International Research Journal of Agricultural Science and Soil Science (ISSN: 2251-0044) Vol. 2(2) pp. 063-067 February 2012
Available online http://www.interesjournals.org/IRJAS
Copyright ©2012 International Research Journals
Full Length Research Paper
Isolation and antigenic characterization of infectious
bronchitis virus from backyard chickens in Yucatan,
Mexico
Ramírez-González S. 1, Gutiérrez-Ruiz E.J. 1*, Aranda-Cirerol F.J. 1, Rodriguez-Vivas R.I.1,
Bolio-Gonzalez M.E.1
1
Universidad Autónoma de Yucatán, Facultad de Medicina Veterinaria y Zootecnia, Depto. de Virología. km 15.5
carretera Mérida-Xmatkuil, Mérida, Yucatán, México.
Accepted 09 January, 2012
Chickens, traditionally raised by yucatecan peasants in the backyard of their houses and feed with
leftovers and table scrapings, provide protein for the most needed people. Respiratory disease has
been reported as one of the most important diseases in these animals. As part of a bigger study
carried out during a year in 30 rural communities of the State of Yucatan, Mexico, with the use of
sentinel chickens for investigating diseases in the backyard system, tracheal and cloacal swab
samples were obtained for the isolation of Infectious bronchitis virus (IBV) in chicken embryos. Four
IBV were isolated from backyard chickens during 2007-2008.The antigenic characterization of the
isolates was carried out using the haemagglutination inhibition test in two directions, and using 5
reference strains for IBV. Two isolates were similar to local variant SIN6/YUC/MEX/1996, one to the
other local variant UADY/YUC/MEX/97, and the last one was related to Massachusetts 41.The fact that
some of the isolations were made from chickens showing respiratory signs strongly suggests that IBV is
at least one of the agents involved in respiratory disease in the chickens kept in the backyard system in
Yucatan, Mexico.
Keywords: Infectious bronchitis virus, antigenic characterization, backyard chickens, haemagglutination
inhibition test.
INTRODUCTION
Keeping animals and edible plants in the backyard of the
houses as a source of food for human beings (known as
the backyard production system), is a traditional activity
Corresponding Author E-mail: gruiz@uady.mx
Tel: +52-999-9423200; fax: +52-999-9423205.
List of Abbreviations
HA.- Haemagglutination, HIT: Haemagglutination inhibition test,
IBV.- Infectious bronchitis virus, NDV.- Newcastle disease virus,
SIN6.- SIN6/YUC/MEX/96 Mexican isolate of IBV, UADY.UADY/YUC/MEX/97 Mexican isolate of IBV, FMVZ-UADY.Facultad de Medicina Veterinaria y Zootecnia, Universidad
Autónoma de Yucatán.
practiced by many ethnic groups in Mexico and many
parts of the world. The main objective of this system,
managed by family members, is to provide food and
sometimes cash to help with the hard economic situation
of peasants (Acosta et al., 1993).
Chickens (Gallus domesticus) represent the most
common species in the backyard system in Yucatan. The
main characteristic of the system is the low cost of
production because the animals are feed with whatever
they can find while roaming free and sometimes,
depending on availability, are supplemented with grains
or local byproducts (Barredo-Pool et al., 1991). Technical
assistance is rare and never constant (Acosta Casanova,
2004), mortality rates for this species is usually high and
the causes of death are only presumed through
information provided by the owners (Honhold et al., 1993;
Gutierrez-Ruiz et al., 2008).
064 Int. Res. J. Agric. Sci. Soil Sci.
Despite the importance of this system, the interest of
researchers is scarce and most work has been done
focusing on economics or general production, with very
few publications, especially on health issues. Previous
studies suggest that respiratory diseases are among the
most important for backyard poultry (Honhold et al., 1993;
Gutierrez-Ruiz et al., 1998; Gutierrez-Ruiz et al., 2008).
The state of Yucatan has been free from velogenic
Newcastle disease and Avian Influenza since 1995
(SENASICA, 2011), but IBV presence has been
demonstrated in commercial and backyard poultry
production (Gutiérrez-Ruiz et al., 1998).
Control of IBV has been carried out through the use
of vaccines but due to the antigenic variation of the virus
this has not always been successful. At present it is
acknowledged that genetic recombinations as well as
mutations occur with IBV, resulting in the emergence of
variant IBV populations (Kusters et al., 1990; Jia et al.,
1995). The haemagglutination inhibition test (HIT) has
been successfully used for antigenic typification of IBV
(Lashgari and Newman, 1984, Gutiérrez-Ruiz et al,
2004). The test is performed using specific antisera and
antigens from each of the reference strains and the
isolates that require characterization (Brown and
Bracewell, 1985).
The objective of the present study was to isolate and
characterize IBV from backyard chickens from the State
of Yucatan, Mexico.
MATERIALS AND METHODS
Samples and communities included
Cloacal and tracheal swabings were collected from
sentinel chickens delivered 12 weeks previously to 120
houses in 30 rural communities chosen randomly from
483 communities, between 100 and 10,000 inhabitants
that exist in Yucatan. On each community 4 houses were
chosen by convenience and 4 sentinel chickens were
delivered to each. The chicks were free of IBV,
Newcastle disease virus and avian influenza
demonstrated by serology and isolation procedures. A
total of 168 cloacal samples and 146 tracheal samples
(pools of the 4 sentinels or resident birds), from the
sentinel chickens (120) and some resident birds were
obtained.
The 30 communities included in the study were:
Ticimul, Uayalceh, Hacienda Oncan, Tepich Carrillo,
Xcucul Sur, Xcunya, Chablecal (municipality of Mérida),
Tamchén (municipality of Uman), Eknakan (municipality
of Acanceh), Cuzamá (municipality of Cuzamá), Telchac
Pueblo (municipality of Telchac pueblo), Sinanché
(municipality of Sinanché), Muna (municipality of Muna),
Kochol (municipality of Maxcanu), Chicán. (municipality
of Tixmeuac), Pencuyut (municipality of Tekax), Kiilinché
(municipality of Tixpeual), Tanya (municipality of Motul),
Xohuayan (municipality of Oxkutzcab), Yotholin
(municipality of Ticul), Cantamayec (municipality of
Cantamayec), Libre Unión (municipality of Libre Unión),
Ticimul and Xanlah (municipality of Chancom),
Pocoboch, Dzonot Ake, San José Montecristo, Santa
Rosa, Chan San Antonio (municipality of Tizimin), and
Panabá (municipality of Panabá).
The samples were placed in glass tubes containing 3
ml PBS with pH 7.2, with 10,000 IU/ml peniciline, 10-mg/ml
estreptomicine and 5000 IU/ml Mycostatine, to inhibit the
growth of fungi and bacteria and preserve the agents of
interest. Samples were identified and placed in a cool box
with ice packs for its transportation to the Virology
laboratory of the Facultad de Medicina Veterinaria y
Zootecnia, Universidad Autónoma de Yucatán for virus
isolation.
Virus Isolation
Five 9-11 day old chicken embryos were inoculated with
0.2 ml of the samples supernatant using the corioallantoic
route according to the procedure described by Gelb Jr. and
Jackwood (1998). Embryos were obtained from a small
flock of leghorn layers kept at the FMVZ-UADY, the birds
were serologically monitored for antibodies to NDV, IBV,
Mycoplasma sinoviae and M. gallisepticum monthly
during the length of the study, and they were always
negative for all antigens.
Inoculated eggs were candled daily with deaths
within the first 24 hours considered non-specific, after
three days, 2 eggs were chilled at +4oC for at least 6 hours,
corio-allantoic fluid was harvested and used for a new
passage. Fluids from each of five passages were tested for
haemagglutination following the method by Alexander et al.
(1983). Samples not showing lesions or mortality and with
no HA activity were discarded after the fifth blind passage.
The corioallantoic fluid from embryos showing lesions
suggestive of IBV like dwarfing, twirling, lack of feathers
development or the presence of urates in the kidney
mesonephros and no HA activity were cloned using the
restrictive dilution method, the corioallantoic fluid of
embryos inoculated with the highest dilution which
produced characteristic lesions was harvested and used for
subsequent tests. The corioallantoic fluids were diluted (102
) in PBS with antibiotics and inoculated into 10 new
embryos for IBV HIT antigen production, according to the
methodology described by Gutiérrez-Ruiz et al. (1998) and
for mono-specific antiserum production, following the
procedure described by Gutiérrez-Ruiz et al. (1998).
Haemagglutination inhibition test for characterization
of IBV isolates
The HIT was run following the procedure described by
Alexander et al. (1983). IBV used as reference strains
Ramírez-González et al. 065
Table 1. Two-way haemagglutination inhibition test results of infectious bronchitis virus isolates from
Yucatan, Mexico and reference strains Massachusets 41 (M41), Connecticut (Conn), Arkansas 99 (Ark),
Holte, CVL/9 and 793B.
Ag
AS
BT
SPF
Xcul
Pen
Mun
Tam
SIN
UADY
M41
ARK
CON
Xcul
Pen
Mun
Tam
SIN
UADY
M41
ARK
CON
3
1*
8
7
3
1
7
3
3
3
2
2
2
8
7
3
2
7
2
3
3
3
3
1
3
2
9
4
1
7
2
3
3
3
1
2
1
2
8
2
2
8
3
2
3
2
7
8
2
3
8
1
2
3
1
3
1
1
3
8
2
3
8
1
3
2
2
1
3
2
3
8
2
2
8
3
3
3
2
2
3
3
2
3
4
3
9
1
3
1
2
4
2
3
4
3
2
2
9
* Titres are expressed in terms of log2
were Massachusetts 41 (M41), Arkansas 99 (Ark),
Connecticut
(Conn),
and
Yucatecan
strains
SIN6/YUC/MEX/96 (SIN6) and UADY/YUC/MEX/97
(UADY) because they have been reported in Mexico
previously. Antisera and antigens from the reference
strains were obtained from the Veterinary Laboratory
Agency, UK and were produced using the same
methodologies.
Antigenic characterization of IBV isolates
HIT were run in both directions using homologous and
heterologous antisera against reference strains and field
isolates.
Antigenic relationships (% similarity in the antigenic
relationship) among reference viruses and field isolates
were calculated using the method by Archetti and Horsfall
(1950), and are presented as r1 and r2 proportions for each
pair of viruses. r1 and r2 values were calculated using the
following formulae:
Aritmetic titre of the serum against heterologous strain
r1 ó r2=--------------------------------------------------------------------Aritmetic titre of the serum against homologous strain
The total antigenic relationships for each pair of
viruses, were calculated as an Average relationship index
(R) using the formulae: R=100 √ (r1 x r2) (Archetti y
Horsfall, 1950; Lashgari y Newman, 1984).
According to Lashgari and Newman (1984), R values
equal or less tan 5 indicate that compared viruses belong
to different serotypes, values above 9 indicate that
viruses are of the same subtype and those with R= 100
represent the same virus type.
RESULTS
No embryo fluids showed haemagglutination of 1%
chicken erythrocytes, therefore the presence of NDV or
Avian influenza virus in the samples obtained was
discarded. Four isolates of IBV were obtained from the
communities of Muna, Xcucul (Mérida), Tamchen (Uman)
and Pencuyut (Tekax).
The isolate from Muna was obtained from young
resident chickens (around 3-4 months of age) which
presented signs of disease like depression, nasal
discharge and tracheal noises, the owners of these birds
reported the death of several animals in the two weeks
prior to our visit. The isolate from Pencuyut was obtained
in the first sampling of the sentinel birds (17 weeks of
age) delivered in the project, the owners reported
respiratory signs two weeks prior to our visit and no
mortality was reported. Isolates from Xcucul and
Tamchem, were obtained from the sentinel birds clinically
healthy and without report of clinical signs from the
owners.
Isolates Xcucul and Pencuyut resulted similar to the
variant virus SIN6/YUC/MEX/1996, the isolate from Muna
is of the same type as the variant virus
UADY/YUC/MEX/97, and the isolate from Tamchen is
related to the Massachusetts 41 virus.
The results of the HIT run in two directions using the
reference strains and the four isolates are presented in
table 1, antigenic relationships of the reference and isoleted
viruses are presented in table 2.
The averages of relationship (R) were calculated for
each pair of viruses. Isolates Xcucul and Pencuyut had
an R of 100% between them, Muna had an R of 50% with
the reference strain UADY, and Tamchen had an R of
066 Int. Res. J. Agric. Sci. Soil Sci.
Table 2. Estimation of antigenic relationships of seven Mexican isolates of infectious bronchitis virus and
reference strains Massachussets 41 (M41), Connecticut (CON), Arkansas 99 (ARK), Holte, CVL/9 and 793B by
analysis of the two-way haemagglutination inhibition test results expressed as ratios (r1 or r2) using the method
by Archetti and Horsfall (1950).
Ag
AS
Xcul
Pen
Mun
Tam
SIN
UADY
M41
ARK
CON
Xcul
Pen
Mun
Tam
SIN
UADY
M41
ARK
CON
1
0.5
0.031
0.008
0.5
0.031
0.031
0.031
0.016
2
1
0.063
0.031
1
0.031
0.063
0.063
0.063
0.015
0.008
1
0.031
0.004
0.25
0.008
0.016
0.016
0.016
0.008
0.016
1
0.016
0.016
1
0.031
0.016
0.5
1
0.016
0.031
1
0. 031
0. 016
0. 016
0. 031
0.008
0.031
1
0.016
0. 031
1
0. 250
0. 016
0. 031
0.031
0.016
0.031
1
0. 125
2
1
0. 031
0. 063
0.008
0.016
0.016
0.008
0. 031
0. 031
0. 016
1
1
0.008
0.031
0.008
0.016
0. 016
0. 016
0. 008
0. 250
1
Table 3. Average relationship index of four Mexican isolates of Infectious
bronchitis virus and refernce strains using the haemagglutination inhibition test
IBV
Xcucul
Pencuyut
Muna
Tamchen
SIN6
UADY
M41
ARK
CON
Xcucul
100
100
2.1
1.1
50
1.6
3.1
1.6
1.1
Pencuyut
100
100
2.2
1.6
100
3.1
4.4
3.2
4.4
100% con M41. Complete results are showed in table 3.
DISCUSSION
Infectious bronchitis virus isolates from Yucatan, Mexico
formed three distinct groups. In a previous study besides
variants SIN6 and UADY other isolates corresponding to
type Connecticut were obtained (Gutiérrez-Ruiz et al.,
2004), different to the present study where a virus of the
M41 type was obtained, which is something expected as
M41 and Connecticut types are the most frequent strains
isolated everywhere they are used in vaccine preparations
(Cavanagh et al., 1992; Gough et al., 1997) and they both
are in vaccine preparations used in commercial poultry in
Yucatan. Commercial birds, like broilers and retired layers,
are from time to time introduced in the backyards of many
communities like the Tamchen where the M41 type virus
was isolated. The low frequency of M41 isolation in this
study can be due to the fact that backyard chickens are not
vaccinated against any disease and probably also that
other types of IBV are prevalent.
Muna
2.1
2.2
100
2.2
0.8
50
1.6
1.6
1.1
Tamchen
1.1
1.6
2.2
100
2.2
1.6
100
1.6
1.6
The other three IBV isolates belong to two different
variant types previously described which are SIN6 and
UADY (Gutierrez-Ruiz, 2004). Due to the fact that no
vaccination is used in backyard chickens it is reasonable to
think that evolutionary pressure is not strong for viruses
circulating in the backyard system. In the past 14 years it
appears that type SIN6 has spread to many communities
most likely through the movement of live poultry which is
usually given away to relative and friends from other
communities or in a smaller scale sold to people from
different towns.
In the case of type UADY, it seems that the virus is not
widespread in the State because it was only isolated from
one community despite the fact that this was originally
isolated from a flock of naked neck chickens kept at the
FMVZ-UADY with the purpose of conservation of the gene,
many birds originated from this flock were sold to people
wanting to raise this kind of bird, whether this type has not
been further isolated because is not very common or by
chance is not known, and it does not indicate its real
importance for the birds health. Further studies are
necessary to determine the pathogenicity and virulence of
Ramírez-González et al. 067
UADY IBV type and if existing vaccines have a
protective effect against this strain; these studies have
been carried out for SIN6 IBV, and have been submitted for
publication.
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