ml of blood from cattle seropositive to the bovine leukemia virus were cultured for 3 days and then tested by radioimmunoassay for antigen production. Infectivity of blood from each animal was also tested by calf inoculation and subsequent serologic detection of bovine leukemia virus transmission by agar-gel immunodiffusion. In a preliminary experiment, blood from each of 3 antigen-positive cattle was inoculated intracutaneously into 2 calves in volumes of 20 or 100 microliter. Blood from each of 4 antigen-negative cattle was similarly inoculated into 3 calves in volumes of 20, 100, or 500 microliter. At the termination of the experiment (8 weeks after inoculation), all 6 calves given blood from antigen-positive cattle had seroconverted, and 11 of 12 calves given blood from antigen-negative cattle had seroconverted. In a 2nd experiment, blood from each of 2 antigen-positive and 2 antigen-negative cattle was inoculated into pairs of calves in volumes of 1, 10, or 20 microliter. At the end of the experiment (12 weeks after inoculation), all calves inoculated with blood from antigen-positive cattle had seroconverted, but only 6 of 12 calves that had been given blood from antigen-negative cattle had seroconverted. The relative infectivity of blood was best illustrated by comparing results from the 1-microliter inoculations. At that volume, the 4 calves given blood from antigen-positive cattle were infected, whereas none of 4 calves given blood from antigen-negative cattle was infected.

In an initial study, 18 calves born to cows persistently infected with bovine leukemia virus (BLV) were tested for infective virus and antibodies at birth, and no infected or seropositive animals were found. Four of these calves were maintained in quarters where infected animals were housed, and 3 of the 4 subsequently became infected. These were probably contact infections acquired during, or at some time after, birth. The remaining 14 calves were kept in isolation pens in a building housing no infected cattle. None of this group was found to be BLV infected during 1 year of observation. In further studies, 15 pregnant cows inoculated with BLV became infected. One abortion, considered to be unrelated to the BLV inoculation, occurred 38 days later. The remaining 14 cows gave birth to 1 dead and 14 live calves. The dead calf and its live twin were seropositive for BLV at birth, indicating that they had been infected in utero. The remaining 13 calves were negative for BLV antibodies at birth and remained so during 1 year of observation.

Four tests for detection of antibodies to bovine leukemia virus (BLV) were compared. The sera that were tested came from cattle in naturally infected commercial dairy herds, cattle that were infected under experimental conditions, and cattle in an isolated BLV-free herd. The tests that were compared included a radioimmunoprecipitation assay (RIA) with p24 antigen, a RIA with glycoprotein (gp) antigen, an agar-gel immunodiffusion (AGID) test with gp antigen, and a virus-neutralization (VN) test that was based on inhibition of BLV-induced syncytia in cell culture. Results of the 4 serologic tests agreed for 96.8% of the sera from cattle in commercial herds. The gp RIA detected the greatest number of positive sera (188); it was followed in turn by the p24 RIA (187), the VN test (183), and the AGID test (176). The gpd RIA titers of the 12 sera that gave negative AGID results were 175 or less. In RIA, the percentage of precipitation of labeled antigen by positive sera was almost always higher with gp antigen than with p24 antigen. Satisfactory sensitivity in the p24 RIA required the acceptance of a low level of antigen precipitation, 15%, as a positive test. In the gp RIA, however, almost all positive sera precipitated at least 50% of the labeled antigen. Nonspecific precipitation of antigen in the RIA by sera from BLV-free cattle ranged from 4% to 10%. Examination of sequential serum samples from 17 experimentally infected cattle showed that BLV antibody was first detected 2 to 8 weeks after inoculation. In 9 cattle, seroconversion was detected simultaneously by all of the tests. Results from the other 8 cattle indicated that seroconversion could be detected first by p24 RIA, followed by the gp RIA and the VN test. The longest interval between RIA seroconversion and AGID seroconversion was 10 days. Monthly tests of sera from 10 laboratory cattle that were infected by contact exposure showed that 7 animals seroconverted in all tests at the same time. Two cattle were positive first in RIA, but the next month they were also positive in the VN and AGID tests. One animal was positive in the RIA and the VN test for 2 months before antibody was detected by AGID.

Persistent levels of serum antibodies to bovine C-type virus were demonstrated by the complement fixation test in cattle of a leukosis herd during an observation period of one and one half years. Using the same method, no antibodies were detected in a control herd.

Reference strains and field isolates of herpesviruses recovered from cattle in the United States were compared by restriction endonuclease (RE) analysis and the indirect fluorescent antibody test. As a result of these comparisons, 5 major biotypes of bovine herpesvirus (BHV) were defined. These types were (i) infectious bovine rhinotracheitis virus (BHV-1), (ii) bovine herpes mammillitis virus (BHV-2), (iii) malignant catarrhal fever (MCF) virus (herpesvirus alcelaphinae), (iv) the group of slow-growth isolates represented by the prototype strain Movar 33/63 (bovine cytomegalovirus candidate), and (v) the syncytia-forming Pennsylvania 47 strain. Bovine herpesvirus-1 and BHV-2 did not cross-react serologically with any other type of BHV tested. A low, but consistent level of serologic cross-reactivity was detected among MCF virus, the Movar group, and Pennsylvania 47. Several nonsyncytial, slow-growth strains, which were recovered from dissimilar clinical syndromes and were serologically related to Movar 33/63, exhibited similar DNA RE cleavage patterns, confirming their identity as members of a single type. There was no isolate from American domestic cattle similar to the African MCF virus, which has been sporadically isolated from exotic ruminants in the United States. The African MCF virus isolated during a MCF epizootic in a United States zoo exhibited some DNA RE cleavage differences in comparison with the MCF virus world prototype strain WC 11, indicating that strain diversity exists within this biotype.

Secretions and excretions from cattle with persistent bovine leukemia virus (BLV) infections were tested for BLV by inoculation into sheep. Development of antibody to the BLV glycoprotein antigen and reisolation of virus from peripheral blood leukocytes were the two criteria used to confirm infection in test sheep. The virus was detected in milk from 4 of 6 cows examined and in colostrum from 1 of 4 cows examined. The virus was not detected in semen from 8 bulls or in nasal secretions, saliva, and urine from 2 cows.

Ten lactating Holstein cows that had been given multiple injections of Listeria monocytogenes (serotype 4B, Scott A strain) via the intramammary route were allotted to 2 groups: group 1 (n = 5) was treated with the synthetic glucocorticoid, dexamethasone (0.04 mg/kg of body weight), for 3 consecutive days, and group 2 (n = 5) served as controls. Two days after the initial dexamethasone injection, the number of L monocytogenes in the milk had increased nearly 15-fold (1.16 log10) over pretreatment values. On day 3, Listeria numbers in the milk had increased by 1.83 log10, compared with pretreatment values. By day 4, Listeria numbers in the milk were approximately 100-fold (2.03 log10) greater than pretreatment numbers. Numbers remained high through day 7 and, by day 11, approached pretreatment numbers. Dexamethasone administration was accompanied by high total WBC and milk somatic cell counts and decreased eosinophil and lymphocyte numbers, and decreased milk production. The increase in shedding of L monocytogenes in the milk may reflect impairment of cell-mediated immune mechanisms and phagocytic cell functions that are critical for sustaining listerial immunity.

Bovine leukosis describes lymphatic cancers of cattle. The most common form of this disease occurs in adult animals and is caused by bovine leukemia virus. Infection is widespread in the United States, especially in dairy cattle, but the virus produces tumors in only a small percentage of infected animals. Nevertheless, bovine leukemia virus has been receiving attention from the dairy industry because of its importance in health certification of cattle or semen intended for export. Another source of concern is whether bovine leukemia virus poses any risk to human health. These problems are discussed in the light of recent technological advances in tumor virus research and specifically regarding our current understanding of the biology of bovine leukemia virus.

The purpose of this study was to examine rates of natural in utero infection with bovine leukemia virus for association with breed, sex, dam age, dam parity and time of maternal seroconversion. Analyses conducted for breed and sex, dam age and parity and time of maternal seroconversion were the FUNCAT procedure for categorical data, Wilcoxon Rank Sums test and Fisher's exact test, respectively. A total of 223 calves born between July 1979, and September 1980, to cows infected with bovine leukemia virus in the University of Florida Dairy Research Unit herd were tested for detectable bovine leukemia virus antibodies prior to the consumption of colostrum. Sera were tested for antibodies by agar-gel immunodiffusion and radioimmunoprecipitation using the glycoprotein-51 antigen. In a group of 125 calves in which in utero infection could be confirmed through serological follow-up (group A), eight calves (6.4%) had precolostral bovine leukemia virus antibodies. For all 223 calves (group B), 18 (8.1%) had detectable bovine leukemia virus antibodies. For calves in group A, no associations were detected between precolostral bovine leukemia virus antibodies and breed (p = 0.66), dam age (p = 0.86), dam parity (p = 0.83), or time of maternal seroconversion to bovine leukemia virus (p = 0.50). However, precolostral bovine leukemia virus antibodies were found in 17.4% of the males and 3.6% of the females in group A (p = 0.11) and in 12.4% of the males and 3.6% of the females in group B (p = 0.04).

Simple, practical, reproductible methods for the detection of bovine leukemia virus (BLV) in peripheral blood leukocytes and BLV-neutralizing antibodies in bovine sera are described. Virus detection is based on syncytium formation in transformed feline (F-81) cells. The use of multiwell culture plates provides practical benefits in testing large numbers of samples. Results obtained in 2 farm herds show that the agreement of serologic and virologic test results may vary considerably from herd to herd.

The infectivity for the bovine leukemia virus (BLV) of bovine blood lymphocytes, with varying lymphocyte nuclear pocket (LNP) prevalence, was studied by intradermal injection of buffy coat leukocytes from cows into sheep. Thirty-five sheep were assigned to 3 groups of 9, 11, and 15, respectively, as recipients of blood leukocytes from individual cows as follows: 9 leukocyte donor cows included 3 BLV-seropositive cows with high (greater than or equal to 2.0%) LNP prevalence (group 1); 3 BLV-seropositive cows with low (less than 0.5%) LNP prevalence (group 2); and 3 BLV-seronegative cows with increased (greater than or equal to 0.5%) LNP prevalence (group 3). The 9 sheep in group 1 became BLV-seropositive by postinoculation day (PID) 57 as determined by the agar-gel immunodiffusion test; 2 of 11 sheep in group 2 became agar-gel immunodiffusion seropositive by PID 40; none of the 15 sheep of group 3 became seropositive by PID 92. Seemingly, there is a real difference in infectivity among BLV-seropositive cattle which is related to LNP prevalence, and seronegative cattle with increased LNP prevalence may be noninfectious, ie, were not carrying covert BLV virogene.

Pairs of newborn calves were exposed to bovine leukemia virus (BLV) when they were given their 1st colostrum feeding. Calves that were given 10(6) BLV-infected lymphocytes in colostrum free of BLV-specific antibody became infected. Calves that were fed 10(7), 10(8), or 10(9) infected lymphocytes in colostrum that contained BLV-specific antibody did not become infected. One of 2 calves inoculated intradermally with 250,000 infected lymphocytes was protected by colostral antibody, but the other was not. Colostral antibody titers in the unprotected calf decreased normally until the calf was 4 months old and then increased markedly; this pattern indicates that the presence of colostral antibody may have prolonged the latent period of the BLV infection.

Fluorescent antibody (FA) studies of tissues from three colostrum deprived calves inoculated intranasally with the SF-4 strain of bovine para-influenza 3 (PI-3) virus indicated that these calves developed a mild upper respiratory infection but infected cells were not identified in the lower respiratory tract. Three other calves inoculated intranasally and intratracheally with PI-3 virus developed more severe clinical signs of infection and virus was identified, by FA techniques, in the upper and lower respiratory tract of all three calves and in the spleen of one calf. PI-3 virus was detected in smears of nasal epithelium from five of six calves at some time during the observation period.