samples were related to two differe

samples were related to two different production systems,
with one production system presenting only one MLVA
type and the other production system presenting 11
different MLVA types found in 15 different farms. The
limited number of samples originated from Mexico could
have influenced the observation of these two clusters. In
Brazil samples were originated from a diagnostic laboratory
that receives clinical specimens from various states
and one frequent type was found in 8 different states.
Furthermore, it was possible to observe heterogeneity of
M. hyopneumoniae within state, in all states analyzed. In
Spain the samples were related to the main pig production
areas, located in Catalonia and Aragon, which can explain
the higher heterogeneity found in that country. Samples
that were obtained from diagnostic laboratories (Brazil,
Mexico, and USA) showed higher heterogeneity in the P97
gene when compared to the isolates originated from Spain.
Spanish samples had limited variation in the P97 locus,
with only five types. The P146 locus had demonstrated
higher variation in both set of samples, strains and clinical
specimens. In total, 17 different types were observed in the
P97 locus, and these types ranged from 2 to 18 repeats. On
the other hand, P146 presented 34 different types ranging
from 7 to 48 repeats. The types that presented more than
30 repeats in P146 were found only in Spain and Brazil.
This observation is in agreement with reports from De
Castro et al. (2006), in which Brazilian samples ranged
from 18 to 44 repeats in the same repeat region.
Nevertheless, no relationship has been demonstrated
between number of repeats and bacterial virulence. It is
important to note that various DNA extraction methods
were used for samples originating in different countries,
and this could have biased the comparison among
countries.
In the results presented by Vranckx et al. (2011) and
Charlebois et al. (2014) the MLVA types for each sample
were not disclosed. Therefore, it was not possible to
compare the number of repeats found in our study with
those previously published. The large number of MLVA
types found in this investigation suggests a large diversity
in M. hyopneumoniae circulating in the swine herds. Also,
evidence of multiple M. hyopneumoniae variants was
observed in single samples (13.8%; 49/355), which was
observed by the amplification of more than one peak in the
capillary electrophoresis analysis. These results suggest
infection with more than one strain in the same pig, as
previously suggested (Vranckx et al., 2011; Charlebois
et al., 2014). The high numbers of M. hyopneumoniae
variants identified in this investigation are in agreement
with other reports which identified clonal variants in the
single herds and in different herds (Nathues et al., 2011;
Vranckx et al., 2012; Charlebois et al., 2014). Clonal
variants present in the same herd showed a low variability
among them, and shared the same number of repeats in
one locus and variable number of repeats in the other
locus. In large production systems, a different pattern of
clonality was observed which is in agreement with the
results of Vranckx et al. (2012).
The M. hyopneumoniae reference strain used to standardize
this MLVA is the parental strain of US232, a
reference strain in the USA (Minion et al., 2004). Both
samples showed the same type (14–21) when evaluated
with this method. This type was only identified in the USA,
which could be explained by the origin of the isolate. There
was not a common MLVA type across all countries.
However, some types appeared to be shared among
countries.
5. Conclusion
The MLVA typing method developed in this study is an
improved assay for differentiation of M. hyopneumoniae
variants in clinical specimens. This assay revealed a high
discriminatory index, suggestive of high diversity of M.
hyopneumoniae in the investigated herds, indicating that
multiple M. hyopneumoniae variants are circulating in
swine herds in the USA, Brazil, Mexico and Spain. Further
analysis of samples collected longitudinally from diverse
geographic locations and clinical presentation is necessary
to investigate if a nonrandom distribution of genotypes is
present among strains.
Competing interest
The authors declare that they have no competing
interest.
Acknowledgments
The authors thank Dr. Anna Perez de Rozas from CReSA
for providing Spanish isolates, Jason Daniels for his help
with sample processing, the UMN-VDL for providing USA
and Mexican samples, and Microvet for its support in
providing samples from Brazil. This project was funded by
the Minnesota Pork Board and the Pork Checkoff, and the
Swine Disease Eradication Center. L.F. Dos Santos was
supported by Capes Foundation, Ministry of Education of
Brazil (proc. No: BEX17617/12-0) and Fundac¸a˜o de Amparo
a` Pesquisa do estado de Minas Gerais FAPEMIG. M.A.S.
Moreira was supported by CNPQ.

ผลลัพธ์ (แอฟริกา) 1:

Twee verskillende monsters is wat verband hou met produksiestelsels,
Production System met een wat net een MLVA
Tipe Ander Produksie Stelsel en die aanbieding van 11
verskillende tipes MLVA gevind in 15 verskillende plase. Die
beperkte aantal monsters afkomstig van Mexiko kan
die opmerking Twee van hierdie groepe beïnvloed. In
Brasilië is monsters afkomstig van 'n Diagnostiese Laboratorium
dat kliniese monsters ontvang van verskillende state
en is een Gereelde Tipe gevind in 8 verskillende state.
Verder was dit moontlik om heterogeniteit van Neem
M. hyopneumoniae binne die staat, in alle lande ontleed. In
Spanje is die monsters wat verband hou met die Hoof Varkproduksie
gebiede, geleë in Katalonië en Aragon, wat kan verklaar
die Hoër heterogeniteit gevind in daardie land. Monsters
wat verkry word uit diagnostiese laboratoriums (Brasilië,
Mexiko, en die VSA) het hoër heterogeniteit in die P97
Gene in vergelyking met die isolate afkomstig van Spanje.
Spaanse Beperk variasie in die monsters gehad P97 Lokus,
met slegs vyf tipes. Lokus het die P146 gedemonstreer
Hoër variasie van in beide monsters, stamme en kliniese
monsters. Altesaam 17 verskillende tipes waargeneem in die
P97 Lokus en hierdie tipe gewissel 2-18 herhaal. Op
die ander kant, P146 Aangebied 34 verskillende tipes wat wissel
7-48 herhaal. Het die tipes wat meer as
30 Herhalings in P146 gevind net in Spanje en Brasilië.
Dit is in ooreenstemming met sigbare tekens verslae van De
Castro et al. (Twee duisend ses), waarin Brasiliaanse monsters gewissel
18-44 in die herhalings Dieselfde Herhaal Streek.
Nietemin, nie het getoon Verhoudings
tussen aantal herhalings en ​​bakteriële virulensie. Dit is
belangrik om daarop te let dat DNA ekstraksie Verskeie metodes
is gebruik om monsters uit verskillende lande,
en dit kan die vergelyking tussen het bevooroordeeld
lande.
In die resultate Aangebied deur Vranckx et al. (2011) en
Charlebois et al. (Die 2014) die tipes MLVA vir elke monster
is nie bekend gemaak. Daarom is dit nie moontlik was om
te vergelyk die aantal herhalings in ons Studie met
dié wat vroeër gepubliseer word. Die groot aantal MLVA
tipes gevind in hierdie ondersoek dui op 'n groot verskeidenheid
in M. hyopneumoniae wat sirkuleer in die varke beeste. Ook,
M. hyopneumoniae Bewyse van verskeie variante is
waargeneem in 'n enkele monsters (13.8%; 49/355), wat
waargeneem deur die versterking van meer as een in die Peak
kapillêre elektroforese Ontleding. Hierdie resultate dui daarop
infeksie met meer as een stam in dieselfde vark, as
voorheen voorgestel (Vranckx et al, 2011th;. Charlebois
. Et al, 2014). Die groot getalle van M. hyopneumoniae
variante wat in hierdie ondersoek is in ooreenkoms
met ander verslae wat die klonale variante in geïdentifiseerde
enkele beeste en beeste in verskillende (Nathues et al, 2011th;.
Vranckx et al, 2012;. Charlebois et al,.. 2014). Klonale
variante teenwoordig in dieselfde kudde het 'n lae veranderlikheid
onder hulle, en die gedeelde Dieselfde aantal herhalings in
een en Lokus Veranderlike aantal herhalings in die ander
Lokus. Produksie in groot stelsels, 'n ander patroon van
Clonality is waargeneem wat in ooreenstemming met die
resultate van Vranckx et al. (Die 2012).
Die M. hyopneumoniae druk gebruik te standaardiseer Verwysing
MLVA dit is die ouers se stam van US232, 'n
Verwysing spanning in die VSA (Minion et al., 2004). Beide
monsters het die dieselfde soort (14-21) toe geëvalueer
met hierdie metode. Tik hierdie is net in die VSA geïdentifiseer,
wat die oorsprong van die isoleer kan wees deur verduidelik. Daar
was nie 'n gemeenskaplike MLVA Tipe in alle lande.
Maar sommige tipes blyk te wees gedeel
lande.
5. Gevolgtrekking
Tik MLVA Die ontwikkel in hierdie studie metode is 'n
verbeterde toets vir differensiasie van M. hyopneumoniae
variante in kliniese monsters. Hierdie toets aan die lig gebring 'n Hoë
diskriminerende indeks, suggestief van groot diversiteit van M.
hyopneumoniae in die ondersoek beeste, wat aandui dat
verskeie variante is wat sirkuleer in M. hyopneumoniae
Swine kuddes in die VSA, Brasilië, Mexiko en Spanje. Verdere
analise van versamelde monsters lengte van uiteenlopende
plekke en kliniese Geographic Aanbieding nodig
om te ondersoek of 'n nonrandom Verspreiding van genotipes is
tans onder stamme.
Mededingende belange
dat hulle geen meeding Verklaar die skrywers
belang.
Erkennings
Die outeurs bedank dr Anna Perez de Dos. uit Cresa
vir die verskaffing van Spaanse isolate, Jason Daniels vir sy hulp
met monster verwerking, UMN-VDL vir die verskaffing van die VSA
en Mexikaanse monsters, en vir Microvet sy steun in
die verskaffing van monsters van Brasilië. Hierdie projek is befonds deur
die Minnesota Vark Raad en die Vark checkoff, en die
Swine siekte uitwissing Center. Dos Santos LF is
deur Capes Stigting, die Ministerie van Onderwys van
Brasilië (processed No:. BEX17617 / 12-0) en Fundac¸a ~ o de Amparo
pesquisa doen Estado de Minas Gerais n `FAPEMIG. MAS
Moreira is deur CNPQ.