Prevalence of anti-Toxoplasma gondii antibody in domestic horses in Japan

The present study is the first report that investigated the seroprevalence of Toxoplasma gondii infection in domestic horses in various prefectures of Japan and analyzed risk factors for seropositivity. We performed a latex agglutination test for riding/racing horses from 11 prefectures in Japan (783 samples) and 4 groups of Japanese native horses (254 samples). The total seroprevalence of anti-T. gondii antibody in horses examined in this study was 4.24% (44/1037). As for riding/racing horses, we did not find a statistically different T. gondii seroprevalence between sampling prefectures. In contrast, seroprevalence of T. gondii in older horses (> 21 years) was significantly higher than that in younger horses (< 5 years and 11–15 years). There was no significant difference in T. gondii seroprevalence between riding/racing horses and Japanese native horses. Logistical regression analysis revealed that age, but not sex and usage, is a significant risk factor of T. gondii infection for domestic horses in Japan. These findings suggest that domesticated horses in Japan can be horizontally infected with T. gondii by ingestion of food or water contaminated with oocysts.     

First description of Coxiella burnetii and Rickettsia spp. infection and molecular detection of piroplasma co-infecting horses in Xinjiang Uygur Autonomous Region,China

Q fever, spotted fever rickettsioses and equine piroplasmosis, are some of the most serious equine tick-borne diseases caused by Coxiella burnetii, Rickettsia spp., Babesia caballi and/or Theileria equi. This study surveyed and molecularly characterized these pathogens infecting horses in ten ranches from XUAR, China using molecular technology. Among 200 horse blood samples, 163 (81.5%) were infected with at least one of the pathogens. Rickettsia spp. was the most prevalent pathogen (n = 114, 57.0%), followed by C. burnetii (n = 79, 39.5%), T. equi (n = 79, 39.5%) and B. caballi (n = 49, 24.5%). Co-infections were observed in 61.3% of positive samples in this study. Statistically significant differences were observed between the sampling regions for C. burnetii, B. caballi and T. equi, and also in different age group for C. burnetii and T. equi. The genotype analysis indicated that C. burnetii htpB, Rickettsia spp. ompA, B. caballi rap-1, B. caballi 18S rRNA, T. equi EMA-1 and T. equi 18S rRNA gene sequences from horses in XUAR were variable. To the best of our knowledge, this study is the first report of C. burnetii and Rickettsia spp. infection and co-infected with piroplasma in horses in China. Overall, this study revealed the high infection rate of the pathogens in horses in XUAR, China. The current findings are expected to provide a basis for better tick-borne disease control in the region.     

Activation of foal neutrophils at different ages by CpG oligodeoxynucleotides and Rhodococcus equi

Toll-like receptor 9 (TLR9) activation stimulates protective immune responses against intracellular pathogens by phagocytes, including neutrophils. This study examined TLR9-mediated neutrophil activation in neonatal foals. Unmethylated CpGs, ligands for TLR9, were used to stimulate equine neutrophils, either purified or in contact with other peripheral blood leukocytes. Rhodococcus equi was used as another stimulus in parallel. TLR9 mRNA was constitutively expressed at a similar level in purified equine neutrophils across different ages from birth to adulthood, and expression was not affected by either CpG or R. equi. Purified foal neutrophils were directly sensitive to CpG stimulation, reflected by enhanced reactive oxygen species generation following fMLP stimulation, and by expressing significantly (P < 0.05) greater mRNA of IFN-γ, IL-8, IL-12p35, and significantly (P < 0.05) decreased TNF-α mRNA. In comparison, purified foal neutrophils stimulated by R. equi showed significantly (P < 0.05) increased mRNA production of IL-6, IL-8, IL-23p19, and TNF-α. Neutrophils co-cultured with other leukocytes expressed a distinct profile of cytokine mRNA than purified neutrophils in response to CpG stimulation, whereas the profile was very similar following R. equi stimulation irrespective of neutrophil purity. When co-cultured with other leukocytes, foal neutrophils were significantly (P < 0.05) activated at birth by B-class CpGs and produced IL-6, IL-8, IL-12p40, and IL-23p19 at similar magnitudes to those at 2 months of age. In foal neutrophils at birth, R. equi significantly (P < 0.05) induced all cytokines stimulated by CpGs (except IL-12p40), as well as TNF-α. Our results indicate that foal neutrophils were sensitive to CpG or R. equi activation as early as at birth, and that B-class CpGs enhanced foal neutrophil functions in vitro.     

Inhibitory effect of terpene nerolidol on the growth of Babesia parasites

Nerolidol is a sesquiterpene present in the essential oils of many plants, approved by the U.S. FDA as a food flavoring agent. Nerolidol interferes with the isoprenoid biosynthetic pathway in the apicoplast of P. falciparum. In the present study, the in vitro growth of four Babesia species was significantly (P < 0.05) inhibited in the presence of nerolidol (IC₅₀s values = 21 ± 1, 29.6 ± 3, 26.9 ± 2, and 23.1 ± 1 µM for B. bovis, B. bigemina, B. ovata, and B. caballi, respectively). Parasites from treated cultures failed to grow in the subsequent viability test at a concentration of 50 µM. Nerolidol significantly (P < 0.05) inhibited the growth of B. microti at the dosage of 10 and 100 mg/kg BW, while the inhibition was low compared with the high doses used. Therefore, nerolidol could not be used as a chemotherapeutic drug for babesiosis.     

Seroprevalence and risk factors associated with Toxoplasma gondii in domestic pigs from Spain

Serum samples from 2970 (1400 sows, 1570 fattening) pigs, from 100 farms in the 10 main swine production regions in Spain were tested for antibodies against T. gondii by the modified agglutination test (MAT). Antibodies to T. gondii (MAT 1:25 or higher) were detected in 492 pigs (16.6%, 9.7% in fattening pigs and 24.2% in sows). The herd prevalence was 85.0% (95% CI: 78–92) and within-farm prevalence ranged from 2.9% to 92.8% (median = 17.6%). Statistically significant differences were observed among sampling regions with seroprevalence significantly higher in pigs from Valencia Community (27.3%), Extremadura (23.3%) and Catalonia (21.2%). A generalized estimating equations model indicated that the risk factors associated with T. gondii seroprevalence were: age, sows compared to fattening pigs (OR = 2.9; 95% CI = 1.83–4.53), lack of rodent control (OR = 1.9; 95% CI = 1.04–3.60) and presence of cats (OR = 1.6; 95% CI = 1.12–2.34). The seroprevalence observed in the present study indicates a widespread, although variable, exposure to T. gondii among domestic pigs in Spain, which might have important implications for public health. Management measures including control of rodents and cats on the farms could help to reduce the observed prevalence levels in Spain.     

Object recognition and generalisation during habituation in horses

The ability of horses to habituate to frightening stimuli greatly increases safety in the horse–human relationship. A recent experiment suggested, however, that habituation to frightening visual stimuli is relatively stimulus-specific in horses and that shape and colour are important factors for object generalisation (Christensen et al., 2008). In a series of experiments, we aimed to further explore the ability of horses (n = 30, 1 and 2-year-old mares) to recognise and generalise between objects during habituation. TEST horses (n = 15) were habituated to a complex object, composed of five simple objects of varying shape and colour, whereas CONTROL horses (n = 15) were habituated to the test arena, but not to the complex object. In the first experiment, we investigated whether TEST horses subsequently reacted less to i) simple objects that were previously part of the complex object (i.e. testing for object recognition) and ii) a novel object (new shape and colour, i.e. testing for object generalisation), compared to CONTROLS. In the second experiment we investigated whether TEST horses reacted to a change in object order and object location. Behavioural reactions to the object, latency to eat, total eating time and heart rate were recorded. Compared to CONTROLS, TEST horses reacted significantly less towards objects, which were previously part of the complex object (e.g. mean heart rate; P = 0.006), indicating object recognition. In contrast to our expectations, TEST horses also reacted significantly less towards the novel object (e.g. mean heart rate; P = 0.018), suggesting that they were capable of object generalisation. We also found that TEST horses showed an increase in exploratory behaviour when objects within the complex object changed order and location (both P < 0.001), whereas there was no increase in heart rate, indicating that the horses were not frightened by the changes. The results demonstrate that it is possible to increase object generalisation in horses by habituating them to a range of colours and shapes simultaneously. This knowledge greatly affects the way in which horses may be trained to react calmly towards frightening objects.     

Effects of enrichment items on activity and social interactions in domestic horses (Equus caballus)

The aim of this study was to investigate the use of items intended to provide enrichment during turnout, both for individual and group kept horses in an attempt to reduce the amount of passive behaviours. The study was divided into two parts, where study 1 involved eight horses rotated through eight individual paddocks, each containing one of seven enrichment items and one paddock being kept without item, functioning as a control. The horses’ item-directed behaviours; passive behaviours or other non-item related activities were scored using instantaneous sampling, every minute for 1 h at the beginning and the end of the turnout period. Study 2 involved six horse groups (3–6 horses) and the same scoring methods and ethogram as in study 1. The four items that the horses interacted the most with during study 1 (straw STRA, ball filled with concentrates CBALL, branches BRAN and scratching pole POLE) are investigated in study 2. In addition, the amount of social interactions was recorded.Both horses kept individually (P < 0.05) and in groups (P < 0.0001) performed significantly more item-directed behaviours towards edible items like STRA and CBALL than other objects. There was, however, no overall relation between the numbers of item-directed behaviours and the number of passive behaviours observed, indicating that the enrichment items did not alone reduce the amount of passive behaviours during turnout periods. Such a reduction was, however, only apparent when horses spent more time eating green leaves growing on the paddock surface (R = ?0.97 study 1, R = ?0.67 study 2, P < 0.0001). Access to STRA in group kept horses also seemed to reduce the amount of agonistic behaviours (P < 0.0001). In conclusion, if grass is not available in paddocks, the provision of roughage reduces the amount of passive behaviours in singly kept horses and it also reduces the risk of agonistic interactions between horses kept in group.     

Evaluation and comparison of four methods of ranking horses based on reactivity

Four methods of ranking horses on reactivity were evaluated and compared: isolation from conspecifics, presentation of a static novel stimulus, traversing a novel stimulus in a runway (isolation, novel stimulus and runway tests, respectively) and assigning subjective emotionality scores. In all tests, horses’ heart rates were recorded and behaviour was videotaped. To be considered a valid test of reactivity, at least one heart rate and one behavioural measurement in the test had to change significantly between treatments (tranquilizer administation versus sham tranquilizer administration), and behavioural measures had to be displayed in at least 75% of the trials. Forty horses performed each of the three tests daily on three different days in a switchback design. Horses were assigned randomly to a daily test sequence, which was maintained throughout the study. In the runway test, no significant difference in heart rate values in tranquilized and non-tranquilized horses was found, and no behavioural attribute was displayed in more than 52% of the trials; therefore it was rejected as a valid test of reactivity. Both isolation and novel stimulus tests produced valid measurements. Mean heart rate was the most precise physiological measure for these tests, and walking and defecation frequency were the most precise behavioural measures for novel stimulus and isolation tests, respectively. Mean heart rates on the novel stimulus and isolation tests were correlated (r_s = 0.79, P < 0.01) indicating that these tests produced similar rankings based on physiological responses. However, behavioural measures ranked horses differently (r_s = 0.27, P < 0.10) on the tests. Rank correlations between mean heart rates and behavioural measures were higher in the novel stimulus (r_s = 0.66, P < 0.01) than the isolation test (r_s = 0.55, P < 0.01), indicating that the novel stimulus test ranked horses based on either physiological or behavioural responses more similarly than did the isolation test. Therefore, the novel stimulus test was considered the more accurate evaluation of reactivity. Subjective emotionality scores were correlated moderately with mean heart rates (r_s > 0.33, P < 0.01) from the novel stimulus and isolation tests and with walking scores (r_s = 0.47, P < 0.01) from the novel stimulus test. Assignment of subjective emotionality scores was not as accurate as the novel stimulus or isolation tests in ranking horses for reactivity. Using physiological data alone, combining physiological and behavioural measurements or using more than one behavioural measurement in reactivity tests may reflect the reactivity of the horse better than a single behavioural measurement.     

Phytochemical and antioxidant properties of unconventional leafy vegetables consumed in southern Africa

Indigenous leafy vegetables possess high horticultural potential based on their long utilisation history by local communities across Africa. Phytochemical and antioxidant properties of 50% aqueous methanol and water extracts of three indigenous as well as two commercial leafy vegetables commonly consumed in southern Africa were evaluated. The total extractable phenolic content was highest for Amarathus dubius (5.16 ± 0.12 mg GAE/g DW) followed by Cleome gynandra (3.94 ± 0.09 mg GAE/g DW). Total flavonoid concentration was highest for A. dubius (3.89 ± 0.28 mg CE/g DW) followed by C. gynandra (2.19 ± 0.11 mg CE/g DW) and Cucurbita maxima (1.55 ± 0.04 mg CE/g DW). No proanthocyanidins were detected in C. maxima and Brassica napus cv Covo whereas low concentrations were recorded in other vegetables. Total saponins were variable across the evaluated extracts, with the highest concentrations recorded for B. napus cv Covo (83.2 ± 16.58 mg DE/g DW). Total iridoid content was highest for C. gynandra (9.14 ± 0.20 mg HE/g DW). More potent DPPH radical scavenging activities were exhibited by 50% aqueous methanol extracts compared to water extracts. A similar trend was observed in the ferric-reducing antioxidant power assay. The antioxidant activity based on the rate of β-carotene bleaching was higher for water extracts compared to 50% aqueous methanol extracts. The indigenous vegetables evaluated in this study had higher levels of phytochemicals and also exhibited more potent antioxidant activity compared to the commercial varieties. These findings not only suggest the importance of the indigenous vegetables in a healthy diet, but also provide a motivation for exploring their horticultural potential.     

Fruit nutritional composition and non-nutritive traits of indigenous South African tree species

Frugivorous animals play a major role in dispersing tropical, and to a lesser extent, temperate tree species. In order to attract potential seed dispersers, plants generally offer a reward of fleshy fruit pulp. Criteria for fruit choice by avian frugivores are influenced by a number of non-nutritive (e.g. fruit size and colour) factors; and nutritional composition of the fruit. There is a paucity of nutritional composition and other fruit trait data of indigenous South African fruit. This information is necessary in order to determine which frugivores are likely to ingest which fruits and consequently act as potential seed dispersal agents. This information would provide us with an understanding of the inter-relationships between indigenous fruit and frugivores in South Africa. Consequently nutritional composition was investigated in various indigenous fruit species that avian frugivores feed on. Fruits were collected from 38 indigenous tree species found in KwaZulu-Natal Afromontane and coastal forests. Pulp was freeze-dried to constant mass and then analysed for sugar, lipid and protein content; and for water content determination. Fruit width in this study ranged from 4 mm (Searsia rehmanniana and Trema orientalis) to 40 mm (Annona senegalensis, Ficus sur and Xylotheca kraussiana). Of the fruits examined in this study 29% were black and 43% were red when ripe. Most (84%) fruit species analysed for sugar content were hexose dominant with 50% being fructose and 34% being glucose dominant. Only 16% of the fruit species analysed were sucrose dominant. Fruits in this study were generally observed to be high (mean: 68.1 ± 3.3%; n = 30) in water content; and low in protein and lipid content respectively (mean: 8.2 ± 0.5%; 9.3 ± 2.2%; n = 30) indicating that these fruit species could be considered as nutrient-dilute. Future studies need to determine the nutritional composition of the remaining indigenous South African fruit in order to develop a comprehensive database as well as examining non-nutritive factors.     

Housing conditions and breed are associated with emotionality and cognitive abilities in riding school horses

Horses’ emotional reactivity is of a major importance in riding schools where a variety of more or less experienced riders are present. Horses’ learning abilities may also be important for work. Previous studies have shown that different intrinsic or extrinsic factors, such as breed, housing conditions, sire, and work may have an influence, and that different facilities present horses with different characteristics. In this study, we tested the reactions of 184 horses from 22 riding schools, all practicing the same type of work, but differing in particular in terms of housing conditions, to 3 emotionality tests and to one instrumental learning task, in order to (1) try and characterize riding schools, and (2) determine how general management could explain some of the potential differences observed between sites.Multivariate analysis conducted on the whole set of data showed that riding schools could be classified into four categories from those where horses showed low emotionality levels and good learning abilities to those where horses showed high emotionality levels and poor learning abilities. Breed, in accordance to previous studies, had an impact on the time to cross a novel obstacle in hand (“Bridge Test”) (Kruskall–Wallis, H (14, N = 184) = 27.08, p < 0.05) while housing conditions (e.g. box housing) influenced emotionality (emotionality index, MW: p < 0.05), when horses were released in the arena. Box housing was associated with more active locomotion patterns (including trot, canter and passage) (MW: p < 0.005). These results underline the importance for riding schools to take into account both the individual characteristics of the horse, and in particular the breed, and to consider the impact of general management including housing conditions, on horses’ reactivity, and its consequences in terms of human security.     

Survivability of indigenous microflora and a Salmonella typhimurium marker strain in poultry mash treated with buffered propionic acid

Buffered propionic acid (BPA) was evaluated as a potential treatment for the elimination of Salmonella spp. in poultry mash. A primary poultry isolate marker strain of Salmonella typhimurium was added as either a broth or in a dry chalk carrier form to poultry mash containing soybean meal as a protein supplement. The mash was supplemented with buffered propionic acid at 2, 4, 6, 8, 10, 20, 30, 50 and 100 g kg⁻¹ diet and samples were enumerated for indigenous aerobic bacteria, fungi and the S. typhimurium marker strain. Total indigenous aerobic bacteria and fungal populations were generally decreased by addition of more than 20 g BPA kg⁻¹, but an addition of 100 g BPA kg⁻¹ mash was usually required to achieve reductions of approximately 90% of indigenous aerobic bacteria and 99% of indigenous fungi. After 7 days of storage, 8 g BPA kg⁻¹ mash also reduced S. typhimurium populations by more than 90% in mash inoculated via chalk, while at least 50 g BPA kg⁻¹ mash was required to provide the same level of reduction in mash inoculated with a liquid culture of S. typhimurium. Although BPA does not appear to be an overly effective antimicrobial agent with respect to indigenous aerobic bacterial populations in animal feed, higher concentrations may have the potential for reducing fungal and Salmonella spp. contamination in poultry mash.     

Comparison of the RE and B1 gene for detection of Toxoplasma gondii infection in children with cancer

Early, accurate and effective diagnosis of toxoplasmosis can make an important contribution to the prevention and control of disease, especially in people who are at risk. In this study, two commonly used genomic repeats of Toxoplasma gondii, RE (GenBank accession number AF146527) and B1, were compared to each other in nested-PCR assay. Five hundred and thirty-five blood samples from children with leukemia were tested for the presence of T. gondii antibodies using enzyme immunoassays. One hundred and ten DNA samples of these patients (50 IgM +, IgG +, 10 IgM −, IgG +, and 50 IgM −, IgG −) were analyzed by nested-PCR. The specificity of two nested PCR assays was determined using the DNA samples of other parasites and human chromosomal DNA. As a result, 82% (41/50) and 68% (34/50) of the IgM +, IgG + samples were positive on duplicate RE and B1-nested PCR analyses, respectively. None of the 10 IgM −, IgG + seropositive samples was detected positive after testing RE and B1-nested PCR assays in duplicate. One (2%) of the 50 seronegative samples was positive by duplicate RE-nested PCR but none of them were positive by duplicate B1-nested PCR. The detection limit of the RE-nested PCR assay was 640 fg of T. gondii DNA whereas this rate for B1-nested PCR was 5.12 pg of the DNA template. No cross-reactivity with the DNA of other parasites and human chromosomal DNA was found. The results indicate that an RE-based nested PCR assay is more sensitive than B1 genomic target, of those tested, for detection of T. gondii. It is noteworthy that in comparison with B1-nested PCR, RE-nested PCR could detect the T. gondii DNA in seronegative samples too.     

Proximate,mineral, fibre,phytate–phosphate,vitamin E,amino acid and fatty acid composition of Terminalia sericea

Terminalia sericea is widely distributed in the African Savannah bushveld. It is one of the indigenous fruit bearing trees put to multiple uses. Research has focused on the phytochemical composition of its root, bark, and leaf extracts that are used in ethnomedicine neglecting the potential of its seed. This study purposed to determine, by chemical analyses, the nutritive value of T. sericea seed. 78.8% of the seed was found to be crude protein (46.2%) and lipid (32.6%). Ash made up 6.90% of the seed mass. Linoleic and oleic acids constituted 68.63% and 14.05%, respectively, of the seed oil. Phosphorus (1121.75 ± 10.39 mg 100 g^− 1 DM) and glutamic acid (8.07 ± 0.13 g 100^− 1 DM) constituted the most concentrated mineral and amino acid, respectively. T. sericea seed could be utilized as a protein source in feeds and foods and could also be exploited as a non-conventional plant oil source of oleic acid and linoleic acid.     

The use of a mirror reduces isolation stress in horses being transported by trailer

Horse trailers are a common form of transportation for horses and ponies and often require the animal to travel alone or with a single companion. The current study investigated the effect of transporting horses alone, in company or with an acrylic safety mirror (measuring 81 cm × 61.5 cm) that provided surrogate companionship. The behavioural and physiological responses of 12 mature horses during a 30-min journey by trailer under the three treatments were compared. Behaviours (vocalisation, eating, head-tossing, pawing, and head-turning) were recorded. In order to assess circulatory changes that occur as part of the response to transport, heart rate (HR), rectal (T_r) and ear-pinna (T_p) temperatures were recorded. When travelling with a live companion significantly less time was spent vocalising (p < 0.001), head-turning (p < 0.001), head-tossing (p < 0.01) and pawing (p < 0.01); eating behaviour increased (p < 0.05). Physiological responses (increases in HR and T_r and decreases in T_p) were also significantly reduced when travelling with a live companion (p < 0.01). Travelling with the mirror did not significantly affect physiological responses compared with travelling alone, but the rise in T_r and fall in T_p was reduced (p = 0.052 and p = 0.051, respectively) and can be considered a trend. When travelling with a mirror significantly less time was spent turning the head (p < 0.01), vocalising (p < 0.05) and head-tossing (p < 0.05); eating behaviour increased (p < 0.05). The only significant difference between travelling with a live companion and a mirror was that the time spent turning the head round was less with a live companion (p < 0.05). The provision of surrogate companionship in the form of a mirror was found to be preferable to travel alone, but where possible a live companion is recommended. Isolation during transportation was found to suppress feeding behaviour. Although peripheral blood flow (T_p) has been used to assess transport stress in other species it has not previously been used in the horse. Further evaluation of this non-invasive measure is now required.     

Rheediinosides A and B,two antiproliferative and antioxidant triterpene saponins from Entada rheedii

Two triterpenoid saponins have been isolated from the seed kernels of Entada rheedii. Their structures have been established using 1D- and 2D-NMR and mass spectrometry as 3-O-β-d-xylopyranosyl-(1 → 3)-O-α-l-arabinopyranosyl-(1 → 6)-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside A, 1) and 3-O-β-d-glucopyranosyl-(1 → 3)-O-[β-d-xylopyranosyl-(1 → 3)-α-l-arabinopyranosyl-(1 → 6)]-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside B, 2). Compounds 1 and 2 were tested for their antiproliferative activity against T98G, A431, PC3 and B16-F1 cell lines, and further for their antioxidant properties. Moderate cytotoxic potency and antioxidant properties were found for these compounds whereas Rheediinoside B was in all assays more active than Rheediinoside A.     

Interactions between Phaedrotoma scabriventris Nixon (Hymenoptera: Braconidae) and Diglyphus isaea Walker (Hymenoptera: Eulophidae), parasitoids of Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae)

Liriomyza leafminer flies represent a serious threat to horticultural production in East Africa. Total field parasitism rates recorded in Kenya are below 5%, with the indigenous ectoparasitoid Diglyphus isaea Walker being one of the key parasitoid species. The International Centre of Insect Physiology and Ecology (icipe), in collaboration with the International Potato Centre (CIP), imported into Kenya the endoparasitoid Phaedrotoma scabriventris Nixon to improve natural control of leafminers. The objective of this study was to investigate the interactions between D. isaea and P. scabriventris when used together for the biological control of Liriomyza species. These interactions were studied under laboratory conditions, using treatments that involved single, simultaneous and sequential releases of the different parasitoid species onto plants infested by L. huidobrensis larvae. While used separately, parasitism rates of D. isaea and P. scabriventris were 30.4 ± 10.9% and 63.6 ± 7.7% respectively. However, when used simultaneously, the total parasitism rate increased to 77.0 ± 5.3%. Although P. scabriventris had no effect on D. isaea, the presence of D. isaea reduced the specific parasitism rate of P. scabriventris. In addition, both parasitoids induced leafminer mortality through larval-feeding and stinging. However, feeding and stinging mortality induced by D. isaea (41.9 ± 9.1%) was significantly higher compared to P. scabriventris (11.9 ± 8.7). Similarly, pupal mortality due to feeding and stinging activity was 49.1 ± 6.5% and 21.6 ± 1.9% when exposed to D. isaea and P. scabriventris respectively. The implication for simultaneous use of both parasitoids in East Africa is discussed.     

Soil microbial biomass influence on growth and biocontrol efficacy of Trichoderma harzianum

Hyphal growth and biocontrol efficacy of Trichoderma harzianum may depend on its interactions with biotic components of the soil environment. Effects of soil microbial biomass on growth and biocontrol efficacy of the green fluorescent protein transformant T. harzianum ThzID1-M3 were investigated using different levels of soil microbial biomass (153, 328, or 517 μg biomass carbon/g of dry soil). Hyphal growth of T. harzianum was significantly inhibited in soil containing 328 or 517 μg biomass carbon/g of dry soil compared with soil containing 153 μg biomass carbon/g. However, when ThzID1-M3 was added to soil as an alginate pellet formulation, recoverable populations of ThzID1-M3 varied, with the highest populations in soil containing 517 μg biomass carbon/g. When sclerotia of Sclerotinia sclerotiorum were added to soils (10 sclerotia per 150 g soil) with ThzID1-M3 (20 pellets per 150 g soil), colonization of sclerotia by ThzID1-M3 was significantly lower in the soil containing the highest level of biomass. Addition of alginate pellets of ThzID1-M3 to soils (10 pellets per 50 g) resulted in increased indigenous microbial populations (total fungi, bacteria, fluorescent Pseudomonas spp., and actinomycetes). Our results suggest that higher levels of microbial soil biomass result in increased interactions between introduced T. harzianum and soil microorganisms, and further that microbial competition in soil favors a shift from hyphal growth to sporulation in T. harzianum, potentially reducing its biocontrol efficacy.     

Can Beauveria bassiana Bals. (Vuill) (Ascomycetes: Hypocreales) and Tamarixia triozae (Burks) (Hymenoptera: Eulophidae) be used together for improved biological control of Bactericera cockerelli (Hemiptera: Triozidae)?

Bactericera cockerelli (Sulc.) is an important pest of solanaceous crops and a vector of the pathogen Candidatus Liberibacter psyllaurous. Biocontrol of this pest has been attempted with either entomopathogenic fungi or the parasitoid Tamarixia triozae (Burks), but prior to this study, their potential impact in combination had not been studied. The aim of the present study was to evaluate T. triozae parasitism rates on B. cockerelli nymphs that were previously infected for different periods of time by three isolates of Beauveria bassiana (Bals.) Vuill. Two native isolates (BB40 and BB42) and one commercial isolate (GHA) were used. The virulence of these isolates was first estimated against B. cockerelli and T. triozae. LC₅₀ values for the native isolates BB40 and BB42 against B. cockerelli were 9.5 × 10⁵ and 2.42 × 10⁶ conidia mL⁻¹ respectively; they were significantly more virulent than isolate GHA with an LC₅₀ of 1.97 × 10⁷ conidia mL⁻¹. However, isolate GHA was significantly more virulent against T. triozae with an LC₅₀ of 1.11 × 10⁷ conidia mL⁻¹ compared with LC₅₀s of 1.49 × 10⁷ and 1.14 × 10⁸ conidia mL⁻¹ for the native isolates BB40 and BB42 respectively. Groups of nymphs were then inoculated with LC₂₀, LC₅₀ or LC₉₀ concentrations of each isolate and presented to T. triozae as hosts either on the day of inoculation or 1, 2, 3, 4, 5, 6 days after inoculation. Subsequent levels of parasitism were recorded. Overall, parasitism rates were similar in inoculated and control nymphs. No parasitism occurred in nymphs 6 days after fungal inoculation. Parasitoids used to parasitize uninoculated B. cockerelli nymphs survived significantly longer (7.8 days) than parasitoids that had been used to parasitize fungus-inoculated nymphs (7.3 days). This suggests an inability of the parasitoid to avoid infection when foraging on inoculated nymphs. In conclusion, although the parasitism rate in control and fungus-treated nymphs was similar, suggesting a combination of both biological control agents is possible, we believe there are also negative implications for the parasitoid because its survival was greatly reduced after attacking infected nymphs.     

Impact of riding in a coercively obtained Rollkur posture on welfare and fear of performance horses

Rollkur, the usually coercively obtained hyperflexion of the horse's neck, is employed as a training method by some dressage riders; however, its use is controversial as it may cause discomfort and adversely affect the horse's welfare. The objectives of this study were to determine: (1) if horses showed differences in stress, discomfort and fear responses as measured by heart rate and behaviour when ridden in Rollkur (R) obtained by pressure on the reins compared to regular poll flexion (i.e. with the nose-line being at or just in front of the vertical; N), and (2) if they showed a preference between the two riding styles when given the choice. Fifteen riding horses were ridden 30 times through a Y-maze randomly alternating between sides. Riding through one arm of the Y-maze was always followed by a short round ridden in R, whereas riding through the other arm was followed by a short round ridden in N. Immediately after the conditioning phase, horses were again repeatedly ridden into the maze; however, riders left it to the horse to decide which arm of the maze to enter. During R, horses moved slower and showed more often behavioural signs of discomfort, such as tail-swishing, head-tossing or attempted bucks (P < 0.05), and 14 of the 15 horses chose significantly (P < 0.05) more often the maze-arm associated with N rather than R. Subsequently, eight of the horses were also subjected to two fear tests following a short ride in N as well as a ride in R. During R, horses tended to react stronger (P = 0.092) to the fear stimuli and to take longer (P = 0.087) to approach them. These findings indicate that a coercively obtained Rollkur position may be uncomfortable for horses and that it makes them more fearful and therefore potentially more dangerous to ride. Further studies need to assess horses’ reaction to gradual training of Rollkur, as opposed to a coercively obtained hyperflexion, in order to decide whether the practice should be banned.