Control of Salmonella Gallinarum (Fowl Typhoid) in Poultry with Phage-based Interventions
Creators: name: Rehman, Saud Ur
Publisher: name: Purdue University Graduate School
Year: 2022
Subjects: Other agricultural, veterinary and food sciences not elsewhere classified

description: The Pakistan poultry industry has developed into the 11thlargest poultry industry in the world and poultry products provide high-quality and affordable protein sources to communities throughout the country. However, <em>Salmonella </em>Gallinarum, the etiological agent for fowl typhoid, is endemic in Pakistan with infections leading to high mortality and substantial economic loss. Currently, <em>Salmonella </em>Gallinarum infectionsin Pakistan poultry are controlled with antibiotics. The continued emergence of antibiotic resistance, however, has led to global initiatives to reduce the use of antibiotics in both human and veterinary medicine. Concurrently, the Pakistan government recently introduced new national policies that limit the use of antibiotics for performance in livestock and poultry production. As such, controlling bacterial infections in poultry without increasing the likelihood of antibiotic use could ensure the sustainability of Pakistan poultry production without posing risks to public health. Toward this end, we hypothesized that <em>Salmonella</em> Gallinarum infections inchickens could be prevented or otherwise controlled through the use of phages. To test this hypothesis, wastewater samples were collected from Lahore, Pakistan and different cities of Indiana, US and processed to isolate bacteriophages. The phages were characterized in terms of morphology, host spectra, lytic capacity, genomic sequencing, and survivability in different environments. Transmission electron microscopy showed these phages belonged to myoviridae (n = 5) and podoviridae (n = 1) families. Spectrum analysis revealed that each phage lysed at least 8 out of 10 different strains of <em>Salmonella </em>Gallinarum and significantly reduced (P &lt; 0.05) <em>Salmonella </em>Gallinarum when co-cultured in liquid medium with the bacterium. Stability of the phages was tested insimulated gastric fluid (SGF; pH= 2.5) andsimulated intestinal fluid (SIF; pH~6.8). Results showed that phage concentrationswere reduced to undetectable levels when exposed to SGF for more than 5 minutes. However, exposure to SIF did not result in appreciable reductions in phage concentrations. To mitigate potential effects of gastric environments, phages were encapsulated using a sodium alginate-based method. In contrast to unprotected phages, encapsulated phages remained viable (~100%) after 30 minutes exposure to SGF. Additionally, encapsulation efficiencies ranged between 90-99%. Encapsulated phages were sequentially incubated in SGF (30 minutes) and SIF(120 minutes) to determine the rate of release of the phages from capsules. All phages were released from capsules after 60 minutes of exposureto SIF. To determine if the phages effectively controlled <em>Salmonella </em>Gallinarum infections in chickens, 100, day-old Jumbo Cornish Rock Cross birds were randomly assigned to one of four treatments: 1) Control 1 (bacterial challenge, no phage treatment); 2) Control 2 (no phage or bacterial challenge); 3) challenged with SalmonellaGallinarum and treated with unprotected phages; and 4) challenged with <em>Salmonella</em> Gallinarum and treated with encapsulated phages. At7 d of age, chicks receiving the bacterial challenge were administered 5 X106CFU (500 μL) of <em>Salmonella</em> Gallinarum. For birds in phage treatment groups, the phages were administered (500 uL; 5 X108 PFU/mL or g) at 0, 12, and 24 hours post-challenge. Six birds from each group were euthanized at 1, 2, and 4 days post-challenge (dpc) and cecal SalmonellaGallinarum concentrations were quantified. At 1 dpc, birds treated with unprotected and encapsulated phages had significantly lower (P &lt; 0.05) SalmonellaGallinarum concentrations(4.36 ± 0.20and 5.05 ± 0.22 logCFU/g, respectively) than those found in untreated birds (5.71 ± 0.13). Likewise, at4 dpc, <em>Salmonella </em>Gallinarum concentrationsin ceca of birds treated with encapsulated and unprotected phages were significantly lower (P &lt; 0.05; 3.26 ± 0.62 and 4.02 ± 0.15 log CFU/g, respectively) than those found in untreated birds(4.65 ± 0.08log CFU/g). A second trial was conducted with higher challenge doses (1 mL at 1× 109CFU) and an additional treatment including a mixture (1:1) of unprotected and encapsulated phages. At1dpc, <em>Salmonella</em> Gallinarum concentrations in the ceca of birds treated with unprotected phages, encapsulated phages, and a mixture of unprotected and encapsulated phages were significantly lower(4.28 ± 0.11, 3.72 ± 0.40, and 3.81 ± 0.36log CFU/g, respectively) than found in those of untreated birds (5.26 ± 0.19log CFU/g). At 2 dpc, concentrations of<em> Salmonella </em>Gallinarumin the ceca of birds treated with unprotected, encapsulated, and a mixture of unprotected and encapsulated phages were significantly lower (P &lt; 0.05; 4.31 ±0.53, 3.96 ±0.61, and 4.38 ± 0.44logCFU/g, respectively) than those found in the ceca of untreated birds (5.72 ± 0.27logCFU/g).However, no significant differences were found in concentrations of <em>Salmonella</em> Gallinarum in the ceca of birds treated with encapsulated phages versus those treated with unprotected phagesor a mixture of encapsulated and unprotected phages. Similarly, at 4 dpc, <em>Salmonella </em>Gallinarum concentrations in the ceca of birds treated with unprotected phages, encapsulated phages, and a mixture of unprotected and encapsulated phages were significantly lower (3.17 ± 0.45, 3.56 ± 0.51, and 3.81 ± 0.54log CFU/g, respectively) than found in those of untreated birds (5.79 ± 0.08log CFU/g). At 7 d post-challenge, concentrations of <em>Salmonella</em> Gallinarum in the ceca of birds treated with mixture of unprotected and encapsulated phages(2.40 ± 0.55log CFU/g) were significantly lower (P &lt; 0.05) than those found in the ceca of untreated birds(7.08 ± 0.19log CFU/g). Similarly, concentrations of<em> Salmonella</em> Gallinarum in the ceca of birds treated with encapsulated and unprotected phages were significantly lower (P &lt; 0.05; 4.29 ± 0.39and 4.60 ± 0.37 log CFU/g, respectively) than those found in untreated birds. Taken together, these data indicate that <em>Salmonella </em>Gallinarum infections could be controlled with phage-based treatments. Additionally, the use of a mixture of unprotected and encapsulated phages may be more effective, presumably by allowing unprotected phages to act immediately in the proximal gastrointestinal tract (GIT; e.g., crop) with encapsulated phages having greater activity once released from capsules in the distal small intestine. While no deleterious effects of the phages were observed on the chickens themselves, continuing studies should more comprehensively assess host-response to phage treatment including potential impact on microbial communities throughout the chicken GIT.