Comparison of the bacterial community structure within the equine hindgut and faeces using Automated Ribosomal Intergenic Spacer Analysis (ARISA)

The horse’s hindgut bacterial ecosystem has often been studied using faecal samples. However few studies compared both bacterial ecosystems and the validity of using faecal samples may be questionable. Hence, the present study aimed to compare the structure of the equine bacterial community in the hindgut (caecum, right ventral colon) and faeces using a fingerprint technique known as Automated Ribosomal Intergenic Spacer Analysis (ARISA). Two DNA extraction methods were also assessed. Intestinal contents and faeces were sampled 3 h after the morning meal on four adult fistulated horses fed meadow hay and pelleted concentrate. Irrespective of the intestinal segment, Principal Component Analysis of ARISA profiles showed a strong individual effect (P<0.0001). However, across the study, faecal bacterial community structure significantly (P<0.001) differed from those of the caecum and colon, while there was no difference between the two hindgut communities. The use of a QIAamp® DNA Stool Mini kit increased the quality of DNA extracted irrespective of sample type. The differences observed between faecal and hindgut bacterial communities challenge the use of faeces as a representative for hindgut activity. Further investigations are necessary to compare bacterial activity between the hindgut and faeces in order to understand the validity of using faecal samples.     

Bioprospecting the American Alligator Peptidome for antiviral peptides against Venezuelan equine encephalitis virus

The innate immune protection provided by cationic antimicrobial peptides (CAMPs) has been shown to extend to antiviral activity, with putative mechanisms of action including direct interaction with host cells or pathogen membranes. The lack of therapeutics available for the treatment of viruses such as Venezuelan equine encephalitis virus (VEEV) underscores the urgency of novel strategies for antiviral discovery. American alligator plasma has been shown to exhibit strong in vitro antibacterial activity, and functionalized hydrogel particles have been successfully employed for the identification of specific CAMPs from alligator plasma. Here, a novel bait strategy in which particles were encapsulated in membranes from either healthy or VEEV-infected cells was implemented to identify peptides preferentially targeting infected cells for subsequent evaluation of antiviral activity. Statistical analysis of peptide identification results was used to select five candidate peptides for testing, of which one exhibited a dose-dependent inhibition of VEEV and also significantly inhibited infectious titers. Results suggest our bioprospecting strategy provides a versatile platform that may be adapted for antiviral peptide identification from complex biological samples.     

A proposal for standardization in forensic equine DNA typing: allele nomenclature for 17 equine‐specific STR loci

In this study, a proposal is presented for the allele nomenclature of 17 polymorphic STR loci (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3 and VHL20) for equine genotyping (Equus caballus). The nomenclature is based on sequence data of the polymorphic region of the STR loci as recommended by the DNA commission of the International Society for Forensic Genetics for human DNA typing. For each STR locus, several alleles were selected and animals homozygous for those alleles were subjected to sequence analysis. The alleles of the 17 STR loci consisted either of simple (10), compound (6) or complex repeat patterns (1). Only a limited number of alleles with the same fragment size showed different repeat structures. The allele designation described here was based on the number of repeats, including all variable regions within the amplified fragment.     

Experimental reproduction of ELEM

An experiment to gain insight into the minimum toxic dose of fumonisins was conducted by feeding ponies rations with known fumonisin concentrations. Naturally contaminated corn screenings (CS) were blended with pellets, corn, and molasses to formulate individual daily diets. One group of 4 ponies was fed a ration with fumonisin B₁ (FB₁) varying from <1 ppm to 22 ppm. A second group of 5 ponies was fed a ration at varying rates containing 8 ppm FB₁ for 180 days. A panel of clinical chemistry parameters was evaluated twice weekly for both groups. One pony in the first group died of equine leukoencephalomalacia (ELEM) after 225 days of which the final 55 days' diet contained 22 ppm FB₁. Approximately 9 days prior to death, this animal experienced elevated liver chemistry values. All 5 ponies in the second group experienced mild, transient, clincal signs; were euthanized at 180 days; and had mild, histopathological brain lesions.     

A mini‐STR typing system for degraded equine DNA

Degraded biological samples are a challenge for testing laboratories. Genotyping success can be improved through the use of mini‐STRs, by which primers are placed adjacent to the repeat motifs to reduce amplicon size. Here, we present a genetic profiling system comprising 13 autosomal and one X‐linked dinucleotide‐repeat markers and the SRY gene based on the internationally accepted equine parentage panel. The markers are divided into two panels with all alleles falling at or below 182 bp. The application of this method significantly increases the ability to profile difficult samples and to provide discriminating results to clients.     

Assessing Youth Perceptions and Knowledge of Ethics at 4-H Horse Shows in Louisiana

Due to the increased awareness of animal welfare issues and sensitized attitudes throughout society, youth animal projects provide a unique opportunity to assess and impact youth understanding and attitudes regarding animal welfare. Animal ethics training is not an uncommon requirement for participants in 4-H livestock programs. However, participants in 4-H horse projects are not universally held to the same standard. The purpose of this study was to assess youth knowledge, attitudes, and confidence in understanding of equine welfare at the 4-H state horse show, as well as to determine the beliefs and observations of unethical practices, and if differences exist between those parameters in youth competitors. The results suggest that 4-H competitors are interested in ethics and believe that unethical behavior rarely occurs within the state 4-H horse project. However, youth report observing common unethical behaviors at the show, suggesting a disconnect between what they believe is unethical and what they actually observe. Educational opportunities exist within these animal projects and should be enhanced to address the gap in understanding.     

The positive aspects of correct negative reinforcement

Abstract

In the scheme of contemporary animal training, horse training is virtually unique because it relies on negative reinforcement (NR) rather than positive reinforcement (PR). Furthermore, horse trainers are largely unaware that they are using NR in training. Instead, they believe in the benevolent nature of the horse and see their task in training as one of improving the balance and gymnastic ability of the horse—outcomes that emerge when the rider is similarly properly balanced. Under these conditions, it is claimed the willing horse will perform its required maneuvers. These beliefs may be associated with several welfare issues and indicate areas requiring future research: 1. The absence of release of pressure, the release of pressure at the wrong times, the use of opposing pressures simultaneously and the absence of shaping procedures are central to the development of acute and chronic stress responses in horses.

2. Resultant conflict behaviors contribute to equine wastage statistics and include behaviors that are dangerous to horses and humans.

3. There is a need for research into the mechanics of NR because it is poorly researched compared to PR.

4. When NR responses are installed correctly, only mild pressures need to be used, and results are obtained in few trials.

5. Many qualified animal trainers misunderstand NR and confuse it with punishment. They believe that PR has positive welfare implications and thus NR being “negative,” has negative welfare implications. So there is a need for horse trainers to understand learning theory and the principles that surround NR.

6. Horse trainers are isolated from advances in animal training. Therefore they increasingly seek knowledge and solutions from the growing number of “horse whisperers” and unqualified “horse psychologists.” This is potentially detrimental for the welfare of the horse and the need is urgent for universities throughout the world to become the knowledge bases for equitation science.

     

Concentrations of fumonisin B1 in feeds associated with animal health problems

Ninety-eight samples of feeds associated with 44 cases of equine leukoencephalomalacia (ELEM) and 83 samples of feed associated with 42 cases of a porcine pulmonary edema syndrome (PPE) were analyzed for fumonisin B₁ (FB₁). For comparison purposes, 51 feed samples not associated with PPE or ELEM were also analyzed. Feed associated with ELEM contained FB₁ ranging from less than 1 g/g to 126 g/g with 75% of the cases having at least 1 sample above 10 g/g. Feeds associated with PPE ranged from less than 1 g/g to 330 g/g with 71% of the cases having at least 1 sample greater than 10 g/g. Quantitation was by high performance liquid chromatography (HPLC)/fluorescence using the fluorescamine derivative with confirmation by thin layer chromatography (TLC) and/or gas chromatography/mass spectroscopy (GC/MS).     

Patterns of excretion of fecal estradiol and progesterone and urinary chorionic gonadotropin in Grevy’s zebras (Equus grevyi): Ovulatory cycles and pregnancy

The ovulatory cycle and pregnancy of the endangered Grevy’s zebra (Equus grevyi) was characterized by analyzing excretion patterns of fecal estradiol and progesterone. Pregnancy was further described by assay of urinary equine chorionic gonadotropin. Courtship and mating behavior were similar to that seen in the domestic horse, but cycle length was longer, ranging from 28 to 35 days. Steroid hormone profiles during pregnancy were likewise similar to the horse, but gestation length was longer (391 and 406 days) in the two mature mares. A young mare was first seen to mate when 1 year 3 months of age, at which time she conceived but then aborted at 101 days’ gestation. Conception occurred after one and possibly two more abortions. A healthy foal was born after a gestation period of 425 days, when the mare was 3 years 4 months old, the youngest age at first foaling to be reported for this species. Equine chorionic gonadotropin (eCG) was first detected between 35 and 40 days’ gestation, as in the horse, returning to baseline at about 195 days. However, in one young mare, eCG was also detectable around the time of ovulation. The eCG antibody may have cross‐reacted with luteinizing hormone, which would have been reaching peak concentrations at about that time. The general agreement of the endocrine results with observed occurrence of mating behavior and of parturition demonstrates that assay of excreted hormones can be used to document puberty, monitor ovarian cycles, and diagnose and monitor pregnancy in Grevy’s zebra mares. Zoo Biol 20:185–195, 2001. © 2001 Wiley‐Liss, Inc.     

Evaluation of the presence of a specific histocompatibility protein on equine embryonic cells

An indirect immunofluorescence assay was used to detect the presence of H-Y antigen on equine blastocysts. A total of 33 blastocyst stage horse embryos were collected 6 to 7 days post-ovulation by trans-cervical flush and were immediately evaluated for the presence of H-Y antigen. Additionally, 17 embryos, were collected and cultured for 72 h to the expanded blastocyst stage and similarly evaluated. Embryos were placed in medium containing monoclonal antibodies to H-Y antigen followed by incubation in medium containing 1/10 (v/v) fluorescein isothiocyanate conjugated goat anti-mouse IgM Fc specific antiserum. Embryos were individually evaluated at 400X to identify cell specific fluorescence. Following evaluation, embryonic sex was independently verified with karyotypes to identify sex chromosomes. Of the 50 embryos evaluated, 29 were evaluated as non-fluorescent and 21 fluorescent. Expression of H-Y antigen was determined to be uniform in those embryos classified as fluorescent. Twenty-three of 28 (82%) readable karyotypes corresponded to the predicted sex. These results indicate a specific histocompatibility antigen is expressed and maintained at the blastocyst stage of development. In addition, no segregation of this protein on specific cell types occurs in this species.