Oral and Poster Abstracts

or secretions of wild animals, rats or dogs. Serovar canicola seems to
have a protective effect in bovine (0,74 times with regard to humans),
being the infection probability by means of secretions of dogs that
remain in the property. From serovar Sejroë any hypothesis could not
settle down, because humans went negative to the serovar. Results
demonstrate the circulation of different serotypes among the animal
species and they highlight the importance of carrying out more
advanced epidemic studies, with the purpose of understanding the
epidemiology of Leptospira in the region.
Key words: leptospira, human, cattle, Toca, epidemiology
720 Comparison of Gene Detection by IS6110 and IS1081
Polymerase Chain Reaction and Isolation by LJ and BM
Media in Tuberculoid Tissues of Slaughtered Cattle
YS. Cho 1 , YH. Jean 2 , IS. Roh 2 , I. Hwang 1 , D. Cho 1 , H. Nam 1 ,
E. Heo 1 , JM. Kim1, HR. Han 3 , SC. Jung 1
1
National Veterinary Research & Quarantine Service, Bacteriology
and Parasitology Division, Anyang, Korea, South
2
National Veterinary Research & Quarantine Service, Animal
Disease Diagnostic Center, Anyang, Korea, South
3
Seoul National University, College of Veterinary Medicine, Seoul,
Korea, South
Objectives of study: Bovine tuberculosis has been diagnosed by the
examination of tuberculoid tissue as well as tuberculin skin test. Bovine
tuberculosis at slaughterhouse is diagnosed by histopathological
finding, polymerase chain reaction, and isolation of Mycobacterium
bovis. This study provides the isolation rate of Mycobacterium bovis
from tuberculoid tissues of slaughtered cattle and more efficient
laboratory methods to diagnose bovine tuberculosis at slaughterhouse.
Materials and Methods: One part of tuberculoid tissues (n = 89) from
39 slaughtered cattle were macerated and treated with 0.75%
hexadecylpyridium chloride as decontaminant. In addition, the other
part of those were treated with 10% formalin and stained with H&E and
acid-fast staining kit. The macerated tissues were incubated on LJ w/o
glycerol and BM media at 37 ˚C for 16 weeks. LJ w/ glycerol media
were also incubated at 30 ˚C for 16 weeks for the isolation of
Mycobacterium avium. DNA was extracted by GuSCN-silica and
IS6110 and IS1081 primers were used for PCR.
Results: M. bovis was isolated in 82 per cent of slaughtered cattle with
tuberculoid tissues (32/39). Sensitivity, specificity, positive predictive
value, and negative predictive value of IS6110 direct PCR (dPCR)
were 69, 71, 92, and 33, and those of IS1081 dPCR were 79, 71, 79, and
29, respectively. IS6110 dPCR showed the highest positive predictive
value, while IS1081 dPCR showed the highest sensitivity. The result by
dPCR accorded with that by H&E staining, but the tuberculoid tissues
were not always detected acid-fast bacilli. M. bovis isolation rate by
BM (77%; 57/74) was higher than that by LJ w/o glycerol (72%;
63/87). Eighty seven per cent of the isolates (72/83) were IS6110
positive, and 86 per cent of those (67/78) IS1081 positive. At 30 ˚C on
LJ w/ glycerol, 16 mycobacteria-like bacilli were isolated from 90
samples, and 14 isolates of those were identified as Mycobacterium
bovis by IS6110 and IS1081 PCR.
Conclusions: The results from IS6110 and IS1081 dPCR were not
always accorded each other. Therefore, the application of both IS6110
and IS1081 PCR could increase the diagnostic accuracy of bovine
tuberculosis. The isolation rate of BM media showed higher than that
of LJ media. The isolates with only IS6110 or IS1081 gene would be
further analyzed to examine new genotype of M. bovis in the future.
Key words: Mycobacterium bovis, bovine tuberculosis, IS6110,
IS1081, LJ media, BM media
721 Outbreak of Acute BVD in Brazilian Beef Cattle:
Clinicopathological Findings and Molecular Characterization
of a BVDV Strain Subtype 1b
M. Lunardi, S. Headley, J. Lisboa, R. Otonel, A. Alfieri, A. Alfieri
Universidade Estadual de Londrina, Departamento de Medicina
Veterinária Preventiva, Londrina, Brazil
When first described, Bovine Viral Diarrhea (BVD) was characterized
as an acute transmissible disease associated with severe leucopenia,
high fever, depression, diarrhea, gastrointestinal erosions, and
hemorrhages. Recently the severe acute form has been related only to
some hypervirulent BVDV-2 strains. This article reports the detection
of BVDV-1b associated with an acute and fatal outbreak of BVD in a
Brazilian herd. Depression, anorexia, watery diarrhea, sialorrhea, and
weakness were observed in six steers. The affected animals died 24
hours to 15 days after the first manifestation of infection was observed.
One of these animals was evaluated for laboratorial, clinical, and
pathological alterations. Blood samples were collected ante mortem by
routine procedures. Selected tissues were fixed in 10% neutral buffered
formalin solution and processed for histopathological evaluation. RNA
extraction was performed following silica/guanidine isothiocyanate
method. The RT-PCR assay was carried out using a set of primers
(103/372) designed from the 5-UTR region of the BVDV genome. The
identities of the RT-PCR products were confirmed by direct sequencing
of the amplicon from one clinical specimen. Sequences were examined
with the software PHRED for quality analysis and the consensus
sequence was determined using the software CAP3. The alignment was
obtained by BioEdit software. A neighbour-joining phylogenetic tree
was constructed using the MEGA program. Laboratory findings were
non-specific; clinically, the animal was weak, with dehydration and
erosive oral lesions. Pathological alterations were predominant at the
tongue, esophagus, and rumen. The RT-PCR assay identified BVDV in
all clinical samples analyzed. The sequence obtained has been
deposited in GenBank (accession number EF406123). Phylogenetic
analysis of BVDV derived from lymph node revealed that this strain
was clustered with other BVDV subtype 1b isolates, sharing high
similarity (96.7%) with the representative subtype 1b isolate Osloss.
The real role played by BVDV-1b in acute diseases and the frequency
of this subtype in the epizootology of acute and severe BVD are not
totally elucidated. Molecular characterization is the only definitive
method to identify the BVDV genotype involved in acute outbreaks of
severe BVD, since both clinical presentation and pathologic findings
can be similar to BVDV-2 infection.
Financial support: CNPq, CAPES, FINEP and FAP/PR
Key words: cattle, BVD, BVDV, 5-UTR
722 Winter Dysentery by Bovine Coronavirus Infection in a
Tropical Country
E. Takiuchi, A. Barry, D. Stipp, R. Otonel, A. Alfieri, A. Alfieri
Universidade Estadual de Londrina, Departamento de Medicina
Veterinária Preventiva, Londrina, Brazil
Winter Dysentery (WD) is an acute diarrhea caused by Bovine
Coronavirus (BCoV) that affects adult cattle during the winter with
high morbidity and low mortality rates. Low temperature and UV light
intensity during the winter lead to high levels of environmental
contamination by BCoV. The WD occurrence in dairy herds results in
economic losses caused by severe reduction in milk production.
Association between BCoV infection and WD was reported in the
USA, Europe, Japan and Canada, being sporadic in tropical areas. This
study reports, the detection of BCoV in an adult cow during an
outbreak of acute diarrhea in Brazil. The outbreak occurred during
June, in Ponta Grossa city, Paraná, Brazil and affect 138 from 154
lactating cows. Three animals died. Most cows had bloody and watery
diarrhea (three to four days), and milk production dropped from 37 to
26 liters/cow/day. Fecal sample was collected directly from large
intestine of a necropsied cow. The sample was negative to C.
perfringens type C, Eimeria spp, Cryptosporidium spp, enteroxigenic
E. coli, rotavirus and bovine viral diarrhea virus. Liquid feces
suspension at 50% (v/v) was prepared in PBS. RNA extraction was
performed with phenol/chloroform/isoamyl alcohol and
silica/guanidinium isothiocyanate methods. PCR and semi-nested
assay were performed according to Takiuchi et al. (2006) with primers
BCoV1/BCoV2 and BCoV3/BCoV2, respectively, with a predicted
251 bp product (N gene). The products were analyzed by
electrophoresis in ethidium bromide stained 2% agarose gel, under UV
light. BCoV amplicons from semi-nested PCR were purified,
sequenced, and similarity checked in BLAST software confirming that
the 251 bp amplicon was from a BCoV strain. This result suggests the
role of BCoV in the etiology of WD in Brazilian dairy herds. High
incidence of WD in cold months is notified in regions with well-defined
seasons. The present case indicates that WD also occurs in tropical
areas where temperatures are higher. The location of city in the South
of Brazil, with temperature normally lower, and the season (winter) in
which the outbreak occurred, perhaps have created favorable
conditions to virus stability facilitating dissemination among confined
cattle. In conclusion, while there are some studies associating BCoV
with outbreaks of WD in other countries, there are a few reports in
South America, Brazil.
Infectious and Zoonotic Deseases (Public Health) 107