Informasi Terseleksi No. 7 Perpustakaan Balai Besar

INFORMASI TERSELEKSI NO. 7

PERPUSTAKAAN

BALAI BESAR PENELITIAN VETERINER

2019

(Bakteriologi, Parasitologi, Toksikologi, Virologi)

Bakteriologi 1 Rodríguez-lópez, P., Bernárdez, M., Rodríguez-herrera, J. J., Comesaña, Á. S., & Cabo, M. L. (2019). Identification and metagenetic characterisation of Listeria monocytogenes - harbouring communities present in food-related industrial environments. Food Control, 95(July 2018), 6–17. https://doi.org/10.1016/j.foodcont.2018.07.023 Abstract The main aim of this study was to localise, identify and characterise the Listeria monocytogenes- harbouring bacterial communities present in food related premises via 16S rRNA gene metagenetic analysis. With this scope, 319 environmental samples coming from a wide variety of surfaces of fish (n=120), meat (n=80) and dairy industries (n=119), were firstly analysed following ISO 11290–1 and ISO 11290–2 norms. Direct L. mono-cytogenes quantification was possible in 9 samples (2.8%) with values between 2.00 and 3.97 log CFU/cm2. After enrichment, an overall L. monocytogenes incidence of 12.54% (n=40) was obtained, being samples from meat industry the most contaminated. Molecular serotyping assays showed that most of the isolates belonged to 1/2b- 3b-7 subgroup, followed by 1/2a-3a and 1/2c-3c. These results combined with AscI and ApaI PFGE macro-restriction patterns, yielded 7 different L. monocytogenes clusters. Nevertheless, no clear ecological relationships could be stablished. High amounts of L. monocytogenes-associated psychrotrophic microbiota were obtained in all cases with values above 9 log CFU/cm2 in some cases. Metagenetic analysis of one representative sample per each food industry type (fish, meat, dairy) demonstrated that Actinobacteria (53.16%) was mostly present in the meat sample whereas Proteobacteria was the most representative phylum in dairy (69.58%) and fish (97.11%) samples. Subsequent operational taxonomic units (OTUs) analysis, showed a wide variety of taxa associated with L. monocytogenes such as spoilage-associated genera (e.g. Psyschromonas or Shewanella), lactic acid bacteria genera (e.g. Lactococcus or Lactobacillus) or pathogenic species such as Yersinia enterocolitica. It was thus demonstrated, that L. monocytogenes is capable to both survive with different bacteria in different ecological niches, highlighting once more the need for proper surveillance schedules so as to guarantee the safety of the food products.

2 Hudson, J. C., Tolen, T. N., Kirsch, K. R., Acuff, G., Taylor, T. M., Lucia, L. M., & Castillo, A. (2019). Comparison of Antimicrobial Treatments Applied via Conventional or Handheld Electrostatic Spray To Reduce Shiga Toxin – Producing Escherichia coli on Chilled Beef Outside Rounds, 82(5), 862–868. https://doi.org/10.4315/0362-028X.JFP-18-399 Abstract The purpose of this study was to compare the efficacy of different antimicrobial interventions applied via either conventional spray (CS) or handheld electrostatic spray (ESS) to reduce Shiga toxin–producing Escherichia coli (STEC) on fresh beef surfaces. Hot-boned outside rounds (ORs) were inoculated within 1 h after harvest with a cocktail of eight isolates consisting of 8 O157 and non-O157 serogroups of STEC (STEC8). ORs were hung on sterile meat hooks at 40C for 36 h to simulate a contaminated full carcass side in the chiller. ORs were then treated with lactic acid (LA; 4.5%, w/v), 3.0% lauric arginate ester

(LAE), 0.8% cetylpyridinium chloride, 200 mg/L peracetic acid, 3 mg/L chlorine dioxide, 5 mg/L ClO2, or tap water by using CS or ESS. Temperatures of LA and peracetic acid were set at 55 and 420C before spraying, whereas all other solutions were applied at room temperature (250C). Pretreatment and posttreatment STEC8-inoculated beef tissue samples were aseptically collected to evaluate the efficacy of interventions by application method (CS or ESS). LA applied with CS achieved the greatest reduction in STEC8 numbers (3.3 log CFU/cm2) compared with all other treatments: 0.2 log CFU/cm2 (tap water) to 2.3 log CFU/cm2 (LAE). Only for LA did a significant difference arise in mean STEC8 reductions between CS and ESS applications (3.2 versus 1.7 log CFU/cm2, respectively). Among the treatments applied with ESS, LAE produced the greatest reduction of STEC8. Antimicrobial interventions applied via conventional wand or cabinet-applied technologies can reduce the O157 and non-O157 STEC on fresh beef carcass surfaces, reducing transmission to beef consumers.

3 Donaghy, J. A., Jagadeesan, B., Goodburn, K., Grunwald, L., Jensen, O. V. E. N., Jespers, A. D., … Quentin, M. (2019). Relationship of Sanitizers , Disinfectants , and Cleaning Agents with Antimicrobial Resistance, Journal ofFood Protection, Vol. 82, No. 5, 2019, Pages 889–902. https://doi.org/10.4315/0362-028X.JFP-18-373 Abstract Sanitizers, disinfectants, and cleaning agents are vital to food hygiene assurance and are a major public health protection measure. Limiting microbial antibiotic resistance is also a global public health priority. Although many factors contribute to the rise in antimicrobial resistance in bacteria infecting humans, antibiotic use in both human clinical settings and for food- producing animals are primary contributors. Some concerns have been raised about the possibility of coselection between food hygiene chemicals and reduced antimicrobial susceptibility. This article reviews available evidence from individual studies purporting to demonstrate a possible risk of antimicrobial resistance development, following biocide usage. Furthermore, the conclusions of several key expert reports and meta-analysis publications were assessed for supportive evidence of a relationship between biocide usage in food production and resistance development. Although many studies report on the isolation of antimicrobial-resistant bacterial strains in food, evidence is lacking on the attribution of this resistance to biocide usage. Also, although a theoretical risk of causality exists, many of the studies performed to demonstrate this are in vitro studies using laboratory-grown or -trained bacterial isolates, challenged with sublethal (below the recommended food industry) disinfectant or sanitizing agent concentrations. The proper use of, and adherence to biocide manufacturer‟s instruction for use, and the avoidance of biocide active agent dilution (e.g., through biofilm presence) must be ensured in food production environments. It is recommended that in situ studies should be performed to further assess causality, ensured a clear differentiation between interpretation of stable antimicrobial resistance and phenotypic adaptation. Furthermore, authorization of new biocidal active substances should take a scientific and risk-based approach regarding the potential for driving microbial resistance.

4 Interest, G. (2019). Response to Questions Posed by the Food and Drug Administration Regarding Virulence Factors and Attributes that Define Foodborne Shiga Toxin – Producing Escherichia coli (STEC) as Severe Human Pathogens, Journal of Food Protection, Vol. 82, No. 5, 2019, Pages 724–767. https://doi.org/10.4315/0362-028X.JFP-18-479

5 Ateya, A. I., & Arafat, N. (2019). Intestinal gene expressions in broiler chickens infected with Escherichia coli and dietary supplemented with probiotic, acidifier and symbiotic. Veterinary Research Communications (2019) 43:131–142. https://doi.org/10.1007/s11259-019-09753-z Abstract In this study, we investigated the effects of probiotic, acidifier and synbiotic supplementation on growth performance, mortality rate, intestinal gene expressions, fecal shedding, and organs colonization induced by Escherichia coli in broiler chickens. Six experimental groups were included; negative control group (NC), positive control group (PC), probiotic group (PR), acidifier group (AC), synbiotic group (SY) and colistin sulfate group (CS). Chickens in groups NC and PC were fed a basal diet, while chickens in groups PR, AC, SY, and CS were fed a basal diet containing probiotic, acidifier, synbiotic and colistin sulfate, respectively from the 1st day to the 28th day of age. At 7 days of age, all groups (not NC) were orally challenged with 0.5 ml (1.0 × 109 CFU/ml) E. coli O78. The dietary supplementation of acidifier and synbiotic were sufficient to quell the devastating effects of E. coli infection in broilers. Growth performances represented by body weight gain, feed intake and feed conversion ratio were significantly improved as well as, mortalities were prevented whilst the ileal pro-inflammatory gene expressions (IL-6, IL-8, IL-13, TLR-4, IFN-γ, LITAF, AvBD-2, and AvBD-9) were significantly downregulated and the anti-inflammatory cyto-kine (IL-10) was significantly increased. In addition, E. coli fecal shedding and organs colonization was significantly diminished. It was concluded that the addition of both acidifier and synbiotic to the diet of broilers infected with E. coli could modulate the intestinal inflammatory responses induced by E. coli infection and minimized the inflammation-induced damage which resulted in improvement in growth performance, prevention of mortalities and reduction of E. coli environmental contamination.

6 Kumar, S., Kumar, S., Singh, R. V., Chauhan, A., Kumar, A., Sulabh, S., & Bharati, J. (2019). Genetic association of polymorphisms in bovine TLR2 and TLR4 genes with Mycobacterium avium subspecies paratuberculosis infection in Indian cattle population. Veterinary Research Communications (2019) 43:105–114. https://doi.org/10.1007/s11259-019-09750-2 Abstract Toll like receptors (TLRs) are pattern recognition molecules involved in cellular recognition of Mycobacterium avium subspecies paratuberculosis (MAP), the infectious agent causing Paratuberculosis (PTB), a notified disease of domestic and wild ruminants. The present study was undertaken to investigate the presence of single nucleotide polymorphisms (SNPs) in TLR2 and TLR4 gene and to evaluate association of these SNPs with occurrence ofPTB in Indian cattle. A total of213 cattle, were subjected to multiple diagnostic tests viz. John in PPD, ELISA test (Indigenous and Parachek kit method), fecal microscopy and fecal culture for detection of MAP infection. Based on screening results 51 animals each were assigned to case and control population. Two SNPs viz. rs55617172, rs41830058 in TLR2 gene and two SNPs viz. rs8193046, rs8193060 in TLR4 gene and were genotyped by PCR-RFLP method. All SNPs were found to be polymorphic except rs41830058 in the case-control population. Both SNPs in TLR4 gene but none in TLR2 genes were significantly associated with the occurrence of PTB in our population. The genotypes in SNP rs8193046 and SNP rs8193060 were significantly (P < 0.01) different in case- control population. These findings suggest that SNPs rs8193046 and rs8193060 are likely a potential marker against MAP infection and a selection programme eliminating AG genotype for rs8193046 and CT genotype for rs8193060 might be beneficial in conferring resistance to MAP infection in Indian cattle population.

7 Pereira, F., Xavier, S., Prieto, F., Rafael, B., Rezende, R. De, Marcus, P., … Alfenas, P. (2019). Biological and molecular characterization of a bacteriophage infecting Xanthomonas campestris pv. campestris, isolated from brassica fields. Archives of Virology, 164(7), 1857–1862. https://doi.org/10.1007/s00705-019-04263-4 Abstract Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot of crucifers. Here, we report a virus that infects Xcc isolated from brassica fields in Brazil. Morphological, molecular and phylogenetic analysis indicated that the isolated virus is a new member of the genus Pbunavirus, family Myoviridae, and we propose the name “Xanthomonas virus XC 2” for this virus. The isolated virus has a narrow host range, infecting only Xcc isolates, and it did not infect unrelated bacteria. These results indicate that the isolated bacteriophage is highly specific for Xcc and may be a potential agent for biological control.

8 Moore, R. J., Scott, P. C., Van, T. T. H., Moore, R. J., Scott, P. C., Spotlight, T. T. H. Van, & Moore, R. J. (2019). Spotlight on avian pathology: Campylobacter hepaticus , the cause of Spotty Liver Disease in layers Liver Disease in layers, 9457. https://doi.org/10.1080/03079457.2019.1602247 Abstract Campylobacter hepaticus was recently identified as the aetiological agent of Spotty Liver Disease (SLD). SLD causes significant health and productivity losses in the Australian egg industry and the disease is present in other countries. Following the isolation and characterization of C. hepaticus, molecular tools and refined culturing methods have been developed to identify the pathogen. It is suspected that the application of these tools will lead to identification of the pathogen in many poultry production systems throughout the world. As C. hepaticus has only recently been identified, little is known about the mechanisms of pathogenesis and, hence, new research needs to be directed towards understanding SLD epidemiology and C. hepaticus virulence mechanisms to inform efforts to develop intervention strategies.

9 Yin, L., Li, Q., Xue, M., Wang, Z., Tu, J., Song, X., … Xue, T. (2019). The role of the phoP transcriptional regulator on biofilm formation of avian pathogenic Escherichia coli The role of the phoP transcriptional regulator on biofilm formation of avian, 9457. https://doi.org/10.1080/03079457.2019.1605147 Abstract PhoP plays an important role as a transcriptional regulator in the two-component phoP/phoQ regulatory system, which is widely present in Gram-negative bacteria. In this study, we used DNA microarray-based technology to evaluate the role of phoP in biofilm formation in avian pathogenic Escherichia coli (APEC). Differences in gene transcription between APEC wild-type and phoP mutant strains were determined. Mutation of the phoP transcriptional regulator affects the expression profile of genes involved in processes such as flagellar assembly, ABC transporters, quorum sensing, and bacterial chemotaxis. Deletion of phoP in APEC reduced biofilm formation, as indicated by crystal violet staining and scanning electron microscopy (SEM). In addition, the phoP gene was found to be associated with changes in bacterial drug resistance and cell-membrane-related properties. This study shows that phoP plays an important regulatory role in APEC biofilm formation, and provides new insights into strategies for preventing and controlling APEC infection.

10 Chaplot, S., Yadav, B., & Jeon, B. (2019). Atmospheric Cold Plasma and Peracetic Acid – Based Hurdle Intervention To Reduce Salmonella on Raw Poultry Meat, 82(5), 878–888. https://doi.org/10.4315/0362-028X.JFP-18-377 Abstract In Canada, Salmonella-related foodborne illness accounts for more than 88,000 cases annually. Poultry products represent one of the major vectors for the zoonotic transmission of Salmonella. The majority of the current disinfection strategies that are applied in the poultry industry involve the use of diverse chemical antimicrobial agents; however, knowledge about the efficacy of novel antimicrobial technologies such as atmospheric cold plasma (ACP) and the potential of hurdle interventions is very limited. The objective of this study was to evaluate the synergetic effect of ACP and peracetic acid (PAA) as a hurdle antimicrobial intervention to reduce Salmonella enterica Typhimurium on raw poultry meat. Raw poultry meat samples were inoculated with Salmonella Typhimurium followed by the application of different treatments consisting of ACP and PAA (100 and 200 ppm) alone as well as in combination. Different hurdle interventions using PAA and ACP treatments resulted in treatments alone with 100 or 200 ppm or ACP treatment alone, resulting in the reduction of Salmonella populations by 0.6, 1.3, significant (P ≤ 0.05) reductions in Salmonella Typhimurium, ranging from 2.3 to 5.3 log CFU/cm2, in comparison to PAA and 2.3 CFU/cm2, respectively. Treatments involving application of PAA followed immediately by ACP and ACP followed by microscopy images indicated that combined treatments resulted in destruction of Salmonella cells with visible cellular PAA resulted in the highest (P ≤ 0.05) reduction in Salmonella by 4.7 and 5.3 log CFU/cm2, respectively. Transmission electron deformation and loss of cellular integrity. Color and moisture content of poultry meat samples were affected; thus, for large-scale application, further research needs to be done for optimizing this hurdle intervention. In conclusion, this study demonstrates the synergistic effect of ACP and PAA and its potential application for the safety of poultry products.

11 Zhao, X., Zhang, Y., Shi, J., Liu, Y., Lu, Y., & Lian, Z. (2019). Biosynthesis of antibacterial compound against multidrug resistant foodborne pathogens by Phomopsis sp . XP-8. Food Control, 95(April 2018), 223–231. https://doi.org/10.1016/j.foodcont.2018.08.007 Abstract Multi-drug resistance of foodborne pathogens has been greatly highlighted in food safety and caused a significant challenge for the development of new antibiotics and safe preservatives. The ethanol extract of Phomopsis sp. XP-8 broth showed significant inhibitory effects on multi-drug resistance of foodborne pathogens. After purification with thin-layer chromatography and liquid chromatographic analysis, the antibacterial compound was identified as methyl quercetin according to FTIR spectrum analysis and ESI- Q-TOF-MS analysis. Via substrate-feeding experiments, the mass flow for the biosynthesis of methyl quercetin was defined as (starting from glucose to phenylalanine): cinnamic acid, p-coumaric acid, quercetin, and methyl quercetin. The study revealed the capability of methyl quercetin to inhibit foodborne multi-drug resistant Escherichia coli, Staphylococcus aureus, and Salmonella shubra, as well as the capability of Phomopsis sp. XP-8 to produce methyl quercetin, showing potential application in food control.

12 Fang, J., Shen, Y., Qu, D., & Han, J. (2019). Antimicrobial resistance pro fi les and characteristics of integrons in Escherichia coli strains isolated from a large-scale centralized swine slaughterhouse and its downstream markets in Zhejiang, China. Food Control, 95(August 2018), 215–222. https://doi.org/10.1016/j.foodcont.2018.08.003 Abstract The pork production chain and raw pork products are among the most important reservoirs of antimicrobial- resistant microorganisms. This study investigated the antimicrobial resistance profiles and integrons and their associated gene cassettes of Escherichia coli isolated from a large-scale centralized swine slaughterhouse in Zhejiang, China and its downstream markets. A total of 300 E. coli isolates were identified from 720 samples. No E. coli isolates were detected on the slaughtering line or at downstream supermarkets. Of the 300 E. coli isolates collected from lairages and downstream open markets, 242 (80.67%) were classified as multi-drug resistant (MDR). In addition, the prevalence of multi-drug resistance was significantly different (p < 0.05) and anti-microbial resistance profiles varied among the different sampling points. Furthermore, of 300 E. coli strains, 76 (25.33%) were positive for class 1 integrons and nine (3.00%) were positive for class 2 integrons. Three different gene cassettes were detected in class 1 integrons and one gene cassette was detected in class 2 integrons. Our study demonstrates that E. coli isolates from lairages and open markets are frequently MDR, and suggests that precautionary measures and hygienic environments are necessary to prevent the spread of antimicrobial- re- sistant microorganisms and their mobile genetic elements through the food chain.

13 Zhou, Q. I., Zhao, Y. U., Dang, H. U. I., & Tang, Y. (2019). Antibacterial Effects of Phytic Acid against Foodborne Pathogens and Investigation of Its Mode of Action, 82(5), 826–833. https://doi.org/10.4315/0362-028X.JFP-18-418 Abstract This study investigated the antimicrobial mechanism of phytic acid (PA) and its antibacterial effects in combination with ethanol. The MIC of PA on Escherichia coli ATCC 11229, Staphylococcus aureus ATCC 6538P, Bacillus subtilis ATCC 6633, and Salmonella Typhimurium CICC 27483 were 0.24, 0.20, 0.26, and 0.28% (w/w), respectively. E. coli ATCC 11229 and S. aureus ATCC 6538P were selected to investigate the mechanism ofPA by analyzing its effects at 1/2MIC and at MIC on the cell morphology, intracellular ATP, and cell membrane integrity. Environmental scanning electron microscope images revealed that PA was able to change the cell morphology and disrupt the intercellular adhesion. PA retarded bacterial growth and caused cell membrane dysfunction, which was accompanied by decreased intracellular ATP concentrations. Flow cytometry analysis further revealed that almost all the bacterial cells were damaged after treatment with PA at its MIC for 2 h. Moreover, PA has a synergistic antimicrobial ability when used in combination with ethanol. These results suggested that PA is effective in inhibiting growth of foodborne pathogens mainly by the mechanism of cell membrane damage and to provide a theoretical basis for the development of natural antimicrobial agents in the food industry.

Parasitologi 1 Hector, E., Elelu, N., Ferrolho, J., Couto, J., Sanches, G., Antunes, S., … Eisler, M. (2019). PCR detection of Ehrlichia ruminantium and Babesia bigemina in cattle from Kwara State, Nigeria: unexpected absence of infection, Parasitology Research (2019) 118:1025–1029. https://doi.org/10.1007/s00436-019-06204-1 Abstract Ticks and tick-borne diseases (TBDs) continue to pose an insidious and ever-present threat to livestock and livelihoods across the globe. Two of the most significant TBDs of cattle in Africa are heartwater and babesioisis, caused by Ehrlichia ruminantium and Babesia bigemina respectively. Both pathogens are endemic in Nigeria. However, to date, little data has been published regarding the number of cattle infected. In this study, blood samples were collected from cattle of the Kwara State, north-central Nigeria. Probe-based quantitative PCR (qPCR) and semi-nested PCR were used to investigate the presence of both pathogens, respectively. Our study found all samples (n = 157) to be surprisingly negative for both B. bigemina and E. ruminantium. These results contribute new information on the current burden of these two pathogens in Kwara State and may be helpful in informing more effective targeting of control strategies in Nigeria.

2 Li, J., He, J., Elsheikha, H. M., Chen, D., Zhai, B., & Zhu, X. (2019). Toxoplasma gondii ROP17 inhibits the innate immune response of HEK293T cells to promote its survival. Parasitology Research (2019) 118:783–792. https://doi.org/10.1007/s00436-019-06215-y Abstract Toxoplasma gondii secretes a group of rhoptry-secreted kinases (ROPs), which play significant roles in promoting intracellular infection. T. gondii rhoptry organelle protein 17 (ROP17) is one of these important effector proteins. However, its role in modulating host cell response during infection remains poorly understood. Here, we reveal that ROP17 (genotype I) induces significant changes in the expression genes and transcription factors ofhost cells. HEK293T cells were transfected with PCMV- N-HA-ROP17 plasmid or empty control PCMV-N-HA plasmid. Transcriptomic analysis revealed 3138 differentially expressed genes (DEGs) in PCMV-N-HA-ROP17-transfected HEK293T cells, including 1456 upregulated, 1682 downregulated DEGs. Also, 715 of the DEGs were transcription factors (TFs), including 423 downregulated TFs and 292 upregulated TFs. Most differentially expressed TFs, whether belong to signal transduction, cancer-related pathways or immune-related pathways, were downregulated in ROP17-expressing cells. ROP17 also decreased alternative splicing events in host cells, presumably via alteration of the expression of genes involved in the alternative splicing pathway. Taken together, our findings suggest a novel strategy whereby T. gondii ROP17 manipulates various cellular processes, including immune response through reprogramming host gene expression to promote its own colonization and survival in the infected host cells.

3 Obregón, D., Cabezas-cruz, A., Armas, Y., Silva, J. B., Fonseca, A. H., André, M. R., … Corona- gonzález, B. (2019). High co-infection rates of Babesia bovis, Babesia bigemina, and Anaplasma marginale in water buffalo in Western Cuba. Parasitology Research (2019) 118:955–967. https://doi.org/10.1007/s00436-018-06194-6 Abstract Water buffalo is important livestock in several countries in the Latin American and Caribbean regions. This buffalo species can be infected by tick-borne hemoparasites and remains a carrier of these pathogens which represent a risk of infection for more susceptible species like cattle. Therefore, studies on the epidemiology of tick-borne hemoparasites in buffaloes are required. In this study, the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale were determined in water buffalo herds of western Cuba. To this aim, a cross-sectional study covering farms with large buffalo populations in the region was performed. Eight buffalo herds were randomly selected, and blood samples were collected from 328 animals, including 63 calves (3– 14 months), 75 young animals (3–5 years), and 190 adult animals (>5 years). Species-specific nested PCR and indirect ELISA assays were used to determine the molecular and serological prevalences of each hemoparasite, respectively. The molecular and serological prevalence was greater than 50% for the three hemoparasites. Differences were found in infection prevalence among buffalo herds, suggesting that local epidemiological factors may influence infection risk. Animals of all age groups were infected, with a higher molecular prevalence of B. bigemina and A. marginale in young buffalo and calves, respectively, while a stepwise increase in seroprevalence ofB. bovis and B. bigemina from calves to adult buffaloes was found. The co-infection by the three pathogens was found in 12% ofanimals, and when analyzed by pair, the co-infections ofB. bovis and B. bigemina, B. bigemina and A. marginale,and B. bovis and A. marginale were found in 20%, 24%, and 26%, respectively, underlying the positive interaction between these pathogens infecting buffaloes. These results provide evidence that tick-borne pathogen infections can be widespread among water buffalo populations in tropical livestock ecosystems. Further studies should evaluate whether these pathogens affect the health status and productive performance of water buffalo and infection risk of these pathogens

4 Rafiqi, S. I., Garg, R., Ram, H., Reena, K. K., Asari, M., Kumari, P., … Banerjee, P. S. (2019). Immunoprophylactic evaluation of recombinant gametocyte 22 antigen of Eimeria tenella in broiler chickens. Parasitology Research (2019) 118:945–953. https://doi.org/10.1007/s00436-018-06198-2 Abstract Gametocyte proteins are being explored as potential vaccine candidates against Eimeria sp. in chicken since they are the components of the resilient oocyst wall. The aimofthis study was to investigate the immunoprophylactic efficacy of recombinant Eimeria tenella gametocyte antigen 22 (EtGam22) in chickens against homologous oocyst challenge. Broiler chicks were subcutaneously immunized individually with 100 μg of recombinant EtGam22 adjuvanted with Montanide ISA 71 VG at 7 days of age and boosted 2 weeks later. The immunized chickens were challenged individually with 1 × 104 sporulated oocysts of E. tenella 1 week post-booster immunization. The anti-EtGam22 IgY and serum cytokine response was measured post-immunization. The results showed that the anti-EtGam22 IgY antibody, serum IFN-γ, IL-2, TGF-β, and IL-4 levels in chickens vaccinated with recombinant protein were significantly increased post-immunization as compared to unimmunized challenged controls (P< 0.05). The peripheral blood lymphocyte proliferation activity was also found significantly higher in EtGam22- immunized group on day 28, i.e., pre-challenge (P< 0.05). Upon homologous oocyst challenge, chickens immunized with rEtGam22 exhibited a significant drop in the total oocyst output per bird (246.78 ± 36.9 × 106, 45.23% reduction) and a significantly higher weight gain (497.7 ± 19.2 g) as compared to unimmunized challenged controls. Taken together, these data indicate that EtGam22 is a potent immunogen for use as a subunit vaccine against cecal coccidiosis in chickens as it induces a diverse and robust immune response involving multiple cytokines and strong antibody titers.

5 Rousseau, A., Villena, I., Dumètre, A., Escotte-binet, S., Favennec, L., Dubey, J. P., … Carbona, S. La. (2019). Evaluation of propidium monoazide – based qPCR to detect viable oocysts of Toxoplasma gondii. Parasitology Research (2019) 118:999–1010. https://doi.org/10.1007/s00436-019-06220-1 Abstract Information on the viability of Toxoplasma gondii oocysts is crucial to establish the public health significance of this environmental transmission stage that can contaminate water and foods. Interest for molecular-based methods to assess viability is growing and the aim of our study was to assess, for the first time, a propidium monoazide (PMA)–qPCR approach to determine the viability of T. gondii oocysts. Untreated and heat-killed (99°C, 5 min) oocysts were incubated with PMA, a photoreactive DNA binding dye, and analyzed by confocal microscopy and flow cytometry to characterize oocysts‟ dye permeability. Different PMA concentrations (50 to 150 μM), incubation temperatures (22, 37, and 45°C), amplicon length, selected targeted gene, and dyes (PMA, PMAxx™) were evaluated to define optimal conditions to discriminate specifically viable oocysts by PMA–qPCR. In theory, PMA binding to DNA would inhibit PCR amplification in dead but not in viable oocysts. Incubation at 22 °C with 100 μMPMA coupled to qPCR targeting a 123-bp sequence ofthe 529-bp repeat element allowed the distinction between viable and heated oocysts. However, the reduction of viability following heating of oocysts at high temperature was slight and, contrarily to reverse transcriptase-qPCR, the qPCR signal was not totally suppressed in heated suspensions. Therefore, PMA– qPCR is able to assess the impact of heating on T. gondii oocysts‟ viability but underestimates the efficacy ofthis treatment. The relevance of this technique to evaluate the efficacy of other inactivation processes and assess exposure of humans to this pathogen requires further investigations.

6 Steinbrink, A., Zotzmann, S., Cunze, S., & Klimpel, S. (2019). Aedes koreicus — a new member of the genus Aedes establishing in Germany ?. Parasitology Research (2019) 118:1073–1076. https://doi.org/10.1007/s00436-019-06232-x Abstract Aedes koreicus, a mosquito species originating from Japan, China, Korea, and parts of Russia, has been sporadically found in Europe since 2008. It is suspected to be a vector ofa variety of viruses and nematodes. In Germany, one individual was found in 2015 in the city of Augsburg, situated in the federal state of Bavaria. Based on morphological and molecular species identification, this study reports a new finding of Ae. koreicus, about 370 km northwest from Augsburg. The sampling point is located in the city of Wiesbaden, in the federal state of Hesse, where four individuals were found over a period of 2 months in 2017. The re- finding of the species in the same location in May and July 2018 suggests that (a) the species was able to reproduce and overwinter at this site, and (b) spreading of non-native mosquito species is an ongoing process in Germany, which requires close monitoring.

7 Vielmo, A., Fátima, H., Pena, J., Panziera, W., Bianchi, R. M., Lorenzo, C. De, … Pavarini, S. P. (2019). Outbreak of toxoplasmosis in a flock of domestic chickens ( Gallus gallus domesticus ) and guinea fowl ( Numida meleagris ), Parasitology Research (2019) 118:991–997. https://doi.org/10.1007/s00436-019-06233-w Abstract Toxoplasmosis is a disease with a worldwide distribution that affects a wide variety of animal species, though with rare descriptions in chickens. We describe the clinical, epidemiological, pathological, and molecular aspects of a toxoplasmosis outbreak in domestic chickens and guinea fowl in southern Brazil. The flock was composed of 47 domestic chickens and 29 guinea fowl. Of these, 22 birds showed clinical signs of lethargy, anorexia, and neurological signs over a clinical course of 24–72 h, and 15 died. Epidemiological data were obtained through fieldwork performed at the chicken farm and necropsies of six birds. Gross lesions were absent at necropsy, and histopathological findings included inflammatory infiltrate of macrophages, lymphocytes, and plasma cells and necrosis in several tissues associated with intralesional Toxoplasma gondii. Immunohistochemistry for T. gondii was positive. Additionally, restriction fragment length polymorphism (RFLP) analysis with 11 markers (SAG1, SAG2 (5′3′SAG2 and alt. SAG2), SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3) and microsatellite (MS) analysis with 15 markers (TUB2, W35, TgMA, B18, B17, M33, IV.1, XI.1, N60, N82, AA, N61, N83, M48, and M102) were performed. PCR-RFLP revealed T. gondii genotype ToxoDB-PCR-RFLP #280, and MS analysis also showed a unique genotype. This is the first description of this genotype in chickens and adds to the evidence suggesting considerable genotypic diversity of T. gondii in Brazil.

8 Alves, B. F., Oliveira, S., Soares, H. S., Fátima, H., Pena, J., Conte-junior, C. A., & Gennari, S. M. (2019). Isolation of viable Toxoplasma gondii from organs and Brazilian commercial meat cuts of experimentally infected pigs. Parasitology Research (2019) 118:1331–1335. https://doi.org/10.1007/s00436-019-06229-6 Abstract The present study evaluated the distribution and viability of Toxoplasma gondii tissue cysts in the organs and Brazilian commercial cuts of experimentally infected pigs. The pigs were infected with 3 × 103 oocysts ofthe T. gondii isolate TgCkBr57 (Type BrII). Mouse bioassays were performed on the brain, retina, tongue, diaphragm, and heart as well as the following muscle cuts: loin (longissimus), coppa (longissimus, spinalis dorsi, rhomboideus), tenderloin (psoas major), outside flat (biceps femoris), topside (semimembranosus), and top sirloin (gluteus medius). Toxoplasma gondii was isolated from the coppa, heart, diaphragm, and tongue of three pigs; from the tenderloin, outside flat, and brain of two pigs; and from the top sirloin and loin ofone pig. Thus, the viability of T. gondii cysts was observed in all of the organs and cuts evaluated (except for the topside and retina), demonstrating the broad distribution of this parasite in pig organs and commercial meat cuts, and the importance of this species as a source of human infection.

9 Chougar, L., Amor, N., & Merella, P. (2019). New insight into genetic variation and haplotype diversity of Fasciola hepatica from Algeria, Parasitology Research (2019) 118:1179–1192. https://doi.org/10.1007/s00436-019-06270-5 Abstract The liver fluke Fasciola hepatica is the main cause of fasciolosis in North Africa leading to significant economic losses and public health problems. In this study, the ribosomal internal transcribed spacer (ITS), cytochrome c oxidase I (COI), the mitochondrial region spanning the COI-trnT-rrnL, and the NADH dehydrogenase subunit I (NADI)markers were used to characterize Fasciola flukes from Algeria. Fasciola appeared widespread from the east to the west of Algeria. Among 1701 sampled cattle from 8 Algerian provinces, 5% were infected. Using morphological and morphometric analysis, one morphotype of Fasciola was observed. Nuclear ITS marker indicated that all collected flukes belong to F. hepatica. Multiple alignments of ITS dataset revealed two haplotypes, one described here for the first time. Analysis of molecular variance (AMOVA) of mitochondrial markers revealed weak population structure in Algeria. Mismatch distributions, neutrality tests, and median-joining network analysis all were compatible with a recent expansion ofAlgerian F. hepatica population. Fasciolosis appeared common in Algerian cattle, it seems that the absence of control strategy coupled to the favorable Mediterranean climate may lead to a reconstruction and dispersion of its populations. This study provides important results concerning the genetic characterization and variability of F. hepatica in Algeria as well as the significant role of cattle importation in shaping its dispersal route worldwide.

10 Grech-angelini, S., Richomme, C., Garam, C. P. De, & Boucher, J. (2019). Identification and molecular characterization of Echinococcus canadensis G6 / 7 in dogs from Corsica , France, Parasitology Research (2019) 118:1313–1319. https://doi.org/10.1007/s00436-019-06261-6 Abstract Recent surveys at slaughterhouses confirmed the presence of three different species of Echinococcus granulosus sensu lato in France: E. granulosus sensu stricto, E. ortleppi,and E. canadensis G6/7. The latter species was only identified on the French Mediterranean island of Corsica, with a high prevalence in pigs and wild boar. In order to investigate the life cycle of E. canadensis in this region, dog feces were collected in 31 municipalities, mainly from individual kennels. The analysis of fecal samples from 259 dogs by multiplex real-time PCR shows no infection by E. granulosus sensu stricto, but three dogs were infected by E. canadensis G6/7. Genetic analyses of mitochondrial genes (cox1, nad1, nad3, atp6) revealed in two dogs a haplotype previously identified in pigs. The third dog was infected by a new haplotype differing only from the two others from dogs by two mutations in the nad3 gene. This latter haplotype is genetically closer to those identified in pigs rather than those from wild boars. Analysis of questionnaires completed by the owners revealed that the sampled dog population was almost exclusively composed of hunting dogs that had been infrequently dewormed. Most of the owners (78%) leave carcasses of hunter- harvested wild boar in close proximity to their dogs. Nevertheless, genetic results seem to indicate that the three dogs were infected due to their consumption of a pig‟s infected viscera following home slaughtering. This study confirms the role of dogs as definitive hosts of E. canadensis G6/7 in Corsica. Further molecular studies, notably in human cases, are needed to assess the zoonotic impact of E. canadensis G6/7 in this region.

11 Mulieri, P. R. (2019). Using ecological niche models to describe the geographical distribution of the myiasis-causing Cochliomyia hominivorax ( Diptera : Calliphoridae ) in southern South America. Parasitology Research (2019) 118:1077–1086 https://doi.org/10.1007/s00436-019-06267-0 Abstract In southern South America, namely Argentina and Chile, Cochliomyia hominivorax (Coquerel) is the main myiasic agent on humans and domestic animals. The distribution pattern of the species is poorly known and the southern limit of its geographic distribution is unclear. The aims of this study are to elucidate the basic environmental factors associated with occurrence of this myiasic species, evaluation of models constructed on the basis of occurrence data based on adult specimen records to predict geographic occurrence of myiasis, evaluation of unsurveyed sites of high potential of occurrence of the species, and recognition and prioritization of areas that need medical control and specific prophylaxis practices related to this pest. The maximum entropy modeling system (Maxent) was used. Maps of potential distribution of C. hominivorax were produced using two different datasets, models obtained with all localities known for the species (combining medical data and taxonomic data) and only-taxonomic models (excluding medical data). The results obtained include an updated compilation of occurrence of the species in Argentina and Chile. Predictive models obtained in this work indicated that large areas of central-eastern territory of Argentina has the potential for C. hominivorax occurrence, probably reaching the parallel 42° S as is indicated by the complete datasets. Only-taxonomic models fail to predict those myiasic cases occurring in the nearer areas of the Andean chains. The main variables associated with the distribution of C. hominivorax were, alternatively, isothermality or minimum temperature of the coldest month. These results provide a new analytical resource of high potential for the prevention of myiasis and to supports further epidemiological studies.

12 Schoener, E. R. (2019). Protozoan parasites in Culex pipiens mosquitoes in Vienna. Parasitology Research (2019) 118:1261–1269. https://doi.org/10.1007/s00436-019-06219-8 Abstract Avian malaria (Plasmodium spp.) and kinetoplastid (Trypanosoma spp.) parasites are common vector- borne pathogens in birds worldwide; however, knowledge about vector competence of different mosquito species is currently lacking. For a pilot project examining vector competence of mosquitoes of the Culex pipiens complex and Culex torrentium for protozoan parasites in the city of Vienna, 316 individual mosquitoes were sampled in the months June–August 2017 around the campus of the Veterinary University of Vienna. Since vector competence for avian Plasmodium can only be ascertained by finding infectious sporozoites in mosquito salivary glands, special emphasis was on examining these, or at least insect thoraxes, which contain the salivary glands. After species identification, the mosquitoes were processed in three different ways to determine the best method of visually detecting protozoan parasites in salivary glands: (1) microscopic examination of individual, fixed and Giemsa-stained salivary glands, (2) microscopic examination of stained sections of individually fixed and embedded mosquito thoraxes and (3) stained sections of individual whole insects. Material from all three groups was also subjected to PCR to detect avian haemo-sporidian and trypanosomatid parasite DNA. PCR was performed on all 316 collected mosquitoes, with 37 pools (n =2–10) of 263 individuals and 53 single individuals in all together 90 PCR reactions. Avian Plasmodium was found in 18 (20%) and trypanosomatid parasites were found in 10 (11.1%) of the examined samples and pools yielded a higher proportion of positives than did individual samples. Six different species ofprotozoan parasites were identified, namely Plasmodium vaughani SYAT05 which was the most common, P. elongatum GRW6, P. relictum SGS1, Trypanosoma avium, T. culicavium and Crithidia dedva. Seventy-seven mosquito salivary glands were dissected and stained with Giemsa solution. Of these, one (1.3%) featured sporozoites and one (1.3%) trypanosomatid parasites. While the trypanosomes were identified as T. avium, the avian Plasmodium species were present in a mixed infection with P. vaughani SYAT05 as the dominant species. In conclusion, mosquitoes of the Culex pipiens complex are very likely vectors of different avian Plasmodium and Trypanosoma species and PCR was the most successful and reliable method for parasite detection in mosquito samples, delivering higher rates and more accurate results. The visual detection of parasite stages in the salivary glands was more difficult and only a few specimens were detected using Giemsa stain and chromogenic in situ hybridization. For further studies on vector competence of different protozoan parasites in mosquitoes, the use of PCR-based methods would be preferable.

Toksikologi

1 Chen, Y., Kong, Q., & Liang, Y. (2019). Three newly identified peptides from Bacillus megaterium strongly inhibit the growth and aflatoxin B 1 production of Aspergillus flavus, 95(July 2018), 41–49. https://doi.org/10.1016/j.foodcont.2018.07.040 Abstract Aspergillus flavus is one of the major moulds that infect crops, and produces the most carcinogenic and toxic compounds-aflatoxins. The previous research demonstrated that a marine bacterium, Bacillus megaterium, inhibited the growth of A. flavus and the biosynthesis of aflatoxin B1 in peanuts. In this study, five characterized peptides with distinct structures in three ion peaks were identified by LC-MS/MS and demonstrated to have anti- A. flavus activities from B. megaterium fermentation broth. Based on the structural information generated from the peptides, several derivatives (different composition and configuration) were synthesized and examined their efficacy in terms of inhibiting growth of A. flavus,

spore germination, and aflatoxin production. Results showed three peptides L-Asp-L-Orn (D1O), L-Asp- L-Asn (D1N) and L-Asp-L-Asp-L-Asn (D2N) could significantly inhibit the growth of A. flavus. The results of confocal microscopy showed that D1O, D1N and D2N can spontaneously enter into the hyphae of A. flavus. Meanwhile, data of quantitative RT-PCR (qRT-PCR), aflatoxin production and spore germination assay demonstrated that after three peptides entered into the cells of A. flavus, they inhibited conidiation and aflatoxin production, but didn't inhibit hyphae vegetative growth and spore germination.

2 Darra, N. El, Gambacorta, L., & Solfrizzo, M. (2019). Multimycotoxins occurrence in spices and herbs commercialized in Lebanon. Food Control, 95(July 2018), 63–70. https://doi.org/10.1016/j.foodcont.2018.07.033 Abstract Aflatoxins and ochratoxin A are regulated in Europe for some spices (Capsicum spp., Piper spp., Myristica fragrans, Zingiber officinale, Curcuma longa) and mixtures of spices containing one or more of these spices. No mycotoxin limits are in force for herbs. A total of 132 samples of spices (94) and herbs (38) purchased from Beirut in Lebanon were analysed for 12 mycotoxins (AFB1, AFB2, AFG1, AFG2, OTA, FB1,FB2, HT-2, T-2, ZEA, DON, NIV) by using a UPLC-MS/MS method based on „dilute and shoot‟ approach. The limits of detection (LOD) ranged from 0.1 μg/kg (ZEA) to 20.5 μg/kg (DON) and limits of quantification (LOQ) ranged from 0.3 μg/kg (ZEA) to 68.2 μg/kg (DON). 80% of analysed samples were contaminated by 1–11 mycotoxins. Total aflatoxins and ochratoxin A were detected in 19 and 30% of spices, 8 and 11% of herbs, respectively. Mean levels of total aflatoxins and ochratoxin A were 168.1 and 7.1 μg/kg in positive spices, 36.1 and 7.0 μg/kg in positive herbs, respectively. 78 and 10% of positive spice samples contained aflatoxin and ochratoxin A at levels higher than the limits, respectively. Total aflatoxin levels higher than the European limits were also measured in some non- regulated spices (allspice, cloves, coriander, fenugreek) and some herbs (rosemary, sage and oregano). Within the non-regulated mycotoxins FB1 was the most occurring (60% in spices, 55% in herbs) followed by FB2 (35% in spices, 18% in herbs), ZEA (30% in spices, 3% in herbs), DON (12% in spices, 3% in herbs), T-2 and HT-2 toxins (3–5%), whereas NIV and AFG2 were never detected. Mean levels of FB1,FB2, ZEA and DON in positive samples of spices were 6432.3, 203.2, 30.6, 1751.4 μg/kg, respectively; in positive samples of herbs they were 2826.3, 214.9, 2.8, 589.7 μg/kg, respectively. The whole results demonstrate the higher susceptibility of spices to mycotoxin contamination with respect to herbs. Comparison of results obtained for samples produced with (81) and without (51) HACCP and GMP showed that the implementation of HACCP and GMP practices seems to be effective in reducing the occurrence of regulated mycotoxins but was ineffective for the non-regulated ones. The samples analysed in this study originated from at least 15 Countries and the results obtained gives indications about the occurrence of mycotoxins in relation to the Country of origin of the samples. The high percentages of positive samples and the high levels of some mycotoxins observed in this study highlight the problem of mycotoxin contamination in spices and herbs consumed in Lebanon. The occurrence of high levels of aflatoxins and OTA in some non-regulated spices and herbs suggests the addition of these matrices in the list of regulated ones. The high number of positive samples and the high levels of fumonisins observed in this study suggest the inclusions of these mycotoxins in the list of regulated mycotoxins for these matrices.

3 Indyk, H. E., Chetikam, S., Gill, B. D., Wood, J. E., & Woollard, D. C. (2019). Development and Application of an Optical Biosensor Immunoassay for Aflatoxin M 1 in Bovine Milk. Food Analytical Methods https://doi.org/10.1007/s12161-019-01621-5 Abstract An automated optical biosensor-based immunoassay exploiting surface plasmon resonance detection for

the quantitation of aflatoxin M1 (AFM1) in milk and milk powders is described. A monoclonal antibody and an immobilized protein–AFM1 conjugate are utilized in a simple inhibition format following aqueous extraction and immunoaffinity clean-up of the sample, thereby avoiding the need for signal amplification techniques. The sensor surface is stable over multiple regeneration cycles, and the technique yields a method detection limit of 0.1 ng g−1, which is five times lower than the European Commission maximum residue limit. The described antibody-based biosensor technique provides the advantages of quantitative

data, automation, and real-time and non-labeled detection of AFM1. The method therefore facilitates routine quantitative threshold-level screening for the identification of potential non-compliance of AFM1 content prior to confirmatory analysis by reference chromatographic methods and may be considered to complement the enzyme-linked immunosorbent assay technique.

4 Hua, S. S. T., Parfitt, D. E., Sarreal, S. B. L., & Sidhu, G. (2019). Dual culture of atoxigenic and toxigenic strains of Aspergillus flavus to gain insight into repression of aflatoxin biosynthesis and fungal interaction. Mycotoxin Research https://doi.org/10.1007/s12550-019-00364-w Abstract Application of atoxigenic strains to compete against toxigenic strains of Aspergillus flavus strains has emerged as one of the practical strategies for reducing aflatoxin contamination in corn, peanut, and tree nuts. The actual mechanism that results in aflatoxin reduction is not fully understood. Real-time RT-PCR and relative quantification of gene expression protocol were applied to elucidate the molecular mechanism. Transcriptional analyses of aflatoxin biosynthetic gene cluster in dual culture of toxigenic and atoxigenic A. flavus strains were carried out. Six targeted genes, aflR, aflJ, omtA, ordA, pksA, and vbs,were downregulated to variable levels depending on paired strains oftoxigenic and atoxigenic A. flavus. Consistent with the decreased gene expression levels, the aflatoxin concentrations in dual cultures were reduced significantly in comparison with toxigenic cultures. Fluorescent images showed fungal hyphae in dual culture displayed green fluorescent, and contacts of live hyphae were seen. A coconut agar plate assay was used to show that toxigenic A. flavus colony produced blue fluorescence under long UV exposure, suggesting that aflatoxin is exported outside fungal hyphae. Furthermore, the assay was applied to demonstrate the potential role of thigmo-regulation in fungal interaction.

5 Beyene, A. M., Du, X., Schrunk, D. E., Ensley, S., & Rumbeiha, W. K. (2019). High - performance liquid chromatography and Enzyme - Linked Immunosorbent Assay techniques for detection and quantification of aflatoxin B 1 in feed samples: a comparative study. BMC Research Notes, 1–6. https://doi.org/10.1186/s13104-019-4538-z Abstract Objective: Comparison was done between high-performance liquid chromatography (HPLC) and a

competitive enzyme-linked immunosorbent assay (ELISA) for detection and quantification of aflatoxin B1 (AFB1) in feed samples. The two procedures were standardized and validated before the actual experiment. Five concentrations (0, 5, 10, 20 and 30 ppb) of feed samples were used for both methods. For the HPLC technique, the samples were extracted in acetonitrile/water (90/10) solution, cleaned-up using solid phase extraction (SPE) column, and derivatized by water/ trifluoroacetic acid/glacial acetic acid (35/10/5) solution before instrument analysis. The samples were extracted in 70% methanol for the ELISA technique.

Results: The two tests showed very strong linearity with correlation coefficient value of > 0.99 using standard solutions. The mean recovery rate was 92.42% (with relative standard deviation (RSD) of 5.97) and 75.64% (RSD = 34.88) for HPLC and ELISA, respectively. There was no statistically significant difference in recovery rate between the two methods. There was a positive correlation (r = 0.84) between

them which indicated that the two techniques can be used to detect and quantify aflatoxin B1 in feed samples. However, there were variations among replicates for the ELISA method, which shows that this method is more applicable for screening purposes.

6 Livak, K. J., & Schmittgen, T. D. (2001). Analysis of Relative Gene Expression Data Using Real- Time Quantitative PCR and the 2-ΔΔCT Method, 408, 402–408. https://doi.org/10.1006/meth.2001.1262 Abstract The two most commonly used methods to analyzed data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determination the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification the PCR signal of the target transcript in a treatment group to that of another sample such as untreated control. The 2-ΔΔCT method is a convenient way to analyze the relative changes inn gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, aswsumtion, and application of the 2-ΔΔCT method. In addition, we present the derivation and applications of two variations of the 2-ΔΔCT method that may be useful in the analysis of real-time, quantitative PCR data.

7 Chandra, H., Kumari, P., & Yadav, S. (2018). Evaluation of aflatoxin contamination in crude medicinal plants used for the preparation of herbal medicine. Oriental Pharmacy and Experimental Medicine. https://doi.org/10.1007/s13596-018-0356-4 Abstract The medical focus is now on phytofoods, drugs and the consumption of medicinal plants is expanding worldwide. Safety and quality of herbal preparations is of great concern. Quality determines reproducible efficacy of herbal drugs and also safety is a concern both for public and health authorities in many countries specially developing ones. The reason attributed is that many contaminants and microbes that may cause quality deterioration and directly harm to the consumers, find entry in the crude herbal materials during collection and storage. The safety of these products is partially compromised due to the microbial presence, especially toxigenic fungi. The present study was designed to investigate the microbiological quality after storage of locally sold raw medicinal plants that were supplied to different pharma units involved in the preparation of herbal and various formulations of ayurvedic drugs. Twenty samples of raw medicinal plants were stored at room temperature for a year and subjected to microbiological evaluation and found that most of the samples loaded with bacterial and fungal contents, do not comply with the FDA regulations. The presence of aflatoxin gave signals of aflatoxin producing fungi i.e. A. flavus and A. parasiticus. So, there is an urgent need of making strategy to control the microbes during pre-harvest and post-harvest procedures. This study is an attempt to emphasize the need for consistent quality assessment of crude herbal drugs for safe therapeutic products suitable for human beings. Our findings may help in instituting public health standards towards production and safety of herbal drugs worldwide.

8 Wang, F., Zuo, Z., Chen, K., Peng, X., & Fang, J. (2018). Selenium Rescues Aflatoxin B 1 -Inhibited T Cell Subsets and Cytokine Levels in Cecal Tonsil of Chickens. Biological Trace Element Research https://doi.org/10.1007/s12011-018-1412-0 Abstract Cecal tonsil is the largest peripheral lymphoid organ of the gut-associated lymphoid tissue executing immune function. To evaluate the protective effect of selenium (Se) on the cecal tonsil of chicken exposed

to aflatoxin B1 (AFB1), 144 1-day-old healthy Cobb chickens were randomly divided into four groups, and fed with basal diet (control group), 0.6 mg/kg AFB1 (AFB1 group), 0.4 mg/kg Se supplement (+Se group), and 0.6 mg/kg AFB1 + 0.4 mg/kg Se supplement (AFB1 + Se group) for 21 days, respectively. The results + + + + + showed that AFB1 significantly decreased the percentages of CD3 ,CD3 CD4 ,CD3 CD8 Tcells, and the CD4+/CD8+ ratio, and suppressed the expressions of IL-2, IL-4, TNF-α, and IFN-γ mRNA in the cecal tonsil. However, Selenium (Se) supplied in the diets restored the percentages of T cell subsets, the + + CD4 /CD8 ratio, and mRNA expressions of cytokines in the AFB1 group to be close to those in the control group, and did not exhibit obvious toxicity to the cecal tonsil. These results indicated that Se exerted protective effect against AFB1 on the functions of cecal tonsil, and also partially uncovered a new

role of Se that could protect cecal tonsil of chickens from immunotoxicity of AFB1.

9 Gil-serna, J., Vázquez, C., & Patiño, B. (2019). Genetic regulation of aflatoxin , ochratoxin A , trichothecene, and fumonisin biosynthesis : A review. International Microbiology https://doi.org/10.1007/s10123-019-00084-2 Abstract Mycotoxins are a significant food safety concern. Aflatoxins, trichothecenes, fumonisins, and ochratoxin A are considered the most important mycotoxins due to their frequent occurrence in food products and their well-known toxicity. The regulation of mycotoxin biosynthesis occurs mainly at transcriptional level, and specific regulators have been described in every biosynthetic cluster. Secondary metabolite production, including mycotoxin synthesis, is also regulated by general regulator pathways affected by light, osmotic stress and oxidative stress, among others. This review is focused on this genetic regulation of mycotoxin biosynthesis by specific genes and global regulators.

10 Cammilleri, G., Graci, S., Collura, R., Buscemi, M. D., Vella, A., Macaluso, A., … Ferrantelli, V. (2018). Aflatoxin M 1 in cow , sheep , and donkey milk produced in Sicily , Southern Italy, (Creppy 2002). Mycotoxin Research https://doi.org/10.1007/s12550-018-0329-y Abstract Samples (n =485) of raw (n = 394) or heat-treated (n = 91) milk ofthree different species (cow, n =170; sheep, n = 133; donkey, n = 84), collected 2013–2016 in Western Sicily (Southern Italy), were analyzed

for aflatoxin M1 (AFM1) by enzyme-linked immunosorbent assay (ELISA). Positive ELISA results were further analyzed by HPLC with fluorescence detection. Both methods had a detection limit for AFM1 in milk of 7 ng kg−1. ELISA yielded 12.9 and 5% positives in cows and sheep milk, respectively, all samples of donkey milk were negative. Levels ofAFM1 were in most cases at 0.007–<0.05 μg kg−1,only two samples (sheep milk) slightly exceeded the European Union maximum level of 0.05 μg kg−1. Only 6% of −1 the samples were positive for AFM1 in a concentration range of 0.008–0.15 μg kg . Only milk samples collected directly from farms were positive. Overall, the levels were much lower than previously reported for Southern Italy cow and sheep milk samples purchased in retail stores. The results of this work indicate a continuous improvement of the feeding techniques on dairy farms ofSouthern Italy, which is essential to ensure consumers‟ protection.

Virology 1 Manning, J. E. (2019). Time to Micromanage the Pathogen-Host-Vector Interface : Considerations for Vaccine Development, 1–20. https://doi.org/10.3390/vaccines7010010 Abstract The current increase in vector-borne disease worldwide necessitates novel approaches to vaccine development targeted to pathogens delivered by blood-feeding arthropod vectors into the host skin. A concept that is gaining traction in recent years is the contribution of the vector or vector-derived components, like salivary proteins, to host-pathogen interactions. Indeed, the triad of vector-host-pathogen interactions in the skin microenvironment can influence host innate and adaptive responses alike, providing an advantage to the pathogen to establish infection. A better understanding of this “bite site” microenvironment, along with how host and vector local microbiomes immunomodulate responses to pathogens, is required for future vaccines for vector-borne diseases. Microneedle administration of such vaccines may more closely mimic vector deposition of pathogen and saliva into the skin with the added benefit of near painless vaccine delivery. Focusing on the „micro‟–from microenvironments to microbiomes to microneedles–may yield an improved generation of vector-borne disease vaccines in today‟s increasingly complex world.

2 Sunwoo, S., Daniel, P., Morozov, I., Elena, G. S., Gaudreault, N. N., Trujillo, J. D., … Richt, J. A. (2007). DNA-Protein Vaccination Strategy Does Not Protect. https://doi.org/10.3390/vaccines7010012 Abstract African swine fever virus (ASFV) causes high morbidity and mortality in swine (Sus scrofa), for which there is no commercially available vaccine. Recent outbreaks of the virus in Trans-Caucasus countries, Eastern Europe, Belgium and China highlight the urgent need to develop effective vaccines against ASFV. Previously, we evaluated the immunogenicity of a vaccination strategy designed to test various combinations of ASFV antigens encoded by DNA plasmids and recombinant proteins with the aim to activate both humoral and cellular immunity. Based on our previous results, the objective of this study was to test the combined DNA-protein vaccine strategy using a cocktail of the most immunogenic antigens against virulent ASFV challenge. Pigs were vaccinated three times with a cocktail that included ASFV plasmid DNA (CD2v, p72, p32, +/−p17) and recombinant proteins (p15, p35, p54, +/−p17). Three weeks after the third immunization, all pigs were challenged with the virulent ASFV Armenia 2007 strain. The results showed that vaccinated pigs were not protected from ASFV infection or disease. Compared to the non-vaccinated controls, earlier onset of clinical signs, viremia, and death were observed for the vaccinated animals following virulent ASFV challenge. ASFV induced pathology was also enhanced in the vaccinated pigs. Furthermore, while the vaccinated pigs developed antigen-specific antibodies, immunized pig sera at the time of challenge lacked the capacity to neutralize virus, and instead was observed to enhance ASFV infection in vitro. The results of this work points to a putative immune enhancement mechanism involved in ASFV pathogenesis that warrants further investigation. This pilot study provides insight for the selection of appropriate combinations of ASFV antigens for the development of a rationally-designed, safe, and efficacious vaccine for ASF

3 Cell, A., Folegatti, P. M., Bellamy, D., Flaxman, A., Mair, C., Ellis, C., … Gilbert, S. C. (n.d.). Safety and Immunogenicity of the Heterosubtypic Influenza A Vaccine MVA-NP + M1 Manufactured on, 1–12. https://doi.org/10.3390/vaccines7010033 Abstract Seasonal influenza infections have a significant global impact leading to increased health and economic burden. The efficacy of currently available seasonal influenza vaccines targeting polymorphic surface antigens has historically been suboptimal. Cellular immune responses against highly conserved Influenza A virus antigens, such as nucleoprotein (NP) and matrix protein-1 (M1), have previously been shown to be associated with protection from disease, whilst viral-vectored vaccines are an effective strategy to boost cell-mediated immunity. We have previously demonstrated that MVA encoding NP and M1 can induce potent and persistent T cell responses against influenza. In this Phase I study, we evaluated the safety and immunogenicity of MVA-NP+M1, which was newly manufactured on an immortalized cell line, in six healthy adult participants. The vaccine was well-tolerated with only mild to moderate adverse events that resolved spontaneously and were comparable to previous studies with the same vaccine manufactured in chick embryo fibroblasts. A significant increase in vaccine-specific T cell responses was detected seven days after immunization and was directed against both antigens in the vector insert. This small Phase I study supports progression of this vaccine to a Phase IIb study to assess immunogenicity and additional protective efficacy in older adults receiving licensed seasonal influenza vaccines

4 Alexyuk, P. G., Bogoyavlenskiy, A. P., Alexyuk, M. S., Turmagambetova, A. S., Zaitseva, I. A., Omirtaeva, E. S., & Berezin, V. E. (2019). Adjuvant activity of multimolecular complexes based on Glycyrrhiza glabra saponins , lipids , and influenza virus glycoproteins. Archives of Virology, 164(7), 1793–1803. https://doi.org/10.1007/s00705-019-04273-2 Abstract Numerous studies have shown that immunostimulatory complexes containing Quil-A saponin and various antigens are effective in stimulating the immune response and can be used as vaccine preparations for animals and humans. However, Quil-A saponin possesses toxicity and haemolytic activity. In the present work, a saponin-containing preparation named “Glabilox” was isolated from the roots of a Glycyrrhiza glabra L. plant by high-performance liquid chromatography (HPLC). The results showed that Glabilox has no toxicity or haemolytic activity and can form stable immunostimulatory complexes. Subcutaneous immunization of mice with an immunostimulating complex containing Glabilox and H7N1 influenza virus antigens stimulated high levels of humoral and cellular immunity. Vaccination of chickens with the same immunostimulating complex protected 100% of the animals after experimental infection with a homologous virus. Comparative studies showed that the immunogenic and protective activity of immunostimulatory complexes containing Quil-A and immunostimulatory complexes containing Glabilox are comparable to each other. The results of these studies indicated that Glycyrrhiza glabra saponins show great promise as safe and effective adjuvants.

5 Tsushima, H. H. D., Fujihara, H. K. N., Kawashimo, T. T. M., & Fuji, T. N. S. (2019). Complete nucleotide sequence of a new potexvirus , ‘ Cnidium virus X ’, isolated from Cnidium officinale in Japan. Archives of Virology, 164(7), 1931–1935. https://doi.org/10.1007/s00705-019-04261-6 Abstract A flexuous virus was detected in a Cnidium officinale plant in Japan showing mosaic symptoms. The virus was assigned to the genus Potexvirus based on analysis of its complete nucleotide sequence. The genomic RNA of the virus was 5,964 nucleotides in length, excluding the 3′-terminal poly(A) tail. It contained five open reading frames (ORFs), consistent with other members of Potexvirus. The ORF sequences differ from those of previously reported potexviruses. Phylogenetic analysis indicated that the polymerase of the virus is closely related to that of strawberry mild yellow edge virus; and the CP, to those of both yam virus X and vanilla virus X. We propose that this virus be designated as “cnidium virus X” (CnVX).

6 Chen, H., Yan, M., Tang, Y., & Diao, Y. (2019). Pathogenicity and genomic characterization of a novel avian orthoreovius variant isolated from a vaccinated broiler flock in China, 9457. https://doi.org/10.1080/03079457.2019.1600656 Abstract Avian orthoreovirus (ARV) infections of broiler flocks cause arthritis/tenosynovitis syndrome and significant economic losses. ARV variants were detected in the USA and Canada. Viral arthritis/ tenosynovitis syndrome has occurred frequently in China in recent years. In this study, a variant ARV strain associated with viral arthritis/tenosynovitis syndrome was isolated from broilers and designated as LY383. Genomic sequence and phylogenetic analysis of the σC nucleic acid and amino acid sequences revealed that the isolate was closely related to ARV field strains Reo/PA/ Layer/01224B/14, Reo/PA/Broiler/1551/13, GA/14602/2014, GA/13569/2013 and GA/13542/ 2013, in cluster V, but distinct from most Chinese field strains or commercial vaccine strains. Experimental challenge showed that the isolate could cause arthritis/tenosynovitis syndrome in broilers, which possessed a high level of maternal antibodies induced by commercial ARV vaccines (S1133, 1733 and T98). Furthermore, viral nucleic acid could be detected in cloacal swabs of all challenged birds throughout the entire test from 5 dpi onward. These results suggest that a novel ARV genotype emerges and might become prevalent in broiler flocks in China.

7 Ladman, B. S., Jr, J. G., Sauble, L. A., & Murphy, M. V. (2019). Protection afforded by avian influenza vaccination programmes consisting of a novel RNA particle and an inactivated avian influenza vaccine against a highly pathogenic avian influenza virus challenge in layer chickens up to 18 weeks post-vaccination, 9457(May). https://doi.org/10.1080/03079457.2019.1605148 Abstract The efficacies of an oil adjuvanted-inactivated reverse genetics-derived H5 avian influenza virus (AIV) vaccine and an alphavirus replicon RNA particle (RP) AIV vaccine were evaluated in commercial Leghorn chickens. Challenge utilized A/turkey/MN/12582/2015, an isolate representing the U.S. H5N2 Clade 2.3.4.4 responsible for the 2015 highly pathogenic avian influenza (HPAI) epornitic in commercial poultry the United States. As part of a long-term, 36- week study, chickens were challenged at seven weeks of age after receiving a single vaccination, at 18 weeks of age following a vaccine prime-single boost, and at 36 weeks of age after a prime- double-boost. All vaccine programmes reduced virus oropharyngeal and cloacal shedding and mortality compared to the non-vaccinated control birds; however, chickens receiving at least one administration of the RP vaccine generally had diminished viral shedding especially from the cloacal swabbings. A detectable serum antibody response and protection were observed through 18 weeks post-vaccination. Our data suggest that, in conjunction with a comprehensive eradication, enhanced biosecurity and controlled marketing plan, vaccination programmes of commercial layer chickens with novel RP vaccines may represent an important tool for preventing HPAI-related mortalities and decreasing viral load during a catastrophic influenza outbreak.

8 Rahman, A. U., & Shabbir, M. Z. (2019). A comparative phylogenomic analysis of avian avulavirus 1 isolated from non - avian hosts: conquering new frontiers of zoonotic potential among species. Archives of Virology, 164(7), 1771–1780. https://doi.org/10.1007/s00705-019-04276-z Abstract A number of avian avulavirus 1 (AAvV 1) isolates have been reported from avian and non-avian hosts worldwide with varying clinical consequences. In this regard, robust surveillance coupled with advanced diagnostics, genomic analysis, and disease modelling has provided insight into the molecular epidemiology and evolution of this virus. The genomic and evolutionary characteristics of AAvV 1 isolates originating from avian hosts have been well studied, but those originating from non-avian hosts have not. Here, we report a comparative genomic and evolutionary analysis of so-far reported AAvV 1 isolates originating from hosts other than avian species (humans, mink and swine). Phylogenetic analysis showed that AAvV 1 isolates clustered in five distinct genotypes (I, II, VI, VII and XIII). Further analysis revealed clustering of isolates into clades distant enough to be considered distinct subgenotypes, along with a few substitutions in several significant motifs. Although further investigation is needed, the clustering of AAvV 1 strains isolated from non-avian hosts into novel subgenotypes and the presence of substitutions in important structural and biological motifs suggest that this virus can adapt to novel hosts and therefore could have zoonotic potential.

9 Vandalen, K. K., Nemeth, N. M., Thomas, N. O., Barrett, N. L., Ellis, J. W., Sullivan, H. J., … Shriner, S. A. (2019). Experimental infections of Norway rats with avian - derived low - pathogenic influenza A viruses. Archives of Virology, 164(7), 1831–1836. https://doi.org/10.1007/s00705-019-04225-w