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]
Trang 118 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) 7 sequence The RT-PCR RFLP typing assay was performed using 2 different sets of primers, amplifying 716 or 933 base pairs of ORF 4, 5, and 6 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, 4 had a RFLP type shared by another commercial vaccine and
a field strain In 4 cases, 2 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 1 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 9 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) 7 (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 7 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
7 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
template A PCR System 9600 thermocyclerewas used with
Trang 2Figure 1. Examples of PRRSV RFLP types Panels A, B, and C are the RFLP of the 716-bp RT-PCR products Panel D is the RFLP
of the 933-bp RT-PCR product Panels C and D include the same PRRSV strains in the same order for comparison of the 2 different typing methods Panel D includes a control sample (type 2-5-2) and samples from 3 different pigs submitted from the same farm at the same
time Each group of 4 lanes contains digested products derived from the same clinical sample, with the MluI product in the first lane,
HincII product in the second, SacII product in the third, and undigested product in the fourth Numbers indicate the typing code assigned
to each sample NT: non-typeable * Typed by using 933-bp RT-PCR typing method.
Trang 3Table 1. Different RFLP types of PRRSV recovered from Ontario farms by the AHL from January 1988 to July 2000.*
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 3 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 7 min, holding at 4 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 5 and part of ORF 4 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,
separately at 37 C for 2 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 2 or more restriction enzyme sites different from a vaccine strain (type 2-5-2) and an intermediate strain has only 1 site different within ORF 5
Figure 1 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 1 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 4 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
Using the 716-bp RT-PCR, 34 different PRRSV RFLP
Trang 4Table 2. Twenty-three farms with different RFLP types of
PRRSV.
98/12/23
132 252
99/12/21
172 132
00/02/17
144*
161
98/08/31
242 153
99/09/20
212 112
98/11/19
99/06/30
99/10/07
252 132 142 142
98/10/14
98/11/18
222 122 122
99/06/09
00/04/13
172 134
132 with deletion
98/06/17
98/12/02
111 112 114
98/03/10
00/01/05
00/02/10
252 174 123 134*
99/06/11
99/07/15
NT†
124 NT
98/07/15
99/07/14
99/08/11
252 134 NT 252
98/12/12
99/01/06
99/10/30
122 124 124 122
98/04/06
99/10/19
264 264 252
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
00/04/19
124 123
00/03/07
122 124
00/05/03
252 123
98/04/15
262 252
Table 2. Continued.
99/04/12 99/09/09 00/05/10
124 124*
124*
124*
99/02/05 00/02/04
252 252 251
00/01/25
262 252
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 9 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 2 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
In 4 cases, 2 different RFLP types were identified from tissues from different pigs that were submitted from the same farm at the same time The coexisting types were
2-1-2 and 2-1-2-2-1-2-2-1-2; 1-3-2-1-2 and 1-7-2-1-2; 1-2-1-2-2-1-2* and 1-2-1-2-4*; and 1-1-4 and 1-6-2* with possible deletion (data not shown) Of the
195 farms that submitted PRRSV PCR-positive samples, 48 submitted samples on more than 1 occasion during the spec-ified time frame Different RFLP types of PRRSV were re-covered from 23 farms (Table 2) The time between sub-missions varied from 1 day to 26 months, with 1 farm having
5 different types of PRRSV In the other 25 farms, the PRRSV RFLP types remained unchanged for up to 20 months (data not shown) Further investigation is needed to
Trang 5determine 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 6 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.
4 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 5 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.
6 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,
R equi is considered the most common cause of severe