The epizootic haematopoietic necrosis virus (Iridoviridae) induces apoptosis in vitro

The epizootic haematopoietic necrosis virus (EHNV) is an iridovirus causing severe disease in different fish species. We investigated the induction of apoptosis during EHNV infection of the epithelioma carp papulosum (EPC) cell line. Apoptosis reveals several characteristic morphological changes, such as chromatin condensation, nuclear fragmentation, cytoplasm membrane disorientation, or mitochondrial changes. During EHNV infection of EPC cells the occurrence of apoptosis was analysed using a fluorescein-isothiocyanate (FITC) conjugate of annexin-V to detect phosphatidylserines that have changed cytoplasm membrane localization. Annexin-V labelling was obvious 12 h after infection. At 54 h after EHNV infection 39% of the investigated EPC cells exhibited fluorescence. Furthermore, EHNV-infected cells were stained with 4'-6'-diamidino-2-phenylindole (DAPI) to detect pycnotic nuclei. Appearance of DAPI-positive nuclei was found beginning at 18 h after infection. At 54 h after EHNV infection approximately 56% of the EPC cells showed fragmented nuclei. Assays to inhibit a protein kinase-dependent (e.g. double-stranded RNA-dependent protein kinase) apoptosis pathway with 2-aminopurine revealed a reduction of EHNV titres, e.g. titres were reduced 1000-fold in the presence of 100 and 200 mM 2-aminopurine. Apoptosis takes place during iridovirus infection in vitro and it seems to involve the activation of protein kinases.     

Characterization of a galectin-like activity from the parasitic nematode, Haemonchus contortus, which modulates ovine eosinophil migration in vitro

The development of eosinophilia is a characteristic feature of helminth infection, although the exact nature of the interaction between eosinophils and parasites remains to be fully defined. Previously, it has been reported that Haemonchus contortus and other nematodes produce eosinophil-specific chemoattractants. This paper describes studies aimed at isolating and identifying the factor(s) responsible. Initial studies showed that soluble extracts of infective larvae (L3) of H. contortus provoked a chemokinetic, rather than chemotactic, response in ovine bone marrow eosinophils in vitro. This activity was inhibited by lactose to a markedly greater extent than sucrose suggesting a galectin-like identity. Lactose affinity chromatography of soluble H. contortus extracts resulted in the isolation a specific bound fraction which retained biological activity. SDS-PAGE gel electrophoresis indicated a single Coomassie-stained band at between 31 and 41kDa. Subsequent, mass spectrometric analysis confirmed that the bound fraction contained a mixture of nematode galectins. The results confirm that H. contortus larvae produce several galectin-like proteins, at least one of which demonstrates eosinophil chemokinetic activity in vitro. The possibility of the parasite-derived factor mimicking the mammalian galectin-9, a known eosinophil chemokine, is discussed.     

Intracranial migration of Eucoleus (Capillaria) boehmi in a dog

A 4‐year‐old, spayed‐female great Dane was referred for surgical treatment of a suspected meningioma, diagnosed on magnetic resonance imaging 10 days prior to presentation. The suspected meningioma was removed via image‐guided stereotactic craniotomy. Histopathological diagnosis was severe, locally extensive, chronic meningoencephalitis with an intralesional nematode egg. The egg was morphologically consistent with Eucoleus boehmi, and aberrant migration into the cranial cavity was the presumed cause of this lesion. Three faecal samples were collected and revealed 4+ E. boehmi eggs. Treatment involved 110 mg/kg fenbendazole (Panacur, Intervet) orally twice daily for 14 days. Nematodes including E. boehmi are a previously un‐recognised source of intracranial disease in dogs, and should be considered as a differential for mass‐like lesions visualised by magnetic resonance imaging.     

Apoptosis inhibition by insulin-like growth factor (IGF)-I during in vitro maturation of bovine oocytes

Recently, significant progress has been achieved in improving the yield of good quality embryos in vitro. However, efforts are still required to recognize the factors and understand the mechanisms of oocyte maturation, which are essential for subsequent embryo development. The aims of the present study were to determine the frequency of apoptosis in oocytes recovered from slaughterhouse ovaries and to investigate whether insulin-like growth factor (IGF)-I action during oocyte maturation in vitro may withhold apoptosis and improve oocyte quality. Only oocytes of proper morphology with homogenous ooplasm and compact cumulus cells were selected for this study. All oocytes recovered from the slaughterhouse ovaries were divided into two groups. One group of oocytes, chosen for apoptosis detection, was examined immediately after recovery. The other group of oocytes was maturated in vitro. Oocytes were maturated with IGF-I supplementation (100 ng/ml). Oocytes without supplementation were used as a control. Apoptosis in oocytes was determined by positive results of TUNEL assay and active caspase labeling. The percentage of apoptotic oocytes detected by TUNEL fell to zero when the maturation medium was supplemented with IGF-I in comparison to the control matured oocytes (0 vs. 9.87%; P<0.05). However, active caspase labeling was only slightly decreased in the IGF-I matured oocytes compared with the control matured oocytes (1.13 vs. 2.08%; P<0.05). The results indicate that IGF-I may serve as an anti-apoptotic factor during oocyte maturation. We suggest that IGF-I may inhibit apoptosis in oocytes at the stage of caspase activation and may prevent further advancement of oocyte apoptosis.     

Dual targeting of EGFR and ERBB2 pathways produces a synergistic effect on cancer cell proliferation and migration in vitro

Members of the epidermal growth factor receptor (EGFR/ERBB) gene family are frequently dysregulated in a range of human cancers, and therapeutics targeting these proteins are in clinical use. We hypothesized that similar pathways are involved in feline and canine tumours and that the same drugs may be of clinical use in veterinary patients. We investigated EGFR and ERBB2 targeting using a panel of feline and canine cell lines. EGFR and ERBB2 were targeted with siRNAs or tyrosine kinase inhibitors (TKIs) and their effect on cellular proliferation, colony formation and migration was investigated in vitro. Here we report that EGFR and ERBB2 combined siRNA targeting produced synergistic effects in feline and canine cell lines similar to that reported in human cell lines. We conclude that dual EGFR and ERBB2 targeting using TKIs should be further evaluated as a potential new therapeutic strategy in feline head and neck and mammary tumours and canine mammary tumours.     

Antiproliferation and colony-forming inhibition activities of recombinant feline interferon (rFeIFN) on various cells in vitro.

The antiproliferative and colony inhibiting activities of recombinant feline interferon (rFeIFN Type I) against various cells in vitro were examined. Feline and canine cells were both sensitive to rFeIFN. To inhibit the growth of feline cells by 50% approximately 5 x 10(2) to 1 x 10(3) U/mL rFeIFN was required and maximum activity was achieved at a concentration of 1 x 10(5) U/mL. Approximately 5 x 10(3) to 5 x 10(4) U/mL rFeIFN was necessary to inhibit the growth of canine cells by 50%. The antiproliferative and colony inhibiting activities of rFeIFN on canine cells appeared to be cell-specific and dose-dependent. However, human, monkey and hamster cells were resistant to rFeIFN. We suggest that rFeIFN might be useful for treatment of feline and some canine neoplastic conditions.     

Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I)

The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class I) were cloned and sequenced for two haplotypes (H4 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs. The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers. The engineered single-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli. Also, variants were made of the single-chain proteins, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV). An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding. The single best-defined, SLA-I restricted porcine CD8(+) T-cell epitope currently known is a 9-residue peptide from the polyprotein of CSFV (J. Gen. Virol. 76 (1995) 3039). Based on results with the CSFV epitope and two porcine haplotypes (H4 and H7), the in vitro refold assay appeared able to discriminate between peptide-free and peptide-occupied forms of SLA-I. It remains to be seen whether the rapid and technically very simple in vitro refold assay described here will prove generally applicable for the screening of virus-derived peptides for SLA-I binding.     

Porcine circovirus type 2 (PCV2) induces cell proliferation,fusion, and chemokine expression in swine monocytic cells in vitro

Granulomatous lymphadenitis is one of the pathognomonic lesions in post-weaning multisystemic wasting syndrome (PMWS)-affected pigs. This unique lesion has not been reported in direct association with viral infection in pigs. The objective of the present study was to evaluate whether porcine circovirus type 2 (PCV2) alone is able to induce functional modulation in porcine monocytic cells in vitro to elucidate its possible role in the development of granulomatous inflammation. It was found that the proliferation activity of blood monocytes (Mo) and monocyte-derived macrophages (MDM) was significantly enhanced by PCV2. During monocyte-macrophage differentiation, the PCV2 antigen-containing rate and formation of multinucleated giant cells (MGC) were significantly increased in MDM when compared to those in Mo. The MDM-derived MGC displayed a significantly higher PCV2 antigen-containing rate than did the mono-nucleated MDM. Supernatants from PCV2-inoculated MDM at 24 h post-inoculation induced an increased tendency of chemotactic activity for blood Mo. At the same inoculation time period, levels of mRNA expression of the monocytic chemokines, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1, also significantly increased in PCV2-inoculated MDM. The results suggest that PCV2 alone may induce cell proliferation, fusion, and chemokine expression in swine monocytic cells. Thus, PCV2 itself may play a significant role in the induction of granulomatous inflammation in PMWS-affected pigs.     

Scrapie-like encephalopathy in a greater kudu (Tragelaphus strepsiceros) which had not been fed ruminant-derived protein.

A 19-month-old greater kudu (Tragelaphus strepsiceros), whose dam had died 15 months earlier with spongiform encephalopathy, required euthanasia after developing severe ataxia and depression with an apparently sudden onset. No macroscopic abnormalities were detected on post mortem examination but a scrapie-like spongiform encephalomyelopathy was apparent on histopathological examination of brain and segments of spinal cord. Negative stain electron microscopy of proteinase K-treated detergent extracts of tissue from the brain stem revealed the presence of scrapie associated fibrils, and a 25 to 28 kDa band comparable with that identified as abnormal PrP (prion protein) from the brains of domestic cattle with spongiform encephalopathy was detected using rabbit antiserum raised against mouse PrP. The animal was born nine months after the statutory ban on the inclusion of ruminant-derived protein in ruminant feeds and, as no other possible sources of the disease were apparent, it appears likely that the infection was acquired from the dam.     

Radiofrequency radiation at 40 kHz induces hepatic injury in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease

In the present study, we examined effects of radiofrequency (RF) radiation at 40 kHz on hepatic injury in Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson disease, which is a heritable disease of copper metabolism in the liver. The activities of ALT and AST in serum of LEC rats exposed to RF radiation for 2 weeks were approximately 3.8-fold and 2-fold higher than those in serum of sham-exposed rats, respectively. Although there were no significant differences in hepatic copper contents between LEC rats exposed to RF radiation for 2 weeks and sham-exposed rats, copper contents in the kidney and serum of exposed LEC rats were approximately 4.2-fold and 12.9-fold higher than those in sham-exposed rats, respectively. Relative O??-scavenging activities in the S-100 fraction of the liver of LEC rats exposed to RF radiation for 2 weeks were 1.6-fold higher than those in sham-exposed rats. No significant differences were observed in activities of AST and ALT in serum and relative O??-scavenging activity in the S-100 fraction of the liver of normal control WKAH rats that were sham-exposed and exposed to RF radiation. No significant differences were observed in copper contents in the liver, kidney and serum of WKAH rats that were sham-exposed and exposed to RF radiation for 2 weeks. The results show that RF radiation at 40 kHz induced hepatic injury in LEC rats.     

Infection with foot-and-mouth disease virus (FMDV) induces a natural killer (NK) cell response in cattle that is lacking following vaccination

Natural killer (NK) cells play a role in innate antiviral immunity by directly lysing virus-infected cells and producing antiviral cytokines such as interferon gamma (IFN-γ). We developed a system for characterizing the bovine NK response to foot-and-mouth disease virus (FMDV), which causes a disease of cloven-hoofed animals and remains a threat to livestock industries throughout the world. IL-2 stimulation of PBMC resulted in poor killing of human K562 cells, which are often used as NK target cells, while lysis of the bovine BL3.1 cell line was readily detected. Depletion of NKp46-expressing cells revealed that 80% of the killing induced by IL-2 could be attributed to NKp46⁺ cells. In order to characterize the response of NK cells against FMDV in vivo, we infected groups of cattle with three different strains of the virus (A24 Cruzeiro, O1 Manisa, O Hong Kong) and evaluated the cytolytic ability of NK cells through the course of infection. We consistently observed a transient increase in cytolysis, although there was variation in magnitude and kinetics. This increase in cytolysis remained when CD3⁺ cells were removed from the preparation of lymphocytes, indicating that cytolysis was independent of MHC-T cell receptor interaction or γδ T cell activation. In contrast, animals monitored following vaccination against FMDV did not exhibit any increase in NK killing. These data suggest that NK cells play a role in the host immune response of cattle against FMDV, and contrast with the suppression of NK activity previously observed in swine infected with FMDV.     

Treatment of rats with heat killed cells (HKC) of Cryptococcus neoformans var. grubii induces cellular activation in spleen and lymphatic nodes

Our previous studies showed that the subcutaneous pretreatment of rats with heat killed cells of Cryptococcus neoformans (HKC) emulsified in complete Freund adjuvant (CFA) promotes protection against an intraperitoneal challenge with viable C. neoformans. In this model, an appropriate activation of adherent peritoneal cells after antigenic treatment is very important for the control of the infection. Here, we investigated the immune response developed in spleen and lymphatic nodes as a result of treatment with HKC-CFA, which might also contribute in the protective phenomenon of this treatment against cryptococcal infection. The results show that, compared with adjuvant alone, rats which received treatment with HKC-CFA presented a greater activation of adherent splenic cells, with up-regulation of major histocompatibility complex class II (MHC II) and CD86 expression and secretion of anticryptococcal metabolites. Furthermore, this treatment also induced an increase in the blastogenic response and the secretion of Th1 and Th2 cytokines by spleen cells in comparison with cells from CFA-phosphate-buffered saline (PBS) treated rats. On the other hand, lymph node cells from animals treated with HKC-CFA presented a rise in the expression of MHCII but not of CD86 with respect to control cells from rats treated with CFA-PBS. These cells also showed a high proliferative response and secretion of Th1-related cytokines, interleukin (IL)-12 and tumor necrosis factor (TNF). These results show that treatment of rats with HKC-CFA is able to induce an early immune response in secondary lymphoid organs, which may contribute to the protective effect induced by this treatment.     

Co-infection of porcine dendritic cells with porcine circovirus type 2a (PCV2a) and genotype II porcine reproductive and respiratory syndrome virus (PRRSV) induces CD4(+)CD25(+)FoxP3(+) T cells in vitro

Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases in the global swine industry. Porcine circovirus type 2 (PCV2) is the primary causative agent, however co-infection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV) is often required to induce the full spectrum of clinical PCVAD. While the specific mechanisms of viral co-infection that lead to clinical disease are not fully understood, immune modulation by the co-infecting viruses likely plays a critical role. We evaluated the ability of dendritic cells (DC) infected with PRRSV, PCV2, or both to induce regulatory T cells (T(regs)) in vitro. DCs infected with PCV2 significantly increased CD4(+)CD25(+)FoxP3(+) T(regs) (p<0.05) and DCs co-infected with PRRSV and PCV2 induced significantly higher numbers of T(regs) than with PCV2 alone (p<0.05). Cytokine analysis indicated that the induction of T(regs) by co-infected DCs may be dependent on TGF-β and not IL-10. Our data support the immunomodulatory role of PCV2/PRRSV co-infection in the pathogenesis of PCVAD, specifically via T(reg)-mediated immunosuppression.     

Atypical parasitic migration and necrotizing sacral myelitis due to Serratospiculoides amaculata in a prairie falcon (Falco mexicanus)

An adult, wild-caught, female prairie falcon (Falco mexicanus) was presented with the chief complaint of anorexia. Radiographic findings included increased densities within the air sacs, and coelomic endoscopy revealed numerous slender worms within the air sacs and on the serosal surfaces of the ovary, oviduct, liver, proventriculus, and ventriculus. The bird seemed to improve for a short period of time with antiparasitic therapy (ivermectin and fenbendazole) and supportive care. Twenty-one days after initial presentation, the bird became recumbent with increasing pelvic limb neurologic deficits and was euthanized. On histopathologic examination, mature nematodes and larvated eggs identified as Serratospiculoides amaculata were found within the subdural space of the distal thoracolumbar and synsacral spinal cord and within the coelomic cavity. This case suggests that S. amaculata can cause clinically significant lesions in its falconiform host with potentially fatal results.     

The bovine viral diarrhea virus (BVDV) NS3 protein, when expressed alone in mammalian cells, induces apoptosis which correlates with caspase-8 and caspase-9 activation

The bovine viral diarrhea virus (BVDV) strains exist as two biotypes, cytopathic (cp) and noncytopathic (ncp), according to their effects on tissue culture cells. It has been previously reported that cell death associated to cp BVDV in vitro is mediated by apoptosis. Here, experiments were conducted to determine the involvement of the NS3 protein in the induction of apoptosis. The NS3- and NS3Delta50 (deleted from the NH2-terminal 50 amino acids)-cDNA encoding sequences of BVDV NADL cp reference strain were cloned into adenoviral vectors (AdV) from which the BVDV gene of interest could be expressed from a tetracycline-responsive promoter. A549tTA cells infected in vitro with NS3 or NS3Delta50-expressing AdV showed cytopathic changes characterized by cell rounding and detachment, and nucleus chromatin condensation. DNA fragmentation assays, cytochrome c release, and activation of cellular caspases performed on these infected cells clearly correlated with the observed cytopathic changes with apoptosis. The BVDV NS3Delta50-induced apoptotic process was inhibited by caspase-8- and -9-specific peptide inhibitors (Z-IETD-FMK and Z-LEHD-FMK). Furthermore, apoptosis was inhibited in cells expressing the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase (HSV2-R1) or hsp70, two proteins which are known to inhibit apoptosis associated with caspase-8 activation and cytochrome c release-dependent caspase-9 activation, respectively. Given that HSV2-R1, a specific inhibitor of the caspase-8 activation pathway, efficiently suppressed apoptosis and also prevented caspase-9 activation, the overall results indicate that the BVDV NS3/NS3Delta50 induces apoptosis initiated by caspase-8 activation and subsequent cytochrome c release-dependent caspase-9 activation.     

The leucine metabolite 3-hydroxy-3-methylbutyrate (HMB) modifies protein turnover in muscles of laboratory rats and domestic chickens in vitro

This study was conducted to assess the in vitro effects of leucine and 3-hydroxy-3-methylbutyrate (HMB) on protein synthesis and degradation in isolated rat and chick muscles. Isolated muscles — extensor digitorum longus (EDL) and soleus (S) in rats and EDL and tibialis anterior (TA) in chicks — were taken out intact and incubated in Krebs—Henseleit buffer with [¹⁴C]phenylalanine. Protein synthesis was then determined by measuring [¹⁴C]phenylalanine incorporation into protein and protein breakdown was estimated by net release of tyrosine from muscle proteins. In all muscles investigated leucine stimulated protein synthesis (average 20%; p < 0.05 versus control) but only slightly reduced proteolysis (average 8%, NS). In contrast, HMB had no significant effect on protein synthesis but consistently decreased protein degradation in the range from −29% (p < 0.01 versus control) in rat extensor digitorus longus to −5% (NS) in rat soleus muscles. HMB appeared to suppress protein degradation more in white muscle fibres than in red muscle fibres (at least in the rat). These data support observations in humans indicating that HMB decreases muscle proteolysis. It is suggested that metabolism of leucine to HMB could be responsible for the reported suppressive effects of leucine on protein breakdown.