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Vaccines and therapeutics using in vitro transcribed mRNA hold enormous potential for human and veterinary medicine. Transfection agents are widely considered to be necessary to protect mRNA and enhance transfection, but they add expense and raise concerns regarding quality control and safety. We found that such complex mRNA delivery systems can be avoided when transfecting epithelial cells by aerosolizing the mRNA into micron-sized droplets. In an equine in vivo model, we demonstrated that the translation of mRNA into a functional protein did not depend on the addition of a polyethylenimine (PEI)-derived transfection agent. We were able to safely and effectively transfect the bronchial epithelium of foals using naked mRNA (i.e., mRNA formulated in a sodium citrate buffer without a delivery vehicle). Endoscopic examination of the bronchial tree and histology of mucosal biopsies indicated no gross or microscopic adverse effects of the transfection. Our data suggest that mRNA administered by an atomization device eliminates the need for chemical transfection agents, which can reduce the cost and the safety risks of delivering mRNA to the respiratory tract of animals and humans.

ResumenAntecedentesEl desarrollo y aplicación de imágenes tridimensionales en cirugía laparoscópica ha brindado los beneficios de la percepción de profundidad, de la que la laparoscopia tradicional carecía. Existen estudios al respecto en poblaciones con experiencia quirúrgica previa y para limitar este sesgo de selección realizamos este estudio en población sin experiencia previa.Material y métodosEstudio experimental, abierto, transversal y comparativo en el que se evaluaron los logros obtenidos con un equipo 2D de cirugía laparoscópica y un equipo 3D, siendo cada sujeto su propio control. Se evaluaron 6 destrezas realizadas en 2D y 3D.ResultadosSe incluyó a un total de 40 participantes, de los cuales 20 comenzaron los ejercicios en 2D y 20 en 3D. En el 72%, de 118 destrezas se mejoró el tiempo de realización con 3D, contra solo 37% en 2D (p=0.000). El porcentaje al final de los ejercicios en el grupo de 3D tanto en el primero como en el segundo ejercicio fue mayor de manera estadísticamente significativa y se observaron menores tiempos al realizar los ejercicios en 3D. Un 52.5% de los participantes prefirieron el equipo 3D, el 15% el 2D y el 32.5% no tuvieron preferencia por ninguno.DiscusiónComo se ha encontrado en diversos estudios, existió mejoría en el desempeño en el grupo 3D. Al ser población sin experiencia quirúrgica previa, se elimina este sesgo del estudio.ConclusionesLa realización de destrezas laparoscópicas en 3D mostró ventajas en comparación con 2D, con mayor porcentaje de finalización, menores tiempos para realizarlas y menor curva de aprendizaje.AbstractBackgroundThe development and application of 3D images in laparoscopic surgery has brought the benefit of in-depth perception that traditional laparoscopic surgery lacked. Previous studies in surgical populations have demonstrated the advantages of 3D technology. To limit bias of the previous experiences of participants, this study was performed in a population without any experience in this area.Material and methodsAn experimental, open, cross-sectional, comparative study between surgical skills achievements using 2D and a 3D laparoscopy equipment, using each subject as their own control. Six skills were evaluated in 2D and 3D modalities.ResultsOf the 40 participants included, 20 began the skills in the 2D modality and then performed them in 3D, and the other 20 began in 3D. Of the 118 skills evaluated there was a time improvement in 72% in the 3D group compared to 37% in the 2D modality (P=.000). The accomplishment percentage using the 3D laparoscopy was greater for both groups. There was a statistically significant difference in the better time for the 3D performed tasks. Just over half (52.5%) of participants preferred 3D laparoscopy, 15% preferred 2D, and 32.5% had no preferences.DiscussionAs other studies have demonstrated, there was improvement in the overall performance using the 3D laparoscope. Bias was limited by using a population without surgical experience.Conclusions3D laparoscopic surgical skills showed superior to 2D, with higher percentages of tasks completion, less time in performing them, and a shorter learning curve.

Background: Peroral endoscopic myotomy has recently been developed and performed on patients with good results. Aims: To evaluate the technical feasibility of peroral endoscopic full-thickness and partial thickness myotomy in a porcine model. Material and methods: Eighteen criollo pigs were randomly assigned to 2 groups: group A (partial-thickness myotomy) and group B (full-thickness myotomy). The mucosal defect proximal to the myotomy site was left open. On the seventh postoperative day the pig was euthanized and follow-up surgical exploration was performed. The duration of each procedure, postoperative progression of the animal, complications, and anatomopathologic findings were registered. Results: The procedure was viable in all the pigs. The mean surgery duration was 81 ± 35.3 min (group A 51.11 ± 11.12, group B 111 ± 22.61; P

ResumenAntecedentesRecientemente la miotomía endoscópica por vía oral ha sido desarrollada y realizada en pacientes, con buenos resultados.ObjetivoEvaluar la factibilidad técnica de la miotomía endoscópica por vía oral de espesor total y parcial en un modelo porcino.Material y métodosDieciocho cerdos criollos se asignaron al azar a 2 grupos: grupo A (miotomía de espesor parcial) y grupo B (miotomía de espesor total). El defecto mucoso proximal al sitio de la miotomía se dejó abierto. Al séptimo día el cerdo fue sacrificado, realizándose exploración quirúrgica de seguimiento. Se registró el tiempo de cada procedimiento, la evolución del animal en el postoperatorio, la presencia de complicaciones y los hallazgos anatomopatológicos.ResultadosEl procedimiento fue factible en todos los cerdos. El tiempo promedio fue de 81±35.3min (grupo A 51.11±11.12, grupo B 111±22.61; P

Nowadays, different mechanical artificial sphincters can be found implanted in human beings, trying to overcome a deficiency in the performance of the natural one. However, they do not take into account the natural anal sphincter’s (AS) dimensions, and autonomous response; they also lack in basic contraction and relaxation properties. In this paper, by addressing the AS behavior, an AS model designed with Matlab/SimMechanics is shown. The model comprises bodies of concentrated mass interconnected by springs. The mass–spring system is arranged in concentric rings where every concentrated mass is interconnected by a spring. Each spring takes specific stiffness, which varies with length, in accordance to an experimental curve. The system described can be loaded or unloaded, describing then the muscle behavior.Each element that forms the model of rings is subject to displacements caused by forces of traction and compression, when a radial force is applied from the center towards the inner ring. The springs of the inner ring experience forces of traction, whereas the springs that connect the body of the inner ring with the outer ring perpendicularly are submitted to compression forces.The data used in the proposed model corresponded to dimensions of the humanAS: width, height, rigidity, stress, tension, basically obtaining an initial deformation behavior according to the sphincter in the passive state. The model remained stable with some mechanical oscillations due to the elastic elements; by modifying one of the parameters, the behavior became unstable and unmanageable. It was verified that it is a sensitive model when modifying the initial conditions that the concrete data requires in case of reproducing the sphincter muscle with particular dimensions.

We have recently provided evidence that Texas feline immunodeficiency virus (FIV-TX) isolates are an emerging subtype sharing a common ancestry with clade B isolates. Specific, pathogen-free cats were infected, intravenously, with 500, 2000 or 8000 TCID 50 of the FIV-TX53 virus to study the acute stage of infection. Infection of cats resulted in lymphadenopathy at 10 days post-infection (p.i.). By 7 weeks p.i., gag specific antibody could be detected from sera of all infected cats. Virus could be detected by culturing PBMC and by nested capsid PCR. A reduction in the absolute numbers of lymphocytes and neutrophils was observed in infected cats although there was no trend identified between this reduction and the viral dose administered. By 11 weeks p.i., the CD4 + /CD8 + T cell ratios from all infected cats had dropped from approximately 2 to below 1. While decrease in the ratio was dependent on the viral dose, the T cell ratios of cats receiving the highest dose had significantly dropped below 1 by 4–7 weeks p.i. This decrease in the ratio was accompanied by a sharp and temporal decline in the absolute CD4 + T cells and a slight increase in the absolute CD8 + T cell numbers with a dramatic expansion of cells with CD8β low chain expression.

In the present study, an outbreak of proliferative dermatitis in musk ox (Ovibos moschatus), Sichuan takin (Budorcas taxicolor tibetana) and domestic Shetland sheep (Ovis aries) in a zoo is described. Skin lesions consisted of severe, persistent, multifocal, proliferative dermatitis in musk ox, and mild, transient, focal, dermatitis in the Sichuan takin and Shetland sheep. Parapoxviruses were isolated from skin lesions, and characterized by restriction enzyme analysis and partial gene sequencing. The results of this investigation indicate that the outbreak of proliferative dermatitis was due to infection by a single parapoxvirus, which is genetically closely related to other orf virus (ORFV) strains but distant to bovine papular stomatitis virus (BPSV) and pseudocowpox virus (PCPV).

The characterization of an orf virus (OV) isolated from skin lesions of a goat kid with severe, persistent, proliferative dermatitis, and designated orf virus-San Angelo 2000 (OV-SA00) strain, is described. The identity of OV-SA00 was confirmed by a combination of methods, including electron microscopy, amplification of specific fragments of viral DNA by polymerase chain reaction, restriction enzyme analysis of viral DNA and gene sequencing. Restriction endonuclease analyses of viral DNA and the protein profile studied by Western blot revealed differences between OV-SA00 strain and the profiles of other OV strains that have been published. The restriction enzyme profile of OV-SA00 was also different from the orf virus vaccine (OV-V) strain used to vaccinate this kid. Comparison of the nucleotide and deduced amino acid sequences indicated that OV-SA00 is closely related to OV-V strain, the Scottish OV strains orf11 and MRI Scab, and the human OV-CE/Shoe strain and more distant to bovine papular stomatitis virus (BPSV) reference strain and the pseudocowpox virus (PCPV)-MNV/Till strain. These results indicate that OV-SA00 is a strain of OV rather than a different parapoxvirus. Further studies are necessary to determine if the severity of orf-induced lesions in this goat kid was the result of individual host susceptibility factors.

Background The development and application of 3D images in laparoscopic surgery has brought the benefit of in-depth perception that traditional laparoscopic surgery lacked. Previous studies in surgical populations have demonstrated the advantages of 3D technology. To limit bias of the previous experiences of participants, this study was performed in a population without any experience in this area. Material and methods An experimental, open, cross-sectional, comparative study between surgical skills achievements using 2D and a 3D laparoscopy equipment, using each subject as their own control. Six skills were evaluated in 2D and 3D modalities. Results Of the 40 participants included, 20 began the skills in the 2D modality and then performed them in 3D, and the other 20 began in 3D. Of the 118 skills evaluated there was a time improvement in 72% in the 3D group compared to 37% in the 2D modality. The accomplishment percentage using the 3D laparoscopy was greater for both groups. There was a statistically significant difference in the better time for the 3D performed tasks. Just over half (52.5%) of participants preferred 3D laparoscopy, 15% preferred 2D, and 32.5% had no preferences. Discussion As other studies have demonstrated, there was improvement in the overall performance using the 3D laparoscope. Bias was limited by using a population without surgical experience. Conclusions 3D laparoscopic surgical skills showed superior to 2D, with higher percentages of tasks completion, less time in performing them, and a shorter learning curve.

Ovine lentivirus (OvLV), a retrovirus, infects and disseminates to various tissue organs via monocytes. The differentiation of infected monocytes into macrophages is a prerequisite for viral replication, and the presence of infected macrophages in tissue organs induces chronic immunopathology such as lymphoid interstitial pneumonia. The pulmonary intravascular macrophage (PIM) is a recently identified mononuclear phagocyte in domestic animal species, including sheep. Recombinant ovine interferon-tau (roIFN-τ), a type I IFN originally named as the ovine trophoblast protein, has potent antiviral activity against OvLV and human immunodeficiency virus and prevents the development of OvLV-associated lung pathology. We investigated and compared the structural features of PIMs in OvLV-infected and/or roIFN-τ–treated 1-month-old lambs using transmission electron microscopy. The PIMs' numerical counts were performed in toluidine blue–stained sections of Epoxy-embedded lung tissues. A reduction in the number of PIMs was observed with OvLV infection and/or roIFN-τ treatment of lambs as compared to the control group (P ≤ 0.05). The majority of the PIMs in OvLV-infected and/or roIFN-τ–treated groups were devoid of their surface coat. The PIMs of OvLV-infected lambs exhibited signs of biosynthetic activation such as expanded rough endoplasmic reticulum, prominent Golgi complexes, and accumulation of secretory vesicles. A few PIMs contained OvLV-like structures. In roIFN-τ–treated OvLV-infected lambs, the lymphocytes had ruffled plasma membranes and were in intimate contact with the PIMs, as is observed during cytotoxic cell-mediated killing of target cells. Most of the PIMs in roIFN-τ–treated OvLV-infected lambs appeared smaller in size. Ovine lentivirus and roIFN-τ, individually or in combination, alter the integrity of the surface coat of PIMs and cause their disappearance from the lungs. Ovine lentivirus infection induces morphological changes that correlate with cytotoxic cell behavior between lymphocytes and PIMs in roIFN-τ–treated or placebo-treated lambs. The loss of PIMs, probably infected with OvLV, either through direct killing by roIFN-τ or indirectly by roIFN-τ–activated cytotoxic T lymphocytes may represent different aspects of therapeutic actions of this cytokine. Anat. Rec. 251:472–485, 1998. © 1998 Wiley-Liss, Inc.

Lentiviruses, a genus of retroviruses, include the agents that cause ovine progressive pneumonia or maedi. Infection is characterized by long incubation periods and insidious, slowly progressive clinical courses resulting in chronic degenerative diseases. The ovine lentiviruses are widespread among sheep in North America, yet their significance to livestock production is currently not well defined. Lentiviruses persist and replicate in the presence of host specific immune responses and cause immune-mediated lesions in several organ systems. Due to this unusual relationship with their host, the diagnosis, control and treatment of these infections is difficult and expensive. Scientific studies on the biology of ovine lentiviruses (OvLV) and their complex relationship with the host are yielding new approaches to the detection of infected animals and methods for study of the epizootiology and control of OvLV-related diseases. We summarize some of the known biological properties of the virus, spectrum of clinical features of the diseases, current concepts of disease pathogenesis, economic importance, and strategies used to diagnose and control OvLV infections.

As a pregnancy recognition signal, sheep trophoblast cells secrete a type I interferon, ovine interferon-tau (OvIFN-tau), which has potent antiviral activity. We studied the effects of a recombinant protein (rOv-IFN-tau) on the replication of ovine lentivirus (OvLV) in goat synovial membrane cells. The amount of provirus DNA, as measured by polymerase chain reaction (PCR), the virus titers, and the number of OvLV-induced syncytia were 76.5%, 82%, and 95%, respectively, lower in cultures treated with rOv-IFN-tau than in placebo-treated controls (p < 0.01). rOv-IFN-tau also reduced OvLV reverse transcriptase activity and protected cells from OvLV-induced cell lysis, but the effect was less dramatic. The antiviral activity increased with the concentration up to a maximum with 256 antiviral units of rOv-IFN-tau per ml.

16. Rock DL, Hagemoser WA, Osorio FA, McAllister HA: 1988, with deletions in the latency and early protein O genes: repliTranscription from the pseudorabies virus genome during latent cation, virulence and immunity in neonatal piglets. J Vet Diagn infection. Arch Virol 98:99-106. Invest O:OOO–OOO. 17. Southern EM: 1975, Detection of specific sequences among 19. Zhu X, Chen J, Young CHS, Silverstein S: 1990, Reactivation DNA fragments separated by gel electrophoresis. J Mol Biol 98: of latent herpes simplex virus by adenovirus recombinants en503-518. coding mutant IE-O gene products. J Virol 64:4489-4498. 18. Wesley RD, Cheung AK: 1995, A pseudorabies virus mutant

Recombinant major core protein p25 (rP25) and transmembrane protein gp40 (rTM) of the ovine lentivirus (OLV) were used as immobilized antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against caprine arthritis-encephalitis virus (CAEV). The sensitivity and specificity of these assays were compared with an agar gel immunodiffusion (AGID) test and a whole-virus ELISA. The results showed that the rTM ELISA was more effective than rP25 ELISA or AGID test in identifying CAEV antibodies in goat populations. The rTM ELISA had similar sensitivity and specificity as the whole-virus ELISA, with an overall concordance of 87.5%. When data for rP25 and rTM were combined, the overall test agreement between whole-virus ELISA and combined recombinant ELISAs increased to 89.3%. The high quantity and purity characteristic of recombinant proteins should make them suitable as routine diagnostic antigens for CAEV and OLV serology.

Prenatal transmission of ovine lentivirus (OvLV) was studied in 85 eyes and their offspring. The animals were from a flock with endemic OvLV infection and 49 (58%) had serum antibodies to OvLV. Blood was collected from all lambs before they nursed. Using the polymerase chain reaction (PCR), OvLV DNA was detected in peripheral blood mononuclear cells of 13 (11%) of 117 lambs, including two sets of twins. Mothers with OvLV-infected lambs (n = 11) were younger (mean, 2.5 years) and had fewer pregnancies (mean, 2.4) than seropositive ewes (3.2 years and 3.2 pregnancies; P < .05). Of mothers with OvLV-positive lambs, 4 had plasma antigenemia (mean, 31.3 +/- 2.1 ng/mL OvLV) in conjunction with indeterminate antiviral antibody responses by immunoblotting. These results suggest that maternal factors (age and parity) and host-virus interactions (antiviral antibody and antigenemia) are important risk factors in prenatal transmission of OvLV.

Four bluetongue virus (BTV)-seronegative heifers and 2 BTV-seropositive heifers were inoculated with the virulent strain UC-8 of BTV-11 between postbreeding days (PBD) 21 and 30. The heifers were observed for 10-18 days after inoculation for clinical signs, and pregnancy was monitored by ultrasound examination of the uterus and by plasma progesterone levels. Blood samples were collected daily after inoculation and processed for virus isolation and titration. Heifers were euthanized between PBD31 and PBD48, and tissues were collected for virologic and pathologic examination. All but 1 heifer inoculated on PBD21 remained pregnant after BTV inoculation, A cystic corpus luteum was found in the ovary of the nonpregnant heifer, but BTV was not isolated from the reproductive tract of this heifer. Three of the inoculated heifers that remained pregnant showed mild multifocal areas of perivascular lymphocytic infiltration in the ovary. BTV was reisolated from spleen and prescapular and peribronchial lymph nodes 10 days after inoculation from 3 of the 4 BTV-seronegative heifers. BTV was also reisolated from the uterus of 1 of the heifers that remained pregnant, but microscopic lesions were not found in this organ. One major cause of infertility in animals is early embryo loss. 16 As a consequence of embryonic death, the length of calving intervals is increased. 36 Data of the Dairy Herd Improvement Association indicate that the economic losses due to an increase in calving in- tervals in the USA was more than $500 million per year 29 In spite of this detrimental effect, little is known about the causes of embryo mortality. In cattle, tricho- moniasis, 3 vibriosis, 3 bovine viral diarrhea, 2,30 and in- fectious bovine rhinotracheitis 4,22,23,31 have been con- sidered causes of embryo mortality. Epidemiologic and in vitro studies suggest that bluetongue virus (BTV) could also be associated with embryo death in cat- tle. 5,6,14 pathogenesis of viral infections of the embryo at this stage of gestation can be carried out with greater as- surance of the events occurring in the uterus.

To better define the relationship between ovine lentivirus (OvLV) infection and respiratory disease, pulmonary leukocytes and postmortem lung specimens from 42 sheep seropositive or at risk for OvLV infection were obtained. The lungs were examined for lesions of lymphoid interstitial pneumonia (LIP), and animals were categorized into five groups by severity of LIP and OvLV serologic status. The presence of OvLV in alveolar macrophages was established by proviral DNA amplification using the polymerase chain reaction (PCR), and the proportion of infected cells was determined by a quantitative focal immunoassay (FIA) and by immunohistochemistry. The concentration of OvLV p25 in serum was measured by capture ELISA. In contrast to animals with mild or no pulmonary lesions, sheep with moderate or severe LIP (17/42) were all seropositive, 71% had antigenemia (greater than 2 ng/mL), and 82% had proviral DNA in 1.5 x 10(5) alveolar macrophages. Of sheep positive by PCR, those with moderate or severe LIP (79%) had an average of 3 infected cells/10(3) alveolar macrophages by FIA. These results implicate alveolar macrophages as important target cells in the pathogenesis of OvLV-induced respiratory diseases.

Bovine papular stomatitis virus (BPSV) and orf virus (ORFV), members of the genus Parapoxvirus of the Poxviridae , are etiologic agents of worldwide diseases affecting cattle and small ruminants, respectively. Here we report the genomic sequences and comparative analysis of BPSV strain BV-AR02 and ORFV strains OV-SA00, isolated from a goat, and OV-IA82, isolated from a sheep. Parapoxvirus (PPV) BV-AR02, OV-SA00, and OV-IA82 genomes range in size from 134 to 139 kbp, with an average nucleotide composition of 64% G+C. BPSV and ORFV genomes contain 131 and 130 putative genes, respectively, and share colinearity over 127 genes, 88 of which are conserved in all characterized chordopoxviruses. BPSV and ORFV contain 15 and 16 open reading frames (ORFs), respectively, which lack similarity to other poxvirus or cellular proteins. All genes with putative roles in pathogenesis, including a vascular endothelial growth factor (VEGF)-like gene, are present in both viruses; however, BPSV contains two extra ankyrin repeat genes absent in ORFV. Interspecies sequence variability is observed in all functional classes of genes but is highest in putative virulence/host range genes, including genes unique to PPV. At the amino acid level, OV-SA00 is 94% identical to OV-IA82 and 71% identical to BV-AR02. Notably, ORFV 006/132, 103, 109, 110, and 116 genes (VEGF, homologues of vaccinia virus A26L, A33R, and A34R, and a novel PPV ORF) show an unusual degree of intraspecies variability. These genomic differences are consistent with the classification of BPSV and ORFV as two PPV species. Compared to other mammalian chordopoxviruses, PPV shares unique genomic features with molluscum contagiosum virus, including a G+C-rich nucleotide composition, three orthologous genes, and a paucity of nucleotide metabolism genes. Together, these data provide a comparative view of PPV genomics.

Orf (contagious ecthyma) is a viral disease of small and wild ruminants, humans, and less frequently other species. In sheep and goats, the disease is characterized by the formation of vesiculo-proliferative lesions in the skin of lips and nostril. Here, a form of generalized orf in 16 goat kids from 2 different locations in west Texas is described. The disease was characterized by multifocal, severe, proliferative dermatitis that persisted from about 2 months of age until the goat kids were euthanized 3 months later. All affected goats were Boer or Boer crosses under 1 year of age. The mean immunoglobulin concentration in sera of affected goats was elevated compared with healthy control goats. Severe to moderate lymphadenomegaly of the nodes draining the areas of the skin affected with orf lesions was present in all 16 goat kids. Suppurative arthritis, chronic fibrinous pneumonia, and premature thymic involution were found in 3, 5, and 7 of the goat kids, respectively. The skin lesions of 3 goat kids were infested with larvae of the opportunistic black garbage fly ( Ophira sp.). The orf virus was identified in skin lesions by isolation in Marbin—Darby ovine kidney cells, electron microscopy, and amplification of viral DNA by polymerase chain reaction. The orf virus was not detected in peripheral blood or lymph node mononuclear cells of any of the goats. Cross-neutralization experiments showed that an ovine orf virus antiserum raised in sheep was more effective in neutralizing a sheep orf virus isolate than a caprine orf virus isolate. The clinical and epidemiological characteristics of these orf cases may be the result of susceptibility factors within some individuals of the Boer breed of goats.

AdUTPase gene ( du) deleted ovine lentivirus (OvLV(Deltadu)) mutant, derived from Visna/maedi virus (VMV) molecular clone KV1772, was constructed. Subsequently, a copy of the optimized green fluorescent protein ( egfp) coding region was fused into the viral pol open reading frame (ORF) at the deleted du locus to generate viral mutant, OvLV(Deltadu-egfp). OvLV(Deltadu) reverse transcriptase (RT) activity and titer of infectious virus in goat synovial membrane (GSM) cell cultures were not affected compared to that of KV1772 and OvLV-85/34 strain (p0.05). By contrast, OvLV(Deltadu-egfp) RT activity and virus titer were lower than for KV1772 and OvLV(Deltadu) (p0.05). OvLV-85/34 RT in sheep monocyte-derived macrophages (SMDM) was higher than that of KV1772, OvLV(Deltadu) and OvLV(Deltadu-egfp) (p0.05). The ability to prevent dUTP mis-incorporation into newly synthesized DNA was disrupted in OvLV(Deltadu) and OvLV(Deltadu-egfp) (p0.05). Immunoprecipitation demonstrated that GFP is expressed by OvLV(Deltadu-egfp) at a low level. OvLV(Deltadu-egfp) retained egfp after 10 passages in cell culture.OvLV(Deltadu-egfp) was re-isolated in GSM cells from peripheral blood mononuclear (PBMN) cells of three of four OvLV(Deltadu-egfp)-inoculated lambs, but by contrast to the in vitro experiments OvLV(Deltadu-egfp) lost the insert. Priming with OvLV(Deltadu-egfp) did not prevent infection with pathogenic OvLV, but cell-associated viremia in a mock-infected contact control lamb was higher than in OvLV(Deltadu-egfp)-primed lambs. OvLV serum antibody titers increased steadily in OvLV(Deltadu-egfp)-inoculated lambs, but in a lamb from which OvLV(Deltadu-egfp) was not reisolated the antibody titer surpassed the negative/positive cut-off value only after challenge with OvLV-85/34. Because OvLV(Deltadu-egfp) is attenuated for pathogenicity in vitro, replicates in vivo and stimulates an antibody response, subsequent experiments need to address the likelihood of using OvLV(Deltadu-egfp) as an attenuated, live-virus vaccine to protect sheep against OvLV-induced disease when challenged with pathogenic OvLV.

Ovine lentivirus (OvLV) belongs to the family Retroviridae and closely resembles the human immunodeficiency virus (HIV). Pulmonary lesions in OvLV-infected sheep consist of lymphoid interstitial pneumonia (LIP) and lymphocytic alveolitis. Similar pulmonary lesions occur in up to 40% of HIV-infected children and in some adults with AIDS. Interferon-tau (IFN-tau), a type I IFN, is produced by trophectoderm of ruminant conceptuses and is the pregnancy recognition signal in these species. To evaluate changes in phenotypes of bronchoalveolar lavage (BAL) cells of OvLV-infected lambs treated with recombinant ovine IFN-tau (rOvIFN-tau), 24 lambs were randomly allocated to one of four groups (n = 6 per group): 1, no virus + placebo (NVP); 2, no virus + rOvIFN-tau (NVI); 3, virus + placebo (VP); 4, virus + rOvIFN-tau (VI). The BAL cells from 3 lambs in each group were labeled with monoclonal antibodies (mAb) to cell surface markers at 16 weeks of treatment, and cells from the remaining 3 lambs in each group were labeled with mAb at 34 weeks of treatment. After labeling, BAL cells were analyzed by flow cytometry. The morphology of BAL cells from all experimental lambs was examined by transmission electron microscopy (TEM). At week 16, no differences in the relative proportions of BAL cell phenotypes were detected among the experimental groups. At week 34, VI lambs had higher proportions of CD8(+), gammadelta(+), MHC class II(+), and L-selectin (LS(+)) BAL cells compared with VP lambs. Higher proportions of CD14(+) and CD44(+) cells were found in VP lambs compared with NVP lambs at 34 weeks. OvLV-like particles were detected only in bronchoalveolar macrophages of VP lambs. In this study, rOvIFN-tau increased the proportions of primary antiviral gammadelta(+) and CD8(+) immune cells in OvLV-infected lambs. This may represent a cellular mechanism to explain the antiviral and therapeutic efficacy of this cytokine, in addition to its direct antiviral effect. However, because the actual number of cells labeled with mAb CD8 was low and some subsets of gammadelta cells may coexpress the CD8 marker, further studies are necessary to better define the role of rOvIFN-tau in the modulation of these cells in vivo.

To evaluate changes in complete blood cell (CBC) counts, haptoglobin and fibrinogen in ovine lentivirus (OvLV)-infected lambs treated with recombinant ovine interferon-tau (rOVIFN-tau), 24 lambs were allocated to one of four groups (n = 6 per group): (1) virus + rOvIFN-tau, VI, (2) virus + placebo, VP, (3) no virus + rOVIFN-tau, NVI, and (4) no virus + placebo, NVP. Three lambs in each group were treated once a day for 12 weeks, and the remaining 3 lambs were treated for 33 weeks. Blood was collected at days 0, 7, and 10 and at weeks 2-10, 12, 32, and 33 to determine CBC counts, as well as haptoglobin and fibrinogen levels. Hematologic values remained within normal limits in all groups. However, hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and packed cell volume (PCV) values decreased (p < 0.05) in the two rOvIFN-tau-treated groups (VI and NVI) compared with the placebo-treated (VP and NVP) groups. Both rOvIFN-upsilon and OvLV had a mild negative effect on neutrophil numbers. Although Hb, MCV, MCHC, PCV, and neutrophil values declined in the rOvIFN-tau-treated lambs compared with the placebo-treated lambs, these values remained within the reference range for sheep. Experimental lambs did not show adverse clinical signs associated with OvLV infection or as a result of rOvIFN-tau treatment. The lack of significant side effects of high-dose rOvIFN-tau in sheep and previous reports of broad-spectrum and cross-species antiviral activity suggest that rOvIFN-tau warrants further investigation as an antiviral therapeutic agent.

The antiviral effects of recombinant ovine interferon-τ (roIFN-τ) were studied in 26 lambs inoculated with ovine lentivirus (OvLV) or mock-infected. Six of the OvLV-infected lambs and three of the mock-infected lambs were treated with 106 antiviral units (AVU) per kg roIFN-τ daily for 30 days starting at day 0 post-inoculation (p.i.) and twice a week thereafter (early treatment). Six of the OvLV-infected lambs and three of the mock-infected lambs were treated with 106 AVU/kg roIFN-τ daily for 30 days starting at day 150 p.i. and twice a week thereafter (late treatment). Six OvLV-infected and two mock-infected lambs were treated either early or late with placebo. Cell-associated viraemia was quantified by an end-point dilution method. The weekly antibody response against OvLV proteins was studied by ELISA. All experimental animals were killed at 27 weeks p.i. and histological sections of lung were scored for the degree of lymphoid interstitial pneumonia (LIP). A 90% reduction in OvLV titres was detected at 4 weeks post-treatment in lambs that received early roIFN-τ treatment (PPP in vivo and reduces the likelihood of development of lentivirus-induced LIP when infected lambs are treated during the initial phases of OvLV infection.

A lentivirus was isolated from 2 goats in Mexico that were seropositive to caprine arthritis encephalitis virus (CAEV) by the agar gel immunodiffusion (AGID) test. The lentivirus was identified as CAEV by the observation of giant multinucleated cells (syncytia) in goat synovial membrane (GSM) monolayers co-cultivated with blood mononuclear (BMN) cells from the seropositive goats, and by amplifying a DNA segment of the CAEV gag gene using the polymerase chain reaction (PCR) technique. Subsequently, cell supernatants from the GSM cells co-cultivated with BMN cells were used to infect 2 CAEV-seronegative goats. These goats seroconverted to CAEV as determined by the AGID test, and CAEV was re-isolated from these goats. One of the goats developed polyarthritis 8 mo after inoculation. Previous serological surveys indicate that infection with CAEV is prevalent among goats in Mexico. To our knowledge this is the first report of CAEV isolation in Mexico. Because of globalization of markets and increased trading among nations, the rapid identification and reporting of diseases such as CAEV are important to prevent the dissemination of these diseases.

Ovine lentivirus (OvLV), a retrovirus, causes lymphoid interstitial pneumonia (LIP) in sheep. Pulmonary alveolar macrophages are believed to play a central role in lung inflammation caused by this virus. The pulmonary intravascular macrophage (PIM), a recently identified inflammatory cell, is under active investigation for its role in lung pathology. Gadolinium chloride (GC), a rare earth lanthanide, has been used in in vivo studies to abrogate macrophage function to understand their role in tissue pathology. We treated mock- and OvLV-infected lambs with GC once a week for 34 weeks to investigate the implications of PIMs in the pathogenesis of LIP using light and electron microscopy. Repeated treatments with GC did not cause any apparent physiological abnormalities in the lambs. Light microscopy on tissue sections showed that GC reduced the number of PIMs in OvLV-infected and mock-infected lambs. Ultrastructural examination of lung tissues from GC-treated lambs revealed electron-dense deposits of GC-like material in endosomes and cytoplasm of PIMs. The majority of the PIMs in GC-treated lambs appeared damaged. Semi-quantitative histological evaluation showed amelioration of the extent of LIP in GC-treated OvLV-infected lambs compared to placebo-treated OvLV-infected lambs. This study stresses the effectiveness of GC in eliminating PIMs and suggests their involvement in the pathogenesis of OvLV-induced LIP. The results also strengthen the usefulness of GC as a tool to study in vivo function of cells such as PIMs that are difficult to isolate for in vitro studies.

Ovine lentivirus (OvLV), a retrovirus, infects and disseminates to various tissue organs via monocytes. The differentiation of infected monocytes into macrophages is a prerequisite for viral replication, and the presence of infected macrophages in tissue organs induces chronic immunopathology such as lymphoid interstitial pneumonia. The pulmonary intravascular macrophage (PIM) is a recently identified mononuclear phagocyte in domestic animal species, including sheep. Recombinant ovine interferon-tau (roIFN-tau), a type I IFN originally named as the ovine trophoblast protein, has potent antiviral activity against OvLV and human immunodeficiency virus and prevents the development of OvLV-associated lung pathology. We investigated and compared the structural features of PIMs in OvLV-infected and/or roIFN-tau-treated 1-month-old lambs using transmission electron microscopy. The PIMs' numerical counts were performed in toluidine blue-stained sections of Epoxy-embedded lung tissues. A reduction in the number of PIMs was observed with OvLV infection and/or roIFN-tau treatment of lambs as compared to the control group (Por = 0.05). The majority of the PIMs in OvLV-infected and/or roIFN-tau-treated groups were devoid of their surface coat. The PIMs of OvLV-infected lambs exhibited signs of biosynthetic activation such as expanded rough endoplasmic reticulum, prominent Golgi complexes, and accumulation of secretory vesicles. A few PIMs contained OvLV-like structures. In roIFN-tau-treated OvLV-infected lambs, the lymphocytes had ruffled plasma membranes and were in intimate contact with the PIMs, as is observed during cytotoxic cell-mediated killing of target cells. Most of the PIMs in roIFN-tau-treated OvLV-infected lambs appeared smaller in size. Ovine lentivirus and roIFN-tau, individually or in combination, alter the integrity of the surface coat of PIMs and cause their disappearance from the lungs. Ovine lentivirus infection induces morphological changes that correlate with cytotoxic cell behavior between lymphocytes and PIMs in roIFN-tau-treated or placebo-treated lambs. The loss of PIMs, probably infected with OvLV, either through direct killing by roIFN-tau or indirectly by roIFN-tau-activated cytotoxic T lymphocytes may represent different aspects of therapeutic actions of this cytokine.

A survey among small ruminant veterinary practitioners and producers of the United States was conducted to determine the most important health problems of sheep and goats and the need for drugs to treat these diseases. Gastrointestinal nematodes and pneumonia were the most important health concerns. Ceftiofur, long acting tetracyclines, penicillins, tilmicosin and enrofloxacin were the antibiotics most needed. The approval of ivermectin and albendazole for goats, and fenbendazole for sheep were among the most common requests for anthelmintics. Veterinarians also stressed the need for drugs to manipulate the estrous cycle, anti-inflammatory drugs, analgesics and anesthetics. Among the viral diseases, lentivirus infections (ovine progressive pneumonia and caprine arthritis encephalitis) and soremouth were the greatest concerns among veterinarians and producers. Both groups indicated that the availability of a generic antiviral drug would be important. The lack of a rabies vaccine was of great concern particularly among goat producers. Extra-label use of drugs in food animals may result in drug residues in tissues that may be hazardous for consumers. Therefore; in order for producers to provide high quality, safe products while remaining competitive in a global market economy, research to determine safety levels and tissue depletion times of new drugs is an urgent need for the sheep and goat industry.

To determine patterns of cell-associated viremia and antibody responses during the early phase of ovine lentivirus (OvLV) infection in sheep.18 neonatal lambs.12 lambs were inoculated intratracheally with OvLV within 24 hours after birth; 6 lambs were inoculated with noninfected cell culture supernatant. Degree of cell-associated viremia was measured every other week for 16 weeks by use of a limited dilution assay. Antibody responses to OvLV transmembrane (TM) and p25 proteins were determined weekly by use of recombinant ELISA. Neutralizing antibody responses were measured before and 8 and 16 weeks after inoculation.Degree of cell-associated viremia peaked between 2 and 6 weeks after inoculation and then decreased. For inoculated lambs, mean anti-p25 titer peaked 5 weeks after inoculation then slowly declined, whereas mean anti-TM and neutralizing antibody titers increased steadily. Over time, mean degree of cell-associated viremia was negatively correlated with mean anti-TM titer. Maximum individual degree of cell-associated viremia was positively correlated with maximum individual anti-TM titer.Results suggest that after experimental inoculation, OvLV replicates actively for several weeks and that an increase in anti-TM titer coincides with a decrease in degree of cell-associated viremia. Although the role antibodies play in protecting against lentivirus infection remains uncertain, understanding the dynamics of the antibody response may have important implications for diagnosis of OvLV infection, and antibodies may prove to be valuable markers for prediction of infection and disease.

Three calves were sensitized with three doses of inactivated BTV-11 UC8 strain and then experimentally infected with the homologous virus. In addition, four BTV-seronegative heifers were also experimentally infected with BTV-11. Granulocyte rich fractions of peripheral blood leucocyte (PBL-GRF) cultures from BTV-sensitized/infected calves and from control unexposed cattle were exposed in vitro with BTV-11. Histamine, leukotriene (LT) C4 and prostaglandin (PG) D2 were assayed in supernatant fluids. Plasma histamine levels increased in BTV-infected heifers from 10.1 +/- 2 ng/ml at Day 0 to 23.1 +/- 6.6 ng/ml at Day 12 following virus exposure. In addition, in this experimental group the concentration of PGF2 alpha (mean 551.97 +/- 243.54 pg/ml) increased significantly (P < or = 0.05) compared with control cattle (mean 467.3 +/- 73.9 pg/ml). Bluetongue virus induced histamine and LTC4 release after in vitro infection of PBL-GRF. Release of LTC4 was significantly (P < or = 0.05) higher in PBL-GRF cultures from sensitized and control animals than in unexposed PBL-GRF cultures. In contrast to these results, PGD2 was not released after BTV infection of PBL-GRF in vitro. The histamine release caused by BTV was virus-specific and mainly mediated by an immunological reaction, since the release was significantly reduced by removal of cell surface immunoglobulins.

Maedi-Visna and ovine progressive pneumonia are diseases of sheep that are caused by ovine lentivirus and characterized by chronic inflammation of the lungs, mammary glands, joints, and central nervous system. Although tremendous progress in research has led to a better understanding of the pathogenesis of these diseases, many questions still remain. Much of the mystery is the result of the complexity of the ovine lentivirus genome and the intricate interactions of the virus with the host during replication. Discoveries in molecular virology are shedding light on these interactions and novel approaches to prevent and control lentivirus infections are being explored. There is hope that some of these approaches will eventually be used to eradicate these diseases.

The teratogenic potential of three bunyaviruses, two California serogroup bunyaviruses, LaCrosse virus and San Angelo virus, and a Bunyamwera serogroup member, Main Drain virus, in sheep was studied following in utero inoculation of ewes in early gestation. Although Main Drain virus appeared to be most teratogenic, all three viruses induced a range of lesions including arthrogryposis, hydrocephalus, fetal death, axial skeletal deviations, anasarca, and oligohydramnios. The teratogenic effects of these viruses are identical to those described in ovine infections by Cache Valley and Akabane viruses. Demonstration of a common bunyaviral tropism for fetal tissue infection that results in congenital brain and musculoskeletal malformations provides evidence that human in utero infection by bunyaviruses could result in similar malformations in human infants.

In order to localize the immunodominant regions, 12 ovine lentivirus (OLV) gag-coding gene fragments were cloned and expressed in Escherichia coli and when tested in a Western blot (WB) assay against a panel of sera collected from US and Italian OLV-infected sheep. The most immunoreactive regions were mapped to the amino-terminal of p25 and carboxyl-terminal of p14. In addition, we found that the reactivity pattern between US and Italian sheep was very similar, suggesting the antigenic domain between US and Italian isolates in the gag gene structures could be conserved. Given the broad immunoreactivity of the amino-terminal of p25, this region could serve as an ideal diagnostic antigen for the serological identification of OLV-infected sheep.