This paper describes the isolation of porcine epidemic diarrhea (PED) virus in Vero and porcine cell cultures, and the influence of age on disease in experimental infection. PED virus was isolated from the small intestine of piglets inoculated with PED samples and cultured in Vero, porcine bladder and kidney cells propagated in collagen-coated tissue culture plates in maintenance medium (MM) containing trypsin. In porcine bladder and kidney cell cultures inoculated with isolated PED virus, cytopathic effects (CPE) including cell fusion were detected. Specific brilliant fluorescence was observed in the cytoplasm of these cells. Two- and 7-day old, and 2-, 4-, 8- and 12-week old specific pathogen-free (SPF) pigs were orally inoculated with PED virus isolated from an outbreak. All 2- and 7-day old pigs inoculated developed severe watery diarrhea from post-inoculation day (PID) 1 and died between PID 3 and 4. Although three of five 2-week old pigs developed diarrhea on PID 1-4, they eventually recovered. In the 4-week old group, three of five pigs had mild diarrhea for 1-2 days. None of the 8- and 12-week old pigs showed any clinical signs. Antibodies against PED virus were detected in all surviving pigs by virus neutralization (VN) test and immunofluorescence assay (IFA). Therefore, there is an age-dependent resistance to pathogenic PED virus infection in pigs.

A sudden outbreak of epidemic diarrhoea of piglets occurred in Japan, the principal features being watery diarrhoea, dehydration and high mortality in newborn animals. The microscopical lesions were villous atrophy in the small intestine, the villous enterocytes being vacuolated and cuboidal in shape. The villus-crypt ratio was severely reduced, varying from 1:1 to 3:1. Transmission electron microscopy showed numerous coronaviruses within the cytoplasm of enterocytes and among microvilli. Specific antigens of porcine epidemic diarrhoea (PED) virus were detected in the cytoplasm of enterocytes by the streptavidin-biotin (SAB) technique. Infected cells, which were most abundant in the villous epithelia of the jejunum and ileum, were present in small numbers in the large intestine, the crypt epithelia, the lamina propria and Peyer's patches. The study suggests that the SAB technique is useful for the diagnosis of PED.

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Since late 2013, after an absence of seven years, outbreaks of porcine epidemic diarrhea virus (PEDV) infection have reemerged and swept rapidly across Japan, resulting in significant economic losses. In this study, we report the emergence, mixed infection, and genetic characterization of 15 novel field PEDV variants with large genomic deletions. The sizes of deletion varied between 582 nt (194 aa) and 648 nt (216 aa) at positions 28–714 (10–238) on the S gene (protein). Among 17 PEDV samples isolated from individual pigs, all of them contained at least two distinct genotypes with large genomic deletions, and 94.1% of them were found to consist of strains with an intact S gene. These variants were found in eight primary and nine recurrent outbreaks, and they might be associated with persistent PEDV infection in the farms. Full-length S and ORF3 genes of eight variants derived from 2 samples were characterized. This is the first report of mixed infections caused by various genotypes of PEDV and would be important for the studies of viral isolation, pathogenesis, and molecular epidemiology of the disease.

ABSTRACT. We examined the susceptibility in vitro of 99 isolates of Mycoplasma (M. bovirhinis, M. bovis, M. alkalescens and M. bovoculi) and one of Acholeplasma laidlawii collected in 2008 and 2009 from Japanese homebred cattle and cows imported from Australia to 13 antimicrobial agents. Mycoplasma species isolated from homebred cattle were susceptible to spectinomycin and fluoroquinolones but resistant to macrolides, tetracyclines, thiamphenicol and flumequine. Kanamycin, lincomycin and chloramphenicol maintained intermediate effectiveness against these species. The isolates from the imported cows maintained sufficient susceptibility to all antimicrobials examined except for erythromycin and thiamphenicol. These results showed that four species of Mycoplasma isolated from Japanese homebred cattle had obviously higher drug resistance compared with previous reports, even within this decade.KEY WORDS: bovine respiratory diseases, drug resistance, Mycoplasma spp.J. Vet. Med. Sci. 72(12): 1661-1663, 2010 Bovine respiratory disease complex (BRDC) caused by several combinations of viral and bacterial infections together with stress is a significant problem that has considerable economic impact on the cattle industry [4,12]. Mycoplasma is a causative agent of BRDC [2,6,7,10,12]. Because vaccination of cattle to prevent Mycoplasma infection has not yet been approved in Japan, other approaches are needed, such as preventing bacterial invasion of farms through hygiene management, preventing respiratory symptoms through nutritional management and antibiotic treatment of cattle with abnormal respiratory manifestations. Although the antimicrobial susceptibility of Mycoplasma species has changed [6,7,10], there has been no report of examination of the susceptibility in Japan since isolates were collected from 1996-1997 [6]. Therefore, this study examined the antimicrobial susceptibility of Mycoplasma strains recently isolated from cattle in Japan.We analyzed 99 isolates of Mycoplasma (M. bovirhinis, M. bovis, M. alkalescens and M. bovoculi) and one of Acholeplasma laidlawii collected in 2008 and 2009 from Japanese homebred cattle that were 15 days to 11 months old at 33 farms in Kyusyu and healthy cows imported from Australia that were 14 to 15 months old. At the time of sampling, the Australian cattle were isolated from other cows because of quarantine restrictions at the destination farm.We examined the minimum inhibitory concentrations (MICs) of 13 antimicrobial agents (kanamycin, spectinomycin, erythromycin, tylosin, tilmicosin, lincomycin, oxytetracycline, chlortetracycline, chloramphenicol, thiamphenicol, flumequine, enrofloxacin and danofloxacin) required to completely suppress the growth of the four species of Mycoplasma and A. laidlawii. The MICs were determined using agar dilution according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [3] and a previous report [5]. Mycoplasma species and A. laidlawii were grown and diluted 1:100 in M broth [9]. Bacterial suspensions (about 10 6 colony formi...

-To determine the teratogenic potential of Aino virus (AINOV) in cattle, pregnant cows and fetal cattle were infected with a fresh isolate of AINOV. Five pregnant cows were inoculated intravenously with the virus at 122 to 162 days of gestation and allowed to give birth. All of the cows developed neutralizing antibodies to the virus, indicating that the cows had been infected with the virus; however, no clinical abnormalities were seen in their six newborn calves, and no specific antibodies to the virus were detected in the precolostral serum of calves. Five fetuses with fetal ages ranging from 132 to 156 days were inoculated in utero with the virus. One weak newborn and four stillborn calves were delivered at gestation days 256 to 263, i.e., less than the standard gestation term; they had congenital abnormalities including arthrogryposis, hydranencephaly and cerebellar hypoplasia. Antibodies specific to AINOV were detected in their precolostral serum. These results demonstrate that AINOV is a potential etiological agent of congenital malformation of cattle. Aino virus / cattle / congenital malformation / experimental infection

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