In vitro effects of Tabernaemontana citrifolia extracts on Haemonchus contortus

Tabernaemontana citrifolia (Apocynaceae) is traditionally used as an anthelmintic preparation for ruminants in Guadeloupe (French West Indies). This study was carried out to evaluate the in vitro effect of this plant against the parasitic nematode of small ruminants Haemonchus contortus. Three extracts (aqueous, methanolic and dichloromethane) of T. citrifolia fruit, leaf and root were tested on four developmental stages of the parasite, using egg hatch assay (EHA), larval development assay (LDA), L3 migration inhibition assay (LMI), and adult worm motility assay (AWM). Compared to the negative control, significant effects were observed for the different parts of T. citrifolia but with differences depending on the parasitic stage; efficacies on the larval development of H. contortus from 88.9% to 99.8% for fruit, from 72.1% to 83.8% for root and from 33.5% to 85% for leaf with dose-dependent effect for the methanolic extract. The root gave the best result on EHA (22.7% efficacy for dichloromethane extract) and AWM (56% efficacy, with dose-dependent effect for dichloromethane extract) and the leaf on LMI (49.4% efficacy). These results suggest that T. citrifolia possess anthelmintic activity against H. contortus. The active ingredients responsible for the activity could be the alkaloid compounds present in the plant parts of the plant.     

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.     

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.     

Assessing the efficacy of Duddingtonia flagrans chlamydospores per gram of faeces to control Haemonchus contortus larvae

The aims were (a) to quantify the number of Duddingtonia flagrans chlamydospores per gram of faeces (CPG) recovered from sheep administered with different oral doses and, (b) to describe the relationship between CPG and eggs per gram of faeces (EPG) on the efficacy to reduce Haemonchus contortus infective larvae. Three doses of chlamydospores per kg BW were orally administered during seven days: (T1) non treated control group, (T2) 1 × 10⁶, (T3) 2.5 × 10⁶ and (T4) 5 × 10⁶. Three lambs, infected with H. contortus, were used per group. Faeces were obtained from the rectum of each lamb during the fungal administration period (days 0–6) and for six days after that period. Four coproculture replicates were made from each animal in days 2, 4, 6, 8 and 10. A higher chlamydospore dose produced higher CPG in faeces (p < 0.05), but a clear dose dependent effect was not found either in the larvae reduction or in the CPG:EPG ratio. When ratios were re-analyzed, independently of the treatment groups of origin, a better efficacy was obtained with a ratio from 5 to 10 CPG:EPG and a higher ratio (>10 per egg) showed a lower reduction efficacy (p < 0.05). The binomial analysis showed that for each unit of increment in CPG:EPG ratio there was a reduction of larvae number until a point (between 5 and 10 CPG:EPG) where no further reduction was detected. The surface response test indicated that the number of larvae was reduced by CPG until possible saturation. The highest CPG:EPG ratios did not necessarily improve efficacy of D. flagrans.     

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.     

Effect of aqueous extracts of Baccharis trimera on development and hatching of Rhipicephalus microplus (Acaridae) eggs

This study evaluated the effects of aqueous extracts of Baccharis trimera (Less.) DC (Asteraceae), colloquially known as carqueja, on egg production, and hatching rate of larvae of Rhipicephalus microplus. Plant samples were collected in Montes Claros, north of Minas Gerais, Brazil. Adult female ticks were distributed into 24 homogeneous groups of 10. The in vitro test was performed by immersing each group in 10 ml solutions of aqueous extracts at 50, 100, 150, or 200 mg of fresh leaves ml⁻¹. These concentrations were compared with distilled water as negative control and a commercial product as positive control and the tests were repeated four times. The carqueja extract at concentrations of 150 and 200 mg of fresh leaves ml⁻¹ showed 100% efficacy in inhibiting egg hatching and therefore could have potential as an acaricide.     

Effect of new ethyl and methyl carbamates on biological parameters and reproduction of the cattle tick Rhipicephalus microplus

The effect of carbamates on engorged female Rhipicephalus microplus ticks and larvae was evaluated using the adult immersion test (AIT) and the larval packet test (LPT), respectively. Seventeen synthetic carbamates different from current commercial acaricides were synthesised at the National Autonomous University of Mexico. None of the carbamates had an effect on the percentage of females laying eggs. Six of the compounds inhibited egg laying up to 65.4% and inhibited egg hatching by up to 100% (p < 0.05). Compared to untreated females, eggs produced by treated females had a dark, dry, opaque appearance and were less adherent. Carbamates LQM 934 and LQM 938 had an effect on larval mortality (p < 0.05). Carbamate LQM 934 showed lethal concentrations (LC) of LC₉₀ = 0.76% and LC₉₉ = 0.87%, while LQM 938 showed concentrations of LC₉₀ = 0.267% and LC₉₉ = 0.305%. The compounds were distributed into three classes of acaricidal activity using the AIT or the LPT. These three classes were as follows: (1) compounds having no apparent effect; (2) compounds that inhibit egg laying and embryo development or (3) compounds that exhibit acaricidal activity to larval ticks.     

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.     

Anthelmintic activity of artesunate against Fasciola hepatica in naturally infected sheep

In light of rapidly spreading triclabendazole resistance alternative fasciocidal drugs are urgently needed. Following up on promising results obtained with artemether in Fasciola hepatica infected sheep, we here report the efficacy and safety of artesunate in sheep with a natural F. hepatica infection. Artesunate was administered intravenously and intramuscularly, adverse events were monitored and drug efficacy was elucidated by means of faecal egg and worm burden reductions. A single 40 mg/kg intravenous dose of artesunate induced an egg count reduction of 68.9% and a worm burden reduction of 77.4%. Intramuscular artesunate at 40 mg/kg reduced faecal egg count and worm burden by 97.6% and 91.9%, respectively; whereas at 60 mg/kg it caused 93.2% and 87.1% reduction in faecal egg count and worm burden, respectively. Three sheep died 24-72 h post-treatment with a double dose of 40 mg/kg intramuscular artesunate, showing lethargy, sialorrhoea, reduced rumination and tremors. Egg and worm burden reductions of 93.3% and 83.9%, respectively, were calculated in the three surviving sheep. In conclusion, the interesting fasciocidal properties of artesunate in sheep warrant further investigations with an emphasis on toxicity studies.     

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.     

Anthelmintic activity of Cocos nucifera L. on intestinal nematodes of mice

In this study, we evaluated the anthelmintic activity of the liquid extracted from the bark of the green coconut (LBGC), as well as butanol extract obtained from LBGC, on mouse intestinal nematodes. Thirty-six naturally infected mice were distributed into six groups receiving the following treatments: Group I: 1000 mg/kg of LBGC; Group II: 2000 mg/kg of LBGC; Group III: 500 mg/kg of butanol extract; Group IV: 1000 mg/kg of butanol extract; Group V: 0.56 mg/kg febendazole; and Group VI: 3% dimethylsulfoxide. The chemical composition of the LBGC and its butanol extract was determined by phytochemical tests. A dose of 1000 mg/kg of butanol extract had 90.70% efficacy in reducing the mouse worm burden (p < 0.05). Phytochemical tests revealed the presence of triterpens, saponnins and condensed tannins in the LBGC and butanol extracts. These results suggest that Cocos nucifera extracts may be useful in the control of intestinal nematodes.     

Quantitative real-time PCR and culture examination of Mycobacterium avium subsp. paratuberculosis at farm level

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease in ruminants and may contribute to Crohn's disease in humans. The aim of this study was to determine the occurrence and quantity of MAP in cattle feces and milk in the Iranian context. In addition, we evaluated the effect of cattle age as well as farming system as risk factors contributing to MAP load. For this, a total sample of 373 consisting of 150 cattle feces (CF), 150 individual cow's milk (ICM), as well as 73 bulk-tank milk (BTM) was collected randomly and regardless of the cattle health status. The samples were assayed using F57 quantitative real-time PCR (qPCR) and culture method. According to the results of qPCR which was found ∼10 times more sensitive than culture assay, MAP was detected in 68.66% (103/150) of the CF, 12% (18/150) of the ICM and 52.05% (38/73) of the BTM samples. In contrast to the previous reports, the quantity of MAP in the BTM (2.03–5.97 log cfu/50 ml) was statistically (p < 0.01) higher than the ICM (0.90–1.97 log cfu/50 ml). Data suggested a direct relation (p < 0.01) between the cattle age and the quantity of MAP in the CF samples, while the relation was not statistically significant (p > 0.05) for the ICM. In addition, MAP load in the BTM samples obtained from traditional farms was significantly (p < 0.01) higher than that of the industrial ones, while the differences in CF and ICM was not significant (p > 0.05).     

Clinically relevant multidrug resistant Salmonella enterica in swine and meat handlers at the abattoir

The presence of multidrug resistant (MDR) Salmonella serotypes in slaughtered swine, carcasses, meat and meat handlers is scarcely evaluated. Recently we demonstrated that diverse Salmonella serotypes are frequently present in swine, pork meat and carcasses, and meat handlers at Portuguese abattoirs. Here we have characterized their antibiotic resistance phenotypes and genotypes, helping elucidate the flow of MDR Salmonella in the food chain. Testing 60 Salmonella isolates from different serotypes, the highest frequencies of resistance were observed for tetracycline (T) [70% (n = 42/60), tet(A)/tet(B)/tet(G)], streptomycin (S) [63% (n = 38/60), aadA2/strA/strB], sulfamethoxazole (Sul) [62% (n = 37/60), sul1/sul2/sul3] and ampicillin (A) [57% (n = 34/60), bla_PSE-1/bla_TEM]. Thirty-seven percent (n = 22/60) carried class 1 integrons and multidrug resistance was frequently observed (63% n = 38/60), including those serotypes common to human infections [S. Typhimurium 78% n = 25/32; S. 4,[5],12:i:- 67% n = 2/3; S. Rissen 75% (n = 3/4); S. London 67% n = 2/3; S. Derby 55%; n = 6/11)]. The emergent S. 4,[5],12:i:- isolates were mostly characterized by ASSuT phenotype [bla_TEM/strA-strB/sul2/tet(B)], typical of the European clone, while for the first time the ST phenotype [strA-strB-tet(A)-tet(B)] was also observed. Moreover, we report a first finding of a MDR phenotype in S. London [ANSSuT; bla_TEM-strA-strB-sul2-tet(A)]. Our findings suggest that the abattoir environment and the slaughter operations seem not only to harbor MDR serotypes that originated in the pig reservoir, but also propagate them through cross-contamination processes, involving meat handlers. The present study suggests a probable relationship between swine and human salmonellosis throughout the food chain, which is of interest for epidemiological, animal health and public health purposes.     

Condensed tannins from Sesbania sesban and Desmodium intortum as a means of Haemonchus contortus control in goats

Two experiments were conducted to determine whether the proanthocyanidin (condensed tannin)-containing forage legumes Desmodium intortum cv Greenleaf and Sesbania sesban (accession 15019) could be integrated into a feeding management strategy as a means of Haemonchus contortus control in goats. The anthelmintic effects of condensed tannin extracts from the two legumes on H. contortus L₃ larvae were studied in an in vitro larval migration inhibition system. The extracts inhibited larval migration in a dose-dependent manner, and at concentrations from 1,000???g/ml condensed tannin, the extract from D. intortum caused a significantly higher inhibition of larval migration than did the corresponding concentrations of the S. sesban extract (P?<?0.01). Prolonged feeding of tanniniferous forage legumes showed that animals receiving D. intortum had the lowest total worm burden, the lowest female to male parasite ratio, the lowest number of eggs in the uterus of each female worm and the lowest per capita fecundity (P?<?0.01). However, there was no change in the performance (weight gain) of parasite-infected goats probably due to incomplete removal of the parasite or prolonged confinement of goats in small pens, which calls for further investigation. However, since there is no single efficient method in control of parasites, based on the obtained data from this experiment, integrated feeding of D. intortum with other suitable method of parasite control is thus suggested.     

A new Brucella canis species-specific PCR assay for the diagnosis of canine brucellosis

Brucellosis is a zoonotic disease that is transmitted from animals to humans, and the development of a rapid, accurate, and widely available identification method is essential for diagnosing this disease. In this study, we developed a new Brucella canis species-specific (BcSS) PCR assay and evaluated its specificity and sensitivity. A specific PCR primer set was designed based on the BCAN_B0548-0549 region in chromosome II of B. canis. The PCR detection for B. canis included amplification of a 300-bp product that is, not found on other Brucella species or, genetically or serologically related bacteria. The detection limit of BcSS-PCR assay was 6 pg/μl by DNA dilution, or 3 × 10³ colony-forming units (CFU) in the buffy coats separated from whole blood experimentally inoculated with B. canis. Using the buffy coat in this PCR assay resulted in approximately 100-times higher sensitivity for B. canis as compared to detect directly from whole blood. This is the first report of a species-specific PCR assay to detect B. canis, and the new assay will provide a valuable tool for the diagnosis of B. canis infection.     

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.     

Anthelmintic and antimicrobial activity of methanolic and aqueous extracts of Euphorbia helioscopia L.

The aim of this study was to evaluate the anthelmintic and antimicrobial efficacy of Euphorbia helioscopia crude extracts. A worm motility inhibition assay and egg hatch assay were used for in vitro study, and a faecal egg count reduction assay was used for in vivo study. The in vitro study revealed anthelmintic effects of crude methanolic extracts of E. helioscopia on live Haemonchus contortus worms as evident from their paralysis and/or death at 8 h after exposure. Different concentrations (12.5 mg?ml^?1, 25 mg?ml^?1 and 50 mg?ml^?1) of aqueous and methanolic extracts were used against H. contortus which exhibited dose-dependent anthelmintic effects on H. contortus. Different extracts of E. helioscopia on percent inhibitory egg hatching was very low as compared to levamisole. The antimicrobial activity of extracts ranging from 100 to 500 mg?ml^?1 screened by disc diffusion method against four selected bacterial (Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas multocida and Escherichia coli) and two fungal strains (Aspergillus flavus and Candida albicans) was also dose dependent with the extract showing more inhibitory effects against S. aureus and E. coli and a minimum inhibitory effect against A. flavus. It is concluded that the entire plant of E. helioscopia possesses significant anthelmintic and antimicrobial activity and could be a potential alternative for treating cases of helminth infections in ruminants.     

Predicting resistance of sheep to Haemonchus contortus infections

In vitro cultures of lymphocytes from worm-free sheep responded to larval and adult H. contortus antigens by undergoing blast transformation. The level of response varied considerably between animals but was heritable and positively correlated with resistance to subsequent primary, secondary and trickle infection by the parasite, but not with age of the sheep older than 5 months. Lymphocyte responses to phytohaemagglutinin and H. contortus antigens were not present at birth in lambs but developed during the first 5 months in the absence of infection. The proportion of the infecting dose of larvae which was inhibited at the early fourth stage correlated with age of the sheep and with the increase in lymphocyte response to H. contortus antigens during primary infections with H. contortus. Sheep which were most susceptible to infection had the lowest lymphocyte responses to H. contortus antigens but had the highest rate of weight gain during infection. It is suggested that man has selected the most productive animals at the expense of resistance to H. contortus. The definition of genetically controlled markers on lymphocytes is seen as a means by which disease resistant strains of animals may be selected.     

Efficiency of entomopathogenic fungi in the control of eggs and larvae of the horn fly Haematobia irritans (Diptera: Muscidae)

The present study assessed the pathogenic effect of isolates E9, IBCB425 and IBCB159 of the Metarhizium anisopliae fungus, JAB06, JAB07 and AM09 of Beauveria bassiana, IBCB133 and CB75 of Isaria fumosorosea (=Paecilomyces fumosoroseus) and CG189 and CG195 of Isaria farinosa (=Paecilomyces farinosus) against eggs and larvae of the horn fly Haematobia irritans. Eggs were inoculated with suspensions containing 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ of the fungal isolates and observed after 48 h to determine viability. In the larvae study, eggs were allowed to hatch into fresh bovine feces that had been treated with 10⁸, 10⁷ or 10⁶ conidia mg feces⁻¹. In both studies, 5 days after initial procedures, all formed pupae were transferred to an incubator at 27 ± 0.5 °C until the emergence of the adult flies was complete. The M. anisopliae isolates did not cause the death of H. irritans eggs, but they did promote the death of larvae that hatched from treated eggs, and therefore increased the total mortality. Isolate E9 promoted 100% mortality of treated larvae at a concentration of 10⁸ conidia ml⁻¹. For the B. bassiana isolates, no activity was observed against insect eggs or larvae. Both I. fumosorosea isolates promoted significant mortality (p < 0.05) of eggs at every concentration of conidia. Isolate CG195 of I. farinosa increased the mortality of larvae and pupae that hatched from treated eggs and promoted significant total mortality (p < 0.05) of the insect at every concentration of conidia.     

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.