Acute phase proteins in experimentally induced pregnancy toxemia in goats

J Vet Diagn Invest 23:57–62 (2011)
Acute phase proteins in experimentally induced pregnancy toxemia
in goats
Félix H. D. González, Fuensanta Hernández, Josefa Madrid, Silvia Martı́nez-Subiela,
Asta Tvarijonaviciute, José J. Cerón, Fernando Tecles1
Abstract. The present work aimed to study the behavior of acute phase proteins (haptoglobin, serum
amyloid A, acid soluble glycoprotein, fibrinogen, and albumin) in fasting-induced pregnancy toxemia in goats
and their relationship with classical indicators of this disorder such as beta-hydroxybutyrate and nonesterified
fatty acids in the blood and decreased urine pH and ketonuria. Twelve adult Murciano–Granadina goats at
the final stage of gestation were used in this experiment. Pregnancy toxemia was induced in 6 goats by fasting
for 72 hr. The other 6 animals were used as control group. Ketonuria was present in 4 out of 5 fasting animals
at 24 hr and in all fasting animals at 48 hr of fasting. Serum nonesterified fatty acids were significantly
increased at 24, 48, and 72 hr of fasting. Beta-hydroxybutyrate and haptoglobin achieved significantly
increased concentrations at 48 hr and 72 hr, respectively, remaining increased during the entire study. Serum
amyloid A, acid soluble glycoprotein, fibrinogen, and albumin were not affected by fasting. In conclusion,
acute phase proteins (including haptoglobin) seemed not to have an advantage over traditional markers in
diagnosis of fasting-induced pregnancy toxemia in goats.
Key words:
Acute phase proteins; beta-hydroxybutyrate; goats; pregnancy toxemia.
bances in ruminants.5 The major APPs in ruminants
that have been described in the literature are haptoglobin (Hp) and serum amyloid A (SAA).16 In goats,
Hp and SAA could be considered as major APPs, while
acid-soluble glycoprotein (ASG) and fibrinogen may be
considered as moderate.10 Previous studies in ruminants suggested a relationship between selected APPs
and lipid mobilization. One study18 reported increased
Hp concentration in cows with fatty liver, compared
with the values in healthy cows (466 mg/l vs. 0.01 mg/l).
In addition, increased Hp levels in cows around
parturition have been described. It has been postulated
to be related to negative energy balance, since cows
with high milk Hp also showed high non-esterified fatty
acid (NEFA) concentration in serum.11 A significant
correlation has been also reported between Hp and
BHB in lactating goats (Trevisi E, D’Angelo A,
Gaviraghi A, et al.: 2005, Blood inflammatory indices
in goats around kidding. Ital J Anim Sci 4:404.
Abstract).
The previous findings reveal a possible relationship
between Hp and markers of negative energy status in
ruminants and led the current authors to study the
APP response in pregnancy toxemia in goats. The
present work was aimed at determining the concentration and evolution of different APPs, such as Hp,
SAA, ASG, fibrinogen, and albumin, in fastinginduced pregnancy toxemia in goats and comparing
them with other traditional biochemical markers such
as BHB, NEFA, glucose, triglycerides, and choles-
Introduction
Pregnancy toxemia is a metabolic disorder that
affects goats during the last period of gestation,
occurring most frequently in animals with high body
condition with 2 or more fetuses.21 Undernutrition,
due to great energy demand by fetuses at the final
stage of pregnancy, and obese body condition are the
main factors that result in decreased glucose and
excessive lipid mobilization, and consequently, ketonemia.2 It has been demonstrated that hypoglycemia
leads to production of ketone bodies in a manner
inversely proportional to the plasma glucose concentrations.26 The diagnosis of this disease is based on
history, clinical signs, and detection of ketone bodies
such as beta-hydroxybutyrate (BHB) in serum or
urine.14 Morbidity may approach 5–20% in farm
goats, and mortality in nontreated animals may be as
high as 80%.21
Acute phase proteins (APPs) have been proposed as
sensitive and rapid indicators of inflammatory disturFrom the Veterinary School, Federal University of Rio Grande
do Sul, Porto Alegre, Rio Grande do Sul, Brazil (González); the
Animal Production Department (Hernández, Madrid) and the
Animal Medicine and Surgery Department (Martı́nez-Subiela,
Tvarijonaviciute, Cerón, Tecles), Veterinary School, University of
Murcia, Murcia, Spain.
1
Corresponding Author: Fernando Tecles, Animal Medicine
and Surgery Department, Veterinary School, University of
Murcia, Campus of Espinardo s/n, Murcia 30100, Spain.
[email protected]
57
58
González et al.
terol. The current study presents an evaluation of
these proteins in the disease and possible practical
applications of APPs in the diagnosis and management of pregnancy toxemia in goats.
Materials and methods
Animals and diet
Twelve healthy Murciano–Granadina multiparous goats
of similar age (mean: 4.3 years, range: 3–5 years) from a
farm in the Murcia Region of Spain were used in the
present experiment. This breed is characterized by milk
production of more than 600 kg of milk per lactation.
Weight ranged between 45 and 55 kg (mean: 48 kg), which
was within breed parameters for female individuals (40–
55 kg). The mean body condition score of the goats was
3.5–4.0 on a scale of 1–5 (Fredricks G: 1993, Using body
condition score to evaluate feeding management. In:
Proceedings of the American Dairy Goat Association
National Convention, p. 78. Portland, OR), indicating that
the goats had adequate fat reserves.
All goats were inseminated after synchronization with 3mg norgestimate ear implants and a 7.5-mg injection of
dinoprosta 13 days after the implant. Forty-five days after
insemination, pregnancy was diagnosed by transabdominal
ultrasonography. Approximately 6 weeks prior to the
scheduled parturition, a new ultrasonography was completed to check that all goats had a minimum of 2 fetuses.
Goats were fed ad libitum on alfalfa hay of 0.6 milk
forage unit (UFL)/kg dry matter (DM), 156 g crude protein
(CP)/kg DM, and 463 g neutral detergent fiber (NDF)/kg
DM plus 250 g of concentrate/day at 2 months gestation,
increasing gradually to 500 g/day per head for the last
4 weeks of pregnancy. The concentrate used (1.05 UFL/kg
DM, 160 g CP/kg DM, and 246 g NDF/kg DM) was a
mixture of corn, barley, oat, cotton, and lupine grains;
sunflower meal; citrus pulp; and dried distillers grains
pellets; and a pellet concentrate, where vitamin and mineral
supplements were included. The concentrate was offered in
2 equal portions at 8:00 AM and 4 PM. The animals had free
access to water.
Approximately 4 weeks prior to the scheduled parturition (at 120 days from insemination), goats were randomly
divided into 2 groups—control group (n 5 6) and fasting
group (n 5 6)—and housed in 2 separated pens with straw
bedding. The control group was fed normally during the
study. After a period of 2 weeks for adaptation to housing
in separated pens, pregnancy toxemia was induced in the
fasting group by allowing access only to water for 72 hr.26
Animals were examined daily to detect clinical signs of
pregnancy toxemia. After 72 hr of starvation, the first
clinical signs were observed; at this point, feed was
reintroduced ad libitum to all animals of the fasting group
using the same diet that was used for the control group. All
procedures were done with the approval of the Ethical
Committee of the University of Murcia (Spain).
Sampling
Blood and urine samples were collected every morning at
10:00 AM in both groups of animals, 1 day before the
induction of fasting, and daily during the following 5 days.
Blood samples were collected in vacuum tubesb with
heparin (5 ml) for fibrinogen analysis and in plain tubes
(10 ml) for the rest of the biochemical analyses. Urine
samples were obtained by voluntary miction or induced by
covering the nose and the mouth of the goat for few
seconds (Ortolani E: 2003, Diagnóstico de doenças
nutricionais e metabólicas por meio de exame de urina
em ruminantes [Diagnostic of nutritional and metabolic
diseases using urine examination in ruminants]. In: Anais
do I Simpósio de Patologia Clı́nica Veterinária da Região
Sul do Brasil, ed. Gonzalez FHD, Campos R, pp. 91–102.
Universidade Federal do Rio Grande do Sul, Porto Alegre,
Brazil). In fasted goats, sampling at 72 hr was performed
prior to feed reintroduction to avoid any influence in the
results. Samples were kept in a thermal box for transport to
the laboratory. Blood in plain tubes was centrifuged (2,000
3 g for 10 min), and serum was stored at 220uC until
biochemical analysis.
Urine and serum metabolites analysis
Determination of the urine pH was made with a digital
pH meter.c Chemical analysis of urine was conducted using
reagent strips.d The following serum metabolites were
determined by commercially available colorimetric methods: glucose,e triglycerides,e total cholesterol,e BHB,f and
NEFA.f
Acute phase proteins analysis
Albumin was measured using a colorimetric technique.e
Serum concentrations of Hp were quantified by a
spectrophotometric method using a commercial kit.g The
method of ASG determination was based on a technique
described in 198917 and modified in 1996.6 Serum SAA
concentration was measured using a commercially available
enzyme-linked immunosorbent assay (ELISA) kitg according to the manufacturer’s instructions. Fibrinogen was
measured in plasma samples by the heating precipitation
method at 56uC.25 All methods for APP determination have
been previously validated at the authors’ laboratory in
goats, giving intra-assay and interassay coefficients of
variation within 1.36–4.47% and 3.69–12.68%, respectively.
Limits of detection were 0.02 g/l for Hp, 0.20 g/l for ASG,
and 3.06 mg/l for SAA.10 All colorimetric methods were
performed in an automatic analyzer.h The final absorbance
in ELISA methods was measured in a microtiter plate
readeri at 450 nm wavelength.
Statistical analysis
Arithmetic means and standard deviations were calculated using routine descriptive statistical procedures.
Kolmogorov–Smirnov test was used to assess normality
of data, giving a nonparametric distribution. Therefore, all
data were normalized by logarithmic transformation before
analysis. To assess differences between control and fasting
groups, 2-way analysis of variance of repeated measures
with Bonferroni posttest was performed.j A P value , 0.05
was considered statistically significant. Correlation between
APPs and other biochemical parameters were analyzed
Acute phase proteins in pregnancy toxemia in goats
59
Table 1. Mean values of urinary pH and units of ketonuria (acetoacetic acid) after the induction of pregnancy toxemia in goats by
fasting for 72 hr (n control 5 6, n fasting 5 5).*
Ketonuria{
Fasting
Minimum
Control
Hours after fasting onset
0
0 [6]
24
0 [6]
48
72
Recovery period
96
120
Urinary pH
Maximum
0 [5]
0 [1]
0 [6]
0 [6]
1+ [1]
2+ [1]
0 [6]
0 [1]
0 [6]
0 [3]
3+
2+
3+
3+
4+
[2]
[2]
[4]
[2]
[2]
3+ [1]
4+ [3]
3+ [2]
Control
Fasting
8.08 (0.06)
8.20 (0.07)
8.02 (0.09)
8.08 (0.20)
8.18 (0.19)
8.38 (0.33)
7.38 (0.59)
5.94{ (0.90)
7.87 (0.07)
7.39 (0.94)
7.84 (0.36)
7.68 (0.84)
* Numbers in parentheses are standard deviations. Numbers in brackets are the number of animals with the indicated ketonuria units.
{ 0 5 negative; 1+ 5 1.47 mmol/l; 2+ 5 3.92 mmol/l; 3+ 5 7.84 mmol/l; 4+ $ 15.68 mmol/l.
{ Significant differences (P , 0.001) between control and fasting goats at the same period.
using Pearson correlation analysis; data obtained from all
animals during the study period were included in this
analysis.
Results
Clinical signs of early pregnancy toxemia started to
appear after 72 hr of fasting, when 5 out of 6 goats
(83.3%) showed depression, muscle tremors, and
staggering gait. At this moment, feed was reintroduced for all the animals. The clinical signs disappeared in all animals within a few hours after feed was
reintroduced. One of the fasted goats kidded the last
day of the experiment, so the values of this goat were
excluded from statistical analysis.
Moderate to severe ketonuria was evident at 24 hr
after feed withdrawal, and the urinary pH decreased
significantly at 72 hr of fasting. During the recovery
period, urinary pH returned to normal in all animals,
whereas ketonuria was present in 2 goats of the
fasting group at the end of the study (Table 1).
Compared with the control group, glucose values
significantly (P , 0.001) decreased in the fasting
group after 48 hr of fasting. NEFA were significantly
(P , 0.01) increased in the fasting goats after 24 hr
and peaked at 48 hr of fasting. BHB was significantly
(P , 0.05) higher in the fasting group than the control
after 48 hr of fasting and peaked at 72 hr. Glucose
and NEFA normalized as soon as feed was reintroduced, whereas BHB was significantly higher in the
Table 2. Mean values of glucose, non-esterified fatty acid (NEFA), beta-hydroxybutyrate (BHB), triglycerides, and cholesterol
after the induction of pregnancy toxemia in goats by fasting for 72 hr (n control 5 6, n fasting 5 5).*
Glucose (mmol/l)
Control
Fasting
Hours after fasting onset
0
2.41 (0.29) 2.31 (0.15)
24
2.01 (0.19) 1.56 (0.71)
48
2.86 (0.44) 1.03{ (0.25)
72
2.65 (0.43) 1.01{ (0.40)
Recovery period
96
2.3 (0.52)
120 1.97 (0.58)
*
{
{
1
NEFA (mmol/l)
Control
0.44
0.51
0.28
0.34
Fasting
BHB (mmol/l)
Control
(0.20) 0.48 (0.19) 0.66
(0.26) 1.23{ (0.52) 0.86
(0.24) 1.50{ (0.39) 0.64
(0.10) 1.38{ (0.30) 0.66
2.43 (0.40) 0.56 (0.26)
2.22 (0.73) 0.52 (0.17)
Fasting
(0.16)
0.70 (0.24)
(0.36)
0.96 (0.37)
(0.15) 2.031 (0.94)
(0.16) 4.43{ (2.19)
Triglycerides (mmol/l)
Control
0.16
0.15
0.24
0.16
(0.02)
(0.02)
(0.09)
(0.05)
Fasting
0.18
0.30
0.20
0.22
(0.01)
(0.16)
(0.10)
(0.14)
Total cholesterol (mmol/l)
Control
1.66
1.79
1.53
1.59
(0.37)
(0.40)
(0.44)
(0.39)
Fasting
1.40
1.46
1.44
1.43
(0.36)
(0.42)
(0.32)
(0.34)
0.71 (0.19) 0.84 (0.27) 2.80{ (1.45) 0.13 (0.04) 0.13 (0.05) 1.71 (0.50) 1.42 (0.52)
0.74 (0.38) 0.70 (0.18) 1.881 (1.38) 0.15 (0.03) 0.15 (0.06) 1.61 (0.41) 1.31 (0.21)
Numbers in parentheses are standard deviations.
Significant difference, P , 0.01, between control and fasting goats at the same period.
Significant difference, P , 0.001, between control and fasting goats at the same period.
Significant difference, P , 0.05, between control and fasting goats at the same period.
60
* UDL 5 under detection limit of the assay. Numbers in parentheses are standard deviations. Numbers in brackets are the number of animals with the recorded SAA concentration
upper detection limit of the assay.
{ Several goats showed SAA under detection limit of the assay, and values were not included.
{ Significant differences (P , 0.05) between control and fasting goats at the same period.
27.96 (0.93)
28.05 (1.98)
29.05 (3.61)
29.31 (3.03)
2.67 (1.03)
3.0 (1.1)
2.67 (1.03)
3.0 (1.67)
1.06 (0.33)
1.12 (0.56)
1.38 (0.29)
1.27 (0.41)
UDL
UDL
UDL
UDL
438.3{ (372.5)
412.6{ (238.1)
Recovery period
96
30.0 (73.4)
120
51.6 (80.1)
(2.18)
(1.89)
(2.37)
(1.52)
30.06
30.71
28.83
29.40
(2.79)
(2.95)
(2.78)
(3.3)
27.96
28.80
27.50
28.31
(1.03)
(1.63)
(1.1)
(1.03)
2.67
2.67
3.0
2.67
(1.03)
(1.63)
(1.1)
(0.45)
2.67
3.33
2.8
2.2
(0.25)
(0.35)
(0.33)
(0.48)
1.09
1.14
1.07
0.97
(0.14)
(0.50)
(0.72)
(0.41)
1.38
1.57
1.14
1.23
UDL
4.29{ (0.10) [3]
4.88{ (1.51) [4]
UDL
UDL
UDL
7.74{ (2.63) [3]
6.95{ [1]
(59.5)
(93.8)
(246.6)
(304.2)
Control
Fasting
Control
Hours in relation to fasting onset
0
41.6 (49.9)
35.0
24
76.6 (100.5)
38.3
48
73.3 (82.3)
361.6
72
111.6 (134.2)
488.3{
Fasting
Control
Fasting
Fasting
Control
Fasting
Control
Albumin (g/l)
Fibrinogen (g/l)
ASG (g/l)
SAA (mg/l)
Hp (mg/l)
Table 3. Mean values of haptoglobin (Hp), serum amyloid A (SAA), acid-soluble glycoprotein (ASG), fibrinogen, and albumin after the induction of pregnancy toxemia in goats
by fasting for 72 hr (n control 5 6, n fasting 5 5).*
González et al.
fasting group throughout the study (Table 2). Triglycerides and total cholesterol values did not show
any significant difference between the control and
fasting group (Table 2).
Haptoglobin values tended to be increased in the
fasting group at 48 hr and were significantly (,0.05)
increased at 72 hr, remaining elevated during the
recovery period (Table 3). The values of ASG,
fibrinogen, SAA, and albumin were not affected by
fasting.
The Pearson coefficient of correlation between Hp
and BHB during the fasting period was 0.84 (P ,
0.05). Correlation between other APPs and metabolites did not show any significance.
Discussion
The first clinical signs of pregnancy toxemia in
sheep usually appear when the blood concentration of
ketone bodies reaches 4 mmol/l.7 In the present
experiment, clinical signs of pregnancy toxemia,
characterized by staggering gait, muscle tremors,
and depression, were evident in the goats after 72 hr
of fasting when the mean concentration of serum
BHB was 4.43 mmol/l.
Ewes and goats with BHB concentrations of 0.86–
1.6 mmol/l are classified as having mild or subclinical
pregnancy toxemia.3,19 According to these criteria,
fasted goats in the current study had subclinical
pregnancy toxemia 24 hr after fasting when BHB
values were 0.96 mmol/l. Also, NEFA were increased,
and ketonuria appeared after 24 hr of fasting, but
clinical signs were not evident at this time.
In the present study, Hp concentrations tended to
be increased at 48 hr of fasting in goats and were
significantly increased at 72 hr. Concentrations of the
other APPs, such as ASG, fibrinogen, SAA, and
albumin, were not affected by the induction of
pregnancy toxemia. SAA showed great variability
with undetectable concentrations at multiple time
points. A few goats (3 goats at 24 hr and 4 goats at
48 hr in the fasting group, and 3 goats at 48 hr and 1
goat at 72 hr in the control group) had a measurable
concentration of SAA but a consistent change
associated with pregnancy toxemia was not observed.
In all instances, SAA concentrations were lower than
10 mg/l, which could be a normal variation for this
species.10 Other authors have reported increases in Hp
without increases in other APP such as alpha 1–acid
glycoprotein after starvation of cows.27 Also, ketosis
did not produce any change in serum transferrin (an
APP) in cows.15 Moreover fasting has been related to
a decrease of inflammation status by decreasing
interleukin-6 in humans.8 These observations and
the lack of increase in other APPs evaluated in the
current study suggest that the increase in Hp observed
Acute phase proteins in pregnancy toxemia in goats
in the present study was not part of an inflammatory
or acute phase response.
Increases in Hp found in goats with pregnancy
toxemia could be related to the changes in lipid
metabolism that occurs in this process. In cows,
nonfeeding induces NEFA mobilization.1,22 When a
fasted period of 3 days was applied to a group of
calves, NEFA mobilization was observed on the first
fasting day.13 In the same study, Hp was also
observed to be increased but lagged behind the rise
in NEFA and was not increased in most calves after
2–3 days of refeeding.13 There are some evidences of a
relationship between Hp and lipids. In humans, Hp is
synthesized by adipocytes as well as by hepatocytes,9
and Hp locus is close to the loci of lipid-related
enzymes such as lecithin:cholesterol acyltransferase
and cholesteryl ester transfer protein.20 Moreover, Hp
is attached to high-density lipoproteins (HDL) and
apolipoproteins (apo) such as apoA-I24 and apoE4 in
humans, and to HDL and very high-density lipoproteins in cows.12
Significant increases in Hp appeared in fasted goats
at the same time as the clinical signs in the present
study. However, it seems that there is no correlation
between severity of clinical signs and Hp concentration because the only goat that did not show clinical
signs had high Hp values (570 mg/l). In addition, Hp
does not seem to be a good marker of subclinical
pregnancy toxemia because other markers such as
plasma NEFA and ketone bodies in urine showed
significant changes at 24 hr. Similar results have been
reported in cows with subclinical ketosis showing
increased concentrations of BHB and normal Hp
concentrations.23
In conclusion, fasting in goats causes a significant
increase in Hp values but not in other APPs.
However, a significant increase in Hp was not
observed until the appearance of clinical signs, so it
should not be used for the diagnosis of subclinical
pregnancy toxemia and seemed not to have clear
advantages over the traditional markers of pregnancy
toxemia such as BHB or ketonuria.
Acknowledgements
This study was supported by the Seneca Foundation
from the Regional Government of Murcia (Spain) and by
the National Council for Scientific and Technological
Development (CNPq) in Brazil.
Sources and manufacturers
a. Lutalyse, Pharmacia and Upjohn Co., Kalamazoo, MI.
b. BD, Franklin Lakes, NJ.
c. Basic 20, Crison Instruments SA, Barcelona, Spain.
61
d. MultistixH 10SG, Siemens Healthcare Diagnostics, Deerfield,
IL.
e. Spinreact SA, Girona, Spain.
f. Randox Laboratories Ltd., Crumlin, United Kingdom.
g. Tridelta Development Ltd., Greystones, County Wicklow,
Ireland.
h. Cobas Mira Plus, ABX Diagnostica, Montpellier, France.
i. PowerWave XS, BioTek Instruments Inc., Winooski, VT.
j. Version 5.0, GraphPad Software Inc., La Jolla, CA.
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