Isolation and identification of septicaemic pathogens in Japanese quails

Introduction

Poultry farming plays a major role in providing subsidiary income and nutritional security to the small-scale farmers of India. Presently in India quails have become the third largest avian species to be reared next to chicken and ducks Asha R.R. (2011) [2] . Japanese quail belongs to the order Galformes, family Phasidae, genus Coturnix and species japonica. In India two species are commonly noticed namely Black breasted quail found in jungle (Coturnix Coromandelica) and the Brown coloured Japanese quail (Coturnix Coturnix japonica), which is raised commercially. In Tamil Nadu, the climate is suitable for quail rearing as they adapt quickly to environmental fluctuations. Quail farming has always been farmer friendly due to its low capital investment, minimal labour cost, short lifespan, good returns from eggs and meat in minimal space requirement (Leonal Rabins and Sethupathi, 2022) [12] . Quail eggs are nutritive with increased amount of lysozyme-a natural antimicrobial enzyme which kills harmful bacteria. Also, quail meat is rich in protein with fewer calories (Chindo and Olowoniya, 2006) [4] and has medicinal qualities with a unique flavour (Jeke et al., 2018) [9] . Quails have better immunity against diseases than chicken and are considered resilient (Khoshbakht et al. 2017) [10] . However, certain management lacunae, specific lethal infections, nutritional deficiencies, non-infectious and parasitic diseases etc., impede quail production. Intensive farming methods to commercially raise quails makes them vulnerable to severe septicaemic infections of concern like Mannheimiasis and Salmonellosis. Other common diseases include ulcerative enteritis, omphalitis, coccidiosis etc. (Shanaway, 1994) [16] . Salmonellosis is a major poultry disease worldwide. It is zoonotic and needs to be constantly surveyed (Shivaprasad and Barrow 2008) [17] . Salmonellosis is a systemic bacterial infection that results in high rates of mortality and morbidity with high productive influence (Barrow and Freitas Neto 2011) [3] . Mannheimiasis is yet another acute septicaemia to which quails are vulnerable as they are to virulent strains of avian pathogenic Escherichia coli strains which cause extra-intestinal infections in various organs.

The present study was conducted to isolate and characterize septicaemic pathogens from diseased quails reared in a private quail farm in Tamil Nadu.

Materials and Methods

Clinically ill and dead quails from a disease outbreak in a private quail farm were investigated. History, clinical signs, and post mortem lesions were suggestive of mixed infections due to septicaemia that had caused sudden mortality with few lesions. Hence it was given least attention by mere burial and disinfection.  History: Problem was said to be present persistently over a period of 3 months and had escalated with increased mortality and severe morbidity by 3 months. Young birds exhibited mortality in the first 3 weeks of life and were stunted.  Clinical signs: Diseased quails showed laboured respiration. Typical signs included somnolence, closed eyes, drooped wings, ruffled feathers and huddling near heat sources. They were anorectic and emaciated. Convalescent birds exhibited acute illness and ruffled feathers. Diarrhoea was distinct sulfur, white or green in colour or bloody in nature. Severely infected quail chicks, (3 weeks) had swollen eyes and joints. (Kumar et al., 2001) [11] .  Post mortem examination: At necropsy a systemic dissection was made and gross changes in different tissues were recorded and suitably collected. There was generalized tissue congestion and hemorrhages in the lungs. The dark friable and bronze coloured enlarged liver was suggestive of fowl typhoid. Spleen was enlarged, in most birds. There was necrotic foci on entire liver parenchyma suggestive of Mannheimiasis. There was catarrhal inflammation of the intestine with petechial hemorrhages. Cheesy caeca cores clogged the ileo-caecal junction. Many birds had hemorrhagic ovarian follicles and unabsorbed yolks. (Erdogrul et al., 2002) [6] .  Sample collection: A total of 73 samples were collected from quails suffering from septic mortalities. During post mortem examination, heart blood was collected aseptically from 10 birds. Deep swab of cloaca, intestinal contents and pieces of ovary, follicles, liver and long bone marrow (femur) were collected for bacteriological examination (Figure 1). [14] Mixed type colonies were re-inoculated in 5ml of Rappaport-Vassiliadis (RV) Medium which has a low pH to select for Salmonella and incubated at elevated temperature of 42 °C. Samples were also inoculated in Nutrient and Brain Heart Infusion broth and incubated at 37°C for 16-18 h at 70rpm/min.  The non-lactose fermenting pale colonies and pink colonies on MacConkey and dewdrop like translucent colonies on BHI agar were stained by Gram's method. All bacterial isolates were phenotypically characterized by biochemical tests viz. IMViC pattern, TSI and urease slants and by sugar fermentation.  Blood examination: Blood was directly collected from the heart and jugular veins of ailing quails and a thin blood smear was prepared. Impression smears were made with cut surfaces of liver and spleen on glass slides, air dried and stained by Leishman's method.  Antibiotic Sensitivity Test: For this, 10 different antibiotics were used against all the isolates obtained viz., E. coli (20%); Mannheimia haemolytica (04%) and Salmonella serovar (59%), as per (Finegold SM and Martin WG, 1982) [7] .AST was performed in Muller Hinton agar by disc diffusion method as per Kirby and Bauer (1982) with FOS: Fosfomycin (300µg), NX: Norfloxacin (10 µg), DoHcl: Doxycycline hydrochloride (30µg), AMP: Ampicillin (10 µg), AMX: Amoxicillin (10 µg), CTX: Cefotaxime (30 µg), E:Erythromycin (15µg), CTR: Ceftriaxone (30mcg) GEN: Gentamicin (30mcg) and CIP: Ciprofloxacin (5µg).Interpretation of the zone of inhibition was done according to (CLSI M02, 2024) [5] .  Genotypic characterization: Detection and confirmation of virulence genes of Salmonella arizonae serovars, Mannheimia spp and APEC strains was also done by PCR. (Zhang et al., 2017) [19] For all samples DNA was extracted from bacterial cultures using the QIAamp DNA Mini kit (Qiagen, Germany) with some modifications. PCR was done as 20µl per reaction using Promega Green amplicon master mix with gene specific primers.  Analysis of the PCR Products: After the completion of reaction cycles, the amplified products were electrophoresed on 1.5% agarose gel stained with ethidium bromide (0.5 µg/ml). The images of stained DNA bands were analyzed under ultraviolet transilluminator and digitized using a gel documentation system (Bio-rad, USA).

Moist litter bound on quail toes

Congested abdominal organs Inflamed congested crop and caecum Hyperemic gonads

Results

In all, 23 Salmonella was isolated from deep cloaca swabs (32%), 11 was from intestinal ulcers (15%) and 9 from liver (12%) as reported from Kerala in India (Ambily and Mini,2014) [1] From 9 different intestines 07 E. coli was isolated (10%), from 03 spleen and 05 ovaries 2 (3%) and 3 (4%) E. coli isolates were obtained. From 05 caecum samples 02 was E. coli (3%). From 02 bone marrow 2 isolates (3%) and from 05 heart blood samples 01 isolate (1%) was positive for M. haemolytica respectively (Table 1). The same is depicted in Figure 2. The results of blood smears stained by Leishman's method, depicted presence of bipolar organisms under oil immersion microscopically. As per cultural characteristics, on Brain Heart Infusion agar Mannheimia haemolytica strains were observed typically as translucent dew drop like colonies and also it grew on Mac Conkey agar as pink, minute, moist colonies and when streaked on Blood agar produced beta haemolysis suggestive of M. haemolytica. The culture was Gram negative cocco-bacilli.

Colonies from BGA when stained was long slender Gramnegative rods and was Salmonella spp. It appeared as translucent colourless colonies with or without black centres on XLD agar also grew on MacConkey agar as colourless non-lactose fermenting colonies. The same was tested by IMViC and on TSI slants for biochemical differentiation (Quinn et al., 1994) [14] . Lactose fermenting colonies on MaConkey agar produced pink colonies and stained as Gram negative rods. All E. coli strains produced metallic sheen on Eosin Methylene Blue agar. Salmonella enterica subsp. arizonae is a Gram-negative, motile, facultative anaerobic, rod-shaped bacterium. It was motile. Biochemical tests gave the following results. Isolates were negative for Oxidase and did not give blue colour on discs. It was positive for catalase with effervescence.

Fermentation tests revealed a rapid lactose fermentation observed in 18-20 hours. Glucose fermentation was normal and produced bubbles due to gas production with yellow slant/butt with and typically red (unfermented) or yellow (non-sucrose-fermenting) butt. There was black discoloration on TSI slant due to production of hydrogen sulphide (H2S) which had a foul rotten egg odour as given in the image. For the IMViC test, isolates were negative for Indole production. They were MP positive and VP negative (Figure 3). They were able to utilize citrate as a carbon source and were positive on green citrate slants changed to indigo blue. They were Lysine decarboxylase positive and negative on Urease slants. These biochemical tests aid to differentiate S. arizonae from other bacteria within the Enterobacteriaceae family. S. enterica subsp. arizonae is defined by its ability to ferment lactose, differentiating it from other Salmonella, but its sugar profile can vary slightly, with sucrose fermentation being rare. The isolates fermented maltose and mannitol, L-rhamnose, Larabinose.as well as trehalose which helped to differentiate from suspected Mannheimia spp. which can ferment arabinose or trehalose as per the biovar though they produce pink colonies on MacConkey agar. All isolates amplified positively for the invA gene which is specific for Salmonella spp., infection (Jahan et al., 2018) [8] Strains of APEC amplified for stx-1; stx-2 and eaeA genes.

Discussion

In quails, incidence of infection with Salmonella. enterica subspecies arizonae is on the rise. Infected adult birds may become asymptomatic carriers, and shed the organism intermittently in their feces. In this study, Salmonellosis was caused by Salmonella arizonae serovar in Japanese quails (Yahi et al., 2024) [18] . It produced mortality rates (up to 68%) in birds of all ages, causing substantial economic loss. Overall screening was done systematically for a period of 6 months.

No relation between age and rate of isolation of Salmonella from birds was observed and these results agree with (Palanisamy and Bamaiyi, 2015) [13] wherein it is reported that there is no significant difference between the prevalence of Salmonella spp. in different age groups of quails.

In the study area poor hygiene, farming mismanagement and rainy season augmented mixed infections (Rahman, 2004) [15] . High rate of moisture and humidity aids growth and invasion by pathogens. Virulence genes such as invA for Salmonella and stx-1; stx-2 and eaeA genes for APEC were detected in all examined isolates (Figure 3). Salmonella infections can be prevented with proper sanitary management techniques. Use of pre and probiotics as an immune supplement has an effective role in enhancement of immunity in healthy and diseased birds.

Conflict of Interest Not available

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References

1. Ambily R, Mini M. Salmonellosis in Japanese quails-a report from central Kerala, India. Int J Sci Res. 2014;3:361-363.
2. Asha RR. Simply poultry science. New Delhi: Alfa Publications; 2011, p. 330-331.
3. Barrow PA, Freitas Neto OC. Pullorum disease and fowl typhoid-new thoughts on old diseases: a review. Avian Pathol. 2011;40(1):1-13.
4. Chindo H, Olowoniya F. Processing and utilization of quail products. In: Proceedings of the National Workshop on Quail Production for Sustainable Household Protein Intake (NAERLS). Zaria: Ahmadu Bello University; 2006. p.11-13.
5. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial disk susceptibility tests. 14th ed. CLSI document M02. Wayne (PA): CLSI; 2024.
6. Erdogrul O, Ozkan N, Cakiroglu E. Salmonella enteritidis in quail eggs. Turk J Vet Anim Sci. 2002;26:321-323.
7. Finegold SM, Martin WG. Bailey and Scott's diagnostic microbiology. 6 th Ed. St. Louis: CV Mosby Company; 1982.
8. Jahan S, Zihadi MAH, Nazir KNH, Islam MS, Rahman MB, Rahman M. Molecular detection and antibiogram of Salmonella spp. of apparently healthy Japanese quails from three farms in Mymensingh. J Adv Vet Anim Res. 2018;5:60-66.
9. Jeke R, Phiri CJ, Musara C. Ethno-medicinal use and pharmacological potential of quail (Coturnix coturnix japonica) meat and eggs and implications for wild quail conservation in Zimbabwe. Cogent Food Agric. 2018;6(1).
10. Khoshbakht R, Seifi S, Karimi A, Khosravi M. Molecular identification of Campylobacter, Arcobacter and Salmonella in Japanese quail (Coturnix japonica) reared in farms of northern Iran. J Food Qual Hazards Control. 2017;4:58-62.
11. Kumar S, Sadana JR, Mishra SK. Studies on clinical signs, growth response and haematological changes in Japanese quail (Coturnix coturnix japonica) infected with Salmonella typhimurium. Indian J Poult Sci. 2001;36:335-337.
12. Leonal Rabins S, Sethupathi S. Production traits of Namakkal quail and Namakkal Gold quail at Tenkasi, Tamil Nadu. Int J Agric Sci. 2022;14(10):11729-11731.
13. Palanisamy S, Bamaiyi PH. Isolation and antibiogram of Salmonella spp. from quails in a farm from Kelantan, Malaysia. J Vet Adv. 2015;5:1191-1198.
14. Quinn PJ, Carter ME, Markey BK, Carter GR. Clinical veterinary microbiology. London: Mosby Year Book Europe Ltd; 1994.
15. Rahman ANMA. Roogbadhi Protikar-In quail palan. Dhaka: Porua Publications; 2004. p.173-175.
16. Shanaway MM. Quail production systems: A review. Rome: Food and Agriculture Organization; 1994, p. 1145.
17. Shivaprasad HL, Barrow P. Pullorum disease and fowl typhoid. In: Saif YM, editor. Diseases of poultry. 12 th Ed. Ames (IA): Blackwell Publishing; 2008, p. 620-636.
18. Yahi HM. The incidence of Salmonella arizonae in different poultry species including quail: isolation and identification from field samples. Assiut Vet Med J. 2024.
19. Zhang W, Liu X, Liu M, Ma B, Xu L, Wang J. Development of a multiplex PCR for simultaneous detection of Pasteurella multocida, Mannheimia haemolytica and Trueperella pyogenes. Acta Vet Hung. 2017;65:327-339.