Clinicopathological effects of Nigella sativa (kalonji) seeds in Escherichia coli infected Psittaculakrameri.

The clinicopathological effects of E. coli infected Psittaculakrameri (ring necked parrots) supplemented with N. sativa seeds in feed were evaluated. Faecal samples from “psittacine” species parrots were collected and E. coli was isolated for cultural, morphological, biochemical and in-vitro studies. A total of 27 parrots were selected and divided into 03 groups (A= control, B= no treatment and C= treatment+ infection) having 09 parrots in each and were fed with N. Sativa seeds @100mg/kg from 3 rd day. The parrots of group B and C were challenged with 0.1ml pathogenic E. coli @10 6 CFU/ml/bird subcutaneously on day 9 th . Clinical signs, mortality and morbidity rates were observed during the trial. For haematology, histopathology and CRP in sera, three birds were slaughtered weekly from each group. Signicant decreased in feed consumption, haematological values like red blood cells, haemoglobin and pack cell volume while signicant increase in clinical signs, morbidity, mortality, liver, heart, kidneys weights and white blood cell were recorded in group B. C-Reactive protein values was signicantly decreased in group C as compared to group B while degenerative changes were seen in liver, heart and kidneys of group B.


Introduction
Parrots are among the most distinguishable and widely distributed of all the birds, referring to more than 350 species of the order Psittaciformes occupying major parts of the tropics (Snyder and McGowan, 2000).Several species of parrots are kept as pets because of their friendly and loving nature, ability to emulate human voices, phenomenal feather coloration and intellect (Gill, 1997).The Rose-Ringed Parakeet (Psittaculakrameri) belongs to the family Psittacidae and it is native to Africa and India. Its size is 38-42 cm as it is a medium sized green bird and its tail accounts 25 cm which is more than half of its length immature birds are di cult to differentiate from females as these juveniles begin developing of red ring when they are three years old. Four subspecies of Psittaculakrameriare currently recognized as P. k. krameri, P. k. parvirostris, P. k. borealis and P. k. manillensis (Butler, 2003). They can take a large variety of feed but usually prefer fruits, vegetables, seeds, cereal grains and oil seed crops (Khan and Ahmad, 1983). E. coli is classi ed into its pathotypes by the help of genes which are responsible for their expression of virulence factors. Avian pathogenic E. coli, Enteropathogenic E. coli and Uropathogenic E. coli are the most commonly described pathotypes of E. coli. APEC is famous for providing massive economic losses to the poultry sector resulting in septicemic and respiratory diseases, while EPEC causes diarrhoea in infants (Nataro and Kaper, 1998). UPEC is responsible for urinary diseases in humans (Johnson et al., 2000). A large number of medicinal plants and their constituents are known for their therapeutic properties. Nigella sativa(N. sativa) is a medicinal plant, belongs to family Ranunculaceae. The seeds of N. sativa are the source of active ingredients of this plant and these are being employed for thousand years. The seeds of N. sativa and their oil have been widely used in the treatment of various disorders throughout the world for centuries and it is an important drug in traditional medicine system. The seeds of N. sativa contain pharmacologically active substances like thymoquinone (30-48%), dithymoquinone (7-15%), thymohydroquinone (1-4%), carvacol (6-12%) and thymol. It also contains large amount of xed oils, proteins, alkaloids, essential oil, mineral elements, carbohydrates, proteins and fats. Thymoquinone is the most active component largely considered for therapeutic ability of N. Sativa (Ghosheh et al., 1999).This plant has been suggested to possess many properties like antimicrobial, antioxidant, anti-in ammatory, antihistaminic, antitumor, antidiabetic, spasmolytic, anticancerous, bronchodilator, hepato-protective, renal protective, gastro-protective, immunomodulatory etc. The photochemical analysis of N. sativashows that the plant is safe to use and possess harmless effects (Ali and Blunden, 2003).

Ethical Approval
The study was approved by University ethical Committee to ensure that no harm is provided to the animals.

Materials And Methods
Bacterial isolationÀ total of 12 faecal samples were taken from Jhang Bazar, Faisalabad 38000, Punjab, Pakistan derby and Ghulam Muhammadabad Faisalabad 38000, Punjab, Pakistan derby. Using standard measures Bacteria needed for the research was obtained from the faecal sample of Psittacine family in a series of steps i.e. Escherichia coli (E. coli). Instantaneously, after the collection of faecal swab, these swabs were inoculated in a liquid medium named as nutrient broth and kept for 24 hours at 37°C for enrichment of these samples. Following the enrichment of the faecal swabs in the nutrient broth, swabs were streaked on the nutrient agar. Swabs were further puri ed on the surface of MacConkey Agar to obtain pink coloured colonies of E. coli.

Identi cation and biochemical characterization of E. coli
Probable identi cation of E. coli was performed using gram's staining and biochemical tests. Slide was observed under the microscope to observe the colonies of Escherichia coli at 40X. Citrate test, Indole test, Lactose fermentation and Methyl red testwere applied for E. coli con rmation. Determination of pathogenicity by Congo red dye by method described by (Ali and Blunden, 2003).

Management and procurement of Parrots
A total of 27 parrots were purchased from local derby of district Faisalabad approximately of same age and they were kept in cages under proper standard managemental conditions. Parrots were divided into 3 different groups A, B and C from 3 rd day of experiment and were inoculated with 0.1ml 10 6 CFU of Avian Pathogenic E. coli (APEC) subcutaneously to group B and C at day 9 of experiment and group A and B were kept separate to avoid contamination.

Treatments of different groups
Parrots were divided into 3 groups from day 3rd day of the experiment group A was kept as control positive group in which parrots were not given any infection and treatment group B was kept as control negative in which parrots were infected with E. coli and not received and treatment and Group C was supplemented with N. sativa at 100mg/kg in feeds. Parrots of group B and C were challenged with pathogenic E. coli at 9 th day of experiment. About 3 birds were slaughtered at weekly basis and blood was collected from the jugular vein from which serum was separated for determination of CRP and Liver, Heart and Kidneys were collected and weighed and organs showing any abnormality were preserved in neutral buffered formalin for histopathology (Bancroft and Gamble, 2008). (Table 1) Hematological parameters such as RBC, WBC, Hb and PCV were studied following (Benjamin, 1978). While absolute and relative weight of internal organs (liver, heart and kidney) were performed and concentration of C-reactive protein was measured according to (Singer et al., 1957). Histopathology was performed following technique described by Bancroft & Gamble (2008). Data thus obtained was analysed statistically using two-way ANOVA and DMR tests.

Results And Discussions
Cultural properties of E. coli Test for presence of E. coli were performed by culturing on various growth media it showed white, greyish and opaque colonies on nutrient agar while on MacConkey agar it showed dark pink coloured colonies which were indicative for E. coli in samples

Morphological characteristics
Microscopic examination of Gram's stained smear of these bacteria revealed Gram negative rod shape with peritrichous agella.

Morbidity and Mortality Rate
Marked variation in days of post infection. Morbidity percentage was 75% and mortality was 50% in group B.The parrots of group C were challenged with E. coli and supplemented with N. sativa seeds in feed. Decreased morbidity percentage was seen in this group as compared to group B. Morbidity percentage was 37.5% and mortality percentage was 25% in group C.
Haematological parameters in E. coli infected Psittaculakrameri supplemented with N. sativaseeds in feed.
In this trial, total erythrocyte count was signi cantly decreased in parrots of group B as compared to that of group A and C. Total erythrocyte count of parrots of group A and C were non-signi cantly different from each otheras given in table (2).
Total leukocyte count was signi cantly increased in parrots of group B as compared to that of group A and C. Total leukocyte count of parrots of group A and C were non-signi cantly different from each otheras given in table (2).

Hemoglobin (Hb) concentration (g/dl)
Hemoglobin concentration was signi cantly decreased in parrots of group B and there was non-signi cant difference among values of hemoglobin of parrots of group A and C (Table 2).

Packed cell volume percentage (%)
Packed cell volume also decreased signi cantly in parrots of group B as compared to that group A and C. Packed cell volume of parrots of group A and C were non-signi cantly different from each other.
Absolute and relative weight of visceral organism E. coli infected Psittaculakrameri supplemented with N. sativaseeds in feed.

Absolute and Relative weight
After E. coli infection, there was a signi cant increase in absolute weights of liver, heart and kidneys of parrots in group B as compared to that of group A and C in this trial. The absolute weights of organs of group C parrots were also signi cantly higher than group A parrots.
After E. coli infection, there was a signi cant increase in relative weights of liver, heart and kidneys of parrots in group B as compared to that of group A and C in this trial. The relative weights of organs of group C parrots were also slightly higher than group A parrots.

C -reactive protein (CRP)
In this experimental trial, CRP concentration was signi cantly increased in parrots of group B and C as compared to that of group A. The CRP concentration in parrots of group C was signi cantly decreased as compared to that of group B.

Histopathology
In E. coli infected group of parrots, hydropic degeneration of hepatocytes with karyolysis of nuclei was observed. At some places, blood vessels were congested. Severe sloughing of cuboidal epithelium with necrotic changes in convoluted tubules and atrophied Bowman's capsule were seen in E. coli infected group of parrots. Accumulation of in ammatory cells and loss of striations were seen in E. coli infected group of parrots (Figure 1).

Conclusion
In the current study, it was concluded that N. sativaseeds supplementation @ 100 mg/kg in feed had antibacterial action against E. coli infection in parrots as it reduced morbidity and mortality rate. The decrease in CRP concentration also showed antibacterial activity of N. sativa seeds. It has no deleterious effects on hematological parameters and physiology of major body organs (liver, heart and kidney) of parrots.

Declarations
Authors' Contribution MW, SN and MA designed the idea, performed experiments and wrote the article. FUR and MSI did analysis and reviewed the article FAK, MKK and MS collected and processed the samples.

Novelty Statement
To the author's knowledge limited research work has been done on Nigella sativa (kalonji) seeds in Escherichia coli infected Psittaculakrameri, so it is a new era to nd the effect of Nigella sativa (kalonji) seeds in Escherichia coli infected Psittaculakrameri to reduce the dose, cost and side effects of the drugs.

Data availability statement
The Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Statement of con ict of interest
The authors have declared no con ict of interest.