Characterization of Porcine Epidemic Diarrhea Virus Isolate US/Iowa/18984/2013 Infection in 1-Day-Old Cesarean-Derived Colostrum-Deprived Piglets.

Porcine epidemic diarrhea virus (PEDV) was first recognized in North America in April 2013 and has since caused devastating disease. The objective of this study was to characterize disease and viral detection associated with an original North American PEDV isolate inoculated in neonatal piglets. Thirty-six 1-day-old cesarean-derived and colostrum-deprived piglets were randomly assigned to the control (n = 16) or challenged group (n = 20); the latter were orogastrically inoculated with 1 ml of US/Iowa/18984/2013 PEDV isolate titered at 1 × 10(3) plaque-forming units per milliliter. Rectal swabs were collected from all piglets prior to inoculation and every 12 hours postinoculation (hpi) thereafter, with 4 control and 5 challenged piglets euthanized at 12, 24, 48, and 72 hpi. One piglet had a positive real-time quantitative polymerase chain reaction test on rectal swab at 12 hpi, and all remaining piglets were positive thereafter, with highest viral quantities detected at 24 and 36 hpi. Diarrhea was evident in 30% and 100% of challenged piglets at 18 and 24 hpi, respectively. Viral antigen was detected in enterocytes by immunohistochemistry in the duodenum and ileum of piglets euthanized at 12 hpi and was apparent throughout the small intestine of all piglets thereafter, with villus height:crypt depth ratios consistently below 4:1. Viremia was confirmed in 18 of 20 pigs at euthanasia. Clinical disease was severe and developed rapidly following infection with an original North American PEDV isolate, with lesions, viremia, and antigen detection possible by 12 hpi.

Vascular lesions in pigs experimentally infected with porcine circovirus type 2 serogroup B.

Vasculitis is a hallmark lesion of the severe form of systemic porcine circovirus-associated disease (PCVAD). In 2 experimental studies with porcine circovirus type 2 serogroup b (PCV2b), 2 pigs developed fatal PCVAD with acute vasculitis, and 5 related pigs developed chronic lymphohistiocytic and plasmacytic peri- and endarteritis. Five of these pigs (1 with acute vasculitis and 4 with chronic vasculitis) had also been inoculated with bovine viral diarrhea virus type 1 (BVDV1) or BVDV1-like virus. Vascular lesions were similar, independent of whether pigs had been inoculated singly with PCV2b or dually with PCV2b and BVDV1 or BVDV1-like virus. The acute vasculitis was accompanied by marked pulmonary and mesenteric edema and pleural effusion. In situ hybridization demonstrated abundant intracytoplasmic porcine circovirus type 2 (PCV2) nucleic acid in endothelial, smooth muscle-like, and inflammatory cells within and around affected arteries. The pigs with lymphohistiocytic and plasmacytic vasculitis had lesions of systemic PCVAD, including multisystemic lymphoplasmacytic and histiocytic or granulomatous inflammation. PCV2 nucleic acid was detected in renal tubule epithelial cells, mononuclear inflammatory cells, and rare endothelial cells in noninflamed vessels in multiple tissues of these animals. The 2 pigs with acute vasculitis had no PCV2-specific antibodies (or a low titer of), whereas the pigs with lymphohistiocytic and plasmacytic vasculitis developed high antibody titers against this virus. These observations suggest that (1) acute vasculitis observed in the current studies is directly caused by PCV2b, (2) chronic vasculitis may in part be mediated by the subsequent immune response, and (3) host factors and viral strain may both contribute to vasculitis in animals infected with PCV2b.

Cutaneous and systemic poxviral disease in red (Tamiasciurus hudsonicus) and gray (Sciurus carolinensis) squirrels.

From September 2005 through October 2006, fibromatosis was diagnosed in 2 red squirrels (Tamiasciurus hudsonicus) and 1 gray squirrel (Sciurus carolinensis). All 3 squirrels had multifocal to coalescing, tan, firm alopecic cutaneous nodules. Two squirrels also had pulmonary nodules. Histologically, the cutaneous nodules had marked epidermal hyperplasia, with ballooning degeneration of keratinocytes, spongiosis, and eosinophilic cytoplasmic inclusions. The dermis was expanded by proliferation of atypical mesenchymal cells with cytoplasmic inclusions. Additional findings included pulmonary adenomatous hyperplasia with cytoplasmic inclusions, renal tubular epithelial hyperplasia with cytoplasmic inclusions, atypical mesenchymal proliferation in the liver, and atypical mesenchymal proliferation with cytoplasmic inclusions in the seminal vesicles. Ultrastructurally, poxviral particles were observed in skin scrapings and sections of cutaneous and pulmonary nodules. Polymerase chain reaction targeting the highly conserved Leporipoxvirus DNA polymerase gene was positive using DNA extracted from the cutaneous lesions of all 3 squirrels. Nucleotide sequence of the 390 base PCR amplicons was closely related to that of other members of the genus Leporipoxvirus. To the authors' knowledge, this is the first report of cutaneous and systemic poxviral disease in American red squirrels with molecular characterization of the squirrel fibroma virus.

Differential ability of human blood monocyte subsets to release various cytokines.

We have shown that two human monocyte subsets can be isolated from the peripheral blood of healthy donors; these subsets possess different morphological, cytochemical, functional, and in vivo trafficking properties [1]. In this report, these two subsets were further characterized. One subset (intermediate monocytes, IM) has been shown to have significantly lower acid phosphatase activity and total cellular protein content as well as lower peroxidase activity when compared with another subset (regular monocytes, RM). The overall activation status of the two subsets (as determined by their alkaline phosphodiesterase activity) was identical. We also examined the capacity of these subsets to release various cytokines with or without polyriboinosinic and polyribocytidylic acid (Poly I:C) stimulation. There was no appreciable difference in their ability to release interferon (IFN), interleukin 1 (IL-1), and prostaglandin E (PGE) without stimulation, while IM produced slightly, but significantly, higher amounts of colony-stimulating factor (CSF) than RM. The amount of IFN released by IM in response to poly I:C was approximately three times higher than the amount of IFN released by RM. IL-1 was also released in higher amounts by IM than by RM in response to poly I:C. IM were also found to release more CSF than RM in response to poly I:C. In contrast, it was noted that IM secrete significantly less PGE response to poly I:C than do RM. These findings indicate that two purified human monocyte subsets, distinguishable by maturation markers, differ significantly in their ability to release various cytokines after stimulation; this difference may be relevant to potential in vivo roles of these immunoregulatory cells.

Effects of adherence, activation and distinct serum proteins on the in vitro human monocyte maturation process.

Elutriator-purified human monocytes were cultured in a serum-free (SF) medium, and various serum proteins and functional activating agents were assessed for their effects on the in vitro maturation of human monocytes to macrophages. Following 3 days of suspension culture in Teflon labware, 60% of the monocytes were easily recovered. When varying concentrations of human AB serum (HuAB) were employed, human monocyte maturation progressed rapidly; the kinetics of this maturation process during cell suspension culture were very similar to the pattern observed following adherence culture. In contrast, when SF medium was employed, a marked retardation of the monocyte maturation process was observed; this could not be attributed to any changes in cell recovery and/or viability. Thus, cells could be maintained in their monocytoid form for 3 days when cultured in SF medium. When HuAB was added after 3 days of culture, human monocyte maturation into macrophages proceeded at a normal rate. We attempted to characterize certain of the serum protein(s) found in HuAB which promoted the monocyte maturation process. Human immunoglobulin G (IgG) was found to be the most potent serum protein in increasing 5'-N activity and decreasing peroxidase activity of suspension cultured monocytes. Immunoglobulin M (IgM) and albumin (Alb) were shown not to have significant monocyte maturation activity. Heat-treated human gamma globulin and IgG purified by high-performance liquid chromatography (HPLC) were shown to have patterns identical with that of untreated HGG and IgG with regard to promoting monocyte maturation; F(ab')2 was not an active maturation promoter, indicating the need for an intact Fc portion of the IgG molecule. Fibrinogen and fibronectin also had maturation promoting activity. Finally, addition of the potent monocyte functional activators, muramyl dipeptide (MDP), polyriboinosinic:polyribocytidilic acid (Poly I:C), and lipopolysaccharide (LPS) had no effect on the monocyte maturation process. Thus, neither cell adherence or activation appear to be critical for the monocyte to macrophage maturation process. Instead, we hypothesize that in addition to proper nutritional support, a group of serum proteins (unified mainly by their ability to interact with monocyte membrane receptors) appear to be the principal promoters of this process.

The effect of anesthetic agents on human immune system function. I. Design of a system to deliver inhalational anesthetic agents to leukocyte cultures in vitro.

A novel system for exposing purified human mononuclear leukocyte subsets or any cultured cell to inhalational anesthetic agents has been devised. Monocytes and lymphocytes are purified by counter-current centrifugal elutriation and put into culture vessels with and without appropriate functional activators. The culture vessels are placed into one of four anesthetic agent exposure chambers, each containing a different concentration of the anesthetic agent to be tested. The system that delivers the anesthetic agent to the culture vessels is essentially the same as the one used in the operating room; the actual levels of anesthetic agent delivered are monitored. In this report, we present evidence that halothane can be successfully tested using the above-cited experimental design; this agent substantially inhibits the secretion of interferon by human mononuclear cells. The ability of monocytes to secrete alpha interferon in response to polyinosinic: polycytidylic acid (poly I:C) was significantly depressed following 4 h in vitro exposure to increasing concentrations of halothane; the secretion of gamma interferon by lymphocytes in response to phytohemagglutinin (PHA) was also depressed, although to a lesser degree. The system presented should allow for the in vitro exploration of the effects of inhalational agents on purified human leukocyte subset functions as well as for the analysis of the effect of these agents on monocyte/lymphocyte interactions.

Driving ability after intravenous fentanyl or diazepam. A controlled double-blind study.

Uncomfortable or moderately painful radiologic diagnostic and therapeutic procedures are being performed increasingly on outpatients. If sedation and analgesia are used, patients cannot drive themselves home or return rapidly to normal activities. This study compares the effect of fentanyl (100 micrograms), diazepam (7.5 mg), and placebo on driving ability of young volunteers as measured by the thacometer. Speed and accuracy were impaired at 30 and 120 minutes by both drugs, and by fentanyl more than diazepam. This study design may be suitable for the assessment of whether patients can drive safely after other analgesic drugs.

Sonographic changes during hepatic interstitial laser photocoagulation. An investigation of three optical fiber tips.

RATIONALE AND OBJECTIVES: Interstitial laser photocoagulation (ILP) destroys tumors thermally, using laser energy delivered from implanted optical fibers. The objectives of the study are to identify a fiber tip/delivered energy combination which produces lesions of useful size, visible on ultrasound (US) during ILP, and to compare ILP lesions and their US images. METHODS: Hepatic ILP was performed at laparotomy in six pigs, using three different fiber tips (cylindrical diffusing, spherical diffusing, plane-cut). US images were obtained during ILP, immediately after ("early" images), and before the animals were killed (2-2.5 hours, "late" images). Actual lesions were assessed histopathologically. RESULTS: Few US changes were seen around cylindrical diffusing and spherical diffusing tips until tip destruction. Plane-cut tips, at 1.5 to 2.0 W, produced prominent US images of the 1- to 2-cm thermal lesions. Early images tended to overestimate necrosis. Late images approximated necrosis. CONCLUSION: For US-controlled ILP, plane-cut tips are better than currently available cylindrical diffusing or spherical diffusing tips. Lesion image growth periods might enable control of lesion size. Further studies are needed to determine the consistency of the described relationship between lesion images and actual lesions.

The colitides.

Diverse signs may be seen on barium enema in the various forms of colitis. Barium enema is only reasonably specific in the diagnosis of Crohn's disease and ulcerative colitis once the other possible diseases have been excluded from consideration by biopsy, microscopy, culture, or therapeutic trial. In particular, campylobacter colitis has proved to be such a common entity that many patients originally thought to have had one attack of ulcerative colitis, ischemic colitis, or Crohn's colitis may never have had these diseases. Many of the relatively specific signs can be seen in a variety of conditions. For example, aphthae in the colon may be seen in Crohn's disease, yersinia enterocolitis, Behçet's syndrome, amebiasis, ischemia, tuberculosis, and campylobacter colitis. Continuity of disease is characteristic in ulcerative colitis. Nevertheless, patches of healing may occur, so that a rare patient may be seen during a resolving attack of ulcerative colitis in whom only scattered patches of active disease can be seen on barium enema. In general, few radiologic signs on barium enema are truly specific for one disease, with penumatosis cystoides coli being an exception to this rule. However, the sensitivity of barium enema with currently available materials for double-contrast techniques is such that radiology continues to be useful at present in diagnosing the colitides and in managing patients.

Porcine reproductive and respiratory syndrome virus does not exacerbate Mycoplasma hyopneumoniae infection in young pigs.

The purpose of this study was to determine if Porcine Reproductive and Respiratory Syndrome (PRRS) virus infection altered the severity of acute Mycoplasma hyopneumoniae (MH) infection in young pigs. Twenty five, 3-week-old male pigs were randomly assigned by litter and weight to one of 3 groups. Groups 1 (PRRS only, n = 5) and 2 (PRRS + MH, n = 10) were inoculated intranasally with PRRS virus (IN-5 isolate, 10(5) TCID50) and viremia in all pigs was confirmed by virus isolation from serum 3 days later. Group 3 (MH only, n = 10) was inoculated at the same time with virus free culture media. Seven days after virus inoculation, Groups 2 and 3 were inoculated intratracheally with MH (strain P-5722-3, 10(7) CCU). All pigs were euthanized and necropsied 28 days later, when maximum lesions of mycoplasmosis occurs. Pigs in group 1 did not cough and had no gross lung lesions, but were still viremic at necropsy. MH was isolated from all pigs in groups 2 (avg. log 5.2 +/- 1.3) and 3 (avg. log 5.1 +/- 1.5), but differences were not significant (P = 0.87). Similarly, there were no differences in average days coughing (8.9 +/- 2.8 v 11.2 +/- 4.5, P = 0.17), grossly pneumonic lung (16.5% v 17%, P = 0.91), or microscopic lung lesion scores (10.1 +/- 2.6 v 11.1 +/- 1.9, P = 0.35) between pigs in groups 2 and 3. Under these experimental conditions, PRRS virus infection did not increase the severity of experimental Mycoplasma hyopneumoniae infection in young pigs.

Antagonism of drug discrimination learning within the conditioned taste aversion procedure.

Animals injected with morphine prior to the presentation of a saccharin-LiCl pairing and the morphine vehicle prior to saccharin alone rapidly acquired the drug discrimination, avoiding saccharin following the administration of morphine and consuming saccharin following its vehicle after only four conditioning trials. Once stimulus control was established, the opiate antagonist naloxone (1 mg/kg) was administered prior to morphine in a test of its ability to antagonize the morphine stimulus. Pretreatment times ranged from 10 to 180 min. Naloxone antagonized the stimulus properties of morphine for all subjects, although there were individual differences in the onset, duration (time course) and degree of antagonism. Together with the rapid acquisition typically reported in this design, the fact that antagonism was demonstrated in the present study suggests that the conditioned taste aversion procedure may be useful in the general assessment of drug discriminations.

Morphine discriminative control is mediated by the mu opioid receptor: assessment of delta opioid substitution and antagonism.

Morphine is an effective training drug in drug discrimination procedures. In subsequent generalization tests in which other opioids are administered, mu opioid agonists selectively substitute for the training drug. Given the relative selectivity of morphine for the mu receptor, such substitution patterns suggest that the mu opioid receptor is mediating the discriminative control of this compound. The present study assessed this selective mediation by examining the ability of the delta opioid agonist SNC80 to substitute for (and the delta opioid antagonist naltrindole to antagonize) morphine stimulus effects in rats trained to discriminate morphine from its vehicle in the conditioned taste aversion baseline of drug discrimination learning. Although morphine and methadone produced dose-related substitution for morphine (10 mg/kg), there was no evidence of substitution for morphine by SNC80 at any dose tested. Further, although naloxone (3.2 mg/kg) completely blocked the discriminative effects of morphine, naltrindole (3.2-10 mg/kg) did not significantly affect the morphine stimulus. These data suggest that the discriminative control established to morphine is mediated by its activity at the mu, but not the delta, receptor.

Mice and rats (laboratory and feral) are not a reservoir for PRRS virus.

Porcine reproductive and respiratory syndrome (PRRS) is caused by an unclassified arterivirus. The syndrome was first reported in the USA in 1987 as epizootics of reproductive failure in sows and respiratory disease in nursery, growing, and fattening pigs. An enzootic form of the disease has now emerged, characterized by interstitial pneumonia and an increased incidence of secondary infections. Because the disease has now become enzootic on many farms, rodents were investigated as a possible reservoir for the infection. Wild rodents from an endemically infected farm were trapped, and virus isolation for PRRS virus (PRRSV) was attempted using porcine primary alveolar macrophage cultures. PRRSV was not isolated from serum and selected pooled tissues (thymus, lung, and spleen) of 14 feral mice and 2 feral rats. Also, transmission experiments were carried out on 3-week-old Balb/c mice and 12-week-old Fischer-344 rats to determine if these species were susceptible to infection. The rodents were inoculated intranasally, orally, and intraperitoneally with a virus proven to transmit PRRS to pigs. Virus isolations from selected pooled tissues (lung, spleen, thymus, and kidney) and from serum were negative, and there were neither gross nor microscopic lesions. Weight gains and white blood cell counts were not significantly different between treated and control groups. These results indicate that rodents are not susceptible to infection with PRRSV and therefore are probably not a reservoir for the disease.

Tissue distribution and genetic typing of porcine circoviruses in pigs with naturally occurring congenital tremors.

Congenital tremors (CT) type A2 is associated with porcine circovirus (PCV) and deficient and abnormal myelin. The aim of this study was to determine the tissue distribution and genetic type of PCV in 1-2-day-old pigs with naturally occurring CT type A2 using in situ hybridization, polymerase chain reaction (PCR), and indirect fluorescent antibody tests on frozen tissue sections. CT-affected and clinically normal pigs were selected from 4 farms in the midwestern USA that were undergoing outbreaks of CT type A2. All CT and most normal pigs were infected with PCV. PCV was widely distributed in tissues of infected pigs and was most common in tissues of the central nervous system and liver. In all infected pigs, there were more PCV-infected cells in brain and spinal cord than in nonneural tissues. CT pigs had many more PCV-infected cells in the brain and spinal cord than did clinically normal pigs because of a more diffuse distribution and a larger proportion of infected cells. The cells most commonly infected with PCV in brain and spinal cord were large neurons. In nonneural tissues, macrophages were the most frequent cell type infected. PCR analysis demonstrated only PCV type 2 and not PCV type 1 in all PCV-infected pigs on all 4 farms.

Salmonella arizonae sepsis in a lynx.

A 4.5-wk-old lynx (Felis lynx) was presented for necropsy with a history of poor growth, mild diarrhea, anemia, and lethargy. The liver was enlarged and had a 7 mm long fracture that resulted in severe intraabdominal hemorrhage and death. Microscopic lesions were indicative of severe ulcerative cystitis and septicemia. Pure cultures of Salmonella arizonae were isolated from the liver, kidney, and spleen. Based on differences in the chronicity of inflammation in the urinary bladder versus other organs, we speculate that chronic cystitis caused by S arizonae lead to septicemic infection.

Characterization of infection with endemic porcine reproductive and respiratory syndrome virus in a swine herd.

Infection of 8-week-old pigs with endemic porcine reproductive and respiratory syndrome (PRRS) virus was detected on a farm that had an epidemic of PRRS in 1989. During the 2.5 years since the original epidemic, reproductive performance of the breeding herd had been within acceptable limits, but mortality had periodically exceeded one-fourth of the pigs in the nursery (195 died of 761 weaned, 25.6%). Investigators attempted to determine the age and humoral immune status of pigs infected with endemic PRRS virus on the farm. Serum obtained from 9 groups of 1- to 18-week-old pigs (10 pigs/group) was examined for PRRS virus by virus isolation. Serum was obtained from 8 sows that had farrowed within the preceding 24 hours. Serum from the sows was obtained weekly until litters were weaned at 3 weeks of age. Serum was obtained from 27 newborn pigs (3 to 4 newborn pigs from each of the 8 sows) prior to intake of colostrum and at weekly or biweekly intervals until the pigs were 20 to 21 weeks of age. Isolation of PRRS virus and indirect fluorescent antibody serologic testing were performed on these serum samples. In another study, serum was obtained for serologic testing from 10 sows in each of 6 parity groups. The PRRS virus was isolated from serum of only 3- to 12-week-old pigs. The 8 sows and their 27 pigs were seronegative for PRRS virus during the 3-week lactation period. By 10 weeks of age, 18 of the 27 suckling pigs were still alive and had seroconverted (titers > or = 1:20).(ABSTRACT TRUNCATED AT 250 WORDS)

[Three cases of postweaning multisystemic wasting syndrome (PMWS) due to porcine circovirus type 2 (PCV 2) in Switzerland]. / Drei Fälle von "Postweaning Multisystemic Wasting Syndrome" (PMWS) hervorgerufen durch das porcine Circovirus Typ 2 (PCV 2) in der Schweiz.

Porcine circovirus type 2 (PCV 2) was found in three five- and nine-week-old pigs from two feeder pig producer farms. Clinical signs were persistent diarrhea and wasting. The animals showed histomorphologic changes characteristic for "Postweaning Multisystemic Syndrome" (PMWS). One animal had the typical basophilic intracytoplasmatic circoviral inclusion bodies in the Peyer's patches of the ileum. Circoviral DNA was detected in the lymphatic organs as well as in the liver. This case report is the first description of PCV 2 in Switzerland.

Pathogenesis of porcine epidemic diarrhea virus isolate (US/Iowa/18984/2013) in 3-week-old weaned pigs.

Porcine epidemic diarrhea virus (PEDV) is associated with clinical diarrhea in naïve swine of all ages. This report describes timing of antibody generation and disease progression following infection with a US PEDV isolate by assessing fecal viral shedding, morphometric analysis of intestinal lesions, and magnitude of immunohistochemical staining. Sixty-three, 3-week-old pigs were randomly allocated into control (n=27) and challenged (n=36) groups. Challenged pigs were administered 1 mL of 1 × 10(3) PFU/mL of US/Iowa/18984/2013 PEDV isolate by oro-gastric gavage. Three control and four challenged pigs were necropsied on days post-inoculation (dpi) 1, 2, 3, 4, 7, and weekly thereafter, until study termination on dpi 35. Clinical disease, fecal shedding, body weight, and temperature were monitored during the study period. Diarrhea was observed in challenged pigs beginning for some on dpi 2, affecting a majority of pigs by dpi 6 and subsiding by dpi 10. Average daily gain was significantly lower (P<0.001) for one week post-infection in challenged pigs. PEDV was detected in feces by PCR on dpi 1 and continued in a subset of pigs until dpi 24. PEDV-specific antigen was detected in villous enterocytes of challenged pigs by immunohistochemistry (IHC) on dpi 1, 2, 3, 4, 7, and 14. Microscopic lesions included severe diffuse atrophic enteritis with significantly reduced (P<0.001) villous length observed on dpi 3, 4, and 7. Under the conditions of this study, fecal shedding of PEDV and IHC staining can precede and continue beyond the observation of clinical signs, thus increasing the risk of viral transmission.