Comparison of selected diagnostic methods for identification of Cryptosporidium parvum and Cryptosporidium andersoni in routine examination of faeces

This study involved the comparison of suitability of different methods for routine diagnostics of Cryptosporidium spp. Two staining methods, one concentration-sedimentation method, seven concentration-floatation methods and one combined floatation-sedimentation method were compared. The methods were tested with two concentrations (1 x 10(5) and 1 x 10(6)/g) of C. parvum and C. andersoni. The methods were evaluated using light microscope, magnification 400x for concentration methods and 1000x for stained samples respectively. Specificity of both staining methods was 95-100%. Ziehl-Neelsen with P < 0.01 is more suitable for identification of C. andersoni and modified Milácek-Vítovec with P < 0.01 for identification of C. parvum. Concerning specificity and sensitivity, the floatation-concentration method by Sheather was found to provide the best results of all selected methods. The merthiolate iodine formaldehyde concentration (MIFC) method was the least specific one. The least suitable method concerning sensitivity and costs was the floatation method with caesium chloride (CsCl) with a specificity of 29%.     

A survey on the feeding of eventing horses during competition

Summary This study aims at the comparison of the actual feeding of horses with the recommendations from the literature, and it studies the effects of feeding and exercise on several blood metabolic parameters before and after exercise. Blood samples were collected from 25 horses during one‐star eventing competitions and evaluated for blood glucose, insulin, lactate, free fatty acids and triglyceride levels. Questionnaires on the feeding practices of the horses were evaluated. The questionnaires revealed that during training, and on tournament days, horses received on average 4.3 kg of concentrate per day (min. 1.54 kg, max. 8 kg). The statistical analysis showed no significant effect of the amount of concentrate fed before exercise on the measured blood values. Oil was supplied as a supplementary energy source to 30% of the horses, but most of them only received very small quantities (0.02–0.4 l/day). Five horses (20%) had no access to salt supplements at all, and eleven horses (45%) had no access to salt on tournament days. Fifteen horses (60%) were supplied with mineral feed. Twenty‐one horses (84%) had daily access to pasture during the training period. During competition, 55% of the horses received roughage ad libitum, compared with 37% during training. The majority of the horses received less roughage on days before the cross‐country competition. It could not be ascertained whether feeding a large amounts of roughage had a beneficial effect on performance, because only a few horses in this study were fed with very restrictive roughage. Feeding of most of the horses was in agreement with the recommendations from the literature, except the need for sodium and chloride. The sodium and chloride need for sport horses may be overestimated in literature and needs to be re‐evaluated.     

Cryptosporidium scrofarum n. sp. (Apicomplexa: Cryptosporidiidae) in domestic pigs (Sus scrofa)

We describe the morphological, biological, and molecular characteristics of Cryptosporidium pig genotype II and propose the species name Cryptosporidium scrofarum n. sp. to reflect its prevalence in adult pigs worldwide. Oocysts of C. scrofarum are morphologically indistinguishable from C. parvum, measuring 4.81–5.96 μm (mean = 5.16) × 4.23–5.29 μm (mean = 4.83) with a length to width ratio of 1.07 ± 0.06 (n = 400). Oocysts of C. scrofarum obtained from a naturally infected pig were infectious for 8-week-old pigs but not 4-week-old pigs. The prepatent period in 8-week-old Cryptosporidium-naive pigs was 4–6 days and the patent period was longer than 30 days. The infection intensity of C. scrofarum in pigs was generally low, in the range 250–4000 oocysts per gram of feces. Infected pigs showed no clinical signs of cryptosporidiosis and no pathology was detected. Cryptosporidium scrofarum was not infectious for adult SCID mice, adult BALB/c mice, Mongolian gerbils (Meriones unguiculatus), southern multimammate mice (Mastomys coucha), yellow-necked mice (Apodemus flavicollis), or guinea pigs (Cavia porcellus). Phylogenetic analyses based on small subunit rRNA, actin, and heat shock protein 70 gene sequences revealed that C. scrofarum is genetically distinct from all known Cryptosporidium species.     

Infectivity, pathogenicity, and genetic characteristics of mammalian gastric Cryptosporidium spp. in domestic ruminants.

Farm ruminants were infected experimentally with four mammalian gastric Cryptosporidium, namely Cryptosporidium andersoni LI03 originated from cattle and three isolates of Cryptosporidium muris from brown rat (isolate RN66), Bactrian camel (isolate CB03) and firstly characterized isolate from East African mole rat (isolate TS03). Sequence characterizations of the small-subunit rRNA gene showed that the LI03 isolate was C. andersoni and the other three isolates belonged to C. muris, although the TS03 isolate showed unique sequence variations (one single nucleotide change and four nucleotide insertions). C. andersoni LI03 was infectious for calves only, whereas lambs and kids were susceptible to C. muris CB03. C. muris TS03 and RN66 were not infectious for any farm ruminants. Infection dynamics including prepatent and patent period and infection intensity of the isolates used differed depending on the host species, but no clinical signs of cryptosporidiosis were observed in any of experimentally infected hosts. Cryptosporidium developmental stages were only detected in infected animals in the abomasum region. Histopathological changes were characterized by dilatation and epithelial metaplasia of infected gastric glands with no significant inflammatory responses in the lamina propria.     

Diversity of microsporidia (Fungi: Microsporidia) among captive great apes in European zoos and African sanctuaries: evidence for zoonotic transmission?

Abstract: Two hundred and seventeen captive great apes (150 chimpanzees, Pan troglodytes; 14 bonobos, Pan paniscus; 53 western gorillas, Gorilla gorilla) and 20 personnel from thirteen European zoos and two African sanctuaries were sampled and examined in order to determine the occurrence ofEnterocytozoon bieneusi and species of Encephalitozoon in faecal specimens and to compare the epidemiological situation between zoos and sanctuaries. Microsporidia were detected at all sampling sites. Sequence analyses of ITS amplicons generated by using microsporidia-specific primers determined the presence ofmicrosporidia in 87 samples including 13 humans; since two cases of simultaneous occurrence of Encephalitozoon cuniculi and Enterocytozoon bieneusi were identified, 89 full-length ITS sequences were obtained, namely 78 Encephalitozoon cuniculi genotype I, five E. cuniculi genotype II, two E. hellem 1A and four Enterocytozoon bieneusi. No Encephalitozoon intestinalis-positive samples were identified. This is the first report of Encephalitozoon species and Enterocytozoon bieneusi genotypes in captive great apes kept under various conditions and the first record of natural infection with E. hellem in great apes. A comparison of zoos and sanctuaries showed a significantly higher prevalence of microsporidia in sanctuaries (P<0.001), raising a question about the factors affecting the occurrence of microsporidia in epidemiologically and sanitarily comparable types of facilities.     

New view on the age-specificity of pig Cryptosporidium by species-specific primers for distinguishing Cryptosporidium suis and Cryptosporidium pig genotype II

Two species of Cryptosporidium are commonly identified in pigs: Cryptosporidium suis and Cryptosporidium pig genotype II. Detection of Cryptosporidium spp. is routinely based on molecular methods such as polymerase chain reaction (PCR) and subsequent restriction fragment length polymorphism (RFLP) or gene sequencing. However, most of these methods are hampered by low sensitivity to mixed infections. As a solution of this problem, novel species-specific primers were designed and tested in the present study. Sensitivity of our primers was identical to genus-specific primers, but more (1:48) mixed infections were detected using these species-specific primers on 477 DNA samples originating from naturally infected pigs of different age categories. Our results show differences in age-dependent susceptibility of pigs to the infection. Whereas C. suis was found in all tested categories of pigs (1-12 week of age and sows), Cryptosporidium pig genotype II was recorded only in animals older than 6 week of age. Usage of species-specific primers could help to better the understanding of epidemiology of pig specific Cryptosporidium spp. and its occurrence, which, on the basis of our results, is underestimated.     

Natural infection with two genotypes of Cryptosporidium in red squirrels (Sciurus vulgaris) in Italy

We investigated the genotypes of Cryptosporidium infecting red squirrels (Sciurus vulgaris L.) in two areas of the Western Alps in Italy. Examination of 141 faecal samples from 70 red squirrels revealed oocysts of Cryptosporidium in 17 animals (24.3%). Based on 18S rRNA gene sequencing, two genotypes of Cryptosporidium species were found: 15 squirrels were positive for the Cryptosporidium ferret genotype and 2 for the Cryptosporidium chipmunk genotype I. The occurrence and intensity of Cryptosporidium infection did not differ between the study areas or sex. More than 85% of the positive animals were adults; however no difference was found between Cryptosporidium infection in the juvenile and adult age groups. Oocysts of the Cryptosporidium ferret genotype measured 5.5 +/- 0.3 x 5.2 +/- 0.2 microm (shape index 1.06) and the Cryptosporidium chipmunk genotype I 5.8 +/- 0.3 x 5.4 +/- 0.3 microm (shape index 1.07). Neonatal and adult CD1 and BABL/c mice inoculated with 1 x 10(3) fresh oocysts of both genotypes did not produce detectable infection.     

New indicator for classification of agroclimatic conditions for the cultivation of catch crops

The realization of the benefits of stubble catch crops (CC) cultivated after the harvest of the main crop requires the ability to produce substantial amounts of biomass before the onset of winter. The growth and biomass production of CC is primarily determined by the time period of sowing and weather conditions of the site. The construction of a new indicator of agroclimatic conditions for the cultivation of CC is described. This indicator includes the possible time period for CC sowing according to the harvest time of the preceding main crop, temperature, and water conditions for emergence and growth. The termination of growth due to low temperatures is considered. The assumptions used for the construction of the indicator, and the relationships to CC biomass were validated with data from field experiments. The resulting maps of regions in the Czech Republic with similar indicator values for three groups of CC differing in temperature demands are shown.     

Kinetics and kinematics of the passage

Reasons for performing study: The load acting on the limbs and the load distribution between fore‐ and hindlimbs while performing specific dressage exercises lack objective assessment. Hypothesis: The greater a horse's level of collection, the more load is shifted to the rear and that during the passage the vertical load on the limbs increases in relation to the accentuated vertical movement of the centre of mass. Methods: Back and limb kinematics, vertical ground reaction force and time parameters of each limb were measured in 6 Grand Prix dressage horses performing on an instrumented treadmill at the trot and the passage. Horses were ridden by their own professional rider. Results: At the passage, horses moved at a slower speed (?43.2%), with a lower stride frequency (?23.6%) and, therefore, higher stride impulses (+31.0%). Relative stance duration of fore‐ and hindlimbs and suspension duration remained unchanged. While at the trot the diagonal limbs impacted almost simultaneously, the hindlimbs always impacted first at the passage; the time dissociation between landing and lift‐off remained unchanged. Because of the prolonged stride duration, stride impulse and consequently limb impulses were higher at the passage in the fore‐ as well as in the hindlimbs (+24.8% and +39.9%, respectively). Within the diagonal limb pair, load was shifted from the forehand to the hindquarters (percentage stride impulse carried by the forehand ?4.8%). Despite the higher impulses, peak vertical forces in the fore‐ and hindlimbs remained unchanged because of the prolonged absolute stance durations in fore‐ and hindlimbs (+28.1% and +32.2%, respectively). Conclusions: Based on the intralimb timing, the passage closely resembles the trot. Compared to other head‐neck positions, the higher degree of collection resulted in a pronounced shift in impulse towards the hindquarters. Despite the higher limb impulses, peak forces acting on the limbs were similar to those observed at the trot. Potential clinical relevance: An understanding of load distribution between fore‐ and hindlimbs in relation to different riding techniques is crucial to prevent wear‐and‐tear on the locomotor apparatus.     

Influence of different head‐neck positions on vertical ground reaction forces,linear and time parameters in the unridden horse walking and trotting on a treadmill

Reasons for performing study: It is believed that the head‐neck position (HNP) has specific effects on the loading pattern of the equine locomotor system, but very few quantitative data are available. Objective: To quantify the effects of 6 different HNPs on forelimb‐hindlimb loading and underlying temporal changes. Methods: Vertical ground reaction forces of each limb and interlimb coordination were measured in 7 high level dressage horses walking and trotting on an instrumented treadmill in 6 predetermined HNPs: HNP1 ‐ unrestrained; HNP2 ‐ elevated neck, bridge of the nose in front of the vertical; HNP3 ‐ elevated neck, bridge of the nose behind the vertical; HNP4 ‐ low and flexed neck; HNP5 ‐ head and neck in extreme high position; and HNP6 ‐ forward downward extension of head and neck. HNP1 served as a velocity‐matched control. Results: At the walk, the percentage of vertical stride impulse carried by the forehand (Iz_fore) as well as stride length and overreach distance were decreased in HNP2, HNP3, HNP4 and HNP5 when compared to HNP1. At the trot, Iz_fore was decreased in HNP2, HNP3, HNP4 and HNP5. Peak forces in the forelimbs increased in HNP5 and decreased in HNP6. Stance duration in the forelimbs was decreased in HNP2 and HNP5. Suspension duration was increased in HNP2, HNP3 and HNP5. Overreach distance was shorter in HNP4 and longer in HNP6. Conclusions: In comparison to HNP1 and HNP6, HNPs with elevation of the neck with either flexion or extension at the poll as well as a low and flexed head and neck lead to a weight shift from the forehand to the hindquarters. HNP5 had the biggest effect on limb timing and load distribution. At the trot, shortening of forelimb stance duration in HNP5 increased peak vertical forces although Iz_fore decreased. Potential relevance: Presented results contribute to the understanding of the value of certain HNPs in horse training.