Effects of gastrointestinal parasites on parasite burden, rectal temperature, and antibody titer responses to vaccination and infectious bovine rhinotracheitis virus challenge.

Thirty-three colostrum-deprived Holstein bull calves (initial BW of 131 ± 4 kg) were used to determine the effect of timing of anthelmintic administration relative to vaccination on antibody titer response to vaccine component antigens. When calves were at least 3 mo of age, they were sorted randomly into individual pens and assigned to 1 of 3 treatment groups, treatments consisted of 1) dewormed 2 wk before vaccination (DPV), 2) dewormed at the time of vaccination (DV), or 3) control, vaccinated but not dewormed (CONT). All calves were inoculated with infective larvae of brown stomach worms (Ostertagia ostertagi) and intestinal worms (Cooperia spp.) on d 1, 7, 10, 14, and 18 for a total dose of 235,710 infective larvae per calf. Calves (DPV and DV) were dewormed on d 21 or 35 with a 10% fenbendazole suspension at 5 mg/kg of BW. On d 35, all calves were vaccinated with a modified-live virus respiratory vaccine containing IBRV (infectious bovine rhinotracheitis virus), BVDV-1 (bovine viral diarrhea virus genotype 1), BVDV-2 (BVDV genotype 2), PI-3 (parainfluenza-3), and BRSV (bovine respiratory syncytial virus). During the 103-d experiment, weekly fecal egg counts, blood, and rectal temperatures were collected and health status was recorded daily. Blood samples were obtained weekly to determine serum neutralizing (SN) antibody titers to IBRV, BVDV-1, BVDV-2, and PI-3 and cytokine levels for IL-4, IL-6, TNF-α (tumor necrosis factor-α), and IFN-γ (interferon-gamma). There was a tendency (P < 0.09) for CONT calves to have greater IL-4 concentrations. By design, control calves had greater (P < 0.01) fecal egg counts during the experiment. All calves developed antibody titers to IBRV, BVDV-1, BVDV-2, and PI-3 by d 15 postvaccination. On d 88, all calves were challenged with IBRV and blood samples were obtained on d 88, 89, 90, 93, 95, 98, 99, and 103. All calves had increased rectal temperatures during the final 7 d of the IBRV challenge. However, the CONT group had greater (P < 0.01) rectal temperatures on each sampling day except d 90 compared with the DPV and DV treatments. Therefore, deworming before or at vaccination reduced parasite burden and decreased rectal temperature increase after an IBRV challenge. Deworming strategy had no effect on antibody response to vaccination or IBRV challenge.

Interferon stimulated genes, CXCR4 and immune cell responses in peripheral blood mononuclear cells infected with bovine viral diarrhea virus.

Non-cytopathic bovine viral diarrhea virus (ncpBVDV) induces immune responses mediated by chemokines and interferon (IFN) stimulated genes (ISGs). Cultured bovine peripheral blood mononuclear cells (PBMC) from ncpBVDV-naïve cattle were used herein to demonstrate that BVDV infection modulates chemokine receptor 4 (CXCR4), CXCL12, IFN-I, ISGs and selected immune cell marker (CD4, CD8, CD14) mRNAs, and that these acute responses to viral infection are reflected in PBMC cultured with serum from heifers carrying fetuses persistently infected (PI) with ncpBVDV. Infection of PBMC with ncpBVDV increased IFN-ß, ISG15, RIG-I, CXCR4, CXCL12, and CD8 mRNA concentrations after 32 h. Culture of PBMC with uterine vein serum from acutely infected heifers, inoculated with ncpBVDV during early gestation to generate PI fetuses, also increased the concentration of CXCR4, RIG-I and ISG15 mRNAs. In vitro PBMC treatment with ncpBVDV or uterine vein serum from acutely infected pregnant heifers activates chemokine, ISG and immune cell responses.

Bovine viral diarrhea virus cyclically impairs long bone trabecular modeling in experimental persistently infected fetuses.

Persistent infection (PI) with bovine viral diarrhea virus (BVDV) has been associated with osteopetrosis and other long bone lesions, most commonly characterized as transverse zones of unmodeled metaphyseal trabeculae in fetuses and calves. This study was undertaken to characterize the morphogenesis of fetal long bone lesions. Forty-six BVDV-naïve pregnant Hereford heifers of approximately 18 months of age were inoculated with noncytopathic BVDV type 2 containing media or media alone on day 75 of gestation to produce PI and control fetuses, respectively, which were collected via cesarean section on days 82, 89, 97, 192, and 245 of gestation. Radiographic and histomorphometric abnormalities were first detected on day 192, at which age PI fetal long bone metaphyses contained focal densities (4 of 7 fetuses) and multiple alternating transverse radiodense bands (3 of 7 fetuses). Day 245 fetuses were similarly affected. Histomorphometric analysis of proximal tibial metaphyses from day 192 fetuses revealed transverse zones with increased calcified cartilage core (Cg.V/BV, %) and trabecular bone (BV/TV, %) volumes in regions corresponding to radiodense bands (P < .05). Numbers of tartrate resistant acid phosphatase positive osteoclasts (N.Oc/BS, #/mm(2)) and bone perimeter occupied (Oc.S/BS, %) were both decreased (P < .05). Mineralizing surface (MS/BS, %), a measure of tissue level bone formation activity, was reduced in PI fetuses (P < .05). It is concluded that PI with BVDV induces cyclic abnormal trabecular modeling, which is secondary to reduced numbers of osteoclasts. The factors responsible for these temporal changes are unknown but may be related to the time required for osteoclast differentiation from precursor cells.

The fetal brain in bovine viral diarrhea virus-infected calves: lesions, distribution, and cellular heterogeneity of viral antigen at 190 days gestation.

Previous studies have shown that the brain is a target of persistent infection with bovine viral diarrhea virus (BVDV) and have demonstrated viral tropism for neurons as well as other endogenous cell types in diverse brain areas. Apart from foci of mild residual inflammation in some postnatal calves, consistent brain lesions, per se, have not been reported. No similar comprehensive studies of the brain have been reported in bovine fetuses. In the current study, 12 BVDV-seronegative heifers were inoculated intranasally with a 2-ml 4.4 log(10) TCID(50)/ml dose of noncytopathic type 2 BVDV at 75 and 175 days of gestation to create persistently and transiently infected fetuses, respectively. In only persistently infected fetuses, encephaloclastic lesions resulting in pseudocysts were observed in the subependymal zone in the region of the median eminence and adjacent corona radiata as well as in the region of the external capsule associated with lenticulostriate arteries. Additionally, areas of rarefaction in white matter were observed at the tips of cerebrocortical gyri and in the external capsule. The distribution of viral antigen was examined by immunohistochemical labeling using the 15C5 anti-BVDV monoclonal antibody. Viral antigen was detected only in calves inoculated at 75 days of gestation, i.e., persistently infected. The pattern of BVDV immunolabeling revealed both similarities and differences compared with previous studies in postnatal calves, suggesting that viral infection in the brain is a dynamic and progressive rather than static process.

Transplacental infection with non-cytopathic bovine viral diarrhoea virus types 1b and 2: viral spread and molecular neuropathology.

Infection with bovine viral diarrhoea virus (BVDV) represents a reproducible natural animal model in which to study mechanisms of transplacental viral infection. In the present study, BVDV-seronegative heifers were challenged intranasally with non-cytopathic BVDV of genotype 1b or 2. Fetuses were retrieved by caesarean section 7-114 days post-challenge of the dam and subjected to virological, histopathological and immunohistochemistry(IHC) studies. Gross and histopathological changes were only seen in fetuses infected at gestational age 75-85 days and retrieved at gestational age 190 days. Viral antigen could be detected in most tissues from 14 days post-infection, but the primary target organs for histopathological changes were brain, liver and spleen. In the brain, microscopical changes included leucomalacia and macrophage infiltration of meninges and neuropil. Viral antigen was detected in neurons, oligodendrocyte precursors and infiltrating macrophages. IHC revealed normal to slightly increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha) in the infected fetuses, with evidence of neuronal apoptosis and induction of inducible nitric oxide synthase (iNOS) and phospho-p38alpha mitogen-activated protein kinase (MAPK). These findings suggest that hypoxia may play only a limited role in the pathogenesis of the neural lesions. By contrast, virus-induced cytokine cascades, as part of the fetal innate immune response, and apoptosis of neurons and glial precursor cells may be central to the development of lesions.

Characterization of a novel pestivirus originating from a pronghorn antelope.

A unique pestivirus, isolated from a pronghorn antelope (Antilocopra americana), was characterized. Serum neutralization studies suggested that this virus was antigenically related to pestiviruses. Genomic characteristics, unique to pestiviruses, indicated that this virus belongs to the Pestivirus genus. These characteristics included the organization of the 5' untranslated region (5'-UTR), the presence and length of a viral Npro coding region, conservation of cysteine residues in Npro, conservation of predicted amino acid sequences flanking the cleavage sites between viral polypeptides Npro and C and between C and Erns and conservation of predicted hydrophobicity plots of Npro protein. While this data indicated the virus belongs to the Pestivirus genus, phylogenetic analysis in 5'-UTR, Npro and E2 regions suggested that it is the most divergent of the pestiviruses identified to date. This conclusion was also supported by the amino acid identity in coding regions. The corresponding values were much lower for the comparison of pronghorn pestivirus to other pestivirus genotypes than only between previous recognized genotypes. These results suggest the virus isolated from pronghorn antelope represents a new pestivirus genotype. It also represents the only pestivirus genotype first isolated from New World wildlife.

Isolation of bovine viral diarrhea virus from a free-ranging mule deer in Wyoming.

A noncytopathic type 1a bovine viral diarrhea virus (BVDV) was isolated from a free-ranging yearling female mule deer (Odocoileus hemionus) from northwestern Wyoming (USA). The mule deer was emaciated, weak, and salivating, and Arcanobacterium pyogenes was cultured from lung abscesses. Bovine viral diarrhea virus was isolated from lung, however, BVDV antigen was not detected by immunohistochemistry. The BVDV genotype was determined by reverse transcriptase polymerase chain reaction and the RNA sequences from the 5'UTR and E2 genes compared with sequences of a type 1a BVDV isolated from cattle from the same area as the deer. The sequences from the deer BVDV were distinct from those of the bovine type 1a BVDV, but similar to other bovine type 1a BVDVs. Seventy-four (60%) of 124 sera collected from mule deer in this area had serum neutralizing antibody titers to type 1a BVDV of > or = 1:32. The high prevalence of seropositive mule deer and isolation of BVDV suggests that this virus circulates in the mule deer population. The isolate described in this report is the second reported BVDV isolate from free-ranging deer in North America and the first from a mule deer.

Identification and phylogenetic comparison of Salem virus, a novel paramyxovirus of horses.

A virus that could not be identified as a previously known equine virus was isolated from the mononuclear cells of a horse. Electron microscopy revealed enveloped virions with nucleocapsid structures characteristic of viruses in the Paramyxoviridae family. The virus failed to hemabsorb chicken or guinea pig red blood cells and lacked neuraminidase activity. Two viral genes were isolated from a cDNA expression library. Multiple sequence alignments of one gene indicated an average identity of 45% as compared to Morbillivirus N protein sequences. A weaker relationship was found with Tupaia paramyxovirus (TPMV) and Hendra virus (HeV) N proteins. In the second gene, multiple open reading frames (ORFs) were identified, corresponding to the arrangement of the P, V, and C ORFs in the Morbillivirus and Respirovirus viruses. Short stretches in the C-terminal regions of the P and C proteins showed limited homologies to viruses in the Morbillivirus genus but no obvious relationship to viruses in other genera. The V ORF translation product contained a highly conserved, cysteine-rich domain that is common to most viruses in the Paramyxovirinae subfamily. Sequencing of P gene cDNA clones confirmed the use of a cotranscriptional editing mechanism for the regulation of P/V expression. Based on the location of its origin it has been named Salem virus (SalV).

Experimental infection of deer with bovine viral diarrhea virus.

In order to determine the susceptibility of deer to infection with bovine viral diarrhea virus (BVDV), four mule deer (Odocoileus hemionus) fawns and one white-tailed deer (O. virginianus) fawn were inoculated intranasally with the New York-1 strain of BVDV originally isolated from cattle. None of the animals developed clinical signs of illness. Virus was isolated from white blood cells from four fawns on one or more occasions from day 2 through day 15 post-inoculation (PI) indicating that infection and systemic spread of BVDV had occurred. In addition, virus was isolated from nasal swabs from three fawns, one to three times, from day 2 through day 8 PI. Four fawns had virus neutralizing antibody titers to two strains of BVDV prior to inoculation and all developed greater than four-fold increases in virus neutralizing antibody titers by 3 wk PI. No gross lesions of bovine viral diarrhea were detected at necropsy approximately 3 mo PI. A variety of nonspecific lesions were detected by histopathology. Based on these findings, mule and white-tailed deer are susceptible to infection with BVDV. Isolation of virus from nasal swabs is evidence that BVDV could be transmitted by deer via direct contact.

Tumor necrosis factor as a mediator of inflammation in influenza A viral pneumonia.

The role of tumor necrosis factor alpha (TNF alpha) in the pathogenesis of influenza A viral pneumonia was examined. CD-1 male mice were challenged intranasally with influenza A virus A/PR/8/34 (H1N1) and administered rabbit anti-mouse TNF alpha-specific-neutralizing antibodies intraperitoneally. The effect of treatment on virus titer, TNF alpha levels, morbidity, mortality, and on pathologic lung lesions were compared with sham-treated controls. The severity of gross and histologic lung lesions positively correlated with the peak bronchoalveolar TNF alpha levels and was ameliorated with anti-TNF alpha treatment. Survivorship was prolonged in mice given a lethal dose of virus by treatment with TNF-alpha neutralizing antibodies. Reduction of TNF alpha levels by treatment with TNF alpha-antibodies did not affect virus titers in the lung. These results suggest that TNF alpha is a mediator of pulmonary inflammation during influenza A viral pneumonia, but may not play a significant anti-viral role in influenza pneumonia.

Influenza A viruses isolated from waterfowl in two wildlife management areas of Pennsylvania.

A survey was conducted at two wildlife management areas of Pennsylvania (USA) to evaluate an antigen capture enzyme-linked immunosorbent assay (AC-ELISA) for the detection of avian influenza viruses (AIV) in cloacal swabs from waterfowl and to determine the influenza A virus subtypes and the distribution of these viruses among waterfowl. We collected 330 cloacal swabs from hunter-killed waterfowl in the fall of 1990 and from cage-captured waterfowl in the summer of 1991. Thirty-one hemagglutinating agents were isolated by chicken embryo inoculation (CEI) of which 27 were influenza A viruses and four Newcastle disease viruses (NDV). The prevalence of AIV infection was 8.2%. Compared to CEI, AC-ELISA was only 15% sensitive and 61% specific. Based on the distribution of AIV by species of waterfowl, mallards (Anas platyrhynchos) and American wigeons (Anas americana) were at equal risk of AIV infection even though most of the AIV isolates came from mallards. Although significant crude effects of sampling site and season on AIV recovery could be established, juvenile age was identified as the primary risk factor of AIV recovery. Twelve AIV subtypes were identified by hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests. The most prevalent subytpes were H4N8 and H6N8. We concluded that AC-ELISA was not useful for the detection of AIV in cloacal swabs from waterfowl and that CEI, HI, and NI tests remain as the method of choice for AIV screening in waterfowl. Based on the results AIV infected preferentially the young which represent the high risk group in waterfowl populations.(ABSTRACT TRUNCATED AT 250 WORDS)

Influenza A virus replication is inhibited by tumor necrosis factor-alpha in vitro.

Treatment of Madin-Darby canine kidney (MDCK) cells with > or = 10 ng/ml of recombinant murine tumor necrosis factor-alpha (rmTNF-alpha) prior to inoculation with influenza A/PR/8/34, (H1N1), resulted in decreased production of infectious virus as determined by plaque assay, inhibition of viral protein synthesis and a reduction in cytopathic effect. The inhibition of viral replication in vitro suggests a role for TNF-alpha in the host's defense against influenza A virus infections.

Virulent avian influenza A viruses: their effect on avian lymphocytes and macrophages in vivo and in vitro.

To investigate the pathogenesis of virulent avian influenza A viruses, the effect of A/turkey/Ont/7732/66 (H5N9) (Ty/Ont), A/tern/South Africa/1961 (H5N3) (Tern/S.A.) and A/chicken/Pennsylvania/1370/83 (H5N2) (Ck/Penn) on avian lymphoid cell populations was examined in vivo. Previous studies have shown that infection of chickens with Ty/Ont resulted in the extensive destruction of lymphoid tissues. In this study, other virulent avian H5 influenza viruses, Tern/S.A. or Ck/Penn, had little or no effect on lymphoid tissues of infected chickens. Therefore the effect of Ty/Ont on lymphoid tissue is a specific activity of this virus only and not of other virulent avian H5 influenza strains. To examine the role of viral replication in the destruction of lymphocytes, in vitro cultures of avian macrophages and lymphocytes were inoculated with Ty/Ont. Macrophages supported the synthesis of viral proteins whereas lymphocytes produced small, but detectable amounts of viral protein; however, infectious virus was not produced by either cell type. Furthermore inoculation of chicken spleen cells with Ty/Ont in vivo and in vitro had a profound effect on the proliferative response of lymphocytes to concanavalin A. These results suggest that Ty/Ont infects macrophages as well as lymphocytes in the chicken, and the effects of the virus on both cell types may well contribute to lymphoid necrosis.

Destruction of lymphocytes by a virulent avian influenza A virus.

Infection of chickens by a virulent avian influenza A virus, A/turkey/Ont/7732/66 (H5N9), was associated with a severe lymphopenia. High titres of infectious virus were found in lymphoid tissues early in infection and were accompanied by severe damage to the lymphocyte populations as demonstrated by histopathological examination. Non-lymphoid cell populations in these tissues were unaffected, as were other organs examined. The viral nucleoprotein was localized by immunoperoxidase staining to lymphocytes in affected tissues early in infection.

Pathological lesions in the lungs of ducks infected with influenza A viruses.

To determine histopathological damage in the respiratory tract, ducks were inoculated with five different influenza A viruses, including viruses virulent for other avian hosts. Lungs were collected for detection of virus and histopathological examination. Small amounts of infectious virus were recovered from lungs, and viral antigens were demonstrated by immunoperoxidase staining with monoclonal antibodies to the viral nucleoprotein. Although clinical signs were not detected, lungs of ducks infected with both virulent and avirulent viruses had mild pneumonia characterized by infiltrates of lymphocytes and macrophages. These findings show that although clinical signs are not evident, ducks may have damage to the respiratory tract during influenza.

Sperm morphology assessment as an indicator of human fertilizing capacity.

Semen samples from 95 men were examined by routine semen analysis and specific histologic staining for sperm morphology. The men were classified into fertile and infertile groups on the basis of clinical evaluation and in vitro testing, using the zona-free hamster egg penetration assay. Thirty men were designated as fertile, as they had fathered children and their sperm showed penetration of greater than 20% of the zona-free hamster eggs with the in vitro fertilization test. Subjects classified as infertile were men from infertile couples whose wives showed no evidence of infertility and whose in vitro fertilization ability was 10% or less. The semen analysis parameters of the fertile and infertile groups were significantly different. Fertile men had mean values of 108 X 10(6) sperm/ml, 61% motile, 64% normal forms (sperm with oval morphology), and 69% penetration in vitro. The mean values for infertile men were significantly lower: 42 X 10(6) sperm/ml, 45% motile, 32% normal forms, and 3.2% penetration in vitro. The importance of the morphology parameter was revealed by comparison of the percentage of penetration with count, motility, and morphology. Penetration correlated best with morphology (r = 0.730) as compared with motility (r = 0.451) and count (r = 0.605). The distribution of abnormalities in the infertile group revealed 81.6% with abnormal morphology (less than 50%), 53.8% with abnormal motility (less than 50%), and 38.5% with abnormal count (less than 20 million/ml). As a single parameter, decreased number of normal forms appears to be a good indicator for clinical infertility if in vitro fertilization testing is not available.