Development of anthelmintic resistance in nematodes from sheep in Australia subjected to different treatment frequencies

Development of anthclmintic resistance in nematodes from sheep in Australia subjected to different treatment frequencies. International Journal for Parasitology13: 125–132. In a three year (1978, 1979, 1980) field experiment in south-eastern Australia Merino weaner sheep were exposed to one of four worm control programmes. Sheep were treated either 50–51 times, 9–12 times, 3–6 times or 1–3 times annually with anthelmintic. Thiabendazole (TBZ) was used throughout 1978 and for the initial dose in 1980, levamisole (LEV) throughout 1979 and 1980. Sheep were replaced annually each December.During 1978 in vitro egg hatch tests indicated that populations of Haemonchus contenus with resistance factors to TBZ of 10·7, 10·0, 2.5 and 1·8 respectively were selected by these treatment frequencies. No resistance to LEV was detected in 1979 but during 1980 in vivo egg count reduction tests showed that highly resistant populations of Ostertagia spp. were selected in sheep dosed either 49 or 11 times. This was confirmed by an in vitro larval paralysis test. Partial resistance occurred where sheep were dosed 5 times but not where one dose was given. The only population of H. conlortus still present at the end of 1980 in sufficient numbers to allow eggs to be tested in vitro was from the sheep dosed once. This had retained its resistance to TBZ.     

Effects of reproduction,genotype and anthelmintic treatment of ewes on Ostertagia spp. populations

Donald A. D., Morley F. H. W., Waller P. J., Axelsen A., Dobson R. J. and Donnelly J. R. 1982. Effects of reproduction, genotype and anthelmintic treatment of ewes on Ostertagia spp. populations. International Journal for Parasitology12: 403–411. Merino and Border Leicester × Merino (BL × M) ewes, nearly all of the same age and reared at the same site, were either unmated or mated to Border Leicester rams. Ewes of each genotype and reproductive status were untreated or were given a single pre-lambing drench with thiabendazole at 50 or 100 mg/kg a week before the start of lambing in spring on pastures at Canberra which had been contaminated during autumn and winter by adult sheep. The two genotypes grazed together within each combination of reproductive status and anthelmintic treatment which grazed separately. Thiabendazole was highly effective in removing both fourth stage larvae and adults of Ostertagia spp., the most abundant genus. Eight weeks after the pre-lambing drench lactating ewes carried larger Ostertagia spp. populations than did unmated ewes of both genotypes, but as a result of reinfection after treatment, differences between drenched and undrenched ewes were not significant. At this time lactating as well as unmated ewes harboured large populations of arrested early fourth stage larvae of Ostertagia spp. acquired during the last 8 weeks, showing that arrest of development is not prevented by lactation. There was strong evidence that some ingested larvae which became arrested in lactating ewes were rejected by unmated ewes. At all stages of the reproductive cycle studied, BL × M ewes were substantially more resistant to Ostertagia spp. infection than Merinos. No persistent benefits in parasite control or in animal production were detected from the pre-lambing drench.     

The impact of refugia on the development of thiabendazole resistance in Haemonchus contortus

Martin P. J., Le Jambre L. F. and Claxton J. H. 1981. The impact of refugia on the development of thiabendazole resistance in Haemonchus contortus. International Journal for Parasitology11: 35–41. Beginning with a mixture of 5% thiabendazole-resistant and 95% non thiabendazole-resistant Haemonchus contortus larvae, worm free sheep were artificially infected with either 10,000, 9000, 7000, 2500 or zero larvae. Four weeks later, the infected sheep were treated with 44 mg/kg of thiabendazole. Seven days after anthelmintic treatment the same sheep were infected with zero, 1000, 3000, 7500 or 10,000 larvae respectively so that each sheep was given a total of 10,000 larvae. The larvae given after thiabendazole treatment are referred to as being in refugia (defined as an area where individual members of a population can escape exposure to a drug). This was repeated for six generations of parasites, maintaining the same proportion of larvae in refugia in each generation. The egg hatch assay for resistance indicated that refugia delayed the development of resistance in Haemonchus contortus. Where none or a small proportion of larvae were in refugia, a rapid increase in resistance occurred. With an increased proportion of larvae in refugia, resistance was slower to develop. Population size, as estimated by faecal egg counts done after anthelmintic treatment and corrected for the number of larvae given in each treatment, was lower at the higher levels of refugia. This was due to a lower level of resistance in the higher refugia lines and consequently a greater effect of the drug. However, the estimate of population size, from egg counts done after the larvae introduced from refugia had matured, were similar in all lines.     

A 4 year observation of gastrointestinal nematode egg counts,nemabiomes and the benzimidazole resistance genotypes of Teladorsagia circumcincta on a Scottish sheep farm

Anthelmintic resistance threatens the sustainability of sheep production globally. Advice regarding strategies to reduce the development of anthelmintic resistance incorporates the outcomes of modelling exercises. Further understanding of gastrointestinal nematode species diversity, and population dynamics and genetics (which may vary between species) is required to refine these models; and field studies combining faecal egg outputs, species composition and resistance genetics are needed to calibrate them. In this study, faecal samples were taken from ewes and lambs on a commercial farm in south-eastern Scotland at approximately 3 t-4 week intervals between spring and autumn over a period of 4 years. Faecal egg counts were performed on these samples, and L₃ were collected from pooled coprocultures. Deep amplicon sequencing was used to determine both the species composition of these L₃ and the proportions of benzimidazole-resistant single nucleotide polymorphisms in the isotype-1 β-tubulin locus of the predominant species, Teladorsagia circumcincta L₃. Despite consistent management throughout the study, the results show variation in gastrointestinal nematode species composition with time and between age groups, that was potentially associated with weather conditions. The F200Y benzimidazole resistance mutation is close to genetic fixation in the T. circumcincta population on this farm. There was no evidence of variation in isotype-1 β-tubulin single nucleotide polymorphisms frequency between age groups, and no genetic evidence of reversion to benzimidazole susceptibility, despite targeted benzimidazole usage. This study highlights the need to include speciation when investigating gastrointestinal nematode epidemiology and anthelmintic resistance, and serves as an example of how genetic data may be analysed alongside species diversity and faecal egg counts, when markers for other anthelmintic classes are identified.     

No Evidence for Resistance to Fenbendazole in Trichostrongylus tenuis,a Nematode Parasite of the Red Grouse

ABSTRACT The parasitic nematode Trichostrongylus tenuis has a detrimental effect on red grouse (Lagopus lagopus scoticus) at the individual and population levels. Treatment using grit coated with the anthelmintic fenbendazole hydrochloride reduces parasite infection and increases grouse density. However, a frequent and low dose of anthelmintic increases selection pressure for parasite resistance, a serious practical and economic problem. We used an egg hatch assay to test resistance of T. tenuis from 12 moors in northern England, which differed in grit treatment intensity. The anthelmintic concentration that prevented 50% and 95% of T. tenuis eggs from hatching (ED₅₀ and ED₉₅, respectively) did not differ among moors and were not related to treatment. We suggest annual monitoring and responsible anthelmintic use to prevent resistance so that medicated grit continues to enhance red grouse management.     

Analysis of putative inhibitors of anthelmintic resistance mechanisms in cattle gastrointestinal nematodes

Effects of the cytochrome P450 inhibitor piperonyl butoxide and the P-glycoprotein inhibitor verapamil on the efficacy of ivermectin and thiabendazole were studied in vitro in susceptible and resistant isolates of the cattle parasitic nematodes Cooperia oncophora and Ostertagia ostertagi. The effects of combined use of drug and piperonyl butoxide/verapamil, respectively, were investigated in the Egg Hatch Assay, the Larval Development Assay and the Larval Migration Inhibition Assay. The effects of piperonyl butoxide and verapamil as inhibitors of thiabendazole and ivermectin responses were particularly marked for larval development, where both inhibitors were able to completely eliminate all differences between susceptible and resistant isolates. Even the lowest concentrations of anthelmintics used in combination with inhibitors caused complete inhibition of development. Differences and/or similarities among responses in different isolates were only obtained in the two other assays: in the Egg Hatch Assay piperonyl butoxide caused a shift in concentration–response curves obtained with thiabendazole to the left for all isolates tested, changing relative differences between isolates. In contrast, an effect of verapamil in the Egg Hatch Assay was only apparent for benzimidazole-resistant isolates. In the Larval Migration Inhibition Assay only ivermectin was tested and piperonyl butoxide shifted the concentration–response curves for all isolates to the left, again eliminating differences in EC₅₀ values between susceptible and resistant isolates. This was not the case using verapamil as an inhibitor, where curves for both susceptible and benzimidazole-resistant isolates shifted to the left in Ostertagia isolates. In Cooperia the picture was more complex with ivermectin-resistant isolates showing a larger shift than the susceptible isolate. Single nucleotide polymorphisms in the β-tubulin isotype 1 gene were investigated. Significantly increased frequencies of resistance-associated alleles were observed for the codons 167 and 200 in one benzimidazole-resistant isolate but not in an isolate selected for benzimidazole resistance at an early stage of selection.     

Trichostrongylus colubriformis: effect of anthelmintics on ingestion and oviposition

The effects of the anthelmintics ivermectin, levaminsole, oxfendazole, piperazine citrate, pyrantel pamoate, tetramisole, and thiabendazole on ingestion and oviposition by Trichostrongylus colubriformis were determined. Six of the compounds reduced in vitro feeding at the tested doses while all drugs reduced in vivo feeding after treatment of the host. Additionally, in vitro or in vivo exposure to most anthelmintics decreased oviposition during subsequent in vitro assay. Invermectin had the most pronounced effect on in vivo and in vitro feeding and egg release. The neuromuscular activities of pharyngeal pumping and egg ejection may be suitable systems for rapid determinations of anthelmintic effects.     

The effect of selection with levamisole on benzimidazole resistance in Ostertagia spp. of sheep

A field population of Ostertagia spp. (predominantly O. circumcincta) in sheep, found to be resistant to oxfendazole, was exposed to selection with1 levamisole in the laboratory and the field. Progeny of the survivors of a single dose of levamisole in penned sheep showed a significantly lower level of resistance to oxfendazole in an anthelmintic assay, and a similarly lower level of resistance to the ovicidal activity of thiabendazole in vitro compared with the progeny of the survivors of a single dose of oxfendazole, although they remained more resistant than a known susceptible strain of 0. circumcincta. The progeny of worms present in grazing sheep after three doses of levamisole at 4-weekly intervals, and subjected to the same tests, behaved in a similar fashion. They also showed a substantial fall in benzimidazole resistance compared with the progeny of worms not exposed to any anthelmintic for 6 months. The results suggested that levamisole selected positively against benzimidazole resistance. In these circumstances some form of alternation in the use of different anthelmintics could delay the development of a high level of resistance and maintain the practical usefulness of existing anthelmintics.     

Selection for thiabendazole resistance in Ostertagia spp. by low efficiency anthelmintic treatment

Thiabendazole (TBZ) treatment of grazing weaners infected with a predominantly susceptible strain of Ostertagia reduced worm burdens by 90%. The surviving sub population had a level of resistance four times greater than the original population when compared to similarly infected, but untreated, weaners. TBZ treated and control weaners were relocated to worm-free pasture so that the contamination on the plots grazed by TBZ treated weaners was from the survivors of the drench. The anthelmintic treated groups were subsequently managed under a two summer drench programme while control groups remained so throughout the experiment. Monitoring both parasite population size and the degree of resistance over the following 2 years indicated that after three anthelmintic treatments, resistance developed to a level where TBZ was ineffective. When compared to control groups, a mean reduction in population size of around 80% was maintained in the TBZ treated groups for 1 year. This difference, however, disappeared by the second year. A similar study over 1 year with a moderately TBZ resistant strain of Ostertagia indicated that TBZ treatment produced no significant reduction in population size but more than doubled the level of resistance. The study indicated the critical need for highly efficient anthelmintic treatment, particularly in situations where, through ecology or management, post treatment contamination is likely to contribute substantially to future generations of worms.     

Resistance of selected lines of Ostertagia circumcincta to thiabendazole, morantel tartrate and levamisole

Le Jambre I. F., Southcott W. H. and Dash K. M. 1977. Resistance of selected lines of Ostertagia circumcincta to thiabendazole, morantel tartrate and levamisole. International Journal for Parasitology7: 473–479. A strain of Ostertagia circumcincta was isolated from a field in which all sheep had been treated in sequence every 7–10 days from September 1970 to January 1974 with either thiabendazole, morantel tartrate or levamisole. Thiabendazole had not been used after the first 15 months. The LD₉₅ for this strain was 88 mg/kg thiabendazole, 6.9 mg/kg morantel tartrate and 5-4 mg/kg levamisole.Another strain of O. circumcincta isolated from an area where anthelmintics had been used much less frequently was divided into four lines for exposure to selection in the laboratory. The first line was selected with 50 mg/kg thiabendazole, the second with 5 mg/kg morantel tartrate, and the third with 3.2 mg/kg levamisole; the fourth line was not selected for drug resistance. After eight generations the three lines selected with thiabendazole, morantel tartrate and levamisole had (Spld)(in95) of > 200, 5.7 and 6.2 mg/kg for the selecting drugs respectively, compared with corresponding values of 20, 2.9, and 1.8 in the unselected line. That is, the field strain had about the same levels of resistance to morantel tartrate and levamisole as the respective laboratory strains selected with these individual drugs. However, the field strain, which had been exposed to thiabendazole for only 15 months, was less resistant to thiabendazole than the laboratory strain selected with this drug. These results show that giving of several drugs in sequence cannot be relied upon to prevent the development of resistance to the individual drugs.The dose responses of adult worms showed low, but significant resistances to morantel tartrate and levamisole and a relatively high resistance to thiabendazole. Levamisole was found to select for inhibition of development with approx. 8.0% of the inhibited larvae showing no dose response above 1.6 mg/kg. Levamisole was also associated with an increase from 0.1 % to 9.0% in the O. trifurcata component of an Ostertagia population.     

Population dynamics of the parasitic stages of Ostertagia spp. in sheep

Callinan A. P. L. and Arundel J. H. 1982. Population dynamics of the parasitic stages of Ostertagia spp. in sheep. International Journal for Parasitology12: 531–535. The development and survival of continuing infections of Ostertagia spp. in weaner sheep were studied in order to develop a general model of the parasitic stages of the life cycle of these sheep nematodes. After 10 days, 13.8% of infective larvae (L₃) given at the rate of 1 dose of 1000 L₃ twice per week (group 1) and 20.8% of L₃ given at the rate of 10,000 L₃ twice per week (group 2) were recovered in the first of the serial nematode counts. In the final counts at 137 days, 7.7 and 0.7% were recovered in these groups. The build up and maintenance of nematode populations was regulated and related to the level of infection. A model in which the death rate of the parasitic stages was a function of the time of exposure to infection and rate of infection was used to describe the serial total nematode counts. During the experiment there was no noticeable trend in numbers of fourth stage larvae (L₄) in nematode counts, the size of adult nematodes, nematode egg counts (EPG) and egg output per female nematode (EPF). After 112 days, liveweight gains were significantly reduced in group 2 only, but increases in wool lengths were significantly reduced in both groups.     

Effects of triflumezopyrim on the reproduction of brown planthopper,Nilaparvata lugens

The brown planthopper, Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a notorious pest on rice in Asia and has evolved serious resistance to traditional chemical insecticides in recent years. Triflumezopyrim, a newly commercialized mesoionic insecticide, exhibited an excellent control effect on sucking insects, including N. lugens. However, the effects of triflumezopyrim on the reproduction of N. lugens have not yet been fully elucidated. In this study, we investigated the impacts of different concentrations of triflumezopyrim (LC₁₀, LC₅₀ and LC₉₀) on the reproduction and the reproduction related genes of N. lugens exposure to the insecticide via systemic and contact route. The results indicated that the preoviposition periods of N. lugens females in the LC₅₀ and LC₉₀ triflumezopyrim-treated groups (♀ t × ♂ t) were prolonged in contact toxicity treatment, compared with control (acetone treatment). The fecundities for three pair combinations (♀ c ×♂ t, ♀ t ×♂ c and ♀ t ×♂ t) treated with LC₅₀ and LC₉₀ concentrations of triflumezopyrim showed a significant decrease compared to water/acetone-treated control via contact and systemic route. No significant effects of LC₁₀ triflumezopyrim on preoviposition period and fecundity were found in contact and systemic toxicity treatments. However, the triflumezopyrim did not influence the egg hatchability. By microscope observation, the LC₅₀ and LC₉₀ triflumezopyrim impended the ovarian development in females. qRT-PCR analysis showed that the vitellogenin (NlVg) relative expression level of female adults exposed to LC₅₀ and LC₉₀ triflumezopyrim decreased by 30.3 – 39.5% in two toxicity treatments. The titers of another reproduction related hormones juvenile hormone III (JH III) in LC₅₀ and LC₉₀ triflumezopyrim-treatments were 27.4% and 25.8% lower than control in contact toxicity treatment and 27.08% and 37.18% lower than control in systemic toxicity treatment. In summary, these results suggested that the median lethal and high lethal concentrations of triflumezopyrim inhibited the reproduction and reduced the expression of NlVg and JH III in N. lugens. This study provided a reference for the efficient and scientific utilization of triflumezopyrim in the field.     

Uptake of thiabendazole and its effects on glucose uptake and carbohydrate levels in the thiabendazole-resistant and susceptible Trichostrongylus colubriformis

Sangster N. C. and Prichard R. K. 1984. Uptake of thiabenzadole and its effects on glucose uptake and carbohydrate levels in thiabendazole-resistant and susceptible Trichostrongylus colubriformis. International Journal for Parasitology14:121–126. Some possible mechanisms of thiabenzadole (TBZ) resistance have been investigated in Trichostrongylus colubriformis. Uptake of TBZ and 3-0-methyl glucose and changes in carbohydrate levels in a TBZ-resistant and a TBZ-susceptible strain of T. colubriformis were measured after in vitro incubation, in the absence or presence of TBZ. The rate of TBZ uptake in adult worms was greater in the resistant strain but the difference was not statistically significant. Final concentrations of TBZ in resistant worms were significantly greater than in the susceptible strain, but there was no difference in TBZ uptake by eggs of either strain. A mechanism of resistance based on decreased drug uptake by resistant parasites is thus unlikely. Resistant worms absorbed 3-0-methyl glucose faster than susceptible parasites, however, this rate was unchanged in either strain when the worms were exposed to TBZ and indicates that the drug does not affect glucose uptake. Levels of glucose, trehalose and glycogen fell during the incubation with the decrease being more pronounced in the resistant strain. When the worms were exposed to TBZ the same changes in carbohydrate levels occurred except for resistant worms maintained anaerobically where levels were higher, showing that TBZ does not affect the rate of carbohydrate utilization.     

Evaluation of Pakistanian Bacillus thuringiensis isolates against Scirpophaga incertulas and Cnaphalocrocis medinalis

A large number of Bacillus thuringiensis (Bt) isolates separated from different ecological regions of Pakistan were characterized for crystal protein gene composition and pesticidal activity against two lepidopteran rice insect pests, the yellow stem borer (Scirpophaga incertulas) and the rice leaf folders (Cnaphalocrocis medinalis). A representative seventeen isolates were selected on the basis of initial screening and further characterization of pesticidal activity was performed according to following criteria; colony and parasporal inclusion morphology, SDS-PAGE, western blot analysis and comparative biotoxicity assays to determine LC₅₀ values. All isolates produced parasporal inclusion bodies and spores in their cells. Immunoblotting results showed that Pakistanian isolates synthesized entomocidal proteins belonging to Cry1A and Cry2A toxin groups. The biological activity of local isolates demonstarted a wide range of LC₅₀ values against both target insects pests. The most potent isolates, INS 1.13, INS 2.25 and NW 4.1 against S. incertulas showed LC₅₀ values of 29.83, 30.37 and 24.77 ng/ml of toxin, respectively. The LC₅₀ values of 57.37 and 73.09 ng/ml of toxin were exhibited by local isolates, INS 2.25 and RL 4.8 against C. medinalis, respectively.     

Nematode parasitism in sheep in North-East England: The epidemiology of Ostertagia species

This investigation was undertaken to determine the epidemiology of a single parasite genus, Ostertagia, in natural parasite populations in sheep raised under intensive conditions in North-East England.Over the 2 years of study, striking differences were found in the overall level of parasitism which were related to different climatic conditions, specifically rainfall. Despite the low source of infection derived from the ewes grazing on clean pasture in 1973 the overall level of parasitism in the lamb crop was high, this was attributed to the ideal climatic conditions for parasite translation during Spring and Summer. Ostertagia spp. was found to survive well over Winter and to initiate early infections in the young lambs in the following year. Despite the two sources of egg contamination, from the ewes and lambs, unusually dry conditions were experienced from March to June which resulted in much lower levels of infection in the second year of investigation.Regulation of the parasite population within the lambs was clearly demonstrated. Inhibition of Ostertagia was recorded somewhat earlier in the season than had been hitherto reported and was considered to be induced by a multiplicity of factors.     

Resistance of selected lines of Haemonchus contortus to thiabendazole, morantel tartrate and levamisole.

A field strain of Haemonchus contortus isolated from sheep at Armidale N.S.W., was found to be resistant to thiabendazole with approximately 20 per cent of the worms surviving a 50 mg/kg dose. The isolate was selected over six generations for resistance to 50 mg/kg thiabendazole. After this time, selection on one line was continued at 50 mg/kg thiabendazole and selection on a second line was extended to include 8·8 mg/kg morantel tartrate. A drug tolerance assay on the third generation of the thiabendazole-morantel tartrate selected line showed the ld_5o to be 5·3 mg/kg and the ld₉₅ to be 18 mg/kg morantel tartrate; the reported ld₅₀ and ld₉₅ for non-resistant strains of H. contortus to morantel tartrate are 2·5 mg/kg and 8 mg/kg respectively. In the fourth generation the thiabendazole and the thiabendazole-morantel tartrate selected lines together with a recently isolated field strain were assayed for resistance to thiabendazole, morantel tartrate and levamisole. The results indicated that the resistance to thiabendazole was probably due to a single gene. Both selected lines were more resistant to thiabendazole than the field strain. The thiabendazole-morantel tartrate selected line was more resistant to morantel tartrate than either of the other two. Resistance to morantel tartrate appeared to be polygenic in nature and due to increased vigour. The lowest dose of levamisole (1·6 mg/kg) killed more than 95 per cent of all strains of worms. There was no significant increase in effectiveness at higher dose rates indicating that surviving worms were resistant to the drug.     

Anthelmintic tolerance in free-living and facultative parasitic isolates of Halicephalobus (Panagrolaimidae)

SUMMARY Studies on anthelmintic resistance in equine parasites do not include facultative parasites. Halicephalobus gingivalis is a free-living bacterivorous nematode and a known facultative parasite of horses with a strong indication of some form of tolerance to common anthelmintic drugs. This research presents the results of an in vitro study on the anthelmintic tolerance of several isolates of Halicephalobus to thiabendazole and ivermectin using an adaptation of the Micro-Agar Larval Development Test hereby focusing on egg hatching and larval development. Panagrellus redivivus and Panagrolaimus superbus were included as a positive control. The results generally show that the anthelmintic tolerance of Halicephalobus to both thiabendazole and ivermectin was considerably higher than that of the closely related Panagrolaimidae and, compared to other studies, than that of obligatory equine parasites. Our results further reveal a remarkable trend of increasing tolerance from fully free-living isolates towards horse-associated isolates. In vitro anthelmintic testing with free-living and facultative parasitic nematodes offers the advantage of observing drug effect on the complete life cycle as opposed to obligatory parasites that can only be followed until the third larval stage. We therefore propose Halicephalobus gingivalis as an experimental tool to deepen our understanding of the biology of anthelmintic tolerance.     

海南省潜伏香蕉果实的炭疽菌对特克多的抗药性

对来自海南省13个地区的67个Colletotrichum musae（Berk. & Curt.）Arx菌株进行抗药性检测,所测菌株均表现出敏感,即未产生特克多抗药性。室内采用高浓度特克多和90～95%致死剂量紫外光进行抗性诱导,获得的抗性菌株均能在1000g/ml特克多的PDA培养基上生长,但EC50值相差很大,最高达130g/ml,而最低为0.87g/ml。抗性菌株对多菌灵和甲基托布津均表现正交互抗药性。连续无毒培养10代后,所有菌株仍可在5g/ml特克多的培养基上生长,表现抗性遗传稳定。抗性菌株的产孢能力和致病力与敏感菌相比并没有下降,表现很高的适应能力。     

An in vitro study of Ocimum sanctum as a chemotherapeutic agent on oral cancer cell-line

Oral squamous cell carcinoma (OSCC) is the most commom cancer in the world. If remain untreated for several years, it may be fatal. Hence, it is important to prevent and treat OSCC at an early stage. In this study the effect of aqueous and dry leaves extract of Ocimum sanctum was observed on Ca9-22 cell line, which is an OSCC cell line. For this, Ca9-22 cell line was cultured and maintained. After 24 h, the cells were treated with aqueous and dry leaves extract of Ocimum sanctum plant. Viability of the cancerous cells were studied by 3-(4, 5-dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and neutral red uptake (NRU) assay. Minimum inhibitory concentrations (MIC), lethal concentration₂₅ (LC₂₅), lethal concentration₅₀ (LC₅₀) and highest permissive concentration (HPC) was calculated by probit computational method. Experimentally, the MIC value was 5 mg/L, whereas the HPC was 30 mg/L of the plant extract in aqueous state. For the dry extract the MIC was 5 mg/L whereas the HPC was 35 mg/L for both MTT and NRU assays. For MTT assay LC values: 7.41 (LC₂₅), 14.79 (LC₅₀) and 26.91 mg/L (LC₇₅) for aqueous extract and 12.58 (LC₂₅), 20.89 (LC₅₀), 29.51 mg/L (LC₇₅) for dry extract. For NRU assay LC values were 10.23 (LC₂₅), 14.79 (LC₅₀) and 20.89 mg/L (LC₇₅) aqueous extract, and 16.59 (LC₂₅), 23.44 (LC₅₀), 30.19 mg/L (LC₇₅) dry extract of the plant. From the above study it was concluded that, Ocimum sanctum have anti-cancerous activity. It can further be used for therapeutic purposes.     

Prevalence of anthelmintic resistance in gastrointestinal nematodes of dairy goats under extensive management conditions in southwestern France

The occurrence of benzimidazole (BZ) and levamisole resistance was investigated in 18 randomly selected dairy goat herds located in southwestern France and characterized by extensive management. On each of the 18 farms, 45 adult goats were randomly allocated into three groups of 15 animals each: an untreated control group, a group that was orally administered fenbendazole (10 mg kg(-1) body weight) and a group that received orally a levamisole drench (12 mg kg(-1) body weight). Individual faecal egg counts and pooled larval cultures were done 10 days after anthelmintic treatment. Naive lambs were infected with larvae obtained from control and fenbendazole treated groups and were necropsied 35 days after infection for worm recovery. Faecal egg count reductions (FERC) were calculated for fenbendazole and levamisole and, when less than 95 per 100, were considered as indicative of anthelmintic resistance. An in vitro egg hatch test (EHT) was conducted with thiabendazole on eggs isolated from pooled faeces of fenbendazole treated goats in nine farms. Faecal egg count reductions indicated the occurrence of benzimidazole resistance in 15 out of 18 farms. Among these farms, nine had EHT values above 0.1 microg thiabendazole ml(-1) confirming the benzimidazole resistance status. Levamisole resistance was detected in two farms through FECR. Based on necropsy results, the prevalence of benzimidazole resistance was higher in Trichostrongylus colubriformis, medium in Haemonchus contortus and lower in Teladorsagia circumcincta. In nine farms the benzimidazole resistance was monospecific whereas multispecific resistance was found in the six remaining farms. A negative relationship was found between FECR for fenbendazole and the average number of anthelmintic treatments given per year on the farm. Despite extensive management including a low number of treatments, the prevalence of benzimidazole resistance was very high suggesting that the repeated and sometimes exclusive use of benzimidazole drugs, even at low frequency, is probably the main cause in developing nematode resistance in dairy goat herds. The importance of other factors such as under-dosing or buying animals already carrying resistant nematodes are discussed.