Oral and Poster Abstracts

three cattle farms each. Leptospirosis was also present. It is notorious
the fact that in dairy cattle different etiological agents could be
involved in reproductive problems, therefore the diagnosis procedures
must be integral, including a set of serologic test that allows to
determinate their presence and to be able to apply adequate and
effective control measures.
Financial support: SAGARPA-CONACyT. (2004/COI/23)
Key Words: abortion, cattle, serology
700 Viability of Mycobacterium Avium Subsp. Paratuberculosis
(MAP) in Baled Grass Silage
J. Khol 1 , V. Beran 2 , P. Kralik 2 , M. Trckova 2 , I. Pavlik 2 ,
W. Baumgartner 1
1 Clinic for Ruminants, Department for Farm Animals and Herd
Health, Vienna, Austria
1 Veterinary Research Institute, Brno, Czech Republic
Objective of the study: Ensiling of grass from areas where livestock or
wild animals infected with paratuberculosis are grazing can be a
possible source of Mycobacterium avium subsp. paratuberculosis
(MAP) infection in domestic ruminants. The results of a preliminary
study about the viability of MAP, the causative agens of
paratuberculosis, in baled grass silage should be presented in this
poster.
Materials and Methods: Seventeen bales of grass silage were spiked
with a suspension containing MAP. Silage samples were collected
periodically for four months to observe MAP viability over time.
Collected samples were tested for MAP by bacterial culture and Real
Time-PCR (Polymerase Chain Reaction).
Results: Viable MAP could not be detected at any sampling date
during the trial by culture, more than 60 % of the analysed samples
were tested positive by Real Time-PCR.
Conclusions: Even though the results of the presented work suggest
that grass silage seems to be an unlikely source of paratuberculosis
infection for livestock, further investigations are needed to elucidate
the reaction of MAP to unsuitable environmental conditions and their
influence on the infectivity of the bacterium.
701 Phylogenetic Position of an Unreported BPV Type Detected
From a Cutaneous Lesion of a Brazilian Cattle Herd
M. Lunardi, M. Claus, A. Alfieri, A. Alfieri
Universidade Estadual de Londrina, Departamento de Medicina
Veterinária Preventiva, Londrina, Brazil
In Brazil, Bovine Papillomavirus (BPV) infections are endemic in
beef and mainly in dairy cattle herds. Despite the high frequency of
BPV infection, the identification of BPV types in Brazilian cattle is
still sporadic. In a prior study, through the analysis of a partial
segment of L1 gene, we could identify the presence of previously
described BPV types and four putative new BPV types associated
with skin warts. The aim of this study was to determine the entire L1
nt sequence of a putative novel BPV type (BPV/BR-UEL2) detected
in Brazil, and thus state its phylogenetic position. As a phylogenetic
analysis employing the FAP amplicon had revealed our isolate as
closest related to BPV-4 (Xi genus), two pair of degenerate primers
were designed by using alignments of L1, L2, LCR regions of
genome of Xi genus representatives. In addition, aiming to obtain the
full L1 gene sequence, the previously described FAP primer pair was
also employed both in the original form as in combination with
designed primers. The PCR amplicons were purified from agarose
gel and submitted to cloning. Plasmid DNA from 2 clones was
sequenced in both directions using M13 forward/ reverse primers.
Sequences were examined with the software PHRED for quality
analysis and the consensus sequence was determined using the
software CAP3. The alignment was obtained with the software
BioEdit. A neighbour-joining phylogenetic tree was constructed
with complete L1 ORF sequences of 38 PVs classified in 18 genera,
and with the entire L1 ORF sequence of BPV/BR-UEL2 isolate,
using the MEGA program v.3.1. A consensus sequence, representing
the terminal portion of L2 gene, the complete sequence of L1 gene
and an initial segment of LCR, could be achieved from three PCR
amplicons. By ORF analysis, it was possible to determine that the L1
ORF encoded protein of the Brazilian isolate consists of 532 aa. The
phylogenetic analysis revealed that the BPV/BR-UEL2 is related
with BPV types held in Xi genus. Besides, this isolate displayed the
102 XXV. Jubilee World Buiatrics Congress 2008
highest L1 nt sequence similarity with BPV type 4 (74%), suggesting
its classification in the Xi genus. The realization of further studies
involving the molecular epidemiology of BPV infections, in
Brazilian cattle herds as much in diverse geographical areas around
the world, become necessary to verify the prevalence of this new
viral type and to check its association with cutaneous lesions.
Financial support: CNPq, CAPES, FINEP and FAP/PR
Key words: BPV; L1 ORF; phylogeny
702 Monitor pProject : Evidence of Some Respiratory Virus
Isolation from Marchigiana Breeding Farms with Respiratory
Disorders, Note 2.
S. Petrin 1 , M. Panicci 1 , S. Briscolini 1 , L. Cucco 1 , M. Ferrari 2 ,
G. Filippini 1 , G. Pezzotti 1
1
Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche,
Perugia, Italy
2
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia
Romagna Brescia, Brescia, Italy
Objectives of study: In the framework of the monitor project,
necropsy has been carried out on 52 dead calves originating from
herds with bovine respiratory disease (Monitor project note 1). This
report describes the results of the bacteriological and virological
investigations carried out on subjects died for respiratory disorders.
Materials and methods: Organs (trachea, lung, bronchial lymph nodes),
were collected from 52 dead animals for bacteriological examination and
viral isolation. The bacteriological examination was performed according
to Quinn (1999); briefly, specimens were streaked on blood agar (BA),
Mc Conkey agar (MC), Mannitol Salt agar (MSA); and incubated for 12-
24 h at 37 °C. Isolates were characterized according to Quinn (1999), and
further identified using the API Systems (Biomerieux). Testing for
Mycoplasma spp. was carried out inoculating PPLO agar and broth,
incubated for 12 days at 37 °C under 5% CO 2, and checked daily. For
virus isolation the samples were diluted, centrifuged and the supernatants
were inoculated into BEK cells. In absence of citopathic effect (CPE), 3
subpassages were made. In positive cases, the identification of the isolated
virus was evidenced with serum neutralization test (SN) using reference
immune serums against IBR and with nested-PCR methods according to
Vilcek S. et al. (1994) to identify BRSV. For the SN tests virus isolates
were diluted serially and mixed with the reference immune serum in 96well
microtiter plates which were held for 90 min at 22 °C. After the
incubation 20,000 BEK cells in E-MEM were added. The plates were
checked for 7 days, and the CPE was evaluated. Virus dilution mixed with
E-MEM represented the control. The virus was identified if titre in the
presence of immune serum was at least 2 log lower than the virus titre in
E-MEM.
Results: The results evidenced the presence of the following bacteria:
M. haemolytica (15 %); P. Multocida (26 %); Mycoplasma spp (4 %);
Negative cases (55 %). From trachea and lung samples of three
animals, CPE was detected in BEK cell cultures whose features were
typical of IBR or BRSV, respectively. They were detected at the first or
second serial passage. IBR and BRSV were identified by SN and nested
PCR tests, respectively.
Conclusions: The results of the investigation demostrated that viruses,
isolated from Marchigiana breeding farms with respiratory disorders
are members of the Herpesviridae and Paramyxoviridae family.
703 Previously Described BPV Types and Putative New Types in
Cutaneous Papillomatosis from Brazilian Cattle Herds
M. Claus, M. Lunardi, A. Alfieri, A. Alfieri
Universidade Estadual de Londrina, Departamento de Medicina
Veterinária Preventiva, Londrina, Brazil
The aim of the current study was to report the identification of
putative new BPV types in skin warts in cattle herds from Paraná
state of Brazil. Papilloma specimens (n=27) were taken individually
from diverse body sites of adult and young bovines, from dairy (n=2)
and beef (n=2) cattle herds from Paraná state, South region of Brazil.
The PCR assay was carried out using the primer pair FAP59 and
FAP64. All PCR products were purified and a direct sequencing was
performed with FAP primers. For the amplicons which the prior
analysis revealed them as a putative new BPV type, a cloning and a
further sequencing, in both directions, was performed employing the
plasmid DNA from two selected clones of each sample. For quality
analysis of chromatogram readings and determination of the