From January 1998 to July 2000, 2,456 clinical samples, including lung, tonsil, lymph node, and serum, from 760 cases submitted to the Animal Health Laboratory, Ontario, Canada, were tes[r]
(1)18 Shadduck JA, Albert DM, Niederkorn JY: 1981, Feline uveal melanomas induced with feline sarcoma virus: potential model of the human counterpart J Natl Canc Inst 67:619– 627
19 Stiles J, Bienzle D, Render JA, et al.: 1999, Use of nested poly-merase chain reaction (PCR) for detection of retroviruses from
formalin-fixed, paraffin-embedded uveal melanomas in cats Vet Ophthalmol 2:113–116
20 Tsatsanis C, Fulton R, Nishigaki K, et al.: 1994, Genetic deter-minants of feline leukemia virus-induced lymphoid tumors: pat-terns of proviral insertion and gene rearrangement J Virol 68: 8296–8303
J Vet Diagn Invest 14:343–347 (2002)
Restriction fragment length polymorphism of porcine reproductive and respiratory syndrome viruses recovered from Ontario farms, 1998–2000
Hugh Y Cai, Hazel Alexander, Susy Carman, Dara Lloyd, Gaylan Josephson, M Grant Maxie
Abstract. From January 1998 to July 2000, 2,456 clinical samples, including lung, tonsil, lymph node, and serum, from 760 cases submitted to the Animal Health Laboratory, Ontario, Canada, were tested for porcine reproductive and respiratory syndrome viruses (PRRSV) using reverse transcriptase polymerase chain reaction (RT-PCR) and RT-PCR product restriction fragment length polymorphism (RFLP) analysis A total of 516 samples from 284 cases were PCR positive for the PRRSV open reading frame (ORF) sequence The RT-PCR RFLP typing assay was performed using different sets of primers, amplifying 716 or 933 base pairs of ORF 4, 5, and of PRRSV Samples from 254 cases were typeable, yielding 34 different RFLP types Of these, 164 cases had 32 different RFLP types of field or intermediate strains, 86 had a pattern similar to a commercial PRRSV vaccine or VR 2332 strain of the virus, had a RFLP type shared by another commercial vaccine and a field strain In cases, different RFLP types were identified from tissues from different pigs that were submitted at the same time from the same farm Of the 195 farms that submitted PRRSV PCR-positive samples, 48 submitted samples on more than occasion during the specified time frame In 23 of those 48 farms, RFLP patterns of PRRSV differed between submissions, whereas in the other 25 farms, the RFLP pattern remained unchanged There were 34 different PRRSV patterns identified from 236 cases using the primer set amplifying 716 base pairs of PRRSV There were 18 cases, consisting of different patterns, typeable only by using the primers amplifying a 933-base pair fragment of the virus
Current methodologies used to detect porcine reproductive and respiratory syndrome virus (PRRSV) infection include 1) virus isolation; (2) antibody detection, e.g., indirect im-munofluorescence, indirect immunoperoxidase monolayer assay, enzyme-linked immunoasorbent assay, and virus neu-tralization; 3) antigen detection, e.g., immunohistochemistry and immunofluorescence tests on tissue samples; 4) viral nu-cleic acid detection, e.g., reverse transcriptase polymerase chain reaction (RT-PCR) for detection and RT-PCR product restriction fragment length polymorphism (RFLP) analysis
for strain typing.3 Primers from the open reading frame
(ORF) (nucleocapsid gene) of PRRSV have been shown to be specific for detection of all North American and
Eu-ropean isolates.2 In addition to detection of the virus,
RT-PCR RFLP typing methods have been developed that make it possible to differentiate different field strains and the
com-mercial vaccine.4 The current study detected and typed
PRRSV using PCR-based methods from clinical samples
ob-From the Animal Health Laboratory, Laboratory Services Divi-sion, University of Guelph, Box 3612, Guelph, Ontario N1H 6R8, Canada
Received for publication June 12, 2001
tained from Ontario swine herds from January 1998 to July 2000
Clinical samples, including lung, tonsil, lymph node, and serum were submitted to the diagnostic lab from pigs with clinical signs of respiratory disease and histologic evidence of interstitial pneumonia or from aborted fetuses Pieces (0.25 g) of frozen tissues were homogenized using an
hom-ogenizerain guanidinium thiocyanateb solution as described
previously.1 The homogenate was frozen and thawed to
break the tissue cells, then subjected to RNA extraction
us-ing a commercial RNA extraction kit.cSerum was subjected
directly to RNA extraction using a commercial RNA extrac-tion kit.d
To detect the virus, a 1-tube RT-PCR was performed by amplifying 433 base pairs (bp) of ORF The primers
(1010PLS/1010PLR) used were described previously,2 with
the exception of a 5⬘addition of a G on the upstream primer
This primer set was reported as being specific for the ORF region of both North American and European strains The
RT-PCR was carried out in a 25-l reaction mix containing
2 mM MgCl2,e 0.2 mM of each dNTP,e 0.4 U/L RNase
inhibitor,e 1.0 U/l MuLV reverse transcriptase,e 0.05 U/l
Taq DNA polymerase,e0.1M of each primer,fand 2l of
(2)(3)Table 1. Different RFLP types of PRRSV recovered from Ontario farms by the AHL from January 1988 to July 2000.* RFLP type (no of cases) % of total RFLP type (no of cases) % of total
111 (1)
112 (5) 114 (9) 122 (12)
123 (9)
124 (21) 132 (16)
132 with deletion (1)
0.39 1.97 3.54 4.72 3.54 8.27 6.30 0.39
174 (3)
212 (4)
214 (1) 222 (2) 242 (1) 251 (1)
252 (86) 254 (1)
1.18 1.57 0.39 0.79 0.39 0.39 33.86 0.39 133 (4)
134 (10) 142 (4)
143 (3)
144 (4) 151 (1) 152 (4) 153 (1)
161 (1) 162 (3) 163 (3) 164 (1) 171 (1) 172 (10)
1.57 3.94 1.57 1.18 1.57 0.39 1.57 0.39 0.39 1.18 1.18 0.39 0.39 3.94
262 (8)
264 (3) 292 (1) 2-10-2 (1) 114† (3) 122† (1) 124† (3) 133† (1) 134† (3) 144† (2) 162† (2)
162† with deletion (1) 164† (2)
3.15 1.18 0.39 0.39 1.18 0.39 1.18 0.39 1.18 0.79 0.79 0.39 0.79 Total RFLP types 43
Total typeable cases 254
* The types in bold have been reported previously.4,5 † Typeable only by ORF5-933bp-RT-PCR typing method
the following thermocycling programs: cDNA synthesis at 42 C for 20 min, inactivation of reverse transcriptase and denaturation at 95 C for min, 35 cycles of denaturation at 95 C for 20 sec, primer annealing at 60 C for 15 sec; and primer extension at 72 C for 20 sec, final extension at 72 C for min, holding at C The PCR product was detected
by electrophoresis in a 1.5% agaroseggel stained with
ethi-dium bromide.h
A previously described RT-PCR method4was modified to
amplify a 716-bp fragment of ORF and part of ORF and
6 (716-bp RT-PCR) The PCR mix was 50l in volume and
contained reagents in the same concentrations as above, with
the exception that 1.5 mM MgCl2, 0.2M of each primer,
and 4l of template were used Hot start was achieved using
a wax beadi to separate the Taq DNA polymerase from the
rest of the reagents The RT-PCR cycle parameters were sim-ilar to above except primer extension time was lengthened to 30 sec If no amplicon was generated by the 716-bp RT-PCR, a second method was used to amplify a 933-bp region, including 47 bp upstream to 170 bp downstream of the above 716-bp fragment (933-bp PCR) The 933-bp RT-PCR was identical to that of 716-bp RT-RT-PCR except for the
primer sequence used, i.e., forward primer 5⬘-GAC ACC
TGA GAC CAT GAG and reverse primer 5⬘-TCT ATG GCT
GAG TAC ACC After 716-bp RT-PCR or 933-bp RT-PCR,
10l of product was digested with MluI, HincII, and SacII
separately at 37 C for hr as described by the manufacturer.j
The samples were electrophoresed through a 2%
high-strength agarosek gel and visualized by staining with
ethi-dium bromide Fragment size was determined against a
100-bp DNA ladder.j The code representing a digested pattern
was assigned according to or adapted from those described
previously.4 For example, a PRRSV strain was assigned as
type 2-5-2 if its RT-PCR amplicon was digested by MluI and generated 279- and 437-bp fragments, by HincII and gen-erated 327- and 389-bp fragments, and by SacII and
gener-ated 53- and 663-bp fragments As defined previously,5 a
field strain has or more restriction enzyme sites different from a vaccine strain (type 2-5-2) and an intermediate strain has only site different within ORF
Figure shows examples of the PRRSV RFLP types iden-tified in this study The different RFLP types of PRRSV recovered from Ontario farms from January 1998 to July 2000 are summarized in Table A total of 2,456 samples from 760 submitted cases were analyzed Of these, 516 (20.3%) samples from 284 cases (37.4%) were PRRSV ORF 7-sequence positive Of these 284 PRRSV-positive cases, 254 were typeable and 30 cases were nontypeable The 254 typeable cases consisted of 34 different RFLP types Of these 254 cases, 86 were type 2-5-2, similar to a commercial
vaccine1 or VR 2332 strain of PRRSV The relationship of
these samples to the time when the herd was vaccinated was not clear because of incomplete herd histories Type 1-4-4,
an RFLP type shared by another commercial vaccinem and
a field strain, was detected in cases Samples from 164 cases had 32 different RFLP types of field or intermediate strains Two strains were identified with patterns similar to 1-3-2 or 1-6-2 separately except all digested and undigested fragments were smaller in size Further study is needed to investigate whether this reflects a natural deletion in the PRRSV genome
(4)Table 2. Twenty-three farms with different RFLP types of PRRSV
Farm Submit date Type
A 98/03/18 98/12/23 132 252 B 99/04/28 99/12/21 172 132 C 99/06/29 00/02/17 144* 161 D 98/01/07 98/08/31 242 153 E 99/09/20 99/09/20 212 112 F 98/10/08 98/11/19 99/06/30 99/10/07 252 132 142 142 G 98/10/05 98/10/14 98/11/18 222 122 122 H 98/02/12 99/06/09 00/04/13 172 134 132 with deletion
I 98/06/07 98/06/17 98/12/02 111 112 114 J 98/02/16 98/03/10 00/01/05 00/02/10 252 174 123 134* K 99/03/08 99/06/11 99/07/15 NT† 124 NT L 98/02/16 98/07/15 99/07/14 99/08/11 252 134 NT 252 M 98/06/11 98/12/12 99/01/06 99/10/30 122 124 124 122 N 98/04/06 98/04/06 99/10/19 264 264 252 O 98/01/30 98/01/30 98/11/27 99/02/04 99/04/08 99/05/11 00/03/30 00/03/30 00/04/27 00/05/16 00/07/13 123 252 252 114 114* 114 132 252 132 252 112 P 98/10/23 00/04/19 124 123 Q 98/10/08 00/03/07 122 124 R 99/03/25 00/05/03 252 123 S 98/02/13 98/04/15 262 252
Table 2. Continued
Farm Submit date Type
T 99/03/02 99/04/12 99/09/09 00/05/10 124 124* 124* 124* U 98/07/20 99/02/05 00/02/04 252 252 251 V 99/03/16 00/01/25 262 252 W 99/11/26 99/11/26 222 212 * Typeable only by ORF5-933bp-RT-PCR typing method † Nontypeable by either ORF5-716bp-RT-PCR or ORF5-933bp-RT-PCR typing methods
types were identified from 236 of the 254 typeable cases In the remaining 18 cases, the PRRSV consisted of different RFLP types, which were typeable only by the 933-bp RT-PCR typing method This suggests that the 716-bp RT-RT-PCR primer sites of these strains may be different from that of the 716-bp RT-PCR typeable strains The codes previously
reported4 were adapted to describe these strain types (those
types marked with an asterisk below)
There were isolates with a previously undescribed
HincII cut pattern One of the new HincII cut patterns was
designated as pattern 9, which consisted of 490 bp, 200 bp, and undetermined small fragment(s); the other was desig-nated as pattern 10, which consisted of 400 bp and 180 bp and undetermined small fragment(s)
This study identified all of the intermediate types
de-scribed previously,5including types 1-5-2, 2-1-2, 2-6-2, and
2-5-4 Also identified from Ontario farms were type 2-2-2, 2-5-1, 2-9-2, and 2-10-2 PRRSV These RFLP types were
similar to a commercial vaccinel (type 2-5-2), with only 1
restriction site different Moreover, some isolates with an RFLP type shared by a field strain and another commercial
vaccinem(type 1-4-4) were also identified The recently
re-ported results of a full-length sequence of a Canadian PRRSV isolate suggest this isolate may have originated from the spread of the vaccine virus to uninfected animals in the
herd, with subsequent reversion to virulence.6Further study
is needed to determine whether some of the strains identified in this study were field strains or intermediate strains of the vaccines
(5)determine whether the management practices influenced the RFLP type
Fifteen different RFLP types of PRRSV have been re-ported previously.4,5This study identified all of these except
type 1-8-2 As well, 17 additional RFLP types of the virus were identified in this study Two strains may have a natural deletion in ORF 5, ORF 4, or ORF This study indicates that many RFLP types of PRRSV exist on Ontario farms The great variety of PRRSV in Ontario suggests that the virus undergoes frequent mutation under field conditions
Sources and manufacturers
a IKA Ultra-Turrax T25, IKA Laboratory Technology, Staufen, Germany
b Fisher Scientific, Nepean, ON, Canada
c RNeasy威Mini Kit, Qiagen, Mississauga, ON, Canada
d Qiamp威Viral RNA Mini Kit, Qiagen, Mississauga, ON, Canada e PE Applied Biosystems, Missisauga, ON, Canada
f Molecular Supercenter, University of Guelph, Guelph, ON, Can-ada
g GIBCOBRL, Burlington, ON, Canada h SIGMA, Oakville, ON, Canada
i AmpliWax娂PCR Gem, PE Applied Biosystems, Missiauga, ON, Canada
j Amersham Pharmacia Biotech, Baie d’Urfe´, PQ, Canada k NuSieve agarose, BioWhittaker Molecular Applications,
Rock-land, ME
l Ingelvac威RespPRRS vaccine, Boehringer Ingelheim Ltd., Bur-lington, ON, Canada
m PRIME PAC威PRRS vaccine, Schering-Plough Animal Health, Omaha, NE
References
1 Chomczynski P, Sacchi N: 1987, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform ex-traction Anal Biochem 162:156–159
2 Mardassi H, Mounir S, Dea S: 1994, Identification of major dif-ferences in the nucleocapsid protein genes of a Que´bec strain and European strains of porcine reproductive and respiratory syn-drome virus J Gen Virol 75:681–685
3 Mengeling WL, Lager KM: 2000, A brief review of procedures and potential problems associated with the diagnosis of porcine reproductive and respiratory syndrome Vet Res 31:61–69 Wesley RD, Mengeling WL, Lager KM, et al.: 1998,
Differen-tiation of a porcine reproductive and respiratory syndrome virus vaccine strain from North American field strains by restriction fragment length polymorphism analysis of ORF J Vet Diagn Invest 10:140–144
5 Wesley RD, Mengeling WL, Lager KM, et al.: 1999, Evidence for divergence of restriction fragment length polymorphism pat-terns following in vivo replication of porcine reproductive and respiratory syndrome virus Am J Vet Res 60:463–467 Wootton S, Yoo D, Rogan D: 2000, Full-length sequence of a
Canadian porcine reproductive and respiratory syndrome virus (PRRSV) isolate Arch Virol 145:2297–2323
J Vet Diagn Invest 14:347–353 (2002)
Detection of Rhodococcus equi by polymerase chain reaction using species-specific nonproprietary primers
Jose´ Miguel Arriaga, Noah D Cohen, James N Derr, M Keith Chaffin, Ronald J Martens
Abstract. Species-specific primers for the polymerase chain reaction (PCR) for the detection of
Rhodo-coccus equi were developed These primers were based on unique DNA fragments produced from R equi
reference strains and field isolates Following random amplification of polymorphic DNA from R equi and
R rhodochrous with a set of 40 arbitrary 10–base pair (bp) primers, a pair of species-specific primers was
designed to detect a unique 700-bp fragment of R equi chromosomal DNA This PCR product was limited to R equi and was not detectable in other Rhodococcus species or in a panel of additional gram-positive and gram-negative bacteria
Rhodococcus equi is an aerobic gram-positive
pleomor-phic bacterium with worldwide distribution.24,33,37 Although
this facultative intracellular pathogen can infect a wide range
of animals, it is primarily a pathogen of foals.24 Nearly all
isolates of R equi from affected foals contain an 85–90-kilobase (kb) plasmid that possesses a gene that encodes a 15–17-kD protein antigen, commonly referred to as the
vir-From the Departments of Large Animal Medicine and Surgery (Arriaga, Cohen, Chaffin, Martens) and Veterinary Pathobiology (Derr), College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4475
Received for publication May 30, 2001
ulence-associated protein antigen (VapA).39 Rhodococcus
equi is being more frequently recognized as a pathogen of
immunocompromised humans, particularly patients with AIDS.13,19,27,28
The primary clinical manifestation of R equi infection in
foals is severe suppurative bronchopneumonia.3,24
Pneumo-nia is an important cause of morbidity and mortality for foals Approximately 9% of all foals in the United States are
affected by pneumonia, and about 12% of these foals die.9
In Texas, respiratory disease is the most common cause of
disease and death in foals.6Although many different
organ-isms have been associated causally with pneumonia in foals,
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