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Control of Salmonella Gallinarum (Fowl Typhoid) in Poultry with Phage-based Interventions

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posted on 2022-07-27, 20:11 authored by Saud Ur RehmanSaud Ur Rehman

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, Salmonella Gallinarum, the etiological agent for fowl typhoid, is  endemic  in  Pakistan  with  infections  leading  to  high  mortality  and  substantial  economic  loss. Currently, Salmonella 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 Salmonella 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 Salmonella Gallinarum and significantly reduced (P < 0.05) Salmonella 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 Salmonella 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 Salmonella Gallinarum  and  treated  with encapsulated phages. At7 d of age, chicks receiving the bacterial challenge were administered 5 X106CFU (500 μL) of Salmonella 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  <  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, Salmonella Gallinarum concentrationsin  ceca  of  birds  treated  with encapsulated and unprotected phages were significantly lower (P < 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, Salmonella 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 Salmonella Gallinarumin the ceca of birds treated  with  unprotected,  encapsulated,  and  a mixture  of  unprotected  and  encapsulated  phages were significantly  lower  (P  <  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 Salmonella 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, Salmonella 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 Salmonella Gallinarum in  the  ceca  of  birds treated  with mixture  of  unprotected  and  encapsulated phages(2.40  ±  0.55log  CFU/g)  were significantly lower (P  <  0.05) than  those  found  in the ceca  of  untreated  birds(7.08  ±  0.19log CFU/g). Similarly,  concentrations of Salmonella Gallinarum  in the  ceca of  birds  treated  with encapsulated and unprotected phages were significantly lower (P < 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 Salmonella 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.

History

Degree Type

  • Master of Science

Department

  • Animal Sciences

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Paul Ebner

Additional Committee Member 2

Alan Mathew

Additional Committee Member 3

Nicole Widmar

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