Distribution of acute phase proteins in the bovine forestomachs and abomasum.
ABSTRACT Acute phase proteins (APPs) are produced mainly by the liver and their concentration is increased during the systemic inflammatory response. Expression of haptoglobin (Hp), serum amyloid A (SAA), lipopolysaccharide-binding protein (LBP) and α-1 acid glycoprotein (AGP) was determined in the mucosa of the normal bovine forestomachs and abomasum by qualitative and quantitative reverse transcriptase-PCR for mRNA and by Western blot analysis and immunohistochemistry for proteins. Although expression of SAA mRNA was evident in the forestomachs and abomasum, SAA protein was identified only in the abomasum. Expression of Hp protein was high in the forestomachs and abomasum, even though expression of Hp mRNA was negligible. The main site of expression of LBP mRNA was the omasum, whereas the highest protein expression was evident in the abomasum. AGP was expressed at low levels in the bovine forestomachs. Western blot analysis revealed a heterogeneous electrophoretic pattern for AGP, LBP and Hp, indicating that different stomach compartments produce isoforms that are different to those expressed by the liver. Expression of APPs by the bovine forestomachs and abomasum may contribute to regulation of the innate immune response against pathogens.
- [show abstract] [hide abstract]
ABSTRACT: The objectives were to establish the origin of 2 acute phase proteins in milk during subclinical bovine mastitis and to characterize the relationship between those proteins in milk and blood. Haptoglobin (Hp) and mammary-associated serum amyloid A (M-SAA3) appear in milk during mastitis, whereas Hp and serum amyloid A increase in serum during mastitis. The concentrations of these proteins were determined in an experimental model using a field strain of Staphylococcus aureus to induce subclinical mastitis in dairy cows. The expression of mRNA coding for these proteins was assessed and the presence of M-SAA3 in mammary tissues was determined using immunocytochemistry. Increases of M-SAA3 and Hp in milk occurred within 12 h of Staphylococcus aureus infusion, with peak concentrations occurring 3 d after infusion of the bacteria. The increase of acute phase proteins in milk (15 h) preceded the increase in serum concentrations of both proteins (24 h). Expression of mRNA for M-SAA3 and Hp increased in both mammary and hepatic tissues 48 h after infusion of the mammary glands. In mammary tissue, the increase of M-SAA3 mRNA was greater than the increase in Hp mRNA expression, whereas in hepatic tissue, the increase in M-SAA3 mRNA was less than that for Hp mRNA. Immunocytochemistry demonstrated that M-SAA3 protein was present within secretory epithelial cells at significantly higher levels in infected mammary glands than in control tissues. These proteins, which have host defense and antibacterial activities, may play a significant role in the early response to invasion of mammary tissues by pathogenic bacteria.Journal of Dairy Science 06/2006; 89(5):1488-501. · 2.57 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The acute phase protein alpha(1)-acid glycoprotein (AGP--Orosomucoid) is a lipocalin with immunomodulatory functions. The present study provides evidence that the plasma glycoforms of AGP inhibit the migration of bovine monocytes in response to classical chemoattractants. The inhibition is specific, since neutrophils are apparently not affected. To investigate the molecular basis of this finding, the expression of the molecules mostly involved in chemotaxis, including CD18, CD11b and CD47 was studied. It was found that the incubation of activated monocytes with acute phase concentration of AGP (0.9 mg/mL) induces a down-regulation of CD18, and has no apparent influence on CD11b and CD47. RT-PCR expression studies on CD18, CD11b and CD47 mRNA revealed that AGP treatment does not modify the expression rate of these genes. Since AGP treatment is related to a down-regulation of CD18 on the surface of the monocytes, the authors suggest that one of its possible functions consists in specifically reducing the firm adhesion phase of bovine monocytes to the endothelium.Veterinary Research 07/2008; 39(5):50. · 3.43 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The major gastrointestinal nematode parasites of ruminants all belong to the Order Strongylida and the family Trichostrongyloidea. Despite this close evolutionary relationship, distinct differences exist in the microenvironmental niches occupied by the developmental stages of the various parasites, which may account for the variable susceptibility of the different parasite species to the immune effector mechanisms generated by the host. In addition, different manifestations of resistance have been observed against the adult and larval stages of the same parasite species, and even against the same parasite stage. In particular, both rapid and delayed rejection of infective larval stages of gastrointestinal nematode parasites has been documented. This review will give an overview of the various manifestations of resistance to gastrointestinal nematode parasites of ruminants, as well as the immune mechanisms and antigens associated with the generation of immunity by the ruminant hosts to these parasites. In addition, a working model is provided aimed at reconciling most of the present knowledge on the different immune responses generated during infection with the various parasite rejection profiles. Extrapolation of these results to field conditions will need to take into account the variability imposed by seasonal changes and management practices, as well as the individual variability in immune responsiveness present in outbred animal populations.Advances in Parasitology 02/2000; 45:181-241. · 3.78 Impact Factor
Distribution of acute phase proteins in the bovine forestomachs and abomasum
Francesca Dildaa, Laura Francesca Pisania, Mizanur Md Rahmana,1, Silvia Modinab,c, Irene Tessarob,
Paola Sartorellia,c, Fabrizio Ceciliania,c, Cristina Lecchia,⇑
aUniversità degli Studi di Milano, Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, via Celoria 10, 20133 Milano, Italy
bUniversità degli Studi di Milano, Dipartimento di Scienze Animali, via Celoria 10, 20133 Milano, Italy
cCISMA, Centro Interdipartimentale di Studi sulla Ghiandola Mammaria, Università degli Studi di Milano, via Celoria 10, 20133 Milano, Italy
a r t i c l ei n f o
Accepted 7 May 2011
Acute phase proteins
Serum amyloid A
a-1 Acid glycoprotein
a b s t r a c t
Acute phase proteins (APPs) are produced mainly by the liver and their concentration is increased during
the systemic inflammatory response. Expression of haptoglobin (Hp), serum amyloid A (SAA), lipopoly-
saccharide-binding protein (LBP) and a-1 acid glycoprotein (AGP) was determined in the mucosa of
the normal bovine forestomachs and abomasum by qualitative and quantitative reverse transcriptase-
PCR for mRNA and by Western blot analysis and immunohistochemistry for proteins. Although expres-
sion of SAA mRNA was evident in the forestomachs and abomasum, SAA protein was identified only in
the abomasum. Expression of Hp protein was high in the forestomachs and abomasum, even though
expression of Hp mRNA was negligible. The main site of expression of LBP mRNA was the omasum,
whereas the highest protein expression was evident in the abomasum. AGP was expressed at low levels
in the bovine forestomachs. Western blot analysis revealed a heterogeneous electrophoretic pattern for
AGP, LBP and Hp, indicating that different stomach compartments produce isoforms that are different
to those expressed by the liver. Expression of APPs by the bovine forestomachs and abomasum may con-
tribute to regulation of the innate immune response against pathogens.
? 2011 Elsevier Ltd. All rights reserved.
The gastrointestinal tract fulfils divergent roles of nutrient
absorption and host defence. The mucosal surfaces of the fore-
stomachs and intestines of ruminants are in continuous contact
with a vast, diverse and dynamic microbial community. Since mi-
crobes can cross the epithelial surface through the intestinal bar-
rier, protective immune responses must be raised against
pathogens, whereas there is a need to tolerate innocuous antigens
from commensal bacteria. Intestinal defences include epithelial-
derived antimicrobial peptides, cytokines and acute phase proteins
(APPs) (Dommett et al., 2005).
APPs belong to a large family of structurally unrelated proteins
that are produced as part of the systemic inflammatory response
and play a role in modulating innate immunity and scavenging
inflammatory by-products (Gabay and Kushner, 1999). They are
produced mainly by the liver after stimulation by pro-inflamma-
tory cytokines (Baumann and Gauldie, 1994), but extrahepatic
expression also has been reported (McDonald et al., 2001;
Upragarin et al., 2005; Lecchi et al., 2009; Rahman et al., 2010).
There is little information on the involvement of the ruminant
gastric mucosa in innate immunity. The mucosa of the three fore-
stomachs (rumen, reticulum and omasum) is lined by stratified,
squamous, keratinised epithelium and the mucosa of the aboma-
sum is lined by simple, glandular, columnar epithelium (Scala
et al., 2011). The rumen contains >1010commensal microbiota/g
contents and the mucosal surface represents an important line of
defence against the penetration of microorganisms (Krause and
There is no organised lymphoid tissue in the mucosa of the fore-
stomachs and abomasum. Scattered lymphocytes and Langerhans’
cells are present in the forestomachs (Josefsen and Landsverk,
1996) and there are scattered mast cells and lymphocytes in the
abomasum (Balic et al., 2000).
A (SAA), lipopolysaccharide-binding protein (LBP) and a-1 acid gly-
coprotein (AGP). Previous studies have identified expression of LBP
(Rahman et al., 2010) and AGP (Lecchi et al., 2009) in the stomachs
and intestinal tract of ruminants. In the present study, we per-
formed a more detailed examination of expression of Hp, SAA, LBP
and AGP in the normal bovine forestomachs and abomasum.
1090-0233/$ - see front matter ? 2011 Elsevier Ltd. All rights reserved.
⇑Corresponding author. Tel.: +39 250318100.
E-mail address: email@example.com (C. Lecchi).
1Present address: Chittagong Veterinary and Animal Sciences University, Depart-
ment of Medicine and Surgery, Zakir Hossain Road, Chittangong 4202, Bangladesh.
The Veterinary Journal 192 (2012) 101–105
Contents lists available at ScienceDirect
The Veterinary Journal
journal homepage: www.elsevier.com/locate/tvjl
Materials and methods
Tissue collection and preservation
Bovine gastric tissues were collected from four clinically healthy animals at a
local abattoir. The tunica mucosa was mechanically separated from the submucosa
with a scalpel and portions of tissue were preserved in RNAlater (Sigma–Aldrich)
and stored at ?80 ?C. Samples for Western blot analysis were collected immediately
into liquid nitrogen and stored at ?80 ?C. Samples for immunohistochemistry were
embedded in Killik (Bio-Optica), frozen in liquid nitrogen-cooled isopentane (Sig-
ma–Aldrich) and stored at ?80 ?C. Unless otherwise stated, all subsequent proce-
dures were conducted at room temperature.
Qualitative and quantitative mRNA expression
Total RNA was extracted using TriZol (Invitrogen) and treated with DNase I
(Invitrogen). Total RNA was quantified using a NanoDrop ND-1000 UV–vis spectro-
photometer. Reverse transcription (RT) was carried out with 1 lg RNA using the
iSCRIPT cDNA Synthesis Kit (BioRad). The cDNA was used as the template for PCRs,
which were performed in 10 lL final volumes containing 1? buffer (Vivantis),
1.5 mM MgCl2, 0.2 mM each deoxynucleotide triphosphate (dNTP), 1 lM each pri-
mer and 0.025 U Taq polymerase (Vivantis). The same primers were used in quali-
tative and quantitative PCR for SAA and LBP, whereas different primers were used
for Hp and AGP (Table 1). PCR conditions were 35 cycles of 94 ?C for 30 s, 60 ?C
for 30 s and 72 ?C for 45 s (Eppendorf Mastercycler). PCR products were visualised
on 1.9% agarose gels stained with ethidium bromide.
Quantitative reactions were performed using 20 lL Eva Green mix (BioRad) and
450 nM each primer (SAA, LBP, Hp_RT, AGP_RT and b-actin; Table 1). Each sample
was tested in duplicate. To evaluate PCR efficiency, fourfold serial dilutions were
prepared from reference samples. The thermal profiles for each target gene were
95 ?C for 90 s, 50 cycles of 95 ?C for 5 s and 60 ?C for 10 s; conditions for melting
curve construction were 55 ?C for 60 s then 80 cycles starting at 55 ?C and increas-
ing 0.5 ?C each 10 s. Results were compared using the D–D Cq method (Giulietti
et al., 2001).
Western blot analysis
Antibodies validated in cattle for detection of APPs by Western blot analysis and
immunohistochemistry are listed in Table 2. Samples for Western blot analysis
were prepared from aliquots of 50–100 mg tissues using protease inhibitors (Sig-
ma–Aldrich), as previously described (Rahman et al., 2008). The protein content
of the supernatant was quantified at A280 nm. Aliquots of 25–50 lg/mL were
separated by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis
(SDS–PAGE) and Western blotted onto nitrocellulose membranes. The membranes
were immunolabelled for the presence of APPs using specific antibodies (Table 2)
and immunoreactive bands were visualised by enhanced chemiluminescence
(ECL) using Immobilon Western Chemiluminescence substrate (Millipore). To
confirm that an equal amount of protein was loaded in each lane, membranes were
stripped and immunolabelled with mouse anti b-actin antibody (1:10,000). Liver ly-
sates were used as positive controls.
Immunohistochemistry for AGP and Hp was performed on cryostat sections
incubated in 0.3% H2O2in methanol (Sigma–Aldrich) for 30 min to block endoge-
nous peroxidase activity. Non-specific binding sites were blocked with 10% normal
goat serum (Sigma–Aldrich) for 30 min and sections were incubated overnight at
4 ?C in the presence of specific antibodies (Table 2). Staining of specific proteins
was carried out with diaminobenzidine (DAB, Vector Laboratories) after incubation
with peroxidase-conjugated secondary antibodies (Sigma–Aldrich). Nuclei were
counterstained with Mayer’s haematoxylin (DDK Italia). Sections were mounted
in Poly-mount (Polysciences) and examined using a Nikon Eclipse E600 microscope.
Bovine liver was used as a positive control. Primary antibodies were omitted for
Expression of acute phase protein mRNA in the bovine forestomachs
Expression of Hp, SAA, LBP and AGP RNA was detected by qual-
itative RT-PCR in the mucosa of the bovine forestomachs and
abomasum (Supplementary Fig. 1). Quantitative RT-PCR was used
to determine the relative expression of Hp, SAA, LBP and AGP
RNA after normalisation against b-actin (Fig. 1). Increased amounts
of SAA and LBP RNA relative to the liver were expressed in the
mucosa of the forestomachs and abomasum; the highest levels of
SAA were detected in the omasum and abomasum, whereas the
highest levels of LBP were detected in the omasum. Expression of
AGP and Hp in the mucosa of the forestomachs and abomasum
was negligible compared to the liver.
Expression of acute phase proteins in the bovine forestomachs and
Hp, LBP and AGP were detected by Western blot analysis in all
samples of mucosa from the forestomachs and abomasum (Fig. 2).
AGP was detected as a low molecular weight (MW) immunoreac-
tive band of 21 kDa expressed mainly in the rumen, reticulum
Sequences of oligonucleotide primers for acute phase proteins.
Name Fragment size (base pairs)Sense Antisense
SAA, serum amyloid A; LBP, lipopolysaccharide-binding protein; Hp, haptoglobin; AGP, a-1 acid glycoprotein. SAA, LBP, Hp, AGP and b-actin primers were designed on the
basis of GenBank sequences (Accession Numbers NM181016.3, NM001038674.1, NM_001040470.1, AM403243 and BC142413.1, respectively). Hp_RT primers sequences are
from Eckersall et al. (2006).
Primary antibodies for Western blotting and immunohistochemistry.
Name Western blot analysisImmunohistochemistry
Primary antibody Incubation timeReferencePrimary antibody Incubation time
Mouse anti-bovine SAA (C100–8)
Mouse anti-human LBP (biG42)
Rabbit anti-bovine Hp
Rabbit anti-bovine AGP
Mouse anti-b-actin (CP01)
1:100 (3.9 lg/mL)
1:200 (5 lg/mL)
1:2,000 (1.37 lg/mL)
1:2,000 (1.65 lg/mL)
McDonald et al. (1991)
Rahman et al. (2010)
Ceciliani et al. (2007b)
Lecchi et al. (2008)
1:200 (13.75 lg/mL)
1:200 (16.5 lg/mL)
SAA, serum amyloid A; LBP, lipopolysaccharide-binding protein; Hp, haptoglobin; AGP, a-1 acid glycoprotein.
F. Dilda et al./The Veterinary Journal 192 (2012) 101–105
and omasum, which corresponded to the expected MW of bovine
deglycosylated AGP (21.3 kDa). In addition, there was a higher
MW band of 43 kDa expressed mainly in the omasum and aboma-
sum, which corresponded to fully glycosylated AGP. Hp was de-
tected as two major bands of 16 and 45 kDa, corresponding to
the a and b chains of human Hp (Levy et al., 2010), respectively,
in the rumen and abomasum. Several high MW bands were also
detected in the forestomachs and abomasum; these were the pre-
dominant immunoreactive bands in the reticulum and omasum
and are likely to be due to association of the 16 and 45 kDa sub-
units with albumin (Eckersall and Conner, 1990). Immunoreactiv-
ity for LBP was evident in the forestomachs and abomasum at
55–60 kDa (corresponding to the expected MW of LBP), with the
highest band intensity in the abomasum. LBP immunoreactivity
was also evident at 18 kDa (low MW bands) and 70–80 kDa (high
MW bands). SAA was detected only in the abomasum, with a
MW of 14 kDa (Fig. 2).
AGP and Hp were localised by immunohistochemistry in the
superficial layers of the stratified squamous epithelium of the fore-
stomachs and in the glandular epithelium of the abomasum
APPs exhibit binding and immunomodulatory properties and
are involved in the first stages of the innate immune response
(Murata et al., 2004; Petersen et al., 2004). Thus, APPs expressed
in the gastric mucosa might contribute to homeostasis of the
host-microbial interface. In this study, APPs were detectable in
the mucosa of the bovine forestomachs and abomasum, suggesting
that these tissues are capable of generating a local acute phase re-
sponse. In view of its large surface area, the mucosa of the rumi-
nant forestomachs and abomasum may be an important source
of APPs during diseases such as sub-acute ruminal acidosis (Gozho
et al., 2005) and abomasal parasitism (Conner et al., 1989).
Hp and SAA are the major APPs released during inflammation in
cattle (Petersen et al., 2004). SAA is a multifunctional protein in-
volved in several immunomodulatory functions, including opsoni-
sation of bacteria (Hari-Dass et al., 2005; Shah et al., 2006;
Molenaar et al., 2009). In the present study, SAA mRNA was dem-
onstrated in the forestomachs and abomasum by quantitative PCR,
but SAA protein could be identified only in the abomasum. Since
SAA is expressed in a spatially and temporally restricted manner
in tissues (McDonald et al., 2001; Molenaar et al., 2009), it is pos-
sible that SAA mRNA accumulates constitutively in gastric epithe-
lial cells, but is translated into active proteins only during
The main activity of Hp is to bind haemoglobin, thus preventing
renal damage and iron loss (Levy et al., 2010), as well as inhibiting
the utilisation of iron by pathogenic bacteria (Eaton et al., 1982).
Expression of Hp protein was high in the forestomachs and aboma-
sum, even though expression of Hp mRNA was negligible. It is pos-
sible that Hp may not be produced locally in the forestomachs or
abomasum, but instead may be produced elsewhere, probably in
the liver, then transported to the gastric mucosa.
LBP is secreted by enterocytes and activates host defences
against bacteria, as well as being involved in endotoxin tolerance
Fig. 1. Relative expression of serum amyloid A (SAA), lipopolysaccharide-binding protein (LBP), haptoglobin (Hp) and a-1 acid glycoprotein (AGP) in the bovine forestomach
mucosa by real-time PCR. Values are expressed as fold change relative to liver and normalised using b-actin as the reference gene. Data are means ± standard error of four
F. Dilda et al./The Veterinary Journal 192 (2012) 101–105
(Vreugdenhil et al., 1999; Zweigner et al., 2006). The main site of
expression of LBP mRNA in the present study was the omasum,
confirming previous findings (Rahman et al., 2010) (Supplemen-
tary Fig. 2), whereas the highest protein expression was evident
in the abomasum.
AGP is a binding protein with immunomodulatory functions
expressed at high levels in the liver and moderate levels in the
mammarygland, salivary glands,pancreas, spleen, lungsand uterus
also shows that AGP is expressed in the bovine forestomachs.
Western blot analysis demonstrated several isoforms of APPs
that differed from those expressed in the liver. It is possible that
glycosylation patterns of APPs are specific for the site of origin
(Ceciliani et al., 2007a; Cooray et al., 2007; Rahman et al., 2010).
Fig. 2. SDS–PAGE and Western blot analysis of the forestomachs and abomasum mucosal lysates. Acute phase proteins were identified after immunostaining using antibodies
listed in Table 1 and detection by electrochemiluminescence.
Fig. 3. Immunolocalisation of a-1 acid glycoprotein (AGP) and haptoglobin (Hp) in the bovine forestomachs and abomasum. Representative images demonstrate AGP (A–E)
and Hp (F–J) localisation in the bovine rumen (A and F), reticulum (B and G), omasum (C and H) and abomasum (D and I). Note the staining of endothelial cells of blood vessels
(arrow). E and J show negative control staining in the rumen (E) and omasum (J). Bar = 100 lm.
F. Dilda et al./The Veterinary Journal 192 (2012) 101–105
The three bovine forestomachs and the abomasum produce
APPs. Although expression of SAA mRNA was evident in the fore-
stomachs and abomasum, SAA protein could be identified only in
the abomasum. Expression of Hp protein was high in the forestom-
achs and abomasum, even though expression of Hp mRNA was
negligible. The main site of expression of LBP mRNA was the oma-
sum, whereas the highest protein expression was evident in the
abomasum. AGP was expressed at low levels in the bovine fore-
stomachs. Western blot analysis demonstrated several isoforms
of APPs that differed from those expressed in the liver. On the basis
of their lipid-binding and immunomodulatory activities, the
expression of APPs in the bovine forestomachs and abomasum
may contribute to regulation of the innate immune response
against bacteria and parasites.
Conflict of interest statement
None of the authors of this paper has a financial or personal
relationship with other people or organisations that could inappro-
priately influence or bias the content of the paper.
The authors acknowledge the important gift of the anti-SAA
antibody from Dr. Thomas McDonald and Dr. Annika Weber,
University of Nebraska. The authors would like to thank Dr. Adrian
J. Molenaar, AgResearch, Ruakura Research Centre, New Zealand,
for helpful suggestions. This work was financed by Grant numbers
PUR2007 and PUR2008.
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
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