Embryonated Eggs Research Articles

Effects of the In Ovo and Dietary Supplementation of L-Ascorbic Acid on the Growth Performance, Inflammatory Response, and Eye L-Ascorbic Acid Concentrations in Ross 708 Broiler Chickens.

Simple SummaryL-ascorbic acid (L-AA), known as vitamin C, is involved in several metabolic process, including bone formation, antioxidant activity, and inflammatory response. It is well documented that the antioxidant capacity and immunity of broilers are enhanced in response to the dietary or in ovo administration of L-AA. The aim of the current study was to determine the effects the in ovo or dietary administration of L-AA on the post hatch performance, plasma nitric oxide, and eye L-AA concentrations of Ross 708 broilers. At 17 days of incubation, the following treatments were administered: non-injected or sham-injected (100 μL of saline) controls, or the injection of either 12 or 25 mg of L-AA suspended in 100 μL of saline. In addition, chicks were fed a commercial diet with or without 200 mg/kg of supplemental L-AA. The results of the current study revealed that the eye concentrations of L-AA were higher in males in comparison to females. Additionally, 12 mg of L-AA decreased plasma nitric oxide levels in 14-day-old male broilers in comparison to all the other in ovo injection treatment groups. Compared to a commercial diet, dietary L-AA lowered feed conversion ratio during the second week of post hatch growth. In conclusion, the in ovo administration of L-AA was more effective in reducing inflammatory response, whereas the dietary source had a greater impact on the live performance of broilers. Further research is needed to determine the aforementioned effects throughout the entire growing phase.Effects of the dietary and in ovo administration of L-ascorbic acid (L-AA) on the performance, plasma nitric oxide, and eye L-AA concentrations of Ross 708 broilers were investigated. At 17 days of incubation, live embryonated hatching eggs were randomly assigned to a non-injected or sham-injected (100 μL of saline) control group, or a group injected with either 12 or 25 mg of L-AA suspended in 100 μL of saline. Chicks received a commercial diet with or without 200 mg/kg of supplemental L-AA and were randomly assigned to each of 6 replicate floor pens in each in ovo injection-dietary treatment combination. Weekly live performance variables through 14 days of post hatch age (doa) and the eye weights in both sexes at 0, 7, and 14 doa were determined. At 0 and 14 doa, plasma nitric oxide levels and eye L-AA concentrations of one bird of each sex in each pen were determined. Dietary supplemental L-AA decreased feed intake and growth between 0 and 7 doa, but from 8 to 14 doa; all birds fed supplemental L-AA had a lower feed conversion ratio. At 14 doa, male chicks had higher eye L-AA concentrations and lower plasma nitric oxide levels when treated in ovo with 12 mg of L-AA. In conclusion, dietary L-AA may be used to improve feed conversion in the second week of broiler post hatch growth. However, the in ovo administration of 12 mg of L-AA can increase male eye L-AA concentrations and is effective in reducing their general inflammatory response.

57-Year-Old Man With Headache, Vomiting, and Gait Instability

57-Year-Old Man With Headache, Vomiting, and Gait Instability

Genetic Evolution of Avian Influenza A (H9N2) Viruses Isolated from Domestic Poultry in Uganda Reveals Evidence of Mammalian Host Adaptation, Increased Virulence and Reduced Sensitivity to Baloxavir.

A (H9N2) avian influenza A viruses were first detected in Uganda in 2017 and have since established themselves in live bird markets. The aim of this study was to establish the subsequent genetic evolution of H9N2 viruses in Uganda. Cloacal samples collected from live bird market stalls in Kampala from 2017 to 2019 were screened by RT-PCR for influenza A virus and H9N2 viruses were isolated in embryonated eggs. One hundred and fifty H9N2 isolates were subjected to whole genome sequencing on the Illumina MiSeq platform. The sequence data analysis and comparison with contemporary isolates revealed that the virus was first introduced into Uganda in 2014 from ancestors in the Middle East. There has since been an increase in nucleotide substitutions and reassortments among the viruses within and between live bird markets, leading to variations in phylogeny of the different segments, although overall diversity remained low. The isolates had several mutations such as HA-Q226L and NS-I106M that enable mammalian host adaptation, NP-M105V, PB1-D3V, and M1-T215A known for increased virulence/pathogenicity and replication, and PA-E199D, NS-P42S, and M2-S31N that promote drug resistance. The PA-E199D substitution in particular confers resistance to the endonuclease inhibitor Baloxavir acid, which is one of the new anti-influenza drugs. Higher EC50 was observed in isolates with a double F105L+E199D substitution that may suggest a possible synergistic effect. These H9N2 viruses have established an endemic situation in live bird markets in Uganda because of poor biosecurity practices and therefore pose a zoonotic threat. Regular surveillance is necessary to further generate the needed evidence for effective control strategies and to minimize the threats.

Predicting Egg Passage Adaptations to Design Better Vaccines for the H3N2 Influenza Virus.

Seasonal H3N2 influenza evolves rapidly, leading to an extremely poor vaccine efficacy. Substitutions employed during vaccine production using embryonated eggs (i.e., egg passage adaptation) contribute to the poor vaccine efficacy (VE), but the evolutionary mechanism remains elusive. Using an unprecedented number of hemagglutinin sequences (n = 89,853), we found that the fitness landscape of passage adaptation is dominated by pervasive epistasis between two leading residues (186 and 194) and multiple other positions. Convergent evolutionary paths driven by strong epistasis explain most of the variation in VE, which has resulted in extremely poor vaccines for the past decade. Leveraging the unique fitness landscape, we developed a novel machine learning model that can predict egg passage substitutions for any candidate vaccine strain before the passage experiment, providing a unique opportunity for the selection of optimal vaccine viruses. Our study presents one of the most comprehensive characterizations of the fitness landscape of a virus and demonstrates that evolutionary trajectories can be harnessed for improved influenza vaccines.

A New Species of Sprentascaris (Nematoda: Raphidascarididae) in Hypostomus commersoni (Pisces: Loricariidae) from Argentina.

To report a new species: Sprentascaris pampayensis, a Raphidascarididae parasite from the Neotropical region. During a parasitological survey, specimens were collected of a new species of Sprentascaris from Hypostomus commersoni Valenciennes from urban lakes in Santa Fe City, Santa Fe Province, in the Pampean region of Argentina. Morphological and morphometric descriptions, and molecular analyses were performed. The new species, S. pampayensis n. sp., is characterized by having slightly extended lateral wings in both sexes, oral structure with three well-developed lips with double papillae, ventrolateral lips with an amphidial pore. Triangular buccal opening with three well-developed lips with flattened extensions at their bases, which end in a pointed manner towards the back. Males have 17 pairs of preanal papillae, 1 pair of adanal papillae, 3 pairs of postanal papillae and 1odd median postanal papilla located between the first and second pairs, unusual shape of caudal papillae with the central tubular part conspicuously elevated. Females have pre-equatorial vulva, smooth-shelled eggs, and mature embryonated eggs. Sprentascaris pampayensis n. sp. differs morphologically and genetically from S. manherti Petter and Cassone, 1984, S. lanfrediae Melo, Santos, Giese, Santos and Santos, 2011 and S. andersoni Malta, Paiva, Elisei, Tavares and Pereira, 2018. Morphological and morphometric comparison also distinguishes it from S. pimelodi Petter and Cassone, 1984, S. hypostomi Petter and Cassone, 1984, S. marano Ramallo, 2009, and S. saltaensis Ailán-Choke, Ramallo and Davies, 2017. Sprentascaris pampayensis n. sp. is a new parasitic nematode of Hypostomus commersoni.

A copro-antigen ELISA for the detection of ascarid infections in chickens

A reliable method of diagnosing the most prevalent helminth infections in chickens is vital for developing effective control strategies. Ascaridia galli and Heterakisgallinarum are phylogenetically close nematode species that can elicit the development of cross-reactive antibodies in chickens. Therefore, an enzyme-linked immunoassay (ELISA) based on Ascaridia galli antigens in faeces of chickens to detect and quantify infections with both A. galli and H. gallinarum was developed. The ELISA utilised polyclonal antibodies that were obtained from rabbits immunised with soluble antigens isolated from A. galli. In two separate experiments, chickens were kept as uninfected controls or were orally infected with either 100 or 1000 of embryonated eggs of A. galli or H.gallinarum. Faecal samples were collected after 28–30 weeks post-infection. The ELISA was then used to quantify the concentration of soluble worm antigens in faecal samples, i.e., the amount of antigen per gram faeces, APG. The APG from infected chickens was significantly higher than non-infected groups in both experiments (P 0.001). Both 100 and 1000 infection dose groups were not significantly different (P = 0.999) in the experiment with H. gallinarum, whereas in the experiment with A. galli, APG was significantly higher in the 1000 infection group (P 0.001). A receiver operation characteristics (ROC) analysis that evaluates the qualitative performance of diagnostics tests was used to calculate the assay parameters within each mono-infection experiment. The result showed that the assay had a high diagnostics accuracy with an area-under-curve (AUC) of 0.99 in detecting infection in A. galli infected chickens and a moderate-high accuracy (AUC = 0.89) in birds infected with H. gallinarum. The diagnostic sensitivity and specificity of the assay at the optimal cut-off point equivalent to Youden index were 93% and 100% for detecting infections in A. galli experiment and 85% and 92% in H. gallinarum experiment, respectively. The correlation between faecal antigen concentration and all worm burden parameters was positive but generally low (r < 0.33), which provided less information about infection intensities. Nonetheless, these results indicate that a reliable and accurate qualitative diagnosis of the two most prevalent intestinal nematodes in chickens can be achieved using a non-invasive copro-antigen ELISA assay.

Characterization and Immunogenicity of Influenza H7N9 Vaccine Antigens Produced Using a Serum-Free Suspension MDCK Cell-Based Platform.

Human infections with avian-origin H7N9 influenza A viruses were first reported in China, and an approximately 38% human mortality rate was described across six waves from February 2013 to September 2018. Vaccination is one of the most cost-effective ways to reduce morbidity and mortality during influenza epidemics and pandemics. Egg-based platforms for the production of influenza vaccines are labor-intensive and unable to meet the surging demand during pandemics. Therefore, cell culture-based technology is becoming the alternative strategy for producing influenza vaccines. The current influenza H7N9 vaccine virus (NIBRG-268), a reassortant virus from A/Anhui/1/2013 (H7N9) and egg-adapted A/PR/8/34 (H1N1) viruses, could grow efficiently in embryonated eggs but not mammalian cells. Moreover, a freezing-dry formulation of influenza H7N9 vaccines with long-term stability will be desirable for pandemic preparedness, as the occurrence of influenza H7N9 pandemics is not predictable. In this study, we adapted a serum-free anchorage-independent suspension Madin-Darby Canine Kidney (MDCK) cell line for producing influenza H7N9 vaccines and compared the biochemical characteristics and immunogenicity of three influenza H7N9 vaccine antigens produced using the suspension MDCK cell-based platform without freeze-drying (S-WO-H7N9), the suspension MDCK cell-based platform with freeze-drying (S-W-H7N9) or the egg-based platform with freeze-drying (E-W-H7N9). We demonstrated these three vaccine antigens have comparable biochemical characteristics. In addition, these three vaccine antigens induced robust and comparable neutralizing antibody (NT; geometric mean between 1016 and 4064) and hemagglutinin-inhibition antibody (HI; geometric mean between 640 and 1613) titers in mice. In conclusion, the serum-free suspension MDCK cell-derived freeze-dried influenza H7N9 vaccine is highly immunogenic in mice, and clinical development is warranted.

An O-Specific Polysaccharide/Tetanus Toxoid Conjugate Vaccine Induces Protection in Guinea Pigs against Virulent Challenge with Coxiella burnetii

Q fever is caused by the bacterium Coxiella burnetii and is spread to humans from infected animals especially goats, sheep and cattle, predominantly when giving birth. There is an effective human vaccine (Q-VAX) against Q fever, and although Q fever is a worldwide problem, the vaccine is only used in Australia due to difficulties associated with its use and the risk of adverse reactions. The desire to protect humans, particularly farmers and abattoir workers, from Q fever prompted the development of a new safe and effective human vaccine without all the difficulties associated with the current vaccine. Candidate vaccines were prepared using purified O-specific polysaccharide (OSP) extracted from the lipopolysaccharide of virulent (phase 1) C. burnetii, strain Nine Mile, which was then conjugated to a tetanus toxoid (TT) carrier protein. Two vaccines were prepared using OSP from C. burnetii grown in embryonated eggs (vaccine A) and axenic media (vaccine B). Vaccines with or without alum adjuvant were used to vaccinate guinea pigs, which were later challenged by intranasal inoculation with virulent C. burnetii. Both vaccines protected guinea pigs from fever and loss of weight post challenge. Post-mortem samples of the spleen, liver and kidney of vaccinated guinea pigs contained substantially less C. burnetii DNA as measured by PCR than those of the unvaccinated control animals. This study demonstrated that a C. burnetii OSP-TT conjugate vaccine is capable of inducing protection against virulent C. burnetii in guinea pigs. Additionally, OSP derived from C. burnetii grown in axenic media compared to OSP from embryonated eggs is equivalent in terms of providing a protective immune response.

Analysis of Cathepsin-L and Cathepsin-B Gene Expression in Embryonated Eggs of Fasciola gigantica and Effect of Various Protease Inhibitors on Miracidial Hatching

Background: Fasciola gigantica is a helminth parasite of veterinary importance and a constraint on the growth and productivity of domestic livestock in the tropical countries of Asia and Africa. Present study aimed at determining the expression of four cysteine protease genes including cathepsin (cat) L-1 D and cat-Bs in the embryonated egg stage of F. gigantica by polymerase chain reaction (PCR). In addition to the analysis of expression of the above genes in the embryonated eggs, inhibition of in vitro hatching of the miracidia from the eggs was carried out using three protease inhibitors viz. serine, cysteine and leucine aminopeptidase inhibitors to understand the possible role of these proteases in the hatching of the miracidia from the eggs. Methods: Cat- L-1D, cat B-1, cat B-2 and cat B-3 genes were amplified by reverse transcriptase and quantitative-PCR, respectively for determining their expression in the embryonated eggs. Inhibition of in vitro hatching of the miracidia from the eggs was carried out by exposing the embryonated eggs to different concentrations of iodoacetamide, phenyl methyl sulphonyl fluoride and bestatin as cysteine, serine and leucine aminopeptidase inhibitors. Result: Results showed that cat L1-D, cat B-1 and cat B-3 genes were expressed at the embryonated egg stage but cat B-2 gene was not expressed at this stage of the parasite. Iodoacetamide and phenyl methyl sulphonyl fluoride (cysteine and serine protease inhibitors) inhibited the miracidial hatching at different concentrations, indicating probable role of cysteine and serine proteases in its hatching process. However, leucine aminopeptoidase does not seem to influence the miracidial hatching from the eggs as bestatin did not inhibit its hatching.

A Temperature-Sensitive Recombinant of Avian Coronavirus Infectious Bronchitis Virus Provides Complete Protection against Homologous Challenge.

ABSTRACTAvian coronavirus infectious bronchitis virus (IBV) is the etiological agent of infectious bronchitis, an acute highly contagious economically relevant respiratory disease of poultry. Vaccination is used to control IBV infections, with live-attenuated vaccines generated via serial passage of a virulent field isolate through embryonated hens’ eggs. A fine balance must be achieved between attenuation and the retention of immunogenicity. The exact molecular mechanism of attenuation is unknown, and vaccines produced in this manner present a risk of reversion to virulence as few consensus level changes are acquired. Our previous research resulted in the generation of a recombinant IBV (rIBV) known as M41-R, based on a pathogenic strain M41-CK. M41-R was attenuated in vivo by two amino acid changes, Nsp10-Pro85Leu and Nsp14-Val393Leu; however, the mechanism of attenuation was not determined. Pro85 and Val393 were found to be conserved among not only IBV strains but members of the wider coronavirus family. This study demonstrates that the same changes are associated with a temperature-sensitive (ts) replication phenotype at 41°C in vitro, suggesting that the two phenotypes may be linked. Vaccination of specific-pathogen-free chickens with M41-R induced 100% protection against clinical disease, tracheal ciliary damage, and challenge virus replication following homologous challenge with virulent M41-CK. Temperature sensitivity has been used to rationally attenuate other viral pathogens, including influenza, and the identification of amino acid changes that impart both a ts and an attenuated phenotype may therefore offer an avenue for future coronavirus vaccine development.IMPORTANCE Infectious bronchitis virus is a pathogen of economic and welfare concern for the global poultry industry. Live-attenuated vaccines against are generated by serial passage of a virulent isolate in embryonated eggs until attenuation is achieved. The exact mechanisms of attenuation are unknown, and vaccines produced have a risk of reversion to virulence. Reverse genetics provides a method to generate vaccines that are rationally attenuated and are more stable with respect to back selection due to their clonal origin. Genetic populations resulting from molecular clones are more homogeneous and lack the presence of parental pathogenic viruses, which generation by multiple passage does not. In this study, we identified two amino acids that impart a temperature-sensitive replication phenotype. Immunogenicity is retained and vaccination results in 100% protection against homologous challenge. Temperature sensitivity, used for the development of vaccines against other viruses, presents a method for the development of coronavirus vaccines.

Modulatory Effects of Arctostaphylos uva-urs Extract In Ovo Injected into Broiler Embryos Contaminated by Aflatoxin B1.

Simple SummaryThe poultry industry is of great importance worldwide; however, mycotoxins contamination, especially aflatoxins, has harmful effects on poultry production. Therefore, finding out new strategies to alleviate aflatoxin contamination is an important trend of research. In our study, we focused on the in ovo injection techniques using the methanolic extract of brae beery “Arctostaphylos uva-ursi” (Ar. uu) to modulate the aflatoxinB1 (AFB1) adverse effects in broiler embryos. As a result, 0.1 g of Ar. uu in ovo injection alleviates AFB1 embryotoxicity by enhancing chicks’ physiological responses and optimizing antioxidant status, which is reflected in a low mortality rate and heavier relative weight of the hatched chicks. The in ovo injection of AFB1 + Ar. uu significantly declined AFB1-induced toxicity in embryos and increased broiler chicks embryo’s survival by 62.5%, increased relative embryo weight by ∼65.25% compared to eggs injected with AFB1 alone. Regarding immune responses, the in ovo injection of Ar. uu enhances the embryo’s humoral immune responses and regulates oxidative stress biomarkers. In general, the in ovo injection of Ar. uu modulates broiler chicks’ embryotoxicity caused by AFB1.In ovo injection of nutrients can modulate the embryo’s physiological responses against aflatoxin B1 (AFB1) embryotoxicity. This hypothesis was tested using in ovo injection of Arctostaphylos uva-ursi (Ar. uu.) methanolic extract. The total polyphenols, total flavonoids, total antioxidant capacity, and GC-MS analysis were all assessed in the Ar. uu. methanolic extract. A total of 180 ten-day-old embryonated eggs were distributed into six groups of 30 replicates each. The first group was used as a control (non-injected), and the second, third, fourth, fifth, and sixth groups were injected with 10 µ double-distilled water (DDW), 500 µL methanol, 0.01 g Ar. uu./500 µL methanol, 50 ng AFB1/10 µL DDW, and 50 ng AFB1 in 10 µ DDW + 0.01 g Ar. uu./500 µL methanol, respectively. The relative embryo weight, residual yolk sac weight, tibia length and weight, and survival were recorded. Total and differential leukocytes, oxidative stress, and humoral immune responses were observed. The residual yolk sac was lower (p < 0.05) in the Ar. uu. group than other groups. The embryonic growth (tibia weight and length) was enhanced in AFB1 + Ar. uu.-injected embryos compared with those injected with AFB1 alone. In conclusion, in ovo injection of Arctostaphylos uva-ursi could modulate AFB1-induced toxicity in chicken embryos.

Enhancement of protective efficacy of innate immunostimulant based formulations against yolk sac infection in young chicks

This study was conducted to characterize and compare the protective effects of various innate immune stimulants against yolk sac infection (YSI) caused by an avian pathogenic Escherichia coli in young chicks. The immune stimulants were administered alone or in various combinations of unmethylated CpG oligodeoxynucleotides (CpG), polyinosinic:polycytidylic acid (Poly I:C), and avian antimicrobial peptides (AMPs). Routes included in ovo or in ovo followed by a subcutaneous (S/C) injection. CpG alone and in combination with Poly I:C, truncated avian cathelicidin (CATH)-1(6-26), avian beta defensin (AvBD)1, and CATH-1(6-26) + AvBD1, were administered in ovo to 18-day-old embryonated eggs for gene expression and challenge studies. Next, CpG alone and the potentially effective formulation of CpG + Poly I:C, were administrated via the in ovo route using 40 embryonated eggs. At 1 day post-hatch, half of each group also received their respective treatments via the S/C route. Four hours later, all chicks were challenged using E. coli strain EC317 and mortalities were recorded for 14 d. The first challenge study revealed that amongst the single use and combinations of CpG with different innate immune stimulants, a higher protection and a lower clinical score were offered by the combination of CpG + Poly I:C. The second challenge study showed that this combination (CpG + Poly I:C) provides an even higher level of protection when a second dose is administered via the S/C route at 1 day post-hatch. The current research highlights the efficacy of a combination of CpG + Poly I:C administered either in ovo or in ovo along with a S/C injection and its potential use as an alternative to antibiotics against yolk sac infection in young chicks.

Apigenin inhibits infectious bronchitis virus replication in ovo.

Infectious bronchitis virus (IBV), for which no effective drugs are available, is among the most important causes of economic loss within the poultry industry. Apigenin is a flavonoid that can be isolated from plants. Apigenin has low toxicity with anti-viral activity. However, the effects of apigenin against IBV remain unclear. Thus, here we investigate the anti-viral effect of apigenin on IBV using 10 day-old embryonated eggs by determining the virus titer by embryo infective doses50 (EID50/mL) and determining IBV genomes copy number (per µL) of allantoic fluid. We found that apigenin protected embryonated eggs from IBV. Additionally, apigenin reduced the log titer of the IBV with a significant correlation of up to 9.4 times at 2 µg/egg. Also, apigenin appears to significantly reduce IBV genomes copy number (per µL) in the allantoic fluid. Apigenin may be a promising approach for the treatment of IBV, since it protects embryonated eggs from IBV in ovo and suppresses viral replication.

Potency of bacterial sialidase Clostridium perfringens as antiviral of Newcastle disease infections using embryonated chicken egg in ovo model.

Background and Aim:Clostridium toxins are widely used as medicinal agents. Many active metabolic enzymes, including sialidase (neuraminidase), hyaluronidase, and collagenase, contribute to the mechanism of action of these toxins. Sialidase from Clostridium perfringens recognizes and degrades sialic acid receptors in the host cell glycoprotein, glycolipid, and polysaccharide complexes. Sialic acid promotes the adhesion of various pathogens, including viruses, under pathological conditions. This study aimed to investigate the potential of C. perfringens sialidase protein to inhibit Newcastle disease virus (NDV) infection in ovo model.Materials and Methods:C. perfringens was characterized by molecular identification through polymerase chain reaction (PCR) and is cultured in a broth medium to produce sialidase. In addition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was conducted to characterize the sialidase protein. In contrast, enzymatic activity and protein concentration were carried out using a neuraminidase assay kit and Bradford to obtain suitable active substances. Furthermore, embryonated chicken egg models were used to observe the toxicity of several sialidase doses. Then, the hemagglutination (HA) titer was obtained, and absolute quantitative reverse transcription–PCR assay was performed to measure the viral replication inhibitory activity of sialidase against NDV.Results:Each isolate had a specific sialidase gene and its product. The sialidase derived from C. perfringens could hydrolyze the sialic acid receptor Neu5Ac (2,6)-Gal higher than Neu5Ac (2,3)Gal in chicken erythrocytes, as observed by enzyme-linked lectin assay. A significant difference (p = 0.05) in the HA titer in the pre-challenge administration group at dosages of 375 mU, 187.5 mU, and 93.75 mU in the competitive inhibition experiment suggests that sialidase inhibits NDV reproduction. Quantification of infective viral copy confirmed the interference of viral replication in the pre-challenge administration group, with a significant difference (p = 0.05) at the treatment doses of 750 mU, 375 mU, and 46.87 mU.Conclusion:The potency of sialidase obtained from C. perfringens was shown in this study, given its ability to reduce the viral titer and copy number in allantoic fluids without adversely impacting the toxicity of the chicken embryo at different concentrations.

Protocatechuic acid: A novel detoxication agent of fumonisin B1 for poultry industry.

Fumonisin B1 (FB1) is a major fusarium mycotoxin that largely contaminates feedstuffs and foods, posing a health risk to both animals and humans. This mycotoxin can enter the human body directly through contaminated food consumption or indirectly by toxins and their metabolites. In a prior study, feed-borne FB1 is one of the leading mycotoxins in breeder eggs, leading to reduced hatchability and gizzard ulceration in chicken progenies. Currently, no effective way is available to remove FB1 from feeds and human-contaminated foods. We hypothesize that FB1 can be reduced to low risk by protocatechuic acid (PCA). To assess the ability of FB1 to be degraded in vivo, 1 ppm of FB1 was treated with PCA, or D-glucose, or silymarin, or anti-FB1 monoclonal antibody. Our study revealed that both D-glucose and PCA exhibited 53.4 and 71.43% degradation, respectively, at 80°C for 2 h, while 35.15% of FB1 detoxification was determined in the silymarin group at 60°C for 0.5 h. A dose-dependent manner was found after treatment with D-glucose or PCA at 80°C for 2 h. As for detoxification of anti-FB1 monoclonal antibody, the 1:3,000 dilution induced significant FB1 detoxification, accounting for 25.9% degradation at 25°C for 2 h. Furthermore, 50 SPF 11-day-old embryonated eggs were divided into 10 groups, with five eggs per group. Post treatment with PCA or D-glucose, or silymarin or anti-FB1 monoclonal antibody, the treated samples were inoculated into albumens and monitored daily until the hatching day. Consequently, 100% of the chickens survived in the D-glucose group and other control groups, except for the FB1 control group, while 80, 80, and 60% hatching rates were found in the PCA-treated group, the anti-FB1 monoclonal antibody-treated group, and the silymarin-treated group. Additionally, both the FB1 group and the silymarin-treated group yielded lower embryo growth than other groups did. Postmortem, lower gizzard ulceration index was determined in the PCA-treated group and the anti-FB1 monoclonal antibody-treated group compared to those of the silymarin-treated group and D-glucose-treated group. Based on the above evidence, PCA is a promising detoxification to reduce FB1 contamination in the poultry industry, contributing to the eradication of mycotoxin residuals in the food chain and maintaining food security for human beings.

Genomic Characterization of CIAV Detected in Contaminated Attenuated NDV Vaccine: Epidemiological Evidence of Source and Vertical Transmission From SPF Chicken Embryos in China.

Live attenuated vaccines have been extensively used to prevent infectious disease in poultry flocks. Freedom from exogenous virus is a high priority for any veterinary vaccines. Recently, attenuated Newcastle disease virus (NDV) vaccines were detected to be contaminated with chicken infectious anemia virus (CIAV) in a routine screening for exogenous viruses. To investigate the possible source of the contamination, we conducted virological tests on a specific-pathogen-free (SPF) layer breeder flock that provide the raw materials for vaccines in this manufacturer. Firstly, CIAV antibodies in serum and egg yolks samples of the SPF laying hens were detected by ELISA assays. The results showed that CIAV antibodies in serum and egg yolks were 62% positive and 57% positive, respectively. Then, DNA was extracted from the NDV vaccines and SPF chicken embryonated eggs, and detected by molecular virology assays. The results showed that three assays for pathogens in embryonated eggs had similar positive rates (35.8%). And the sequences of CIAV from SPF embryos and NDV vaccines consisted of 2,298 nucleotides (nt) with 100% homology. The new full-length genome of CIAV was designated SDSPF2020 (Genbank accession number: MW660821). Data showed SDSPF2020 had the sequence similarities of 95.8–99.6% with reference strains, and shared the highest homology with the Chinese strain HLJ15125. These results strongly suggested that exogenous CIAV contamination is most likely caused by wild virus infection in SPF flocks and vertical transmission to chicken embryos. Collectively, this study illustrated that vertical transmission of CIAV from a SPF layer breeder flock to embryos was a non-neglible way for exogenous virus contamination in vaccine production.

Rare presentations of ocular enterobiasis - Case reports.

Enterobius vermicularis, also known as pinworm or threadworm, is a large intestinal nematode which has a high prevalence among children and peripubertal age in our country. Transmission usually occurs by autoinfection like finger contamination of the embryonated eggs deposited by the gravid female worm on the perianal and perineal region. Globally, only a few reports are there regarding the isolation of the parasite from extra-intestinal sites. These are two rare case reports of ocular enterobiasis. The first case was a middle-aged female and the second one was a 14-year-old girl, both of whom were referred from other tertiary care hospitals to Calcutta School of Tropical Medicine and who presented with discharge of live motile worms from their eyes (conjunctiva). In both the cases, identification was done by saline wet mount and direct microscopy of a gravid female worm. Plano-convex embryonated eggs were also observed. The oval embryonated eggs, plano-convex in shape, and the gravid female, with its cervical alae near the anterior end and straight thin pointed tail, were identified under the microscope. Although E. vermicularis is a very common large intestinal parasitic infestation of children and adolescents, it can also rarely be isolated from unusual sites, which should be taken into account for effective diagnosis and treatment.

NDV as an Oncolytic Agent - Study in Cancer Cell Lines

Newcastle disease virus (NDV) exhibits oncolysis in its natural form. This oncolytic virus (OV) has the potential to specifically infect, propagate, and lyse cancer cells while sparing the normal cells. This study was aimed to screen for oncolytic NDV strain isolated from poultry. A total of ten velogenic NDV strains were propagated in 10 day old embryonated SPF chicken eggs and allantoic fluid of these infected eggs was collected for further study. The virus enumeration was carried out by hemagglutination assay (HA) and end point dilution method. The cytopathic effect of ten NDV strains on cancer cell lines like MDA-MB-231, MCF-7, PC3, and A549 along with normal control cell line HEK293 was determined by MTT assay 72 hours post infection. These cell lines were infected with three doses (1, 0.1, and 0.01 MOI). DNA laddering effect of the screened NDV isolate was studied after infecting all cancer and normal cells at MOI 1. Morphological changes in MDA-MB-231 on infection with the screened NDV isolate were analyzed using H&amp;E hematoxylin and eosin staining. The screened NDV isolate showed the maximum cytopathic effect i.e. 61.55% on MDA-MB-231 at MOI 1 but had no potent cytotoxic effect on HEK293. DNA laddering effect was observed which confirmed the mode of death to be apoptosis. All the observed morphological changes in MDA-MB-231 were typical of the cytopathogenic effects of NDV on cancer cell lines. In conclusion, the screened oncolytic NDV shows effective oncolysis against MDA-MB-231 cell line. However, further study is required to determine the exact mode of action involved.

Feed Composition and Isolate of Histomonas meleagridis Alter Horizontal Transmission of Histomonosis in Turkeys. Proof of Concept

Outbreaks of histomonosis in turkeys are typically initiated by the ingestion of contaminated embryonated eggs of Heterakis gallinarum, potentially present in earthworms and mechanical vectors. Once an outbreak is started, infected turkeys can transmit the disease by horizontal transmission. Factors influencing horizontal transmission of histomonosis are poorly understood. Replication of horizontal transmission in experimental conditions has not been consistent, presenting an obstacle in searching for alternatives to prevent or treat the disease. Two pilot experiments and three validation experiments were conducted in the present study. In pilot experiment 1, one isolate of Histomonas meleagridis (named Buford) was used. Turkeys were fed a low-nutrient density diet corn-soy based (LOW-CS) and raised in floor pens. In pilot experiment 2, another isolate of H. meleagridis was used (named PHL). Turkeys were fed a low-nutrient density diet with the addition of wheat middlings (LOW-WM) and raised in floor pens. In experiment 3, conducted on floor pens, both isolates and diets were used in different groups. In experiment 4, turkeys were raised on battery cages and only the PHL isolate was used. Both diets (LOW-WM and LOW-CS) were used, in addition to a diet surpassing the nutritional needs of young poults (turkey starter, TS). In experiment 5, conducted in battery cages, only the PHL isolate was used, and the LOW-WM and TS diets were in different groups. The horizontal transmission was achieved only with the PHL isolate from all experiments. The transmission rate varied among experimental diets, with the TS diet having the lowest transmission rate in experiments 4 and 5. Variation was observed between experiments and within experimental groups.

Asymptomatic isolated intramuscular cysticercosis in diabetic patient: a case report

Cysticercosis is an endemic disease in multiple areas of the world, caused by ingestion of embryonated eggs of Taenia solium. We reported a case of a 66-year old diabetic male presenting with decrease of consciousness and “rice grain” calcification in both hemithorax and neck soft tissue region, without findings of cysticercosis in computed tomography (CT) scan. Cysticercosis most commonly affects the central nervous system, causing neurocysticercosis (NCC), while solitary intramuscular cysticercosis without involvement of the central nervous system is rare. Treatment of cysticercosis is indicated for symptomatic patients, including NCC surgery is indicated in patients with neurovascular compromise caused by growth of cyst. Recognizing the signs and symptoms of cysticercosis is crucial for diagnosis and treatment in endemic areas where Taenia solium is prevalent.