The invasion process of bovine erythrocyte by Babesia divergens: knowledge from an in vitro assay

ABSTRACT: Babesia divergens is a tick-transmitted apicomplexan parasite for which asexual multiplication in its vertebrate hosts is restricted to erythrocytes. Current knowledge of invasion of these target cells is limited. An efficient in vitro invasion assay was set up to gain access to this information. Parasites prepared from infected RBC, lysed by electroporation, and mixed with bovine RBC in a selected synthetic medium (RPMI 1640 supplemented with calcium) were able to establish subsequent cultures with parasitemia ranging from 6 to 14%. Free parasites remaining in the invasion medium could be eliminated by Percoll gradient and culture could be pursued with the freshly invaded erythrocytes. In this way, the invasion time window could be shortened to obtain a synchronised start of the culture or to study the kinetics of invasion. With this assay we demonstrate that 1) erythrocyte invasion by B. divergens is a rapid process since 70% of the invasion-competent parasites invaded the RBC in less than 45 s; 2) all invasion-competent parasites achieved invasion within 10 min of contact; 3) one erythrocyte could be invaded concomitantly by two merozoites; 4) despite a synchronous start, the parasite population evolved heterogeneously resulting in a progressive loss of synchronisation. Western blot analysis of proteins collected from invasion medium were performed with sera from animals experimentally infected with B. divergens and highlighted several proteins. The dose-dependent, inhibitory effects of these sera on B. divergens invasion suggest that these proteins might be involved in the invasion process. Further investigations are required for their characterisation.     

Treatment of feline otitis externa due to Otodectes cynotis and complicated by secondary bacterial and fungal infections with Oridermyl auricular ointment

A blinded randomized study was conducted on 24 cats to confirm the presence of bacterial and/or fungal secondary infections associated with otoacariasis and to verify the efficacy of Oridermyl, an acaricidal/antibiotic/antifungal/anti-inflammatory ointment, for treatment of the primary infestation and secondary infections. Sixteen cats were treated once daily for 10 d; 4 cats were not treated and 4 were treated with a placebo ointment. On Days 0 and 10, ears were swabbed for counts of bacteria and yeasts, for bacterial culture and sensitivity, and examined for determination of the degree of clinical otitis. Auricular secretions were removed for mite counts on Day 10, except for 8 treated cats that were done on Day 30. There was a high number of bacteria and yeasts in most cats and Oridermyl treatment significantly decreased those numbers. Staphylococci were the most frequently isolated bacteria. No live ear mites were found in cats treated with Oridermyl or the placebo ointment.     

Effects of feeding a high omega‐3 fatty acids diet in dogs with naturally occurring osteoarthritis

The aim of this randomized, placebo‐controlled and double‐blinded trial was to compare the effect of a veterinary therapeutic diet (VTD) rich in omega‐3 fatty acids (omega‐3) from fish origin to a regular diet used as control (CTR) over a period of 13 weeks in dogs afflicted by naturally occurring osteoarthritis (OA). Thirty privately owned dogs were selected. Dogs had lameness confirmed by an orthopaedic examination, had stifle/hip OA and had locomotor disability based on the peak of the vertically oriented ground reaction force (PVF) measured using a force platform. At Baseline, all owners were asked to determine 2–5 activities of daily living that were the most impaired. Activities were scores (0–4) in accordance with severity using case‐specific outcome measures (CSOM). The PVF was also measured. Dogs (15/group) were then randomly assigned to receive either the CTR or the VTD. The CSOM was completed twice weekly. The recording of PVF was repeated at Week 7 and 13. The VTD‐fed dogs showed a significantly higher PVF at Week 7 (p < 0.001) and at Week 13 (p < 0.001) when compared to Baseline. From Baseline to Week 13, VTD‐fed dogs had a mean (± SD) change in PVF recording of 3.5 ± 6.8% of body weight (%BW) compared with 0.5 ± 6.1%BW (p = 0.211) in CTR‐fed dogs. This change in primary outcome was consistent with an effect size of 0.5. Conversely, dogs fed the CTR did not show significant change in PVF measurements. At the end of the study, the CSOM was significantly decreased (p = 0.047) only in VTD fed dogs. In lame OA dogs, a VTD that contains high level of omega‐3 from fish origin improved the locomotor disability and the performance in activities of daily living. Such nutritional approach appears interesting for the management of OA.     

Real-time PCR quantification and spatio-temporal distribution of airborne inoculum of Mycosphaerella graminicola in Belgium

Two kinds of propagules play a role in Mycosphaerella graminicola dissemination: splash-dispersed pycnidiospores and airborne sexual ascospores. A method based on real-time polymerase chain reaction (PCR) assay and using Burkard spore traps was developed to quantify M. graminicola airborne inoculum. The method was tested for its reliability and applied in a spore trap network over a 2-year period in order to investigate the spatio-temporal distribution of airborne inoculum in Belgium. At four experimental sites, airborne inoculum was detected in both years. A seasonal distribution was observed, with the highest mean daily quantities (up to 351.0 cDNA) trapped in July and with clusters detected from September to April. The first year of trapping, a mean daily quantity of 15.7 cDNA of M. graminicola airborne inoculum was also detected in the air above a building in a city where the spatio-temporal distribution showed a similar pattern to that in the field. Mean daily quantities of up to 60.7 cDNA of airborne inoculum were measured during the cereal stem elongation and flowering stages, suggesting that it contributes to the infection of upper leaves later in the season. Most detection, however, tended to occur between flowering and harvest, suggesting significant production of pseudothecia during that period. Variations in mean daily quantities from 1.0 to 48.2 cDNA were observed between sites and between years in the patterns of airborne inoculum. After stem elongation, the quantities detected at a site were positively correlated with the disease pressure in the field. Quantities trapped at beginning of the growing season were also well correlated with the disease level the previous year. Multiple regressions revealed that some factors partly explain the daily variations of airborne inoculum.     

Individual heterogeneity in erythrocyte susceptibility to Babesia divergens is a critical factor for the outcome of experimental spleen-intact sheep infections

Susceptibility of sheep erythrocytes to Babesia divergens was investigated in vitro and a high inter-individual variability in their ability to support parasite population development was demonstrated, with some individuals having refractory red blood cells (RBC). As neither changes in growth conditions nor the use of different B. divergens strains influenced the level of susceptibility, the main factor postulated for this variability is the erythrocyte itself. Sheep therefore represent an excellent in vitro model to study the parasite-erythrocyte interaction. In addition, the existence of refractory RBC should help in the identification of the erythrocyte components required for B. divergens development. Experimental infections were carried out on spleen-intact sheep characterized by refractory or fully susceptible erythrocyte types. These differences translated into the successful infection of only those animals with susceptible erythrocytes: infected animals showed no clinical signs, but maintained an asymptomatic persistent infection, as usually observed in the natural bovine host. Sheep therefore represent model organisms that can allow us to study interactions between B. divergens and its vertebrate host at different levels of biological organisation, from the target cell to the intact animal, and represent an experimental infection model of concomitant immunity. Only a low percentage (13%) of the sheep population tested possessed susceptible erythrocytes and the potential role of sheep as a natural host or reservoir of B. divergens is discussed.     

Tocopherols and Tocotrienols in Barley Oil Prepared from Germ and Other Fractions from Scarification and Sieving of Hulless Barley

Two cultivars of hulless barley (Doyce and Merlin) were scarified to abrade the outer layers of the kernels (germ, pericarp, and aleurone). The resulting scarification fines fractions were then separated into four particle-size subfractions using sieves. Each of the size subfractions was then extracted with hexane to produce a barley oil, and the levels of free phytosterols, tocopherols, and tocotrienols in the various barley oils were compared. For both cultivars, the fraction with the largest particle size (0.717–1.410 mm) had the highest oil yields (11–12%). Visual examination of this fraction indicated that it consisted almost entirely of small fragments (≈1 mm) of the germ portion of the kernel. The levels of tocopherols were highest in the largest particle-size fraction and their proportion decreased in the fractions with decreasing particle size. In contrast, the levels of tocotrienols were very low in the largest particle-size fraction and increased in the fractions with decreasing particle sizes. Intact germ was also prepared by hand-dissection, extracted, and analyzed. The results indicate that the ≈1 mm germ fragments obtained by scarification-sieving consisted almost entirely of germ fragments, but these fragments represented only 17.5 and 23.7% of the total mass of the germ, from Merlin and Doyce, respectively. These results also suggest that it may be possible to control the concentrations of tocopherols and tocotrienols in barley oil by controlling the particle size of the feedstock used to extract the oil. Germ fragments isolated by such processes could potentially be used as functional food ingredients or extracted to yield oils enriched in health-promoting phytosterols, tocopherols, or tocotrienols.     

Do cannibalistic fish possess an intrinsic higher growth capacity than others? A case study in the Asian redtail catfish Hemibagrus nemurus (Valenciennes, 1840)

This study relied on the day‐by‐day analysis of bioenergetics and prey size preference in larvae and juveniles of Hemibagrus nemurus selected at random, held in isolation (28.5°C, 12L:12D) and feeding ad libitum on conspecifics [21 fish, 12–46 mm total length (TL), dry mass (DM) of 1–145 mg]. Several traits concur to make young H. nemurus highly cannibalistic: (1) high predation capacities (largest prey = 88% and 70% TL in fish of 8 and >40 mm TL, respectively), (2) opportunistic feeding behaviours with no marked size preference and (3) an unusual combination of high food intake (>100% DM at 1 mg DM) and very high gross conversion efficiency (>0.70). A growth model was constructed from the top performances of fish feeding maximally (16 of 21 fish) and indicated that the risk of cannibalism would be high and permanent except for meal frequencies over five daily meals. Cannibals emerging spontaneously under communal rearing (6 fish L^?1, three daily meals) grew more slowly or just at the same rate as predicted by the growth model constructed from siblings selected at random. This indicates that the individuals that become cannibals do not possess higher intrinsic capacities for growth than others.