Impact of dietary-nucleotides and Saccharomyces cerevisiae-derivatives on growth-performance, antioxidant-capacity, immune-response, small-intestine histomorphometry, caecal-Clostridia, and litter-hygiene of broiler-chickens treated with florfenicol.

Stress in poultry production is energy-demanding. Nucleotides and yeast cell-wall products are essential nutrients for broiler performance, gut function, and immune response. Antibiotics, like florfenicol, negatively affect the immune system. A total of 600 one-d-old broiler chickens (Cobb-500) were weighed and randomly allotted into four groups with three replicates each. The control group (G1) received the basal diet, G2 received a diet supplemented with a combination of nucleotides and Saccharomyces cerevisiae derivatives (250 g/Ton), G3 received the basal diet and medicated with florfenicol (25 mg/Kg body weight) in drinking water for 5 days, while G4 received a combination of nucleotides and Saccharomyces cerevisiae-derivatives (250 g/Ton) and medicated with florfenicol in drinking water. Growth performance criteria were recorded weekly. Blood, intestinal contents, small-intestine sections, and litter samples were collected to measure birds' performance, carcass yields, leukocytic counts, antioxidant capacity, antibody titres, phagocytic index, caecal Clostridia, intestinal histomorphometry, and litter hygiene. Nucleotide-supplemented groups (G2 and G4) revealed significant (p ≤ 0.05) improvements in feed conversion, and body weight, but not for carcass yields in comparison to the control. Dietary nucleotides in G2 elevated blood total proteins, leucocytic count, antioxidant capacity, and phagocytic index, while they lowered blood lipids and litter moisture and nitrogen (p ≤ 0.05). Dietary nucleotides in G4 ameliorated the immunosuppressive effect of florfenicol (p ≤ 0.05) indicated in reducing caecal Clostridia, improving duodenal and ileal villi length, and increasing blood albumin and globulin levels, and phagocytosis%. Supplementing diets with nucleotides and yeast products has improved the immune system and provided a healthier gut for broilers.

Impact of buffered sodium butyrate as a partial or total dietary alternative to lincomycin on performance, IGF-1 and TLR4 genes expression, serum indices, intestinal histomorphometry, Clostridia, and litter hygiene of broiler chickens.

BACKGROUND: Sodium butyrate (SB) is a short-chain fatty acid and a safe antibiotic alternative. During 35 days, this study compared the impact of coated SB (Butirex C4) and lincomycin (Lincomix) on broiler growth, gut health, and litter hygiene in 1200 one-day-old Ross-308 broiler chicks that were randomly assigned into 5-dietary groups with 5-replications each. Groups divided as follows: T1: Basal diet (control), T2: Basal diet with buffered SB (1 kg/ton starter feed, 0.5 kg/ton grower-finisher feeds), T3: Basal diet with 100 g/ton lincomycin, T4: Basal diet with buffered SB (0.5 kg/ton starter feed, 0.25 kg/ton grower-finisher feeds) + 50 g/ton lincomycin, and T5: Basal diet with buffered SB (1 kg/ton starter feed, 0.5 kg/ton grower-finisher feeds) + 50 g/ton lincomycin. Birds were housed in a semi-closed deep litter house, where feed and water were available ad libitum. Results were statistically analyzed using ANOVA and Tukey's post hoc tests. RESULTS: Combined dietary supplementation with SB and lincomycin (T4 and T5) significantly enhanced body weights, weight gains, feed conversion ratio, and profitability index. Also, carcasses in T4 and T5 exhibited the highest dressing, breast, thigh, and liver yields. T5 revealed the best blood biochemical indices, while T3 showed significantly elevated liver and kidney function indices. T4 and T5 exhibited the highest expression levels of IGF-1 and TLR4 genes, the greatest villi length of the intestinal mucosa, and the lowest levels of litter moisture and nitrogen. Clostridia perfringens type A alpha-toxin gene was confirmed in birds' caeca, with the lowest clostridial counts defined in T4. CONCLUSIONS: Replacing half the dose of lincomycin (50 g/ton) with 0.5 or 1 kg/ton coated SB as a dietary supplement mixture showed the most efficient privileges concerning birds' performance and health.

Influence of dietary Bacillus coagulans and/or Bacillus licheniformis-based probiotics on performance, gut health, gene expression, and litter quality of broiler chickens.

Probiotics are non-pathogenic microorganisms that are potentially important non-antibiotic alternatives. This study aimed to compare novel multi-strain and single-strain Bacillus probiotics and their respective influences on broiler chickens' performance, gut health, litter quality, immune response, and NBN and TLR gene expression. A total of 1200 Arbor-Acres 1-day-old broiler chicks were randomly allocated into three treatments (T1 was a control, T2 was supplemented with a combined Bacillus coagulans (2 × 109 cfu/g) and Bacillus licheniformis (8 × 109 cfu/g) probiotic strains (0.2 kg/ton of feed), and T3 was supplemented with Bacillus licheniformis (3.2 × 109 cfu/g) probiotic (0.5 kg/ton of feed) with eight replicas of each. Supplementing the broiler diet with either the single-strain (T3) or the multi-strain (T2) Bacillus-based probiotic raised the overall birds' body weight, body weight gain, feed conversion ratio, and European production efficiency factor compared to the control (T1), with a significant enhancement achieved by the multi-strain Bacillus product (P = 0.005). T2 and T3 exhibited significantly improved cholesterol, Alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and alkaline phosphatase levels than the control (P ≤ 0.05). The transcript levels of both NBN and TLR genes were upregulated in the liver in the T2 and T3 groups. The T2 group experienced significant reductions in gut bacterial counts, especially for Clostridia, and recorded the lowest litter moisture and nitrogen. In conclusion, supplementing broiler diets with probiotics of multiple Bacillus strains increased production profitability by promoting bird growth, improving feed intake, enhancing gut mucosa and immune organs, and upregulating genes responsible for immunity. All these inhibit the overgrowth of enteric pathogens and sustain litter quality.

Immune Cell Reaction Associated with Coenurus cerebralis Infection in Sheep with Particular Reference to ELISA as a Diagnostic Tool.

Sturdy is a disease caused by Coenurus cerebralis (C. cerebralis) that typically affects the brain and spinal cord of sheep. So, this study aimed to detect the pathological, hematological and immunological changes caused by C. cerebralis in sheep. On examination, a total of 17 sheep out of 30 sheep (56.7%) from various regions in Egypt were found infected with C. cerebralis from May to August 2019. Each cyst was extracted from the sheep brain; in addition, tissue specimens were taken from the brain tissues for histopathological examination. The hematological profile was analyzed. Enzyme-Linked Immunosorbent Assay's (ELISA) specificity and sensitivity were evaluated using cystic fluid and protoscolices antigens (Ag). The cell-mediated immunity against the C. cerebralis cyst was also assessed via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) to show alterations in mRNA expression of the Tumor Necrosis Factor-alpha (TNF-α) and gamma Interferon (IFN-γ) cytokines qRT-PCR. In histopathological sections, cerebral tissue showed an areolar cyst wall with many protoscolices attached to the tissue. The affected part showed prominent necrosis together with inflammatory cells' aggregation. Hyperplastic proliferation of the ependymal cells was a common finding. The infected sheep exhibited significantly lower total erythrocyte numbers (ER), hemoglobin levels (Hb), packed cell volume (PCV), platelet numbers (PN) and segmented cell numbers compared to apparently healthy sheep. Despite the sensitivity for the indirect ELISA being 100% for both of the Ags (fluid and scolex), the evaluation of ELISA specificity using the two antigen (Ag) preparations showed specificities of 46.2% and 38.5% for fluid and scolex Ag, respectively. Meanwhile accuracy ranged from 76.7% and 73.3% for the fluid and scolex Ags, respectively, that showed the priority was directed to the fluid to be used as an ideal sample type for ELISA. Levels of TNF-α and IFN-γ were significantly elevated in infected sheep compared to non-infected control ones. In conclusion, C. cerebralis is a serious disease infecting sheep in Egypt revealing economic losses. Although this investigation supports preliminary information about the prevalence, pathological and serological characterization of C. cerebralis, further sequencing and phylogenetic analysis is needed to understand better the T. multiceps epidemiology in ruminants and canines in Egypt.

Evaluation of the Effects of Silver Nanoparticles Against Experimentally Induced Necrotic Enteritis in Broiler Chickens.

BACKGROUND: Clostridium perfringens-associated necrotic enteritis (NE) is a serious problem affecting broiler production. A major global challenge is to reduce the use of antibiotics in poultry industry due to their negative impacts on public health. One alternative is to use nanoparticles (NPs) to overcome bacterial resistance to antibiotics. Silver nanoparticles (Ag NPs) showed strong antimicrobial activity. METHODS: A total of 120 Cobb broiler chicks (1-day old) were obtained for this study and were divided into 4 equal groups at age of 14 days (30 birds each); each group was subdivided into 3 equal replicates (10 birds each). The groups were designated as follows: G1, infected; G2, infected and treated with Ag NPs; G3, treated with Ag NPs; and G4, negative control. Birds were infected with 4×108 colony forming unit (CFU)/mL/bird C. perfringens type A for 2 successive days. In the treated groups, Ag NPs (mean diameter 15 nm; total dose 150 µg/bird) were administered via crop gavage. During the observation period (5 weeks), bird performance and immune organ indexes were recorded. Serum samples were collected for immunological evaluation, and tissue samples were collected for histopathology and estimation of Ag NPs residues. RESULTS: Treatment with Ag NPs reduced the colonization of C. perfringens in the intestine and ceca, decreased the severity of clinical signs and reduced mortalities in comparison with infected non-treated group. Ag NPs treatment alleviated pathological lesions in the intestine and liver, but their residues were found in the muscles. CONCLUSION: Ag NPs have a positive impact on gut health integrity while having no impact on immune organs. Ag NPs have some residues in muscles; therefore, further studies are needed on the concentration and size of Ag NPs, the route of administration, and withdrawal time to ensure the safety of chicken meat for human consumption.

Prevalence, genetic, and biochemical evaluation of immune response of police dogs infected with Babesia vogeli.

BACKGROUND AND AIM: Babesia species are tick-borne protozoan parasites of apicomplexan type which infect the erythrocytes of dogs it ranges from subclinical to severe cases, depending on different factors such as immune status, age, and presence of other co-infections with the Babesia species. Hence, this study aimed to identify the protozoan parasites infecting police dogs of different breeds, ages, and both sexes in Egypt. Concerning molecular detection of Babesia vogeli using conventional polymerase chain reaction sequencing and phylogenetic analysis, followed by the assessment of immunological and biochemical status of infected dogs. MATERIALS AND METHODS: The blood of 242 police K9 dogs was collected. The age, breed, sex, and health status with clinical signs of dogs were recorded. Hematological, biochemical, and oxidative stress analyses of the blood were performed together with gene expression analysis using two genes (gamma interferon [IFN-γ] and tumor necrosis factor-alpha [TNF-α]). The identification of the causative agent was performed using molecular analysis of the 18S ribosomal RNA (rRNA). The 18S rRNA region of canine Babesia spp. was successfully amplified, and sequencing data were deposited in GenBank (accession number: MT565474.1), which resembled those of B. vogeli. RESULTS: The results of blood samples screening revealed that of the 242 blood samples, 62 were positive for B. vogeli infection. The infection rate in male dogs was higher than that in female dogs. The police dogs were classified into the following three groups of dogs: (1st group) healthy, (2nd infected with B. vogeli, and mixed infection of B. vogeli and Ehrlichia canis). The oxidative stress biomarkers levels in B. vogeli infected dogs were greater than that of healthy dogs. Likewise, IFN-γ and TNF-α level in B. vogeli infected dogs were elevated in infected dogs. CONCLUSION: Our findings demonstrated that B. vogeli had completely adverse effects on the health condition of the police dogs that may lead to death in some dogs.

Epidemiological and genetic characteristics of asymptomatic canine leishmaniasis and implications for human Leishmania infections in Egypt.

Leishmaniasis is a neglected zoonotic disease that poses significant veterinary and public health risks in developing countries. Dogs act as a reservoir host for leishmaniasis transmitted to humans. A total of 108 human cases of cutaneous leishmaniasis (CL) were identified in the Al-Houd Al-Marsoud Hospital in Cairo, Egypt, during 2018. Blood samples and skin biopsies were collected for further examination. Blood samples from 96 asymptomatic dogs were collected. All samples were subjected to molecular and phylogenetic analysis. Quantitative RT-PCR was used to measure the expression of genes related to mTOR signalling and inflammation in blood and tissue samples. The distribution pattern of human cases pointed to an endemic focus in North Sinai (66.67%). The prevalence of asymptomatic canine leishmaniasis was 66.60%. Histopathological examination of human skin lesions revealed a severe granulomatous inflammatory reaction, necrosis and ulceration. Moreover, leishmanial amastigotes could be detected in human tissue samples. Phylogenetic analysis revealed 100% identity of human isolates to Leishmania tropica (MN453682), and dog isolates to Leishmania infantum (MN453673), with 94.9% similarity between the two isolates. Gene expression related to mTOR signalling and inflammation in both species' samples confirmed a significant alteration of EIF4EBP1, CCR4 and INF-γ expression compared with control groups. In Egypt, increased incidence of asymptomatic carrier dogs acting as a significant reservoir host for Leishmania poses a public health hazard. Findings warrant further epidemiological investigation of CL in Egypt, as well as additional study of parasite differentiation and gene regulation.

Advanced immunological studies on Cephalopina titillator with special references to the epidemiological uses of Dot-ELISA in camel sera.

Cephalopina titillator (C. titillator) is a common worldwide nasal bot fly larval infestation of camels, which belongs to the family Oestridae. This study aimed to evaluate two new immunologic diagnostic techniques; indirect-ELISA and Dot-ELISA, for the screening of C. titillator infestation in camels. Thirty slaughtered camel heads were examined carefully for the presence of C. titillator larvae. One hundred, third-stage larvae (L3), were dissected for the collection of their salivary glands, for the preparation of the salivary gland antigen. Blood samples were obtained for hematological and serological examinations. Results revealed a true prevalence of C. titillator in the sampled camels being 80% (24/30). Infested camels showed a significant reduction in leukocytes (P < 0.0001) and neutrophils (P = 0.045), and a significant increase in eosinophils and monocytes (P < 0.0001). The serological examination estimated apparent prevalence as 80% (24/30) and 90% (27/30) by Dot-ELISA and indirect-ELISA, respectively. Dot-ELISA revealed 100% sensitivity, specificity, and accuracy. While, indirect-ELISA displayed 100% sensitivity, 50% specificity, and 90% accuracy. Dot-ELISA exhibited perfect agreement with the gold standard test, so it could be considered an ideal, simple, and accurate immunologic screening technique for the detection of C. titillator in camels.

Evaluation of tumor necrosis factor-alpha (TNF-α); gamma interferon (IFN-γ) genes and oxidative stress in sheep: immunological responses induced by Oestrus ovis (Diptera: Oestridae) infestation.

This study aimed to evaluate the cell mediated immune responses against Oestrus ovis (O. ovis) in sheep through measurement of the changes in mRNA expression of the tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) cytokines using quantitative Real time-PCR (qRt-PCR). Also; to detect the role of Oestrus ovis infestation in the oxidative stress markers in sheep. Fifty sheep head were examined in Cairo abattoir from the period of May to August 2019. Sera were separated and collected for measurement of nitric oxide, zinc and malondialdehyde (MDA). While TNF-α and IFN-γ mRNA were extracted from nasal mucosa. Levels of IFN-γ and TNF-α were significantly higher in infested sheep than that in non-infested one. Also, oxidative stresses were indicated by high level of nitric oxide as one of reactive oxygen species (ROS) and serum MDA as oxidative stress marker and low antioxidant capacity (zinc concentration in serum) in infested sheep. The obtained results indicated that measurements of TNF-α and IFN-γ cytokines using qRT-PCR could be used as an association and reproducible quantitative method for the diagnosis of O. ovis infestation in sheep.

Emergence of ß-lactamase- and carbapenemase- producing Enterobacteriaceae at integrated fish farms.

BACKGROUND: Epidemiological studies suggested that determinants for antibiotic resistance have originated in aquaculture. Recently, the integrated agriculture-aquaculture system has been implemented, where fish are raised in ponds that receive agriculture drainage water. The present study aims to investigate the occurrence of ß-lactamase and carbapenemase-producing Enterobacteriaceae in the integrated agriculture-aquaculture and the consequent public health implication. METHODS: Samples were collected from fish, fishpond water inlets, tap water, outlet water, and workers at sites of integrated agriculture-aquacultures. Samples were also taken from inhabitants of the aquaculture surrounding areas. All samples were cultured on MacConkey agar, the Enterobacteriaceae isolates were tested for susceptibility to cephalosporins and carbapenems, and screened for blaCTX-M-15, blaSHV, blaOXA-1, blaTEM, blaPER-1, blaKPC, blaOXA-48, and blaNDM. Strains having similar resistance phenotype and genotype were examined for the presence of Incompatible (Inc) plasmids. RESULTS: A major proportion of the Enterobacteriaceae isolates were resistant to cephalosporins and carbapenems. Among the 66 isolates from fish, 34 were resistant to both cephalosporin and carbapenem groups, 26 to carbapenems alone, and 4 to cephalosporins alone. Of the 15 isolates from fishpond water inlets, 8 showed resistance to both groups, 1 to carbapenems alone, and 5 to cephalosporins alone. Out of the 33 isolates from tap water, 17 were resistant to both groups, and 16 to cephalosporins alone. Similarly, of the 16 outlet water isolates, 10 were resistant to both groups, and 6 to cephalosporins alone. Furthermore, of the 30 examined workers, 15 carried Enterobacteriaceae resistant strains, 10 to both groups, and 5 to cephalosporins alone. Similar strains were isolated from the inhabitants of the aquaculture surrounding areas. Irrespective of source of samples, strains resistant to all examined antibiotics, carried predominantly the carbapenemase gene blaKPC either alone or with the ß-lactamase genes (blaCTX-M-15, blaSHV, blaTEM, and blaPER-1). The isolates from fish, water, and workers harboured a wide-range of multi-drug-resistance Inc. plasmids, which were similar among all isolates. CONCLUSION: The present findings suggest transmission of the resistance genes among Enterobacteriaceae strains from different sources. This reiterates the need for control strategies that focus on humans, animals, water, and sewage systems to solve the antibiotic resistance problem.

Isolation and characterization of E. coli strains causing intramammary infections from dairy animals and wild birds.

The study was conducted to estimate the prevalence of Escherichia coli (E. coli) in sub-clinically mastitic (SCM) animals, and in wild and migratory birds which may act as reservoir disseminating such pathogen. Farm hygiene, management and milking procedures were listed through a questionnaire. Thirty lactating cows and 15 lactating buffaloes from five small-scale dairy farms were randomly selected and screened for subclinical mastitis (SCM) using California Mastitis Test (CMT) and somatic cell count (SCC). In addition, 80 teat skin swabs, 5 drinking water samples and 38 wild and migratory bird faecal matter were also collected. All samples were processed for E. coli isolation by culturing on Levine's Eosin Methylene Blue (L-EMB) agar, followed by purification and biochemical identification. Positive samples were subjected to molecular identification and serotyping. In addition, the presence of extended-spectrum beta-lactamase (ESBL) and carbapenemase-producing E. coli have been reported by antimicrobial sensitivity testing. Escherichia coli were isolated from 7.7%, 50% and 50% of the positive CMT cows' quarters, cows' composite and buffaloes' composite milk samples, respectively. In addition, 14% of cows' teats, 20% of water samples, 70% of faecal matter from wild bird, and 33.3% of faecal matter from migratory waterfowls were carrying E. coli. Serotyping, antibiotic-resistant pattern and phylogenetic analysis have pointed the bearable implication of milking hygiene and wild birds in disseminating E. coli strains causing intramammary infections.

A sensitive serodiagnostic tool for the detection of active infection of zoonotic visceral and nasopharyngeal linguatulosis.

AIM: This study aimed to evaluate the different serological techniques for early diagnosis of acute concurrent infections with linguatulosis in the definitive host (dogs) and an intermediate host (goats). This evaluation compared between the gold standard (GS) test (GS; examination of nasal and fecal samples in dogs and examination of lymph nodes in goats), sandwich enzyme-linked immunosorbent assay (S-ELISA), and indirect ELISA. MATERIALS AND METHODS: Fifty goats and fifty dogs were examined for the presence of Linguatula serrata nymphs and adults, respectively, besides the collection of blood samples from the examined animals for serologic testing. RESULTS: In goats; GS, S-ELISA, and indirect ELISA showed positivity in 32 (64%), 28 (56%), and 39 (78%) samples, respectively. In dogs; GS, S-ELISA, and indirect ELISA showed positivity in 25 (50%), 25 (50%), and 30 (60%) samples, respectively. S-ELISA displayed significant higher agreement with the GS test (≥0.83) than indirect ELISA (≤0.67) in both hosts. Infection with linguatulosis showed significant relation with the age of goats and dogs and the sex of goats (p<0.05). CONCLUSION: S-ELISA displayed more sensitivity and specificity for the detection of concurrent infections with linguatulosis in both dogs and goats than indirect ELISA, which could detect the prior infections. Similarly, these assays could be used for diagnosis of concurrent infections with linguatulosis in human, especially the chronic ones.

New Insight of Apparently Healthy Animals as a Potential Reservoir for Clostridium Perfringens: A Public Health Implication.

INTRODUCTION: Clostridium perfringens is commonly found in the gastrointestinal tract of animals and humans and continues to cause one of the most prevalent foodborne diseases in man. MATERIAL AND METHODS: A total of 355 samples were examined for the occurrence of C. perfringens: rectal swabs from cattle, sheep, and goats, fresh stool samples from diarrhoea sufferers having been in contact with these animals, irrigation water and soil samples from the husbandry sites, and pre-harvesting fresh produce from farms irrigated with the sampled water. All samples were collected from Cairo and Giza governorates, Egypt. PCR analysis was carried out with positive isolates using the α-toxin gene. Sequence analysis of the gene of C. perfringens isolates was performed using the neighbour-joining approach. Bootstrap analysis was executed with 1,000 resamplings. RESULTS: 174 C. perfringens strains were isolated with a 49.01% prevalence. The highest prevalence of C. perfringens in apparently healthy animals was found in sheep (65.45%) followed by goats (58%), buffaloes (55%), and cattle (47.1%). Its prevalence in humans being in contact with these animals was 47.5%. The bacterium's isolation from the soil and irrigation water was achieved in 40% and 31.7% of samples, respectively, posing a risk, particularly when the water and soil contact food in the field, shown by the fresh produce isolation of 40%. A significant relationship between the prevalence of C. perfringens in animal and environmental samples was identified (P < 0.05). A significant relationship was identified neither between animal species and C. perfringens prevalence, nor between the environmental source and C. perfringens prevalence (P > 0.05). All isolates were positive for the α-toxin gene by PCR. The sequence analysis and the phylogenetic relationship of the α-toxin genes from different samples revealed that C. perfringens from faeces of apparently healthy cattle, buffaloes, sheep, and goats is a significant threat in places where it can contaminate the soil and water. In addition, the sequence of C. perfringens from humans suffering from diarrhoea was found in the same cluster with the sequence from cows, goats, and sheep. CONCLUSION: The role of apparently healthy animals in transmitting C. perfringens to humans, either through being in direct or indirect contact via water or soil in the cultivation of vegetables and fruits, was demonstrated.