[The therapy of equine sarcoid with a non-specific immunostimulator--the epidemiology and spontaneous regression of sarcoids]. / Zur Therapie des Equinen Sarkoids mit einem unspezifischen Immunstimulator-Beitrag zur Epidemiologie und zur spontanen Regression des Sarkoids.

20 sarcoid-affected horses from a practice in the northern Jura were used in this experiment. The mean age of the 20 horses was 3.9 years at the time of the first observation of sarcoid tumors. On the average, 4.4 tumours were noted per horse. 10 of the horses were treated in a double-blind study with an unspecific immunostimulant (Baypamun P), 10 others received a placebo. One single tumour only was treated per horse. The injections were given under and around the sarcoid. In eight out of the 20 horses all tumours regressed totally or for more than 50% of their initial size. Five of these had received placebo, three the immunostimulant. Four animals showed a modest, but measurable reduction in tumour size (3 immuno-stimulant, 1 placebo) and in the remaining eight horses (4:4) no reduction or even an increase in tumour size was observed. The phenomenon of tumour regression was very probably due to spontaneous regression and horses which had been observed to develop sarcoid within the last three months had significantly more regressions than animals with older tumours (p < 0.05). The haplotype of the equine leucocyte antigens was thought to predispose 12 of the 20 horses for the sarcoid. However, the ELA-type did not measurably influence the phenomenon of tumour regression.

Tumour suppressor gene p53 in the horse: identification, cloning, sequencing and a possible role in the pathogenesis of equine sarcoid.

The tumour suppressor protein p53 enhances the genetic stability of the cell and plays a critical role in tumour suppression. Equine p53 was analysed by sequencing exons 5 to 9, a region which includes most known mutations and all the mutational hotspots in the species that have been investigated. The fragment was amplified, cloned and sequenced from genomic and complementary DNA. A comparison of the predicted amino acid sequences between the horse and other species resulted in identities between 66 per cent with the clawed frog and 92 per cent with the cat. Using the single strand conformation polymorphism technique, exons 5 to 8 amplified from sarcoid tissue and peripheral leucocytes of 28 sarcoid-affected and 11 healthy horses were screened for mutations. No mutations were identified, suggesting that the frequency of p53 mutations in equine sarcoid might be low. However, the high incidence of bovine papillomavirus (BPV) infection in equine sarcoid may indicate the functional inactivation of p53 by BPV-encoded E6 protein.

Experimental treatment of equine sarcoid using a xanthate compound and recombinant human tumour necrosis factor alpha.

A xanthate compound with antiviral and antitumoural activities, tricyclodecan-9-yl-xanthogenate (D609) in combination with the potassium salt of the lauric acid (KC12) and, in a further investigation, the above-mentioned substances together with recombinant human TNF alpha (rh-TNF alpha), were tested on equine sarcoid tumours for therapeutic efficacy. A pilot investigation on 5 healthy horses showed that the compounds were well-tolerated; apart from a local, temporary oedema at the injection site, no other clinical symptoms were observed after subcutaneous administration of volumes from 0.1 to 10 ml per injection. The tested concentrations of D609 and KC12 (5 mg/ml solution) and of rh-TNF alpha (50 micrograms/ml) were used for the treatment experiments. The repeated injections of the compounds to 11 sarcoid affected horses were also well-tolerated, except by one horse. In this case the treatment had to be interrupted after two injections because of severe reaction, i.e. fever and lameness due to oedemas. Five horses (n = 6 sarcoids) were treated by local, subcutaneous injection of D609 and KC12 under the tumour at intervals of 3 weeks. On one periocular sarcoid the compounds were applied as an ointment. After a follow-up period of 18 months, 5 tumours did completely regress and one remained unchanged. The periocular tumour showed a reduction in size. Five horses (n = 9 sarcoids) were then treated with a combination of D609, KC12 and rh-TNF alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the frequency and associations of equine leucocyte antigens in sarcoid and summer dermatitis.

The equine leucocyte antigen (ELA) types and the clinical diagnosis for equine sarcoid and summer dermatitis were evaluated in 2026 horses representing five breeds. Data were analysed in unrelated animals and in family material. In the case of equine sarcoid, a strong association was observed between the ELA class II DW13 antigen and its effect on Swiss (cP < 0.001), French (cP < 0.0001) and Irish (cP < 0.01) Warmblood horses. The class I antigen A3 occurred more frequently in sarcoid-affected French horses (cP < 0.001). These results confirm our earlier findings (Gerber et al. 1988). Among Freiberger horses, which lack the ELA DW13 and A3 specificities, a breed-specific class I antigen, ABe108, displayed an increased frequency (cP < 0.05) in the affected group. Among Arabian horses, a tendency for increased frequency of the A1 antigen was observed in the affected animals, but the number of affected horses is too small for statistical significance. The Mendelian segregation in diseased half-siblings by ELA DW13 heterozygous stallions showed a strong association (P < 0.0001) between the inherited DW13 antigen and susceptibility to the sarcoid effect. In the case of summer dermatitis, previously published data (Marti et al. 1992) have been extended. The ELA types in four multiple-case families, founded by the same stallion, were analysed for an association with the effect of sarcoid. Eight out of nine ELA-typed affected offspring inherited the paternal haplotype A15, DW23 in contrast to nonaffected offspring where three out of 12 displayed these antigens (P < 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Immune functions of veal calves fed low amounts of iron.

Immune functions were studied in male calves fed milk replacer (MR) containing 10 or 50 mg iron (Fe)/kg. Calves fed 10 mg Fe/kg MR developed marked hypoferremia and anemia, whereas serum-Fe and haemoglobin concentration of calves fed 50 mg Fe/kg MR were normal. Growth performance was reduced, while feed/gain ratio, incidence of infections (especially pneumonias), febrile body temperatures and antibiotic treatments were higher in calves fed 10 than 50 mg Fe/kg MR (p < 0.05). Whereas antibody production (to horse erythrocytes) and lymphocyte stimulation (by mitogens) were not significantly altered, cell-mediated immunity (measured as cutaneous delayed-type hypersensitivity reaction to dinitrofluoro-benzene), number of neutrophils with phagocytic capacity, activity of the Fe-containing enzyme myeloperoxidase, blood serum IgG concentration and the number and diameter of germinal centres as a measure of the number and production of B-cells in cervical superficial lymph nodes in calves fed 10 mg Fe/kg MR were reduced when compared with calves fed 50 mg Fe/kg MR (p < 0.05). In conclusion, severe Fe deficiency caused reduced growth performance, associated with and partly due to higher incidence of infections because of defective immune reactions.

DNA of bovine papillomavirus type 1 and 2 in equine sarcoids: PCR detection and direct sequencing.

Nucleotide sequences of bovine papillomavirus (BPV) DNA amplified by the polymerase chain reaction (PCR) from samples of equine sarcoid skin tumours were determined. All naturally occurring sarcoids (n = 58 tumours from 32 horses and 2 donkeys) contained BPV-DNA. All but 3 of the genome fragments belonged to the BPV type 1 strain (BPV-1); the remaining were BPV type 2. Similar results were obtained with cutaneous bovine papillomas used as controls (n = 20). One of the horses, carrying 2 sarcoids, was particularly interesting; one tumour contained BPV-1 DNA whilst the other sarcoid yielded BPV-2 DNA, suggesting that horses are not immune to super-infection. BPV-DNA was even amplified from the sarcoid samples which had yielded negative results in previous investigations when DNA isolated from the lesions was used in Southern blot hybridization with BPV probes. In addition, there was no detectable BPV-DNA in any equine or bovine tissue examined other than sarcoids or cutaneous bovine papillomas. Biopsies of normal skin surrounding lesions yielded exclusively negative results. The described nucleotide differences represent a natural genomic variation of this BPV type between geographically distant locations. The identical variations recovered from cattle and horses in Switzerland, a finding of great epidemiological interest, strongly suggest that a uniform variant of BPV-1 is one of the etiologic agents of equine sarcoid and bovine papilloma in a given region.

Characterization of BPV-like DNA in equine sarcoids.

The DNA from equine sarcoid samples from New York State and Switzerland was isolated and probed with bovine papillomavirus type 1 (BPV-1) to determine if BPV genomes were present. Twelve of 13 sarcoids from New York State and 17/20 sarcoids from Switzerland contained DNA that hybridized to the BPV-1 probe. Restriction enzyme analysis of the positive samples demonstrated restriction fragment profiles characteristic of BPV-1 in 22 sarcoids and restriction fragment profiles characteristic of bovine papillomavirus type 2 (BPV-2) in 7 sarcoids. In addition, three tissues histologically diagnosed as pyogranulomatous dermatitis, fibropapilloma, and fibrosarcoma contained BPV-like DNA. Tissues with BPV-1-like and BPV-2-like DNA contained an average of 285.7 (21 to 808) and 125.8 (2 to 762) BPV-like genomes per cell, respectively. Minor differences in the restriction fragment profiles of the BPV-like DNA and evidence for partial BPV-like genomes were found in some sarcoids. BPV-like DNA was not detected in lymphocyte DNA from sarcoid-affected horses. These results confirm previous observations and support the hypothesis that bovine papillomavirus, or a very similar virus, is linked to the cause of equine sarcoid.

Association between equine leucocyte antigens (ELA) and equine sarcoid tumors in the population of Swedish halfbreds and some of their families.

The distribution of equine leucocyte antigens (ELA) in Swedish Halfbreds affected by sarcoid tumors was determined and compared with that of control horses of the same breed. ELA-haplotype A3W13 appeared more frequently in affected horses, resulting in a chi 2 value of 4.45 (P = 0.034) for A3 and 9.05 (P = 0.0026) for W13, respectively. The relative risk factor (RR) could be estimated to 2.13 and 3.00 for A3 and W13, respectively. The etiology fraction (EF) was calculated to 28% and 37% for A3 and W13, respectively. Thus, in the population of Swedish Halfbreds approximately 40% (at least) of the disease appeared to be associated with the genetic background of the affected horse. Family studies established that ELA are codominantly expressed and inherited as simple Mendelian traits and that sarcoids among offspring are significantly associated with one of the parental haplotypes (P = 0.00942). This parental haplotype does not always include A3W13. These results confirm and extend previous results from other breeds and strongly suggest the existence of a predisposition for sarcoids among horses, that is due to an autosomal, dominant, ELA-linked gene with incomplete penetrance. In extension, this indicates a multifactorial etiology of equine sarcoids (additional non-MHC gene(s) and/or environmental factors).

Evidence for linkage between the caprine leucocyte antigen (CLA) system and susceptibility to CAE virus-induced arthritis in goats.

The distribution of caprine leucocyte antigens (CLA) in goats from four different breeds (n = 546) affected by caprine arthritis-encephalitis virus (CAEV)-induced arthritis were determined and compared breed for breed with those of infected but clinically healthy controls (n = 402). Differences in frequencies of some of the CLA specificities between the affected and control groups were found, but after correction of the ordinary P values for number of observed alleles, only the CLA Be7 specificity in the Saanen breed showed a significant deviation at the 0.05 probability level. Animals of the Saanen breed carrying this specificity are less prone to develop arthritis after CAE virus infection than goats lacking this specificity. Eleven groups (multiple-case families or halfsibling groups with at least two informative diseased offspring/group) were analyzed for manifestation of the disease and segregation of the parental haplotypes. The results of the maximum likelihood test of association (P less than 0.005) and the calculated high lod score value of 5.70 give evidence for linkage between the locus encoding the determined class I CLA alleles and a hypothetical locus (i) coding for genes responsible for arthritis resistance/susceptibility. The particular class I CLA allele associated with the disease susceptibility varied from family to family, however. These data provide the first evidence that CAE virus-induced arthritis in the goat is genetically influenced by the MHC system; they also suggest that susceptibility/resistance genes are not directly associated with the determined class I gene products but rather are in close genetic linkage.

Equine leukocyte antigens: relationships with sarcoid tumors and laminitis in two pure breeds.

Frequencies of equine leukocyte antigen distribution were determined by complement-mediated cytotoxicity testing among populations of Thoroughbred and Standardbred horses, including animals affected with equine sarcoid and laminitis. A highly significant association is described between the presence or history of sarcoid lesions in Thoroughbreds and the expression of the major histocompatibility complex (MHC)-encoded antigens, W3 and B1. No association was found between antigenic expression frequencies and laminitis in either breed. These findings suggest that a strong relationship exists between the equine MHC and a predisposition to sarcoid.

Equine leucocyte antigens in sarcoid-affected horses.

The distribution of equine leucocyte antigens (ELA) in horses affected by equine sarcoid tumours was determined and compared with unaffected controls. ELA-haplotype W3,B1 occurred more frequently in affected riding horses of Irish, Swiss and French background. The combined data for the three breeds resulted in a chi 2 value of 20.35 (P less than 0.0005 after correction). Simultaneously, ELA-specificity W11 was more frequently found in horses of Irish background, while W5 was found in Swiss and French horses with sarcoids. The combined data for haplotype W3,B1 and/or W5 specificity demonstrated, in the genetically closely-related Swiss and French horses, a highly significant difference in the occurrence of these haplotypes between affected horses and healthy controls (chi 2 = 28.69, P less than 0.00001 after correction). Thus, the results strongly suggest that the predisposition of horses to sarcoid is associated with or linked to the major histocompatibility complex.

Equine leukocyte antigen system. I. Serological studies.

Lymphocytotoxic alloantibodies have been recognized in primiparous mare sera and colostra using the two-step microcytotoxicity test. The antigens detected on the leukocyte membrane do not occur on the erythrocytes. After testing on a cell panel from unrelated horses, absorptions with leukocytes were carried out. The subsequent retesting of the serum reagents on a cell panel including cells from 24 families rendered possible a grouping of preliminary monospecific reagents characterizing 18 antigen specificities on the cell membrane.

Lymphokines. VI. Factors in human and other heterologous sera inhibiting the migration of guinea pig macrophages.

Different heterologous sera including human, rabbit, bovine and fetal calf serum (FCS) showed a strong inhibiting effect on the migration of guinea pig peritoneal macrophages (GPPM), compared to the migration of GPPM in homologous guinea pig serum. The inhibiting effect of these sera on the migration of horse monocytes on the other hand was much less marked. Fractionation of human and rabbit serum showed the 4 S fraction to be most inhibitory on GPPM migration. The migration inhibiting effect of heterologous sera on GPPM was prevented by addition of homologous (GP) serum, by absorption of the sera by various guinea pig cells, by heating at 56 degrees and by addition of alpha-L-fucose. Human sera were found to be strongly cytotoxic for 51Cr-labelled guinea pig erythrocytes and lymphocytes, but not for horse lymphocytes. Upon absorption with guinea pig erythrocytes, lymphocytes and kidney cells, the cytotoxicity of the human sera was strongly reduced. Accordingly, the role of heterophilic antibodies and/or of heat-labile MIF-like factors in heterologous sera has to be considered in al macrophage migration experiments where heterologous sera are being used. These factors may differ from the heat-stable MIF activity generated upon antigen- or mitogen-induced stimulation of cultured lymphocytes.

Lymphokines. II. Use of horse monocytes as indicator cells for human MIF.

Peritoneal exudate macrophages in guinea pigs and peripheral blood monocytes in man are the most readily available cells sensitive to the migration-inhibiting factor(s) (MIF) induced by tuberculin or insoluble concanavalin A in supernatants of stimulated lymphocyte cultures. The scarcity of MIF-sensitive cells is probably the main reason for the unsatisfactory results obtained with direct and indirect MIF tests when using white blood cells as indicator cells. Isolated horse monocytes represent an alternative sensitive source of indicator cells for human MIF assays, whereas guinea pig peritoneal exudate macrophages appear to be less sensitive and to show large individual variations in sensitivity to human MIF. The species specificity of MIF from various origins shows various patterns and is briefly discussed.

Lymphokines. I. Use of insoluble concanavalin A for the production of migration inhibitory factor in guinea pig lymphocyte cultures.

Incubation of guinea pig lymph node lymphocytes with insoluble concanavalin A (Con A) in serum-free medium yields sizable amounts of migration inhibition (MIF) and mitogenic factors (MF). Various controls confirmed that the activities detected in supernatants from such cultures are not due to free soluble Con A released during culture. Whereas guinea pig MIF does not act upon human and horse monocytes, guinea pig MF stimulated human but not horse lymphocytes. Upon stimulation by insoluble Con A, guinea pig MIF and MF appear to be produced essentially by T lymphocytes. The technique proposed for producing lymphokines appears convenient for further purification and biochemical characterization of culture supernatants.