Awad A. Shehata

The effect of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro.

The use of glyphosate modifies the environment which stresses the living microorganisms. The aim of the present study was to determine the real impact of glyphosate on potential pathogens and beneficial members of poultry microbiota in vitro. The presented results evidence that the highly pathogenic bacteria as Salmonella Entritidis, Salmonella Gallinarum, Salmonella Typhimurium, Clostridium perfringens and Clostridium botulinum are highly resistant to glyphosate. However, most of beneficial bacteria as Enterococcus faecalis, Enterococcus faecium, Bacillus badius, Bifidobacterium adolescentis and Lactobacillus spp. were found to be moderate to highly susceptible. Also Campylobacter spp. were found to be susceptible to glyphosate. A reduction of beneficial bacteria in the gastrointestinal tract microbiota by ingestion of glyphosate could disturb the normal gut bacterial community. Also, the toxicity of glyphosate to the most prevalent Enterococcus spp. could be a significant predisposing factor that is associated with the increase in C. botulinum-mediated diseases by suppressing the antagonistic effect of these bacteria on clostridia.

Detection of Glyphosate Residues in Animals and Humans

In the present study glyphosate residues were tested in urine and different organs of dairy cows as well as in urine of hares, rabbits and humans using ELISA and Gas Chromatography-Mass Spectroscopy (GC-MS). The correlation coefficients between ELISA and GC-MS were 0.96, 0.87, 0.97and 0.96 for cattle, human, and rabbit urine and organs, respectively. The recovery rate of glyphosate in spiked meat using ELISA was 91%. Glyphosate excretion in German dairy cows was significantly lower than Danish cows. Cows kept in genetically modified free area had significantly lower glyphosate concentrations in urine than conventional husbandry cows. Also glyphosate was detected in different organs of slaughtered cows as intestine, liver, muscles, spleen and kidney. Fattening rabbits showed significantly higher glyphosate residues in urine than hares. Moreover, glyphosate was significantly higher in urine of humans with conventional feeding. Furthermore, chronically ill humans showed significantly higher glyphosate residues in urine than healthy population. The presence of glyphosate residues in both humans and animals could haul the entire population towards numerous health hazards, studying the impact of glyphosate residues on health is warranted and the global regulations for the use of glyphosate may have to be re-evaluated.

Field Investigations of Glyphosate in Urine of Danish Dairy Cows

In the present study, thirty dairy cows from each of eight Danish dairy farms were investigated for excretion of glyphosate in urine. Blood serum parameters indicative of cytotoxicity as alkaline phosphatase (AP), glutamate dehydrogenase (GLDH), glutamate oxaloacetate transaminase (GOT), creatinine kinase CK), nephrotoxicity, (urea, creatine), cholesterol and the trace elements as manganese (Mn), cobalt (Co), selenium (Se), copper (Cu) and zinc (Zn) were investigated. All cows excreted glyphosate in their urine but in varying concentrations. Increased levels of GLDH, GOT and CK in cows from all farms demonstrate a possible effect of glyphosate on liver and muscle cells. High urea levels in some farms could be due to nephrotoxicity of glyphosate. Also the unexpected very low levels of Mn and Co were observed in all animals which could be explained due to a strong mineral chelating effect of glyphosate. In contrast the mean levels of Cu, Zn and Se were within the normal reference range. In conclusion, this study gives the first documentation to which extent Danish dairy cattle are exposed to Glyphosate and its impact on blood parameters.

Detection of Glyphosate in Malformed Piglets

Glyphosate residues in different organs and tissues as lungs, liver, kidney, brain, gut wall and heart of malformed euthanized one-day-old Danish piglets (N= 38) were tested using ELISA. All organs or tissues had glyphosate in different concentrations. The highest concentrations were seen in the lungs (Range 0.4-80 μg/ml) and hearts (Range 0.15-80 μg/ml). The lowest concentrations were detected in muscles (4.4-6.4 μg/g). The detection of such glyphosate concentrations in these malformed piglets could be an allusion to the cause of these congenital anomalies. Further investigations are urgently needed to prove or exclude the role of glyphosate in malformations in piglets and other animals.

Efficacy of early treatment with toltrazuril in prevention of coccidiosis and necrotic enteritis in chickens

In the present study, efficacy of the toltrazuril treatment for prevention of coccidiosis and necrotic enteritis was tested. Ninety-six 14-day-old commercial broiler chickens were caged and divided into eight groups (n=12), designated groups 1 to 8. Chickens of groups 1 to 6 were inoculated orally at 18 days of age with 25,000 oocysts of Eimeria tenella and 75,000 oocysts of Eimeria brunetti. At 22 days of age, chickens of groups 1 to 6 were infected with 109 colony-forming unit Clostridium perfringens. Chickens of group 1 were treated with 75 parts/106 toltrazuril in drinking water for 8 h on two consecutive days up to 12 h before Eimeria infection, while chickens of groups 2 to 5 were treated with the same dose of toltrazuril at 12 h, 36 h, 60 h and 84 h after Eimeria infection, respectively. The non-treated group 6 served as a positive control. Chickens in group 7 were treated with toltrazuril at 17 and 18 days of age, and those of group 8 remained uninfected and non-treated as a negative control. The feed conversion ratio was higher in the positive control compared with other groups. The mortality rates were 16.8% and 41.7% in the late toltrazuril-treated (at 84 h) and infected non-treated chickens, respectively. Lesions scores of necrotic enteritis or coccidiosis in infected, non-treated chickens were significantly more severe compared with negative controls (P<0.01) and late toltrazuril-treated (at 84 h) chickens (P<0.05). In conclusion, application of toltrazuril before Eimeria challenge protected chickens from coccidiosis and indirectly from successive necrotic enteritis caused by C. perfringens infection.

Development of a recombinant ELISA using yeast (Pichia pastoris)-expressed polypeptides for detection of antibodies against avian influenza A subtype H5

Two truncated sequences (designated P1 and rHA1) of influenza A virus subtype H5 haemagglutinin (HA) were cloned and expressed in yeast Pichia pastoris (P. pastoris). These polypeptides were used in an indirect recombinant ELISA (rELISA) for detection of H5 antibodies in poultry. Serum samples obtained from broiler chickens vaccinated with commercial inactivated vaccine (H5N2) and control negative sera from non-vaccinated chickens against influenza were tested using rP1-ELISA, rHA1-ELISA, whole H5N1-ELISA, Western blot, agar gel immunodiffusion (AGID) and haemagglutination inhibition (HI) tests. The rHA1-ELISA proved to be highly sensitive and specific. To study the validity of rHA1-ELISA, a total of 179 serum samples obtained from commercial broiler chickens vaccinated previously with commercial H5N2 inactivated vaccines, were tested by rHA1-ELISA, commercial ELISA (cELISA) and HI. The relative sensitivity and specificity between rHA1-ELISA, and HI tests were 100% and 70%, respectively, and between cELISA and HI were 100% and 57%, respectively. The agreement ratio between rHA1-ELISA and HI was 84.9% and between cELISA and HI tests was 76.5%. Serum samples obtained from ducks vaccinated with commercial inactivated H5N2 were tested by rHA1-ELISA and the results showed significant reactivity with duck sera. In conclusion, the results demonstrate the potential applicability of the rELISA for the determination of antibodies to H5 influenza virus in chickens and ducks.

Antagonistic effect of different bacteria on Clostridium botulinum types A, B, D and E in vitro

Clostridium botulinum is an anaerobic, Gram-positive, spore-forming rod that produces a potent neurotoxin. Seven types (A, B, C1+2, D, E, F and G) of C botulinum are recognised, based on the antigenic specificity of the botulinum neurotoxin (BoNT) produced by each strain. Types A, B, E and F cause human botulism, while types B, C and D cause disease in farm animals (Goonetilleke and Harris 2004, Simpson 2004, Radostits and others 2007, Popoff and Bouvet 2009). Animals most commonly affected are wild fowl and poultry, cattle, horses and some species of fish. There are three major recognised disease entities in humans, food-borne botulism, infant botulism and wound botulism . Recently, Rodloff and Kruger (2012) suggested that a new form of chronic, visceral botulism may exist that affects both human beings and animals. The antagonism between C botulinum and bacterial members of the microecosystem …

Safety and Efficacy of a Metabolic Drift Live Attenuated Salmonella Gallinarum Vaccine Against Fowl Typhoid

SUMMARY. Fowl typhoid (FT), a systemic disease that results in septicemia in poultry, is caused by Salmonella enterica serovar Gallinarum biovar Gallinarum (SG). Mortality and morbidity rates from FT can reach up to 80%. Attenuated live Salmonella vaccine candidates have received considerable attention because they confer solid immunity, and they can produce systemic and mucosal immunity in the gut when administered orally. In the present study, five metabolic drift (MD) mutants with a single- (designated SG-Rif1, SG-Sm6) or double-attenuating marker (designated SG-Rif1-Sm4, SG-Sm6-Rif10, and SG-Rif1-Sm10) were isolated. The relative colony sizes to wild-type strain after 24 hr at 37 C incubation were 50%, 40%, 30%, 30%, and 20%, respectively. The probability of a back mutation can almost be excluded because the reduced colony sizes were stable after at least 50 passages on culture media. The safety and immunogenicity were evaluated in susceptible 1-day-old commercial layer chickens. After oral administration of 108 colony-forming units (CFU), all developed MD mutants proved to be safe and did not cause death of any infected birds during 15 days postvaccination, whereas chickens receiving 106 CFU SG wild-type strain showed a high mortality rate (40%). Vaccination of commercial layer chicks with SG-Rif1, SG-Sm6, SG-Rif1-Sm4, and SG-Sm6-Rif10 MD mutants could protect chickens against challenge by homologous wild-type strain; however, SG-Rif1-Sm10 could not protect against challenge, indicating hyperattenuation. In conclusion, vaccination with SG MD mutant vaccine appears to be safe and offers protection against FT in chickens. RESUMEN. Seguridad y eficacia de una vacuna variante metabólica de Salmonella Gallinarum viva atenuada contra la tifoidea aviar. La tifoidea aviar, una enfermedad sistémica que produce septicemia en aves de corral, es causada por Salmonella enterica serovar Gallinarum biovar Gallinarum (SG). Las tasas de mortalidad y morbilidad de la tifoidea aviar pueden alcanzar hasta el 80%. Candidatos de vacunas vivas atenuadas de Salmonella han recibido una atención considerable porque confieren inmunidad sólida, y pueden producir una inmunidad sistémica y en la mucosa intestinal cuando se administran por vía oral. En el presente estudio, se aislaron cinco mutantes metabólicas con un solo marcador (designadas SG-Rif1 y SG-Sm6) o con marcador de atenuación doble (designadas SG-Rif1-Sm4, SG-Sm6-Rif10 y Rif1 SG–Sm10). Los tamaños de colonia relativos a la cepa de tipo silvestre después de 24 h con incubación a 37 C fueron de 50%, 40%, 30%, 30%, y 20%, respectivamente. La probabilidad de una mutación en reversa pudo excluirse porque las colonias con tamaños reducidos fueron estables después de al menos 50 pases en medios de cultivo. La seguridad y la inmunogenicidad se evaluó en aves de postura comerciales susceptibles de un día de edad. Después de la administración oral de 108 unidades formadoras de colonias (UFC), todas las mutantes metabólicas MD desarrolladas demostraron ser seguras y no causaron la muerte de las aves infectadas dentro de los quince días después de la vacunación, por su parte, las aves que recibieron 106 UFC de S. Gallinarum de tipo silvestre mostraron una alta tasa de mortalidad (40%). La vacunación de ponedoras comerciales con las mutantes metabólicas SG-Rif1, SG-Sm6, SG-Rif1 Sm4, y SG-SM6-Rif10 pudo proteger a los pollos contra el desafío con cepas silvestres homólogas, sin embargo, la vacuna SG-Rif1-SM10 no pudo proteger contra el desafío, indicando hiperatenuación. En conclusión, la vacunación con vacunas mutantes metabólicas de S. Gallinarum parece ser segura y ofrece protección contra la tifoidea aviar en pollos.

Necrotic enteritis in chickens: development of a straightforward disease model system

The interaction between Eimeria species and Clostridium perfringens was investigated in two different necrotic enteritis (NE) models: 120-day-old broilers were used in two separate experiments consisting of six groups (n=10) each. Besides controls, chickens were infected with coccidia on study day (SD) 18 (Eimeria maxima and Eimeria acervulina (experiment 1) or Eimeria tenella and Eimeria brunetti (experiment 2) and/or a NetB toxin positive C perfringens strain (both experiments: SD 14 or SD 22, respectively)). Body weight, feed intake, mortality rate, clinical disease, Eimeria species oocyst excretion and C perfringens counts were recorded. NE and coccidiosis specific lesion scores were assessed (SD 24 and SD 30). In coinfected groups, NE-typical clinical signs occurred. Coccidiosis-specific lesions were most severe in coinfected groups (significant for E tenella, P<0.05). Most pronounced NE lesions occurred in coinfected chickens compared with C perfringens monoinfected groups (experiment 2, C perfringens infections on SD 22: P<0.05). In experiment 2, E tenella antibody levels were (non-significantly) higher in coinfected groups than in Eimeria species monoinfected groups. Thus, infection with E tenella and Eimeria brunetti followed by C perfringens inoculation is regarded as an easy to handle and suitable model for investigations into NE of chickens.

Oral Application of Charcoal and Humic acids to Dairy Cows Influences Clostridium botulinum Blood Serum Antibody Level and Glyphosate Excretion in Urine

The present study was initiated to investigate the influence of oral application of charcoal, sauerkraut juice and humic acids on chronic botulism in dairy cows. A total of 380 Schleswig Holstein cows suffering from chronic botulism were fed daily with 400 g/animal charcoal for 4 weeks (1-4 weeks of study), 200 g/animal charcoal (5-10 weeks of study), 120 g/animal humic acid (11-14s week of study), 200g charcoal and 500 ml Sauerkraut juice/animal (13-16 weeks of study), 200 g charcoal and 100 mL Aquahumin /animal (15-18s week of study), 100 g charcoal and 50 mL Aquahumin (19-22 weeks of study) followed by 4 weeks without any supplementation. Bacteriological and immunological parameters investigated included C. botulinum and botulinum neurotoxins (BoNT) in faeces, C. botulinum ABE and CD antibodies, positive acute phase proteins (APPs) haptoglobin and LPS-binding protein (LBP) using serum ELISA, negative APP paraoxanase by its enzymatic activity and glyphosate in urine by ELISA. Neither BoNT nor C. botulinum was detected in feacal samples. From week six until four weeks before the end of the study, there was a significant reduction in antibody levels. All supplementation, except low doses of charcoal (200g /animal) alone, led to a significant reduction of C. botulinum ABE and CD antibody levels. There also was a significant reduction of glyphosate in urine following supplementation with a combination of 200g charcoal plus either 500 mL sauerkraut juice or humic acid. Haptoglobin, paraoxanase and LBP were significantly increased by the 24th week of the study. The positive APPs and C. botulinum antibodies were significant negative correlations. In conclusion, a charcoal-sauerkraut juice combination and humic acids could be used to control chronic botulism and glyphosate damage in cattle.