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Laishevtsev A.I., Zulkarneev E.R., Pimenov N.V., Kiseleva I.A., Bagandova K.M., Mizaeva T.E., Aleshkin A.V. Biological properties of the Salmonella phage BF-1356 strain and its effectiveness in phage therapy of chick-en pullorosis. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2024, Vol. 59, № 4, p. 799-813.
(how to cite this article)

doi: 10.15389/agrobiology.2024.4.799eng

UDC: 619:578

 

BIOLOGICAL PROPERTIES OF THE Salmonella phage BF-1356 STRAIN AND ITS EFFECTIVENESS IN PHAGE THERAPY OF CHICKEN PULLOROSIS

A.I. Laishevtsev1, 2 ✉, E.R. Zulkarneev3, N.V. Pimenov4, I.A. Kiseleva5, K.M. Bagandova5,  T.E. Mizaeva5, A.V. Aleshkin5

1TsBO Microecologiya, 42/1, Bolshoi Bulvar, territory of Skolkovo Innovation Centre, Moscow, 143026 Russia, е-mail a-laishevtsev@bk.ru (✉ corresponding author);
2Federal Scientific Centre Skryabin and Kovalenko All-Russian Institute of Experimental Veterinary RAS, 24/1, Ryazansky prospect, Moscow, 109428 Russia;
3Plague Control Center of Rospotrebnadzor, 10/4, ul. Pogodinskaya, Moscow, 119435 Russia, е-mail elzz89@mail.ru;
4Skryabin Moscow State Academy of Veterinary Medicine and Biotechnology, 23, ul. Akademika Skryabina, Moscow, 109472 Russia, е-mail pimenov-nikolai@yandex.ru;
5Gabrichevsky Moscow Research Institute for Epidemiology and Microbiology, 10, ul. Admirala Makarova, Moscow, 125212 Russia, е-mail irina6804@mail.ru, kallybagandova@mail.ru, toita.mizaeva@bk.ru, andreialeshkin@googlemail.com

ORCID:
Laishevtsev A.I. orcid.org/0000-0003-1658-1949
Bagandova K.M. orcid.org/0000-0001-5166-9677
Zulkarneev E.R. orcid.org/0000-0002-5920-8098
Mizaeva T.E. orcid.org/0000-0002-0046-9226
Pimenov N.V. orcid.org/0000-003-1658-1949
Aleshkin A.V. orcid.org/0000-0002-0532-1378
Kiseleva I.A. orcid.org/0000-0001-7206-4220

Final revision received March 30, 2023

Accepted June 20, 2023

 

Salmonella Pullorum is a rare but significant pathogen of the genus Salmonella, respon-sible for considerable damage to the poultry industry and economic losses due to high poultry mortality. The potential use of bacteriophage therapy as an adjunctive treatment for poultry has been the subject of considerable debate in recent times. This study demonstrates, for the first time, the therapeutic efficacy of virulent bacteriophage Salmonella phage BF-1356 administered 12 hours after oral infection of cross Cobb 500 chickens (Gallus gallus L.) with a highly virulent strain Salmonella Pullorum № 732-VIEV. The objective of this study was twofold: firstly, to char-acterize Salmonella phage BF-1356 and its interaction with the host bacterial strain in vitro; sec-ondly, to evaluate the efficacy of phage therapy for pullorosis in vivo on the Cobb 500 chicken model. Salmonella strain (Salmonella Pullorum № 732-VIEV) was isolated in 2017 at a poultry farm in the Moscow region. The virulent bacteriophage Salmonella phage BF-1356 from the col-lection of the Gabrichevsky Research Institute of Epidemiology and Microbiology (Moscow, Russia), was used for phage therapy. The purified bacteriophage exhibited a titer of at least 1011 PFU/ml. The sterility of the phage preparation was verified through the incubation of 0.1 ml of phage lysate in 5 ml of fluid thioglycollate medium (HiMedia, India) at 32.5±2.5 and 22.5±2.5 C for 14 days. The host range of bacteriophage lytic activity was determined using 164 bacterial strains of Salmonella enterica isolated in the Russian Federation between 2010 and 2018. The lytic activity was evaluated visually by a lysis zone at the points of bacteriophage application on indica-tor Petri dishes with 1.5 % Mueller-Hinton agar which contained 200 µl of a mid-log-phase cul-ture of the tested strains (106 CFU). The adsorption of Salmonella phage BF-1356 on Salmonella Typhimurium strain B-1025 was evaluated. The latent period and burst size of Salmonella phage BF-1356 were determined by monitoring the dynamic changes in the number of phage particles during the replicative cycle on Salmonella Typhimurium B-1025. In order to evaluate the effect of pH on bacteriophage titer, known concentrations of purified bacteriophages were incubated in buffers at different pH values. Salmonella-free 14-day-old chickens of cross Cobb 500 (n = 40) were used as laboratory objects. The chickens were randomly divided into three groups. Ten chick-ens were used to control housing conditions (I control group not exposed to infection and phage therapy), 20 chickens were used to determine the efficacy of the phage after its use for therapeu-tic purposes and to assess further Salmonella carriage (experimental group), and 10 chickens were used as infection control (II control group infected with Salmonella without bacteriophage ad-ministration). The 14 day-old chicks in the experimental and II control groups were orally infected with Salmonella Pullorum № 732-VIEV at a dose of 3.8½108 cells in 0.4 ml which corresponded to approximately 5 LD50 (the acute infectious dose that guaranteed the modelling of pullorosis in-fection with the complete death of control group subjects). The administration of bacteriophage was initiated upon the onset of symptoms indicative of the infectious process. The chicks in the experimental group were administered bacteriophage orally at a dose of 1010 PFU/ml in 0.2 ml. The presence of phages and Salmonella cells was promptly identified following the act of defeca-tion. The quantity of phage particles was subsequently determined by the double-layer agar meth-od. To confirm the efficacy of phage therapy, the birds in the experimental group were slaugh-tered for pathological and anatomical autopsy and collection of sectional material for laboratory studies and the identification of Salmonella cultures from organs. In the control group, patholog-ical autopsy were conducted as the birds died. Salmonella phage BF-1356 demonstrated specific lytic activity against common Salmonella serovars, including Pullorum, Enteritidis, Typhimurium, Cholerasuis and Infantis. The mean latent period for bacteriophage was 40 minutes, with an aver-age of 67 viral particles released per cell. The bacteriophage demonstrated stability at pH levels between 5 and 8, with a slight decrease in titer observed at pH 3.6 within the first hour. A single oral administration of bacteriophage BF-1356 in a titer of 1010 PFU/ml 12 hours after infection of Cobb 500 chickens with Salmonella Pullorum strain № 732 resulted in 0 % mortality in the popula-tion and a significant reduction in the consequences of the pathological process in the chickens after phage therapy. The chickens in the control group exhibited mortality within a period of 2-6 days. The structure of the pathological lesions was consistent with that observed in salmonellosis, a diagnosis that was subsequently confirmed in laboratory conditions. Despite the absence of a complete elimination of the pathogen from the organism, a notable reduction in the isolation of the pathogen from organs and tissues occurred on day 7, particularly under the infection with 5 LD50. Therefore, the prototype agent based on Salmonella phage BF-1356 demonstrated 100 % therapeutic efficacy and 75-80 % microbiological efficacy. The data obtained indicate the potential of the studied bacteriophage strain for the creation of therapeutic and prophylactic means against pullorosis to be used in poultry farming and in the epizootic control.

Keywords: pullorosis, virulent bacteriophages, adsorption, spectrum of lytic activity, in vivo tests, salmonellosis, generalized form.

 

REFERENCES

  1. Pimenov N.V., Laishevtsev A.I. Rossiyskiy zhurnal sel’skokhozyaystvennykh i sotsial’no-ekonomicheskikh nauk, 2017, 4(64): 257-269 CrossRef (in Russ.).
  2. Pimenov N.V., Laishevtsev A.I. Pimenova V.V. Rossiyskiy zhurnal sel’skokhozyaystvennykh i sotsial’no-ekonomicheskikh nauk, 2017, 2(62): 282-289 CrossRef (in Russ.).
  3. Barrow P.A., Freitas Neto O.C. Pullorum disease and fowl typhoid — new thoughts on old diseases: a review. Avian Pathology, 2011, 40(1): 1-13 CrossRef
  4. Allen H.K., Levine U.Y., Looft T., Bandrick M., Casey T.A. Treatment, promotion, commotion: antibiotic alternatives in food-producing animals. Trends in Microbiology, 2013, 21(3): 114-119 CrossRef
  5. Chen C., Li J., Zhang H., Xie Y., Xiong L., Liu H., Wang F. Effects of a probiotic on the growth performance, intestinal flora, and immune function of chicks infected with Salmonella pullorum. Poultry Science, 2020, 99(11): 5316-5323 CrossRef
  6. Zhang Y., Ding Y., Li W., Zhu W., Wang J., Wang X. Application of a novel lytic podoviridae phage Pu20 for biological control of drug-resistant Salmonella in liquid eggs. Pathogens, 2021, 10(1): 34 CrossRef
  7. Le Bouquin S., Bonifait L., Thépault A., Ledein T., Guillon F., Rouxel S., Souillard R., Chemaly M. Epidemiological and bacteriological investigations using whole-genome sequencing in a recurrent outbreak of pullorum disease on a quail farm in France. Animals, 2021, 11(1): 29. CrossRef
  8. Li X., Nie C., Liu Y., Chen Y., Lv X., Wang L., Zhang J., Yang W., Li K., Zheng C., Jia Y., Ning Z., Qu L. The genetic architecture of early body temperature and its correlation with Salmonella Pullorum resistance in three chicken breeds. Frontiers in Genetics, 2020, 10: 1287 CrossRef
  9. Pan Z.M., Wang X.Q., Zhang X.M., Geng S.Z., Chen X., Pan W.J., Cong Q.X., Liu X.X, Jiao X.N., Liu X.F. Changes in antimicrobial resistance among Salmonella enterica subspecies enterica serovar Pullorum isolates in China from 1962 to 2007. Veterinary Microbiology, 2009, 136(3-4): 387-392 CrossRef
  10. Gao P., Ma C., Sun Z., Wang L.F., Huang S., Su X.Q., Xu J., Zhang H.P. Feed-additive probiotics accelerate yet antibiotics delay intestinal microbiota maturation in broiler chicken. Microbiome, 2017, 5: 91 CrossRef
  11. Sulakvelidze A., Alavidze Z., Morris Jr. J.G. Bacteriophage therapy. Antimicrobial Agents and Chemotherapy, 2001, 45(3): 649-659 CrossRef
  12. Summers W.C. Bacteriophage therapy. Annual Review of Microbiology, 2001, 55: 437-451 CrossRef
  13. Pimenov N.V., Laishevtsev A.I. Sovremennye aspekty bor’by s sal’monelleznoy infektsiey v ptitsevodstve [Modern aspects of the fight against salmonella infection in poultry farming]. Moscow, 2017 (in Russ.).
  14. Pimenov N.V. Rossiyskiy zhurnal sel’skokhozyaystvennykh i sotsial’no-ekonomicheskikh nauk, 2013, 23(11): 16-23 (in Russ.).
  15. Aleshkin A.V., Volozhantsev N.V., Svetoch E.A., Kiseleva I.A., Rubal’sky E.O., Afanas’ev S.S., Borzilov A.I., Zatevalov A.M., Vasil’ev D.A., Zolotukhin S.N., Zeigarnik M.V., Galimzyanov Kh.M., Rubal’sky O.V. Bacteriophages as probiotics: phage-based probiotic dietary supplement in prophylaxis against foodborne infections. Infekc. Bolezni (Infectious Diseases), 2016, 14(2): 31-40 CrossRef
  16. Kropinski A.M., Mazzocco A., Waddell T.E., Lingohr E., Johnson R.P. Enumeration of bacteriophages by double agar overlay plaque assay. In: Bacteriophages. Methods in molecular biology, vol. 501. M.R. Clokie, A.M. Kropinski (eds.). Humana Press, 2009: 69-76 CrossRef
  17. Haines M.E., Hodges F.E., Nale J.Y., Mahony J., van Sinderen D., Kaczorowska J., Alrashid B., Akter M., Brown N., Sauvageau D., Sicheritz-Pontén T., Thanki A.M., Millard A.D., Edouard E., Galyov E.E., Clokie M.R. Analysis of selection methods to develop novel phage therapy cocktails against antimicrobial resistant clinical isolates of bacteria. Frontiers in Microbiology, 2021, 12: 613529 CrossRef
  18. Pajunen M., Kiljunen S., Skurnik M. Bacteriophage φYeO3-12, specific for Yersinia enterocolitica serotype O:3, is related to coliphages T3 and T7. Journal of Bacteriology, 2000, 182 (18): 5114-5120 CrossRef
  19. Capra M.L., Quiberoni A., Reinheimer J. Phages of Lactobacillus casei/paracasei: response to environmental factors and interaction with collection and commercial strains. Journal of Applied Microbiology, 2006, 100(2): 334-342 CrossRef
  20. Metodicheskie ukazaniya po patomorfologicheskoy diagnostike bolezney zhivotnykh, ptits i ryb v veterinarnykh laboratoriyakh [Guidelines for pathomorphological diagnostics of diseases of animals, birds and fish in veterinary laboratories]. Moscow, 2000 (in Russ.).
  21. Metodicheskie ukazaniya 4.2.2723-10. Laboratornaya diagnostika sal’monellezov, obnaruzhenie sal’monell v pishchevykh produktakh i ob’‘ektakh okruzhayushchey sredy [Guidelines 4.2.2723-10. Laboratory diagnostics of salmonellosis, detection of salmonella in food products and environmental objects]. Moscow, 2010 (in Russ.).
  22. Federation of Animal Science Societies. Guide for the care and use of agricultural animals in agricultural research and teaching (4th ed.). American Dairy Science Association, 2010.
  23. Ravindran V. Feed enzymes: the science, practice, and metabolic realities. Journal of Applied Poultry Research, 2013, 22(3): 628-636 CrossRef
  24. Payne R.J., Jansen V.A. Understanding bacteriophage therapy as a density-dependent kinetic process. Journal of Theoretical Biology, 2001, 208(1): 37-48 CrossRef
  25. Kasman L.M., Kasman A., Westwater C., Dolan J., Schmidt M.G., Norris J.S. Overcoming the phage replication threshold: a mathematical model with implications for phage therapy. Journal of Virology, 2002, 76(11): 5557-5564 CrossRef
  26. Atterbury R.J., Van Bergen M.A.P., Ortiz F., Lovell M.A., Harris J.A., De Boer A., Wagenaar J.A., Allen V.M., Barrow P.A. Bacteriophage therapy to reduce Salmonella colonization of broiler chickens. Journal of Applied and Environmental Microbiology, 2007, 73(14): 4543-4549 CrossRef
  27. Gonçalves G.A.M., Donato T.C., Baptista A.A.S., de Oliveira Corrêa I.M., Garcia K.C.O.D., Andreatti Filho R.L. Bacteriophage-induced reduction in Salmonella Enteritidis counts in the crop of broiler chickens undergoing preslaughter feed withdrawal. Poultry Science, 2014, 93(1): 216-220 CrossRef

 

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