d
, C.P
Article history:
Received 24 August 2010
Accepted 12 May 2011
Infectious bovine keratoconjunctivitis (IBK) is a highly contagious ocular disease of cattle caused by
the organism from one animal to another. Ultraviolet radiation and
animal stress favor the emergence and spread of this disease. Also,
other factors are implicated such as season, breed, eye pigmenta-
tion and mechanical irritation (dust, grass, weeds, plant awns,
etc.) (Snowder et al., 2005). Pathogens such infectious bovine
oped based on formulations containing piliated strains of Mb or
purified Mb pili. It was reported that pili were organelles special-
ized on adherence of the bacterium to target tissues and the anti-
bodies directed against them prevented attachment to the bovine
corneal epithelium (Annuar and Wilcox, 1985). Pugh and Hughes
(1975) reported that a vaccine prepared with purified Mb pili with-
out adjuvant, inoculated subconjunctivally, induced an acceptable
immune response in calves. Nayar and Saunders (1975) reported
that in the lacrimal secretions of calves with severe IBK there were
elevated levels of IgA against Mb antigens, suggesting that local
(presumably ocular) vaccination might be beneficial.
⇑ Corresponding author. Tel.: +54 3496 420639/421037x128.
E-mail address: virginiazbrun@yahoo.com.ar (M.V. Zbrun).
1 Tel.: +54 3472 425001x149.
Research in Veterinary Science 93 (2012) 183–189
Contents lists availabl
Research in Vete
w.e

2 Tel.: +54 1147 246200/6300x6400.

1. Introduction
Infectious bovine keratoconjunctivitis is a highly contagious
ocular disease of cattle caused by Moraxella bovis. Even though it
does not produce mortality, the economic impact on the livestock
farmer by reduced weight gain and eye disfigurement is substan-
tial. The morbidity rate can approach 80%, reaching its maximum
in the third or fourth week after of the onset of clinical cases (Blood
et al., 2002).
Several risk factors may predispose the bovine eye to MB colo-
nization/ocular damage. The face fly (Musca autumnalis) acts as
mechanical vector of the agent (Kopecky et al., 1986), transferring
rhinotracheitits virus, Mycoplasma spp. and Moraxella bovoculi are
associated with IBK lesions (Angelos et al., 2007a).
Variable responses to experimental vaccines have been re-
ported. Hughes and Pugh (1971) indicated that repeated intramus-
cular (IM) injection of viable Mb organisms reduced to some
degree the incidence of ocular infections, but did not reduce the
severity of the disease in cattle that develop keratitis. Following
this early research, many other studies reported the use of differ-
ent immunogens designed to prevent eye infection by Mb and
the development of conjunctival and corneal lesions associated
with IBK (Lepper and Moore, 1992; Funk et al., 2009; Angelos
et al., 2004, 2007b). New kinds of vaccines against IBK were devel-

Keywords:
Immunoglobulin A
Bovine tears
Experimental vaccines
Moraxella bovis

0034-5288/$ - see front matter 2011 Elsevier Ltd. Al
doi:10.1016/j.rvsc.2011.05.008

Moraxella bovis (Mb). Parenterally administered immunogens used to prevent the disease do not offer
complete protection possibly because they stimulate a poor ocular mucosal secretory response, in which
locally secreted immunoglobulin-A (sIgA) is one of the main components. The principal aim of this study
was to evaluate by an indirect enzyme linked immunosorbent assay (ELISA), the local ocular mucosal sIgA
response against Mb purified pili, produced after intranasal inoculation of experimental vaccines. Pili
were adjuvanted by several different adjuvants (QuilA, Marcol Arlacel, Marcol Span, microencapsulated
pili with PLGA polymers). Results were compared to sIgA response produced by adjuvant placebo inoc-
ulations and by IBK natural infection. Significantly higher anti-pili IgA response (p < 0.05) was detected
in calves vaccinated intranasally with pili QuilA and pili Marcol Span compared to control calves,
although this specific immune response did not seem to be related to protection against Mb infection
or typical IBK lesion development.
2011 Elsevier Ltd. All rights reserved.

a r t i c l e i n f o a b s t r a c t

Evaluation of anti-Moraxella bovis pili immunoglobulin-
intranasal vaccination of cattle
M.V. Zbrun a,⇑, G.C. Zielinski b,1, H.C. Piscitelli b,1, C. Descarg
L. Hermida c,2
a Departamento de Salud Pública Veterinaria, Facultad de Ciencias Veterinarias, Universida
Provincia de Santa Fe, Argentina
b Instituto Nacional de Tecnología Agropecuaria EEA M, Juárez, Ruta 12 Km 3, M. Juárez
c Centro de Investigación y Desarrollo en Química, Instituto Nacional de Tecnología Industria
journal homepage: ww

l rights reserved.

A in tears following
a b,1, L.A. Urbani b,1, M.V. Defain Tesoriero c,2,
Nacional del Litoral, Kreder 2805, Esperanza, C.P. S3080HOF,
. 2580, Provincia de Córdoba, Argentina
l (INTI), Parque Tecnológico Migueletes, Buenos Aires, Argentina
e at ScienceDirect
rinary Science
l sevier .com/locate / rvsc

that the administration of antigens by an intrapalpebral route of
Pili were purified following a previously published procedure
against 0.15 M NaCl containing 0.05 M Tris–HCl, pH 8.0 at 8 C

for 24 h. The pilus aggregates that formed were separated from sol-
uble contaminants by centrifugation at 12,000g for 60 min and the
pellet dissolved in a small amount of PBS buffer pH 7.4.
Protein concentration was measured through the method re-
ported by Lowry et al. (1951). To evaluate the purity of purified pili,
a SDS–PAGE was performed using 15% of polyacrylamide as de-
scribed by Laemmli (1970).
2.1.3. Vaccine formulation
2.1.3.1. Chemicals. Saponin from Quillaga bark (QuilA), Mineral oil
(Marcol), Sorbitan Sesquioleate (Arlacel), Sorbitan monooleate
(Span 80) were purchased from Sigma–Aldrich Chemical Co (St.
Louis, MO, USA). Polyoxyethylene (20) sorbitan monooleate
(Tween 80) Fluka was obtained from Sigma–Aldrich Chemical
Co (St. Louis, MO, USA. The Poly(D,L-Lactide-co-Glycolide) 50:50,
Resomer RG 503H, was purchased from Boehringer Ingelheim
(Ingelheim, Germany).

(Ruehl et al., 1988). Briefly, M. bovis was inoculated onto 5% equine
blood agar plates, being incubated aerobically at 37 C for 18 h.
Once the lawn was developed, organisms were harvested and sus-
pended in 0.15 M ethanolamine buffer (Eth), being homogenized at
30,000 rpm for 10 min in an ice bath. After two washings at 10,000
and 12,000g at 4 C for 30 and 60 min, respectively, the pellet was
discarded and pili in supernatant were slowly precipitated with
ammonium sulphate at 10% saturation. The pili were collected by
centrifugation at 12,000g for 60 min, the pellet was dissolved in
the Eth buffer and insoluble contaminants were removed by cen-
trifugation at 12,000g for 60 min. The supernatant was dialyzed

inoculation could stimulate the CALT leading to the production of
specific local ocular IgA (Knop and Knop, 2007).
The objective of this study was to evaluate the ability of differ-
ent pilin-adjuvant combinations to stimulate anti-pilin sIgA in bo-
vine lacrimal fluid from beef calves exposed to natural infection
with Mb and differentiate this immune response from antibodies
produced by IBK natural infection.
2. Material and methods
2.1. Vaccines elaboration
2.1.1. Strain
M. bovis strain 1194-04, pili serotype G (Zielinski et al., 1998)
was used. It belongs to the Strain Bank of the Laboratory of Bacte-
riology (EEA-INTA Ms. Juárez). Pili expression was identified by the
formation of characteristic agar corroding colonies (‘‘pitting fac-
tor’’) on blood agar (Ruehl et al., 1988).
2.1.2. Purification of M. bovis pili

Studies in mice inoculated intranasally with antigens showed
increased IgA, produced by B-cells from NALT (Nasal Associated
Lymphoid Tissue) (Zuercher, 2003). The NALT located below the
nasal pharyngeal epithelium, contains aggregates of lymphoid fol-
licles (B cell areas), interfollicular areas (T cells), macrophages and
dendritic cells. This system would be in close proximity to CALT
(Lymphoid Tissue Associated with the Conjunctiva) and LDALT
(Lacrimal Drainage Associated Lymphoid Tissue), so that stimula-
tion of NALT or CALT could elicit a response, resulting in an in-
creased sIgA in tears (Knop and Knop, 2000). It was hypothesized

184 M.V. Zbrun et al. / Research in Veterinar

2.1.3.2. Adjuvanting of . Four different adjuvants were utilized:
three surfactants blends and microspheres.

2.1.3.2.1. Surfactants blends. Adyuvant ‘‘QuilA’’: a 10 mg/ml
aqueous stock solution of QuilA was mixed with PBS at 7% (v/v);
Adyuvant ‘‘Marcol Arlacel’’ consisted in a mixture of Marcol 85%,
Arlacel 80 13% and Tween 80 2% (% v/v); Adyuvant ‘‘Marcol Span’’
consisted in Marcol 94 5%, Span 80 3.5% and Tween 80 2% (% v/v).
Vaccines were prepared emulsifying the aqueous suspension of
pili (200 lg/ml) on the adjuvants at 40:60 proportions v/v at room
temperature. Placebos consisted in an emulsion of PBS buffer
(without antigens) on the three different adjuvants at the same
proportions as used for vaccines.
2.1.3.2.2. Microspheres. The microspheres containing purified
pili were prepared as follows. PLGA (polylactic-coglicolyc acid)
microspheres were obtained following the procedure of double
emulsion and solvent evaporation method as described by Igartua
et al. (1998). Briefly, purified pili purification was used as the inter-
nal phase (W1). The organic phase (O), consisting in 25 ml of a
solution of PLGA 50:50 in dicloromethane (O) was emulsified with
5 ml of W1 using a high shear homogeinizer (Heidolph 900X),
with or without 0.5% PVA (polyvinilic alcohol) for the preparation
of Pili 17-PLGA or Pili 18-PLGA microspheres, respectively. The
resulting emulsion (W1/O) was poured into 300 ml of the external
phase (W2), consisting on 0.5% PVA being emulsified for 5 min at
22,000 rpm. Evaporation of solvent was carried out by vigorous
agitation of emulsion W1/O/W2 at room temperature. The micro-
spheres obtained were collected by centrifugation (10.000g;
15 C; 15 min) washed, lyophilized and stored at 4 C.
The protein content of microspheres was determined by a pre-
viously reported method. It involved alkaline hydrolysis of the
microspheres and determination of the protein recovered using
the Micro BCA Protein assay kit (Pierce, Protein Research Prod-
ucts) (Blanco and Alonso, 1998). Briefly, 200 mg of lyophilized
microspheres were shaken with 2 ml of a 5% (w/v) sodium dodecyl
sulfate (SDS) in 0.1 M NaOH solution (SDS/NaOH) for 24 h at room
temperature. Following centrifugation (10.000g; 15 C, 15 min),
the protein content was determined in the supernatant. Results
are presented as ‘encapsulation percentage’ values (lg protein/
100 mg microspheres).
2.2. Experimental design
2.2.1. Animals
Recently weaned 6–8 month old Angus calves were used in this
study. The animals belonged to the bovine herd of the Experiment
Station of Marcos Juárez, National Institute of Agriculture Technol-
ogy (Marcos Juárez, Argentina). They were maintained on pasture
with concentrated feed, and managed in a single group. This herd
was never vaccinated against IBK but usually presents high preva-
lence of clinical cases of IBK annually. Calves received no other vac-
cination than mandatory foot and mouth disease inactivated viral
vaccine. Animals were managed according to usual outdoors, open
field livestock management practices, not being treated under
painful or cruel procedures.
Prior to vaccination, the animals were bled and an eye swab was
collected using a sterile cotton swab rolled over the dorsal and
ventral surfaces of the conjunctiva for bacteriological Mb studies.
Swabs were inoculated onto 5% equine blood agar and incubated
aerobically at 37 C. Suspected Mb colonies were isolated and inoc-
ulated onto a new blood agar plate for purification. On suspected
cultures, organisms were studied for Gram reaction, oxidase, cata-
lase, Kliegler iron agar, sugar attack and gelatinase (Pugh and
Hughes, 1975). Only culture negative animal with no clinical signs
of IBK were included in the experiment.
y Science 93 (2012) 183–189

2.2.2. Experimental treatments
The calves were randomly divided into nine experimental
groups of five animals per treatment, according to kind of experi-

imental vaccines, treatments values of OD were higher (p < 0.05)

mental vaccine or placebo received, being individually ear tagged
(Table 1). During each sampling animals were eye cultured for iso-
lation of Mb and observed for lesions compatible with IBK. Animals
culture positive for Mb that concomitantly showed typical IBK le-
sions (epiphora, keratitis, corneal ulcerations, keratoconus) were
classified as IBK+.
2.3. Doses and routes of inoculation
Calves were immobilized in a squeeze chute for vaccine inocu-
lation. A device was designed for inoculation of experimental vac-
cines as spray into the nostrils. It consisted in a canula used for oral
administration of parasiticides connected to an automatic syringe
in one side and to the top of a device developed by B&D for
administration of intranasal vaccine in humans that shooted
spray-form injections, on the other side. For microencapsulated
pili, a total of 6 ml of the experimental vaccine were inoculated
(3 ml/nostrail) with a total of 210 lg of antigen/nostrail. Respect
to the other experimental vaccines, each animal received intrana-
sally 6 ml of inoculums (3 ml/nostrail) with a total of 500 lg of
antigen/animal.
Calves were inoculated by the corresponding vaccine or placebo
at day 0 and day 15. After the booster vaccination animals were
observed and sampled every 30 days during 5 months. (Fig. 1).
2.4. Sample collection
At each sampling, samples of tears (1–2 ml) were taken by man-
ual stimulation of conjunctiva and collected with a plastic dropper.
These samples were frozen at 20 C until used in the ELISA assay.
Calves were also observed for ocular lesions and conjunctivally
swabbed for bacteriological studies. Individual animals showing
IBK lesions were recorded as well as animals culture positive to
Mb in each sampling.
Table 1
Experimental groups and route of administration.
Experimental group Antigen Adjuvant Route of administration
1 Pilin PLGA 17 Intranasal
2 Pilin PLGA 18 Intranasal
3 Pilin QuilA Intranasal
4 Pilin Marcol Arlacel Intranasal
5 Pilin Marcol Span Intranasal
6 Placebo QuilA Intranasal
7 Placebo Marcol Arlacel Intranasal
8 Placebo Marcol Span Intranasal
9 Control – –
M.V. Zbrun et al. / Research in Veterinar

2.5. Serological evaluation
The concentration of anti-Mb pili secretory IgA in tears was
evaluated by an indirect-ELISA test based on the methodology de-
scribed by Dominguez et al. (2002) and Moore and Rutter (1987)
with modifications, using purified pili as antigen adsorbed onto a
96-well plastic plate (Costar, Corning). Tears of an animal with
high titers of IgA anti-M. bovis selected in previous studies and Fe-
tal Calf Serum (Sigma F2442) were used as positive and negative
controls respectively. Briefly, 96-well plates (Corning) were coated
with 1 lg/well Mb purified pili in coating buffer (0.5 M carbonate
bicarbonate buffer, pH 9.6). After overnight incubation at 37 C
the plates were incubated with blocking buffer (PBS-Tween Oval-
bumin 1%). After 1 h at 37 C the plates were washed with PBS-T.
Calves tears diluted 1:25 in blocking buffer were added to individ-
ual wells in duplicate. After 2 h at 37 C the plates were washed

than their respective placebos from day 45 to day 135 post immu-
nization. The antibody titers generated by inoculation of pili-QuilA
were higher than pili-M.Span.
In general, no significant differences were observed between OD
values of pili-M.Span and pili-PLGA microencapsulated treatments
with their placebos (p > 0.05). Only pili-17 PLGA and pili-18 PLGA
showed significantly higher antibody titers than (p < 0.05) with
their control groups in day 15 and day 45 post immunization
(Fig. 2D).
3.2. Relationship between isolations, lesions and IBK+ with groups that
presented IgA Mb specific conversion
Culture positive, lesioned and IBK+ calves were detected along
the trial, although no differences in proportions were found be-
tween treatments (p > 0.05).
Fig. 3 shows proportions of animals with Mb isolation, detected
lesions, IBK+ and Mb pili IgA+. In Fig. 3A and B (groups inoculated
with pili-QuilA and pili-M.Span) respectively, it can be observed
that proportion of calves with IgA positive conversion were higher
than the groups corresponding to placebos (p < 0.05) (Fig. 3C and
D). However, percentages of animals with Mb isolation, lesions or
IBK+ were not different (p > 0.05).
In Fig. 3A and B, it can be observed that anti Mb pili antibodies
increased from day 15 to the end of the trial, while for placebo

three times and 100 ll/well of sheep anti-bovine IgA HRP conju-
gated (Bethyl Laboratories), diluted in PBS-T OVA 1% (1/1500)
was added. Following 3 h incubation at 37 C plates were washed
with PBS-T. Then 100 ll/well of chromogen (o-Phenylenediamine
dihydrochloride, Sigma P6662) was added according to the manu-
facturer’s instructions. The reaction was stopped with 50 ll/well of
5 NH2SO4 after 30 min and read at 490 nm in a Labsystems Multis-
kan ELISA reader.
2.6. Statistical analysis
Results from each observation concerning optical density (OD)
of IgA antibodies in tears were analyzed by Analysis of Variance
(ANOVA) using SPSS software. The reaction cutoff point was deter-
mined by ELISA on paired samples by the T0–Tx (T0 = day 0,
Tx = day 15, 45 or corresponding with the subsequent samplings).
Tear samples were considered positive (antibodies conversion)
when Tx showed double value of OD when compared to T0
(Dominguez et al., 2002; Zamorano et al., 2002; Villarreal-Ramos
et al., 1998). Also, antibody levels produced after natural infection
were evaluated by the Kruskal–Wallis and Mann Whitney test. The
samples that were positive for Mb IgA specific antibodies were
analyzed in relation to subsequent Mb isolation or lesions by the
same tests. Differences were considered significant when p values
were lower than 0.05.
3. Results
3.1. ELISA results of anti-pili Mb
Significant differences in OD values between groups inoculated
with different vaccines (p < 0.05) and between samplings (p < 0.05)
were detected. Mean differences between inoculated groups and
their respective placebos are presented in Fig. 2. The ELISA test
used showed that immunization with pili-QuilA and pili-M.Span
was able to induce IgA specific antibodies in tears. For both exper-
y Science 93 (2012) 183–189 185

groups a small proportion of animals showed the same antibody
response at the end of the samplings (Fig. 3C and D).

186 M.V. Zbrun et al. / Research in Veterinar

4. Discussion
The main purpose of this work was to study the capability of
several formulations of experimental vaccines containing purified
Mb pili with four different adjuvants, inoculated intranasally, to in-
duce a local mucosal immune response in calves measured as spe-
cific sIgA in tears that could suggest a protective response. Specific
anti Mb antibodies in lacrimal secretions were predominantly of
Fig. 1. Experimenta
Fig. 2. Titers curves of average OD obtained for each experimental vaccine group. Each grou
significant differences.

y Science 93 (2012) 183–189

the IgA class, indicative of a local production. It is known the pro-
tective function of this antibody isotype in terms of toxin neutral-
ization and attachment inhibition of antigens (Bishop et al., 1982;
Pedersen, 1973; Mach and Pahud, 1971).
The method used to assess specific Mb pili antibody was an
indirect ELISA, which was adapted to the conditions required by
this experimental work. In previous trials conducted by Domin-
guez et al. (2002), the IgG response of commercial bacterins against
l design.
p inoculated with experimental vaccines was compared with its placebo. ⁄Indicate

+

M. bovis was measured, utilizing as antigen inactivated soma of Mb
adsorbed onto ELISA plates. In contrast, for this work purified Mb
pili was adsorbed onto ELISA plates and experimental vaccines
Fig. 3. Percentage of animals that showed positive isolations for Mb, ocular lesions, IBK
line indicates the trend of specific antibodies.
M.V. Zbrun et al. / Research in Veterinar

were used to stimulate a specific immune response. In previous
studies, it was observed that ELISA was a suitable test to investi-
gate antibody response directed to Mb antigens in cattle sera and
lacrimal secretions as well (Angelos et al., 2004; Bishop et al.,
1982).
This trial allowed us to demonstrate that intranasally inocu-
lated Mb pili was able to stimulate a specific immune response
measured as locally secreted IgA. Since specific IgA antibodies were
almost not detected either in placebo inoculated animals nor in
uninoculated controls, it can be assumed that titers found in pili
inoculated treatments were produced by experimental inocula.
The experimental groups inoculated with PLGA microencapsu-
lated pili (Pili17 and Pili18) did not generate high titers of IgA
(Fig. 2D), contradicting results obtained with the same type of anti-
gen presenting system (PLGA microcapsules), although prepared
with different type of antigens (Wyatt and Frederick, 2006; Sinha
and Aman, 2003; O’Brien et al., 1996). These unexpected results
could be attributed to several issues. One could be that the inocu-
lation was not correctly sprayed; therefore, the microspheres did
not reach the nasal mucosa. Additionally, the microspheres size
could not have been sufficiently small, so that their uptake by anti-
gen presenting cells could have been affected. The immune re-
sponse is dependent on the size of the particles presented to
antigen presenting cells, the smaller the particle, the higher the im-
mune response (Vila et al., 2005). Several authors (O’Brien et al.,
1996; Eldridge et al., 1991) demonstrated that successful mucosal
immunization required microspheres smaller than 10 lm that
could be ingested by antigen-presenting cells (Meclean et al.,
1998). In our case, although there were microspheres smaller than
10 lm in the preparations measured by scanning electron micros-
copy (SEM) (data not shown) most of the particles were of a larger
size and presented aggregates that could be seen in the SEM
preparations.

In contrast to the results obtained with mucoadherent poly-
mers, QuilA used as adjuvant produced a very different immune re-
sponse in animals inoculated with antigens when compared to
animals inoculated with placebo (Fig. 2A). This finding is interest-
and seroconvertion of specific IgA (IgA+) in Pili-QuilA and Pili-M.Span groups. Black
y Science 93 (2012) 183–189 187

ing because experimental vaccines containing pili-QuilA produced
high titers of specific sIgA anti Mb pili during four out of five sam-
plings (120 days). Previous studies evaluated the effects of deriva-
tives of Quillaja saponaria showing major increments of
immunoproliferative cells (Chavali et al., 1987; Plohmann et al.,
1997; Lacaille-Dubois et al., 1999). Animal trials also demonstrated
that the use of QuilA as an adjuvant in veterinary formulations
generated an excellent response from the immune system (Dalsg-
aard, 1987).
A specific immune response was also observed in the groups
that received pilin in Marcol Span as adjuvant (Fig. 2B). While
the conversion of specific IgA was observed on the fourth sampling
(day 75), the magnitude of the increase was smaller than the one
observed in the QuilA adjuvanted group. In contrast, in the group
of animals inoculated with pilin in Marcol Arlacel, differences re-
spect to control and placebo groups were observed, but the gener-
ation of specific antibodies titers was not high nor it was constant
in time. The immunostimulatory properties of this adjuvant was
reported for antigens inoculated subcutaneously (Garaicoechea et
al., 2008; Snodgrass et al., 1982). In our experiment probably the
route of administration was not adequate for this adjuvant, not
allowing the contact between the antigen with the nasal mucosa,
thus avoiding the stimulation of local immune response.
The Mb isolations and presence of lesions were variable and
most of the times were not concomitantly found in the same ani-
mal. It is known that Mb can be isolated from healthy eyes and also
that once IBK lesion are developed, chances to culture the organ-
ism diminish dramatically. In consequence, finding IBK+ animals
(lesioned plus culture positive) is a rather rare event. It can be ob-
served that, as long as sIgA titers increase along the trial for pili
QuilA and pili M.Span vaccinated groups, the number of Mb iso-
lates decrease (Fig. 3A and B). This phenomenon might be related

gested that subcutaneous vaccination using conventional

adjuvants could be a reasonable choice for IBK vaccine develop-
ment, but could not disregard the possibility that local response
plays an important role in IBK protection. Furthermore, they sug-
gested that the benefit of augmented local ocular IgA mediated
protection required further study, since it could be the way to re-
duce local immune mediated ocular injury following attraction of
neutrophils to the site of infection, that occurs during IBK lesion
development.
In our study, statistical analysis found no relationship between
Mb isolates/lesions and antibody conversion, indicating that in-
crease in IgA titers were probably due to experimental vaccines.
If so, this new ability to elicit an augmented local immune re-
sponse, sIgA mediated, should be tested in further works using
experimental and natural infection models with known Mb strains
as challenge.
5. Conclusions
The method used in this study (ELISA) to measure immunoglob-
ulin-A in tears in calves demonstrated the ability to detect the im-
mune stimulation after the inoculation of experimental vaccines
intranasally.
Considering these results, immunization of cattle with pili-Qui-
lA and pili-M.Span have a good potential for increasing specific IgA
in tears and could be useful as a preventive measure against the
disease. Therefore, it would be necessary to conduct further exper-
iments to evaluate potential protective capacity against M. bovis
infection.
Acknowledgments
This work was supported by a grant from The National Agency
of Science and Technology, FONCyT, PICT 08 05237. We also would
like to thank Lic. Beatriz Masiero and Dr. Marcelo Signorini for their
generous collaboration with the design and statistical analysis of
this work.
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