Progress 07/15/24 to 07/14/25

Outputs
Target Audience:In this reporting period (1st year), we have reached/communicated with owners and workers of allselected BFs/ICLFs (Backyard Farms/Integrated Crop-livestock farms)and arranged our visiting schedule. To determine theprevalence and potential reservoirs of Avibacterium paragallinarum (AP) and Infectious Bronchitis Virus (IBV) in above mentionedfarmswith poultry components, various farm samples were collected. During thesamples collection and laboratory analysis, all participating students and the postdoctoral fellow were trained for handling biosafety level-2 pathogens. In addition, we also provided primary knowledge of these animal pathogens to the farm workers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students and a postdoctoral fellow are involved in this project, and they have learned microbiological techniques including culture, molecular analysis of isolation, identification and 16S sequencing, and data analysis. In addition, several undergraduate students were also trained to help graduate students and a postdoctoral fellow/researcher by preparing media, sample preparation and performed DNA isolation. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We are planning to continuethe farm visit and collect more farmsamples for microbiological and molecular analysis for confirmation of the targeted pathogens and determine the sources of the pathogens. We will also keep collecting clinical samples from the clinical laboratory ofMaryland Veterinary Division. We will alsocomparethecolonization or infection withAvibacterium paragallinarum(AP)and/or Infectious Bronchitis Virus (IBV)in chicks of ICLFs determining theproperties of isolated pathogensusing16S sequencing. We are also aiming to perform poultrytrials using our probiotic or prebiotic or their combination. We are also planning to analyze our data and prepare the manuscripts.

Impacts
What was accomplished under these goals? To achieve the goal -1, we visited each selected farm siteseveraltimes of a single production cycle and collected samples including chick delivery-box paper liners (if it is available), nasal swab, fecal, floor, feed water, open ground, grass, fecal from wild birds, animal and rodent, flies etc.Water samples werecollected: one from the tank and another from the final dispenser lines. Surface/floor swabs werecollected from the interior or exterior sides of the house with sterile wet gauze pads and disinfectant neutralizers. Each indoor and outdoor environmental samples werecollected from areas around or on the ventilation fans. Feed samples werecollected directly from trucks every time a new truck load arrives at the farm.We collected the samples aseptically and in sterilized plastic bags and carried them to the lab using ice containing cool box. In the lab, we have performed microbiology culture and selected the presumptive position colonies for further culture and molecular confirmation. Solid samples werehomogenized at the laboratory before evaluating forAvibacterium paragallinarum(AP)isolation by culture and PCR detection for both AP andInfectious Bronchitis Virus (IBV). Our collaborator, Maryland Veterinary Doctor, also provided clinical samples which were positive with either AP or IBV or both.All samples wereenriched in BHI broth with supplementas well as serial diluted and directly plated on chocolate agar plate.Genomic DNA wasextracted from all collected samples (both farm samples and clinical specimens) and suspected colonies for AP from culture using the AllPrep DNA/RNA Mini Kit (Qiagen, Valencia, CA) following the manufacturer's recommendations.Simultaneously, we havealso preparedthe poultry trials usingour developed probiotic,prebiotic and their combination in preventing AP or IBV infections.

Publications