In Vivo Effect of Novel Lytic Phage Cocktail against Salmonella Pullorum

Rida Haroon Durrani1, Ali Ahmad Sheikh1*, Masood Rabbani1, Muti-ur-Rehman Khan2, Aneela Zameer Durrani3, Salman Ashraf1, Muhammad Anas Naeem1, Muhammad Usman3 and Tauqeer Mahmood4 1Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore 54000 2Department of Pathology, University of Veterinary and Animal Sciences, Lahore 54000 3Department of Clinical Medicine, Faculty of Veterinary Sciences, University of Veterinary and Animal Sciences, Lahore 54000 4Poultry Research Institute, Rawalpindi 44000, Pakistan.


INTRODUCTION
W ith the increase in the global population (7.9 billion), poultry has become the fastest-growing livestock sector to meet the expanding nutritional needs and provide a livelihood to rural communities. The poultry industry is anticipated to contribute $322.5 billion to the market, with Asia being the largest egg-producer with 64% of the output (FAO). The universal success of poultry products is owed to their inexpensive availability, consumer convenience, nutritional properties, and absence of any social and religious taboos (Petracci et al., 2019). Despite advancements, avian salmonellosis continues to cause significant production and economic losses, especially in developing countries (Berhanu and Fulasa, 2020).
Pullorum disease (PD) is a septicemic disease of avian species caused by Salmonella enterica subsp. enterica serovar Gallinarum biovar Pullorum that often manifests in young birds (2-4 weeks). Whereas atypical signs (asymptomatic carriers) are manifested in mature stock with loss of productivity leading to reduced hatchability O n l i n e

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Antibiotic prophylaxis is a common veterinary practice in the poultry and calf-rearing industry in many underdeveloped countries as a surrogate for inadequate managemental practices, but at the cost of escalating antibiotic resistance and drug residues (active metabolites) in the food chain at slaughter, if no withdrawal requirements are followed (Mohsin et al., 2019). The decreased efficacy of available antibiotics has led to renewed interest in lytic bacteriophages and derived products. There are numerous studies on the use of single phage or mixture of phages against pathogens of veterinary importance cited in literature includes S. Enteritidis, S. Typhimurium, C. jejuni, C. coli, S. aureus, P. aeruginosa, E. coli K1 + , E. coli O2, enteropathogenic E. coli, E. coli O157: H7 and A. pyogenes in chickens, cows, dogs, calves, sheep, and steers (Morris Jr et al., 2001). Keeping this in view, the present study was designed to test the in vivo efficacy of a four phage cocktail against Salmonella Pullorum to investigate the influence of delivery routes and schedule among experimental birds. For this purpose, log 10 -reduction of bacterial count in intestinal tissue was used as an indicative parameter.

Shed preparation
The shed was disinfected with Medisep®, which is a quaternary ammonium compound (QAC) and fumigated with 40% of formalin per cubic meter of space. Following fumigation, the shed was sealed tight for the next 48 h (FAO Guidelines for Cleaning and Disinfection of Poultry Farms).

Experimental birds and their maintenance
Day-old SPF (Salmonella) Hyline w-36 chicks (n=360) were procured from a commercial (avg. 40-45 g/bird). Rapid slide agglutination test was performed to ensure that the birds were seronegative.
The conventional floor space was provided at 100 -200 cm 2 /bird. For the first three days, the temperature was maintained at 33-35ᵒC. The temperature was re-adjusted every three days as per the managemental guidelines (FAO, 2021a). The light intensity was kept uniform at 30-50 lux with 60% relative humidity. The air quality was maintained at 4 m 3 /kg/hour from the west to east direction (Hy-Line w-36 Managemental Guide).
Ready-made starter feed was offered to the birds during the trial. The feed and water intake were measured as 13-16 g/bird/day and 20 mL/bird/day, respectively.
The feed and water were tested prior to being Salmonella negative via PCR. Chickens were cared for using protocols approved by the Ethical Review Committee for Animal Handling of the University of Veterinary and Animal Sciences, Lahore.

Experimental design
The birds in the positive control group were given an oral challenge with S. Pullorum (10 5 CFU/mL per 60 g of body weight). The birds in the negative control group remained unchallenged and untreated. Five experimental birds from the negative group were euthanized at 0, 7, 14 and 21 days to ensure sterility of the experiment (via PCR). The birds in the third control group were given phage cocktail via all three delivery routes to monitor shedding (i.e., internalization) and the occurrence of adverse reactions (if any). The birds in the fourth group were given phage cocktail as prophylaxis to monitor the effectiveness of the cocktail at preventing systemic infection or reducing the clinical severity of disease (upon challenge). The birds in the fifth group received challenge infection prior receiving phage cocktail 6 h post-infection (PI) to evaluate the impact of the cocktail as immediate therapy. The sixth group also received challenge infection prior to phage treatment, but with a 36h delay assessing the effect of the phage cocktail during active infection ( Table I). The number of birds in all experimental subsets was 30 and were placed in different sheds with similar housing requirements. 05 birds from each treatment group on the 3 rd , 6 th and 9 th day PI, were euthanized via cervical dislocation, and intestinal tissue (pooled sample ileum/cecum) was collected aseptically for estimation of log 10 -reduction in Salmonella count. The quantitative enumeration of bacteria was carried out following method of Cappuccino and Sherman (2014).

Host bacterial strain and bacteriophage cocktail
The challenge bacterial strain, Salmonella Pullorum (SP-1628), was confirmed by conventional and molecular techniques at the national reference laboratory for poultry diseases (NRLPD). The phage cocktail was concoction of four lytic phages, isolated from sewage/sludge samples procured from commercial poultry units. The cocktail demonstrated optimal activity against motile serovars, such as Salmonella Enteritidis and Salmonella Typhimurium, tolerance to moderately high temperatures above 62ᵒC with a log10 of 8.73 PFU/mL, and activity in acidic and alkaline conditions. At 150 min, the cocktail reduced the bacterial count by log 10 2.93 with 100 MOI. The growth kinetics of all four monophage suspensions (SalØ_ABF37, SalØ_RCMPF12, SalØ_MCOH26 and SalØ_DNLS42) showed an average latent time between 33 to 46.7 min with burst sizes given 73, 97, 75 and 132 PFU CFU -, respectively. All isolates were revealed to possess dsDNA during nucleic acid characterization.

Statistical analysis
The data obtained from experimental trials was collected and organized into Microsoft Excel spread sheets (Office 2016). To check for significant differences in bacterial concentrations as a result of delivery routes and timing data was analyzed with generalized linear model (GLM) using Minitab 17.0.1. Differences were considered statistically significant at (P ≤ 0.05). Post-hoc mean separation was used to test for pairwise differences among experimental groups.

Control group
The birds of positive control group developed nonspecific signs like depression, weakness, somnolence, loss of appetite, drooping of wings, huddling, dehydration and ruffled feathers post 36 h of challenge infection. Gasping, white pasty diarrhea, and pasting of the vent feathers were also seen at 72 h PC. The bacterial count was enumerated to be log 10 7.17 CFU in intestinal content (ileum/cecum).
5% mortality was observed in negative control group birds under normal circumstances (i.e., drowning in drinker). The birds remained free of infection for whole the duration of trial. 46.67% (drinking water) and 53.3% (feed additive) of birds shed bacteriophage in their feces. No bacteriophage shedding was observed in experimental birds that received cocktail intranasally. All the birds remained clinically normal and maintained a healthy weight of between 60 -73 g/bird.

Prophylactic group
In the prophylactic group, a significant (P ≤ 0.05) log 10 reduction in S. Pullorum was seen in comparison to the untreated positive control group with a mean log 10 7.17 CFU. The pairwise comparison was significant (P ≤ 0.05) for all delivery routes, with higher log 10 -reduction seen in the oral groups than recipients with nasal delivery. On 3 rd dpc, log 10 1.43 (96.32%) and log 10 1.92 (98.81%) CFU reduction was observed in birds that received cocktail through drinking water and as feed additive, while log 10 0.85 CFU (86%) were recorded for nasal delivery subjects. Subsequently, on the 6 th dpc, the counts were log 10 1.17 (93.33%) and log 10 1.629 (97.65%) CFU for oral delivery routes and no reduction in bacterial count was observed for nasal delivery route. The efficacy of phage cocktail as prophylaxis did not yield efficient log 10reduction on 9 th dpc, by only reducing bacterial numbers by log 10 0.334 (53.67%) and log 10 0.44 (63.93%) CFU for oral delivery routes, i.e., by water and as a feed additive with no log 10-reduction seen in experimental birds treated with phage cocktail via nasal delivery. The delivery of phage cocktail as pre-challenge choice resulted in reducing the clinical severity of disease with 15% mortality in immunocompromised chicks.
A significant (P ≤ 0.05) log 10 reduction was seen in the second delivery schedule of phage therapy, i.e., immediate therapy in comparison to the untreated positive control group with a mean log 10 7.17 CFU. Non-significant (P ≥ 0.05) pairwise comparison was seen between delivery Phage Based Biocontrol of White Bacillary Diarrhea

O n l i n e F i r s t A r t i c l e
routes. The phage cocktail delivery by oral routes, i.e., by water and as a feed additive, modulated S. Pullorum count by log 10 0.85 (86%) and log 10 1.02 (90.53%) CFU on the 3 rd dpc. The efficacy of phage delivery as immediate therapy did not reduce bacterial count on 6 th and 9 th dpc, with 53.33% mortality in immunocompromised birds. The group in which the treatment was delayed to 48 h generated similar results in terms of bacterial reduction with non-significant (P ≥ 0.05) pairwise comparison recorded for the delivery routes. A reduction of log 10 1.2 (93.78%) and log 10 1.55 (97.21%) CFU was seen in experimental birds treated with a phage cocktail mixed in water and feed on the 3 rd dpc. On the 6 th dpc, the efficacy of phage cocktail mixed in water and feed was reduced to log 10 0.77 (83.33%) and log 10 0.66 (78.26%), respectively. On 9 th dpc, the phage cocktail only reduced bacterial incidence to log 10 0.319 (52.13%) and log 10 0.228 (40.86%) CFU in oral delivery routes, i.e., by water and as a feed additive, respectively. 93.3% experimental birds that received cocktail through nasal route died with no significant reduction in bacterial numbers suggesting that nasal delivery did not modulate bacterial incidence in presence of active infection.
Summing up, the logarithmic reduction was concurrent with the GLM which affirmed that delivery schedules are significantly different in reducing bacterial incidence. However, the oral delivery routes, on the other hand, are non-significant i.e., they produced similar log 10reduction in bacterial count, with nasal delivery being least effective out of all delivery routes, as seen in Figure 1.

DISCUSSION
Salmonellosis and poultry have been linked epidemiologically and economically since the commercialization of poultry industry. Although PD or white bacillary diarrhea, caused by S. Pullorum is largely eliminated from developed countries, but PD remains an under-reported problem in many under-developed countries with free-range backyard and intensively reared commercial poultry. The experiment carried out in the present study resulted in significant reduction in bacterial incidence for oral and nasal phage delivery. The findings of the present study are supported by literature where several routes of administration have been shown to influence the impact of therapy (Andreatti-Filho et al., 2007;Borie et al., 2009;Carlos et al., 2009;Hong et al., 2013;Huff et al., 2006;Rozema et al., 2009).
The number of days also had a significant effect on bacterial reduction. The timing of delivery although yielded significant effects in all experimental groups, but phage delivery 72 h before and 48 h after challenge showed significant results, whereas the group that received immediate treatment of cocktail produced the lowest reduction in bacterial numbers. The present study is in accord with several studies cited in literature that suggest delivery of phages too early may result in low threshold of multiplication but can significantly reduce the clinical severity and delay attachment or colonization (Hong et al., 2013;Lim et al., 2011;Wagenaar et al., 2005).
The positive control group of study had an incidence that reached 100% where 46% birds exhibited acute mortality within 36 h of challenge, while the rest expired within 72-96 h post-challenge. The results are in accord with the study of Carlos et al. (2009) who suggested a 100% incidence of infection in positive control group with their 3 phage cocktail lowered S. Enteritidis incidence to 80% alone and 75.5% in presence of a competitive exclusion product broilact (Carlos et al., 2009).
The feces from cocktail control group also showed that the candidate isolates passed down the gastrointestinal system upon re-isolation of lytic bacteriophages with 3 different plaque morphologies i.e., 3 out 4 strains survived in the digestive tract while one lost its viability. This is a significant finding in regard to the in vivo application of lytic phages, as the particles were able to survive and replicate in host as cited by (Colom et al., 2015;Toro et al., 2005).
The use of quadcocktail in the present study was to reduce the possibility of selection of resistance against a particular candidate bacteriophage. For this purpose, cocktails having two or more bacteriophage isolates are chosen as combating tool cited in literature. Additionally, using cocktails is efficient to reduce the risks associated with the multiple dose and continuous administration of bacteriophages and to make this methodology more costeffective and practical under commercial prospects (Bielke et al., 2007;Borie et al., 2008).
The results obtained in this preliminary study can lay foundation for cost-effective futuristic approach O n l i n e

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Phage Based Biocontrol of White Bacillary Diarrhea 5 towards biocontrol of salmonellosis with further work can be established in terms of stability and formulation parameters for efficacious delivery and greater retention.

CONCLUSION
The results of this study are a preliminary effort to measure the efficacy of bacteriophages in controlling bacterial infections in chickens. As reported above, lytic bacteriophages significantly reduced the concentrations of target bacteria in the experimentally infected birds. As such, the application of phages in such a manner could provide clinicians and animal producers with additional means to treat bacterial infections.