Comparison of virus isolation, immunofluorescence and DNA probe hybridization for detection of pseudorabies virus in experimentally infected pigs.

Virus isolation, immunofluorescent staining and DNA probe hybridization, three techniques for the detection of pseudorabies virus (PRV) have been compared in pigs experimentally infected with the Thailand CB-1 strain PRV. The virus isolation and DNA hybridization detection demonstrated a good correlation in detecting infection in live animals by nasal swabbing. In white blood cells an earlier detection was seen with the DNA-hybridization techniques. Consistent results with all the three techniques tested were found in organ materials as nasal mucosa and tonsils as well as in the olfactory bulb.     

Limitations of in situ hybridization for the detection of bluetongue virus in blood mononuclear cells

In situ nucleic acid hybridization was tested for the ability to detect bluetongue virus (BTV) nucleic acids in blood mononuclear cells. A standard protocol was devised and applied to the demonstration of BTV genetic sequences in cultured bovine mononuclear cells that had been infected in vitro. In situ hybridization using biotinylated single-stranded RNA probes, in the presence of 50% formamide at 50 C, demonstrated an intense, positive signal in 0.001-0.01% of the BTV-infected cultured mononuclear cells. The protocol was applied to isolated mononuclear cells from an experimentally infected heifer. No infected cells were observed by this method, although the blood specimens were obtained during peak viremia.     

Comparison of some storage and isolation methods to recover bluetongue virus from bovine blood.

Bovine blood containing bluetongue virus was stored as whole blood with EDTA (4 degrees C), as washed cellular components (4 degrees C), and as washed cellular components with 10% DMSO (-70 degrees C). Periodic isolation attempts were made over a period of 330 days in four cell lines and embryonating chicken eggs (intravenous inoculation). Bluetongue virus was successfully isolated in all systems from most samples throughout the test period. There appeared to be more variation amongst days of attempted isolation within systems than between systems.     

Detection of cattle infected with bovine viral diarrhea virus using nucleic acid hybridization.

A ribonucleic acid (RNA) hybridization assay to identify cattle infected by bovine viral diarrhea virus (BVDV) is described. The RNA probe was derived from the coding region at the 3' end of the genome of the NADL strain of BVDV. Total RNA from infected cell cultures or peripheral blood leukocytes from suspect animals was extracted and applied to nylon membranes with a slot blot apparatus. Peripheral blood leukocytes were tested concurrently for BVDV by virus isolation. The results of hybridization and virus isolation were in agreement for 92% of the cases. When compared with virus isolation, hybridization had a sensitivity of detection of 59.5% and a specificity of 95%. Cross-reactivity to RNA extracts of border disease virus-infected cells was noted. No cross-reactivity was detected to other common bovine viruses (bovine herpesvirus-1, bovine respiratory syncytial virus, parainfluenza-3 virus, and bluetongue virus), to viruses classified in related families (equine arteritis virus and Venezuelan equine encephalitis virus), or to viruses having similar genomic organization (dengue virus type 2 and Japanese encephalitis virus).     

In vitro production of antibodies to bovine virus diarrhoea virus by peripheral blood mononuclear cells from cattle immunized by infection

A method for in vitro production of antibodies to bovine virus diarrhoea virus (BVDV) by peripheral blood mononuclear cells (PBMC) was developed. The PBMC were cultured in microtitre plates coated with detergent-solubilized BVDV and the supernatants were tested in an enzyme-linked immunosorbent assay which detects IgG antibodies to BVDV. Following incubation of PBMC with an optimal concentration of pokeweed mitogen for 5 days, antibodies to BVDV were detected in culture supernatants of PBMC from immune cattle, but not in supernatants of PBMC from seronegative cattle, from persistently BVDV-infected cattle or from a 5-day-old calf that received BVDV antibodies via colostrum. This antibody-production assay may therefore be used as a tool to discriminate between passively acquired antibodies and those actively induced.     

Bovine virus diarrhoea virus induces in vitro a proliferative response of peripheral blood mononuclear cells from cattle immunized by infection.

The ability of bovine peripheral blood mononuclear cells (PBMC) to mount a proliferative response to bovine virus diarrhoea virus (BVDV) in vitro was examined. After culturing PBMC in the presence of a non-cytopathic strain of BVDV for 6 days, the magnitude of PBMC proliferation was measured as incorporation of radiolabelled thymidine, present during the last 18 h. Live, but not heat-inactivated, BVDV evoked a proliferative response of PBMC obtained from cattle seropositive to the virus. However, PBMC from seronegative or persistently BVDV-infected animals were not stimulated by BVDV. The presence of live BVDV did not alter the proliferative response of PBMC to stimulation with concanavalin A.     

Comparison of polymerase chain reaction assays with bacteriologic culture, immunofluorescence, and nucleic acid hybridization for detection of Leptospira borgpetersenii serovar hardjo in urine of cattle

OBJECTIVE: To compare sensitivity and specificity of various polymerase chain reaction (PCR) assays for detection of Leptospira borgpetersenii serovar hardjo in bovine urine and to compare results of the optimal PCR assay with results of immunofluorescence, nucleic acid hybridization, and bacteriologic culture. ANIMALS: 6 heifers. PROCEDURE: Heifers were exposed to serovar hardjo type hardjo-bovis by conjunctival instillation of 10(6) leptospires on 3 successive days. Urine samples were collected before and after infection. Sensitivity and specificity of 5 PCR assays were compared, to determine the optimal assay for use with bovine urine samples. The optimal PCR assay was then compared with results of bacteriologic culture, nucleic acid hybridization, and immunofluorescence. RESULTS: A PCR assay with the best combination of specificity (100%) and sensitivity (91%) was selected for comparison with the other diagnostic tests. Sensitivity for nucleic acid hybridization was 55%, whereas sensitivity for bacteriologic culture and immunofluorescence was 89 to 93%. CONCLUSIONS AND CLINICAL RELEVANCE: Bacteriologic culture, PCR, and immunofluorescence were sensitive for detection of L borgpetersenii serovar hardjo type hardjo-bovis in urine specimens of cattle, but a single technique was not the most sensitive for each animal tested. Therefore, the use of 2 techniques in combination is warranted for maximal sensitivity for diagnosis.     

Detection of bluetongue virus RNA by in situ hybridization: comparison with virus isolation and antigen detection.

An in situ nucleic acid hybridization (ISH) technique was developed to detect bluetongue virus (BTV) RNA in cell culture. The sensitivity of the ISH technique was compared with virus isolation (VI) and antigen detection, using an indirect fluorescent-antibody (IFA) or an enzyme immunocytoassay (EICA) technique, for detection of 5 BTV serotypes indigenous to the United States. The VI was the most sensitive technique, detecting BTV early after infection of the cells. The IFA and EICA were of similar sensitivity; BTV antigen could be detected shortly after demonstration of virus by isolation. The sensitivity of ISH for detection of BTV-17 was equivalent to that of antigen detection. The ISH was not as sensitive as VI or antigen detection when assaying for the other BTV serotypes.     

Hepatic ultrasonography and blood changes in cattle with experimentally induced hepatic abscesses.

Hepatic abscesses were induced experimentally in 5 steers by inoculating Fusobacterium necrophorum via ultrasonography-guided, percutaneous catheterization of the portal vein. Hepatic ultrasonography was performed to determine the onset and progression of abscessation. Blood samples were collected before and after inoculation for performing leukocyte counts and hepatic function tests. Ultrasonographic evidence of liver abscesses was observed as early as 3 days after inoculation. Abscesses appeared as hyperechoic centers (cellular debris and pus) surrounded by hypoechoic or anechoic areas (fluid). Increases in rectal temperature, leukocyte counts, fibrinogen, globulin, bilirubin, gamma-glutamyltransferase, and sorbitol dehydrogenase concentrations were detected. Hepatic dysfunction was evidenced by decrease in serum albumin concentration and low sulfobromophthalein clearance. The ultrasonographic diagnosis of abscesses correlated well with necropsy findings.     

Measurement of lymphoblast proliferative capacity of stimulated blood mononuclear cells from cattle with chronic paratuberculosis.

Concanavalin A (conA) blast proliferation as a quantitative measure of lymphoblast proliferative capacity by blood mononuclear cell supernatants was measured in cattle naturally infected with Mycobacterium paratuberculosis and in healthy control cattle. Blast cell proliferation was significantly reduced in infected animals, compared with control cattle when blood mononuclear cells were stimulated with conA. Proliferation was significantly greater than media control when M bovis purified protein derivative and johnin were used to stimulate cells from the infected group. After sensitizing control and affected cattle with M paratuberculosis bacterin (live M bovis and keyhole limpet hemocyanin in Freund's incomplete adjuvant), infected animals had no difference in blast cell proliferative capacity with the mycobacterial antigens and conA stimulation, whereas healthy animals had significantly increased blast proliferation in response to all the sensitizing antigens. The blast cell proliferative capacity in infected animals with keyhole limpet hemocyanin stimulation was increased significantly after sensitization; however, it remained significantly less than that in the sensitized control group. These data indicate that cattle naturally infected with M paratuberculosis probably produce suboptimal interleukin-2 (IL-2) activity in response to a potent IL-2 inducer (conA) and fail to optimize IL-2 activity when sensitized with a potent immunogen (keyhole limpet hemocyanin).     

Replication of bluetongue virus and epizootic hemorrhagic disease virus in pulmonary artery endothelial cells obtained from cattle,sheep, and deer

OBJECTIVE: To compare replication of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in pulmonary artery endothelial cells (ECs) obtained from juvenile cattle, sheep, white-tailed deer (WTD; Odocoileus virginianus), and black-tailed deer (BTD; O hemionus columbianus). SAMPLE POPULATION: Cultures of pulmonary artery ECs obtained from 3 cattle, 3 sheep, 3 WTD, and 1 BTD. PROCEDURE: Purified cultures of pulmonary artery ECs were established. Replication, incidence of infection, and cytopathic effects of prototype strains of BTV serotype 17 (BTV-17) and 2 serotypes of EHDV (EHDV-1), and (EHDV-2) were compared in replicate cultures of ECs from each of the 4 ruminant species by use of virus titration and flow cytometric analysis. RESULTS: All 3 viruses replicated in ECs from the 4 ruminant species; however, BTV-17 replicated more rapidly than did either serotype of EHDV. Each virus replicated to a high titer in all ECs, although titers of EHDV-1 were significantly lower in sheep ECs than in ECs of other species. Furthermore, all viruses caused extensive cytopathic effects and a high incidence of cellular infection; however, incidence of cellular infection and cytopathic effects were significantly lower in EHDV-1-infected sheep ECs and EHDV-2-infected BTD ECs. CONCLUSIONS AND CLINICAL RELEVANCE: There were only minor differences in replication, incidence of infection, and cytopathic effects for BTV-17, EHDV-1, or EHDV-2 in ECs of cattle, sheep, BTD, and WTD. It is not likely that differences in expression of disease in BTV- and EHDV-infected ruminants are attributable only to species-specific differences in the susceptibility of ECs to infection with the 2 orbiviruses.     

Comparison of two methods for detection of transmissible gastroenteritis virus in feces of pigs with experimentally induced infection

An indirect, double-antibody sandwich-type ELISA for detection of transmissible gastroenteritis virus (TGEV) was developed, using a solid phase of rabbit hyperimmune serum and a pool of 3 antipeplomer monoclonal antibodies to trap and to detect the virus, respectively. The technique was used to detect viral antigen in feces of pigs that had been infected with the virulent Miller strain, the attenuated Purdue strain, or the Erica strain (a Dutch field isolate) of TGEV. The results were compared with those of a solid-phase immunosorbent electron microscopy (SPIEM) technique for virus detection. Both techniques detected shedding of virulent virus in feces obtained from pigs on the first or second day after infection, and virus excretion continued for 6 to 8 consecutive days. Virus shedding started later in pigs infected with the attenuated Purdue strain of TGEV and lasted only 2 to 4 days. In comparison with the 2 virulent strains, infection with the attenuated strain appeared to be limited to a smaller portion of the small intestine. Of 242 fecal specimens that were tested by use of ELISA and SPIEM, 119 had positive results in both tests. Additionally, virus could be detected by ELISA in 21 and by SPIEM in 16 specimens. Fecal specimens obtained from pigs before infection always reacted negatively by ELISA for TGEV antigen; there was no cross-reactivity with fecal specimens containing porcine rotavirus or porcine epidemic diarrhea virus. The ELISA and SPIEM were found to be specific and sensitive for the detection of TGEV in feces.     

Comparison of dot blot hybridization, polymerase chain reaction, and virus isolation for detection of bovine herpesvirus-1 (BHV-1) in artificially infected bovine semen.

Bovine semen samples spiked with bovine herpesvirus 1 (BHV-1) were used to compare dot blot hybridization, polymerase chain reaction (PCR), and virus isolation for detection of BHV-1 in bovine semen. The PCR amplification used primers targeting the BHV-1 thymidine kinase gene and a nucleic acid releasing cocktail (GeneReleaser); the PCR product was used as the DNA probe in dot blot hybridization; virus isolation was done in primary bovine fetal testis (BFT) cell cultures. Semen diluted 1:20 in tissue culture medium had the least cytotoxicity and inhibition of viral cytopathic effects in BFT cells, allowing detection of 1 TCID50/100 microL of BHV-1 suspension by virus isolation. The presence of foreign DNA such as bovine sperm DNA or salmon sperm DNA increased the sensitivity of dot blot hybridization in detecting BHV-1, allowing detection of 20,000 TCID50/100 microL of neat semen. The inhibition of PCR amplification of BHV-1 DNA in bovine semen was eliminated by diluting the samples 1:20 in tissue culture medium. The best PCR amplification was obtained when semen was diluted 1:20 and when a reaction buffer of pH 9.0, with 1.0 mM MgCl2 was used. Under these conditions, the PCR followed by ethidium bromide staining of agarose gels could detect 1 TCID20/100 microL of sample, whereas PCR followed by Southern blot hybridization could detect 0.01 TCID50/100 microL of sample.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accelerated binding of autoantibody to red blood cells with increasing anaemia in cattle experimentally infected with Theileria sergenti

Anaemia is the most important clinical manifestation in cattle infected with Theileria sergenti. In order to determine the mechanism of red blood cells (RBC) destruction in anaemic cattle, we studied the binding of autoantibody (IgG) to RBC during the development of anaemia in T. sergenti infection. The low levels of IgG-bound RBC before the development of anaemia were triggered in proportion with the progression of anaemia and parasitaemia. Our results suggest an accelerated destruction of RBC in anaemic cattle by IgG-dependent phagocytosis.     

Use of a polymerase chain reaction assay to detect infection with Eperythrozoon wenyoni in cattle.

OBJECTIVE: To determine whether a polymerase chain reaction (PCR) assay could be used to detect Eperythrozoon wenyoni in the blood of cattle. DESIGN: Prospective study. ANIMALS: 95 cattle from various herds in Alabama and Georgia and 96 bulls enrolled in Auburn University's Alabama Beef Cattle Improvement Association Bull Test program. PROCEDURE: Blood samples were collected by means of venipuncture of the median caudal vein and submitted for a CBC and PCR assay. Blood smears were made immediately after blood collection and examined by means of light microscopy. RESULTS: Three of 95 cattle from herds in Alabama and Georgia and 5 of 96 bulls enrolled in the Bull Test program had positive PCR assay results. Organisms were seen in blood smears from only 5 of these 8 animals. Organisms were not seen in blood smears from any animals for which results of the PCR assay were negative. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that a PCR assay may be an effective method for detecting E wenyoni infection in cattle and that the PCR assay may be a more sensitive test than evaluation of blood smears.     

Peripheral blood mononuclear cell supernatants from asymptomatic dogs immunized and experimentally challenged with Leishmania chagasi can stimulate canine macrophages to reduce infection in vitro

Leishmania chagasi is the causative agent of visceral leishmaniasis in both humans and dogs in the New World. The dog is the main domestic reservoir and its infection displays different clinical presentations, from asymptomatic to severe disease. Macrophages play an important role in the control of Leishmania infection. Although it is not an area of intense study, some data suggest a role for canine macrophages in parasite killing by a NO-dependent mechanism. It has been proposed that control of human disease could be possible with the development of an effective vaccine against canine visceral leishmaniasis. Development of a rapid in vitro test to predict animal responses to Leishmania infection or vaccination should be helpful. In this study, an in vitro model was established to test whether peripheral blood mononuclear cell (PBMC) supernatants from dogs immunized with promastigote lysates and infected with L. chagasi promastigotes could stimulate macrophages from healthy dogs in order to control parasite infection. PBMC from a majority of the immunized and experimentally infected dogs expressed IFN-gamma mRNA and secreted IFN-gamma when stimulated with soluble L. chagasi antigen (SLA) in vitro. Additionally, the supernatants from stimulated PBMC were able to reduce the percentage of infected donor macrophages. The results also indicate that parasite killing in this system is dependent on NO, since aminoguanidine (AMG) reversed this effect. This in vitro test appears to be useful for screening animal responses to parasite inoculation as well as studying the lymphocyte effector mechanisms involved in pathogen killing by canine macrophages.