Resistance to anticoccidial drugs : Alternative strategies to control coccidiosis in broilers

Manuscripts documenting the occurrence of resistance against all commonly used anticoccidial drugs abroad, together with the high incidence of clinical coccidiosis in the field (60-90% of flocks) in the Netherlands, were the reasons to start investigations on the occurrence of anticoccidial drug resistance of Dutch and other European Eimeria spp. field isolates. The results showed a high degree of resistance against most tested anticoccidial drugs. The research performed in this thesis was preceded by an extensive literature review focusing on anticoccidial products and alternative strategies. Based on this literature study one alternative coccidiosis control strategy, namely a mannanoligosaccharide (a prebiotic), was chosen for further experimental studies in broiler chickens. Additionally, two other products i.e. ibuprofen (a non-steroidal anti-inflammatory drug (NSAID)) and a mucolytic enzyme (protease) were also used in experimental research aiming at assessing their anticoccidial potential. Ibuprofen was chosen because as a cyclooxygenase inhibitor it was expected to reduce the pathology of coccidiosis lesions through impairment of the production of prostaglandins, and because it was reported that another NSAID (indometacin) was able to reduce oocyst shedding. The mucolytic enzyme was studied as it was speculated that a protease might block the attachment of Eimeria to the mucus layer through its degradation. In view of the poor anticoccidial effect found in the studies performed with mannanoligosaccharide, ibuprofen and the mucolytic enzyme, further research focused on vaccination against chicken Eimeria, which despite some drawbacks has shown to be highly effective against outbreaks of clinical coccidiosis. Another reason to focus on vaccination was the inefficacy of rotation and shuttle programs to solve the coccidiosis resistance problem, which is explained by the fact that resistance is stable even in the absence of drug selection pressure. A welcome side effect of vaccination is the improved sensitivity of Eimeria spp. field isolates for anticoccidial drugs reported by some researchers. This phenomenon may play a key role in reducing the anticoccidial drug resistance problem. However, large-scale field studies documenting this were lacking. Therefore, the relation between the coccidiosis prevention program (vaccination with a live attenuated anticoccidial vaccine or anticoccidial drugs in feed) and anticoccidial drug sensitivity profiles of Eimeria spp. field isolates for diclazuril and monensin was studied. The results showed that use of vaccination was associated with an increased sensitivity of Eimeria spp. field isolates to anticoccidial drugs. Coccidiosis vaccines could be produced more efficiently if cross protection between Eimeria spp. would be relevant enough to reduce the number of species included in the vaccine and/or reduce the vaccination dose. Therefore, cross protection studies between an E. acervulina vaccine line and E. tenella, and E. maxima were performed. The results showed complete protection against E. acervulina, partial cross protection against E. tenella but no cross protection against E. maxima. The thesis is finalized with a summarizing discussion with conclusions and perspectives for future research

Resistance to anticoccidial drugs : Alternative strategies to control coccidiosis in broilers

Manuscripts documenting the occurrence of resistance against all commonly used anticoccidial drugs abroad, together with the high incidence of clinical coccidiosis in the field (60-90% of flocks) in the Netherlands, were the reasons to start investigations on the occurrence of anticoccidial drug resistance of Dutch and other European Eimeria spp. field isolates. The results showed a high degree of resistance against most tested anticoccidial drugs. The research performed in this thesis was preceded by an extensive literature review focusing on anticoccidial products and alternative strategies. Based on this literature study one alternative coccidiosis control strategy, namely a mannanoligosaccharide (a prebiotic), was chosen for further experimental studies in broiler chickens. Additionally, two other products i.e. ibuprofen (a non-steroidal anti-inflammatory drug (NSAID)) and a mucolytic enzyme (protease) were also used in experimental research aiming at assessing their anticoccidial potential. Ibuprofen was chosen because as a cyclooxygenase inhibitor it was expected to reduce the pathology of coccidiosis lesions through impairment of the production of prostaglandins, and because it was reported that another NSAID (indometacin) was able to reduce oocyst shedding. The mucolytic enzyme was studied as it was speculated that a protease might block the attachment of Eimeria to the mucus layer through its degradation. In view of the poor anticoccidial effect found in the studies performed with mannanoligosaccharide, ibuprofen and the mucolytic enzyme, further research focused on vaccination against chicken Eimeria, which despite some drawbacks has shown to be highly effective against outbreaks of clinical coccidiosis. Another reason to focus on vaccination was the inefficacy of rotation and shuttle programs to solve the coccidiosis resistance problem, which is explained by the fact that resistance is stable even in the absence of drug selection pressure. A welcome side effect of vaccination is the improved sensitivity of Eimeria spp. field isolates for anticoccidial drugs reported by some researchers. This phenomenon may play a key role in reducing the anticoccidial drug resistance problem. However, large-scale field studies documenting this were lacking. Therefore, the relation between the coccidiosis prevention program (vaccination with a live attenuated anticoccidial vaccine or anticoccidial drugs in feed) and anticoccidial drug sensitivity profiles of Eimeria spp. field isolates for diclazuril and monensin was studied. The results showed that use of vaccination was associated with an increased sensitivity of Eimeria spp. field isolates to anticoccidial drugs. Coccidiosis vaccines could be produced more efficiently if cross protection between Eimeria spp. would be relevant enough to reduce the number of species included in the vaccine and/or reduce the vaccination dose. Therefore, cross protection studies between an E. acervulina vaccine line and E. tenella, and E. maxima were performed. The results showed complete protection against E. acervulina, partial cross protection against E. tenella but no cross protection against E. maxima. The thesis is finalized with a summarizing discussion with conclusions and perspectives for future research

In vivo screening of five phytochemicals/extracts and a fungal immunomodulatory protein against colibacillosis in broilers

Five phytochemicals/extracts (an extract from Echinacea purpurea, a ß-glucan-rich extract from Shiitake, betaine [Betain™], curcumin from Curcuma longa [turmeric] powder, carvacrol and also a recombinant fungal immunomodulatory protein [FIP] from Ganoderma lucidum) cloned and expressed in Escherichia coli were investigated for their anticolibacillosis potential in three chicken experiments, which were conducted in floor pens. Birds that were inoculated with E. coli intratracheally were treated with the phytochemicals/extracts or the FIP and compared with doxycycline-medicated and non-medicated infected broilers. Non-medicated and non-infected birds were used as negative controls. Mortality, colibacillosis lesions and body weight gains were used as parameters. Considering the sum of dead birds and chickens with generalized colibacillosis per group, there was no significant difference between the positive control groups and birds treated with phytochemicals/extracts or the FIP. In contrast, doxycycline-treated birds showed significantly lower mortality and generalized colibacillosis. Moreover, none of the phytochemicals/extracts and the FIP improved recovery from colibacillosis lesions, while all doxycycline-treated broilers recovered completely. The negative control birds and doxycycline-treated groups consistently showed the highest weight gains. Pulsed-field gel electrophoresis of reisolates showed that they were genetically indistinguishable from the inoculation strain. In conclusion, none of the tested phytochemicals/extracts and the FIP significantly reduced the E. coli-induced mortality and generalized colibacillosis, and nor did they improve recovery from colibacillosis lesions

Dietary protease can alleviate negative effects of a coccidiosis infection on production performance in broiler chickens

Two experiments were conducted to determine the effect of dietary protease on coccidiosis infection, production performance, the intestinal mucus layer thickness, and brush border enzyme activity using broilers challenged with Eimeria spp. laboratory isolates (Eimeria acervulina, E. maxima and E. tenella). In the first study the protease was Supplied at a concentration of 2.5 x 10(4) amylase-DU per kg feed. Broiler chickens were housed in cages and were infected at 10 days of age with 10(4.0) E. acervulina, 10(3.8) E. maxima or 10(3.3) E. tenella sporulated oocysts. Coccidial lesion scores, oocysts shedding, sporulation assessment and daily weight gain were used as parameters to quantify the effect of the protease. In the second study the effects of the protease (supplied at a concentration of 2.5 x 10(4) amylase-DU per kg feed) on the thickness of the mucus adherent layer and sucrase-isomaltase activity (SIA) of three regions (duodenum, jejunum and caecum) of the intestinal tract were determined. In experiment 1, no significant interaction between dietary enzyme supplementation and single Eimeria spp. challenge was observed on body weight gain. However, protease addition to the diet resulted in a significant (P=0.046) higher weight gain after comparing all supplemented and non-supplemented groups. E. maxima infected chickens showed a significant lower body weight gain in comparison with the other Eimeria infected groups. Coccidial lesions were not significantly affected by the dietary protease supplementatio

In vivo screening of five phytochemicals/extracts and a fungal immunomodulatory protein against colibacillosis in broilers

Besides the anticoccidial drug resistance problem, increasing consumer concerns about food safety and residues have propelled the quest for alternative prevention and control strategies amongst which phytotherapy has gained appeal due to a renewed interest in natural medicine.Objective: The objective was in vivo screening of four phytochemicals/extracts and a fungal immunomodulatory protein (FIP) against an Eimeria acervulina infection in broilers.Animals and methods: Four phytochemicals/extracts (extract from Echinacea purpurea, betaine (Betain™), curcumin, carvacrol (two different doses)), and a recombinant FIP from Ganoderma lucidum cloned and expressed in Escherichia coli were investigated for their anticoccidial potential. The experiment was conducted in a battery cage trial with 54 cages of eight birds each. Broilers infected with E. acervulina (a low and high infection dose of 104 and 105 sporulated oocysts, respectively) and treated with the phytochemicals/extracts or the FIP were compared with broilers treated with the anticoccidial salinomycin sodium (Sacox®) and with an untreated uninfected and an untreated infected control group. Coccidiosis lesion scores, body weight gains and oocyst shedding were used as parameters.Results: The results showed a coccidiosis infection dose effect on the mean coccidiosis lesion scores. The phytochemicals/extracts and the FIP failed to reduce coccidiosis lesion scores and oocyst shedding, while salinomycin efficiently controlled the E. acervulina infection and enabled significantly higher body weight gains.Conclusion: In conclusion, the selected phytochemicals/extracts and the FIP did not reduce the lesions of an experimentally induced E. acervulina infection

Angiogenetic balance in human melanoma: expression of VEGF, bFGF, IL-8, PDGF and angiostatin in relation to vascular density of xenografts in vivo

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