Detection and semi-quantification of Strongylus vulgaris DNA in equine faeces by real-time quantitative PCR

Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available to differentiate among strongyle species in a faecal sample is by identifying individual L3 larvae following a two week coproculture procedure. The aim of the present study is to overcome this diagnostic obstacle by developing a fluorescence-based quantitative PCR assay capable of identifying S. vulgaris eggs in faecal samples from horses. Species-specific primers and a TaqMan probe were designed by alignment of published ribosomal DNA sequences of the second internal transcribed spacer of cyathostomin and Strongylus spp. nematodes. The assay was tested for specificity and optimized using genomic DNA extracted from identified male worms of Strongylus and cyathostomin species. In addition, eggs were collected from adult female worms and used to evaluate the quantitative potential of the assay. Statistically significant linear relationships were found between egg numbers and cycle of threshold (Ct) values. PCR results were unaffected by the presence of cyathostomin DNA in the sample and there was no indication of PCR inhibition by faecal sources. A field evaluation on faecal samples obtained from four Danish horse farms revealed a good agreement with the traditional larval culture (kappa-value=0.78), but with a significantly higher performance of the PCR assay. An association between Ct values and S. vulgaris larval counts was statistically significant. The present assay can reliably and semi-quantitatively detect minute quantities of S. vulgaris eggs in faecal samples.     

Horses grazing with cattle have reduced strongyle egg count due to the dilution effect and increased reliance on macrocyclic lactones in mixed farms

Strongyle infection is an important issue in horse breeding. It impairs horse health and performance, with young horses being the most sensitive. Strongyle control has long relied on the systematic use of chemical treatments. However, expanding anthelmintic resistance among strongyles calls for alternative options. Mixed grazing is assumed to reduce strongyle load on the pasture as the result of a dilution effect. This has been shown in small ruminants grazing with cattle, but the putative benefits of co-grazing between horses and cattle have not yet been evaluated. Here, we conducted field surveys and face-to-face interviews on 44 farms from two contrasted saddle-horse production areas, Normandy and northern Massif Central, to compare equine strongyle management practices between specialized systems and mixed horse-cattle systems. Our goals were (i) to quantify breeders’ awareness of the putative benefits associated with the co-grazing of horses and cattle, (ii) to establish whether mixed farming was associated with different strongyle management strategies and (iii) to test whether strongyle egg excretion was reduced in horses grazed with beef cattle. Every breeder relied on systematic calendar treatments, and only 8 out of the 23 mixed breeders were aware that co-grazing of horses with cattle could be used as part of their strongyle control strategy. Management practices were similar across both systems in Normandy. In Massif Central, mixed breeders formed a distinct cluster from their specialized counterparts: deworming was less frequent and stocking density was higher in mixed farms, while specialized breeders seemed more willing to integrate herd and plot management into control strategies. Faecal egg counts measured in horses from Massif Central were significantly reduced when horses were grazed with cattle. This was the result of an increased reliance on macrocyclic lactones in mixed farms (P < 0.01) and a significant dilution effect (P < 0.01). When considering a subsample of horses treated with macrocyclic lactones only, young horses grazed with cattle had 50% fewer strongyle eggs excreted in their faeces than horses grazed in equine-only pastures (P < 0.01). This is the first evidence of the benefits of mixed grazing with cattle as an alternative to control strongyle infection in horses, although this promising alternative remains largely unknown by horse breeders.     

Facial hair whorls (trichoglyphs) and the incidence of motor laterality in the horse

Several species demonstrate obvious motor laterality (sidedness, handedness) in their motor function. Motor laterality in the horse affects locomotion and subsequently equine performance during training and may have inherent safety implications for equitation. Some of the most commonly used identification features in the horse are hair whorls (trichoglyphs), since their specific location and character vary to some degree in every horse. We investigated the relationship between the hair flow of single facial hair whorls and the incidence of lateralised motor bias in 219 horses when under saddle in ridden work. The horses exhibited significant differences in motor preferences with 104 left-lateralised (LL) horses, 95 right-lateralised (RL) horses compared to only 20 well-balanced (WB) horses (chi(2)=36.9, d.f.=2, P<0.01). There was also a significant difference in the frequency distribution of single facial hair whorl patterns in the horses consisting of 114 horses with counter-clockwise (CC) whorls, 82 horses with clockwise (C) whorls and 23 horses, which had radial (R) whorls (chi(2)=38.87, d.f.=2, P<0.01). Overall there was a statistically significant association between motor behaviour and facial hair whorl patterns in the horses (chi(2)=69.4, d.f.=4, P>0.001). The RL horses had significantly more C facial hair whorls and the LL horses had significantly more CC facial hair whorls than would be expected purely by chance alone (P<0.05). The findings may provide trainers with a useful tool when attempting to identify simple, non-invasive and reliable predictors of motor laterality in the horse. Furthermore, given that efficient targeted training of performance horses during ridden work may produce WB equine athletes, the findings could assist trainers when designing individual-specific training programmes for young horses.     

The site distribution of adult strongyle parasites in the large intestines of horses in tropical Australia

In a study of gastro-intestinal parasites occurring in 57 horses in north Queensland, the locations where adult strongyle parasites were found in the large intestines were recorded in order to determine the preferred sites of these parasites. Differences were noted in the intensity of infection between the three sections of the large intestines with the ventral colon being the most heavily infected and having the highest number of species, followed by the dorsal colon and the caecum. The distribution of the various strongyle species between these sections of the intestines was not random. Many species showed preferences for particular sections. Changes were noted in site preferences of some species. These changes were related to season but not the age, sex and breed of the animal, nor the environment in which the animals were kept. These seasonal changes are discussed in relation to the epidemiology of equine strongyle infections in the area. There seems to be no differences in the basic site preference behaviour between strongyle species occurring in horses in the tropics and those from the temperate regions.     

Prevalence of antibodies to Neospora sp. in horses from Alabama and characterisation of an isolate recovered from a naturally infected horse [corrected

An IFAT was used to determine the prevalence of Neospora-specific IgG antibodies in serum from Alabama horses. Serum samples (n = 536) were from asymptomatic horses routinely submitted for equine infectious anaemia virus infection testing. We also subjected a 13-year-old horse with CNS disease to necropsy examination for isolation and in vitro cultivation of protozoal organisms. In antemortem tests, this horse was positive for antibodies to Neospora sp. in the IFAT and western immunoblot. Results of the prevalence survey indicated that IgG antibodies to Neospora were present in 62 (11.5%) of the 536 serum samples. Endpoint titres for the positive samples were 1:50 (35/6.5%), 1:100 (19/3.5%), 1:200 (7/1.3%) and 1:1600 (1/0.2%). Tachyzoites were first seen in cultured bovine turbinate cells 32 days after inoculation with spinal cord homogenates from the horse with CNS disease. Tachyzoites reacted with known N. caninum-positive serum from horses, cows, dogs and mice, but did not react with murine anti-Toxoplasma gondii or equine anti-Sarcocystis neurona serum. Ultrastructural features of tachyzoites and results of comparison of tachyzoite immunodominant proteins revealed that they were identical to those of N. hughesi, a species described recently from a naturally infected horse. The isolate recovered from the naturally infected horse in the present study (designated NA1) is thought to be an isolate of N. hughesi, although confirmation of this awaits additional molecular characterisation. These results provide some additional evidence that N. hughesi is a valid species and that Neospora infections in horses may occur in widely separated geographic regions of the United States.     

Analysis of somatic and salivary gland antigens of third stage larvae of Rhinoestrus spp. (Diptera, Oestridae)

Larvae of Rhinoestrus spp. (Diptera, Oestridae) infect nasal and sinus cavities of horses, causing a nasal myiasis characterized by severe respiratory distress. Presently, the diagnosis of horse nasal botfly relies on the observation of clinical signs, on the post mortem retrieval of larvae or on molecular assays performed using pharyngeal swabs. The present study was carried out to characterize larval somatic proteins and salivary glands of Rhinoestrus spp. in a preliminary assessment towards the immunodiagnosis of equine rhinoestrosis. Out of the 212 necropsied horses 13 were positive for the presence of Rhinoestrus spp. larvae. The analysis of the sera from the infected animals by Western blotting assay showed the presence of a specific host humoral immune response against Rhinoestrus spp. larvae and proved that the salivary glands are the major immunogens in horse nasal botflies.     

Prevalence of strongyle nematodes in naturally infected ponies of different ages and during different seasons of the year in Louisiana

One hundred and seventeen ponies were surveyed for the seasonal prevalence of strongyloid parasites, particularly cyathostomes, and for host- and age-related differences in these infections. For 56 ponies, all stages of the cyathostome life cycle, both mucosal and luminal, were enumerated. Total numbers of cyathostomes and percentage of developing larvae (DL) encysted in the mucosa remained constant in all 4 seasons of the year, whereas a significant increase in the percentage of adults in the cyathostome population occurred in fall. In yearling ponies, encysted early third-stage larvae constituted a significantly lower percentage of the cyathostome population, and DL and adults formed a significantly higher percentage, compared with those stages in older ponies, 2-5 yr of age. More species of cyathostomes were present in yearling ponies than in older ponies. Significant differences occurred in fecal egg counts at different seasons of the year, even though adult cyathostome and large strongyle numbers remained constant. Twenty-four species of cyathostomes were found year-round, and 2 rare species were found in only 2 or 3 seasons of the year. Prevalences for these 24 species were not significantly different during any season, although 5 species had significant differences in intensity levels of infection in certain seasons of the year. Three species of large strongyle (strongylinae) adults had significant seasonal variations in intensities. These were Strongylus edentatus and S. vulgaris, which occurred in significantly higher numbers in summer and fall, and Triodontophorus brevicauda, which was more numerous in spring and summer. Fourth- and fifth-stage larvae of S. vulgaris recovered from the mesenteric vasculature were significantly more numerous in winter and spring than in other seasons.     

Gastrointestinal helminths of ponies in Louisiana: a comparison of species currently prevalent with those present 20 years ago

A survey in Louisiana of gastrointestinal helminths recovered at necropsy from 117 ponies with minimal exposure to anthelmintics between 1989 and 2000 is compared with a survey conducted 20 yr earlier in the same region. An objective of this study was to determine whether species diversity has been affected by the advent and use of the macrocyclic lactone (ML) parasiticides and by the increased anthelmintic pressure on the helminth species infecting the general equine population. Twenty-six cyathostome species and 8 strongyle species were recovered. Two cyathostome species that were not found before, Cylicostephanus asymetricus and C. bidentatus, and 1 species of large strongyle, Oesophagodontus robustus, were added to the list of species found in Louisiana. All cyathostome and large strongyle species found previously were still present. But prevalences and intensities were significantly reduced for almost all large and small strongyle species. Prevalences and intensities of Oxyuris equi adults and larvae were reduced, whereas the prevalence of Parascaris equorum remained constant. The tapeworm Paranoplocephala mamillana was added to the list of parasite species found in Louisiana. Anoplocephala perfoliata remained the most common cestode. This species was found at the same level of intensity but increased slightly in prevalence. Anoplocephala magna was found less frequently than previously. The overall diversity of species remained reatively unchanged. The reasons for the differences in intensity and prevalence of strongyles between these 2 periods are unknown but might be related to the development and use of the broad-spectrum ML anthelmintics in the intervening period, a difference in the population of equids surveyed, different techniques used to identify the parasites, or differences in numbers of parasites identified (or to all).     

Are sainfoin or protein supplements alternatives to control small strongyle infection in horses?

The spread of anthelmintic resistance in equine strongyle nematodes has become a major problem, advocating for the development of alternative control for strongyles. Our study consisted of both in vivo and in vitro experiments. We investigate for the first time the efficacy of a short-term consumption of tannin-rich sainfoin (Onobrychis viciifolia) or extra proteins in naturally infected horses. We used 30 horses allocated into three groups of 10 individuals that received for 18 days either (i) a tannin-rich diet with 70% DM sainfoin pellets (Sd), (ii) a protein-rich diet with 52% DM Italian rye-grass pellets and 18% DM grinded linseed expeller (Pd), or (iii) a control diet with 45% DM barley and 25% DM cereal-based pellets (Cd). The three diets were isoenergetic, covering 94% of animal energy requirements on average, and the Sd and Pd diets were isoproteic and provided extra proteins (227% of protein requirements v. 93% for the Cd diet). Pd and Cd were compared to test for benefits of receiving extra proteins, while Sd and Pd were compared to account for the effect of sainfoin secondary metabolites. There were no between-diet differences in faecal egg counts (FEC) or in worm burden evaluated from worm counts in faeces of drenched horses at the end of the experiment. However, coprocultures from the faeces collected in each group at the beginning and at the end of the experiment suggested a lower rate of strongyle larval development in the Sd group at the end of the experiment (Sd=8.1%, Pd=30.5%, Cd=22.6%). In vitro tests using sainfoin solutions evidenced the influence of sainfoin on strongyle larval development: adding 29% of sainfoin pellets to faeces reduced the strongyle egg development into infective larvae by 82% (P<0.001) and using solutions with sainfoin concentrations higher than 7.5 mg/ml reduced egg hatching by 37% (P<0.05). The short-term use of tannin-rich plants in horse diet could thus constitute a promising strategy to reduce the risk of infection by strongyles at pasture.     

Cross reaction of recombinant equine infectious anemia virus antigen to heterologous strains and application for serological survey among horses in the field

Cross reactivity of equine infectious anemia virus (EIAV) antigen prepared using a recombinant baculovirus containing the p26 gene of strain P337-V70 was examined by the agar gel immunodiffusion (AGID) test and enzyme-linked immunosorbent assay (ELISA). Serum samples serially collected from 13 horses experimentally infected with six different EIAV strains (two or three horses per strain) were subjected to the test. Positive reactions were observed in the AGID test and ELISA before or soon after the first feverish period and continued persistently in most of the horses. The results with recombinant antigens were essentially the same as those with the virion antigen prepared from horse cell cultures both in the AGID test and ELISA. The reactivities of the antigens were further compared using serum samples collected from horses in 1999 in certain districts of Mongolia where equine infectious anemia has been prevalent, and from horses in Japan in 1973 when EIA had not been eliminated completely from Japanese horses. These results were completely concurrent. Generally, recombinant antigens have high specificity but low cross reactivity to heterologous strains. However, the present study showed that the recombinant EIAV p26 antigen has cross reactivity to the heterologous strain and is useful for diagnosis of EIA in the field.     

Development of Bacteroides 16S rRNA gene TaqMan-based real-time PCR assays for estimation of total, human, and bovine fecal pollution in water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r2= 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.     

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.     

Occurrence of Gasterophilus intestinalis and some Parasitic Nematodes of Horses in Sweden

A survey was performed on the occurrence of some internal parasites in 461 horses (1–30 years old) slaughtered from October 1992 to September 1993 at the Linköping abattoir in central Sweden. Macroscopical examination was carried out specifically for parasites of the tear ducts and conjunctival sacs of the eyes, and of selected parts of the alimentary tract and car-dio-vascular system. The following parasites were found in selected parts of the large intestine: encapsulated cyathostome larvae (in 35.6% of the horses), and mature strongyle worms (17.4%); in the stomach: Gasterophilus intestinalis (12.3% during October-June) and Habronema muscae (1.1%); and in the conjunctival sac: Thelazia lacrymalis (3.1%). Significantly more encapsulated cyathostome larvae were found during January to June than during other times of the year, and horses aged 1–5 years harboured significantly more larvae than older horses. Severe damage to the cranial mesenteric artery and its main branches was noticed in 16 (6.1%) out of 263 horses specifically examined. However, the 4th stage larva of Strongylus vulgaris was only recovered in 6 (2.3%) of the horses. Quantitative and qualitative faecal egg counts were done on 412 and 384 of the horses, respectively. Eggs of strongyles, Parascaris equorum and Strongyloides westeri were found in 78.1%, 1.9% and 0.2% of the faecal samples, respectively. Highest prevalence of strongyle eggs was found during July to September. Third stage larvae of the following nematodes were recovered from faecal cultures: subfamily Cyathostomum sensu lato (78.1%) Triodontophorus spp. (6.5%), Strongylus vulgaris (3.6%), Trichostrongylus axei (1.3%), while Gyalocephalus sp., Oesophagodontus sp., Poteriostomum sp. and Strongylus edentatus each comprised less than 0.5%.     

Prevalence and abundance of equine strongyles (Nematoda: Strongyloidea) in tropical Australia

A postmortem survey of 57 horses in tropical northern Queensland revealed 41 (89%) infected with intestinal strongyles. Thirty-five strongyle species (8 large strongyles and 27 small strongyles [Cyathostominae]) were recorded of which 9 species are reported from Australia for the first time. The 14 most prevalent small strongyles were Cyathostomum catinatum (in 76% of horses), Cyathostomum coronatum (65%), Cyathostomum pateratum (33%), Cyathostomum labiatum (30%), Cylicostephanus calicatus (70%), Cylicostephanus longibursatus (67%), Cylicostephanus goldi (43%), Cylicostephanus minutus (26%), Cylicocylus nassatus (67%), Cylicocyclus leptostomus (41%), Cylicocylus insigne (41%), Cylicocyclus radiatus (33%), Cylicocyclus brevicapsulates (22%), and Poteriostomum imperidentum (24%). The remaining cyathostomes were each found in less than 15% of horses. The 4 most common large strongyles were Triodontophorus serratus (30%), Strongylus vulgaris (28%), Strongylus equinus, and Strongylus edentatus (both 22%). The number of species of small strongyles per horse showed a marked variation (mean 10.3, range 2-21) but bore no relationship to either the total number of strongyles per horse, age, sex, and breed of horse, or season. Total number of strongyles per horse (mean 15,890, range 20-165,000) was less than in recent surveys in Europe and the U.S.A. Most horses had low worm burdens, whereas a very small number were heavily infected. Ninety-seven per cent of the total strongyle counts were small strongyles. Strongylus species contributed just over 1%. Small numbers of large strongyles per horse were usual with T. serratus (mean 570), S. vulgaris (mean 330), and S. equinus (mean 330) the most numerous.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the fecal bacteria communities of forage-fed horses by pyrosequencing of 16S rRNA V4 gene amplicons

The diversity of the equine fecal bacterial community was evaluated using pyrosequencing of 16S rRNA gene amplicons. Fecal samples were obtained from horses fed cool-season grass hay. Fecal bacteria were characterized by amplifying the V4 region of bacterial 16S rRNA gene. Of 5898 mean unique sequences, a mean of 1510 operational taxonomic units were identified in the four fecal samples. Equine fecal bacterial richness was higher than that reported in humans, but lower than that reported in either cattle feces or soil. Bacterial classified sequences were assigned to 16 phyla, of which 10 were present in all samples. The largest number of reads belonged to Firmicutes (43.7% of total bacterial sequences), Verrucomicrobia (4.1%), Proteobacteria (3.8%), and Bacteroidetes (3.7%). The less abundant Actinobacteria, Cyanobacteria, and TM7 phyla presented here have not been previously described in the gut contents or feces of horses. Unclassified sequences represented 38.1% of total bacterial sequences; therefore, the equine fecal microbiome diversity is likely greater than that described. This is the first study to characterize the fecal bacterial community in horses by the use of 16S rRNA gene amplicon pyrosequencing, expanding our knowledge of the fecal microbiota of forage-fed horses.     

Sialic acid species as a determinant of the host range of influenza A viruses

The distribution of sialic acid (SA) species varies among animal species, but the biological role of this variation is largely unknown. Influenza viruses differ in their ability to recognize SA-galactose (Gal) linkages, depending on the animal hosts from which they are isolated. For example, human viruses preferentially recognize SA linked to Gal by the alpha2,6(SAalpha2,6Gal) linkage, while equine viruses favor SAalpha2,3Gal. However, whether a difference in relative abundance of specific SA species (N-acetylneuraminic acid [NeuAc] and N-glycolylneuraminic acid [NeuGc]) among different animals affects the replicative potential of influenza viruses is uncertain. We therefore examined the requirement for the hemagglutinin (HA) for support of viral replication in horses, using viruses whose HAs differ in receptor specificity. A virus with an HA recognizing NeuAcalpha2,6Gal but not NeuAcalpha2,3Gal or NeuGcalpha2,3Gal failed to replicate in horses, while one with an HA recognizing the NeuGcalpha2,3Gal moiety replicated in horses. Furthermore, biochemical and immunohistochemical analyses and a lectin-binding assay demonstrated the abundance of the NeuGcalpha2,3Gal moiety in epithelial cells of horse trachea, indicating that recognition of this moiety is critical for viral replication in horses. Thus, these results provide evidence of a biological effect of different SA species in different animals.     

Development of Bacteroides 16S rRNA Gene TaqMan-Based Real-Time PCR Assays for Estimation of Total, Human, and Bovine Fecal Pollution in Water

Bacteroides species are promising indicators for differentiating livestock and human fecal contamination in water because of their high concentration in feces and potential host specificity. In this study, a real-time PCR assay was designed to target Bacteroides species (AllBac) present in human, cattle, and equine feces. Direct PCR amplification (without DNA extraction) using the AllBac assay was tested on feces diluted in water. Fecal concentrations and threshold cycle were linearly correlated, indicating that the AllBac assay can be used to estimate the total amount of fecal contamination in water. Real-time PCR assays were also designed for bovine-associated (BoBac) and human-associated (HuBac) Bacteroides 16S rRNA genes. Assay specificities were tested using human, bovine, swine, canine, and equine fecal samples. The BoBac assay was specific for bovine fecal samples (100% true-positive identification; 0% false-positive identification). The HuBac assay had a 100% true-positive identification, but it also had a 32% false-positive rate with potential for cross-amplification with swine feces. The assays were tested using creek water samples from three different watersheds. Creek water did not inhibit PCR, and results from the AllBac assay were correlated with those from Escherichia coli concentrations (r² = 0.85). The percentage of feces attributable to bovine and human sources was determined for each sample by comparing the values obtained from the BoBac and HuBac assays with that from the AllBac assay. These results suggest that real-time PCR assays without DNA extraction can be used to quantify fecal concentrations and provide preliminary fecal source identification in watersheds.     

Equine learning behaviour

Scientists and equestrians continually seek to achieve a clearer understanding of equine learning behaviour and its implications for training. Behavioural and learning processes in the horse are likely to influence not only equine athletic success but also the usefulness of the horse as a domesticated species. However given the status and commercial importance of the animal, equine learning behaviour has received only limited investigation. Indeed most experimental studies on equine cognitive function to date have addressed behaviour, learning and conceptualization processes at a moderately basic cognitive level compared to studies in other species. It is however, likely that the horses with the greatest ability to learn and form/understand concepts are those, which are better equipped to succeed in terms of the human-horse relationship and the contemporary training environment. Within equitation generally, interpretation of the behavioural processes and training of the desired responses in the horse are normally attempted using negative reinforcement strategies. On the other hand, experimental designs to actually induce and/or measure equine learning rely almost exclusively on primary positive reinforcement regimes. Employing two such different approaches may complicate interpretation and lead to difficulties in identifying problematic or undesirable behaviours in the horse. The visual system provides the horse with direct access to immediate environmental stimuli that affect behaviour but vision in the horse is of yet not fully investigated or understood. Further investigations of the equine visual system will benefit our understanding of equine perception, cognitive function and the subsequent link with learning and training. More detailed comparative investigations of feral or free-ranging and domestic horses may provide useful evidence of attention, stress and motivational issues affecting behavioural and learning processes in the horse. The challenge for scientists is, as always, to design and commission experiments that will investigate and provide insight into these processes in a manner that withstands scientific scrutiny.     

Comparison of the Fecal Microbiota of Healthy Horses and Horses with Colitis by High Throughput Sequencing of the V3-V5 Region of the 16S rRNA Gene

The intestinal tract houses one of the richest and most complex microbial populations on the planet, and plays a critical role in health and a wide range of diseases. Limited studies using new sequencing technologies in horses are available. The objective of this study was to characterize the fecal microbiome of healthy horses and to compare the fecal microbiome of healthy horses to that of horses with undifferentiated colitis. A total of 195,748 sequences obtained from 6 healthy horses and 10 horses affected by undifferentiated colitis were analyzed. Firmicutes predominated (68%) among healthy horses followed by Bacteroidetes (14%) and Proteobacteria (10%). In contrast, Bacteroidetes (40%) was the most abundant phylum among horses with colitis, followed by Firmicutes (30%) and Proteobacteria (18%). Healthy horses had a significantly higher relative abundance of Actinobacteria and Spirochaetes while horses with colitis had significantly more Fusobacteria. Members of the Clostridia class were more abundant in healthy horses. Members of the Lachnospiraceae family were the most frequently shared among healthy individuals. The species richness reported here indicates the complexity of the equine intestinal microbiome. The predominance of Clostridia demonstrates the importance of this group of bacteria in healthy horses. The marked differences in the microbiome between healthy horses and horses with colitis indicate that colitis may be a disease of gut dysbiosis, rather than one that occurs simply through overgrowth of an individual pathogen.