Common, emerging, vector-borne and infrequent abortogenic virus infections of cattle.

This review deals with the aetiology and the diagnosis of bovine viral abortion. While the abortion rates on beef and dairy cattle farms usually do not exceed 10%, significant economic losses because of abortion storms may be encountered. Determining the cause of abortions is usually a challenge, and it generally remains obscure in more than 50% of the necropsy submitted foetuses. Bovine viral diarrhoea virus and bovine herpesvirus-1 are the most common viruses causally associated with bovine abortions in farmed cattle globally. Rift Valley fever virus and bluetongue virus are important insect-transmitted abortogenic viruses. The geographic distribution of these two viruses is primarily dependent on the distribution of the insect vector, but direct transmission is possible. Recent global warming and subsequent insect vector expansion, coupled with the increase in international trade of animals and animal products, have been important factors in recent geographic advances of those two viruses. Bovine herpesviruses-4 and 5 in cattle, as well as other less frequent vector-borne viruses including epizootic haemorrhagic disease virus, Aino virus, Wesselsbron virus and lumpy skin disease virus, are discussed.

Lack of detection of a putative retrovirus associated with haemic neoplasia in the soft shell clam Mya arenaria.

Haemic neoplasia (HN) is a leukemia-like disease that affects at least 20 species of marine bivalves including soft shell clam, Mya arenaria. Since the disease was discovered in 1969, the etiology remains unknown. A retroviral etiology has been suggested based on the detection of reverse transcriptase activity and electron microscopic observation of retroviral-like particles using negative staining. To date, however no virus isolate and no retroviral sequence from HN has been obtained. Moreover, transmission of the disease by cell-free filtrate from affected clams has not been reproduced. In the current study, we reinvestigated the association of HN with a putative retrovirus. Sucrose gradient centrifugation followed by assessment of reverse transcriptase activity, electrophoretic analysis of protein and RNA, and electron microscopic examinations of fractions corresponding to retroviral density were employed. Detection of retroviral pol sequences using degenerate RT-PCR approaches was also attempted. Our results showed visible bands at the expected density of retrovirus in HN-positive and HN-negative clam tissues and both with reverse transcriptase activity. Electron microscopy, RNA analysis, protein analysis, and PCR systems targeting the pol gene of retroviruses did not however provide clear evidence supporting presence of a retrovirus. We point out that the retrovirus etiology of HN of Mya arenaria proposed some 25 years ago should be reconsidered in the absence of a virus isolate or virus sequences.

Reverse transcriptase activity in tissues of the soft shell clam Mya arenaria affected with haemic neoplasia.

Since all retroviruses possess reverse transcriptase (RT) enzyme, reverse transcriptase activity has been the main supportive evidence of retroviral etiology of haemic neoplasia (HN) in soft shell clams, Mya arenaria. The objective of the present study was to search for a putative retrovirus in various tissues of diseased clams following quantification of RT activity (biochemical indicator of retroviral infection). The clams were assessed by flow cytometry (FCM) for diagnosis of HN. RT activity was quantified by TaqMan-product enhanced reverse transcriptase (TM-PERT) assay in four different organs, gonad, gills, digestive gland, and mantle, at various stages of HN. The digestive gland, the organ with the highest RT activity, and haemocytes, the target cell of HN, were assessed by EM for presence of retroviruses. All organs were assessed by histology. The results of this study demonstrated that although all organs of healthy clams have some background RT activity, the activity observed in most of organs of diseased clams was significantly increased (p<0.05). An association was observed between the degree of neoplastic cell infiltration and the level of RT activity. Digestive gland showed the highest and most consistent RT activity in both healthy and diseased clams. No evidence for the existence of a retrovirus like particle was found by positive staining EM. The presence of RT activity without indications of retroviral particles in digestive gland and haemocytes suggests a probable endogenous source of RT.

Reverse transcriptase activity associated with haemic neoplasia in the soft-shell clam Mya arenaria.

Reverse transcriptase (RT) activity has been reported in bivalves affected by haemic neoplasia (HN). Since all retroviruses have RT, detection of RT activity was regarded as evidence for the retroviral etiology of HN. This study investigates the relationship between RT levels and the progress of HN as indicated by percentages of tetraploid cells in soft-shell clams Mya arenaria. The percentages of tetraploid cells were estimated by flow cytometry, and the RT levels were quantified using TaqMan product-enhanced RT (TM-PERT) assay. Results demonstrated that the amount of RT was positively correlated with the percentage of tetraploid cells circulating in clam haemolymph (R2 = 0.974, p < 0.001). Compared to HN-negative clams (<5% tetraploid cells), 2 stages with significantly elevated levels of RT activity were observed: the first stage at approximately 10 to approximately 20% tetraploid cells, and the second at approximately 30 to approximately 80% tetraploid cells (p < 0.01). These data support the well established fact from mammalian models that transformed cells express high levels of non-telomeric RT. The observed increase in RT levels at approximately 30% tetraploidy coincides with previously reported p53 gene expression. Taken together, this could indicate that using RT levels as an indicator of HN, > or = 30% tetraploidy is the stage at which the disease process undergoes a change, and perhaps becomes irreversible.

Fluorescence spectroscopy monitoring of the conformational restraint of formaldehyde- and glutaraldehyde-treated infectious bursal disease virus proteins.

Interaction of native proteinaceous antigens during the recognition and the effector phases of an immune response leads to antigenic conformational modifications which may elicit additional specific immune response. Protein cross-linking and conformation restraining formaldehyde and glutaraldehyde have been extensively used in vaccine preparation, but the relative efficiencies of conformational restraint at concentrations similar to those used in vaccine preparation have not been investigated. We addressed this issue by comparing the extent of conformational restraint of virus proteins in formaldehyde- and glutaraldehyde-treated virus preparations by monitoring the fluorescence intensities (I320) of infectious bursal disease virus preparations (IBDV) and those of untreated virus during thermal denaturation. Formaldehyde was found to cause no detectable conformational restraint at 0.01% and only very weak restraint at 1%, while glutaraldehyde caused very strong conformational restraint at 0.01%. It is proposed how conformational restraint of proteinaceous antigens may alter ensuing immunity.

Detection of feline coronavirus RNA in feces, tissues, and body fluids of naturally infected cats by reverse transcriptase PCR.

A nested reverse transcriptase PCR (RT-nPCR) was developed for the detection of feline coronavirus (FCoV) RNA in the feces, tissues, and body fluids of infected cats. The RT-nPCR was targeted to the highly conserved 3'-untranslated region of the viral genome and will detect most, if not all, feline coronaviruses in the field. With the RT-nPCR, FCoV RNA was detected in plasma samples from experimentally infected cats as early as 2 days postinoculation. FCoV RNA was also detected in serum, plasma, or ascitic fluid samples from 14 of 18 cats (78%) with naturally occurring feline infectious peritonitis (FIP). The use of RT-PCR for FIP diagnosis is limited because of the occurrence of apparently healthy FCoV carriers. These asymptomatic cats shed the virus in the feces and, in a number of cases, also had detectable virus in the plasma. Because of the nature of FCoV infections, our RT-PCR assay with plasma or serum cannot be used to establish a definite diagnosis of FIP. However, this assay does provide a new means to identify asymptomatic FCoV carriers. As such, RT-nPCR will be of use to screen cats before their introduction into FCoV-free catteries. Moreover, this assay provides an important tool to study the epidemiology of FCoV.

The use of ELISA for detection of the antibody-induced conformational change in a viral protein and its intermolecular spread.

Antibody-induced conformational changes of proteins have been recently frequently suggested to explain a variety of observations. In spite of the fundamental importance of this phenomenon for both in vivo and in vitro antigen-antibody interactions, it is not generally accepted because of the lack of conclusive evidence. This report utilizes a novel approach to the study of antibody-induced antigenic conformational changes. Pairs of monoclonal antibodies (mAb) were used to induce and to assess conformational changes in potato virus X (PVX) protein. Blocking ELISA with native and glutaraldehyde treated virus was used to detect conformational changes. Double antibody sandwich (DAS) ELISA was designed to investigate possible inter-molecular spread of conformational changes. Detection of one way blocking in a blocking ELISA, with a pair of mAbs reacting to non-overlapping epitopes, suggested conformational change as the mechanism of blocking. The putative conformational change was confirmed when the one way blocking was prevented using conformationally restrained virus. Inter-molecular spread of the conformational change among the molecules of PVX protein was demonstrated in DAS-ELISA, when capture mAb inhibited binding of detecting mAb in the absence of steric hindrance. Unlike X-ray crystallography, the methodology utilized in this study indicates directly the significance of a changed conformation to antibody binding.

Current approaches to vaccine preparation.

Numerous conventional vaccines for animal use are currently available, and many of these vaccines have been instrumental in the control of infectious diseases of major economic importance. A vaccine has even been instrumental in global eradication of smallpox, an important human disease. However, many of the current vaccines are deficient in efficiency, potency, or safety. It has been recognized that the conventional methodologies are a limitation to further vaccine development. Introduction of monoclonal antibodies, recombinant DNA, and protein engineering techniques has facilitated a rather rapid increase in the knowledge of pathogenetic mechanisms, as well as of protective antigens at the molecular level. This knowledge provides the basis for development of a new generation of vaccines. As a rule, these vaccines contain purified immunogens, or even isolated epitopes, identified and prepared by molecular biological techniques. The efforts to find better delivery systems and better adjuvants accompany the research on vaccines.

The participation of antibody in spontaneous and antibody-dependent cell-mediated cytotoxicity in the pig.

Treatment of porcine lymphocytes with trypsin reduced their spontaneous cell-mediated cytotoxicity (SCMC) activity against target cells persistently infected with transmissible gastroenteritis virus (PK15-TGE cells), but had no effect on antibody-dependent cell-mediated cytotoxicity (ADCC). SCMC activity was partially restored to trypsin-treated lymphocytes by incubation in RPMI-1640 medium or in medium containing F(ab')2 fragments of rabbit anti-porcine immunoglobulin, but not by brief incubation in autologous serum. F(ab')2 fragments of anti-porcine immunoglobulin did not block the SCMC reaction, but ADCC was greatly reduced by this reagent. Thus SCMC and ADCC mediated by porcine lymphocytes against PK15-TGE target cells clearly involved two distinct mechanisms in terms of antibody participation and sensitivity to trypsin.

Characterisation of the effector cells in antibody-dependent and spontaneous cell-mediated cytotoxicity in swine against target cells infected with transmissible gastroenteritis virus.

When porcine peripheral blood leucocytes were fractionated, lymphocytes were the most active effectors in both antibody-dependent cell-mediated cytotoxicity (ADCC) and spontaneous cell-mediated cytotoxicity (SCMC), although both polymorphs and macrophages showed some activity in ADCC. Adsorption of lymphocytes to antibody-sensitised or unsensitized PK-15-transmissible gastroenteritis (TGE) cells caused similar reductions in ADCC and SCMC effector activities. Over 60 per cent of the target-binding lymphocytes were non-specific esterase positive large lymphocytes, which did not form erythrocyte (E)-rosettes, and about 30 per cent were non-specific esterase positive medium sized lymphocytes, which formed low avidity E-rosettes. The remainder were non-specific esterase negative small lymphocytes, some of which formed high avidity E-rosettes. None of the eluted lymphocytes stained for surface immunoglobulin and all formed low avidity erythrocyte-antibody rosettes. Porcine killer and natural killer cells resembled in many respects those described in humans and rodents.

The tissue distribution of spontaneous cell-mediated cytotoxicity effector lymphocytes in swine.

Peripheral blood lymphocytes, intraepithelial lymphocytes from the small intestine and lymphocytes from the thymus, spleen, mesenteric lymph nodes and Peyer's patches from 5 young adult pigs were used as effector cells in a spontaneous cell-mediated cytotoxicity chromium release assay against PK-15 cells persistently infected with transmissible gastroenteritis virus as targets. Both peripheral blood and intraepithelial lymphocytes caused marked specific chromium release, while the lymphocytes from the remaining tissues were inactive in spontaneous cell-mediated cytotoxicity.

The role of interferon in spontaneous cell-mediated cytotoxicity in pigs.

Specific release of 51Cr and the production of interferon (IFN) increased in parallel in a spontaneous cell-mediated cytotoxicity (SCMC) assay in which uninfected PK-15 cells or PK-15 cells persistently infected with transmissible gastroenteritis virus (PK-15-TGE cells) were used as targets, and peripheral blood lymphocytes (PBL) from a young adult pig were used as effector cells. Higher levels of both specific 51Cr release and IFN were obtained in the assays containing PK-15-TGE cells. Co-cultivation of PBL from newborn piglets with PK-15-TGE cells yielded similar levels of IFN to those produced by co-cultivation of adult PBL and PK-15-TGE cells, but lower levels of IFN were produced by co-cultivation with uninfected PK-15 cells. Pretreatment of adult PBL with IFN augmented their SCMC effector activity for both PK-15 and PK-15-TGE cells in the 51Cr release assay. Pretreatment of the PK-15-TGE target cells with IFN did not affect their release of 51Cr in the SCMC reaction, while IFN pretreatment of PK-15 targets protected them against SCMC. In a single cell cytotoxicity assay the effects of IFN pretreatment on the effector adult PBL and on the PK-15 and PK-15-TGE target cells were confirmed, and SCMC incompetent PBL from neonatal piglets were rendered cytotoxic by pretreatment with IFN. PBL from newborn piglets bound to either target cell with the same frequency as PBL from SCMC competent adult pigs, and IFN pretreatment of either effector or target cells had no effect on target-binding frequency.

The effect of adoptive transfer of mononuclear leukocytes from an adult donor on spontaneous cell-mediated cytotoxicity and resistance to transmissible gastroenteritis in neonatal piglets.

The purpose of this study was to attempt to establish spontaneous cell-mediated cytotoxicity effector activity in the intraepithelial lymphocytes of neonatal piglets by adoptive transfer of mononuclear leukocytes from an adult donor and to determine the effect of transfer on the resistance of piglets to transmissible gastroenteritis. Cytotoxicity was determined by a chromium release assay using PK-15 cells persistently infected with transmissible gastroenteritis virus as targets. The experimental animals were inbred miniature pigs, in which a high degree of uniformity in lymphocyte defined histocompatibility complex antigens was demonstrated by the mixed lymphocyte reaction. Adoptive transfer of 8 X 10(7)-4 X 10(8) adult pig leukocytes established effector activity in eight recipient piglets, and leukocytes labelled with fluorescein isothiocyanate homed to the epithelium of the small intestine. When four recipients of 5 X 10(8) adult leukocytes were challenged with transmissible gastroenteritis virus, the onset of diarrhea was delayed for 24 h and the diarrhea was usually milder than in four untreated control piglets. It was concluded that the adoptive transfer of leukocytes with spontaneous cell-mediated cytotoxicity effector activity, which homed to the small intestinal epithelium, may have contributed to an increased resistance to transmissible gastroenteritis.

Antibody-dependent and spontaneous cell-mediated cytotoxicity against transmissible gastroenteritis virus infected cells by lymphocytes from sows, fetuses and neonatal piglets.

The purpose of this study was to investigate the ontogeny in the pig of effector lymphocytes mediating antibody-dependent and spontaneous cell-mediated cytotoxicity against target cells infected with transmissible gastroenteritis virus. The same activities were also studied in sows in late pregnancy and early lactation. Peripheral blood lymphocytes and intraepithelial lymphocytes collected from piglets during the first week of life failed to mediate cytolysis as determined by chromium release assays, but significant activities developed during the second week and usually increased further by the sixth week. Peripheral blood lymphocytes collected from fetuses on the 109th gestation day failed to produce spontaneous cell-mediated cytotoxicity and only reacted in antibody-dependent cell-mediated cytotoxicity when contaminated with macrophages. The cytotoxic activities of peripheral blood lymphocytes from sows were lowest at parturition. It was concluded that impaired lymphocyte cytotoxicity in newborn piglets and parturient sows may contribute to their relatively high susceptibility to transmissible gastroenteritis.

Antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against cells infected with porcine transmissible gastroenteritis virus.

The objective of this study was to determine whether porcine peripheral blood leukocytes and intestinal intraepithelial leukocytes can mediate antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against target cells infected with transmissible gastroenteritis virus. Peripheral blood leukocytes collected from six young adult pigs and intraepithelial leukocytes from a further five pigs were used as effector cells in chromium release assays against PK-15 cells persistently infected with transmissible gastroenteritis virus. Both peripheral blood leukocytes and intraepithelial leukocytes were capable of mediating antibody-dependent cell-mediated cytotoxicity and spontaneous cell-mediated cytotoxicity against PK-15 transmissible gastroenteritis cells. While the peripheral blood leukocytes mediated lower levels of specific 51Cr release in spontaneous cell-mediated cytotoxicity than in antibody-dependent cell-mediated cytotoxicity, the intraepithelial leukocytes were more effective in spontaneous cell-mediated cytotoxicity than antibody-dependent cell-mediated cytotoxicity.

[Effect of various breeding conditions on the development of respiratory syndrome in calves]. / Vliv ruzných podmínek odchovu na vznik respiracního syndromu u telat.

In two experimental rearing houses with new-born non-colostral calves, separated from their mothers at birth and artificially fed colostrum or colostral whey, the epizootological situation was very favourable throughout the time of observation. The respiratory syndrome occurred in both cases as a result of the long-term worsening of microclimatic conditions and failure to adhere to the principle of the all-in all-out system. The etiology of the disease was of a complex character, involving virus PI-3, IBR, and bacteria, and its arising reminded of the exacerbation of latent virus infection. The primary role of the PI-3 virus in the arising of the disease was documented by complex laboratory examination. As distinct from the large-capacity calf-house with continual operation where the PI-3 virus and the same bacteria participated in the arising of the respiratory syndrome, which was not controlled, in the experimental calf-houses it was sufficient to adjust the microclimatic conditions, to disinfect the stables, and to adhere strictly to the priniciple of the all-in all-out system. The proposed method of the rearing of "minimum-morbidity calves" appears to be promising for use in the prevention of respiratory infections in calves and its further testing in large-capacity calf-houses is considered as desirable.