Monitoring chronic infection with a field strain of Aleutian mink disease virus

Aleutian mink disease virus (AMDV) readily spread within farmed mink and causes chronic infections with significant impacts for welfare and economy. In the present study a currently circulating Danish AMDV strain was used to induce chronic experimental infection of farmed mink.PCR was used to detect viral DNA in full blood, organs, faeces and oro-nasal swabs weekly for the first 8 weeks and then biweekly for another 16 weeks after AMDV challenge inoculation of wild type mink. The mink (n = 29) was infected and seroconverted 2–3 weeks after AMDV inoculation and AMDV antibodies persisted during the maximum experimental period of 24 weeks. Viraemia and faecal excretion of viral DNA was detected in the mink (n = 29) at various and intermittent time intervals. Excretion of viral DNA in oro-nasal swabs was detected for 1–8 weeks in 21 mink. This highlights the risk of transmitting AMDV between infected farms.PCR was successfully used to detect viral DNA in organs 8, 16 and 24 weeks after AMDV inoculation with only minor differences between these weeks which is of diagnostic interest.This AMDV challenge model was also used to mimic natural infection of susceptible sapphire mink. Four of 6 sapphire mink were infected indirectly via the AMDV inoculated wild type mink whereas the other 2 sapphire mink remained uninfected.     

Gene expression studies of host response to Salmonid alphavirus subtype 3 experimental infections in Atlantic salmon

Salmonid alphavirus subtype-3 (SAV-3) infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM), cohabited (CO) or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi). Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs) of both IM and CO groups, with changes appearing first in the pancreas (2 wpi) in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-α, Mx and ISG15. IFN-γ and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-α, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3ε, CD4, CD8, TCR-α and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.     

Nonisotopic detection of Loma salmonae (microspora) in rainbow trout (Oncorhynchus mykiss) gills by in situ hybridization

Loma salmonae, a microsporidian parasite of salmonids of the genus Oncorhynchus, is a significant cause of economic loss in pen-reared chinook salmon (O. tschawytscha). Final stages of L. salmonae infections are easily recognized by the xenomas that form in the gills during sporogony. However, early prexenoma stages of infection (3 weeks or less after infection) are difficult to detect on histologic slides. An L. salmonae-specific single-stranded DNA probe labeled with digoxigenin was used to detect these prexenoma stages of L salmonae by in situ hybridization in experimentally infected rainbow trout. This method allows detection of the parasite in the gills only 2 weeks after infection, providing a sensitive and specific way of detecting L. salmonae during the early stages of infection.     

牛病毒性腹泻病毒感染牛外周血单核细胞对IFN-α、β、γmRNA转录的影响

为了解牛病毒性腹泻病毒（BVDV）感染对干扰素（IFN）mRNA转录时相的影响,探讨宿主-病毒之间的相互关系,用非致细胞病变（noncytopathic,NCP）和致细胞病变（cytopathic,CP）型BVDV感染临床健康BVDV检测阴性的荷斯坦奶牛外周血单核细胞（PBMC）,利用实时荧光定量PCR技术对感染后IFN-α、β、γmRNA转录水平的变化进行定量分析。结果表明,CP型和NCP型BVDV感染PBMC后,Ⅰ型IFN（IFN-α、β）均呈现出不同程度的转录水平上调,且差异极显著（P〈0.01）;只有IFN-α在CP型BVDV感染后4,12h（P〈0.5）出现转录下调。IFN-γ在整个感染过程中均呈现出不同程度的转录水平上调,且差异显著（P〈0.05）。这表明2种生物型BVDV感染可引起PBMC中IFN mRNA转录水平升高。     

Immune response of sheep to bluetongue virus: in vitro induced lymphocyte blastogenesis

Sheep were inoculated with 2 ml of 10(7) plaque forming units per ml of purified prototypes of the four United States serotypes (10, 11, 13 and 17) of bluetongue virus. Nine weeks following the initial inoculation, a challenge inoculation with homologous virus was done. Animals were followed for virus isolation and evidence of cell-mediated immunity by weekly lymphocyte stimulation tests (LST). Two dilutions (10 micrograms/ml and 1 microgram/ml) of pure virus from each of the purified serotypes were used as antigen as were the phytomitogens phytohemagglutinin, Concanavalin A, and pokeweed mitogen. LST data were analyzed by the analysis of variance method and reported as counts per minute and stimulation index (SI). Significant SI were observed following primary and secondary challenge with both homologous and heterologous virus. There was evidence of lymphocyte perturbations characterized by a sharp decrease in response to mitogens following primary and secondary challenge lasting for one week followed by a significant increase in blastogenesis three to four weeks after inoculation of virus. These results provide evidence that cell-mediated immunity is evident in bluetongue infection, that there is cross reactivity between viral serotypes and that BTV infection leads to perturbations in lymphocyte function including suppression of responses. An increase in the blastogenic response to phytomitogens correlated with viral clearance.     

Alloimmunity does not protect from challenge with the feline immunodeficiency virus

Immune responses against polymorphic host molecules incorporated into lentiviral envelopes during cell budding have induced protection against primate immunodeficiency virus infection. Dendritic cells (DCs) express high levels of MHC molecules and are infectable by lentiviruses. Therefore, in this pilot study we addressed the hypothesis that immunization of cats with allogeneic DC would induce immune responses that protect against challenge with the feline immunodeficiency virus. Two groups of 3 cats each received 3 subcutaneous injections of allogeneic or autologous DC, and were then challenged with viruses propagated in the immunizing DC. Infection status and lymphocyte parameters of cats were assessed during 6 weeks after challenge. MHC II antigens were incorporated into viral particles as identified by Western blot; and antibodies reactive with MHC class II antigens were detected in the serum of cats immunized with allogeneic but not autologous DC. After challenge, all cats had proviral DNA in blood leukocytes from 2 weeks post-challenge onward and seroconverted. Cats immunized with allogeneic DC maintained higher total and CD21(+) lymphocyte concentrations, and higher CD4(+)/CD8(+) lymphocyte ratios; however, these differences were not significantly different from cats that received autologous DC immunizations. Plasma viral load was not significantly different between groups of cats (p=0.204). These results suggest that immunization of cats with allogeneic DC does not induce protective immunity against FIV infection.     

Dog response to inactivated canine parvovirus and feline panleukopenia virus vaccines

Inactivated canine parvovirus (CPV) and inactivated feline panleukopenia virus (FPV) vaccines were evaluated in dogs. Maximal serologic response occurred within 1-2 weeks after vaccination. Antibody titers then declined rapidly to low levels that persisted at least 20 weeks. Immunity to CPV, defined as complete resistance to infection, was correlated with serum antibody titer and did not persist longer than 6 weeks after vaccination with inactivated virus. However, protection against generalized infection was demonstrated 20 weeks after vaccination. In unvaccinated dogs, viremia and generalized infection occurred after oronasal challenge with virulent CPV. In contrast, viral replication was restricted to the intestinal tract and gut-associated lymphoid tissue of vaccinated dogs. Canine parvovirus was inactivated by formalin, beta-propiolactone (BPL), and binary ethylenimine (BEI) in serum-free media; inactivation kinetics were determined. Formalin resulted in a greater loss of viral HA than either BEI of BPL, and antigenicity was correspondingly reduced.     

Antigen dose and humoral immune response correspond with protection for inactivated infectious pancreatic necrosis virus vaccines in Atlantic salmon (Salmo salar L)

An enduring challenge in the vaccinology of infectious pancreatic necrosis virus (IPNV) is the lack of correlation between neutralizing antibodies and protection against mortality. To better understand the immunological basis of vaccine protection, an efficacy trial including Atlantic salmon (Salmo salar L.) vaccinated with a high antigen (HiAg) or low antigen (LoAg) dose vaccine was carried out in a cohabitation challenge model using the highly virulent Norwegian Sp strain NVI015. To pinpoint the immunological basis of vaccine protection, pathogenic mechanisms of IPNV were unraveled in control fish while obtaining feedback on mechanisms of protection in the vaccinated fish. During the incubation period, infection rates were highest in control fish, followed by the LoAg group with the lowest infections being in the HiAg group. Although both the liver and pancreas are target organs prone to tissue damage, infection in the liver was delayed until acute infection in most fish. A correlate of pathology determined as the cutoff threshold of viral copy numbers linked to tissue damage in target organs was estimated at ≥ 10^7.0, which corresponded with an increase in mortality. The kinetics of IFNα and Mx expression suggests that these genes can be used as biomarkers of IPNV infection progression. Mechanisms of vaccine protection involved reducing infection rates, preventing infection of the liver and reducing virus replication in target organs to levels below the correlate of pathology. Overall, the study shows that antigen dose corresponds with vaccine efficacy and that antibody levels can be used as a signature of protective immunity against pathological disease and mortality.     

Social isolation may influence responsiveness to infection with bovine herpesvirus 1 in veal calves

An experiment was performed to develop a model to study the impact of stress on responsiveness to infection with bovine herpesvirus 1 (BHV1) in veal calves. Social isolation after previous group-housing was used as a putatively stressful treatment. Group-housed specific pathogen-free veal calves (n=8) were experimentally infected with BHV1 at the age of 12 weeks. Half of the calves were socially isolated at the time of infection. Clinical, virological and serological responses to BHV1, and adreno-cortical reactivity to exogenous ACTH were examined. In comparison with group-housed calves, calves socially isolated at the time of infection showed a diminished clinical and fever response, and delayed viral excretion after primary infection with BHV1. Four weeks after social isolation, basal cortisol levels before, and the integrated cortisol response after administration of a low dose of ACTH, were significantly depressed in socially isolated calves. The results suggest that social isolation in veal calves influences the response to an experimental BHV1 infection. A possible mechanism is discussed.     

Response of sheep to challenge infection with Fasciola hepatica

Sheep were infected with 100 metacercariae of Fasciola hepatica and reinfected 16 weeks later with a further 100 metacercariae. Serum samples were taken weekly for 36 weeks after primary infection. Serum was assayed for the presence of the enzymes glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (gamma-GT), as indicators of liver and bile duct damage respectively, and for levels of precipitating antibody. Antibody and GLDH levels rose following the primary infection but fell after patency had been reached . A peak in gamma-GT activity was associated with the onset of patency. After the challenge infection levels of both enzymes rose substantially and there were persistent fluctuations in activity. Antibody levels did not rise markedly following challenge but fluctuated at low levels until autopsy, 20 weeks after challenge. There was no resistance to challenge judged by worm recoveries at autopsy. It is suggested that the presence of adult flukes in the bile ducts suppresses the antibody response to challenge infection. Tissue damage, which is shown by fluctuations in GLDH and gamma-GT levels after adult flukes have become established in the bile ducts, is considered to be due to the feeding activity of adult flukes and the deposition of immune complexes in the liver parenchyma.     

Piscine orthoreovirus (PRV) infects Atlantic salmon erythrocytes

Piscine orthoreovirus (PRV) belongs to the Reoviridae family and is the only known fish virus related to the Orthoreovirus genus. The virus is the causative agent of heart and skeletal muscle inflammation (HSMI), an emerging disease in farmed Atlantic salmon (Salmo salar L.). PRV is ubiquitous in farmed Atlantic salmon and high loads of PRV in the heart are consistent findings in HSMI. The mechanism by which PRV infection causes disease remains largely unknown. In this study we investigated the presence of PRV in blood and erythrocytes using an experimental cohabitation challenge model. We found that in the early phases of infection, the PRV loads in blood were significantly higher than in any other organ. Most virus was found in the erythrocyte fraction, and in individual fish more than 50% of erythrocytes were PRV-positive, as determined by flow cytometry. PRV was condensed into large cytoplasmic inclusions resembling viral factories, as demonstrated by immunofluorescence and confocal microscopy. By electron microscopy we showed that these inclusions contained reovirus-like particles. The PRV particles and inclusions also had a striking resemblance to previously reported viral inclusions described as Erythrocytic inclusion body syndrome (EIBS). We conclude that the erythrocyte is a major target cell for PRV infection. These findings provide new information about HSMI pathogenesis, and show that PRV is an important factor of viral erythrocytic inclusions.     

The kinetics of feline leukaemia virus shedding in experimentally infected cats are associated with infection outcome

Feline leukaemia virus (FeLV) infection in felids results mainly from oronasal exposure to infectious saliva and nasal secretions, but the potential for viral transmission through faeces and urine has not been completely characterized. In order to assess and compare potential FeLV transmission routes, we determined the viral kinetics in plasma, saliva, faeces and urine during early experimental FeLV infection (up to week 15 post-exposure) in specific pathogen-free cats. In addition to monitoring p27 antigen levels measured by ELISA, we evaluated the presence of infectious particles by cell culture assays and quantified viral RNA loads by a quantitative real-time TaqMan polymerase chain reaction. RNA load was associated with infection outcome (high load-progressive infection; low load-regressive infection) not only in plasma, but also in saliva, faeces and urine. Infectious virus was isolated from the saliva, faeces and urine of infected cats with progressive infection as early as 3-6 weeks post-infection, but usually not in cats with regressive infection. In cats with progressive infection, therefore, not only saliva but also faeces and to some extent urine might represent potential FeLV transmission routes. These results should be taken into account when modelling FeLV-host interactions and assessing FeLV transmission risk. Moreover, during early FeLV infection, detection of viral RNA in saliva may be used as an indicator of recent virus exposure, even in cats without detectable antigenaemia/viraemia. To determine the clinically relevant outcome of FeLV infection in exposed cats, however, p27 antigen levels in the peripheral blood should be measured.     

Foetal cross-protection experiments between type 1 and type 2 bovine viral diarrhoea virus in pregnant ewes

A flock of 82 non-pregnant ewes was split into three immunisation groups and given an intranasal dose of either cell culture medium, or a type 1 or a type 2 bovine viral diarrhoea virus (BVDV-1 or BVDV-2). Two months later the flock was reconstituted and after a further three weeks, the ewes were bred to pestivirus negative rams after synchronisation of oestrus using progesterone sponges. Fifty-five ewes were segregated into three challenge groups, each of which comprised ewes from different immunisation groups. At 7 weeks gestation, one challenge group was given an intranasal dose of cell culture medium, whilst the other two were given intranasal doses of either BVDV-1 or BVDV-2, using the same inocula as for the immunisations. Three weeks later, the ewes were killed and their foetuses tested for the presence of BVDV-1 and BVDV-2. The results showed that immunisation of six ewes without subsequent challenge did not lead to infection of any of their 11 foetuses. Challenge with BVDV-1 or BVDV-2 in the absence of immunisation lead to 15 out of 15 or 11 out of 14 foetuses becoming infected, respectively. Immunisation with the homologous virus to that used for challenge resulted in complete protection of 32 foetuses from 15 ewes. Heterologous protection was one way. All 12 foetuses from ewes immunised with BVDV-1 were protected from challenge with BVDV-2, whereas 18 foetuses from ewes immunised with BVDV-2 were all infected after challenge with BVDV-1. This provides evidence that a recent exposure to infection with one pestivirus does not necessarily induce foetal protection against another. The one-way result suggests that factors other than antigenic differences are involved in cross-protection.     

Duration of immunity in dogs vaccinated against leptospirosis with a bivalent inactivated vaccine

Duration of immunity in dogs induced with current commercial inactivated leptospirosis vaccines and evaluated against experimental infection, to date, has hardly been documented. The purpose of the present work was to assess the duration of immunity in dogs that is attainable with a commercial inactivated bivalent leptospirosis vaccine. For this purpose, young dogs were vaccinated twice followed by challenge with either Leptospira interrogans serovar canicola or L. interrogans serovar icterohaemorrhagiae 5 weeks, 27 weeks or 56 weeks after the second vaccination. For assessment of the duration of immunity, titres of agglutinating serum antibodies were measured before and after challenge, and the effects of challenge on a variety of parameters were determined including reisolation of challenge organisms from blood, urine and kidney. Both challenge strains induced a generalised infection in control dogs, the canicola strain being most virulent. From the results with different parameters it appeared that the two vaccinations induced a high rate of protection from generalised infection with canicola and icterohaemorrhagiae at 5, 27 and 56 weeks after the second vaccination. In addition, after 56 weeks, still a high level of immunity against renal infection with sv. canicola and, as a consequence, urinary shedding of sv. canicola bacteria, was demonstrated. It was, therefore, concluded that with this vaccine, using this vaccination schedule, a duration of immunity of 1 year can be attained against infection with both serovars.