Specific immune response of mares and their newborn foals to Actinobacillus spp. present in the oral cavity

Oral swab samples, serum and colostrum was taken from 15 mares and 14 of their foals, within 24 h of birth. The presence of antibody against Actinobacillus spp. isolated from the oral cavity was investigated using agar gel immunodiffusion. Antibodies against 48 out of the 77 Actinobacillus isolates from all horses in the study were present in the respective sera of 13 mares and 9 foals. In 11 mother-foal pairs, the antibody content of the foal serum was similar to that of the mare, and in 9 cases this was reflected in the antibody content of colostrum from the mare. The results indicate that an immune response to Actinobacillus spp. colonising the oral cavity is present in many adult horses and that this immune response can be transferred from mother to foal via colostrum.     

Clinical survey of antibodies against red blood cells in horses after homologous blood transfusion

Serum samples of 20 horses were evaluated for antibodies against RBC after homologous blood transfusion. Transfusion-associated antibodies against RBC were detected in 10 horses. Antibodies recognizing horse blood group antigens Aa, Ae, Db, and Dc were identified. Antibodies against Aa were found in all samples from Aa-negative horses that were transfused with Aa-positive RBC. Antibodies against Aa persisted for at least 1 year after transfusion. Antibodies against Ae were detected in 7 of 8 horses transfused with Ae-positive RBC. Initial appearance and persistence of antibodies against Ae differed among the horses; antibodies were initially detected 1 week to 154 weeks after transfusion and disappeared as early as 4 weeks after transfusion. Antibodies against Db or Dc were detected in less than or equal to 33% of the horses that lacked Db or Dc antigens and were transfused with Db- or Dc-positive RBC. Antibodies against Db and Dc were initially detected in sera later than were the A-system antibodies. Three mares with transfusion-associated antibodies subsequently produced healthy offspring. Two foals had RBC antigens corresponding to their dam's alloantibodies; maternal colostrum with antibodies against Aa was withheld from the Aa-positive foal. The Db-positive foal remained healthy after nursing the mare with serum antibodies against Db.     

Immunoglobulin isotypes in sera and nasal mucosal secretions and their neonatal transfer and distribution in horses

OBJECTIVE: To determine concentrations of IgA and IgG subclasses in serum, colostrum, milk, and nasal wash samples of adult horses and foals. ANIMALS: Seven 2-year-old Welsh ponies, 27 adult mixed-breed horses, and 5 Quarter Horse mares and their foals. PROCEDURE: Serum was obtained from ponies and adult horses. Colostrum and milk were obtained from mares and serum and nasal wash samples from their foals immediately after parturition and on days 1, 7, 14, 28, 42, and 63. Nasal wash samples were also obtained from 23 adult horses. Concentrations of immunoglobulins were determined by use of inhibition ELISA. To determine transfer of maternal isotypes to foals, concentrations in colostrum and milk were compared with those in foal serum. Serum half-lives of isotypes in foals were also determined. RESULTS: IgGb was the most abundant isotype in serum and colostrum from adult horses, whereas IgA was the predominant isotype in milk. The major isotype in nasal secretions of adult horses and foals > or = 28 days old was IgA, but IgGa and IgGb were the major isotypes in nasal secretions of foals < or = 14 days old. Serum half lives of IgGa, IgGb, IgG(T), and IgA in foals were 176, 32, 21, and 3.4 days, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The early immunoglobulin repertoire of neonatal foals comprised IgGa, IgG(T), and IgA; endogenous synthesis of IgGb could not be detected until 63 days after birth. The restricted repertoire of immunoglobulins in foals may influence humoral immune responses to vaccination.     

Enzyme-linked immunosorbent assay for diagnosis of Corynebacterium (Rhodococcus) equi infection in foals

An enzyme-linked immunosorbent assay (ELISA) was used to diagnose Corynebacterium (Rhodococcus) equi infection in foals. In tests done with different antigen-extraction procedures (sodium dodecyl sulfate, sodium deoxycholate, polyoxy-ethylene [9] p-tert-octylphenol, polyoxy-ethylene [9-10] p-tert-octylphenol, sonification, homogenization, and heat treatment at 121 C), Tween 20 was a satisfactory reactive antigen. Using hyperimmune rabbit sera or infected foal sera, we investigated the specificity and the sensitivity of the ELISA with the Tween 20 antigen of the different serotypes or of the isolates. Corynebacterium equi strain ATCC 6939 antigen had the best activity for detecting antibodies to C equi in foals. Sera from 218 healthy horses, 11 healthy foals, 17 healthy newborn foals, a foal with suspected C equi infection, and 5 infected foals were evaluated for antibodies to C equi, using ELISA. The optical density values of 206 healthy horses, 17 healthy newborn foals, and 9 healthy foals were less than 0.1. Infected foal sera, except from foal 3, and serum from a foal with suspected C equi infection had higher optical density values. Using ELISA, specific antibodies against C equi were detected in a naturally infected 6-week-old foal after the foal had a rapid increase in the number of bacteria in the feces and after the initial development of clinical signs of illness at 5 weeks of age. Therefore, ELISA was useful for the early diagnosis of C equi infection in foals.     

Effect of withholding macromolecules on the duration of intestinal permeability to colostral IgG in foals

OBJECTIVE: To quantify absorption of colostral IgG by healthy neonatal foals and to test the hypothesis that delayed ingestion of macromolecules prolongs the duration of intestinal permeability to immunoglobulins (Ig) in newborn foals. ANIMALS: Thirteen mixed breed foals. PROCEDURE: Foals were randomly assigned to two treatment groups, which were fed either a glucose-electrolyte solution or a commercial milk replacer for 12 h after birth, before being fed a known amount of colostral IgG. A control group was fed a known amount of colostral IgG from birth. The efficiency of IgG absorption was calculated following determination of plasma IgG concentration for each foal. RESULTS: Foals given colostrum immediately after birth transferred approximately 51% of ingested IgG into their vascular space. Delayed colostral ingestion significantly reduced the amount of IgG absorbed by foals. Withholding macromolecules for 12 h had no effect on the subsequent efficiency of IgG absorption. CONCLUSIONS: Colostrum should be supplied to foals within 12 h of birth for best uptake of Ig. The type of fluid administered to foals before the ingestion of colostrum does not influence subsequent absorption of Ig, suggesting that the process of gut closure in foals is not mediated by a finite capacity for macromolecular uptake.     

Antibodies to Aqx toxin of Actinobacillus equuli in horses and foals

Actinobacillus equuli is found in the normal oral flora of horses, but has been associated with several diseases, and particularly with the usually fatal septicaemia in neonatal foals which is thought to be associated with a failure of the passive transfer of immunoglobulins via the colostrum. The Aqx protein of A equuli, belonging to the RTX family of pore-forming toxins, is also cytotoxic to horse lymphocytes. The presence of antibodies to Aqx was investigated in sera from individual horses from different regions; the sera from adult horses and foals 24 hours after birth reacted with Aqx, and sera from foals sampled shortly after an intake of colostrum also reacted with Aqx, but sera from foals taken before an intake of colostrum did not react with Aqx.     

Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE

In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine V(H)186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine lambda(1) chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or isolated equine light chains, IgGc and IgA from horse serum, or the native mAb B1-8delta, expressing the same heavy chain variable regions and light chains. One of the mAbs (alphaIgE-132) recognized the recombinant equine IgE, but did not recognize any protein in equine serum, i.e. native IgE. A total of 16 mAbs detected a serum protein of approximately 210,000Da on Western blots, corresponding to the expected MW of native IgE. In addition, one of the mAbs (alphaIgE-176) detected a protein of 76,000Da under reducing conditions, most likely the equine IgE heavy chain. According to binding inhibition studies, the equine IgE specific mAbs recognize at least two different epitopes of the equine IgE. In an ELISA using two anti-IgE mAbs which recognized different epitopes, no significant differences in the concentration of total serum IgE could be detected between adult Icelandic horses with IgE-mediated type I allergy (summer eczema) and healthy control animals. In Icelandic horse foals, no serum IgE could be measured 6 months post partum. All anti-IgE mAbs recognized a small population (1.3+/-0.5%) of leukocytes from adult Icelandic horses by surface immunofluorescence, but no cells could be detected in foal blood. The stained leukocytes from adult horses could be enriched by magnetic cell sorting and contained 32% basophils, 53% monocytes and/or large lymphocytes, 13% small lymphocytes and 2% eosinophils.     

The transfer of maternal IgE and other immunoglobulins specific for Trichostrongylus colubriformis larval excretory/secretory product to the neonatal lamb

The transference of immunoglobulins from six New Zealand Romney ewes to their lambs was examined. Immunoglobulin levels were determined in ewe plasma, colostrum and lamb plasma shortly after birth and before the lambs fed, in lamb plasma 2 days after birth, and lamb plasma, ewe plasma and milk 30 days after parturition. Levels of total IgE, and IgE, IgG1, IgG2, IgM, and IgA with specificity for Trichostronglus colubriformis third stage larval secretory/excretory products (TcL3E/S) were determined. Mean levels of total IgE were three times higher in colostrum than in parturient ewe plasma while only trace amounts were detected in milk at 30 days after birth (107.7, 34.3, and 0.2U ml(-1), respectively, differences between means P< or =0.01). Mean total IgE in lamb plasma rose from being undetectable before suckling to levels comparable to those of the ewes by 2 days after birth (21.7U ml(-1)) and then declined to low levels by 30 days (0.4U ml(-1)). Total IgE levels in lamb plasma were significantly correlated with levels in ewe plasma and colostrum (r=0.91, P< or =0.01; r=0.96, P< or =0.003, respectively). The transference of TcL3E/S-specific IgE, IgG1 and IgA was substantial with mean levels of these antibodies in lamb plasma at 2 days comparable to that in parturient ewe plasma (absorbance levels in lamb plasma of 0.283, 0.537, and 0.334, respectively). Proportionally less maternal IgM and IgG2 appeared to be transferred to the lambs (absorbance of 0.112 and 0.081, respectively). Levels of TcL3E/S-specific IgE and IgG1 in lamb plasma at 2 days were significantly correlated with levels in parturient ewe plasma and colostrum (r=0.89 and 0.82, 0.85 and 0.96; all P< or =0.05, respectively). These results indicate that IgE is concentrated in ewe colostrum and that substantial amounts of maternal IgE are transferred to lambs via colostrum. Further, the results suggest that humoral immunity against gastro-intestinal nematode parasites and potentially other parasites in colostrum-fed lambs may approximate that of the ewe. The implications of the transference of humoral immunity through colostrum in ruminants for the passive protection and the development of active immunity against parasites remains to be fully explored.     

Absorption of bovine colostral immunoglobulins G and M in newborn foals

The uptake of colostral IgG and IgM, their serum half-lives, and the rates of endogenous synthesis of IgG and IgM were evaluated in 6 newborn foals fed bovine colostrum (principals) and 6 foals allowed to suckle their dams (controls). The principal foals were fed 400 ml of bovine colostrum (IgG, 10,000 mg/dl and IgM, 200 mg/dl) at 2-hour intervals, from 2 to 20 hours after foaling (total dose, 4 L). Serum IgG and IgM concentrations were determined by single radial immunodiffusion from birth to 98 days of age. At foaling, principal foals had no detectable serum equine IgG, but 1 control foal had serum equine IgG of 185 mg/dl. After ingestion of colostrum, there was no significant difference in the maximal serum bovine IgG concentration (range, 1,350 to 3,300 mg/dl) in the principal foals, and maximal serum equine IgG concentration in the control foals (range, 500 to 6,000 mg/dl). The calculated biological bovine and equine IgG half-life in the principal and control groups was 9.4 and 26 days, respectively. Endogenous IgG synthesis was first detected in 1 principal foal at 3 days of age, but was detected first between 28 and 42 days in the other principal foals. Starting on day 56 there was no significant difference in serum equine IgG concentration between groups. At foaling, foals in both groups had low equine IgM concentrations. In the control foals, there was marked individual variation in the increases in equine IgM concentration (range, 5 to 73 mg/dl) after ingestion of colostrum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum antibodies in mares and foals to Actinobacillus equuli whole cells, outer membrane proteins, and Aqx toxin

Actinobacillus equuli is carried in the alimentary tract of mares and can cause severe septicemia of neonatal foals. A hemolytic subspecies, A. equuli subsp. haemolyticus, and a non-hemolytic subspecies, A. equuli subsp. equuli, have been identified. Hemolytic strains produce the RTX toxin Aqx. The purpose of this study was to demonstrate sequentially in two sets of mare-foal pairs antibodies to A. equuli whole bacterial cells, outer membrane proteins, and recombinant Aqx and to compare the transfer of antibodies to these antigens between mares and their foals. Two mare/foal sets of sera were evaluated. Cohort A consisted of 18 mare-foal pairs obtained in the spring of 2005. Cohort B consisted of 10 mare-foal pairs obtained in the spring of 2006. For both sets, mare and foal sera were obtained immediately after foaling and prior to nursing (time 0) as well as at 12 and 24h and daily thereafter for 7 days. For Cohort B, sera were also obtained 30 days after birth. At parturition all mares had detectable antibodies to A. equuli whole cells and outer membranes; however, of those mares, two in Cohort A had undetectable antibodies to Aqx and their foals likewise had undetectable anti-Aqx antibodies. Antibodies against whole cells, outer membrane proteins, and Aqx were readily transferred from mares to foals. In most cases, there were significant correlations (p<0.05) between antibodies against whole cells, outer membrane proteins, and Aqx in mares' sera at the time of parturition and foal sera 24 after birth. Antibodies against the three antigen preparations had declined insignificantly (p>0.05) by day 30.     

Serum levels of the immunoglobulins IgG and IgG(T) in horses

Levels of the immunoglobulins IgG and IgG(T) in serum in Norwegian horses of the breeds “Døle” and “Fjord” were determined by the quantitative radial immunodiffusion test.No significant differences were apparent between the 2 Norwegian breeds. The immunoglobulin levels were approximately in the same range as previously reported for Shetland ponies.Immunoglobulins could not be detected in the newborn foal. As early as 24 hrs. after birth the mean immunoglobulin level was within the adult range. After a drop during the first month of life, the immunoglobulins increased. IgG(T) rose more rapidly and to a higher level than IgG.In 2 year old horses, IgG(T) was significantly higher than in adults, while IgG was significantly lower. IgG(T) seems to be a very important immunoglobulin in foals and young horses.     

Microcytosis, hypoferremia, hypoferritemia, and hypertransferrinemia in standardbred foals from birth to 4 months of age

At birth, 24 Standardbred foals were assigned at random to 1 of 2 groups and were given a placebo supplement (group 1) or an iron supplement (248 mg of iron/treatment; group 2). Foals were given iron supplement or placebo 4 times during the second and third weeks after birth. Hematologic variables and general health were monitored until foals were 4 months old. Mean PCV in foals of both groups decreased during the first 2 weeks after birth, but values remained within adult horse reference ranges. During the first 6 weeks after birth, foal erythrocytes were smaller than adult horse erythrocytes, but foal erythrocyte glucose-6-phosphate dehydrogenase activity was greater than that in adult horses. At every measurement, indices of anisocytosis were lower in foals, compared with adult horse reference values, suggesting that foals have a homogeneous population of microcytic erythrocytes during early foalhood. In 2-week-old foals of both groups and in 4-week-old placebo-treated foals, mean serum iron concentration was lower than that in adult horses. In foals at birth and during the first 4 months, total iron-binding capacity values were above the adult reference range. In newborn foals, transferrin saturation percentage values decreased to below the reference range in foals from 2 weeks to 4 months after birth. When foals were born, serum ferritin concentration values were above the adult horse reference range, but decreased to within the reference range by the time foals were 1 day old. From 2 through 6 weeks after birth, foal ferritin concentration values were below the adult reference range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lyophilized hyperimmune equine serum as a source of antibodies for neonatal foals

In a study with 15 neonatal foals (5 per treatment group), foals were fed within 4 hours of birth as follows: 250 ml of colostrum, 250 ml of lyophilized serum reconstituted at 5 times the original concentration, or 250 ml of a mixture (1:1) of colostrum and lyophilized serum. Foal serum samples were tested for immunoglobulin (Ig)G concentration and titrated for anti-equine rhinovirus 1 and anti-equine influenza A1 and A2 antibodies at 0 and 24 hours after foals were born. Except in a foal which had suckled the dam before treatment, there was no evidence of IgG or specific viral antibodies in the samples taken at birth. There were no significant differences found in the serum IgG concentrations and antibody titers among the 3 treatment groups. Seemingly, IgG was absorbed efficiently from both serum and colostrum, so that the use of reconstituted lyophilized serum as a prophylactic measure of conferring passive immunity to a newborn foal deserves serious consideration.     

Colostral transfer of bovine immunoglobulin E and dynamics of serum IgE in calves

The role of IgE in protective immunity is becoming understood, therefore the colostral transfer of IgE and the age-dependent changes of IgE levels may be important for neonatal immunity. To investigate this question, serum samples were collected from range-fed Hereford cows and their calves from birth through 9 months of age. The sera were assayed for total IgE by enzyme-linked immunosorbent assay (ELISA). Calves were found to have significant levels of IgE during the first week postpartum, indicating colostral transfer of IgE. Thereafter, serum levels declined rapidly within 3 weeks from birth. The IgE levels began to increase after 12 weeks of age, and in some cases reached adult levels. The passive transfer of maternal IgE through colostrum may be important in providing early protection from disease, especially against intestinal parasites.     

Interpretation of the detection of Sarcocystis neurona antibodies in the serum of young horses

Horses that are exposed to Sarcocystis neurona, a causative agent of equine protozoal myeloencephalitis, produce antibodies that are detectable in serum by western blot (WB). A positive test is indicative of exposure to the organism. Positive tests in young horses can be complicated by the presence of maternal antibodies. Passive transfer of maternal antibodies to S. neurona from seropositive mares to their foals was evaluated. Foals were sampled at birth (presuckle), at 24h of age (postsuckle), and at monthly intervals. All foals sampled before suckling were seronegative. Thirty-three foals from 33 seropositive mares became seropositive with colostrum ingestion at 24h of age, confirming that passive transfer of S. neurona maternal antibodies occurs. Thirty-one of the 33 foals became seronegative by 9 months of age, with a mean seronegative conversion time of 4.2 months. These results indicate that evaluation of exposure to S. neurona by WB analysis of serum may be misleading in young horses.     

A Fresh Look at the Process of Arriving at a Clinical Prognosis. Part 3: Neonatal Illness

Determining the prognosis in a sick newborn foal is complicated by the following three factors: the normal transition from maternal/placental dependence to independence, intrauterine conditions before birth, and the birth process itself. In addition, there are several vulnerabilities and unique characteristics of the neonatal foal that are not shared by older foals or adult horses. However, the same principles of assessing homeostasis apply to establishing a prognosis in a sick neonate, as previously described for the adult. Overall, survival rates for septic critically ill foals generally vary between 60% and 80%. In noncritical yet seriously ill foals—a diverse group sometimes referred to as the “sick, nonseptic foal”—survival rates typically range from 75% to 95% with proper medical care. Long-term survival rates are lower for all categories of sick foals, and subsequent athletic performance may be adversely affected by sepsis or septic arthritis/osteomyelitis.     

Complement activity and selected hematologic variables in newborn foals fed bovine colostrum

Serum complement activity and selected hematologic variables were evaluated in 5 newborn foals fed bovine colostrum (principal group) and 6 foals allowed to nurse their dam (control group). Also, bovine colostrum was evaluated for anti-equine antibodies. Precolostral serum hemolytic and conglutinating complement activities were low and increased similarly in foals of both groups to reach adult values between 1 and 3 weeks after birth. Bovine colostrum strongly agglutinated, but did not hemolyse principal foals' RBC and blood containing all known equine blood group alloantigens. Hemolysis was not detected after administration of bovine colostrum. Physiologic anemia developed in foals of principal and control groups during the first week of life. Erythrocyte osmotic fragility in foals of the principal group prior to and after the ingestion of colostrum remained unchanged. However, at 36 hours after birth, there was a significant decrease in erythrocyte osmotic fragility in foals fed homologous colostrum.     

Quantitative Determination of Equine Alkaline Phosphatase lsoenzymes in Foal and Adult Serum

Automated and semiautomated assays were developed and validated for the determination of equine alkaline phosphatase isoenzymes including intestinal (IALP), bone (BALP), and liver (LALP). The addition of levamisole selectively inhibited more than 97%of LALP while inhibiting only 55% of IALP. Because these percentages were highly reproducible in an automated system, the IALP activity could be calculated in a sample. Bone alkaline phosphatase isoenzyme was selectively precipitated by adding an equal volume of wheat germ agglutinin (5 mg/mL), incubating for 30 minutes at 37C, and centrifugating. The LALP activity was determined from the supernatant fluid and BALP activity was calculated by subtraction from total ALP activity. The within-run coefficient of variation for determination of BALP activity was 4.7%.These assays were used to identify and quantify the isoenzymes present in pony foal sera through the first 21 days of life, in horse foal sera before colostrum ingestion and during the first 21 days of life, and in adult horse and pony sera. Intestinal ALP activity was not found in sera of any of the foals or adult ponies or horses. A majority of serum ALP activity of newborn foals is of bone origin (80 to 92%)which decreases markedly over the first 21 days. In adults, only 17.9% (51.2 ± 18.1 U/L) of serum ALP is derived from bone. The absolute LALP activity in foal serum is similar to that in adults.     

Hypogammaglobulinemia predisposing to infection in foals.

Measurement of serum immunoglobulins in 46 foals less than 2 weeks old revealed 9 foals with hypogammaglobulinemia. The hypogammaglobulinemia was attributed to failure in transfer of immunoglobulins from dam to foal via colostrum. Three of the affected foals did not nurse at all, or only slightly, and 2 of these died of infections within a few days after birth, whereas the 3rd foal did not grow as well as normal foals. Six of the affected foals nursed in an apparently normal manner, and 5 of these had nonfatal respiratory infections between 2 and 5 weeks of age. Analysis of serum samples from surviving foals demonstrated that immunoglobulins were eventually produced. One other foal examined had hypogammaglobulinemia at 57 days of age, an age when the foal should have produced large amounts of immunoglobulin independent of passive transfer. This foal had simultaneous infections and hypogammaglobulinemia, but eventually produced normal amounts of immunoglobulin. Cellmediated immunity was normal at 3 months of age. This condition was designated transient hypogammaglobulinemia and was thought to be due to a temporary inability to make immunoglobulins.     

Transfer of tumour necrosis factor-α via colostrum to foals

This study aimed to determine whether TNF-α is transferred to equine neonates via colostrum and the relationship between TNF-α and IgG concentrations in the equine neonate. Colostrum, presuckle and postsuckle foal serum samples were collected from healthy mares and their foals. Equine TNF-α ELISA and IgG SRID kits were used to determine the concentrations of TNF-α and IgG, respectively. Statistical analysis was performed using the Spearman rank correlation. TNF-α concentrations in all presuckle foal serum were below the limit of detection in 15/16 foals and increased in postsuckle foal serum to a mean concentration of 7.7 x 10(4) pg/ml. TNF-α concentrations in postsuckle foal serum and colostrum showed significant correlation (rho=0.668; P=0.005). However, TNF-α and IgG concentrations in colostrum or postsuckle foal serum did not correlate (rho<-0.016; P>0.05). Ratios of TNF-α/IgG in colostrum or postsuckle foal serum showed significant correlation (rho=0.750; P=0.0008). These results indicate that TNF-α is transferred to the foal via colostrum absorption and may play a role in early immunity.