




































Highlights in BioScience
ISSN:2682-4043
DOI:10.36462/H.BioSci.202509

Research Article
Open Access

1 Department of Aquatic Animal Health, Faculty

of Fishery Sciences, West Bengal University

of Animal and Fishery Sciences, Chakgaria,

Kolkata 700094, West Bengal, India.
2 NITTE-GOK COE AQUAMARIN, NUCSER,

NITTE (Deemed to be University), Paneer

Campus, Deralakatte, Mangalore 575018,

Karnataka, India.
3 State Referral Laboratory for Aquatic Animal

Health, Tamil Nadu Dr J.Jayalalithaa Fisheries

University, Madhavaram Milk Colony, Chennai

600051, Tamil Nadu, India.
4 Aquatic Animal Health and Environment Divi-

sion, ICAR-Central Institute of Brackishwater

Aquaculture, Raja Annamalai Puram, Chennai

600028, Tamil Nadu, India.

* To whom correspondence should be
addressed: abrahamtj1@gmail.com

Editor: Alsamman M. Alsamman, International
Center for Agricultural Research in the Dry Areas
(ICARDA), Cairo, Egypt.

Reviewer(s):
Hayder Tawfeeq Qaddoori, Middle Technical
Univarsity, Technical insitute of Baqubah,
Department of Nursing, Dayala - Iraq.

Abdulaziz Ascandari, African Genome Center,
Mohammed VI Polytechnic University, Ben Guerir,
Morocco.

Received: July 26, 2025

Accepted: September 29, 2025

Published: October 18, 2025

Citation: Sinha P, Abraham TJ, Sen A, Sharon J,
Das R, Rajeshwar BN, Uma A, Patil PK. Influence
of in-feed oxolinic acid therapy on the brain
histoarchitecture and virulence factors of
Streptococcus agalactiae infecting Nile tilapia
Oreochromis niloticus . 2025 Oct. 18;8:bs202509

Copyright: © 2025 Sinha P 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
author and source are credited.

Influence of in-feed oxolinic acid therapy on the brain histoarchitecture
and virulence factors of Streptococcus agalactiae infecting Nile tilapia
Oreochromis niloticus

Priyanka Sinha1
>< �, Thangapalam Jawahar Abraham*1

><�, Arya Sen1
>< �,

Joshi Sharon1
>< �, Ratnapriya Das1

>< �, B. Naveen Rajeshwar2
><�, Arumugam

Uma3
><�, Prasanna Kumar Patil4 >< �

Abstract

Streptococcosis and its management are major constraints to tilapia aquaculture.
The present study assessed the effect of oral administration of 12 mg oxolinic acid
(OA)/kg fish/day for 7 uninterrupted days against Streptococcus agalactiae LCR1
(Sa) infection and its influence on histopathological anomalies in the forebrain,
optic tectum region of the mid-brain and granular cell layer of the cerebellum of
Oreochromis niloticus. Besides, attempts were made to understand how the OA impacts
the glycosyltransferases and CAMP factor of S. agalactiae through molecular docking.
The LD50 of Sa was 1.26 × 108 cells/fish. Sa infection was apparent in OA-treated
and untreated groups, and the brain tissues exhibited the progression and reversal of
meningitis. The forebrain exhibited thickening of the meninx primitiva, vacuolation,
and degeneration in the brain parenchyma and meninx primitiva. Inflammatory changes
such as meningitis and mononuclear cell infiltration were documented. The midbrain
had edematous optic tectum, loosening of connective tissue, and leukocyte infiltration.
In the granular cell layer, cerebellum changes such as spongiform encephalopathy
and necrosis indicate region-specific damage. However, the OA effectively reduced
the severity of brain tissue damage, possibly by its binding affinities and upsetting
the activities of glycosyltransferases and CAMP factor, as confirmed by molecular
docking. These results confirmed that OA can cross the blood-brain barrier and interact
with virulence proteins to reduce the Sa infection in the brain. Furthermore, the results
underscore its responsible use in aquaculture, as OA is a critically important human
medicine and ought to be used as a secondary treatment.

Keywords: Aquaculture, streptococcosis, virulence proteins, antibiotic therapy, meningitis,

histoarchitecture.

Introduction
Aquaculture is swiftly intensifying, significantly contributing to global food production, eco-

nomic development, and nutritional security. In 2022, aquaculture contributed about 59% of the

total fish production of 185.4 million tonnes. Cichlids, including tilapias, ranked third in the ma-

jor farmed fish group with a contribution of 10.6% [1]. Tilapias are highly favourable species for

aquaculture due to their adaptability, rapid growth, disease resistance, and low production costs [2].

However, intensive tilapia aquaculture has led to a rise in diseases, particularly bacterial infections

like streptococcosis, motile Aeromonas septicemia (MAS), vibriosis, and columnaris, which pose

significant challenges to farming [3]. Streptococcosis, caused by Streptococcus agalactiae (Sa) and

S. iniae, is one of the most important bacterial diseases in tilapia aquaculture, causing substantial

economic losses globally [2; 4]. It can lead to meningoencephalitis in tilapia, characterised by several

neurological disorders and brain lesions [5; 6; 7; 8]. Studies indicated that Sa causes extensive

brain damage, particularly in regions controlling swimming activities, due to bacterial invasion

and acute inflammatory responses [6; 8; 9]. Control measures for streptococcosis include proper

farm location selection, good aquaculture practices, antibiotics, immunostimulants, and vaccines [2].

Antimicrobials like amphenicols, quinolones, and tetracyclines are widely used, but their use raises

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https://creativecommons.org/licenses/by/4.0/
mailto:sinhapriyanka1197@gmail.com
https://orcid.org/0009-0005-1867-5422
mailto:abrahamtj1@gmail.com
https://orcid.org/0000-0003-0581-1307
mailto:arya.sen2@gmail.com
https://orcid.org/0000-0001-8991-7875
mailto:ashjosh9@gmail.com
https://orcid.org/0009-0009-6799-9473
mailto:ratnapriyadas2015@gmail.com
https://orcid.org/0000-0002-6414-7840
mailto:naveen.rajeshwar@nitte.edu.in
https://orcid.org/0000-0002-4496-9492
mailto:uma@tnfu.ac.in
https://orcid.org/0000-0002-9429-4075
mailto:PK.Patil@icar.org.in
https://orcid.org/0000-0002-1924-5863
http://bioscience.highlightsin.org/


Sinha P et al., 2025 Effect of oxolinic acid therapy

Figure 1. The histopathological changes indicating meningitis (*) in the

forebrain of Streptococcus agalactiae LCR1 challenged and oxolinic

acid (OA) treated and untreated Oreochromis niloticus juveniles. [A]

Control, [B] Day post-injection (DPI) 1 OA treated and [C] untreated;

[D] DPI 7 OA treated and [E] untreated; [F] DPI 14 OA treated and

[G] untreated; and [H] DPI 21 OA treated and [I] untreated. DMP:

Damage of meninx primitiva; FH: Focal haemorrhage; IMC: Infiltration

of mononuclear cells; LS: Lifting of superficial layer; LSE: Localized

spongiform encephalopathy; MP: Meninx primitiva (meningitis);

TMP: Thickening of meninx primitiva; DBP: Degeneration of brain

parenchyma; and V: Vacuolation x200, H&E staining.

concerns about antimicrobial resistance (AMR), particularly
in regions like China and India [10; 11; 12; 13].

The quinolone antibiotic, oxolinic acid (OA) is not on the
list of approved drugs for use in finfish production by the Food
and Drug Administration in the United States [14]. It develops
resistant bacterial strains that may threaten human health [10; 15].
However, the European Union, Japan, and other Asian countries
have recommended it to prevent bacterial infections at 12 mg/kg
fish/day for 7 days [11; 16]. The antibiotics of the quinolone
family are categorized as critically important and the highest
priority antimicrobial group for human use. Such medicines
should not be used to treat infectious diseases in food-producing
animals. Yet, these medicines are proposed for a second-line
treatment in food-producing animals, complying with the national
legislation in force, when no other alternatives are available [15;
17; 18].

Several studies reported the effectiveness of OA in controlling
fish bacterial pathogens and the onset of diseases [19; 20; 21; 22].
However, more research is needed to evaluate its mode of ac-
tion, safety in tropical fish species, and efficacy against several
mesophilic fish pathogens to ensure sustainable aquaculture prac-

Figure 2. The histopathological changes in the optic tectum (OT) region of the

brain of Streptococcus agalactiae LCR1 challenged and oxolinic acid (OA)

treated and untreated Oreochromis niloticus juveniles. [A] Control, [B] Day

post-injection (DPI) 1 OA treated and [C] untreated; [D] DPI 7 OA treated

and [E] untreated; [F] DPI 14 OA treated and [G] untreated; and [H] DPI 21

OA treated and [I] untreated. EOT: Edematous optic tectum; IL: Infiltration

of leucocytes; IMC: Infiltration of mononuclear cells; LDC: Loosened dense

connective tissue; LS: Lifting of superficial layer; LSE: Localized spongiform

encephalopathy; MP: Meninx primitiva; SLC: Spacing in loose connective tissue;

TMP: Thickening of meninx primitiva; and V: Vacuolation x200, H&E staining.

tices [23]. The infection of Sa relies on several virulence factors,
which encompass adhesion, invasion, and infection. Numerous
virulence factors contribute to invasion and colonisation [24; 25].
The relationship between antibiotics, virulence proteins, and the
ability of bacteria to infect the brain is complex, involving factors
like the blood-brain barrier (BBB) and host immune responses.
For example, glycosyltransferases and the CAMP factor play
crucial roles in pathogenesis, as highly pathogenic strains of Sa
reportedly produce glycosylated serine-rich repeats and glycosyl-
transferases [26; 27]. The extracellular protein, such as CAMP
factor, induces pore formation in target cells and heightens the
pathogenicity of Sa [24; 25; 28].

However, there is a lack of scientific literature focusing on
the specific effects of aquaculture antibiotics on brain-infecting
bacteria, such as Sa, and their virulence proteins. Also, the mis-
use of antibiotics can lead to the emergence of AMR, which is a
significant public health concern [10; 15]. Therefore, to combat
Sa infections, understanding the roles of important virulent pro-
teins and their interaction with antibiotics is essential. This study
delved into the efficacy of OA, an antibiotic recommended for
second-line treatment in food-producing animals [18], to curtail
the effect of Sa-infection on the brain histoarchitecture of Nile
tilapia Oreochromis niloticus, and to comprehend how OA im-
pacts the invasion of Sa into the fish brain by molecular docking

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Sinha P et al., 2025 Effect of oxolinic acid therapy

using glycosyltransferases and CAMP factor, to plan for further
course of research and specific mitigation strategies.

Materials and methods
Experimental fish and bacterial strain and its pathogenicity

Farm-raised young and healthy Oreochromis niloticus were
chosen regardless of sex and adapted in circular tanks of 500 L
capacity for 15 days before the experimentation [23]. Strepto-
coccus agalactiae LCR1 (National Centre for Biotechnology
Information (NCBI) accession number OP752129) was obtained
from the State Referral Laboratory for Aquatic Animal Health,
Madhavaram, Chennai, India. Following earlier descriptions, the
bacterium was assessed for its pathogenicity against O. niloticus
(n = 10 for each challenge dose from 105 to 109 and control, in
triplicate) by intramuscular challenge [29; 30]. The mean lethal
dose (LD50) was calculated from the mortality data using the
method established by Reed and Muench [31]. Using the broth
dilution method, the minimum inhibitory concentration (MIC) of
OA was calculated [32]. Before the challenge, inocula from the
kidney of healthy tilapia (n = 2) were plated onto brain heart in-
fusion agar (BHIA) to ascertain that the stocks were disease-free
[5].

Efficacy of oxolinic acid against Streptococcus agalactiae infection
The European Medicines Evaluation Agency (EMEA) rec-

ommended a dose of 12 mg OA/kg fish/day for 7 uninterrupted
days to control bacterial infection in fish [16]. The medicated
feed to feed the experimental fish at 2% body weight (BW) was
set by emulsifying 0.6 g of OA powder (O0877-25G; CAS-No:
14698-29-4; Sigma-Aldrich, India) in 5 mL vegetable oil. This
emulsion was then top-coated onto the commercial floating pellet
feed (1 kg) and admixed thoroughly. Correspondingly, a control
feed with binder but without OA was prepared [21]. The analy-
sis of OA in medicated feed by liquid chromatography-tandem
mass spectrometry (LC-MS/MS) revealed 96±1% of the incorpo-
rated dose [23]. The efficacy study utilized O. niloticus weighing
17.56 ± 0.89 g (n = 225) and distributed randomly in nine thor-
oughly cleaned polypropylene tanks (L58 × H45 × B45 cm).
The rearing water (80 L) in each tank was conditioned for three
days, stocked with 25 accustomed tilapias, and properly covered
and marked. About half of the water was exchanged to remove
feed and faecal wastes. The fish stocks were divided into three
groups, in triplicate. The control, designated as group 1, received
a 0.1 mL saline injection intramuscularly at the dorsal fin base.
All fish of groups 2 and 3 were injected intramuscularly with
S. agalactiae LCR1 at 1.32 × 107 cells/fish. Post-injection, the
fish were placed in their respective tanks. Control feed was of-
fered to groups 1 and 3 throughout the experimental period of
21 days. OA-medicated feed at 2% BW was served to group 2
continuously for 7 days post-injection (DPI) and then switched
to a control feed [21; 23]. Water quality was continuously mon-
itored, and any leftover feed, if any, was removed 1 hour after
each feeding. Observations on mortality, infection symptoms,
and behaviour were recorded daily. Inocula from the freshly dead

Figure 3. The histopathological changes in the granular cell layer of the

cerebellum (GIC) of the brain of Streptococcus agalactiae LCR1 challenged

and oxolinic acid (OA) treated and untreated Oreochromis niloticus juveniles.

[A] Control, [B] Day post-injection (DPI) 1 OA treated and [C] untreated; [D]

DPI 7 OA treated and [E] untreated; [F] DPI 14 OA treated and [G] untreated;

and [H] DPI 21 OA treated and [I] untreated. LSE: Localized spongiform

encephalopathy; V: Vacuolation; LGC: Lysis of granular cells of the cerebellum;

and N: Necrotised area x200, H&E staining.

Sa-challenged tilapia brain were plated onto BHIA to establish
Sa-infection [5].

Histopathology and evaluation of pathological anomalies
On 0, 1, 7, 14, and 21 DPI, two fish were randomly sampled

from each replicate tank, euthanized by adding clove oil at 100
µL/L, and dissected prudently. The cranial bone was removed
carefully to expose the brain. The precisely collected whole-
brain tissue samples were fixed for 24 hours in Bouin’s fixative,
processed, and embedded in paraffin wax. Sections of 5 µm
thickness were sliced, processed, and double-stained using hema-
toxylin and eosin [33]. The brain sections were examined under
an Olympus microscope (Model: BX51) at 20× magnification
to identify abnormalities in tissue arrangement. For quantitative
analysis, images were captured and processed using an SCO-
LUX camera (16 MP) and ToupTek ToupView software (Version
x64).

The histopathological abnormalities were evaluated based on
circulatory, progressive, regressive, and inflammatory changes.
Following the observations, reaction indices (RI) were calculated
as a product of the score value and the importance factor. The
score value, ranging from 0 to 6, was assigned based on the
severity and degree of damage: no change (0), mild occurrence
(2), moderate occurrence (4), and severe occurrence (6). The
importance factor, ranging from 1 to 3, was assigned based on
the pathological significance of each alteration, categorized 1:
minimal pathological importance, easily reversible post-stressor

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Sinha P et al., 2025 Effect of oxolinic acid therapy

Figure 4. Homology modelled 3D structures [A1 and B1] and Ramachandran

plots [A2 and B2] of glycosyltransferase (AAM99596) and CAMP factor

(AKI96324) of Streptococcus agalactiae, respectively, computed using SWISS-

MODEL.

administration, 2: moderate pathological importance, reversible
if the stressor is neutralized, 3: marked pathological importance,
generally irreversible with potential loss of organ function. For in-
stance, for mild vacuolation, which has a pathological importance
factor of 2, and an observed severity score of 1.65 depending on
damage intensity, the RI would be 3.30 (i.e., 1.65 × 2). Alter-
ations with a higher RI value signified greater severity [34]. The
qualitative scores of the brain histological changes, expressed as
mean ± standard deviation of six observations, were analysed
by a non-parametric Kruskal–Wallis test. The Mann–Whitney
U test was conducted for pairwise comparisons in IBM-SPSS
Version 22.0 at a significance level of P < 0.05.

In silico and molecular docking of Streptococcus agalactiae
virulence factors
Protein and ligand modelling for docking studies

The virulence proteins of S. agalactiae, such as the glycosyl-
transferase enzyme encoded by the iagA gene involved in BBB
invasion, and an invasin protein, CAMP factor [24; 25; 27; 28],
were retrieved from the NCBI database for further detailed anal-
ysis and understanding. The corresponding FASTA protein se-
quences were subsequently subjected to homology modelling
using the ExPASy SWISS-MODEL server. Oxolinic acid (OA)
was considered the ligand, and its 3D structure was obtained from
the PubChem database in .sdf format and then conveniently con-
verted to .pdb format using Open Babel software.

Molecular docking
The molecular docking and interactions between OA and the

target virulence proteins of S. agalactiae were analysed using
MGL Tools and AutoDock Tools 1.5.7 [35], employing a grid
box size of 126 Å in the X, Y, and Z dimensions, and the Lamar-
ckian Genetic Algorithm for docking simulations. The docking
results and molecular interactions were visualised using PyMOL
Edu version 2.5. The docking score, representing the interac-
tion energy with appropriate scaling factors, was used to assess
the binding affinity of the ligand. Furthermore, specific amino
acid interactions between the ligand and target proteins were
computed and outlined. Lower binding free energy may indicate
stronger ligand-protein interaction, while hydrogen bond strength
may categorise donoracceptor distances as strong (2.2–2.5 Å;
mostly covalent), moderate (2.5–3.2 Å; mostly electrostatic), and
weak (3.2–4.0 Å; electrostatic), as per Jeffrey [36].

Results
Brain histopathology and evaluation of pathological anomalies

The LD50 of S. agalactiae and the MIC of oxolinic acid (OA)
were 1.26 × 108 cells/fish and 12.50 µg/mL, respectively. In
the forebrain of the control fish, the tissue structure was nor-
mal, with a well-organized epithelial layer, the meninx prim-
itiva, and mononuclear cells present in the brain parenchyma
(Figure 1 A). The histopathological analyses of OA-treated and
untreated S. agalactiae challenged O. niloticus revealed three
types of reaction patterns: progressive, regressive, and inflam-
matory (Figure 1 B–I; Table 1). The identified progressive
change was thickening of the meninx primitiva, while regressive
changes included superficial layer lifting, localized spongiform
encephalopathy, vacuolation, and degeneration of the meninx
primitiva and brain parenchyma. Inflammatory changes were
characterized by meningitis and mononuclear cell infiltration. On
DPI 1, both groups displayed mild progressive, regressive, and
inflammatory changes, including meninx primitiva thickening
with high RI values. The damage, particularly regressive and in-
flammatory changes, intensified on DPI 7. The OA-treated group
showed reduced regressive changes and evidence of healing in
the brain parenchyma, whereas the untreated group experienced
significant damage on DPI 14. By DPI 21, the OA-treated group
was nearly healed except for mild meningitis, while the untreated
group continued to exhibit moderate brain damage.

In the midbrain, the normal structure consisted of an outer
meninx primitiva and underlying connective tissue above the op-
tic tectum (Figure 2 A). The histopathological changes included
circulatory alterations such as edematous optic tectum, regressive
changes like superficial layer lifting, loosening of connective
tissue, and vacuolation, as well as inflammatory changes such as
mononuclear cell infiltration (Figure 2 B–I; Table 1). On DPI 1,
both groups exhibited mild circulatory, inflammatory, and regres-
sive changes. On DPI 7, circulatory damage peaked in the OA-
treated group, while regressive changes worsened in the untreated
group. A gradual reduction in damage over time was noted in the

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Sinha P et al., 2025 Effect of oxolinic acid therapy

Table 1. Reaction indices* based on the major histopathological changes in the brain of Streptococcus agalactiae LCR1 challenged and oxolinic acid (OA)-fed
Oreochromis niloticus juveniles.

Reaction pattern Histopathological changes IF DPI 1 DPI 7 DPI 14 DPI 21

OA treated Untreated OA treated Untreated OA treated Untreated OA treated Untreated

Forebrain

Progressive Thickening of MP 1 0.80±0.171ab 1.02±0.551ab 1.06±0.271a 1.12±0.211A 0.57±0.251b 0.72±0.301 A B 0.27±0.121c 0.57±0.102 B

Regressive Lifting of SL 1 0.52±0.451ab 1.07±0.531A 0.62±0.241a 0.98±0.371A 0.25±0.121ab 1.05±0.432 A 0.23±0.051b 0.47±0.192 B

LSE 2 2.10±1.181ab 1.67±1.541A 2.60±0.491a 3.80±1.211 B 1.07±0.211bc 1.63±0.392 A 0.83±0.461c 1.27±0.391A

Vacuolation 2 1.93±0.641ab 1.13±0.631A 1.93±0.651a 3.47±1.042 B 1.03±0.591ab 1.73±0.621 A 0.67±0.331b 1.33±0.552A

Damaged MP 3 3.05±1.261ab 2.95±1.221A B 3.05±0.481a 5.55±2.541A 1.70±0.921ab 3.55±1.721 A B 1.00±0.491b 2.20±0.622 B

Degeneration of BP 3 3.00±1.001ab 1.90±1.381A 3.00±0.801b 5.70±1.612 B 1.05±1.321a 3.15±0.732C 0.75±0.371a 1.90±0.822A

Inflammatory Meningitis 2 3.57±0.411a 1.70±1.271A 3.57±1.241b 3.90±1.051 B 1.13±0.391a 2.03±0.961 A 0.70±0.351a 1.43±0.372A

Infiltration of MNC 2 2.17±0.351a 1.23±0.731A 2.17±0.601b 3.10±1.161 B 0.67±0.431c 1.50±0.901 A 0.43±0.391c 1.47±0.352A

Mid-brain (Optic tectum)

Circulatory Edematous OT 1 0.70±0.171a 0.72±0.171A 0.73±0.211a 1.42±0.182 B 0.73±0.161a 1.00±0.241C 0.37±0.141b 0.55±0.181A

Regressive Lifting of SL 1 0.80±0.131a 0.70±0.231A 0.68±0.171a 1.50±0.372 B 0.43±0.231b 1.02±0.282C 0.38±0.121b 0.75±0.102A

Loosening of DCT 1 0.83±0.081a 0.72±0.121A 0.67±0.151a 1.18±0.332 B 0.75±0.211a 1.10±0.321 A B 0.47±0.101b 0.72±0.182A

Spacing of LCT 1 0.85±0.211a 0.85±0.141A 0.73±0.261ab 1.43±0.592 B 0.65±0.141ab 1.15±0.342 B 0.47±0.181b 0.83±0.182A

Vacuolation 2 1.30±0.581a 1.60±0.281A 1.50±0.451a 3.13±0.702 B 1.30±0.451a 2.50±1.062A B 0.97±0.231a 1.13±0.241C

Inflammatory Infiltration of LC 2 1.73±0.241a 1.63±0.291ab 1.27±0.451ab 1.30±0.681A B 0.83±0.611b 1.73±0.332 A 0.57±0.151b 1.03±0.232 B

Cerebellum (Granular cell layer)

Regressive LSE 2 2.57±0.231a 2.40±0.181A 2.20±0.331a 4.20±0.332 B 0.47±0.101b 1.93±0.532 A 0.90±0.281c 1.30±0.282C

Lysis of GC 3 3.75±0.311a 3.70±0.241A 2.30±0.591b 5.25±0.682 B 0.65±0.121c 3.10±1.212 A 1.25±0.351d 1.80±0.661C

Necrotized area 3 5.45±0.581a 5.30±0.411A 1.85±0.581b 5.45±0.962A 1.35±0.161b 3.25±1.362 B 1.35±0.311b 1.80±0.421C

* As per the descriptions of Bernet et al. (1999). No changes were noted in the control group. 1-2: Values sharing a common numerical superscript for a specific

row among the treatment groups for a specific day differed insignificantly (P>0.05). a-d: Values sharing a common alphabetical superscript within a row for

the treated group on different DPI differed insignificantly (P>0.05). A-C: Values sharing a common alphabetical superscript within a row for the untreated

group on different DPI differed insignificantly (P>0.05). DPI: Day post-injection. IF: Importance factor; MP: Meninx primitiva; SL: Superficial layer; LSE:

Localized spongiform encephalopathy; BP: Brain parenchyma; MNC: Mononuclear cells; OT: Optic tectum; DCT: Loosened dense connective tissue; LCT:

Loose connective tissue; LC: Leucocytes; GC: Granular cells of the cerebellum.

OA-treated group, whereas the untreated group presented more
severe and persistent damage. By DPI 21, both groups showed
decreasing damage, with near-normalization of architecture in
the OA-treated group. In the granular cell layer of the cere-
bellum, both groups experienced regressive changes, including
spongiform encephalopathy with vacuolation and necrosis, which
decreased with time. On DPI 21, the treated group displayed
minimal vacuolation, while the untreated group showed moder-
ate damage (Figure 3 B–I; Table 1). Overall, the OA-treated
group exhibited less damage, improved recovery, and reduced
mortalities (26%; P > 0.05) compared to the untreated group
(32%).

Homology modelling and molecular docking
Using the SWISS-MODEL server, three-dimensional (3D)

models of glycosyltransferase and CAMP factor from S. agalac-
tiae were constructed based on sequence similarity with identified
structural templates. It is generally important to note, in a rather
typical and somewhat broadly consistent and commonly recog-
nised manner, that this modelling approach essentially follows
a broadly conventional and widely accepted procedure overall.

The models generated from templates with the highest sequence
identity are presented in (Figure 4 A1, B1). This approach, in
many ways, essentially leverages the inherent structural similarity
among proteins of the same family, which usually tend to share
broadly comparable and functionally related three-dimensional
architectures. The outcomes of the homology modelling are,
more or less, conveniently summarised in (Table 2). The Global
Model Quality Estimation (GMQE) and Qualitative Model En-
ergy Analysis (QMEAN) scores were high, corresponding to
higher sequence identity with templates. The generated mod-
els exhibited optimal quality, as validated by SWISS-MODEL,
with MolProbity scores above 0.66, and were considered ex-
cellent. Nearly 98% of residues were located in the favoured
regions of the Ramachandran plot (Figure 4 A2, B2). The ligand
OA exhibited notable binding affinities toward both glycosyl-
transferase and CAMP factor, with docking scores of –4.99 and
–4.31 kcal/mol, respectively. These interactions were supported,
in a fairly straightforward and somewhat broadly descriptive
and methodologically consistent manner, by multiple hydrogen
bonds and close-contact residues, suggesting generally stable and
favourable binding conformations (Table 3; Figure 5 A, B).

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Sinha P et al., 2025 Effect of oxolinic acid therapy

Table 2. Homology modelling data of glycosyltransferase AAM99596 and CAMP factor AKI96324 of Streptococcus agalactiae computed using SWISS MODEL.

Characteristics Glycosyltransferase AAM99596 CAMP factor AKI96324

Template ID A0A4V0HA53.1.A 5h6i.1.A

Sequence identity (%) 71.69 100

QMEAN 0.90 0.86

GMQE 0.96 0.80

MolProbity score 0.91 0.70

Ramachandran plot – Favoured region (%) 96.97 98.12

Ramachandran plot – Outlier region (%) 0.00 0.00

QMEAN: Qualitative Model Energy Analysis; GMQE: Global Model Quality Estimation.

Discussions
In terms of virulence, the Sa strain, isolated from the kid-

ney of Asian seabass Lates calcarifer, was borderline avirulent
to weakly virulent [7; 37; 38] with a moderately high MIC for
OA. Sa affects the brain using several virulence factors to allow
the infection process [4; 38; 39]. In this study, Sa resulted in
systemic pathological alterations, including inflammation, haem-
orrhages, and meningitis in challenged fish, as in previous re-
search [5; 6; 7; 8; 9]. The Sa-infected fish also exhibited aberrant
swimming, marked by circular movement and C-shaped body
bending. It could be due to the possible neurological problem, as
the midbrain reportedly controls fish swimming by transmitting
axons to innervate the spinal cord’s primary and secondary motor
neurons [9; 40]. Further, abnormal swimming behaviours due
to streptococcal infections were linked to brain fluid accumula-
tion [7]. In this work, we isolated Sa from the brain tissues of
challenged tilapias on BHIA, indicating a breach of the BBB. Sa
employ various strategies to survive in the bloodstream, colonize
brain vasculature, and cross the BBB [41]. The results indicated
that the Sa can enter the brain parenchyma by destroying the host
BBB and causing localized spongiform encephalopathy. Several
earlier studies confirmed the establishment of Sa [6; 8; 42] or
biofilm formation [43] in fish brains. When the bacteria enter the
circulatory system, they survive within macrophages, allowing
them to evade the immune system and spread to the central ner-
vous system (CNS) [8; 44]. Such macrophages reportedly act as
a vehicle for Sa, allowing it to cross the BBB and gain access to
the CNS [6; 8].

The current investigation revealed progressive alterations
such as thickening of the meninx primitiva, suggesting bacte-
rial meningitis similar to earlier studies [5; 6; 7; 9]. Regressive
changes in the forebrain, midbrain, and cerebellum of the current
study were in line with the earlier findings. They reported that
bacteria can permeate brain parenchymal regions and cause lo-
calised spongiform encephalopathy, characterised by focal haem-
orrhages and vacuolization, mostly in the midbrain and hindbrain.
Also, Gram-positive cocci in the granular cell layer were ob-
served, causing lysis of granular cells, and brain lesions, includ-

Figure 5. Protein–ligand interaction of Streptococcus agalactiae virulence

factors: [A] glycosyltransferase and [B] CAMP factor against oxolinic acid, visu-

alised using the PyMOL tool. Note: green structures represent ligands; coloured

ribbons depict the target protein; yellow dotted lines indicate interaction sites.

Distances between the ligand and target at the interaction sites are measured in

angstroms (Å).

ing haemorrhages, neuronal necrosis, and inflammation in regions
governing swimming in tilapia [9]. Sa infection also disrupted
cerebral bioenergetics by impairing mitochondrial complex IV
and creatine kinase activities, leading to energy imbalance [45].
All these investigations pointed out that Sa is neurotropic, seri-
ously harming neurological function and influencing swimming
behaviour and survival of fish. The inflammatory responses in
the forebrain and mid-brain of Sa-infected tilapias corroborated
earlier studies [6; 7; 8; 9; 46], which documented meningitis and
several significant brain alterations. Sa reportedly triggers an
inflammatory response, which impairs phagocytosis, cytokine
and chemokine levels, contributing to oxidative stress and disrup-
tion of the BBB [45; 47] and interfering with the immune system
[48]. The resulting neuroinflammation can be characterized by
immune cell migration and the production of pro-inflammatory
cytokines, which drive the immune response [6].

Antibiotics like ampicillin, colistin sulfate, sulfadimethoxine-
ormetoprim, and florfenicol are effectively used against Strepto-
coccus spp. Moreover, the use of β-lactams, fluoroquinolones and
macrolides has been highlighted as effective against meningitis-
causing S. pneumoniae [49; 50; 51; 46; 52]. In this study, the
RI values increased during the initial period of infection and

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Sinha P et al., 2025 Effect of oxolinic acid therapy

Table 3. Molecular docking summary of glycosyltransferase AAM99596 and
CAMP factor AKI96324 of Streptococcus agalactiae against the ligand oxolinic
acid using the AutoDock tool.

Proteins Affinity Ligand–Protein interaction Distance (Å)

Glycosyltransferase -4.99

A:ASN1̀97:OD1–:UNL1̀:H 2.3

A:LYS1̀93:NZ–:UNL1̀:O 2.7

A:ASN1̀97:OD1–:UNL1̀:O 3.0

A:LYS1̀93:NZ–:UNL1̀:O 3.2

CAMP factor -4.31

A:LYS2̀05:NZ–:UNL1̀:O 2.9

A:ARG2̀04:N–:UNL1̀:O 3.1

A:ARG2̀04:NH1–:UNL1̀:O 3.2

gradually reduced with time, indicating the fish’s adaptive and
immune responses to healing the brain lesions. While the effects
were almost similar, the OA-treated tilapia showed lower RI. The
OA-treatment facilitated quicker recovery, indicating that OA can
cross the BBB and lessen the Sa-infection. The glycosyltrans-
ferase and the CAMP factor play crucial roles in the pathogenesis
of Sa. The infection may influence bacterial interactions with the
host, create specific pores in host membranes, modify proteins
and carbohydrates, and disrupt cell integrity, leading to cell lysis
[24; 53]. The lesser impact of Sa-infection in the brain suggested
that the OA can exhibit binding affinities with virulent proteins,
as confirmed by interactions with glycosyltransferases and the
CAMP factor, and to prevent the activities of Sa. In the molecular
docking study, our observations on the shorter bond distances
and number of interactions with multiple hydrogen bonds and
close-contact residues suggested stable and favourable binding
conformations. These results supported the potential of OA as a
strong inhibitor of the virulent proteins of Sa. Our study further
noted an insignificant difference in mortalities due to the tested
strains moderately high MIC for OA. Strikingly, the 7 days of
treatment at 12 mg OA/kg fish/day inhibited the Sa strain, miti-
gated the severity of brain damage, and improved fish survival.
Thus, these biologically meaningful results proved the protective
effect of OA against the progression of Sa-infection and ensuing
brain histopathological changes. However, future studies are
warranted on the expression of glycosyltransferase, CAMP factor
and other virulence factors using qRT-PCR or Western blotting to
provide direct evidence of their involvement in Sa pathogenesis
and OAs mechanism of action.

Conclusions
Our results demonstrated that understanding the role of viru-

lence proteins of S. agalactiae in pathogenesis and interactions
with drugs can offer scope for new therapeutic strategies, such as
targeting such factors or blocking interactions with host cells and
developing targeted therapies that can disrupt the bacterial toxin’s
activity. In this study, oxolinic acid (OA) therapy in S. agalac-
tiae-infected tilapia revealed a protective effect by reducing the

mortalities, brain damage and histopathological alterations. The
treated fish unveiled less damage in the forebrain, midbrain, and
cerebellum, with tissue architecture almost normalized on DPI
21. The results implied that OA is efficacious and can be used
in treating tilapia against S. agalactiae infection. However, the
World Health Organisation stated that quinolones, including OA,
are critically important human medicines, and they should at no
time be used in animals produced for food as a primary treatment
agent. Based on the results, we advocate the use of OA as a
therapeutic agent for second-line treatment in food-producing
animals following the national and international criteria. Yet, a
prudent and responsible use of antibiotics, targeted therapeutic
applications for treating S. agalactiae-induced meningitis in fish
and implementing long-term AMR and residue surveillance to
safeguard public health are obligatory.

Data Availability Statement
All relevant data are within the paper and supplementary

materials.

Funding
The work was supported by the Indian Council of Agricultural

Research, Government of India, New Delhi, under the All-India
Network Project on Fish Health vide Grant F. No. CIBA/AINP-
FH/2015-16 dated 02 June 2015.

Competing interests
The authors declare that they have no competing interests.

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Highlights in BioScience Page 10 of 10 October 2025|Volume 8

http://bioscience.highlightsin.org/

	Abstract
	Introduction
	Materials and methods
	Experimental fish and bacterial strain and its pathogenicity
	Efficacy of oxolinic acid against Streptococcus agalactiae infection
	Histopathology and evaluation of pathological anomalies
	In silico and molecular docking of Streptococcus agalactiae virulence factors

	Results
	Brain histopathology and evaluation of pathological anomalies
	Homology modelling and molecular docking

	Discussions
	Conclusions
	Data Availability Statement
	Funding
	Competing interests

