An Experimental Infection of Pseudomonas fluorescens and Its Effects on Hematology and Histopathology of Nile Tilapia, Ethiopia

doi:10.21203/rs.3.rs-4880553/v1

Nile tilapia (Oreochromis niloticus) one of the commercially important fast-growing and well-adapted freshwater fish is cultivated extensively worldwide (FAO 2018). The present study was conducted on 36 apparently healthy Nile Tilapia (130 ± 10 g. body weight and average 19.5 cm length) collected from the National Fisheries and Aquatic Life Research Center (NFARC), Sebeta. Fish were adapted for one week in the experimental greenhouse of NFARC before starting the experiment Fish were divided into two groups (18 fish per each), inoculated intraperitoneally using 0.2 ml of saline, and the pathogen, Pseudomonas fluorescens at a concentration of 3 x 106 CFU/ml. The inoculated fish were observed for 21 days for pathogenicity. The clinical signs observed on the second day post injection were loss of balance, excessive mucus secretions on skin and gills, ascites with slightly protruded reddish vent, hemorrhages all over the body surface, frayed fins, and an empty intestine. This study showed that Pseudomonas infection caused 55.4% (10) mortality, with severe alterations in RBC and WBC counts with severe histopathological changes in muscle, gills, liver, kidney, spleen, and intestine. The histopathological changes observed include hemorrhages, tubular cell necrosis, congestion, mononuclear cell aggregation and infiltration in the kidney; hepatocyte vacuolization in liver; lamellar fusion in gills; sloughed and mucosal necrosis in intestine; mononuclear cell aggregation and infiltration in muscle and hemosiderin accumulation in spleen.

Nile tilapia

Pseudomonas flourescens

hematology

histopathology

Nile tilapia, O. niloticus is one of the commercially important fast growing and well-adapted freshwater fish cultivated extensively all over the world (FAO. 2018). It is the second most important freshwater fish after carps with an annual global production of 6.4 MT (Nicholson et al., 2020). Although Nile tilapia is resistant to diseases (Abdel-latif & Asmaa, 2020), they are subjected to infection by a wide variety of pathogens especially bacterial pathogens which cause serious diseases in both wild and cultured fish (Maldonado-Miranda et al., 2022). Pseudomonas bacteria are opportunistic gram-negative bacteria, they occur naturally in aquatic environments, as part of normal gut flora of healthy fish. However, under unfavorable conditions, they become pathogenic causing disease outbreaks (Angelini, 1988; Tohamy et al., 2015 & Vanamala et al., 2022). The disease is characterized by fin rot, hemorrhages, skin darkness, detached scales, abdominal ascites, and exophthalmia (Austin, 2007 & Khalil et al., 2014). The infection reduces RBC count, hemoglobin, and hematocrit and increases the level of WBC counts (Martins et al., 2008). The present study was carried out to determine the effect of Pseudomonas fluorescens on the hematological and histopathological changes in O. niloticus in culture ponds

2.1. Experimental setup: Thirty-six (36) apparently healthy Nile tilapia with an average weight of 130 g ± 10 and an as average length of 19.5 cm obtained from the fish ponds of NFARC were transported to the Greenhouse of NFARC, Sebeta for experimentation. The fish were adapted for two weeks in the holding fiberglass tank (1m x 75 cm x 75 cm) with 1000 l dechlorinated water and were fed at 4% of body weight with a commercial diet containing 30% crude protein (ElSaidy, 2005).

2.2, Isolation and Identification of Pseudomonas fluorescens strain.

Bacterial pathogens were isolated from the muscle tissue, liver, kidney, and spleen as per the method of Buller (2014) and Nair et al. (2021). They were then identified using biochemical methods up to the species level based on colony morphology and arrangement, staining characteristics, and conventional biochemical tests (Austin & Austin, 2016). Further identification of the species was done by API 20E (Buller. 2014). The species identified were: Pseudomonas aeruginosa, P. putida, and P. flourescens. From the isolates identified, the dominant strain Pseudomonas fluorescns was selected to study its effects on the hematology and histopathology of Nile tilapia.

2.3. Experimental design: The fish were divided into two groups, each group with eighteen fish. The first group was injected with 0.2 ml of physiological saline as a control. The second group was injected with 0.2 ml of bacterial pathogen, Pseudomonas fluroscens with a concentration of 3x10⁶ CFU/ml. Fish were observed daily for clinical signs, abnormal behavior, and mortalities if any for a period of twenty-one days post-infection.

2.4. Hematological analysis

The blood sample was taken three times, one before pathogen injection, 2nd and 4th week after the injection Blood film was prepared according to the method described by Bittencourt et al. (2003).Hemoglobin (Hb) was estimated by the Cyanomethemoglobin method (Lavanya et al., 2011) using Hemocor-D reagent. Hematocrit (Hct) was determined by centrifuging the blood-laden duplicate hematocrit capillaries for each sample in a capillary centrifuge (REMI RM-12C, India) at 10,000g for 10 min at room temperature. An improved Neubauer hemocytometer was used to determine the total blood counts i.e.: RBC and WBC in blood mixed with Natt-Herrick’s stain solution (Natt & Herrick, 1952). The number of cell counts was determined as described by Pal et al. (2008) and Parida et al. (2011). Total RBC count per mm3 = 200×50×N⇒10,000N (N = number of RBC counted, dilution factor = 200) and Total WBC count per mm3= {(20×1×L) ÷ (0.4)} cells⇒50×L (L = number of WBC counted dilution factor = 50). Erythrocyte indices such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated as per the method of Bain et al. (2006).

1.1. Histopathology

The infected muscle, liver, spleen, kidney, gills, and intestinal tissues were considered for the study. The tissues were collected during the first, second, and fourth week post-infected fish from each group and fixed in 10% neutral-buffered formalin for 24 hrs. After clearing the tissues in alcohol series, they were embedded in paraffin wax and prepared tissue blocks which were then sectioned in five-micron thickness. Sectioned tissues were stained in hematoxylin and eosin (Bancroft, 1996 & Roberts, 2004).

2. Data analysis

Data collected were analyzed using Ch-square test to study the significance of different variables using SPSS (2004)

The clinical signs were observed during the second day of the post-injection. Fish showed loss of balance, excessive mucus secretion on skin and gills, detachment of scales, and ulceration in the skin. Fish showed mortalities up to 55.5%.

Postmortem examination

The infected fish showed abdominal dropsy with reddish ascites exudates, liver paleness and enlarged in the some fish, the gallbladder changed in coulor to blue and empty intestine.

5.1. Hematological studies:

Table 1

Hematological parameters of Nile tilapia during the experimental Pseudomonas flurescens infection
Period (week) Groups	RBCs10 / mm	Hb (g/dl)	WBCs 10 / mm	MCV	MCH	MCHC
1 control	1.86(3.-5.5)	10.7(11.0–16.0)	12.8(4.0 10.0)	85( 12.36-528.57)	57.9(5.07-120.86)	74(19.84–87.73)
Pseudomonas infection	1.64(3.5–5.5)	11.7(11.7–16.0)	19.5(4.010.0)	98.3( 12.36-528.57)	71.2(5.07-120.86)	72.5(15–45)
2 control	2.17(3.5–5.5)	10.8(11.0–16.0)	13(4.0–10.0)	108.7( 12.36-528.57)	62.9(5.07-120.86)	53.2(15–45)
Pseudomonas infection	1.95(3.5–5.5)	13.5(11.0–16.0)	17(4.0–10.0)	42( 12.36-528.57)	69.1(5.07-120.86)	16.43(15–45)
4 Control	2.17(3.5–5.5)	13.8(11.7–16.0)	12.6(4.0–10.0)	95.7( 12.36-528.57)	57.9(5.07-120.86)	63.2(15–45)
Pseudomonas infection	1.51(3.5–5.5)	10.2(11.0–16.0)	17 (4.0–10.0)	67.7( 12.36-528.57)	67.1(5.07-120.86)	99.2(15–45)

As shown in Table (1). Red blood cells counts and Hemoglobin estimation showed a significant decrease in the infected group when compare with control group along period of the experiment. As well, the infected Nile tilapia showed a significant increase in WBCs counts, lymphocytes, MCV, MCH, MCHC and HCT along period of the experiment when compare with control group (Zafar, et al., 2023)

3.1 Histopathological

Kidney of Nile tilapia infected with P. fluorescens showing a) hemorrhagic lesions, b) mononuclear cell aggregation, c) kidney tubule cell necrosis, d) congestion and mononuclear cell infiltration (X 250).

Liver of Nile tilapia infected with P. fluorescens: a) liver hepatocytes, b) vacuolization c) mononuclear cell aggregation (black arrow), d) liver hepatocytes vacuolization (black arrow) and congestion (arrow X250).

Figure of intestine

Intestines of Nile tilapia infected with P. fluorescens: a) Intestine sloughed (black arrow), b) necrotized mucosa (red arrow), c) intestine mucosal necrosis (black arrow)

Figure of gills

Gill of Nile tilapia infected with P. fluorescens: (a) gill lamellar fusion (black line), b) necrotized lamellae (red arrow), c) gill mononuclear cell aggregates and lamellae necrosis x10, d) gill congestion e) thickened lamellae with mononuclear cells (red line)

Figure muscles

Muscle of Nile tilapia infected with P. fluorescens: a) muscle mononuclear cell aggregation and infiltration (black arrow), b) muscle necrosis (black line), c) mononuclear cell infiltration (red arrow)

A) Photomicrograph of spleen.

Nile tilapia infected with P. fluorescens: a) spleen hemosiderosis (arrow),b)spleen no demarcation between red and white pulp and hemosiderin accumulation (arrow)

3.1.1. General pathology

Spleen

The spleen exhibited hemosiderin accumulation, a mix of white and red pulp, and mild focal necrosis during the 2nd and 4th weeks.

Gills

The gills showed lamellar fusion, thickening, congestion, mononuclear cell aggregation, necrosis, and hyperplasia in both primary and secondary lamellae.

Liver

The liver exhibited lesions, vacuolization, necrosis, and bile duct proliferation. By the 2nd and 4th weeks, there was increased vacuolization and necrosis of hepatocytes, along with mononuclear cell aggregation and some eosinophils.

Muscles

The muscles showed lesions, necrosis, mononuclear cell aggregation, and infiltration.

Kidney

The kidney displayed congestion, tubule cell desquamation, mononuclear cell aggregation, hemorrhage, hydropic degeneration of tubular epithelial cells, and distorted glomeruli with increased cellularity.

Intestine

The intestine showed congestion, shortening and sloughing of villi, and mononuclear cell aggregation during the 2nd and 4th weeks.

Aquaculture is a novel technology and quickly growing sector in Ethiopia and has special attention in the country to increase food and security. It is subject to different environmental stressors such as water quality and bacterial infections during intensification. Pseudomonas spp is one of the most pathogenic bacteria causing large culture fish mortality. In the present study, Nile tilapia was collected from the aquaculture pond for experiments. The Infected Nile Tilapia showed external signs and symptoms such as loss of balance, scales, and skin; excessive mucus secretion on gills and skin, skin ulceration, and ascites with protruded reddish vent. The body showed hemorrhages with ragged and rotten tails and fins. Internally, fish show abdominal dropsy with reddish ascites exudates.

Hematology: The decreased hemoglobin and hematocrit levels in infected fish may be attributed to Pseudomonas species, which are known to cause anemia. This response could result from the disruption of red blood cell production and the destruction of hematopoietic organs (Omoregie, 1998; Nannu et al., 2015). This aligns with reports by Haney (1992), who observed reduced hemoglobin and hematocrit levels in several fish infected with Pseudomonas and Aeromonas bacterial pathogens. This study provides the first evidence of an experimental infection of Pseudomonas fluorescens and its effects on the hematology and histopathology of Nile tilapia in Ethiopia. The gills exhibited fused lamellae and congestion caused by basal hyperplasia and necrosis, resulting in the fusion of primary and secondary lamellae. The bacteria secrete an extracellular hyperplasia-inducing factor, which can lead to lesions like epithelial hyperplasia associated with lamellae fusion (Tomay, 2015; Keerthika et al., 2023). Fish kidneys are key hematopoietic and osmoregulatory organs. In this experiment, the kidneys exhibited necrosis of the tubular cells, congestion, tubular cell desquamation, mononuclear cell aggregation, hemorrhage, mononuclear cell infiltration, hydropic degeneration of the tubular epithelial cells, and distorted glomeruli with increased cellularity. Similar results were reported by Faheem et al. (2015) and Waris Sidra et al. (2023). In the present studies, the liver of Nile tilapia infected with P. fluorescens showed hepatocyte vacuolization (red line) and mononuclear cell aggregation (black arrow). Additionally, hepatocyte vacuolization (black arrow) and congestion (arrow X250) were observed, similar to the findings of Hal and M. (2020). The intestines of infected Nile tilapia, stained for histopathology, displayed sloughing (black arrow), necrotized mucosa (red arrow), and mucosal necrosis, consistent with the results reported by Hal and M. (2020). In the present studies, the muscle of Nile tilapia infected with P. fluorescens showed (a) muscle mononuclear cell aggregation and infiltration (black arrow) and (b) muscle necrosis (black line) and mononuclear cell infiltration (red arrow), which are similar to the findings of El-Sharaby (2018). The spleen of infected fish exhibited (a) hemosiderosis (arrow) and (b) a lack of demarcation between red and white pulp with hemosiderin accumulation, consistent with the results reported by Mahmoud et al. (2018)

The study demonstrated that Pseudomonas fluorescens is primarily pathogenic to Nile tilapia (Oreochromis niloticus), causing significant hematological and histological changes in the infected fish. These changes emphasize the aggressive nature of the bacteria. The clinical manifestations have potentially disruptive effects on the survival and overall production of Nile tilapia in aquaculture and fisheries. This information is crucial for maintaining fish health, improving fish production, and ultimately benefiting the economy of aquaculture farmers and the nation as a whole.

Acknowledgements

I extend my special thanks to Dr. Asegedach Sirak, Dr. Marshet Adugna, Tewoderos Arga and Solomon Getechew for their outstanding technical assistance in pathology laboratory work. I am also grateful to Prof Natarajan p, Dr. Genet Abe and Kassaye Belkaw for their insightful discussions, encouragement, and constructive comments, which greatly improved the proposal's content. Appreciation is extended to the Ethiopian Institute of Agricultural Research, National Fisheries and Aquatic Life Research Center, Biology Department of Hawassa University, and Batu Fishery and other Aquatic Life Research Center for providing laboratory space during fieldwork. Additionally, I thank Tsige Adimkew from Sebeta for his invaluable help in collecting samples.

Authors’ contributions

Alemu Kebede, Tewoderos Arega, and Zufan Bedewi carried out fish and bacterial sampling activities. Alemu Kebede, Tewoderos Arega, Nebiyu Kassa, and Mershet Adugna performed the laboratory work. AK drafted the manuscript, while Prof. Natarajan and TRC revised it and gave final approval for the submitted version.

Funding

This research was partially funded by the Animal Health Institute and the Department of Aquatic Science, Fisheries, and Aquaculture at Hawassa University, Ethiopia. The funding sources did not influence the study design, data collection, analysis, or interpretation, the writing of the report, or the decision to submit the article for publication.

Data Availability

The datasets used and analyzed during the current study are available from The corresponding author on reasonable request.

Ethics approval

This dissertation work was conducted following the fish handling and sample collection guidelines established by Hawassa University, College of Natural and Computational Science, in 2023. The study protocol was approved by the Research Ethics Committee of the same institution. Additionally, the fish were euthanized and rendered unconscious during sample collection to ensure animal welfare. The euthanasia procedure followed the "American Veterinary Medical Association Guideline for Euthanasia" published in 2020. The fish were euthanized by transecting the spinal cord behind the skull, as described by Austin (2016)

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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No competing interests reported.

An Experimental Infection of Pseudomonas fluorescens and Its Effects on Hematology and Histopathology of Nile Tilapia, Ethiopia

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.2, Isolation and Identification of Pseudomonas fluorescens strain.

2.4. Hematological analysis

1.1. Histopathology

2. Data analysis

3. Results

5.1. Hematological studies:

3.1.1. General pathology

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

Status:

Version 1