Apoptosis of peripheral blood leukocytes from rabbits infected with non-haemagglutinating strains of rabbit haemorrhagic disease virus (RHDV)

The report demonstrates that the induction of apoptosis in peripheral blood granulocytes and lymphocytes of rabbits infected with three non-haemagglutinating RHDV strains (English Rainham, German Frankfurt, and Spanish Asturias) is a crucial determinant of the pathogenesis of rabbit haemorrhagic disease. Apoptosis was measured by flow cytometric detection of caspase activity. These studies demonstrated that the investigated RHDV (rabbit haemorrhagic disease virus) viral strains affected leukocyte apoptosis to varying degrees. Enhanced leukocyte apoptosis was detected between 4 and 36h after infection and was more pronounced in lymphocytes than in granulocytes. The data presented here thus provide a preliminary understanding of the kinetics of apoptosis in leukocytes of rabbits infected with RHDV.     

Hepatic lesions in young rabbits experimentally infected with rabbit haemorrhagic disease virus

Twenty young rabbits (eleven 2-week-old and nine 4-week-old) were experimentally infected with rabbit haemorrhagic disease virus (RHDV) to clarify susceptibility. They were killed chronologically up to 96 hours post-inoculation (PI) and examined for lesions. All inoculated rabbits were clinically normal, but grossly minute white or grey spots were detected throughout the liver. Histologically, the lesions consisted of aggregates of lymphocytes, macrophages and heterophils, with or without acidophilic bodies and necrotic hepatocytes. Immunohistochemically, RHDV antigens were found in the degenerated hepatocytes and in macrophages. The cellular aggregates were considered to be a reaction to necrotic hepatocytes infected with RHDV. It was concluded that some hepatocytes are susceptible to RHDV in young rabbits.     

White and red blood cells picture in rabbits experimentally infected with RHD virus

Four strains of RHDV assigned as haemagglutinating (Vt97 and Hartmannsdorf) and non-haemagglutinating (Pv97 and 9905) antigenic variants were examined for dynamic changes in the values of white and red blood cells indexes. The study showed differences among strains examined that were not depending on haemagglutination property.     

Immunohistological diagnosis of rabbit haemorrhagic disease in experimentally infected rabbits in Spain.

The immunoreactivity of routinely processed liver and lung tissue samples obtained from rabbits inoculated with tissue explants from naturally infected animals when antisera directed against parvovirus from different species (canine, feline and porcine) as well as a RHD virus antiserum were employed has been tested by different immunoperoxidase methods. Cross-reactivity between RHD-virus antigens and parvovirus antigens was present. Best results were obtained with RHD and canine parvovirus antisera with the ABC method. The immunoreactivity in the liver was found in hepatocytes, Kupffer and bile duct cells. In the lung, it was exclusively observed in intravascular macrophages.     

Phagocytosis of neutrophils in rabbits infected with antigenic variants of RHD (rabbit haemorrhagic disease) virus

The present study was aimed at determining changes in chosen elements of phagocytosis in rabbits infected with 3 antigenic variants of RHD - Hartmannsdorf, Pv97 and 9905, which differed in haemagglutination ability. The animals were tested for phagocytosis parameters, and the results revealed that the examined strains showed the differences. These variations regarded mainly Pv97 strain, as the intensity of the changes were 5 times stronger in comparison to strain Hartmannsdorf and 9905. As all of the strains examined are signified as antigenic variants, we have stated that this feature does not determine their immunological picture. The results suggest the existence of immunological dissimilarities among strains of the RHD virus, which was revealed for the first time in antigenic variants.     

Nephropathogenesis of chickens experimentally infected with various strains of infectious bronchitis virus

Four-day-old specific-pathogen-free chickens were inoculated by eyedrop with four different strains (Gray, JMK, CV56b, and Wolgemuth) of infectious bronchitis virus (IBV). Birds were monitored clinically and euthanatized at 1, 4, 7, and 14 days postinfection and tissues were collected for virus isolation, histopathologic examination, in situ hybridization (ISH), and immunohistochemistry (IHC). Clinical disease was severe in chickens infected with Wolgemuth, but no overt disease was observed with the other strains. Virus was isolated from the kidneys of chickens infected with the Gray-, CV56b-, and Wolgemuth-strains of IBV. Histologically, interstitial nephritis was evident in chickens infected with these same 3 strains. However, viral nucleic acid and antigen were detected only with Wolgemuth-infected kidneys by ISH and IHC. These results indicate that the pathological changes in kidneys from chickens infected with Gray and CV56b may not have resulted from the cytolytic action of the virus.     

Study of the persistence of Aujeszky's disease (pseudorabies) virus in peripheral blood mononuclear cells and tissues of experimentally infected pigs

The presence of Aujeszky's disease virus (ADV) in peripheral blood mononuclear cells (PBMC) and tissues of experimentally infected pigs was studied. Vaccinated and unvaccinated pigs were inoculated with different doses of Aujeszky's disease NIA-3 strain. Pigs were periodically bled and PBMC were used for virus isolation and PCR detection of virus. Tissues were obtained at the time of death (8 weeks post-inoculation) and used for ADV genome detection by PCR. ADV genome was amplified from PBMC during the acute phase of infection and, in some experimental groups, up to 38 days post-inoculation (PI). The virus was sporadically detected by virus isolation performed from PBMC. In neural tissues, ADV was constantly amplified from the trigeminal ganglia and the olfactory bulb of persistently infected pigs (euthanised 8 weeks PI). In other tissues, the viral genome was rarely detected in lymph nodes and tonsils, and, occasionally, in the bone marrow. Our results indicated that PBMC are not an appropriate source for detecting ADV persistence, since inconsistent results were obtained throughout the experiments. In neural tissues, the olfactory bulb turned out to be as important a target for ADV persistence as the trigeminal ganglia. Viral genome detection in the bone marrow indicated that this tissue may play a role in the establishment of a persistent infection.     

Immunofluorescence of bovine virus diarrhea viral antigen in white blood cells from experimentally infected immunocompetent calves.

A study to evaluate the detection of bovine virus diarrhea viral antigen using immunofluorescence testing of white blood cells was conducted. Five colostrum-deprived calves were inoculated intravenously with a cytopathic strain of the virus. Lymphocyte and buffy coat smears were prepared daily for direct immunofluorescent staining for detection of antigen. Lymphocytes were separated from heparinized blood using a Ficoll density procedure. Buffy coat smears were prepared from centrifuged blood samples collected using ethylenediaminetetraacetic acid as an anticoagulant. Bovine viral diarrhea virus antigen was detected by immunofluorescence between 3 and 11 days postinfection in lymphocyte smears and 3 to 12 days postinfection in buffy coat smears. Isolation of virus from both lymphocytes and buffy coat preparations correlated with detection of immunofluorescence. Serum neutralizing antibody to bovine virus diarrhea virus was detected on day 10 postinfection. Buffy coat smears were as sensitive as lymphocyte smears for the detection of antigen by immunofluorescence. It appeared that immunofluorescent staining of white blood cells was an effective method of detecting bovine virus diarrhea viral antigen.     

Cytometric analysis of lymphocytes T and B in rabbits infected with non-haemagglutinogenic strains of RHD virus (rabbit haemorrhagic disease)--Rainham, Frankfurt and Asturias

The present paper refers to the cytometric analysis of lymphocytes T (with receptor CD5+), Th (with receptor CD4+), Tc/Ts (with receptor CD8+), lymphocytes CD25+ and lymphocytes B with receptor CD19+ in rabbits experimentally infected with strains of RHD virus--Rainham, Frankfurt and Asturias, not having haemagglutinogenic capacities, which makes them unique, as haemagglutinogenic capacity is a classic and typical property of most strains of this virus. The study was performed in the dynamic system, drawing blood samples from animals at hour 0, namely before the administration of the viral antigen, and then at 4, 8, 12, 24 and 36 h after the infection. The study indicated that Rainham and Asturias strains of RHD virus cause a similar amount of changes as the most immunogenic haemagglutinogenic strains CAMP V-561 and CAMP V-562 of the RHD virus do. In contrast, the Frankfurt strain of the RHD virus is characterised with 5-6-fold lower reactivity in this respect and is most similar to the least immunogenic haemagglutinogenic strain CAMP V-558 of the RHD virus.     

Lymphocyte subpopulations in peripheral blood of lambs experimentally infected with bovine respiratory syncytial virus

The lymphocyte subpopulations of peripheral blood of normal lambs and lambs experimentally infected with bovine respiratory syncytial virus (RSV) were analysed by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with bovine RSV was characterized by a significant rise in SBU-T8+ (CD8+ or cytotoxic) T cells and a significant reduction in SBU-T4+ (CD4+ or helper) T cells and B (LCA p220+) lymphocytes (P less than 0.05). The helper/suppressor (CD4/CD8) ratio was reduced from 3.91 on the day of experimental infection to 1.13 on 10 days after experimental infection (P less than 0.001). The total number of SBU-T4+ (CD4+) and B cells returned to pre-inoculation values 14 days after experimental infection but the helper/suppressor ratio remained depressed up to 21 days post-inoculation.     

Indications of immunodepression in chickens infected with various strains of Marek's disease virus

The effect of infection by various strains of Marek's disease virus (MDV) on the immune function of 3-week-old chickens was examined. MDV strains of low (CU-2, RB-7) and high (RB-3, MD-5, and MD-11) pathogenicity were compared with prototype JM-10 strain of moderate pathogenicity. Mortality, whole body weight, relative weights of lymphoid organs, histopathology, humoral antibody responses to thymus-dependent and -independent antigens, and in vitro lymphocyte responses to mitogen stimulation were investigated at 1, 2, and 3 weeks postinfection. MDV strains of high pathogenicity significantly depressed responses at 3 weeks postinfection, seeming to indicate the ability of these viruses to induce severe immunodepression. However, the fact that the moderately pathogenic and even some of the low-pathogenicity strains induced immunodepression suggests that other viral mechanisms are also important in its determination.     

Early detection of viral excretion from experimentally infected goats with peste-des-petits ruminants virus

We observed 15 goats for 9 days after subcutaneous infection with 10(3) TCID(50) with isolates of peste-des-petits ruminants virus from Africa and India and five concurrent, uninfected control goats. Typical clinical signs of the infection were present in all 15 infected goats by day 8 and in most by day 6 and some signs were present by day 4. However, 6 out of 15 goats already have detectable virus shedding by day 3 and four more were shedding by day 4 and every goat had virus shedding for at least 1 day before the recognition of clinical signs. This experiment indicates that incubatory carriers therefore might play a role in the transmission of PPRV among small ruminants.     

Suppression of immunological responses in rabbits experimentally infected with bovine leukemia virus

Ten 2- to 4-month-old rabbits were inoculated subcutaneously with bovine leukemia virus (BLV)-infected bovine or sheep cells. By 6 weeks after inoculation all ten rabbits had converted to BLV antibody-positive, and BLV or BLV antigen was detected in lymphocytes from most of the rabbits tested, although there were few antigen-producing cells. Three rabbits showed continuous respiratory symptoms after infection and one died with pneumonia. Humoral immune responses against mouse serum were significantly suppressed in BLV-infected rabbits compared with non-infected control rabbits. The lymphocyte blastogenesis response was also suppressed in BLV-infected rabbits. At the time of necropsy, six rabbits showed pulmonary lesions; however, none of the BLV-infected rabbits had tumors during an observation period of over 1 year.     

Kinetics of phytohemagglutinin response in chickens infected with various strains of Marek's disease virus

Phytohemagglutinin (PHA) response of whole blood lymphocytes from white leghorn inbred line 7(2) chickens infected with various strains of Marek's disease virus (MDV) was monitored sequentially for 6 weeks postinfection. A significant difference between JM and GA strains was shown. A two-phase depression in the PHA response was observed in chickens infected with the JM strain. Early depression occurred 1 week postinfection and was followed by recovery a week later. The second depression occurred at 4 weeks postinfection and lasted until the end of the experiment. The GA strain-infected group, on the other hand, began to show depression 4 weeks postinfection, and most chickens died within a short time thereafter. PHA response of chickens infected with strain Md11/75C, attenuated in cell culture from highly virulent strain Md11, was almost the same as that of control chickens.     

牛病毒性腹泻病毒感染MDBK细胞和家兔前后炎症因子的表达变化

为了解牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)感染宿主后炎症因子的变化,以Madin-Darby牛肾细胞(Madin-Darby bovine kidney cells,MDBK)和家兔为宿主,选用炎症反应关键酶COX-2和炎症因子如IL-8、MIP-3α、GRO-α作为研究指标。通过设计上述炎症因子的特异性引物,分别从MDBK、家兔脾脏组织和小肠上皮组织中分别扩增目的片段,建立实时荧光相对定量PCR方法检测感染前后COX-2和IL-8、MIP-3α、GRO-αmRNA的相对表达水平。结果表明,在BVDV感染MDBK细胞后,COX-2和IL-8、GRO-αmRNA表达量均显著高于未感染前(P<0.01);当BVDV感染家兔后小肠和脾细胞中COX-2和IL-8、GRO-α表达量均显著高于未感染前(P<0.05);但只有在脾细胞中,MIP-3αmRNA表达量显著高于未感染前(P<0.01)。说明BVDV感染宿主的过程中可引起COX-2和IL-8、GRO-α基因表达量的升高,推测MIP-3α在脾脏抗BVDV感染过程中发挥着重要的作用。     

Lymphocyte subpopulations and apoptosis of immune cells in rabbits experimentally infected with a strain of the RHD virus having a variable haemagglutination capacity

The paper describes the immunological response in the matter of percentage of T cells (receptor CD5+) and subpopulations (Th with receptor CD4+, Tc/Ts with receptor CD8+, T with receptor CD25+) and B cells with receptor CD19+, as well as the percentage of apoptotic granulocytes and lymphocytes, in rabbits experimentally infected with the Hagenow strain of the RHD virus. The material chosen for the experiment is special, as among all strains of RHD virus, there are only two strains which carry the variable haemagglutination capacity of red cells. The results of the study show that the Hagenow strain gives an untypical picture of T and B lymphocytes, whereas the results in inducing apoptosis seems to corespond with previous data, confirming the inclusion of apoptosis from 4 h p.i. and the intensity of the phenomenon being higher in granulocytes.     

Aujeszky's disease (pseudorabies) virus detection in cerebrospinal fluid in experimentally infected pigs

The presence of Aujeszky's disease virus in cerebrospinal fluid of experimentally infected pigs was studied using the techniques of virus isolation and PCR. Pigs, some of which were previously vaccinated against Aujeszky's disease, were inoculated with different doses of the Aujeszky's disease NIA-3 strain. At the time of death or sacrifice, a sample of cerebrospinal fluid was taken and tested for the presence of virus using the mentioned techniques. Virus was isolated only from one sample, while it was detected by PCR in most of them. The higher sensitivity of the PCR technique and the possible presence of antiviral antibodies in the cerebrospinal fluid are reasons that can be argued to explain this fact. By PCR, the virus was detected more efficiently when digested cerebrospinal fluid cells were used as DNA source than when using whole cerebrospinal fluid, suggesting that the virus could be cell-associated. Aujeszky's disease virus could not be detected by PCR in pigs which survived the acute phase of the infection and were euthanased at 8 weeks post-inoculation, when they were latently infected. This indicated that the cerebrospinal fluid is not an adequate sample for the diagnosis of latency. Since Aujeszky's disease virus was detected from most of the tested samples, we believe that this could be an adequate procedure for the quick diagnosis of Aujeszky's disease.     

Changes in peripheral blood leucocytes of sheep experimentally infected with Mycoplasma agalactiae

Contagious agalactia is a serious disease of small ruminants affecting mainly mammary glands, joints and eyes. In sheep, the main aetiological agent is Mycoplasma agalactiae (Ma) whose abilities to persist in the target organs are known. Since there is no information on the effect of acute and chronic Ma infection on circulating leucocytes, the present study was designed to monitor granulocytes, monocytes, T and B lymphocytes, by flow cytometry, in female lactating sheep nasally infected with Ma. A profound depletion of leucocytes was observed from day 5 to day 34 post infection (p.i.). In particular, while the granulocytes returned to baseline levels by day 12 p.i., the monocytes remained significantly low until day 20 p.i. The infection caused a prolonged depletion of peripheral T lymphocytes (both CD4⁺ and CD8⁺) while B lymphocytes remained unaltered throughout the study. Mycoplasma agalactiae was detected by real-time PCR in several anatomical sites (ear, nose and milk) from day 2–5 p.i. until the end of the study (i.e., day 50 p.i.) while a transient bacteraemia was observed from day 5 to day 12 p.i. The leucopenia observed following intranasal Ma infection is likely due to leucocyte infiltration within the target organs.