Isolation and Immunopathological Evaluation of <i>Chromobacterium violaceum</i> from Diarrheic Chickens

Ikram Abbas Al Sammaraa; Roua J. Mohammed; Mohammed Saad Qasim; Linah I. Lateef

doi:10.12688/f1000research.173368.1

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Research Article

Isolation and Immunopathological Evaluation of Chromobacterium violaceum from Diarrheic Chickens

[version 1; peer review: 2 not approved]

Ikram Abbas Al Sammaraa¹, Roua J. Mohammed ^2,3, Mohammed Saad Qasim⁴, Linah I. Lateef⁴

PUBLISHED 04 Mar 2026

Author details Author details

¹ Department of Zoonotic Unit, College of Veterinary Medicine, University of Baghdad, Iraq, baghdad, baghdad, 10010, Iraq
² Microbiology, University of Baghdad, Iraq, baghdad, baghdad, 10010, Iraq
³ Department of Zoonotic Unit, University of Baghdad, Iraq, baghdad, baghdad, 10010, Iraq
⁴ Microbiology, College of Veterinary Medicine, University of Baghdad, Iraq, baghdad, baghdad, 10010, Iraq

Ikram Abbas Al Sammaraa
Roles: Project Administration

Roua J. Mohammed
Roles: Methodology, Writing – Review & Editing

Mohammed Saad Qasim
Roles: Investigation, Resources, Software

Linah I. Lateef
Roles: Formal Analysis

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Fallujah Multidisciplinary Science and Innovation gateway.

Abstract

Background

Chromobacterium violaceum is a rare but potentially pathogenic bacterium that can infect humans and animals. Limited information is available about its occurrence in poultry. This study aimed to isolate and identify C. violaceum from diarrheic chickens and to evaluate its immunological impact in a mouse model.

Methods

Fifty chicken diarrhea fecal samples were cultured and typed by biochemical tests, Vitek 2 Compact System, and 16S rRNA gene PCR sequencing. Killed bacterial antigens were prepared and administered to mice to measure immune responses (IgG and IL-6 levels by ELISA). The Aloe vera extract (100 mg/ml) was co-administered with the antigen to evaluate its immunomodulatory effect. A histopathological study of the liver, kidney, spleen, and intestine following experimental infection was conducted to detect tissue alterations.

Results

C. violaceum was cultured and diagnosed using molecular methods. Pathological changes in the liver, kidney, and intestine, such as necrosis and inflammatory cell infiltration, were significantly observed in infected mice. Immunological analysis revealed antibody response alterations and organ lesion scoring compared to controls.

Conclusions

This is the initial report on isolating and identifying C. violaceum from Iraqi poultry. The findings highlight its pathogenicity, zoonotic potential, and impact on signaling surveillance and preventive control in poultry production.

Keywords

Chromobacterium violaceum; Poultry infection; Bacterial isolation; PCR identification; Mice model; Pathogenicity; Immunological response; Zoonotic potential

Corresponding author: Roua J. Mohammed

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2026 Abbas Al Sammaraa I et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Abbas Al Sammaraa I, Mohammed RJ, Saad Qasim M and I. Lateef L. Isolation and Immunopathological Evaluation of Chromobacterium violaceum from Diarrheic Chickens [version 1; peer review: 2 not approved]. F1000Research 2026, 15:352 (https://doi.org/10.12688/f1000research.173368.1) First published: 04 Mar 2026, 15:352 (https://doi.org/10.12688/f1000research.173368.1) Latest published: 04 Mar 2026, 15:352 (https://doi.org/10.12688/f1000research.173368.1)

Introduction

Chromobacterium violaceum is a beta-proteobacterium that presents smooth-surfaced violet-colored colonies. It is a non-sporing bacillus and a facultative anaerobe (Kumar, 2012). It is motile because of a single centrally placed polar flagellum, one to four lateral flagella, and surface-associated pili (Ravichandran et al., 2018). C. violaceum is oxidase-positive and catalase-positive (Antony et al., 2013). It is predominantly prevalent, especially in tropical and subtropical areas (Batista and Da Silva, 2017). C. violaceum infections in humans are primarily linked with cases that involve patients with weakened immune systems. This is a normal bacterium found in water and soil. However, the most common entry into the bloodstream and subsequent systemic infections it induces occur through weakened skin, e.g., wounds or cuts, through which the bacterium can enter from contaminated surfaces or water reservoirs (Alisjahbana et al., 2021). There have been some case reports of complications due to C. violaceum, including bacterial hemophagocytic syndrome, brain abscesses, conjunctivitis, chronic granulomatosis, diarrhea, endocarditis, meningitis, neutropenic sepsis, osteomyelitis, pneumonia, puerperal sepsis, and urinary tract infections (Lin et al., 2016). It infects various species in mammals, including pigs, sheep, dogs, buffaloes, and monkeys (Liu et al., 2011).

Innate immunity is activated as a reaction to infection, developing granulomas in the liver. Granulomas consist of clusters of immune cells that the immune system has brought in to “wall off” the infection. The chemokine expression pattern of the granulomas is significant in the organization of the immune response (Amason et al., 2024). Neutrophils are considered one of the first lines of defense against C. violaceum infection. However, neutrophils may be insufficient to eliminate the pathogen in isolation. Repairing the infection will require a stronger immune response (Harvest et al., 2023). Granulomas are structured complexes by which immune cells (macrophages, lymphocytes) are walled off in reaction to a persistent pathogen such as C. violaceum. In contrast to humoral immunity employed in pathogen neutralization, cellular immunity is a host defense barrier for infection control and containment (Harvest et al., 2023). The bacteria also produce violacein, a purple antimicrobial pigment. There is also evidence to support the possibility that violacein can modulate host immune response to infection because violacein has immunostimulatory activity based on the stimulation of immune and inflammatory response pathways (Venegas et al., 2019; Lafta & Sadeq, 2024). Infection of poultry with C. violaceum is not uncommon, and neither its clinical presentation nor its immune responses to infection are fully understood. These immune mechanisms are central to formulating efficient vaccines and therapeutic approaches to combat and prevent transmission of C. violaceum in poultry targets. The main objective of the present study is to isolate Chromobacterium violaceum from poultry and investigate its immune response in mice.

Materials and methods

Samples and bacterial isolation

Samples were collected from various poultry farms in Baghdad, yielding 50 specimens from two-week-old chickens that showed signs of diarrhea. These samples were carefully transported in a cold container to the laboratory within two hours. Each fecal sample was placed in a sterile test tube that contained 1 g of the sample, 10 ml of normal saline, and 0.1 ml of the sample suspension. This mixture was then inoculated onto MacConkey agar (HiMedia, India) and Salmonella Shigella agar (HiMedia, India). The inoculated media were incubated at 37°C for 24 to 48 hours—the Vitek 2 compact system was used for diagnosing the bacteria and antibiotic sensitivity test.

Molecular identification of Chromobacterium violecium

DNA extraction

Transfer 1 to 2 ml of cultured cells into a 2 ml tube. Centrifuge the cells at 13,000 rpm for 1 minute to pellet them. DNA extraction was performed using the G-spin kit (Intron Biotechnology, cat. no. 17045). Electrophoresis was conducted to analyze the DNA fragments post-extraction and to assess the results of the PCR interaction in the presence of standard DNA, allowing for the identification of the product size of the PCR interaction on the Agarose gel.

Purity and concentration of DNA

The concentration and purity of DNA are assessed through absorbance measurements of micro-volume samples utilizing the Nano Drop spectrophotometer (Nabi/Korea). Purity is evaluated using the 260/280 ratio, with optimal purity falling between 1.80 and 2.00.

Primers

The primers were lyophilized and dissolved in ultrapure ddH2O to create a stock solution with a final 100 pmol/μl concentration. This stock solution was stored at -20°C to prepare a working concentration of 10 pmol/μl. The working primer solution was made by combining 10 μl of the stock with 90 μl of ddH2O, resulting in a final volume of 100 μl. The primers were sourced from IDT (Integrated DNA Technologies, USA). Target size for amplification was 1250 bp using the primer sets of 16S rRNA 5'-AGAGTTTGATCCTGGCTCAG-3' and 5'-TACGGTTACCTTGTTACGACTT-3' (Mohammed & Al-Samarraae, 2021).

PCR Amplification Analysis

PCR amplification was performed in a 25 μl volume in the presence of iNtRON's Maxime PCR PreMix Kit that included Taq polymerase, PCR buffer, gel loading buffer, and dNTPs. The mixture consisted of 1.5 μl template DNA, one μl each primer at a concentration of 10 pmol, and 16.5 μl distilled water. Amplification was performed using a Mastercycler (Eppendorf, Germany). The PCR process began with the initial denaturation at 94°C for five minutes. Subsequently, it proceeded with a denaturation at 94°C for 45 seconds, an annealing at 56°C, an extension at 72°C for one minute, and a final extension at 72°C for seven minutes. The products of PCR amplification of 16S rRNA forward and reverse primers were separated on agarose gel electrophoresis (1.5% agarose, 5 volts/cm²) in 1x TBE buffer for 1 hour and 30 minutes. The molecular value standard was a DNA marker (100 bp) of Bioneer (Korea), and a gel documentation system to observe.

Sequencing and sequence alignment

The PCR products were analyzed through 2% agarose gel electrophoresis and visualized under ultraviolet light (302 nm), after staining with either ethidium bromide or Red Stain. Macrogen Korea carried out gene sequencing. A homology search was performed utilizing the Basic Local Alignment Search Tool (BLAST), accessible through the National Center for Biotechnology Information (NCBI) at (http://www.ncbi.nlm.nih.gov) and via the BioEdit program.

Antigen preparations

The C. violaceum Killed Whole Cell Antigen (CKA) was constituted as described in reference (Haq et al., 2022). Bacteria were cultivated on nutrient agar at 37°C for 48 hours, then exposed to Gram staining and microscopic examination for their morphological integrity. The bacteria were washed with PBS (pH 7.2) three times after 3000 rpm centrifugation for 20 minutes and treated with a 0.3% formalin PBS (pH 7.2) solution. The combination was incubated at 37°C for one to two hours and then stored overnight at 4°C. Following three more washes using PBS (pH 7.2), the bacterial suspension was centrifuged for the second time at 3000 rpm for 20 minutes to prepare the CKA. The colony-forming units of the CKA were enumerated by the McFarland tube method, and protein concentration was quantified based on reference (Kielkopf et al., 2020).

Preparation of Aloe Vera extract

Aloe Vera extract was obtained from a local supplement manufacturing company (Alemad Factory) and prepared at a concentration of 100 mg/ml.

Experimental design

Twenty-four Swiss mice weighing 14-15 grams were randomly divided into eight groups. The first group was immunized with a subcutaneous injection of 1.5 × 109 CFU/ml of the CKA antigen. The second group was immunized with the same amount of CKA antigen combined with 100 mg/ml of aloe vera subcutaneously. As a negative control, the third group was given subcutaneously 1 milliliter of PBS (pH 7.2). On day 14 post-immunization, the antigen's initial and second booster doses were administered. After immunization, serum samples were collected on day 21 to measure IgG and IL-6 levels using the Elabscience enzyme-linked immunosorbent assay (ELISA) kit tailored for mice (Elabscience, China), following the manufacturer's protocol. Optical density was measured at a wavelength of 450 nm (Socimed Sarl, France), and the concentrations of IgG and IL-6 were determined through interpolation from a standard curve, with results expressed in pg/mL. A pilot study was conducted to determine infectious dosages for C. violaceum using the McFarland tube method, preparing three dilutions (1.5 × 10⁸, 3.0 × 10⁸, and 6.0 × 10⁸ CFU/ml) with a total of 24 mice divided into three groups of eight. Group 1 was infected orally with 1.5 × 10⁸ CFU (0.1 ml), Group 2 received 3.0 × 10⁸ CFU (0.1 ml orally), and Group 3 was also infected with 3.0 × 10⁸ CFU (0.1 ml orally). Clinical symptoms and mortality rates were observed over three days post-infection, confirming that the infectious dose was 1.5 × 10⁸ CFU/ml. Mice infected with C. violaceum at 21 days post-immunization (1.5 × 10⁸ CFU/ml, orally) were monitored for clinical signs every 6 to 8 hours over a week. Ten days after infection, the serum was taken and the mice euthanized for histopathological examination of the vital organs; liver, kidney, spleen, and intestine were collected and fixed in a 10% formalin solution immediately after extraction (Snyder et al., 2022).

Statistical analysis

The Statistical Packages of Social Sciences (SPSS) program was used to detect the effect of different groups and periods on study parameters. LSD-Least significant difference (two-way) was used to compare the means significantly in this study (George & Mallery, 2019).

Results

Isolation

The analysis of fifty fecal samples from diarrhea cases revealed that seven isolates, representing 14%, tested positive, as indicated in Table 1.

Table 1. Rates of C. violaceum isolates.

Months of study	Samples No.	Isolates No.	Positive percentage %
May 2024 – September 2025	50	7	14%

Identification

All strains of C. violaceum manifested as black colonies on SS agar due to their ability to produce H2S after 24 hours (Figure 1). On MacConkey agar, the isolates appeared as pink colonies due to lactose fermentation (Figure 1) and non-producing pigmentation on nutrient agar (Figure 1). Additionally, all isolates tested positive for oxidase and catalase.

Figure 1. C. violaceum on SS agar, MacConkey agar, and Nutrient agar.

The Vitek 2 Compact system was employed to identify and assess the antimicrobial susceptibility of C. violaceum, yielding a 98% probability of identification, as illustrated in Table 2.

Table 2. Vitek 2 compact system and antimicrobial susceptibility for C. violaceum.

Organism Quantity:
Selected Organism: Chromobacterium violaceum
Source:														Collected:
Comments:
Identification Information						Analysis Time:				6.50 hours			Status:			Final
Selected Organism						98% Probability				Chromobacterium violaceum
Selected Organism						Bionumber:				0001000041440210
Biochemical Details
2	APPA	-	3	ADO	-	4	PyrA	-	5	IARL	-	7	dCEL	-	9	BGAL	-
10	H2S	-	11	BNAG	-	12	AGLTp	-	13	dGLU	+	14	GGT	-	15	OFF	-
17	BGLU	-	18	dMAL	-	19	dMAN	-	20	dMNE	-	21	BXYL	-	22	BAlap	-
23	ProA	-	26	LIP	-	27	PLE	-	29	TyrA	-	31	URE	-	32	dSOR	-
33	SAC	-	34	dTAG	-	35	dTRE	+	36	CIT	+	37	MNT	-	39	5KG	-
40	ILATk	-	41	AGLU	-	42	SUCT	+	43	NAGA	-	44	AGAL	-	45	PHOS	+
46	GlyA	-	47	ODC	-	48	LDC	-	53	IHISa	-	56	CMT	-	57	BGUR	-
58	O129R	+	59	GGAA	-	61	IMLTa	-	62	ELLM	-	64	ILATa	-

When standard DNA is present, the PCR interaction's outcome allows for the product size differentiation resulting from the PCR process on the Agarose gel (Figure 2).

Figure 2. Gel electrophoresis of genomic DNA extracted from C. violaceum, utilizing a 1% agarose gel at a voltage of 5 V/cm for 30 minutes.

The concentration of C. violaceum DNA was measured at 42.5 ng/ml, with a purity value of 1.937. One isolated strain was examined to identify the presence of 16S rRNA genes. As a result, the strain showed a positive amplification of 1250 bp on an agarose gel, while no amplification was detected in the negative control (Figure 3).

Figure 3. displays a PCR product with a band size of 1250 bp. The electrophoresis was conducted on a 1.5% agarose gel at a 5 volts/cm² voltage using 1x TBE buffer for 1 hour and 30 minutes.

L: represents the DNA ladder (100 bp). S1: C. violaceum.

The NCBI website (http://www.ncbi.nlm.nih.gov) was used to analyze the sequences. There was 98% homology between the C. violaceum reference strains and the isolated strains in GenBank (Accession No. MW282912.1) Figure 4 Table 3.

Figure 4. C. violaceum homologous with the referenced strain on GenBank.

Table 3. The Level of IL-6 in immunized mice (n=6).

Mouse_ID	Group	Treatment	Day	IL6_pg_ml	Age_weeks	Sex
G1-CKA_M1	G1-CKA	G1-CKA	Day21	32.753	8–9 weeks	Male
G1-CKA_M2	G1-CKA	G1-CKA	Day21	38.543	8–9 weeks	Male
G1-CKA_M3	G1-CKA	G1-CKA	Day21	36.832	8–9 weeks	Male
G1-CKA_M4	G1-CKA	G1-CKA	Day21	33.946	8–9 weeks	Male
G1-CKA_M5	G1-CKA	G1-CKA	Day21	35.348	8–9 weeks	Male
G1-CKA_M6	G1-CKA	G1-CKA	Day21	32.475	8–9 weeks	Male
G1-CKA_M1	G1-CKA	G1-CKA	Day10	240.214	8–9 weeks	Male
G1-CKA_M2	G1-CKA	G1-CKA	Day10	280.587	8–9 weeks	Male

IL-6 measurement

The results regarding IL-6 levels across the various groups (G1 and G2) indicated no significant differences (P<0.05) at 21 weeks post-immunization, with measurements of 34.98 ±0.97 pg/ml for G1 and 35.37 ±0.54 pg/ml for G2 when compared to the control negative group, which had a level of 30.93 ±0.62 pg/ml. Furthermore, Group 3 (control negative) demonstrated a significantly elevated IL-6 concentration (P<0.05) ten days following infection with C. violaceum, reaching 945.06 ±9.87 pg/ml. Additionally, both G1 and G2 showed significant increases in IL-6 levels (P<0.05) ten days post-infection, measuring 287.54 ±13.75 pg/ml and 302.11 ±14.68 pg/ml, respectively, as detailed in ( Table 3) and (Figure 5).

Table 4. The Level of IgG in immunized mice (n=6).

Mouse_ID	Group	Treatment	Day	IgG_pg_ml	Age_weeks	Sex
G1-CKA_M1	G1-CKA	G1-CKA	Day21	32.75	8–9 weeks	Male
G1-CKA_M2	G1-CKA	G1-CKA	Day21	38.546	8–9 weeks	Male
G1-CKA_M3	G1-CKA	G1-CKA	Day21	36.835	8–9 weeks	Male
G1-CKA_M4	G1-CKA	G1-CKA	Day21	33.949	8–9 weeks	Male
G1-CKA_M5	G1-CKA	G1-CKA	Day21	35.348	8–9 weeks	Male
G1-CKA_M6	G1-CKA	G1-CKA	Day21	32.475	8–9 weeks	Male
G1-CKA_M1	G1-CKA	G1-CKA	Day10	80.213	8–9 weeks	Male
G1-CKA_M2	G1-CKA	G1-CKA	Day10	80.581	8–9 weeks	Male

Figure 5. The level of IL-6 & IgG In immunized groups.

IgG measurement

The findings related to IgG levels among the different groups (G1 and G2) revealed significant variations (P<0.05) at 21 weeks after immunization. Specifically, G1 presented an average of 34.98 ±0.98 pg/ml, while G2 showed a higher average of 55.43 ±0.49 pg/ml, in contrast to the negative control group, which recorded a level of 11.93 ±0.70 pg/ml. Additionally, Group 2 exhibited a notably increased IL-6 concentration (P<0.05) ten days post-infection with C. violaceum, reaching 105.61 ±2.15 pg/ml. In comparison, G1 attained a level of 84.19 ±1.60 pg/ml, whereas G3 reached only 32.39 ±1.16 pg/ml, as outlined in (Table 4) and (Figure 5).

Histopathological examination

Histopathological examination after 10-day post-challenge showed that all groups under study were affected with different histopathological changes: The G1 CKA: the liver section revealed normal parenchyma with well-conserved hepatic cords and sinusoids, normal liver histology, no pathological lesions identified (Figure 6). The spleen section showed the presence of well-formed granulomas—collections of epithelioid histiocytes (elongated, pale cytoplasm) often surrounded by lymphocytes and increased vasculature with some fibrous septae (Figure 7). The kidney section showed histologically normal renal cortex, and no apparent pathology was present in this section (Figure 8). The intestinal section showed intact villous architecture with slight development of subepithelial (Gruenhagen's) spaces at the villus tip, extremely mild mucosal injury (Figure 9). The G2 CKA- Aloe vera: the liver section showed mild portal inflammation with lymphocytic infiltrates in and around portal tracts, no significant lobular inflammation or hepatocyte necrosis (Figure 10). The spleen section revealed Lymphoid hyperplasia or possible lymphoma and dense lymphocytic infiltrate (Figure 11). The kidney section showed no obvious lesions such as necrosis, inflammatory cell infiltration, fibrosis, or glomerular sclerosis (Figure 12). The intestinal section showed moderate subepithelial space formation and epithelial lifting to the sides of villi (Figure 13). The G3 Control negative (infected = C+): the liver section showed dense infiltrate of lymphocytes in the portal area extending into the periportal area, and interface hepatitis is seen (piecemeal necrosis), scattered lobular inflammation, and mild hepatocyte ballooning (Figure 14). The spleen section showed preserved follicular hyperplasia, tubules appeared dilated with cytoplasmic vacuolization, and moderate lymphocytic infiltration in interstitial areas (Figure 15). The kidney section revealed Diffuse lymphocytic infiltration, focal necrosis, and congestion in zones, glomerular structure observed to be mildly disturbed, severe damage to parenchymal tissue evident (Figure 16). The intestinal section revealed massive epithelial lifting and few villi with denuded tips, demonstrating advancing mucosal injury (Figure 17)

Figure 6. G1 The liver section revealed normal parenchyma, normal liver histology, and no pathological lesions identified by H&E stain. 100x.

Figure 7. G1The spleen section revealed the presence of well-formed granulomas—collections of epithelioid histiocytes by H&E stain. 100x.

Figure 8. G1The kidney section showed histologically normal renal cortex, and no apparent pathology was present in this section by H&E stain. 100x.

Figure 9. G1The intestinal section showed intact villous architecture with slight development of subepithelial (Gruenhagen's) spaces at the villus tip by H&E stain. 100x.

Figure 10. G2 The liver section showed mild portal inflammation with lymphocytic infiltrates by H&E stain. 100x.

Figure 11. G2 The spleen section revealed Lymphoid hyperplasia or possible lymphoma and dense lymphocytic infiltrate by H&E stain. 100x.

Figure 12. G2 Normal renal histology without significant pathological changes.by H&E stain. 100x.

Figure 13. G2 The intestinal section showed moderate subepithelial space formation and epithelial lifting to the sides of the villi by H&E stain. 100x.

Figure 14. G3 the liver section showed dense infiltrate of lymphocytes in the portal area extending into the periportal area, and interface hepatitis is seen (piecemeal necrosis by H&E stain. 100x.

Figure 15. G3 The spleen section showed follicular hyperplasia, tubules appeared dilated with cytoplasmic vacuolization, and moderate lymphocytic infiltration in interstitial areas by H&E stain. 100x.

Figure 16. G3 The kidney section revealed Diffuse lymphocytic infiltration, focal necrosis, and congestion in zones by H&E stain. 100x.

Figure 17. G3The intestinal section showed intact villous architecture with slight development of subepithelial (Gruenhagen's) spaces at the villus tip by H&E stain. 100x.

.

Discussion

Chromobacterium violaceum is involved in opportunistic infections among humans and other animals (Hammerschmitt et al., 2017); however, it is also found in avian hosts, especially chickens. A critical literature review indicates that there have been no reports of isolating C. violaceum from chickens, even from diarrhea in feces. Thus, this study is the first report on isolating C. violaceum from chicken diarrhea cases. The results show that among 50 fecal samples of chickens presenting with diarrhea, 7 C. violaceum isolates were obtained. These results are in concordance with other research where this bacterium has been isolated from other household animals; it has been found in visceral abscesses, pleuropneumonia, and septicemia in pigs (Liu et al., 1989), sheep (Carrasco et al., 1996), dogs (Crosse et al., 2006), cattle (Ajithdoss et al., 2008), and horses (Hammerschmitt et al., 2017). The bacterium was separated to the Genus level by the routine methods, whereas Vitek2 and PCR were employed to differentiate the species. This study's result detected black and non-pigmented colonies on SS agar. This is consistent with Sivendra et al.'s (1975) study, in which pigmented and non-pigmented colonies of C. violaceum water isolates were detected. The VITK2 test was 98% likely, whereas C. violaceum was 98% identical with GenBank MW282912.1 for the presence of the 16s rRNA gene. The findings concur with research by Ibrahim et al. (2020). Isolate C. violaceum VITK2 was the one with a 99.9% matching rate. In the meantime, PCR analysis also indicated the 16S rRNA gene sequence to be 96.58%-99.31% identical to C. violaceum ATCC 12472, C. violaceum NBRC 12614, and C. violaceum JCM 1249 strains of other species within the same genus.

The current study showed that IL-6 levels in all the immunized groups were not statistically different (P<0.05) at the 21st week of immunization. There was an increase in ten days post-infection with C. violaceum. In contrast, there was a difference (P<0.05) ten days post-infection with C. violaceum in the negative control group. These actions can be justified by the induction of inflammatory cytokines and inhibition of the macrophage and neutrophil microbicidal functions and neutralization of IL-6 in infected test animals that could lead to the reduction of mortality (Mohammed, 2025c; Ulich et al., 1991). From the findings, the IgG antibodies in all the immunized groups had an increased antibody titer (P<0.05) 21 days post-immunization. There was also an increase in the antibody level in all groups vaccinated ten days after bacterial infection relative to the control. All these results agree with findings made in (Oladejo and Adebolu, 2013), where they noted that the initial antibody response to the E. coli O157 antigen increased by the first dose and once again reached its peak after booster administration. On the heels, Al-Samarraae and Kareem (2018) reported a very marked rise in IgG titers (P<0.05) after 35 days of vaccination with sonicated Salmonella Typhimurium (KWCSA-ST) and Lactobacillus acidophilus (KWCSA-LBA). This was consistent with the findings of (Mohammed & Al-Samarraae, 2021), who reported that Citrobacter WCSA and LPS Ag-vaccinated rabbits induced elevated humoral (IgG) and cellular (DTH and IL-6) immune responses.

The histopathological results highlight the immunoprotected function of immunization in averting undue damage to tissue in infection: Group 1 (G1), which received the killed antigen alone, exhibited a disease, compared to the other groups, whose immune response was significantly more severe. This agrees with studies that show killed bacterial vaccines to cause immunity without causing excessive tissue damage (Cavallaro et al., 2020). Group 2 (G2), which was vaccinated with the killed antigen and Aloe vera, had heightened immunity with greater inflammation of the spleen and liver, but not excessive tissue damage. The immunomodulatory action of Aloe vera likely resulted in this increased immunity without inflicting so much destruction, evidenced by studies showing that Aloe vera stimulates immunity without exposing toxic side effects (Vijayalakshmi et al., 2015; Nasir et al., 2019). In Group 3, infected with C. violaceum and considered (C+), there were severe histopathological changes in the liver, kidney, spleen, and intestine. The tissue damage in specific organs reflects the inability of the immune system to contain the infection without prior immunization. The above observations highlight the role of immune priming (through vaccination or prior exposure) in preventing severe infection and associated organ injury. This highlights the importance of vaccine immunity and the need for pre-exposure immunity to effectively combat bacterial infections like C. violaceum (Zhao et al., 2020; Deng et al., 2017).

Conclusion

The study is the initial reported isolation of Chromobacterium violaceum from diarrheic poultry. The study demonstrates that immunization with antigens of C. violaceum, in combination with Aloe vera as an immunomodulator, increases humoral and cellular immunity, which confers protection against infection. The study shows that C. violaceum can potentially injure poultry health, and active immunization may significantly prevent severe infection and associated tissue damage. Additional research is recommended to study vaccination efficacy in larger poultry flocks and the bacterium's role in avian disease.

Ethical approval

This study received local ethical permission through the local animal care and use committee at the College of Veterinary Medicine at the University of Baghdad (Number P-G\582,6\3\2025).

Human participants statement

This study involved no human participants, and therefore the Declaration of Helsinki does not apply.

Informed consent statement

Since the study included animals only, an informed consent statement is not.

Data availability

The data underlying this article are available on Zenodo and can be accessed at the following DOIs: https://doi.org/10.5281/zenodo.17597195; https://doi.org/10.5281/zenodo.17812758 (Mohammed, 2025a; Mohammed, 2025b). This dataset was made available under the Creative Commons Attribution 4.0 International (CC BY 4.0) license.

Acknowledgements

N/A.

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Crosse PA, Soares K, Wheeler JL, et al.: Chromobacterium violaceum infection in two dogs. J. Am. Anim. Hosp. Assoc. 2006; 42(2): 154–159. PubMed Abstract | Publisher Full Text
Deng S, Wang X, Liu J, et al.: Intestinal barrier damage during bacterial infections: Histopathological and molecular insights. Clin. Exp. Immunol. 2017; 188(3): 183–194. PubMed Abstract | Publisher Full Text | Free Full Text
George D, Mallery P: IBM SPSS Statistics 26 step by step. Routledge; 2019. Publisher Full Text
Hammerschmitt ME, Rolim VM, Snel GGM, et al.: Chromobacterium violaceum infection in a horse. J. Comp. Pathol. 2017; 156(4): 334–338. PubMed Abstract | Publisher Full Text
Haq RIU, Parray OR, Nazir QUA, et al.: Immune and oxidative response against sonicated antigen of Mycoplasma capricolum subspecies capripneumonia—a causative agent of contagious caprine pleuropneumonia. Microorganisms. 2022; 10(8): 1634. PubMed Abstract | Publisher Full Text | Free Full Text
Harvest CK, Abele TJ, Yu C, et al.: An innate granuloma eradicates an environmental pathogen using Gsdmd and Nos2. Nature Communications. 2023; 14(1). PubMed Abstract | Publisher Full Text | Free Full Text
Ibrahim Y, Abouwarda A, Assar N: Identification and characterization of a soil isolate of Chromobacterium violaceum from Egypt with potential to cause disease. The Egyptian J. Med. Microbiol. 2020; 29(1): 153–160. Publisher Full Text
Kielkopf CL, Bauer W, Urbatsch IL: Methods for measuring the concentrations of proteins. Cold Spring Harbor Protocols. 2020; 2020(4). Publisher Full Text
Lafta IJ, Sadeq Z: Pseudomonas aeruginosa is an effective indicator for screening of quorum-sensing inhibition by plant extracts. The Iraqi J. Vet. Med. 2024; 48(1): 54–62. Publisher Full Text
Lin YD, Majumdar SS, Hennessy J, et al.: The spectrum of Chromobacterium violaceum infections from a single geographic location. Am. J. Trop. Med. Hyg. 2016; 94(4): 710–716. PubMed Abstract | Publisher Full Text | Free Full Text
Liu CH, Chu RM, Weng CN, et al.: An acute pleuropneumonia in a pig caused by Chromobacterium violaceum. J. Comp. Pathol. 1989; 100(4): 459–463. PubMed Abstract | Publisher Full Text
Liu DX, Didier PJ, Plauche GB: Chromobacterium violaceum infections in 13 non-human primates. J. Med. Primatol. 2011; 41(2): 107–114. PubMed Abstract | Publisher Full Text | Free Full Text
Snyder JM, Radaelli E, Goeken A, et al.: Perfusion with 10% neutral-buffered formalin is equivalent to 4% paraformaldehyde for histopathology and immunohistochemistry in a mouse model of experimental autoimmune encephalomyelitis. Vet. Pathol. 2022; 59(3): 498–505. PubMed Abstract | Publisher Full Text | Free Full Text
Mohammed RJ, Al-Samarraae IAA: Investigating the effect of three antigens of Citrobacter freundii on the rabbit’s immune response. Iraqi J. Vet. Med. 2021; 45(1): 56–62. Publisher Full Text
Mohammed RJ: The impact of Quercetin–Berberine complex and Clove extract on mice induced asthmatic condition. Agricultural Biotechnology Journal. 2025a; 17(3): 385–406. Publisher Full Text
Nasir M, Khan F, Faqir H, et al.: Aloe vera as an adjuvant in immunization: Its potential role in immune modulation. Immunol. Lett. 2019; 208: 32–38. Publisher Full Text
Oladejo BO, Adebolu TT: Mechanisms of immunity to Escherichia coli O157: H7 in albino rats: Role of homologous antibodies. European Journal of Biology and Medical Science Research. 2013; 1(2): 31–39. Reference Source
Ravichandran V, Zhong L, Wang H, et al.: Virtual screening and biomolecular interactions of CviR-based quorum sensing inhibitors against Chromobacterium violaceum. Front. Cell. Infect. Microbiol. 2018; 8. PubMed Abstract | Publisher Full Text | Free Full Text
Sivendra R, Lo HS, Lim KT: Identification of Chromobacterium violaceum: Pigmented and non-pigmented strains. J. Gen. Microbiol. 1975; 90(1): 21–31. PubMed Abstract | Publisher Full Text
Ulich TR, Yin S, Guo K, et al.: Intratracheal injection of endotoxin and cytokines. II. Interleukin-6 and transforming growth factor beta inhibit acute inflammation. PubMed. 1991; 138(5): 1097–1101. Reference Source
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Zhao Z, Xie T, Liu F, et al.: Pathogenesis and immune responses in bacterial infections: A histopathological and immunological study on Chromobacterium violaceum. Infect. Immun. 2020; 88(7): e00247–e00220. Publisher Full Text
Mohammed RJ: Isolation and Immunopathological Evaluation of Chromobacterium violaceum from Diarrheic Chickens. [Data set]. Zenodo. 2025b. Publisher Full Text
Mohammed RJ: Isolation and Immunopathological Evaluation of Chromobacterium violaceum from Diarrheic Chickens; The ARRIVE guidelines 2.0: author checklist. [Data set]. Zenodo. 2025c. Publisher Full Text

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¹ Department of Zoonotic Unit, College of Veterinary Medicine, University of Baghdad, Iraq, baghdad, baghdad, 10010, Iraq
² Microbiology, University of Baghdad, Iraq, baghdad, baghdad, 10010, Iraq
³ Department of Zoonotic Unit, University of Baghdad, Iraq, baghdad, baghdad, 10010, Iraq
⁴ Microbiology, College of Veterinary Medicine, University of Baghdad, Iraq, baghdad, baghdad, 10010, Iraq

Ikram Abbas Al Sammaraa
Roles: Project Administration

Roua J. Mohammed
Roles: Methodology, Writing – Review & Editing

Mohammed Saad Qasim
Roles: Investigation, Resources, Software

Linah I. Lateef
Roles: Formal Analysis

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

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Published: 04 Mar 2026, 15:352

https://doi.org/10.12688/f1000research.173368.1

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Abbas Al Sammaraa I, Mohammed RJ, Saad Qasim M and I. Lateef L. Isolation and Immunopathological Evaluation of Chromobacterium violaceum from Diarrheic Chickens [version 1; peer review: 2 not approved]. F1000Research 2026, 15:352 (https://doi.org/10.12688/f1000research.173368.1)

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Reviewer Report 20 Apr 2026

Lucia Galuppo, Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy

Delia Gambino, Istituto Zooprofilattico Sperimentale della Sicilia Adelmo Mirri (Ringgold ID: 18325), Palermo, Sicily, Italy

Not Approved

https://doi.org/10.5256/f1000research.191178.r475589

General Evaluation
This article presents a study on the isolation and immunopathological evaluation of Chromobacterium violaceum in chickens with diarrhea, integrating microbiological, molecular, and immunological approaches. The topic is potentially interesting and could contribute to the understanding of understudied ... Continue reading

General Evaluation
This article presents a study on the isolation and immunopathological evaluation of Chromobacterium violaceum in chickens with diarrhea, integrating microbiological, molecular, and immunological approaches. The topic is potentially interesting and could contribute to the understanding of understudied microorganisms in the poultry context. However, the study has several limitations that reduce its scientific validity, particularly regarding the rationale, methodological description, and interpretation of the results. Specifically, the work tends to suggest a pathogenic role for the microorganism without providing sufficient supporting evidence.

Abstract
The abstract is well-structured; however, the scientific rationale is not sufficiently clear, and the conclusions do not reflect the data presented. Furthermore, the abstract could be improved by including more precise quantitative data, particularly regarding immunological results.

Introduction
The introduction provides a general description of the microorganism and its biological characteristics, which is informative from a microbiological perspective. However, its relevance to the poultry context is limited. There is no adequate literature review on the presence or role of C. violaceum in chickens or poultry farms. Some claims, such as the frequency of infection in chickens, are not supported by the literature. The immunological section appears rather general and unrelated to the specific objectives of the study.

Materials and Methods
The Materials and Methods section describes an integrated approach that includes bacterial cultures, biochemical identification, and molecular analysis, as well as the use of an animal model for immunological evaluation. The sampling description is not entirely clear, as the type of sample collected is not precisely specified; no information is provided on the animals' clinical conditions; and it is not stated whether other potential etiological agents of diarrhea were considered or excluded. The description of the molecular methods is presented as a working protocol and lacks important details regarding sequencing, the number of samples analyzed, and the data analysis methods. There are also some technical inconsistencies, such as in the concentration of the agarose gels. The histopathological analysis section is not described in detail, making it difficult to assess the study's reproducibility.

Results
The results report an isolation rate of 14%, which may be of interest. However, this result is not accompanied by clinical or epidemiological information that would allow for a correct interpretation. The identification of the microorganism using different methodologies is a positive aspect, but some phenotypic descriptions are unclear or inconsistent. Molecular and sequencing results are not clearly presented, and the figures do not always support the conclusions, particularly regarding the presence of controls in PCR analyses.
Regarding immunological data, the presentation could be improved. There are some inconsistencies in the units of measurement used, and the histopathological analysis is described qualitatively, while a quantitative or standardized assessment of the lesions is lacking. These are important data that limit the possibility of making comparisons between groups.

Discussion
A significant issue arises regarding data interpretation. The study tends to suggest a pathogenic role for C. violaceum in chickens based on its isolation, but does not provide sufficient evidence to demonstrate a correlation with the observed disease. Other potential factors or etiological agents that could explain the symptoms are not considered, nor is the possible role of the bacterium as an environmental contaminant or component of the microbiota discussed.

Conclusions
The conclusions summarize the main findings of the study, but are stronger than the data support. The work should be presented as a preliminary study, highlighting an interesting potential isolation, but requiring further investigation to clarify the biological significance of the findings.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Microbiology, animal health, zoonoses, antibiotic resistance.

We confirm that we have read this submission and believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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Reviewer Report 06 Apr 2026

Amro Hashish, Iowa State University, Ames, Iowa, USA

Not Approved

https://doi.org/10.5256/f1000research.191178.r466944

Review of Manuscript Entitled "Isolation and Immunopathological Evaluation of Chromobacterium violaceum from Diarrheic Chickens."
General comments:
The manuscript needs careful revision for English proofreading. Please, re-check all abbreviations.
Major Concerns:
1. The central premise of ... Continue reading

Review of Manuscript Entitled "Isolation and Immunopathological Evaluation of Chromobacterium violaceum from Diarrheic Chickens."
General comments:
The manuscript needs careful revision for English proofreading. Please, re-check all abbreviations.
Major Concerns:
1. The central premise of the study is insufficiently justified. The authors aim to investigate C. violaceum in poultry; however, the current literature does not support a defined clinical or pathological role for this organism in avian species. To date, C. violaceum is not recognized as a poultry pathogen in established avian literature, and reports of its occurrence in birds are extremely limited or absent. Before investigating immune responses, the study should first establish whether this organism has any biological or pathological significance in poultry. This would require evidence of causality, such as reisolation following experimental infection, demonstration of clinical signs, or histopathological lesions consistent with infection. Without such evidence, the organism may represent environmental contamination or a commensal rather than a true pathogen.
2. The objective of the study is not clear and not matching the materials and methods section
Introduction:
1. The introduction lacks a critical review of the organism in the context of poultry. Specifically, the authors did not include or discuss any previous reports of C. violaceum in avian species, including its potential detection in microbiome or metagenomic studies. A comprehensive introduction should address whether this organism has been previously identified in poultry production systems (e.g., water sources, gut microbiota, or environmental samples), even in a non-pathogenic context. The absence of such discussion weakens the rationale for the study and limits its scientific grounding.
2. Authors stated that “Infection of poultry with C. violaceum is not uncommon” this is an overstatement and not supported as there is almost no previous report highlighting the imp of this bacteria in poultry.
3. There is misplaced immunological depth in the intro that is more general immunology.

Materials and methods:
Sample and bacterial isolation:
A table describing the samples and associated meta-data “especially the clinical picture of chickens” should be added to the materials and methods. What kind of sample? “Authors needs to explain “Feces, cloacal swab…”. Please, add reference that you followed to process the sample or conduct the bacterial isolation?
The first line of the DNA extraction part reads more as instruction to do something rather than materials and methods section of scientific journal. Please, rephrase. The part related to Electrophoresis is misplaced and should be placed in a separate heading following the PCR.
The primers’ section should be merged under PCR amplification and cycling conditions with recommendation to trim some parts e.g. preparation of primer’s stock and working solution.
In some parts of the materials and methods gel conc is 1.5% and in other parts was described as 2%? Please, re-check.
Sequencing and sequence alignment section: this part lacks many details. please, list the purification method before sequencing “from amplicon/gel”, which primer “F or R or both” were used for sequencing, how many samples were submitted for sequencing, brief details if sequencing with Sanger. Software used to analyze the data “Trimming ….”
A graph describing the study design should be added for easy understanding of the design.
The study to test the immunogenicity should be separated from the pilot study to determine the infective dose.
Materials and methods lack any data related to Histopathology
Did author try to isolate any other bacterial agents that might explain the diarrhea? Or investigate any other causes for diarrhea “infectious or non”?
Results:
Table 1 should include data about the samples yielded positive results.
Fig 2: It is not clear what authors want to tell from Fig2, and the legend does not tell anything.
Figure 3: where are the lanes for Pos and Neg controls?
Results section for PCR and Sequencing needs improvement “the acc # that showed highest similarity is for another bacteria.

Discussion:
The absence of previous reports of C. violaceum in the literature might indicate non-significance of the bacteria in avian hosts rather than gab in the body of knowledge.
Furthermore, the inference of causation based solely on the isolation of the organism is scientifically unsound. Isolation alone does not establish pathogenicity or a causal relationship with disease. Demonstrating causation requires additional evidence, such as reproducibility of disease following experimental infection, consistent pathological findings, and exclusion of other potential etiological agents. This issue represents a fundamental limitation that directly impacts the interpretation and validity of the study’s conclusions.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Molecular Diagnostics - Bacterial Genomics - Genomic Data analysis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

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VERSION 1 PUBLISHED 04 Mar 2026

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Version 1 04 Mar 26	read	read

Amro Hashish, Iowa State University, Ames, USA
Lucia Galuppo, Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy

Delia Gambino, Istituto Zooprofilattico Sperimentale della Sicilia Adelmo Mirri (Ringgold ID: 18325), Palermo, Italy

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Reviewer Report

5 Views

20 Apr 2026 | for Version 1

Lucia Galuppo, Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy

Delia Gambino, Istituto Zooprofilattico Sperimentale della Sicilia Adelmo Mirri (Ringgold ID: 18325), Palermo, Sicily, Italy

5 Views Cite this report Responses(0)

Not Approved

General Evaluation
This article presents a study on the isolation and immunopathological evaluation of Chromobacterium violaceum in chickens with diarrhea, integrating microbiological, molecular, and immunological approaches. The topic is potentially interesting and could contribute to the understanding of understudied microorganisms in the poultry context. However, the study has several limitations that reduce its scientific validity, particularly regarding the rationale, methodological description, and interpretation of the results. Specifically, the work tends to suggest a pathogenic role for the microorganism without providing sufficient supporting evidence.

Abstract
The abstract is well-structured; however, the scientific rationale is not sufficiently clear, and the conclusions do not reflect the data presented. Furthermore, the abstract could be improved by including more precise quantitative data, particularly regarding immunological results.

Introduction
The introduction provides a general description of the microorganism and its biological characteristics, which is informative from a microbiological perspective. However, its relevance to the poultry context is limited. There is no adequate literature review on the presence or role of C. violaceum in chickens or poultry farms. Some claims, such as the frequency of infection in chickens, are not supported by the literature. The immunological section appears rather general and unrelated to the specific objectives of the study.

Materials and Methods
The Materials and Methods section describes an integrated approach that includes bacterial cultures, biochemical identification, and molecular analysis, as well as the use of an animal model for immunological evaluation. The sampling description is not entirely clear, as the type of sample collected is not precisely specified; no information is provided on the animals' clinical conditions; and it is not stated whether other potential etiological agents of diarrhea were considered or excluded. The description of the molecular methods is presented as a working protocol and lacks important details regarding sequencing, the number of samples analyzed, and the data analysis methods. There are also some technical inconsistencies, such as in the concentration of the agarose gels. The histopathological analysis section is not described in detail, making it difficult to assess the study's reproducibility.

Results
The results report an isolation rate of 14%, which may be of interest. However, this result is not accompanied by clinical or epidemiological information that would allow for a correct interpretation. The identification of the microorganism using different methodologies is a positive aspect, but some phenotypic descriptions are unclear or inconsistent. Molecular and sequencing results are not clearly presented, and the figures do not always support the conclusions, particularly regarding the presence of controls in PCR analyses.
Regarding immunological data, the presentation could be improved. There are some inconsistencies in the units of measurement used, and the histopathological analysis is described qualitatively, while a quantitative or standardized assessment of the lesions is lacking. These are important data that limit the possibility of making comparisons between groups.

Discussion
A significant issue arises regarding data interpretation. The study tends to suggest a pathogenic role for C. violaceum in chickens based on its isolation, but does not provide sufficient evidence to demonstrate a correlation with the observed disease. Other potential factors or etiological agents that could explain the symptoms are not considered, nor is the possible role of the bacterium as an environmental contaminant or component of the microbiota discussed.

Conclusions
The conclusions summarize the main findings of the study, but are stronger than the data support. The work should be presented as a preliminary study, highlighting an interesting potential isolation, but requiring further investigation to clarify the biological significance of the findings.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

No
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Microbiology, animal health, zoonoses, antibiotic resistance.

We confirm that we have read this submission and believe that we have an appropriate level of expertise to state that we do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Respond to this report

Responses (0)

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Reviewer Report

9 Views

06 Apr 2026 | for Version 1

Amro Hashish, Iowa State University, Ames, Iowa, USA

9 Views Cite this report Responses(0)

Not Approved

Review of Manuscript Entitled "Isolation and Immunopathological Evaluation of Chromobacterium violaceum from Diarrheic Chickens."
General comments:
The manuscript needs careful revision for English proofreading. Please, re-check all abbreviations.
Major Concerns:
1. The central premise of the study is insufficiently justified. The authors aim to investigate C. violaceum in poultry; however, the current literature does not support a defined clinical or pathological role for this organism in avian species. To date, C. violaceum is not recognized as a poultry pathogen in established avian literature, and reports of its occurrence in birds are extremely limited or absent. Before investigating immune responses, the study should first establish whether this organism has any biological or pathological significance in poultry. This would require evidence of causality, such as reisolation following experimental infection, demonstration of clinical signs, or histopathological lesions consistent with infection. Without such evidence, the organism may represent environmental contamination or a commensal rather than a true pathogen.
2. The objective of the study is not clear and not matching the materials and methods section
Introduction:
1. The introduction lacks a critical review of the organism in the context of poultry. Specifically, the authors did not include or discuss any previous reports of C. violaceum in avian species, including its potential detection in microbiome or metagenomic studies. A comprehensive introduction should address whether this organism has been previously identified in poultry production systems (e.g., water sources, gut microbiota, or environmental samples), even in a non-pathogenic context. The absence of such discussion weakens the rationale for the study and limits its scientific grounding.
2. Authors stated that “Infection of poultry with C. violaceum is not uncommon” this is an overstatement and not supported as there is almost no previous report highlighting the imp of this bacteria in poultry.
3. There is misplaced immunological depth in the intro that is more general immunology.

Materials and methods:
Sample and bacterial isolation:
A table describing the samples and associated meta-data “especially the clinical picture of chickens” should be added to the materials and methods. What kind of sample? “Authors needs to explain “Feces, cloacal swab…”. Please, add reference that you followed to process the sample or conduct the bacterial isolation?
The first line of the DNA extraction part reads more as instruction to do something rather than materials and methods section of scientific journal. Please, rephrase. The part related to Electrophoresis is misplaced and should be placed in a separate heading following the PCR.
The primers’ section should be merged under PCR amplification and cycling conditions with recommendation to trim some parts e.g. preparation of primer’s stock and working solution.
In some parts of the materials and methods gel conc is 1.5% and in other parts was described as 2%? Please, re-check.
Sequencing and sequence alignment section: this part lacks many details. please, list the purification method before sequencing “from amplicon/gel”, which primer “F or R or both” were used for sequencing, how many samples were submitted for sequencing, brief details if sequencing with Sanger. Software used to analyze the data “Trimming ….”
A graph describing the study design should be added for easy understanding of the design.
The study to test the immunogenicity should be separated from the pilot study to determine the infective dose.
Materials and methods lack any data related to Histopathology
Did author try to isolate any other bacterial agents that might explain the diarrhea? Or investigate any other causes for diarrhea “infectious or non”?
Results:
Table 1 should include data about the samples yielded positive results.
Fig 2: It is not clear what authors want to tell from Fig2, and the legend does not tell anything.
Figure 3: where are the lanes for Pos and Neg controls?
Results section for PCR and Sequencing needs improvement “the acc # that showed highest similarity is for another bacteria.

Discussion:
The absence of previous reports of C. violaceum in the literature might indicate non-significance of the bacteria in avian hosts rather than gab in the body of knowledge.
Furthermore, the inference of causation based solely on the isolation of the organism is scientifically unsound. Isolation alone does not establish pathogenicity or a causal relationship with disease. Demonstrating causation requires additional evidence, such as reproducibility of disease following experimental infection, consistent pathological findings, and exclusion of other potential etiological agents. This issue represents a fundamental limitation that directly impacts the interpretation and validity of the study’s conclusions.

Is the work clearly and accurately presented and does it cite the current literature?

No
Is the study design appropriate and is the work technically sound?

No
Are sufficient details of methods and analysis provided to allow replication by others?

No
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

No
Are the conclusions drawn adequately supported by the results?

No

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Molecular Diagnostics - Bacterial Genomics - Genomic Data analysis

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Respond to this report

Responses (0)

[1] Kumar M: Chromobacterium violaceum: A rare bacterium isolated from a wound over the scalp. Int. J. Appl. Basic Med. Res. 2012; 2(1): 70–72. PubMed Abstract | Publisher Full Text | Free Full Text

[2] Ajithdoss DK, Porter BF, Calise DV, et al.: Septicemia in a neonatal calf associated with Chromobacterium violaceum. Vet. Pathol. 2008; 46(1): 71–74. Publisher Full Text

[3] Alisjahbana B, Debora J, Susandi E, et al.: Chromobacterium violaceum: A review of an unexpected scourge. Int. J. Gen. Med. 2021; 14: 3259–3270. PubMed Abstract | Publisher Full Text | Free Full Text

[4] Al-Samarraae IAA, Kareem AA: The immune response of rabbits immunized by Salmonella typhimurium and Lactobacillus acidophilus. The Iraqi J. Vet. Med. 2018; 42(1): 28–34. Publisher Full Text

[5] Amason ME, Harvest CK, Beatty CJ, et al.: Chemokine expression profile of an innate granuloma. elife. 2024; 96425.1. Publisher Full Text

[6] Antony M, Ashiq M, Salim S, et al.: Inhibition of violacein synthesis in Chromobacterium violaceum DSTS-1 mutant. Int. J. Adv. Biotechnol. Res. 2013; 4(1): 151–157. Reference Source

[7] Batista JH, Da Silva Neto JF: Chromobacterium violaceum pathogenicity: Updates and insights from genome sequencing of novel Chromobacterium species. Front. Microbiol. 2017; 8. PubMed Abstract | Publisher Full Text | Free Full Text

[8] Carrasco L, Astorga R, Méndez A, et al.: Acute pleuropneumonia in Barbary sheep (Ammotragus lervia) associated with Chromobacterium violaceum. Vet. Rec. 1996; 138(20): 499–500. PubMed Abstract | Publisher Full Text

[9] Cavallaro M, Ciccia F, Luca A, et al.: Immunization with killed bacterial vaccines: Histopathological effects in different tissues. J Immunol Res. 2020; 2020: 4216892. Publisher Full Text

[10] Crosse PA, Soares K, Wheeler JL, et al.: Chromobacterium violaceum infection in two dogs. J. Am. Anim. Hosp. Assoc. 2006; 42(2): 154–159. PubMed Abstract | Publisher Full Text

[11] Deng S, Wang X, Liu J, et al.: Intestinal barrier damage during bacterial infections: Histopathological and molecular insights. Clin. Exp. Immunol. 2017; 188(3): 183–194. PubMed Abstract | Publisher Full Text | Free Full Text

[12] George D, Mallery P: IBM SPSS Statistics 26 step by step. Routledge; 2019. Publisher Full Text

[13] Hammerschmitt ME, Rolim VM, Snel GGM, et al.: Chromobacterium violaceum infection in a horse. J. Comp. Pathol. 2017; 156(4): 334–338. PubMed Abstract | Publisher Full Text

[14] Haq RIU, Parray OR, Nazir QUA, et al.: Immune and oxidative response against sonicated antigen of Mycoplasma capricolum subspecies capripneumonia—a causative agent of contagious caprine pleuropneumonia. Microorganisms. 2022; 10(8): 1634. PubMed Abstract | Publisher Full Text | Free Full Text

[15] Harvest CK, Abele TJ, Yu C, et al.: An innate granuloma eradicates an environmental pathogen using Gsdmd and Nos2. Nature Communications. 2023; 14(1). PubMed Abstract | Publisher Full Text | Free Full Text

[16] Ibrahim Y, Abouwarda A, Assar N: Identification and characterization of a soil isolate of Chromobacterium violaceum from Egypt with potential to cause disease. The Egyptian J. Med. Microbiol. 2020; 29(1): 153–160. Publisher Full Text

[17] Kielkopf CL, Bauer W, Urbatsch IL: Methods for measuring the concentrations of proteins. Cold Spring Harbor Protocols. 2020; 2020(4). Publisher Full Text

[18] Lafta IJ, Sadeq Z: Pseudomonas aeruginosa is an effective indicator for screening of quorum-sensing inhibition by plant extracts. The Iraqi J. Vet. Med. 2024; 48(1): 54–62. Publisher Full Text

[19] Lin YD, Majumdar SS, Hennessy J, et al.: The spectrum of Chromobacterium violaceum infections from a single geographic location. Am. J. Trop. Med. Hyg. 2016; 94(4): 710–716. PubMed Abstract | Publisher Full Text | Free Full Text

[20] Liu CH, Chu RM, Weng CN, et al.: An acute pleuropneumonia in a pig caused by Chromobacterium violaceum. J. Comp. Pathol. 1989; 100(4): 459–463. PubMed Abstract | Publisher Full Text

[21] Liu DX, Didier PJ, Plauche GB: Chromobacterium violaceum infections in 13 non-human primates. J. Med. Primatol. 2011; 41(2): 107–114. PubMed Abstract | Publisher Full Text | Free Full Text

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Isolation and Immunopathological Evaluation of Chromobacterium violaceum from Diarrheic Chickens

Abstract

Background

Methods

Results

Conclusions

Keywords

Introduction

Materials and methods

Samples and bacterial isolation

Molecular identification of Chromobacterium violecium

Sequencing and sequence alignment

Antigen preparations

Preparation of Aloe Vera extract

Experimental design

Statistical analysis

Results

Isolation

Table 1. Rates of C. violaceum isolates.

Identification

Figure 1. C. violaceum on SS agar, MacConkey agar, and Nutrient agar.

Table 2. Vitek 2 compact system and antimicrobial susceptibility for C. violaceum.

Figure 2. Gel electrophoresis of genomic DNA extracted from C. violaceum, utilizing a 1% agarose gel at a voltage of 5 V/cm for 30 minutes.

Figure 3. displays a PCR product with a band size of 1250 bp. The electrophoresis was conducted on a 1.5% agarose gel at a 5 volts/cm2 voltage using 1x TBE buffer for 1 hour and 30 minutes.

Figure 4. C. violaceum homologous with the referenced strain on GenBank.

Table 3. The Level of IL-6 in immunized mice (n=6).

IL-6 measurement

Table 4. The Level of IgG in immunized mice (n=6).

Figure 5. The level of IL-6 & IgG In immunized groups.

IgG measurement

Histopathological examination

Figure 6. G1 The liver section revealed normal parenchyma, normal liver histology, and no pathological lesions identified by H&E stain. 100x.

Figure 7. G1The spleen section revealed the presence of well-formed granulomas—collections of epithelioid histiocytes by H&E stain. 100x.

Figure 8. G1The kidney section showed histologically normal renal cortex, and no apparent pathology was present in this section by H&E stain. 100x.

Figure 9. G1The intestinal section showed intact villous architecture with slight development of subepithelial (Gruenhagen's) spaces at the villus tip by H&E stain. 100x.

Figure 10. G2 The liver section showed mild portal inflammation with lymphocytic infiltrates by H&E stain. 100x.

Figure 11. G2 The spleen section revealed Lymphoid hyperplasia or possible lymphoma and dense lymphocytic infiltrate by H&E stain. 100x.

Figure 12. G2 Normal renal histology without significant pathological changes.by H&E stain. 100x.

Figure 13. G2 The intestinal section showed moderate subepithelial space formation and epithelial lifting to the sides of the villi by H&E stain. 100x.

Figure 14. G3 the liver section showed dense infiltrate of lymphocytes in the portal area extending into the periportal area, and interface hepatitis is seen (piecemeal necrosis by H&E stain. 100x.

Figure 15. G3 The spleen section showed follicular hyperplasia, tubules appeared dilated with cytoplasmic vacuolization, and moderate lymphocytic infiltration in interstitial areas by H&E stain. 100x.

Figure 16. G3 The kidney section revealed Diffuse lymphocytic infiltration, focal necrosis, and congestion in zones by H&E stain. 100x.

Figure 17. G3The intestinal section showed intact villous architecture with slight development of subepithelial (Gruenhagen's) spaces at the villus tip by H&E stain. 100x.

Discussion

Conclusion

Ethical approval

Human participants statement

Informed consent statement

Data availability

Acknowledgements

References

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Figure 3. displays a PCR product with a band size of 1250 bp. The electrophoresis was conducted on a 1.5% agarose gel at a 5 volts/cm² voltage using 1x TBE buffer for 1 hour and 30 minutes.