Modulation of allospecific CTL responses during pregnancy in equids: an immunological barrier to interspecies matings?

Maternal immune recognition of the developing conceptus in equine pregnancy is characterized by the strongest and most consistent alloantibody response described in any species, a response directed almost exclusively against paternal MHC class I Ags. This work investigated the cellular immune response to paternal MHC Ags in pregnant and nonpregnant horses and donkeys, and in horses carrying interspecies hybrid mule conceptuses. We observed profound decreases in classical, MHC-restricted, CTL activity to allogeneic paternal cells in peripheral blood lymphocytes from both horse mares and donkey jennets carrying intraspecies pregnancies, compared with cells from nonpregnant controls. This is the first evidence in a randomly bred species for a generalized systemic shift of immune reactivity away from cellular and toward humoral immunity during pregnancy. Surprisingly, mares carrying interspecies hybrid mule conceptuses did not exhibit this transient, pregnancy-associated decrease in CTL activity. The failure of interspecies pregnancy to down-regulate cellular immune responses may be a heretofore-unrecognized, subtle barrier to reproductive success between species.     

Comparative testis morphometry and seminiferous epithelium cycle length in donkeys and mules

The mule (Equus mulus mulus) is a sterile hybrid domestic animal that results from the breeding of a male donkey (Equus asinus) to a female horse (Equus caballus). Usually, spermatogenesis in mules does not advance beyond spermatocytes. In the present study, we performed a comparative and more accurate morphometric and functional investigation of the testis in donkeys and mules. Due to the smaller testis size, lower seminiferous tubule volume density, and fewer germ cells, the total length of seminiferous tubules in mules was significantly smaller than in donkeys. However, the percentage of seminiferous tubules containing germ cells (spermatogonia and spermatocytes) in mules was approximately 95%. The total number of Sertoli cells per testis observed in donkeys and mules was very similar. However, the total number of Leydig cells in mules was approximately 70% lower than in donkeys. At least in part, this difference was probably related to the lower number of germ cells present in mule seminiferous tubules. Although spermatogenesis in mules did not advance beyond secondary spermatocytes/newly formed round spermatids, germ cell associations in the seminiferous epithelium and pachytene spermatocytes nuclear volume in donkeys and mules were similar. The duration of spermatogenesis was estimated using intratesticular injections of tritiated thymidine. Each spermatogenic cycle in donkeys lasted 10.5 days. A similar value was found in mules ( approximately 10.1 days). Considering that the entire spermatogenic process takes approximately 4.5 cycles to be completed, its total duration in donkeys was estimated to last 47.2 days. The results found for mules suggest that the mechanisms involved in the determination of testis structure and function are probably originated from donkeys. Also, the data found for mules suggest that their seminiferous tubules are able to sustain complete spermatogenesis. In this regard, this species is a potential model for transplants of germ cells originated from donkeys and horses or other large animals.     

Comparison of five tests for the serologic diagnosis of myiasis by Gasterophilus spp. larvae (Diptera: Gasterophilidae) in horses and donkeys: a preliminary study

Abstract. Sera from 41 horses and 159 donkeys, from twelve States of México, were tested to ascertain anti-Gasterophilus circulating antibodies by double immunodiffusion (DD), counterimmunoelectrophoresis (CIE), indirect haemagglutination (IH), thin layer immunoassay (TIA) and diffusion-in-gel ELISA (DIG-ELISA) methods using crude somatic antigen from third instar larvae of G.intestinalis (DeGeer). At necropsy, 33/41 horses and 24/159 donkeys were found to be parasitized by G.intestinalis and/or G.nasalis (L.). Gasterophilus intestinalis was the species most commonly found in the equines. Analysis of the sera from the infested animals by DD showed positive results of 21.2% in horses and of 8% in donkeys. Screening the sera with CIE gave sensitivities of 69.7% in horses and of 32% in donkeys. Examination of the sera by IH showed positive results of 87.9% and of 48% in horses and donkeys, respectively. Testing the sera with TIA gave sensitivities of 93.9% in horses and of 96% in donkeys. Analysis of horses' sera by DIG-ELISA showed a sensitivity of 93.9%.     

Culicoides in relation to transmission of African Horse Sickness virus in The Gambia

Twelve light trap collections made near overnight shelters of horses and donkeys in four villages in the Central River Division of The Gambia captured fourteen species of biting midge of the genus Culicoides . Five species new to The Gambia were identified. This brought the number of recognized species of Culicoides (after a revision of C. schultzei ) to twenty-nine in The Gambia. Species known or suspected as vectors of African horse sickness virus (AHSV) and bluetongue virus (BTV) comprised 83% of female captures, 65% of captures being C. imicola or its sibling species , C. miombo . Captures of female Culicoides in the late dry season were almost as large as in the early dry season, despite the extreme heat and dryness at this time of the year.
Tests on batches of formalin-preserved female midges, using AHSV or BTV antigen capture ELISAs, did not show the presence of any virus amongst 2286 females in 240 aliquots. Nearly all Gambian equines are reportedly seropositive to AHSV and these results suggest that virus challenge from Culicoides vectors may be a factor in the health of Gambian horses and donkeys.     

Bloodmeal analysis in Culicoides midges collected near horses,donkeys and zebras in the Eastern Cape,South Africa

An upsurge in African horse sickness (AHS) in the Eastern Cape, South Africa, from 2006 led to an epidemiological reassessment of the disease there. Light trapping surveys carried out near horses, donkeys and zebras in 2014–2016 collected 39 species of Culicoides midge (Diptera: Ceratopogonidae) that are potential vectors of AHS. To establish if these midges fed on equids, DNA sequences were obtained from the gut contents of 52 female midges (35 freshly blood‐fed, 13 gravid and four parous), representing 11 species collected across 11 sites. Culicoides leucostictus fed on all three equids. Culicoides bolitinos, Culicoides imicola and Culicoides magnus fed on both horses and donkeys. Culicoides onderstepoortensis fed on donkeys, and Culicoides similis and Culicoides pycnostictus fed on zebras. Bloodmeals from cows, pigs, warthogs, impalas and a domestic dog were also identified in various species, but none of the midges tested had fed on birds. These results contribute to knowledge of the vectorial capacity of several species of Culicoides with regard to AHS in the Eastern Cape and point to potential reservoir hosts, of which donkeys, zebras and domestic dogs have previously been found to harbour AHS. Blood‐fed midges were also obtained throughout winter, indicating the potential for endemic AHS in the province.     

First detection of anti-Besnoitia spp. specific antibodies in horses and donkeys in Italy

Among Apicomplexa protozoa infecting equids, Besnoitia spp., Toxoplasma gondii and Neospora spp. represent important issues from a sanitary and zootechnical viewpoint. However, only scarce epidemiological data are available on the spread of the infections in horses and donkeys in Europe. Therefore, a serosurvey was planned to estimate the prevalence of these Sarcocystidae species in Italian equids. Serum samples from 268 horses and 18 donkeys raised in Italy were collected and serologically analyzed to detect anti-Besnoitia spp., anti-T. gondii and anti-Neospora spp. antibodies: an approach based on an initial screening by in-house ELISA followed by a confirmatory WB was used. Two horses (0.7%) and four donkeys (22.2%), showed antibodies anti-Besnoitia spp. Ten horses (3.7%) resulted positive to T. gondii and one of these (0.4%) was seropositive also to Neospora spp. This is the first detection of anti-Besnoitia spp. specific antibodies in Italian horses and donkeys. The study confirmed the circulation of Besnoitia spp. among equids in Europe. Low prevalence of T. gondii and Neospora spp. in horses raised in Italy was reported. Nevertheless, it is noteworthy to consider that consumption of horse meat could represent a source for human toxoplasmosis.     

Lethal white foals in matings of overo spotted horses

Overo is a variable pattern of white coat color spotting which occurs in several breeds of horses in the United States. Occasionally, when overos are crossed inter se white foals are born which die soon after birth. Both intestinal tract abnormalities and isoerythrolysis have been reported in these foals. This report presents data which show that neonatal isoerythrolysis (NI) is not involved in the death of the white foals. Further research is needed to define the nature of the lethal anomaly, as well as the genetics, of overo and lethal white foals.     

Two variants in the KIT gene as candidate causative mutations for a dominant white and a white spotting phenotype in the donkey

White spotting phenotypes have been intensively studied in horses, and although similar phenotypes occur in the donkey, little is known about the molecular genetics underlying these patterns in donkeys. White spotting in donkeys can range from only a few white areas to almost complete depigmentation and is characterised by a loss of pigmentation usually progressing from a white spot in the hip area. Completely white‐born donkeys are rare, and the phenotype is characterised by the complete absence of pigment resulting in pink skin and a white coat. A dominant mode of inheritance has been demonstrated for spotting in donkeys. Although the mode of inheritance for the completely white phenotype in donkeys is not clear, the phenotype shows similarities to dominant white in horses. As variants in the KIT gene are known to cause a range of white phenotypes in the horse, we investigated the KIT gene as a potential candidate gene for two phenotypes in the donkey, white spotting and white. A mutation analysis of all 21 KIT exons identified a missense variant in exon 4 (c.662A>C; p.Tyr221Ser) present only in a white‐born donkey. A second variant affecting a splice donor site (c.1978+2T>A) was found exclusively in donkeys with white spotting. Both variants were absent in 24 solid‐coloured controls. To the authors’ knowledge, this is the first study investigating genetic mechanisms underlying white phenotypes in donkeys. Our results suggest that two independent KIT alleles are probably responsible for white spotting and white in donkeys.     

Identification and genetics of horse lymphocyte alloantigens

Six hundred horses were tested with lymphocytotoxic antisera derived from 550 parous mares and 58 antisera produced by alloimmunization with horse blood cells. Seven equine lymphocyte specificities were identified using correlation analysis of the test data, absorption analysis and lysostripping. These specificities are expressed on lymphocytes and platelets, but not on red blood cells (RBC). Therefore, these specificities do not appear to be products of any of the eight known blood group systems of the horse. The distribution of these specificities in 113 Thoroughbred horses and 57 Arabian horses is presented. Two specificities are subtypic to two other specificities reported here. Family studies indicated that all of these specificities are products of one genetic system. However, it is not clear whether the system consists of one or more loci.     

Identification and genetics of horse lymphocyte alloantigens

Six hundred horses were tested with lymphocytotoxic antisera derived from 550 parous mares and 58 antisera produced by alloimmunization with horse blood cells. Seven equine lymphocyte specificities were identified using correlation analysis of the test data, absorption analysis and lysostripping. These specificities are expressed on lymphocytes and platelets, but not on red blood cells (RBC). Therefore, these specificities do not appear to be products of any of the eight known blood group systems of the horse. — The distribution of these specificities in 113 Thoroughbred horses and 57 Arabian horses is presented. Two specificities are subtypic to two other specificities reported here. Family studies indicated that all of these specificities are products of one genetic system. However, it is not clear whether the system consists of one or more loci.Paper No. 80-4-83 of the Kentucky Agricultural Experiment Station.     

Epidemiologic aspects of Taylorella equigenitalis

Contagious equine metritis (CEM) is a sexually transmissible disease in mares. Although the disease is commonly diagnosed by culturing the causative bacterium Taylorella equigenitalis (T. equigenitalis) . false negative results do occur. A recently developed Polymerase Chain Reaction (PCR) assay, however, appeared to be much more sensitive, with initial results indicating an unexpected high incidence of the agent in selected horses. In this study, samples from 107 randomly selected mares with no clinical signs of CEM submitted for conventional culture were all negative for T. equigenitalis . but in the PCR-assay 54 (49%) were positive for Taylorella -DNA. Positives in the PCR-assay were found in all breeds tested, even in horses imported from the isolated population in Iceland. These findings suggest that T. equigenitalis was present long before it was first isolated in 1977, The high incidence of Taylorella in horse populations without apparent clinical signs of CEM, the occurrence of incidental clinical case and the known variability between strains, all indicate that Taylorella is endemic in the horse population. In order to explore whether the organism is present in species other than the horse, we also used the PCR-assay on clinically health donkeys (n = 14), zebras (n = 15), Przewalski horses (n = 2) and cows (n = 21). All the animals showed negative results except one of the Przewalski horses, and one cow that was repeatedly found to give positive reaction. We also found that the fertility of 7 stallions with cultures positive for Taylorella (6 used in an AI-program and 1 by natural breeding) was not affected, as shown by the normal range of foaling rates in mares inseminated or bred by these stallions. The overall results may be interpreted to mean that Taylorella is of limited significance in horse breeding.     

A comparison of the mineral composition of milk of domestic and captive wild equids (Equus przewalski, E. zebra, E. burchelli, E. caballus, E. assinus)

Milk samples were obtained in early and/or late lactation from Przewalski horses, Hartmann's zebras, Grant's zebras, domestic horses, ponies and a mule mare made pregnant by embryo transfer. Samples were compared for their content of total solids, ash, calcium, phosphorus, magnesium, sodium, potassium, copper, zinc and iron. Milk from the Przewalski horses, Hartmann's zebra and the domestic horse had similar mineral composition and the content of minerals was higher in early than in late lactation. Milk from the domestic mule contained the lowest concentration of calcium, phosphorus and zinc but the highest concentration of magnesium, sodium and potassium. Milk from the Grant's zebras contained more sodium than potassium, unlike milk from Przewalski horses, Hartmann's zebras or domestic horses in which there was more potassium than sodium.     

Genetic diversity in a feral horse population from Sable Island, Canada

The present-day Sable Island horse population, inhabiting an island off the eastern coast of Canada, is believed to have originated mainly from horses confiscated from the early French settlers in Nova Scotia in the latter half of the 18th century. In 1960, the Sable Island horses were given legal protected status and no human interference has since been allowed. The objective of this study was to characterize the current genetic diversity in Sable Island horses in comparison to 15 other horse breeds commonly found in Canada and 5 Spanish breeds. A total of 145 alleles from 12 microsatellite loci were detected in 1093 horses and 40 donkeys. The average number of alleles per locus ranged from 4.67 in the Sable Island horse population to 8.25 in Appaloosas, whereas the mean observed heterozygosity ranged from 0.626 in the Sable Island population to 0.787 in Asturcons. Various genetic distance estimates and clustering methods did not permit to support that the Sable Island horses originated from shipwrecked Spanish horses, according to a popular anecdote, but closely resemble light draft and multipurpose breeds commonly found in eastern Canada. Based on the Weitzman approach, the loss of the Sable Island horse population to the overall diversity in Canada is comparable or higher than any other horse breed. The Sable Island horse population has diverged enough from other breeds to deserve special attention by conservation interest groups.     

Seroprevalence of Toxoplasma gondii in equids from Southern Spain

Antibodies to Toxoplasma gondii were determined in serum samples from 616 equids (454 horses, 80 mules and 82 donkeys) in a cross-sectional study of 420 herds in Andalusia (Southern Spain), the region with the highest number of equids in Spain. Antibodies to T. gondii were found in 10.8% horses, 15.0% mules and 25.6% donkeys by using the modified agglutination test (MAT) at a cut-off of 1:25. Herd seroprevalence for horses, mules and donkeys was 14.7% (48/327), 23.9% (11/46) and 34.0% (16/47), respectively, and 75 herds (17.8%) had at least one seropositive animal. Significant differences in T. gondii seroprevalence were observed among species, with donkeys having the highest seroprevalence and horses the lowest (P=0.04). Seroprevalence was significantly higher in herds with presence of domestic ruminants. This study is the first report of the presence of T. gondii antibodies in equine species in Spain and the first reporting T. gondii infection in donkeys in Europe. The presence of antibodies is indication of contact with the parasite and therefore, consumption of equine meat could be a potential source of human infection in Spain.     

Genome-Wide Detection of Copy Number Variations among Diverse Horse Breeds by Array CGH

Recent studies have found that copy number variations (CNVs) are widespread in human and animal genomes. CNVs are a significant source of genetic variation, and have been shown to be associated with phenotypic diversity. However, the effect of CNVs on genetic variation in horses is not well understood. In the present study, CNVs in 6 different breeds of mare horses, Mongolia horse, Abaga horse, Hequ horse and Kazakh horse (all plateau breeds) and Debao pony and Thoroughbred, were determined using aCGH. In total, seven hundred CNVs were identified ranging in size from 6.1 Kb to 0.57 Mb across all autosomes, with an average size of 43.08 Kb and a median size of 15.11 Kb. By merging overlapping CNVs, we found a total of three hundred and fifty-three CNV regions (CNVRs). The length of the CNVRs ranged from 6.1 Kb to 1.45 Mb with average and median sizes of 38.49 Kb and 13.1 Kb. Collectively, 13.59 Mb of copy number variation was identified among the horses investigated and accounted for approximately 0.61% of the horse genome sequence. Five hundred and eighteen annotated genes were affected by CNVs, which corresponded to about 2.26% of all horse genes. Through the gene ontology (GO), genetic pathway analysis and comparison of CNV genes among different breeds, we found evidence that CNVs involving 7 genes may be related to the adaptation to severe environment of these plateau horses. This study is the first report of copy number variations in Chinese horses, which indicates that CNVs are ubiquitous in the horse genome and influence many biological processes of the horse. These results will be helpful not only in mapping the horse whole-genome CNVs, but also to further research for the adaption to the high altitude severe environment for plateau horses.     

Lymphocyte alloantigens of the horse. III. ELY-2.1: a lymphocyte alloantigen not coded for by the MHC

A new polymorphic locus of the horse which has several unusual properties is described. The suggested name for the locus is ELY-2 . The gene product of one allele at this locus, designated ELY-2.1 , has been identified with antisera raised as a result of pregnancy. Antibody to ELY-2.1 was first detected on day 55 after conception in the serum of a mare in first pregnancy. This early onset of antibody is similar to that seen for antibody to ELA antigens, and suggests that the source of the antigenic stimulus may be the tissue of the equine endometrial cups.
The antisera identifying ELY-2.1 are cytotoxic and kill all peripheral blood lymphocytes from horses carrying the antigen. ELY-2.1 is a cell surface molecule expressed on lymphocytes, erythrocytes, and platelets. Other cell types have not been investigated. The overall phenotypic frequency of ELY-2.1 in several horse breeds was 16 %. The ELY-2.1 antigen is controlled by an autosomal, dominant gene which is not coded by the ELA region (the major histocompatibility complex of the horse), nor is it identical to the ELY-1 locus, which codes for another cell surface alloantigen of equine lymphocytes. Stimulator cells carrying ELY-2.1 did not induce proliferation of ELY-2.1 negative responder cells in mixed cultures of horse lymphocytes. Attempts to raise alloantisera to other alleles of the ELY-2 locus through immunization with lymphocytes were unsuccessful. It is possible that the alternate allele(s) does not code for a gene product which is expressed. The function and biochemical nature of the ELY-2.1 molecule are unknown.     

Isolation and chemical composition of the leucocytes from donkey, horse, mule and pig.

1. Leucocytes from the blood of adult and young donkeys (Equus asinus L.), adult horses (Equus caballus L.), adult mules (Equus asinus x Equus caballus) and adult pigs (Sus scrofa L.) were obtained in a high degree of purity (99.9%) using Na2-EDTA-dextrans mixtures. 2. Sialic acids were released, purified, identified and determined from both non-delipided and delipided leucocytes. 3. N-glycolylneuraminic was the predominant sialic acid. N-acetylneuraminic acid and N,O-diacetyl-neuraminic acid were also found in all materials. Except in pig, other unidentified sialic acid(s) were also detected. 4. The concentration of total sialic acids (microgram/mg protein) is different according to the species, and in donkey species according to the age. 5. Galactose, glucose, mannose, fucose and (in a less amount) ribose were determined. Their total content is about 2-3-fold that of hexosamines. 6. There is a higher cholesterol content in adult donkey leucocytes than in those of young ones. 7. Total lipids, cholesterol or phospholipid contents are similar among the leucocytes of the above-mentioned species. 8. The similarities are marked in the electrophoresis patterns of proteins and glycoproteins for the donkey, mule and horse samples. 9. The molecular weights for leucocytes proteins were estimated as ranging between 230,000 and 20,000; and for the main protein bands, between 120,000 and 22,000.     

Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei,a Tier 1 select agent

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/μL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.     

Genetic characterization of Pompeii and Herculaneum Equidae buried by Vesuvius in 79 AD

DNA extracted from the skeletons of five equids discovered in a Pompeii stable and of a horse found in Herculaneum was investigated. Amino acid racemization level was consistent with the presence of DNA. Post‐mortem base modifications were excluded by sequencing a 146 bp fragment of the 16S rRNA mitochondrial gene. Sequencing of a 370 bp fragment of mitochondrial (mt)DNA control region allowed the construction of a phylogenetic tree that, along with sequencing of nuclear genes (epsilon globin, gamma interferon, and p53) fragments, gave us the possibility to address some questions puzzling archaeologists. What animals—donkeys, horses, or crossbreeds—were they? And, given they had been evidently assigned to one specific job, were they all akin or were they animals with different mitochondrial haplotypes? The conclusions provided by molecular analysis show that the Pompeii remains are those of horses and mules. Furthermore one of the equids (CAV5) seems to belong to a haplotype, which is either not yet documented in the GenBank or has since disappeared. As its characteristics closely recall those of donkeys, which is the out group chosen to construct the tree, that appears to have evolved within the Equidae family much earlier than horses, this assumption seems to be nearer the truth. J. Cell. Physiol. 199: 200–205, 2004© 2003 Wiley‐Liss, Inc.