Concurrent infections of Newcastle disease virus and Escherichia coli in layer chicken and their pathobiology

Introduction

The livestock sector is an important subsector of agriculture in the Indian economy and contribute 12.99% growth rate. The contribution of livestock in total agriculture and allied sector gross value added has increased to 30.23% in 2022-23. India ranks second in egg production and 5 th in the meat production in the world (G.O.I CAGR-2014). A significant proportion of farmers involved in the poultry production for their economic status. Animal husbandry sector contributes to about 4.5%-5.5% of the total gross domestic product (GDP) in India. The poultry and livestock production contribute major protein sources to the human population and improving climate change (O.I.E 2017) [10] . The contribution of poultry sector to Indian economy is 1.2% of India's GDP. However, the poultry population facing lot of challenges with colibacillosis and Newcastle disease that landed in severe economic loss to the farming community. ND as a viral disease-causing heavy mortality and low egg production. The birds affected with ND commonly seen with gastrointestinal hemorrhage and ulcers. Because of economic values the ND is designated as a reportable disease to OIE (O.I.E 2017) [10] . Colibacillosis cause high mortality in poultry in all age group of birds. The major clinical findings observed in the colibacillosis including poor growth, low egg production and poor egg quality. The diagnosis of E. coli is based mostly on the culture studies from the infected tissue samples. (Abalaka et al. 2017) [1] Abalaka. The confirmation of ND virus was carried out using HI test (Sajo et al. 2023; Ganesan et al. (1992) [14,8] In this outbreak case, the clinical signs exhibited by the infected poultry population, the laboratory studies for the confirmations of ND virus with E. coli and P.M findings were carried out.

Case details

Continuous mortality i.e. 302 birds out of 1200 in a layer poultry with cage system of rearing at the age of 18 weeks and again at 35 weeks of age with peak egg production was reported in a layer poultry farm in R. Sitapura village of Odisha state, India. This farm was running with a flock size of 1200 pullets procured from a local commercial farm and it faced the outbreak of ND and coli septicemia at the age of 18 weeks and at the age of 35 weeks. The birds were fed with commercial feed and untreated bore water. The pullets were said to be vaccinated with Lasota strain by the commercial farmer twice during grower stage. The chicken at pullet stage and the at the layer stage were examined for their clinical manifestations. The birds suffered with, nasal discharge, low intake of feed, high mortalities and morbidities in the pullet stage and in laying stage additional clinical signs with low egg production and leathery eggs. The mortality started at the age of 18 weeks and the PM studies, serological study for ND virus and the culture studies for E. coli confirmed the prevalence of both NDV and E. coli. The chicken was treated with antibiotics as per antibiogram during pullet stage and were vaccinated with NDV R2b mesogenic strain. The chicken recovered from the infection's and started laying eggs. However, a second outbreak occurred and the etiological agents were again confirmed by clinical signs, P.M findings, culture studies for coli septicemia and H.I studies for NDV.

Clinical studies: Clinical studies on the ailing population of the pullets and the layer status exhibited cough, mucopurulent nasal discharge, high mortality and morbidity in both pullet and layers, and in addition low egg production in the layer stage.

Pathological examination (PM)

Post mortems were performed both in pullet and layer stages. The examinations revealed the lesions of proventricular hemorrhage (Figure 1), cecal hemorrhage (Figure 2), intestinal ulcers, necrotic duodenal loop (Figure 3), splenitis (Figure 4), mucoid trachea (Figure 5), hydro pericardium (Figure 6), friable liver& cholecystitis (Figure 7), petechial hemorrhages in the heart muscles (Figure 8), fibrin deposits in the heart (Figure 9) and in liver (Figure 10), necrotic cecal tonsil (Figure 11), congested comb and brain (Figure 12), egg yolk/salpingo-peritonitis (Figure 13), pedunculated ovarian follicles/salpingitis (Figure 14).

Bacteriological examination

Cultural isolation studies were carried out by using the homogenized E. coli infected tissue materials i.e. trachea, spleen and intestinal contents in MacConkey's agar media and stained with Gram's staining method. Biochemical confirmation studies carried out with catalase, triple sugar iron, Indole, oxidase and citrate tests as per the methods of Olutiola et al. (1991) [11] . The HI test was performed both in pullet and the in layer stage as per the procedures of World Organization for Animal Health (WOAH, 2021) [15] .

Treatment

Based on the clinical history, P.M findings, bacterial culture and serology, the outbreak was diagnosed as Newcastle disease virus with Escherichia coli as a concurrent infection in the layer poultry. The birds were treated with antibiotic as per antibiogram studies and the birds recovered from the clinical infections after a period of 4 weeks post treatment period during the first outbreak. The second outbreak which occurred in these birds at the age of 35 weeks were again confirmed for the above infections with the clinical signs, P.M findings, bacterial culture and HI tests for NDV.

Discussion

Newcastle disease and Escherichia coli combined infection were reported for their challenging approach in the poultry farming practices (Abalaka et al. 2017, 2021) [1] Abalaka. Badau et al. 2024 [4] ; Abalaka et al. (2017) [1] reported the failure of protection chain by various diseases like Infectious bursal disease (IBD) and Avian influenza (HPAI). Qamar-un-Nisa et al. (2018) [13] reported the concurrent infection of ND virus with Escherichia coli infection in broilers, where the infection of E. coli enhanced the degree of severity of clinical signs, gross lesions and death rates i.e.25% which is in agreement with the present case studies. Bitrus et al. (2025) [5] reported the PM lesions in the outbreak of ND virus with colibacillosis in a 7 weeks old pullets which included respiratory related lesions, cloudy air sacs, splenitis, mottled liver, inflamed cecal mucosa, proventricular hemorrhages, petechial hemorrhage of the intestinal mucosa, lesions in the ovarian follicle, oviduct, peritoneum, abdominal cavities and hydro pericardial syndrome. The HI titre values at 18 weeks of age revealed 0 log 2 being below 4 log 2 titre values which is considered as the minimum protective titre values against the lethal effect of field ND virus. Bitrus et al. (2025) [5] suggested these values for minimum protection against field ND virus, which coincides with the finding of this study. The birds were immunized again after this outbreak with R2b mesogenic strain of NDV. However, second outbreak again occurred at the age of 35 weeks with high mortality and the egg production reached 0 level with a greater number of leathery eggs. [5] . Amyleigh Watts and Paul Wigley (2024) [3] reported the P.M finding such as swollen head, air sacculitis, exudate in lungs, nephritis lesions, salpingo-peritonitis, caseous perihepatitis, spotted liver lesions, pericarditis often with fibrinous deposits in the E. coli infected poultry population. The above P.M findings coincides with the finding of the present studies. Oshadu et al. (2025) [12] reported the clinical signs of retarded growth, low egg yield, poor-quality eggs, nasal discharge, congested combs and wattles, with the PM findings of pericarditis, friable liver, misshapen and pedunculated ovarian follicles, necrotic cecal tonsils, frothy lungs, egg yolk peritonitis, petechial hemorrhages in the papillae of the proventriculus, mucoid trachea, pale spleen, empty crop, and live worms in the intestine. Further the authors reported HI titre values of the infected layer poultry of one year age with ND virus and it ranged between 1:16-1:1024, where the authors considered 1:128 as the minimum protective titre values to avoid drop in egg production against the effect of ND virus. Ganesan et al. (1992) [8] reported HI titre values ranged between 0-40 in layer poultry with drop in egg production due to mild form of Newcastle disease and the recovery of the layer birds for restoration of the egg production status by boosting the HI titre values by vaccinating the layer population with RDVK mesogenic vaccine.

In this case study at the age of 18 weeks, mortality started among the pullets. On an average 18-22 pullets were brought for PM studies for a period of 10 days and the scattered mortality reported for a further period of 20 days. After confirmation of the diseases for ND virus and Escherichia coli infections, the birds were immunized with mesogenic R2b ND virus strain followed by antibiotic treatment and calcium supplementation therapy successfully. However, a second outbreak occurred at the age of 35 weeks in this group of birds when the birds were in peak egg production. Vaccination failure occurs due to inadequate cold chain facilities and lack of monitoring of vaccine seroconversion in birds and a lack of effective implementation of farm biosecurity measures. (Edward and Ogbonna 2023) [6] . Sajo et al. (2023) [14] observed poor seroconversion of HI antibody titre values by performing HI test by ND virus vaccines with live attenuated vaccines in the birds already vaccinated and reported severe form of ND in the vaccinated birds which shed the virus continuously in the droppings. The endemic status of the NDV could be a result of emergence of new NDV sub types (Alsahami et al. 2018) [2] . Badau et al. (2024) [4] suggested the lesions in the affected birds i.e. the petechial lesions in the intestine, the proventricular hemorrhages in the papillae and congested cecal mucosa could be due to the acute stage of viscerotropic velogenic virus infection. In this case the birds were provided with bore water continuously. The bore water studies by culture examination confirmed the E. coli infection and it is suggested that the E. coli infection exacerbated the field ND virus of velogenic nature. The E. coli was confirmed in these episodes by lesions and culture studies and it was attributed as one of the major pathogenic factors for the development of lesions in the ovarian system, pericardium and kidneys. Even though the farm personal was advised to provide sanitized water supply after the first outbreak at the age of 18 weeks, the coli septicemia occurred which could be attributed to sanitary insufficiency by violating the biosecurity norms recommended which included delayed disposal of the droppings, improper feed storage, separation of the infected birds, good personnel hygiene records. Prevention of these diseases not achieved by the failure in comprehensive biosecurity, and regular monitoring of both ND virus and E. coli organisms by serology and culture studies. The repeated occurrences of both NDV and coli septicemia suggested a high virulent nature of NDV strain involvement as the area is endemic for ND virus and the disease intensity was precipitated by E. coli infections because of the contaminated water source.

Conclusion

The case study revealed the concurrent infection status of ND virus with Escherichia coli in the affected chicken in both outbreaks at the age of 18 and 35 weeks. These studies emphasize the importance of disease surveillance, monitoring, vaccine potency, vaccination protocol, biosecurity measures to prevent the outbreak of ND complicated with E. coli infections.

Conflict of Interest Not available

Financial Support

References

1. Abalaka SE, Sani NA, Idoko IS, Tenuchu OZ, Oyelowo FO, Ejeh SA, et al. Pathological changes associated with an outbreak of colibacillosis in a commercial broiler flock. Sokoto J Vet Sci. 2017;15(3):95-102.
2. Alsahami A, Ideris A, Omar A, Ramanoon SZ, Sadiq MB. Sero prevalence of Newcastle disease virus in backyard chickens and herd level risk factors of Newcastle in poultry farms in Oman. Int. J Vet Sci. Med. 2018;6(2):186-191.
3. Watts A, Wigley P. Avian pathogenic Escherichia coli: An overview of infection biology, antimicrobial resistance and vaccination. Antibiotics (Basel). 2024;13(9):809.
4. Badau SJ, Musa MA, Yoksa DT. Assessment of Newcastle disease cases in Maiduguri based on gross necropsy findings from 2013 to 2023: A retrospective study. Sahel J Vet Sci. 2024;21(2):19-26.
5. Bitrus AA, Dagare JM, Oladele OO, Nguket DB, Gurumyen GY, Tanko PN, et al. Outbreak of Newcastle disease with colibacillosis in seven-week-old pullets: Diagnosis and management. Sokoto J Vet Sci. 2025;23(1):54-59.
6. Edward I, Ogbonna I. Assessment of biosecurity measures of poultry farmers in commercial poultry farms in Ikot-Ekpene and Uyo LGA, Akwa-Ibom State. J Sustain Vet al.lied Sci; 2023. DOI: 10.54328/covm.josvas.2023.001
7. Ekiri AB, Armson B, Adebowale K, Endacott I, Galipo E, Alafiatayo R, et al. Evaluating disease threats to sustainable poultry production in Africa: Newcastle disease, infectious disease, and avian infectious bronchitis in commercial poultry flocks in Kano and Oyo States, Nigeria. Front Vet Sci. 2021;8:730159. DOI: 10.3389/fvets.2021.730159
8. Ganesan PI, Appaji Rao VN, Venugopal K. Effect of immune status to Ranikhet disease on production performance in layer poultry. Indian Vet J. 1992;69:199202.
9. Government of India. Computer and Auditor General of India report. New Delhi; 2014.
10. World Organisation for Animal Health (OIE). Animal Population Data. Paris: WOAH; 2017.
11. Olutiola PO, Famurewa O, Sonntag HG. Biochemical reactions of microorganisms. In: An introduction to General Microbiology: A practical approach. Heidelberg: Heidelberger Verlagsanstalt und Druckerei GmbH; 1991, p. 157-78.
12. Oshadu DO, Kogi CA, Biallah MB, Magaji DA, Oladele OO. Management of colibacillosis, Newcastle disease and Ascariasis in a flock of one year-old layer chickens. Sahel J Life Sci FUDMA. 2025;3(3):406-412.
13. Nisa MUN, Younus M, Rehman MU, Maqbool A, Umar S. Pathological alterations during co-infection of Newcastle disease virus with Escherichia coli in broiler chicken. Pakistan J Zool. 2018;50(5):495-503.
14. Sajo MU, Hamisu TM, Saidu AS, Haruna NM, Shettima YM, El-Yuguda AD, et al. Comparative analysis of Newcastle disease virus shedding from naturally infected breeds of poultry in Maiduguri, Nigeria. Sahel J Vet Sci. 2023;20(3):33-37.
15. World Organisation for Animal Health (WOAH). Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Newcastle disease. Paris: WOAH; 2021.