Bacteriophage therapy in effective control of E.Coli infections in poultry

Published: August 5, 2019

By: Dr. Ajay Chalikwar / Zonal Technical Manager, Provet Pharma PVT. LTD. Chennai, India.

INTRODUCTION

The poultry market in India is a fast growing and demanding market.
India's poultry industry has transformed from a mere backyard activity into a major commercial activity in just four decades.
India is now the world's 4^th largest egg producer and the 5^th largest producer of broilers.
The Indian poultry market witnessed an increasing trend over the past five years, growing at a compound annual growth rate of 11.39%.
India's per capita consumption of the poultry meat is estimated at around 3.1kg/year, which is low compared to the world average of around 17 kg per year.
The per capita availability of egg is 69/year in India which is also low compared to the world average 179/year. (Source: Poultry entrepreneurship programme 2019)
This is putting tremendous pressure on birds in terms of weight gain, feed conversion efficiency, disease challenge and antimicrobials’ resistance.
Major challenges are bacterial, viral, metabolic, mycotoxins, etc.
E.Coli is emerging as major infections in broilers, layers and breeders and irrespective of good water sanitation programme it is commonly encountered in almost all the poultry operations at some time period including hatchery, meat processing plant, etc.
Let’s discuss regarding E.Coli infections in poultry and its effective control through use of non-conventional therapy like BACTERIOPHAGE.

BRIEF ABOUT E.Coli

E.Coliis a Gram-negative, optional anaerobic, rod-shaped, non-spore forming, coliform bacterium belongs to Enterobacteriaceae.
These strains of E.Coli falls under APEC (Avian pathogenic E.Coli) and most of the strain found in poultry are extra-intestinal.
In poultry, mostly systemic diseases are seen unlike mammals, where gut related diseases are commonly seen with E.Coli.
Most strains are motile having peritrichous flagella.
The organism survives freezing and persists for extended periods at cold temperatures (1).
Reproduction of most strains is inhibited by a pH of less than 4.5 or greater than 9, but the organism is not killed.
It is commonly seen as secondary infection to other bacterial/viral infections or sometimes as primary infectious agent but recent trends shows that they have ability to cause disease on their own.
It is a typically localized or systemic infection when host defences have been impaired.
Most frequent cause of mortality.
Even can be isolated from dead birds.
Normally poultry house dust contains 10⁵to 10⁶CFU/gm of E.Coli inside the houses and outside up to 40 feet area under dry conditions but wetting reduces the count by 84-97%.
E.Coli produce variety of diseases in poultry like Omphalitis, salphingitis, egg yolk peritonitis, colisepticemia, Colibacillosis, cystic degeneration of ovarian follicles, cellulitis, coligranuloma, arthritis, sternal bursitis, etc.

Economic impact of APEC is mainly due to

Mortality
Morbidity
Slower growth rates
Poor FCR
Decrease in egg production
Carcass downgrading
Spreads antimicrobial resistance to other bacteria

Antigenic structures and toxins of E.Coli

Antigens are classified according to Kauffmann scheme (2).
It contains 3 major antigens like O (somatic), H (flagellar), K (capsular), F (Pilus).
Currently, there are approximately 180-O, 60-H and 80-K antigens (3)
The O antigen determines serogroup and addition of the H antigen and sometimes K antigen determines serotype (4).
APEC doesn’t produce enterotoxins unlike mammalian species.
Immune response in poultry is directed against O antigen (5).
Common serotypes found are O1, 02, 018, 035, 036, 078, 0111 (6)

Virulence factors involved

Adhesins- It may be fimbrial or non fimbrial (7), F1 fimbriae may be expressed during initial colonization whereas P fimbriae are expressed later when the organism is in the lower respiratory tract or body tissues.
Bacteria are killed rapidly following expression of F1 fimbriae by macrophages (8).
Iron acquisition methods- Ability of APEC to obtain iron is well documented (9).
Protectins – Resistance to APEC to complement is related to K1 capsule (10) , smooth LPS layer (11) , Outer membrane protein (12) , Increased serum survival gene (ISS) in APEC has its role in complement resistance with a CoIV plasmid which increase virulence in day old chicks by 100 times (13).

Factors that increase host susceptibility to E.Coli infection

Virus- Adenovirus (14), Avian Influenza virus (15), Newcastle disease virus (16), Infectious Bronchitis virus (17), Avian Metapneumo virus (18), Infectious bursal disease (19), Marek’s disease virus (20), Chicken infectious anaemia virus (21).
Bacteria – Mycoplasma Gallisepticum, Pasteurella Multocida, Staphylococcus sp., Streptococcus sp. (22).
Others – Invasion of skin barrier (23), Excess ammonia in sheds (24), mycotoxins like Ochratoxin (25) and Fumonison (26), young age group (27), stress of various origin (28), sex- male are more prone (29), contaminated water and feed (30), dry dusty condition (31), feed/water restriction (30), parasitic infection (32), Intestinal Coccidiosis (33), Caecal coccidiosis (34), inadequate ventilation (35), dysbacteriosis, relatively high ammonia (36) overcrowding, poor litter condition, temperature extremes and high temperature and high humidity (1).
Nutritional – Hypervitaminosis E (37), Hypervitaminosis A & Vitamin A deficiency (38), excess Iron & Selenium.
Adult house flies acts as mechanical vectors and harbour organism in their body and cause horizontal transfer of antibiotic resistance and virulence genes.
Low incubation period – 3-5 days.

Bacteriophage therapy in effective control of E.Coli infections in poultry - Image 1

Bacteriophage therapy in effective control of E.Coli infections in poultry - Image 2

Bacteriophage therapy in effective control of E.Coli infections in poultry - Image 3

Resistance of APEC (E.Coli) to heavy metals, disinfectants and antibiotics

APEC has ability to acquire resistance to Chlorohexidine, Formaldehyde, hydrogen peroxide, and quaternary ammonium compounds through acquiring resistance often encoded by large R plasmids (47).
APEC Inc H12 plasmid, PAPEC-O1-R plasmid confers resistance to silver, copper sulphate, Benzolkonium chloride following transfer of plasmid to recipient strain by conjugation (48).
APEC Inc F IIA plasmid confers resistance to aminoglycosides, quaternary ammonium compounds, phenolic, β lactam antibiotics and silver (49).
APEC Inc A/C plasmid confers resistance to aminoglycosides, sulpha drugs, quaternary ammonium compounds, phenolic, tetracycline and mercury (50).
It has inherent ability to form biofilm on abiotic surfaces like plastic/metal waterlines (51) and protect itself from getting killed by the sanitizers.
Certain plasmids may also contribute to APEC’s acid and bile tolerance, affecting survival of APEC in the bird or elsewhere (52)
But there is some regional variations on certain strains of APEC as per as resistance is concerned, some strains are still susceptible (53)

E.Coli Control through use of bacteriophage

Bacteriophage (54) is a non-pathogenic virus belonging to family with vast majority (about 96%) of known phages belongs to the Myoviridae, Podoviridae and Siphoviridae (55).
They are known as bacterial parasites because they lack the cell structure and enzyme systems necessary for food uptake, protein synthesis or construction of new particles, and as incomplete, organisms can only replicate in a live cell (56).
Positive results of the use of bacteriophages in fighting bacterial infections have contributed to the development of research on the potential use of viruses that destroy bacteria in treatment of diseases in both human and animals (57).
Their specificity and range of activity is determined by the presence of receptors located on the surface of bacterial cells, among which we can distinguish LPS fragments, fimbriae and other surface proteins (58).
No mortality was observed in chickens treated with 10⁸ PFU of an E. coli bacteriophage mixture, another positive effect of the treatment was the absence of visible clinical symptoms (59).
Due to the specificity of bacteriophages for particular bacteria, manifested as the ability to infect only one species, serotype or strain and this mechanism of action does not cause destruction of the commensal intestinal microflora.
It should be cocktail of more than one phages (at-least 3-4) in sufficient concentration to get synergistic effect.

Bacteriophage has 2 life cycles

Lytic-

Here phage act like typical virus, it hijack its host cell and uses cell’s resources to make lots of new phages, thereby causing lysis of the cell and die subsequently.

In lytic phase the PHAGE will attach, enter inside the cell, copies DNA and synthesize protein, assembles new phages and cause lysis of the host cell and release of daughter phages capable of infecting other cells (60).

Lysogenic-

It allows a PHAGE to reproduce without killing its host.

Some phages can use the lytic life cycle but few phages can switch between the two cycles.

In lysogenic life cycle there will be attachment of phage to host cell, injecting DNA into host cell, integration with bacterial chromosome and becoming prophage and finally cell division along with host cell.

Under the right conditions, the prophage can become active and come back out of bacterial chromosome and triggering lytic life cycle and start killing host cell (61).

Bacteriophage therapy in effective control of E.Coli infections in poultry - Image 4

Advantage of cocktail of phages

Phage 1 –The lead phages – It has a very virulent activity and neutralize a bacterial population within few hours of inoculation against wide spectrum of E.Coli in poultry.
Phage 2 – The associate phage – It has lower virulence than lead phage but has very wide spectrum of activity and very stable, this phage can cover the bacterial population which invaded action of lead phage.
Phage 3- The guard phage – It is similar to associate phage but it can attach to a different king of receptor and different mode of action in lytic action compared to lead and associate phage and it is mainly added to ensure protection against resistant E.Coli.

Synergistic action of these 3 phages will ensure complete bactericidal action.

CONCLUSION

Bacteriophage therapy is a very good solution as preventive measure in chick in first week itself and as therapeutic thereafter as per the infection pressure.
Some authors suggest that similar effects of bacteriophage therapy like antibiotics will be seen preventing early development of colibacillosis in chicks (63).
There are evidences that bacteriophages have the ability to penetrate the blood-brain barrier, and confirmed that the bacteriophages had a prophylactic effect in addition to the therapeutic effect (64).
Bacteriophage can be given as orally, through aerosol spray or parenterally or along with antibiotics in case of severe E.Coli challenge (64).
Bacteriophage can be given along with all other live and inactivated vaccines (except live E.Coli vaccine).
Bacteriophage can enter in blood circulation upon oral administration and controls E.Coli systemically (64).

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