Evaluation of the in vitro growth-inhibitory effect of epoxomicin on Babesia parasites

Epoxomicin potently and irreversibly inhibits the catalytic activity of proteasomal subunits. Treatment of proliferating cells with epoxomicin results in cell death through accumulation of ubiquinated proteins. Thus, epoxomicin has been proposed as a potential anti-cancer drug. In the present study, the inhibitory effects of epoxomicin on the in vitro growth of bovine and equine Babesia parasites were evaluated. The inhibitory effect of epoxomicin on the in vivo growth of Babesia microti was also assessed. The in vitro growth of five Babesia species that were tested was significantly inhibited (P < 0.05) by nanomolar concentrations of epoxomicin (IC₅₀ values = 21.4 ± 0.2, 4 ± 0.1, 39.5 ± 0.1, 9.7 ± 0.3, and 21.1 ± 0.1 nM for Babesia bovis, Babesia bigemina, Babesia ovata, Babesia caballi, and Babesia equi, respectively). Epoxomicin IC₅₀ values for Babesia parasites were low when compared with diminazene aceturate and tetracycline hydrochloride. Combinations of epoxomicin with diminazene aceturate synergistically potentiated its inhibitory effects in vitro on B. bovis, B. bigemina, and B. caballi. In B. microti-infected mice, epoxomicin caused significant (P < 0.05) inhibition of the growth of B. microti at the non-toxic doses of 0.05 and 0.5 mg/kg BW relative to control groups. Therefore, epoxomicin might be used for treatment of babesiosis.     

Characterization of the immune response of domestic fowl following immunization with proteins extracted from Dermanyssus gallinae

Dermanyssus gallinae is the most significant ectoparasite of European poultry egg laying production systems due to high costs of control and associated production losses as well as adverse effects on bird welfare. In this study, soluble proteins were extracted from unfed D. gallinae (DGE) using a urea-based detergent and ultra-filtration, passed through a 0.22 μm filter and blended aseptically with adjuvant. One group of laying hens was immunized with DGE and adjuvant (Montanide ISA 50 V) whilst another group (Control) received physiological saline and adjuvant. All birds were immunized on two occasions, 21 days apart. Antibody response to immunization was determined by ELISA and western blotting using immunoglobulins (Igs) extracted from egg yolk. DGE immunization of hens resulted in a significant (P < 0.05) IgY response compared to controls, although there was no significant difference in IgM response between treatments. A number of proteins were identified by western blotting using IgY antibodies from DGE immunized birds, most prominently at 40 and 230 kDa. Analysis of proteins from approximately corresponding bands on SDS-PAGE confirmed the identity of tropomyosin, whilst other proteins showed high sequence homology with myosin and actin from other arachnid and insect species. Immunization of hens with DGE resulted in a 50.6% increase in mite mortality (P < 0.001) 17 h after feeding when tested by an in vitro mite feeding model. Data in this study demonstrate that somatic antigens from D. gallinae can be used to stimulate a protective immune response in laying hens. Further work is needed to identify other proteins of interest that could confer higher protection against D. gallinae, as well as optimization of the vaccination and in vitro testing protocol.     

Characterization of the in vitro core surface proteome of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host–pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host–pathogen interactions.     

Anthelmintic activity of Cocos nucifera L. against sheep gastrointestinal nematodes

The development of anthelmintic resistance has made the search for alternatives to control gastrointestinal nematodes of small ruminants imperative. Among these alternatives are several medicinal plants traditionally used as anthelmintics. This work evaluated the efficacy of Cocos nucifera fruit on sheep gastrointestinal parasites. The ethyl acetate extract obtained from the liquid of green coconut husk fiber (LGCHF) was submitted to in vitro and in vivo tests. The in vitro assay was based on egg hatching (EHT) and larval development tests (LDT) with Haemonchus contortus. The concentrations tested in the EHT were 0.31, 0.62, 1.25, 2.5 and 5 mg ml⁻¹, while in the LDT they were 5, 10, 20, 40 and 80 mg ml⁻¹. The in vivo assay was a controlled test. In this experiment, 18 sheep infected with gastrointestinal nematodes were divided into three groups (n = 6), with the following doses administered: G1—400 mg kg⁻¹ LGCHF ethyl acetate extract, G2—0.2 mg kg⁻¹ moxidectin (Cydectin^®) and G3—3% DMSO. The worm burden was analyzed. The results of the in vitro and in vivo tests were submitted to ANOVA and analyzed by the Tukey and Kruskal–Wallis tests, respectively. The extract efficacy in the EHT and LDT, at the highest concentrations tested, was 100% on egg hatching and 99.77% on larval development. The parameters evaluated in the controlled test were not statistically different, showing that despite the significant results of the in vitro tests, the LGCHF ethyl acetate extract showed no activity against sheep gastrointestinal nematodes.     

Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy

Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.     

Effect of coconut oil and garlic powder on in vitro fermentation using gas production technique

An in vitro gas technique trial was conducted to investigate the effect of coconut oil (Co), garlic powder (G) and their mixtures on in vitro fermentation. Incubation was carried out using rumen fluid obtained from swamp buffaloes. The experimental design was a completely randomized design (CRD). The dietary treatments were ratio of Co and G supplementation at 0:0, 16:0, 8:4, 4:8 and 0:16 mg with rice straw as a roughage source. Cumulative gas production was recorded at 0, 2, 4, 6, 8, 12, 18, 24, 36, 48, 60 and 72 h of incubation. In vitro true digestibility (IVTD) was determined after 48 h incubation. Cumulative gas production at 72 h was significantly lowest (P < 0.05) at Co:G, 16:0 mg. Garlic powder supplementation at 16 mg decreased (P < 0.05) NH₃–N concentration and increased (P < 0.05) in vitro true digestibility (IVTD) while supplemented coconut oil at 16 mg decreased (P < 0.05) IVTD. Total volatile fatty acids (VFAs) were lowest (P < 0.05) by garlic powder supplementation at 16 mg. However, supplementation of Co:G, 8:4, 4:8 and 0:16 mg tended to increase the proportion of propionate, decrease C2:C3 ratio and reduce (P < 0.05) methane (CH₄) production. Protozoal population was significantly lowest (P < 0.05) at Co:G, 8:4 mg. Moreover, application of quantitative PCR to quantify predominant cellulolytic bacteria (16S rRNA) and fungi (18S rRNA) targets revealed that treatments did not have an effect on Ruminococcusflavefaciens and total fungi population. However, it was found that supplementation of Co:G at 8:4 mg increased Ruminococcusalbus population (P < 0.05). Based on this study, it suggests that supplementation of Co:G at 8:4 and 0:16 mg could improve ruminal fluid fermentation in terms of volatile fatty acid profile, reduced methane losses and reduced protozoal population.     

Purification of cattle oviduct specific proteins and their effect on in vitro embryo development

The purpose of this study was to determine the effect of oviduct specific proteins as a media supplement for in vitro embryo development in cattle. The proteins were extracted from oviducts of cows and precipitated by ammonium sulfate (30%, 40%, 50% and 60%) followed by dialysis in 50 mM Tris–HCl (pH 7.0) buffer. The dialyzed proteins were fractionated into acidic, basic and neutral fractions using SP sephadex cation exchange and DEAE sephadex anion exchange column chromatography respectively. Cow oviduct specific proteins (cOSPs) constituting all the extracted proteins were used as media supplement in three different concentrations (10, 50 and 100 μg/ml) for in vitro maturation, fertilization and culture (IVMFC) of cow oocytes. Acidic, basic and neutral (unbound) fractions were also used as media supplement in three different concentrations (10, 30 and 50 μg/ml) for IVMFC. Cumulus oocytes complexes were collected from slaughterhouse ovaries, washed thoroughly and cultured in maturation media for 24 h in 5% CO₂ at 38.5 °C with maximum humidity. In vitro matured oocytes were co-incubated with in vitro capacitated sperm in Fert-BO media at 38.5 °C for 18 h in 5% CO₂. The fertilized oocytes were washed and cultured in embryo development media for cleavage. After 40–42 h cleavage was observed and embryos were put in the replacement media for further development. The cleavage rates (%) for cOSPs were observed as 68.24±2.46, 69.28±2.05, 61.77±0.93 and 42.62±1.31 at concentrations of 0, 10, 50 and 100 μg/ml respectively. Rates of blastocyst stage development were 14.49±3.61, 21.17±2.77, 14.66±1.06 and 11.98±1.84. These results indicate that addition of cOSP at10 μg/ml increased blastocyst formation as compared to other concentrations (0, 50 and 100 μg/ml). Although acidic, basic and neutral fractions seemed to have no major effect on cleavage rate, but both acidic and neutral fraction of oviduct specific proteins improved the cleavage rate at 30 μg/ml concentration and basic fraction improved the blastocyst formation at 10 μg/ml concentration.     

Screening of microfilaricidal effects of plant extracts against Dirofilaria immitis

Canine dirofilariasis is a common tropical parasitic disease of companion animals, caused by infestation of Dirofilaria immitis filarids within the pulmonary arteries and extending into the right heart. Increased reports of adverse reactions elicited by current microfilaricidal agents against D. immitis such as neurological disorders, circulatory collapse and potential resistance against these agents, warrant the search for new agents in forms of plant extracts. The use of plant extracts in therapeutic medicine is commonly met with scepticism by the veterinary community, thus the lack of focus on its medical potential. This study evaluated the presence of microfilaricidal activities of the aqueous extracts of Zingiber officinale, Andrographis paniculata and Tinospora crispa Miers on D. immitisin vitro at different concentrations; 10 mg/ml, 1 mg/ml, 100 μg/ml, 10 μg/ml and 1 μg/ml within 24 h, by evaluation of relative microfilarial motility as a measure of microfilaricidal activity. All extracts showed microfilaricidal activity with Z. officinale exhibiting the strongest activity overall, followed by A. paniculata and T. crispa Miers. It is speculated that the microfilaricidal mechanism exhibited by these extracts is via spastic paralysis based upon direct observation of the microfilarial motility.     

Impacts of different spices on in vitro rumen dry matter disappearance,fermentation and methane of wheat or ryegrass hay based substrates

Two completely randomised experiments compared the impact of different spices, each at 30 mg/g substrate DM, on the in vitro dry matter disappearance (IVDMD), methane, ammonia and volatile fatty acids (VFA) from either wheat flour (wheat) (experiment 1) as highly or hay based mixtures (experiment 2) as moderately fermentable substrates. Experiment 1 tested the effects of adding cinnamon, clove, coriander, cumin and turmeric, individually, on the rumen fermentation during their in vitro incubations with wheat for 24 h. All spices reduced methane (P < 0.05) from wheat as compared to the spice-free wheat (control). Coriander reduced methane more followed by turmeric, cumin and cinnamon than the control. The IVDMD of wheat did not differ for most spices except cinnamon where IVDMD was reduced (P < 0.05) as compared to the control. Whilst, rumen ammonia was greatest for cumin (P < 0.01) followed by coriander and turmeric, it was lowest for clove (P < 0.01). The spices did not affect the pH and total VFA of rumen fluid for wheat. However, the presence of clove reduced acetic acid (P < 0.05). Experiment 2 tested the effect of only three (coriander, cumin and turmeric) spices individually or together on the in vitro fermentation of hay based mixtures during 96 h of incubation. Whilst all the individual spices or their mixture reduced methane from the hay based mixtures (P < 0.05), the extent of this reduction was highest for turmeric, second highest for coriander and lowest for cumin. It appeared that most spices modified methane emission from wheat as a single ingredient or hay based mixtures without having detrimental effect on diet disappearance or fermentation in vitro. However, the extent of this modification depended on the type of a spice and substrate. Therefore, careful selection of a spice to suit specific feeds would be essential before their in vivo use to modify the fermentation efficiency of ruminant diets.     

Yeast extract, brewer’s yeast and spirulina in diets for Labeo rohita fingerlings affect haemato-immunological responses and survival following Aeromonas hydrophila challenge

A feeding trial was conducted for 60 days to study the immunomodulatory role of three different immunostimulants yeast extract (YE), brewer’s yeast (BY) and spirulina (SP) in Labeo rohita fingerlings. Four hundred and fifty fingerlings (avg. wt 3.35 ± 0.15 g) were randomly distributed in ten treatments and fed with either of ten iso-nitrogenous and iso-caloric semi-purified diets, prepared with three incremental levels (1%, 2% and 4%) of different immunostimulants except the control. Growth parameters did not vary significantly (p > 0.05) among the experimental groups. Haematology and serum parameters was performed before Aeromonas hydrophila challenge whereas respiratory burst activity was analysed following challenge. The respiratory burst activity, total leucocyte count, serum total protein and globulin was significantly higher (p < 0.05) in YE 1% supplemented group. The survival (%) after challenging with A. hydrophila was also highest in the YE fed groups. The results indicate that among the different sources and levels of immunostimulants, YE at lower inclusion level is more effective in promoting the immune status of L. rohita fingerlings.     

Lack of prolonged activity of lavender essential oils as acaricides against the poultry red mite (Dermanyssusgallinae) under laboratory conditions

Managing the poultry red mite, Dermanyssus gallinae (De Geer) by conventional means (i.e., synthetic acaricides) has become increasingly problematic. As a possible alternative, research has identified several plant essential oils that are toxic to D. gallinae. However, essential oils are highly volatile and any acaricidal effect they exert could be short-lived in practice.This study investigated the short-lived toxicity of six lavender essential oils to D. gallinae. In sealed Petri-dishes, mites were exposed to filter papers impregnated with essential oil at a concentration of 0.14 mg/cm³. When filter papers were used immediately after impregnation, 66–90% D. gallinae mortality was observed after 24 h, depending upon the essential oil used. If impregnated filter papers were left in a fume cupboard for 24 h prior to use, mortality rates of D.gallinae fell to 11% or less.     

In vitro sensitivity of the amphibian pathogen Batrachochytrium dendrobatidis to antifungal therapeutics

Chytridiomycosis, a skin disease caused by Batrachochytrium dendrobatidis, has caused amphibian declines worldwide. Amphibians can be treated by percutaneous application of antimicrobials, but knowledge of in vitro susceptibility is lacking. Using a modified broth microdilution method, we describe the in vitro sensitivity of two Australian isolates of B. dendrobatidis to six antimicrobial agents. Growth inhibition was observed, by measurement of optical density, with all agents. Minimum inhibitory concentrations (µg/ml; isolate 1/2) were – voriconazole 0.016/0.008; itraconazole 0.032/0.016; terbinafine 0.063/0.063; fluconazole 0.31/0.31; chloramphenicol 12.5/12.5; amphotericin B 12.5/6.25. Killing effects on zoospores were assessed by observing motility. Amphotericin B and terbinafine killed zoospores within 5 and 30 min depending on concentration, but other antimicrobials were not effective at the highest concentrations tested (100 µg/ml). This knowledge will help in drug selection and treatment optimization. As terbinafine was potent and has rapid effects, study of its pharmacokinetics, safety and efficacy is recommended.     

Expression of Mycoplasma bovis variable surface membrane proteins in the respiratory tract of calves after experimental infection with a clonal variant of Mycoplasma bovis type strain PG45

The pathomorphological findings and the expression and distribution of variable surface protein antigens (Vsp) of Mycoplasma (M.) bovis were characterised immunohistochemically in lungs of eight calves following inoculation with a Vsp A-expressing clonal variant of M. bovis type strain PG45. Within 48 h post inoculation (p.i.) an innate immune response dominated by macrophages and neutrophils develops. The monoclonal antibodies (mAbs) 1A1 and 1E5 detected M. bovis Vsp antigens in paraffin tissue sections of seven calves. Vsp antigens were widely distributed and were already present at day two p.i. within macrophages and other lung compartments. Taken together, the results demonstrate that the bovine is unable to eliminate M. bovis during the time period examined. Based on the different immunohistochemical labelling patterns obtained with the mAbs, the results also support the speculation that the in vivo variability of Vsps together with immunological factors may contribute to the chronicity of pulmonary disease.     

Characterization of betaine aldehyde dehydrogenase (BetB) as an essential virulence factor of Brucella abortus

The pathogenic mechanisms of Brucellosis used to adapt to the harsh intracellular environment of the host cell are not fully understood. The present study investigated the in vitro and in vivo characteristics of B. abortus betaine aldehyde dehydrogenase (BetB) (Gene Bank ID: 006932) using a betB deletion mutant constructed from virulent B. abortus 544. In test under stress conditions, including osmotic- and acid stress-resistance, the betB mutant had a lower osmotic-resistance than B. abortus wild-type. In addition, the betB mutant showed higher internalization rates compared to the wild-type strain; however, it also displayed replication failures in HeLa cells and RAW 264.7 macrophages. During internalization, compared to the wild-type strain, the betB mutant was more adherent to the host surface and showed enhanced phosphorylation of protein kinases, two processes that promote phagocytic activity, in host cells. During intracellular trafficking, colocalization of B. abortus-containing phagosomes with LAMP-1 was elevated in betB mutant-infected cells compared to the wild-type cells. In mice, the betB mutant was predominantly cleared from spleens compared to the wild-type strain after 2 weeks post-infection, and the vaccination test with the live betB mutant showed effective protection against challenge infection with the virulent wild-type strain. These findings suggested that the B. abortus betB gene substantially affects the phagocytic pathway in human phagocytes and in host cells in mice. Furthermore, this study highlights the potential use of the B. abortus betB mutant as a live vaccine for the control of brucellosis.     

Salmonella Antimicrobial Activity of Selected Strains of Enterolactobacillus Species Isolated from the Gastrointestinal Tract of the Horse

The gastric mucosa and the mucosa of the right and left dorsal colon were biopsied in each of the 15 horses, and a total of 45 samples were collected. Mucosal samples were cultured using a Lactobacillus enrichment broth. While numerous Lactobacillus strains were identified, Lactobacillus reuteri was the most common organism identified. Sixteen strains of Lactobacillus reuteri were selected for antimicrobial testing. Salmonella antimicrobial activity was identified in six out of 16 strains tested. Organisms with Salmonella antimicrobial activity were cultured from the stratified squamous epithelium of the stomach and the mucosa of the right and left dorsal colon.     

Isolation and characterization of a bovine isolate of Neospora caninum with low virulence

Neospora caninum tachyzoites were isolated from the brain of an asymptomatic naturally infected calf with precolostral-specific antibodies. The new isolate, named Nc-Spain 1H, was identified as a member of the N. caninum species based on its internal transcribed spacer 1 (ITS-1) sequence and was genetically characterized using microsatellite markers. Multilocus analysis showed that Nc-Spain 1H was genetically different from other N. caninum isolates. We compared the in vitro tachyzoite yield and viability rate of the Nc-Spain 1H and Nc-1 isolates in a plaque assay. The lower tachyzoite yields displayed by Nc-Spain 1H were complemented with a significantly lower viability rate. Moreover, in an in vitro tachyzoite–bradyzoite stage conversion assay, the percentage of Nc-Spain 1H bradyzoite conversion was similar to that of the cystogenic isolate Nc-Liv, with the exception that Nc-Spain 1H produced only intermediate bradyzoites. The pathogenicity of Nc-Spain 1H was examined in BALB/c mice, and the results demonstrated that Nc-Spain 1H failed to induce clinical signs or mortality and that no parasite DNA was detected in the brain during the chronic stage of infection. In a pregnant mouse model, Nc-1 infection resulted in high transplacental transmission, leading to a high neonatal mortality rate over time. In contrast, the offspring survival rate from Nc-Spain 1H-infected dams was almost 100%, and N. caninum DNA was detected in only one pup. These data show that Nc-Spain 1H appears to be a low virulence isolate and may be a suitable candidate for live vaccine development.     

Effects of dietary Crotalaria pallida seeds on the health and performance of laying hens and evaluation of residues in eggs

The effect of three dietary concentrations of Crotalaria pallida (C. pallida) seeds (0, 1, 2, and 3% w/w) of their normal diet were investigated in commercial laying hens during a 35 day feeding trial. All concentrations of C. pallida decreased body weight and feed intake (P < 0.05). Egg mass production and average egg weight were decreased by feeding of ≥2% C. pallida seeds (P < 0.05). All concentrations of C. pallida increased relative lung weight and serum activity of ALT, AST and LDH (P < 0.05); 3% C. pallida seeds decreased liver weight (P < 0.05). Analysis of the C. pallida seeds for dehydropyrrolizidine alkaloid content detected usaramine and its N-oxide at a total alkaloid concentration of 0.18% (dry weight). Usaramine was also detected in the eggs of all hens fed C. pallida seeds.     

Molecular detection of Theileria and Babesia infections in cattle

This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina.