Identification of canine coronavirus strains from feces by S gene nested PCR and molecular characterization of a new Australian isolate.

A nested PCR (nPCR) assay for the detection of canine coronavirus (CCV) in fecal samples is described. The target sequence for the assay was a 514-bp fragment within the spike (S) glycoprotein gene. The sensitivity of the assay is extremely high, detecting as little as 25 50% tissue culture infective doses per g of unprocessed feces. A clinical trial using dogs challenged orally with CCV SA4 and CCV NVSL was used to compare viral isolation and the nPCR assay as detection techniques over a 2-week period of infection. Virus isolation detected CCV shedding from day 4 to 9 postchallenge, while the nPCR assay detected CCV shedding from day 4 to 13 postchallenge. Cloning and sequencing of the nPCR assay product enabled investigation of the evolutionary relationships between strains within the S gene. The simple and rapid procedure described here makes this assay an ideal alternative technique to electron microscopy and viral isolation in cell culture for detection of CCV shedding in feces. The described assay also provides a method of identifying new strains of CCV without the complicated and time-consuming practice of raising antibodies to individual strains. This is illustrated by the identification, for the first time, of an Australian isolate of CCV (UWSMN-1).

Canine coronavirus in Australian dogs.

OBJECTIVE: To estimate the frequency of serum antibodies (IgG and IgM) to canine coronavirus (CCV) in the Australian dog population and evaluate the role of CCV as a causative agent of gastroenteritis. DESIGN: A serological survey of antibodies to CCV among different dog populations. PROCEDURE: The development and characterisation of an indirect ELISA for the detection of antibodies (IgG and IgM) to CCV was undertaken. Sera collected from both diarrhoeal and non-diarrhoeal dogs from various populations throughout Australia were tested for these antibodies to CCV. RESULTS: Serum samples (1396) collected from 1984 to 1998 were tested for the presence of IgG antibodies to CCV. Samples were divided into two categories on the basis of the number of dogs housed together. The groups were either an open population containing dogs housed as groups of three or less, or kennel populations. Sera from 15.8% of the open population and 40.8% of kennelled dogs were positive for CCV antibodies. The prevalence of antibodies varied from zero to 76% in kennelled dogs. About 23% of 128 dogs positive for IgG antibodies to CCV were also positive for IgM antibodies to CCV, indicating recent CCV infection. Of those dogs that were presented with clinical signs of gastroenteritis such as diarrhoea and vomiting (n = 29), 85% were positive in the IgM ELISA and 85.7% in the IgG ELISA for antibodies to CCV. In comparison, for those dogs presented without any history of gastroenteritis only 15% were positive for IgM and 30% positive for IgG. CONCLUSION: Serological evidence indicates that infection with CCV in dogs is widespread throughout the Australian mainland. The prevalence of antibodies varies greatly among different populations, with an average of 40.8% positive in kennelled populations and 15.8% in the open population.

Replication of Australian porcine isolates of Ileal symbiont intracellularis in tissue culture.

Ileal symbiont intracellularis (ISI) isolated from Australian cases of PIA and PHE was replicated in the rat ileum enterocyte cell line IEC 18. The number of ISI within cells varied, as did the number of ISI infected within the monolayer. At 24 h post infection a large number of cells were infected with approximately 100 ISI per cell. At the termination of infection, fewer IEC 18 cells were infected but ISI had replicated to fill the cell cytoplasmic space. Numerous foci of infected cells were visible in the monolayer, containing as many as 15 densely infected cells. Division of ISI infected cells indicated the transmission of ISI in the cytoplasm to daughter cells. This suggests that the replication of ISI in culture appears to be reasonably cell dependent. No cytopathic effects were observed in the infected cultures.

The prevalence of F107 fimbriae and their association with Shiga-like toxin II in Escherichia coli strains from weaned Australian pigs.

A total of 480 haemolytic Escherichia coli (HEC) strains from weaned pigs were tested using an oligonucleotide probe to determine the prevalence of F107 fimbriae in Australia. Of these, 62% were positive. F107 was detected in serogroups O141, O138, O8, O45, O139, O157 and O98 but not in O149 nor O147. 81% of E. coli strains not producing other fimbriae (K88, 987P, K99 or F41) were positive for F107; 5% of strains with K88 fimbriae also had F107. Serological investigation of the expression of F107 fimbriae indicated that serogroups O141, O138, O8, O45 and O157 produced variant F107ac. Variant F107ab was found on O139 strains only. The F107 fimbrial subunits of both variants had a molecular weight of approximately 16 kDa. A total of 350 of the HEC strains were tested to determine the prevalence of the Shiga-like toxin II (SLT II) gene. 29.0% of these strains were positive. SLT II was detected in serogroups O141, O138, O149, O98, O45, O8 and O157 but not in O139. 25% of these strains were positive for both F107 and SLT II.

Detection of ileal symbiont intracellularis in porcine faecal samples by polymerase chain reaction.

Ileal Symbiont Intracellularis (ISI), the organism causing proliferative enteritis (PE) in pigs was detected in faeces by the application of polymerase chain reaction (PCR). The assay based on a 319 base pair DNA fragment was used on faecal and mucosal samples derived from pigs either affected or unaffected with PE. As few as 10(3) ISI could be detected in pig faeces spiked with ISI. No amplification product was detected in the faeces of unaffected pigs but faeces of confirmed clinical cases were positive. This method offers an accurate, sensitive, easy to perform alternative to monoclonal antibody tests or histological examination post-mortem for the presence of ISI in pig herds.

Enzyme-linked immunosorbent assay for measuring ileal symbiont intracellularis-specific immunoglobulin G response in sera of pigs.

Proliferative enteritis (PE) is a common intestinal disease on pig farms. The disease is caused by ileal symbiont (IS) intracellularis (Campylobacter-like organisms) bacteria. An enzyme-linked immunosorbent assay (ELISA) was developed to measure IS intracellularis-specific immunoglobulin G (IgG) response in the sera of pigs. The antigen used in the ELISA was filtered, percoll gradient-purified IS intracellularis extracted from the intestines of pigs affected with proliferative hemorrhagic enteropathy. The antibody responses of pigs challenged with intestinal homogenates from pigs affected with proliferative hemorrhagic enteropathy containing IS intracellularis or percoll-gradient purified IS intracellularis were low and variable. The low IgG titers measured in challenged pigs support previous findings that IgG plays a minor role in the immune response of pigs to IS intracellularis. On a farm in which infection was endemic, pigs seroconverted at between 7 and 24 weeks of age. High IgG titers, indicative of maternally acquired antibody, were present in 3-week-old pigs. The IgG titers in piglets were lowest at 6 weeks of age, which approximates the age of onset of clinical disease. These results suggest that IgG plays a role in determining the susceptibilities of pigs to natural infection. Measurements of seroconversion by the ELISA might aid in epidemiological investigations of PE in naturally infected herds. However, the variable antibody responses in experimentally challenged pigs would seem to limit its usefulness as an antemortem diagnostic test for PE.

[Detection with nonradioactively labeled probes of the toxin genes of different E. coli pathotypes from swine]. / Nachweis von Toxingenen verschiedener E. coli Pathotypen beim Schwein mit nichtradioaktiv markierten Sonden.

We tested hemolytic E. coli from 86 pigs with edema disease or colidiarrhea. They were tested serologically and with nonradioactive digoxigenin-dUTP labelled probes for the presence of enterotoxin or Shiga-like-toxin genes. By slide-agglutination we detected 38 cases with E. coli O149:K88, 28 with E. coli O139:82B and 20 with E. coli O141. E. coli of serogroup O149:K88 isolated from diarrheic pigs, reacted with the probes for LT and STb genes. Edema disease E. coli O139:82B reacted with the SLTII probe. E. coli O141, isolated from colidiarrhea or edema disease showed a diversity of toxin gene patterns. All the E. coli O141 from diarrheic pigs reacted with the probes for LT and STap in addition to SLTII. No strains isolated from pigs with edema disease possessed any of these enterotoxin genes. Gene probe technique confirmed the serological method as useful tool for diagnosing E. coli O149:K88 and O139:82B as ETEC or VTEC, respectively. On the other hand only the demonstration of toxin genes with probes could explain the pathological findings in the pigs shedding E. coli of serogroup O141.

Adhesive fimbriae associated with porcine enterotoxigenic Escherichia coli of the O141 serotype.

Enterotoxigenic Escherichia coli of the O141 serotype, isolated from piglets with postweaning coliform enteritis but producing none of the characterized adhesive fimbriae, was examined for fimbrial production by transmission electron microscopy. Two strains that produced numerous fimbriae were chosen for further characterization. The fimbriae were isolated and purified and had a subunit molecular weight of 17,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using antiserum raised against this protein, we have shown it to be specific for the 17,000-molecular-weight band by immunoblotting and to be directed against the fimbriae by immunoelectron microscopy. These fimbriae were not produced when the bacteria were grown at 18 degrees C and did not show any mannose-resistant hemagglutination of the erythrocytes tested. We propose that these are a new type of adhesive fimbriae associated with porcine enterotoxigenic E. coli of the O141 serotype.

Detection of leptospires in pig kidney using DNA hybridisation.

DNA hybridisation detected leptospiral organisms in homogenised kidneys from experimentally infected pigs, and in homogenates of pig kidneys collected at abattoirs. The technique is easy to perform and had some advantages over cultural and histological methods, in permitting the rapid survey of many kidneys simultaneously. Leptospires added to a homogenate of uninfected kidney could be detected at 10(2) organisms ml-1 by DNA hybridisation, but the technique appeared to be less sensitive than culture.

Detection of enterotoxigenic Escherichia coli in piggeries in Victoria by DNA hybridisation using K88, K99, LT, ST1 and ST2 probes.

Rectal swabs collected from piglets with diarrhoea from commercial pig farms were examined for the presence of enterotoxigenic Escherichia coli (ETEC) using DNA hybridisation methods. The probes specifically detected genes for the K88 and K99 fimbrial antigens and the heat-labile and heat-stable enterotoxins. DNA hybridisation methods detected more ETEC than could be detected by either enzyme-linked immunosorbent assay (ELISA) or slide agglutination methods, and also offered the opportunity to test for fimbrial antigens and toxins concurrently. The DNA hybridization method was shown to be applicable to ETEC detection in mixed growths cultured directly from rectal swabs to filters. The method eliminates the need for toxin tests using animals and enables very large numbers of samples to be investigated. The use of toxin probes has revealed large numbers of ETEC with uncharacterized fimbrial antigens.

Prevalence of enteroviral and parvoviral antibodies in pig sera.

Surveys of serum antibodies to enterovirus serotype 8 and parvovirus were conducted in pigs three to 21 weeks old using the virus neutralisation (VN), enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition tests. Antibody levels to enterovirus were at their lowest at four to eight weeks old, increased at 14 to 16 weeks to high levels which were maintained until 21 weeks. Specific immunoglobulin G (IgG) values measured by ELISA paralleled VN values and were similar in two separate farm surveys. Measurement of immunoglobulin M (IgM) levels indicated that enterovirus infection occurred about four weeks old. Sera obtained from a large number of geographically separated farms, from abattoir-bled animals and from colostrum-deprived piglets raised for 33 weeks from birth in an isolated environment were tested by VN. Results revealed that porcine enterovirus serotype 8 is spread widely throughout northern Victorian piggeries and high antibody levels prevail. In contrast to the enterovirus antibody results, parvovirus antibody levels measured in piglets declined from high levels at three to four weeks to undetectable levels from 13 weeks old.

Restricted translation of the genome of the flavivirus Kunjin in vitro.

Virion RNA of Kunjin virus was translated in rabbit reticulocyte lysates at a rate (7000 daltons/min) approaching that observed previously in vivo. As many as 18 polypeptides were translated and shown by tryptic peptide mapping to be closely related to one another and to contain some of the elements of envelope (E) and most of the elements of core (C) proteins of Kunjin virus. None of the products resolved in gels were precipitated by antiserum to purified E protein, but the larger products were precipitated by an antiserum against all the Kunjin virus-specified proteins. No evidence was obtained of translation of the non-structural proteins, despite attempts to retain or perturb the structure of the virion RNA. As many as 50% of the amino acid sequences in the peptide maps were not identifiable.

Transcriptional control of endogenous virus genes in murine lymphocytes.

The expression of endogenous retrovirus in murine lymphocytes is under genetic control and also depends on the differentiation state of the lymphocytes. We have used a cDNA probe complementary to induced virus RNA to quantify transcription of virus sequences in lymphocytes from mitogen-stimulated lymphocytes of the AKR, 129/J and Balb/c mice. Balb/c lymphocytes show the clearest case for induction of new virus sequences in response to stimulation. All strains including 129/J show expression of virus sequences in unstimulated control lymphocytes. The data indicate that mitogen induction of endogenous retrovirus is regulated at the transcriptional level.

Foetal calf serum acts as an inducer of endogenous C-type virus in mouse lymphoid cells.

Several B-lymphocyte mitogens have been previously characterized as efficient inducers of endogenous C-type viruses in mouse spleen cell cultures. We now report that foetal calf serum is also capable of inducing C-type virus release in such cultures. While virus induction by B-cell mitogens was found to be serum independent, the combined effects of serum and mitogens were found to be additive and, with some serum batches, synergistic. The kinetics of induction of virus release by serum was very similar to the established pattern using mitogens. The effect of serum was concentration-dependent. The serum lipoprotein fraction prepared by density ultracentrifugation contained virus-inducing activity. By co-cultivation with mink CCL64 cells, stable lines of mouse xenotropic C-type virus could be recovered from cultures which contained serum, serum lipoprotein fraction or mitogens, but not from control cultures. Preliminary evidence indicates that human sera contained a similar virus-inducing activity in the lipoprotein fraction.