Ovine anti-rabies antibody production and evaluation

In view of the disadvantages of human and equine rabies immunoglobulin still there is urgent needs for safe and cost-control anti-rabies immunoglobulins especially for person who have been severely exposed (categories III) to the virus. Our attempt to produce a less immunogenic and cheaper anti-rabies immunoglobulin affordable for those people living in developing countries, has been harnessed the ovine as a bioreactor instead the horse. The animals have been intramuscular immunized, and the plasma processed with 5% caprylic acid to yield IgG with purity of 95%. Moreover, antibody apparently indicated that the titer and neutralizing indexes were harmonized, especially at higher antibody dilution. The results showed that three immunized sheep were produced about 7000 IU of purified anti-rabies antibody. Sheep's IgG has low immunogenic effect than human and horse antibodies when injected into the mouse. Pure concentrated ovine antibody may serve as a possible alternative to currently available anti-rabies human or equine immunoglobulin.     

Jacalin: its binding reactivity with immunoglobulin A from various mammalian species

Jacalin, a recently described lectin, has been demonstrated to have unique binding properties for some forms of human immunoglobulin A (IgA). The lectin has now become commercially available, immobilized to a beaded agarose support. This laboratory undertook a short study to determine whether this immobilized jacalin demonstrated any binding capacity for IgA from various mammalian species, with a view to utilizing this new form of affinity chromatography to simplify the purification of IgA from these species. Utilizing the chromatographic conditions recommended by the manufacturer, the bound jacalin demonstrated no IgA reactivity from pig, goat, horse, cow or dog.     

Characterization of a homogeneous paraprotein from a horse with spontaneous multiple myeloma syndrome

A novel myeloma paraprotein has been isolated from a horse with a lymphoid tumor. The protein was a euglobulin and consequently was readily isolated from serum in pure form and high yield by simple dilution in distilled water. The purified intact protein had a molecular weight of 150,000 and was composed of heavy and light chains, both of which had blocked amino-termini and were thus not susceptible to amino-terminal sequence analysis. The amino acid compositions of these respective chains corresponded to those of comparable chains from immunoglobulins of other species. Peptide maps of paraprotein light chains prepared by high pressure liquid chromatography corresponded in part to those of normal pooled equine light chains. The identification of this paraprotein as an equine AI (aggregating immunoglobulin) protein was confirmed by serological analysis using a specific antiserum. The relationship of this particular protein to other members of the immunoglobulin family was further demonstrated by the production of an anti-idiotypic antiserum individually specific for this molecule.     

Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p‐hydroxydiazepam [p‐OH‐DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p‐OH‐DZP was significantly less in the horse. Inhibition assays with a CYP‐specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos‐7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.     

Comparative expression of liver cytochrome P450-dependent monooxygenases in the horse and in other agricultural and laboratory species

The apoprotein expression and the catalytic activities of cytochrome P450s involved in the biotransformation of xenobiotics were investigated in horse liver microsomes and compared with those of food producing (cattle, pigs, broiler chicks, and rabbits) and laboratory species (rats). Western blot analysis revealed the presence of proteins immunorelated to rat CYP 1A, CYP 2B, CYP 2E, and CYP 3A subfamilies in hepatic microsomes from horses and from any other examined species. With the exception of the N-demethylation of N-nitrosodimethylamine in broiler chicks, all the recorded interspecies differences were quantitative in nature. Equine preparations proved the most active in the biotransformation of the CYP 1A substrates ethoxy- and methoxyresorufin and the least active in the metabolism of aminopyrine and ethoxycoumarin. On a comparative basis, large differences were observed in the rate of the in vitro metabolism of model substrates between "minor" (rabbits, horses) and "major" food producing species. Taken in due consideration the limitations of the in vitro approach, results from this study reinforce the conclusion that studies on drug efficacy and residue depletion should be performed in each target species.     

Immunodeficiency associated with lymphosarcoma in a horse.

Immune system dysfunction and immunoglobulin deficiency was diagnosed in a 2-year-old horse with disseminated lymphosarcoma. Prolonged (35 days) parenteral nutrition was delivered to support the horse during a period in which immune function studies could be performed. Correction of nutritional compromise by use of parenteral nutrition did not correct the immunoglobulin deficiency, and results of lymphocyte phenotype testing did not indicate abnormal proportions of leukocytes. Lymphoblast transformation studies were suggestive of a circulating immunosuppressive factor in the horse's serum. Normal cell function was detected when the cells were stimulated in precolostral equine serum.     

Change of antibody levels to ferritin in the sera of foals after birth: Possible passive transfer of maternal anti‐ferritin autoantibody via colostrum and age‐related anti‐ferritin autoantibody production

Antibody (immunoglobulin G (IgG), IgM or IgA) levels relative to ferritin in six foal sera (three male and three female) after birth (day 0 and 2, 6, 10, 20, 28, 36, 40, 52 and 56 weeks of age) were semi‐quantitatively measured with normalization with antibody activity to ferritin in one adult horse serum. After addition of horse spleen ferritin to the serum sample, the complex formed between antibodies to ferritin in the serum and ferritin was co‐immunoprecipitated using antibody to horse spleen ferritin. Antibody classes of the co‐immnoprecipitate were detected with antibodies specific for horse IgG, IgM or IgA heavy chain. Six adult horse serum samples were found to have ferritin‐binding activities in all immunoglobulin classes examined. Although ferritin antibody activities (IgG, IgM and IgA) were scant in the foal sera before sucking colostrum (day 0), their activities increased at 2 weeks of age. IgG antibodies showed a biphasic response and IgM antibody activity increased up to 40 weeks of age. Antibody (IgG, IgM and IgA) activities to ferritin in three colostrum samples were significantly higher than in adult horse serum samples. These results demonstrate that antibody to ferritin in foal serum is derived from colostrum after birth and is produced thereafter.     

Single-dose pharmacokinetics of detomidine in the horse and cow

The pharmacokinetics of detomidine, a novel analgesic sedative, was studied in the major target species after high (80 micrograms/kg) i.v. and i.m. doses. In addition, drug residues in some organs were determined. Concentrations were measured using a sensitive, detomidine-specific radio-immunoassay method. Rapid absorption following i.m. dosing occurred. Absorption half-lives were 0.15 h (horse) and 0.08 h (cattle). The mean peak concentration in the horse (51.3 ng/ml) was achieved in 0.5 h and in the cow (65.8 ng/ml) in 0.26 h. The areas under the concentration curve after i.m. dosing were 66% (horse) and 85% (cow) of the corresponding i.v. values. Distribution was rapid with half-lives of 0.15 h (horse, i.v.) and 0.24 h (cow, i.v.). The apparent volume of distribution was higher after the i.m. dosing (horse 1.56 l/kg, cow 1.89 l/kg) than after i.v. dosing (horse 0.74 l/kg, cow 0.73 l/kg). Elimination half-lives were 1.19 h (horse) and 1.32 h (cow) for the i.v. dose and 1.78 h (horse) and 2.56 h (cow) for the i.m. dose. Total clearances ranged from 6.7 (horse, i.v.) to 12.3 (cow, i.m.) ml/min/kg. Renal clearances were less than 1% of the total clearances showing negligible excretion of the drug in urine and suggesting elimination by metabolism. A cross-reacting metabolite in urine corresponded to less than 1.5% of the detomidine dose's immunoreactivity. High-dose detomidine increased urine flow significantly. Excretion of detomidine in milk in cattle was extremely low. No detectable amounts were present 23 h after dosing.(ABSTRACT TRUNCATED AT 250 WORDS)     

Agammaglobulinemia in a horse with evidence of functional T lymphocytes.

Agammaglobulinemia was diagnosed in a 1-year-old Thoroughbred horse on the basis of the following observations: (1) absence of serum immunoglobulins M, A, and G(T); (2) small amounts of serum immunoglobulin G (16 mg/100 ml); (3) absence of specific antibody in the serum of the horse following immunization and challenge exposure to 2 antigens; (4) absence of plasma cells, primary follicles, and germinal centers in a lymph node removed after antigenic stimulation; (5) absence of "natural" serum antibodies to rabbit-erythrocytes which were easily detectable in age-matched control horse serums; and (6) increased susceptibility to infections. There was evidence of functional cell-mediated immunity which included a skin response to injected phytolectins, skin response to antigen challenge following sensitization, and in vitro proliferative response of lymph node cells to phytohemagglutinin. An intact cell-mediated immune response was also supported by the observation that the horse lived to 17 months of age without antibody production, whereas horses with an absence of both antibody production and cell-mediated immunity (combined immunodeficiency) die by 4 months of age without immunologic intervention. The known features of agammaglobulinemia in this horse are similar to those in sex-linked agammaglobulinemia in persons and are unique among the immunodeficiences described in other animals.     

Gastrointestinal stromal tumour and hypoglycemia in a Fjord pony: Case report

Background

Neoplasia may cause hypoglycemia in different species including the horse, but hypoglycemia has not previously been reported in the horse associated with gastrointestinal stromal tumours.

Case presentation

A case of a gastrointestinal stromal tumour in a Fjord pony with severe recurrent hypoglycemia is presented. The mechanism causing the hypoglycemia was not established.

Conclusion

This case indicates that a gastrointestinal stromal tumour may cause hypoglycemia also in the horse.     

A Study on the Presence of Ferritin-binding Proteins in Fetal Horse Plasma

In mammal circulation, ferritin-binding proteins (FBPs) are thought to be involved in clearance of circulating ferritin after complex formation with it through receptor-mediated uptake. However, there is no report on fetal FBP in fetal circulation. Although iron concentrations of fetal horse plasma were higher than those of adult horse plasma, plasma ferritin concentrations and ferritin-binding activities were found to be significantly lower in fetus than in adult. FBPs were purified from fetal or adult horse plasma on horse spleen ferritin-Sepharose 4B affinity column. Partially affinity-purified fetal horse plasma FBPs were mainly separated into 65 and 41 kDa bands in addition to minor bands with higher molecular masses ranged from 102 to 140 kDa on SDS-PAGE under reducing condition. The adult horse plasma FBPs were separated into 74, 54 and 28 kDa bands, and the 74 and 54 kDa bands reacted with antibodies specific for horse IgM and IgG heavy chains, respectively, by immunoblotting analyses. On the other hand, no antibodies to horse immunoglobulin classes detected any bands in fetal horse plasma FBPs. The affinity-purified adult and fetal horse plasma FBPs did not contain fibrinogen as a plasma specific FBP, probably due to its lower affinity to the ligand ferritin. These results demonstrate the presence of FBPs which are different from adult horse plasma FBPs including anti-ferritin autoantibodies in fetal plasma.     

Enzyme-linked immunosorbent assay for detection of equine infectious anemia antibody to purified P26 viral protein

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of equine infectious anemia (EIA) antibody in horse sera. Purified P26 viral protein was the antigen; alkaline phosphatase linked to rabbit anti-horse immunoglobulin G was the conjugate. The ELISA detected EIA antibodies in horse sera as early as 11 to 14 days after experimental inoculations. There was full agreement between the results of ELISA and the agar-gel immunodiffusion tests on EIA proficiency test sera. The ELISA readily detected EIA antibody in horse sera that had weak positive reactions on agar-gel immunodiffusion.     

Comparison of Sarcocystis neurona isolates derived from horse neural tissue

Sarcocystis neurona is a protozoan parasite that can cause neurological deficits in infected horses. The route of transmission is by fecal-oral transfer of sporocysts from opossums. However, the species identity and the lifecycle are not completely known. In this study, Sarcocystis merozoites from eight isolates obtained from Michigan horses were compared to S. neurona from a California horse (UCD1), Sarcocystis from a grackle (Cornell), and five Sarcocystis isolates from feral opossums from Michigan.Comparisons were made using several techniques. SDS-PAGE analysis with silver staining showed that Sarcocystis spp. from the eight horses appeared the same, but different from the grackle isolate. One Michigan horse isolate (MIH6) had two bands at 72 and 25kDa that were more prominent than the UCD1 isolate and other Michigan horse isolates. Western blot analysis showed that merozoites of eight of eight equine-derived isolates, and the UCD1 S. neurona isolate had similar bands when developed with serum or CSF of an infected horse. Major bands were seen at 60, 44, 30, and 16kDa. In the grackle (Cornell) isolate, bands were seen at 60, 44, 29, and 16kDa. DNA from merozoites of each of the eight equine-derived isolates and the grackle-derived isolate produced a 334bp PCR product (Tanhauser et al., 1999). Restriction fragment length polymorphism (RFLP) analysis of these horse isolates showed banding patterns characteristic for S. neurona. The grackle (Cornell) isolate had an RFLP banding pattern characteristic of other S. falcatula species. Finally, electron microscopy examining multiple merozoites of each of these eight horse isolates showed similar morphology, which differed from the grackle (Cornell) isolate. We conclude that the eight Michigan horse isolates are S. neurona species and the grackle isolate is an S. falcatula species.     

Analysis of propionylpromazine and its metabolites in horse urine

The metabolism of propionylpromazine in the horse was studied. Although propionylpromazine is not currently approved or recommended for use in horses, it has been used illegally to alter their performance. Propionylpromazine hydrochloride was administered intramuscularly at clinical and subclinical doses. Three metabolites were detected in urine. The major metabolite was identified as 2-(1-hydroxypropyl) promazine sulfoxide. The detection of this metabolite in routine drug testing has been described.     

Pitfalls in immunofluorescence testing in dermatology. III. Pemphigus-like antibodies in the horse and direct immunofluorescence testing in equine dermatophilosis

Indirect immunofluorescence testing for pemphigus-like antibodies was performed on 79 horses: 28 horses with various nonpemphigus dermatologic diseases, 21 horses with various nondermatologic diseases, and 30 normal horses. Pemphigus-like antibodies were detected in 6 horses: 3 normal horses with titers of 1:40, 2 horses with dermatophilosis at titers of 1:10 and 1:80, and 1 horse with lymphosarcoma at a titer of 1:320. It was concluded that equine pemphigus-like antibodies are a potential source of misinterpretation and misdiagnosis in indirect immunofluorescence testing. Direct immunofluorescence testing for whole immunoglobulin, IgG, IgM, and IgA was performed on skin lesions from 2 horses with dermatophilosis. Diffuse intercellular deposition of whole immunoglobulin and IgG was found in both horses. It was concluded that equine dermatophilosis is a potential source of misinterpretation and misdiagnosis in direct immunofluorescence testing.     

A Comparative Review of Vitamin E and Associated Equine Disorders

Vitamin E is a primary chain‐breaking antioxidant that prevents cyclic propagation of lipid peroxidation. Across species, vitamin E is essential for normal neuromuscular function by acting as a potent antioxidant, as well as by modulating the expression of certain genes, inhibiting platelet aggregation and stabilizing plasma membranes. This review focuses on vitamin E structure, absorption, metabolism, current equine dietary recommendations, the interplay between antioxidants and exercise, a discussion of the necessity of vitamin E supplementation in the horse above the Nutritional Research Council (NRC) 2007 requirements, and a review of equine diseases that are associated with a vitamin E deficiency. Particular emphasis is placed on the proteins involved in vitamin E absorption, transport, and metabolism as potential candidates for vitamin E‐associated diseases across species.     

Effects of dietary horse beans (Vicia faba) on colostrum and milk composition and milk yield in sows

In an experiment sows received 0, 17 and 34% horse beans in the diet. Horse beans replaced the protein mixture (soybean meal, fishmeal, and meat and bone meal) by one half or in full.The composition of colostrum was altered by the dietary horse beans. An increase in dietary horse beans significantly reduced dry matter contents (P ≤ 0.05) and protein contents (P ≤ 0.001). As to the various protein fractions, total whey protein and albumin showed the greatest decline (P ≤ 0.001), followed by immunoglobulin (P ≤ 0.01) and beta lactoglobulins (P ≤ 0.05). Casein and alpha lactalbumin contents were not altered significantly.The total fat content of colostrum was not influenced by dietary horse beans, but the percentages of C 18:1 increased (P ≤ 0.001) and C 18:2 decreased (P ≤ 0.001) with increasing amounts of horse beans in the diet.The effect of horse beans on the composition of sow milk was insignificant, although there was a tendency to higher dry matter and protein content in milk from sows fed diets without horse beans.Horse beans at both levels had a negative effect on milk yield (linear trend: P ≤ 0.05).     

Detection of equine immunoglobulin-secreting cells by a plaque assay.

A protein A-hemolytic plaque assay was applied to detect immunoglobulin (Ig)-producing cells in horse peripheral blood, using pokeweed mitogen as a B lymphocyte activator. A maximum number of Ig-secreting cells was obtained when horse peripheral blood lymphocytes were cultured in a medium containing horse serum. The number of Ig-secreting cells in young horses (2 years old) was lower than that in adult horses (6 to 23 years old). In addition, the plaque formation was unchanged from blood samples kept at 4 degrees C for 24 hours, while blood samples kept for 72 hours did not yield plaques. These results indicate that the plaque assay is a reliable and useful method for detecting Ig-secreting cells in the peripheral blood of the horse.     

Supplemental fat in the diet of horses...is it advantageous?

Feeding fat-supplemented diets to horses has drawn considerable interest. One of the advantages of such diets is that the energy density is increased, so that less feed is needed to meet energy requirements. In addition, adding fat to the diet enhances the contribution of fat oxidation to energy production, thus sparing muscle glycogen. The 'spared' glycogen is available for energy metabolism when the acutely exercising horse reaches a point of oxygen deficit and must rely on anaerobic metabolism. This appears to be beneficial for both aerobic and anaerobic performance. Fats are readily digested by the horse. Vegetable oils are more palatable to horses than animal fats, but the palatability of fat-rich diets may decrease in the long term.