Isolation of animal viruses from farm livestock waste, soil and water.
Ten porcine enteroviruses, 2 porcine adenoviruses and 1 coronavirus were isolated directly from 32 samples of slurry collected from a pig fattening house. Concentration of the same samples by adsorption with the polyelectrolyte PE-60 yielded 24 porcine enteroviruses and 3 porcine adenoviruses. A porcine enterovirus was isolated, following PE-60 concentration, from 1 to 6 slurry samples from a sow farrowing house. No virus was isolated from 12 samples of slurry from dairy cows nor from 6 slurry samples from a calf-rearing unit. A porcine enterovirus was isolated from soil samples, after concentration with PE-60, collected 1, 2 and 8 days after pig slurry was spread on hay stubble. Two porcine enteroviruses were isolated by membrane filtration from 26 samples of surface run-off from land on which pig slurry was routinely spread, and 2 bovine enteroviruses were isolated from cattle feedlot run-off after adsorption to layers of talc and celite followed by hydroextraction. A porcine enterovirus was also isolated from 1 of 33 samples of surface water collected on farms on which pig slurry was routinely spread on the land, but no virus was isolated from 36 samples of ground water from the same farms. The surface water and ground water samples were concentrated by talc-celite adsorption and hydroextraction. (+info)
A novel internal open reading frame product expressed from a polycistronic mRNA of porcine epidemic diarrhoea virus may not contribute to virus attenuation.
Cell-culture-adapted (ca) porcine epidemic diarrhoea virus (PEDV) contains three internal open reading frames (I ORF) within the nucleocapsid protein gene and lacks the downstream counterpart of porcine transmissible gastroenteritis virus ORF7 or feline infectious peritonitis virus ORF6a. To confirm whether such features also exist in wild-type (wt) PEDV, the 3' 1800 nucleotides of its genome were sequenced and were found to be identical to those of ca virus. The coding potential of I-1 ORF was ascertained by transient expression in Vero cells followed by immunofluorescence using antipeptide sera. The I-1 protein was synthesized as a 12 kDa non-phosphorylated PEDV-specific protein that was not present in detectable amounts in virions. However, a low copy number of I-1 in the virion would suggest it is a structural component. Nevertheless, identical nucleotide sequences and gene expression strategies of attenuated ca virus and its virulent parent, wt PEDV, demonstrate that the 3' 1800 nucleotides or the genes and gene products encoded therein may not contribute to virus attenuation. (+info)
In situ hybridization for the detection and localization of porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets.
Detection and localization of porcine epidemic diarrhea virus (PEDV) was studied by in situ hybridization with a nonradioactive digoxigenin-labeled probe in formalin-fixed, paraffin-embedded tissues from 10 naturally infected piglets. A 377-base pair cDNA probe for viral RNA encoding the membrane proteins of PEDV cell-culture-adapted strain V215/78 was generated by the reverse transcription polymerase chain reaction. In the retrospective study of pigs from herds with diarrhea, the 10 piglets naturally infected with PEDV had positive signals for PEDV by in situ hybridization. When intestinal tissues were hybridized with the PEDV probe, a strong signal was seen in the villus enterocytes of jejunum and ileum but not in the cecum and colon. Positive cells typically had dark brown reaction products in the cytoplasm. Scattered epithelial cells along the ileal Peyer's patches dome areas contained viral RNA. In one piglet, hybridization signal was also found in the duodenum. PEDV was not demonstrated in tissues outside of the intestinal tract. These findings indicate that jejunal and ileal villus enterocytes are the main target of PEDV replication during epizootic outbreaks of the disease. (+info)
Detection of Australian gill-associated virus (GAV) and lymphoid organ virus (LOV) of Penaeus monodon by RT-nested PCR.
A highly sensitive test based on reverse transcription followed by nested polymerase chain reaction (RT-nPCR) was developed to detect the Australian yellow-head-like viruses, gill-associated virus (GAV) and lymphoid organ virus (LOV) of Penaeus monodon. The RT-nPCR detected viral RNA in as little as 10 fg lymphoid organ total RNA isolated from GAV-infected P. monodon. Amplification of serial dilutions of a GAV cDNA clone showed that the nested PCR was sufficiently sensitive to detect a single genome equivalent using a DNA template. The specificity and sensitivity of the RT-nPCR was also demonstrated using experimentally infected P. (Marsupenaeus) japonicus, where GAV sequences could be amplified from lymphoid organ and haemocyte RNA as early as 6 h post infection (p.i.), and from gills by 24 h p.i. In contrast, transmission electron microscopy (TEM) identified nucleocapsids and virions in lymphoid organ cells and haemocytes from Days 3 and 6 p.i., respectively, while there was no evidence of infection in gill cells at any time. The practical application of the RT-nPCR was demonstrated by screening healthy wild-caught P. monodon broodstock. The high prevalence (>98%) of broodstock that were positive by RT-nPCR suggests that LOV is endemic in northern Queensland. In addition, results with lymphoid organ, gill and haemocyte RNA suggest that small gill biopsies may be best suited to the non-sacrificial testing of valuable broodstock. The speed and sensitivity of the RT-nPCR make it a useful adjunct to TEM for diagnosing LOV/GAV infection of P. monodon, with the additional benefit that screening of gill biopsies may facilitate selection of LOV-free broodstock. (+info)
Acute undifferentiated neonatal diarrhea in beef calves. I. Occurence and distribution of infectious agents.
Beef calves in a 48-cow herd were studied during one calving season from birth to ten days of age to determine the presence or absence of potentially enteropathogenic bacteria, viruses, and/or chlamydia in both normal and diarrheic calves. Calves were born and raised outside in large pens unless the ambient temperature was below minus 10 degrees F when calving was done inside. Fecal swabs, fecal aliquots, and nasal swabs were taken from each calf at 32, 128 plus or minus 3, and 248 plus or minus 3 hours of age and as soon after the onset of diarrhea as possible. Diarrhea was defined as that condition in which the feces contained less than 10% dry matter. Enteropathogenic Escherichia coli in feces were identified using the ligated gut loop procedure in calves and by feeding broth cultures to colostrum fed lambs seven to 16 hours old. Potentially enteropathogenic viruses were detected using a variety of methods which included tissue culture, fluorescent antibody, hemadsorption, and electron microscope techniques. Of the 40 calves studied, 32 (80%) developed diarrhea before ten days of age. Twenty-two strains of Escherichia coli which caused dilation of calf ligated intestinal loops were isolated from 11 scouring calves and from one normal calf. Nine out of ten strains of Escherichia coli which dilated ligated loops also caused diarrhea when fed to colostrum-fed lambs seven to 16 hours old. Using antibody technique a Reo-like virus was detected in the feces of 15 calves before, during, and after the onset of diarrhea. Four calves excreted both loop dilating strains of E. coli and Reo-like virus in the feces before ten days of age; in all cases the loop dilating E. coli were isolated from the feces prior to the demonstration of Reo-like virus. A Corona-like virus was also demonstrated in three of the 15 calves infected with Reo-like virus and a noncytopathogenic strain of bovine virus diarrhea virus was isolated from two of the 15 calves infected with Reo-like virus. A loop dilating strain of Citrobacter was isolated from one diarrheic calf. There was no consistent pattern of onset or duration of diarrhea in calves which excreted different infectious agents. Salmonella species, infectious bovine rhinotracheitis virus, parvovirus, adenoviruses, parainfluenza-3 virus, and Chlamydia species could not be demonstrated in any of the calves or their dams. No potentially enteropathogenic agents could be demonstrated in 11 of the 32 calves which scoured. These findings emphasize the complexity of the infectious aspect of the neonatal diarrhea syndrome and illustrate the difficulty in making an etiological diagnosis in field outbreaks of the calf scours complex. (+info)
Protective effect of immunoglobulins in serum and milk of sows exposed to transmissible gastroenteritis virus.
Experimental exposure of susceptible pregnant sows by various routes to the gut-origin transmissible gastroenteritis virus stimulated production of milk and serum antibodies. These antibodies neutralized the cytopathic effect of transmissible gastroenteritis virus propagated in cell culture. This in vitro neutralizing antibody resided in the IgG and IgA immunoglobulin classes. On the other hand, protection for baby pigs resided in the IgA class of milk immunoglobulin of sows exposed orally or intramammarily but not of sows exposed intramuscularly to the virus. (+info)
Differential detection of transmissible gastroenteritis virus and porcine epidemic diarrhea virus by duplex RT-PCR.
Transmissible gastroenteritis (TGE) and porcine epidemic diarrhea (PED) are highly contagious enteric diseases of piglets. The clinical signs of these diseases are very similar and include watery, yellowish diarrhea. Thus, the effective differential detection of TGE virus and PED virus is required. In the present study, a duplex reverse transcription-polymerase chain reaction (RT-PCR) was established for the differential detection of TGE and PED viruses. The primers were designed for the S gene of each virus. RNA was extracted from the intestines and stool samples that were collected from the swine with diarrhea. The RT-PCR test could detect both TGE and PED viruses with 2 TCID50/200 microl. Among 90 clinical samples, 7 TGE viruses and 2 PED viruses were detected by the duplex RT-PCR. This duplex RT-PCR may be a useful diagnostic method for the rapid, specific, and sensitive differential detection of TGE and PED viruses using clinical samples. (+info)
The sialate-4-O-acetylesterases of coronaviruses related to mouse hepatitis virus: a proposal to reorganize group 2 Coronaviridae.
Group 2 coronaviruses are characterized within the order Nidovirales by a unique genome organization. A characteristic feature of group 2 coronaviruses is the presence of a gene encoding the haemagglutinin-esterase (HE) protein, which is absent in coronaviruses of groups 1 and 3. At least three coronavirus strains within group 2 expressed a structural protein with sialate-4-O-acetylesterase activity, distinguishing them from other members of group 2, which encode an enzyme specific for 5-N-acetyl-9-O-acetylneuraminic acid. The esterases of mouse hepatitis virus (MHV) strains S and JHM and puffinosis virus (PV) specifically hydrolysed 5-N-acetyl-4-O-acetylneuraminic acid (Neu4,5Ac2) as well as the synthetic substrates p-nitrophenyl acetate, 4-methylumbelliferyl acetate and fluorescein diacetate. The K(m) values of the MHV-like esterases for the latter substrates were two- to tenfold lower than those of the sialate-9-O-acetylesterases of influenza C viruses. Another unspecific esterase substrate, alpha-naphthyl acetate, was used for the in situ detection of the dimeric HE proteins in SDS-polyacrylamide gels. MHV-S, MHV-JHM and PV bound to horse serum glycoproteins containing Neu4,5Ac2. De-O-acetylation of the glycoproteins by alkaline treatment or incubation with the viral esterases resulted in a complete loss of recognition, indicating a specific interaction of MHV-like coronaviruses with Neu4,5Ac2. Combined with evidence for distinct phylogenetic lineages of group 2 coronaviruses, subdivision into subgroups 2a (MHV-like viruses) and 2b (bovine coronavirus-like viruses) is suggested. (+info)