The effect of heat stress on clinicopathological changes and immunolocalization of antigens in experimental Streptococcus agalactiae infection in Red hybrid tilapia ( Oreochromis spp . )

Aim: To understand the influence of environmental temperature on streptococcosis, heat stress associated pathological changes in acute streptococcosis in red tilapia using various routes of infection was investigated. 9 Materials and Methods: Red hybrid tilapia were inoculated with 10 CFU/ml of agalactiae using intraperitoneal (IP), immersion (IM), and immersion cut (IC) which were maintained at 34C for 24 hours while the positive control groups were infected using similar routes but maintained at 28C and the negative control was at 34C without infection. Samples from the gills, brain, eyes and kidney were taken for bacterial isolation, polymerase chain reaction (PCR), histopathology and immunohistochemistry (IHC). Results: Diseased fish showed skin haemorrhage, fin haemorrhage and exophthalmia with more signs in IP route of infection followed by IC and lastly IM at 34C. The bacteria were isolated and detected with PCR in all the organs of fish from 4 hour post-challenge (hpc). The lesions were noticed earlier and severe in heat stressed groups from 4 hpc with 50% mortality in IP group while for all, the bacterium was isolated from 4 hpc in the brain. IHC test also detected the antigen in the tissues as early as 4 hpc with intense staining in the heat stressed group, IP followed by IC and IM routes. Positive immunostaining were observed in the thrombi, meningeal vessels, choroid and interstitial haemorrhagic areas. Conclusion: The severe lesions observed in the brain, eye and kidney under heat stress reveals its contributory role to the mortality pattern and severe pathological changes associated with streptococcosis in fish.


Introduction
ture, low dissolved oxygen concentration, poor water quality (high ammonia or nitrite concentrations) Red hybrid tilapia (Oreochromis spp.) were first salinity and alkalinity (pH>8) [5,6].Many researchers introduced in Malaysia in mid 1980's.Tilapia were had implicated some of these stress factors like oxygen considered to be resistant to diseases but in 1997, there and nitrite levels [7] and water temperature [8] to was heavy mortality of about 300 to 400 g weight influence the mortality pattern due to streptococcal tilapia that were kept in floating net cages in Sungai infection especially that by Streptococcus iniae [9].In Pahang [1].Cages of Kenyir, Pedu and Pergau Lakes in Malaysia however, information on the effect of these Malaysia also experienced this problem in the mid year environmental stressors on the mortality due to S. between April to July of 1997 and the laboratory tests agalactiae had been inferences from epidemiological revealed that the causative agent was Gram-positive investigations that reported higher mortality during the bacteria, Streptococcus agalactiae [1].Ever since in high temperature seasons [10].Malaysia, studies on S. agalactiae infection in fish had Previous studies had revealed that the sources of centred on the pathology and disease outbreaks [2,3,4] infection of S. agalactiae and S. iniae in most farms are with very little attention on the possible role of mainly from newly introduced fish, faeces of infected environmental stressors especially temperature on the fish and infection via contaminated water [11].Other disease manifestation.
means of infection could be through wounds or abrasions Most fish and aquatic animals are often subjected of the skin [12] especially in high stock density farms.to a broad variety of stressors because their homeo-Since the pathogenesis of fish S. agalactiae infection is static mechanisms are dependent on the surroundings.still poorly understood [13], the role of environmental In captive fish, the stress includes physical and mental stress and skin abrasions which are often encountered trauma associated with capture, transport, handling, in the field need to be investigated.Therefore, this crowding,malnutrition, fluctuation in water temperastudy investigates the heat stress associated pathological changes in acute S. agalactiae infection in Red hybrid tilapia using various routes of infection especially in the presence of skin abrasion.

Materials and Methods
agalactiae infection in fish) [3; 4] from three fish every 4 h within the 24 h.The tissues samples were subjected Fish: A total of 120 Red hybrid tilapia (Oreochromis to bacterial culture, PCR, histopathology and IHC.sp.) weighed approximately 100 -150 g were obtained from the Aquaculture Extension Centre, Department of

Bacteria isolation and identification
Bacteria culture: The swab from the organs that were Fisheries Malaysia, Bukit Tinggi, Pahang.Prior to collected every 4 h were immediately streaked onto experiment, tanks were disinfected and cleaned.The source of water was dechlorinated and continuously blood agar plates [14] and incubated at 30C for 18 h.aerated.Water quality was monitored prior to onset of The suspected colonies of S. agalactiae were subcultured the experiment.The fish used, were screened for onto a new blood agar and incubated at 30C for 18 h in bacteria particularly for S. agalactiae and external order to obtain a pure culture.Gram stain test were parasites in order to make sure that the fish are healthy.
performed to identify Gram-positive cocci in chain or The fish were fed ad libitum with commercial feed.
paired and catalase test negative organisms.Finally, the However throughout the experiment, the fish were off colonies were further characterized using the commerfeed.
cialized test kit, API rapid ID 32 Strep® (bioMerieux SA, France).Bacteria: S. agalactiae isolated from outbreak case was used for the study [1] highest dilution (10 ) to lowest dilution (10 ) where 1 GAG TTA CAA AGG ACG AC] and STAUR 6 [AGC ml of cultured broth S. agalactiae was serially added TCA GCC TTA ACG AGT AC], and cycling conditions into 9 ml of peptone water respectively.Then 1 ml from were as follows; 1 cycle at 94C for 4 min, followed by the highest dilution was continuously diluted into 34 cycles at 94C for 1 min, 52C for 1 min, 72C for 1 min another dilution till the lowest dilution.About 0.1 ml of and finally elongation at 72C for 10 min [22].each dilution was poured and spread onto the blood Histopathology: The gills, brain, kidney and eyes that agar and incubated in normal incubator at 37C for 24 h.
were collected every 4 h post-challenge (hpc) were 25 to 250 colonies were counted before the concenroutinely processed and stained with haematoxylin and tration was expressed as colony forming unit per eosin (H&E).millilitre (CFU/ml).The last concentration of live S. agalactiae was recorded.
Immunohistochemistry (IHC): Serial 4 µm thick sections were cut from wax blocks onto silane-coated Experimental design: The experiment was conducted 9 glass slides.The slides were dried for 15 min at 56by challenging the fish with 10 CFU/ml of live S.
60C, dewaxed in xylene and rehydrated through a agalactiae using different routes of infection.The fish graded alcohol series.The slides were washed with (n=120) were divided into 5 groups.Group 1 (n=24) phosphate-buffered saline-Tween 20 (PBST) for 10 were exposed to S. agalactiae through IP route and min.Endogenous peroxidase activity was blocked with transferred into the 6 L tank, Group 2 (n=24) were freshly prepared 3% hydrogen peroxide for 5 min in exposed to S. agalactiae through IM bath (9 L of water room temperature and rinsed and washed with PBST + 1 L of S. agalactiae broth) for 10 min before for 2 min.In enhancing the tissue to be immunotransferred into the 6 L tank and Group 3 (n=24) were reactive, the heat-mediated antigen retrieval with exposed to S. agalactiae through IM bath (9 L of water citrate solution in microwave oven was used.Sections + 1 L of S. agalactiae broth) for 10 min with the body of were blocked with blocking buffer 1% normal serum fish being incised (0.5 cm) at the caudal part before (bovine serum albumin) in PBST, and then sections being transferred into the 6 L tank.Group 4 (n=24) was were incubated with tilapia anti S. agalactiae with the the positive control which were challenged with dilution of 1:50 for at least 1 h at 37C in an incubator.similar dose but maintained at 28C and group 5 as the Then, the procedure was followed by rinsing and negative control group without challenge and washing with PBST for 5 min.Sections were incubated maintained at 34C.The fish were gradually acclimaagain at 37C for 30 min with secondary anti-tilapia IgM tised to the higher temperature before being maintained monoclonal-HRP antibody (Aquatic Diagnostic Ltd, at 34C for the bacterial challenge.The fish were kept UK) with the dilution of 1:100.The slides were rinsed for 24 h off fed and the clinical signs were observed and washed with PBST for 5 min before DAB was continuously in the 24 h duration of the experiment.applied 1 ml diluents to a 1 drop DAB for the The brain, eyes and kidney were collected more than development of the color change.Once the sections the gills (the major predilection tissues for S. became brown, the slides were immediately rinsed mucoid faecal cast, erratic swimming, c-shaped body with distilled water and the slides were stained using curvature, imbalance and dull.The clinical signs Mayer's haematoxylin solution for the background detected in the fish that were challenged by IP injection colour.All the slides were analyzed and captured using followed by IC and lastly by IM of live S. agalactiae.image analyzer NIS-Elements D 3.2 (Nikon, Japan) The clinical signs were observed as early as 2 hpc in most of the fish in the IP group while the IC and IM Statistical analysis: The lesion scoring data obtained groups exhibited similar clinical signs at 4 h later.The was analysed using Kruskal-Wallis analysis of internal lesions observed during post mortem every 4 variance in Statistix ver.9.0 (Analytical software, hpc were enlarged and congested spleen, pale and USA).Only values that were below the stated P < 0.05 haemorrhagic liver, empty gut with engorged gall were considered significant and all-pairwise comparibladder, congested kidney and soft brain (Fig. 1).The sons tests were performed.
positive control group had similar pattern but the Results clinical features were observed at 4 hpc.

Clinical and macroscopic findings:
The clinical signs Mortality: Within 24 h, about 50% mortality was observed during 24 hpc with S. agalactiae were recorded in IP group only while other groups and the haemorrhage eyes, operculum, fin and/or body, long negative controls had no mortality within this period.Bacteria isolation and identification: Smooth transparent reliable method for detecting streptococcosis in tilapia.pin-point colonies were stained blue/purple (Gram-Histopathology: S. agalactiae induced acute positive) cocci in chain or pairs and catalase negative inflammation in the brain, eye and kidney while that further tested for other biochemical tests.API rapid ID observed in the gills were not remarkable.Histological 32 Strep® (bioMerieux SA, France) was used to lesions were severe in IP followed by IC and confirm the identification and strain of the organism.
lastly IM for every 4 hpc.The meninges of telecephalon Based on the API result, the bacterium was S.
and cerebellum were infiltrated with inflammatory agalactiae with 99.9% identification.S. agalactiae cells, which in some cases were accompanied by was isolated in all the tissues examined in IP group haemorrhage and mild infiltration of eosinophilic from 4 hpc while in IM the isolation was 12 hpc.In IC granular cells, which is located dorsal to the fourth the isolation was 8 hpc.In all the routes, the bacterium ventricle.The lesions were more severe in IP12 was isolated from 4 hpc in the brain.On the other hand, compared to IM12 and IC12.Kidney tissue showed in the control group S. agalactiae was isolated in all the various degree of degeneration.Large areas of cellular tissues examined in IP group from 4 hpc while in IM the infiltration and numerous lesions were seen in kidney isolation was 12 hpc in the kidney and the eye.In IC tissue.The lesions in the kidney included tubular necrosis, group the isolation was 8 hpc in the brain and eye haemorrhage and infiltration of the inflammatory cells.

(Table-1).
Most of the kidney tissues that were infected showed haemorrhage in haemopoietic tissue and infiltrated by PCR: Based on Fig. 2, all the colonies that were lymphocytes.The lesions were more severe in the IP12 subcultured are positive for S. agalactiae at 1457-bp.
where massive infiltration of inflammatory cells, PCR was used to confirm the bacterial isolation as it is a  tubular degeneration and haemorrhage can be seen the capillaries in all the organs but more widely compared to IM12 and IC12.Multiple granuloma-like distributed in the kidney.There was no immunolesions were observed with leukocytic infiltration around staining observed in the gills in all the routes.This it.Eye lesions were characterized by a mild cellular immunostaining increased in intensity and distribution infiltrate, composed of macrophages and eosinophilic as the post-challenge period increased in the IP route granular cells.These findings were observed in the followed by IC and IM.The immunostainings were choroid tissues and also in periorbital tissues.The observed in the thrombi, meningeal vessels, choroid lesions in the eye can be seen at 12 hpc whereby some and renal interstitial haemorrhagic areas.The presence of the pigment cells surrounded the bases of the of cocci-shaped structures with brown colour and/or a photoreceptors were sloughed off.The lesions could be brown staining with a diffuse distribution was found seen clearly in IP16 followed by IC12 and lastly IM12.
within macrophages, associated with inflammatory The meninges were congested and thickened with the reactions in the kidney, blood vessels lumen and wall presence of eosinophilic granular cells in the brain and and some within thrombi in the eye especially in choroid, it is associated with the loss of orientation and swimming there was also presence of the immunolabelling in the abnormalities.There is significant difference (P< 0.05) meningeal blood vessel both in the lumen and the wall in the lesions recorded between IP, IC routes when (Fig. 4).On the other hand, the immunostaining compared to IM route in most of the organs.However, followed the same pattern but was observed as from 8 there is no significant difference between IP to IC in hpc (Table-2).most of the organs.On the other hand, there is no significant difference in most of the organs at P > 0.05

in different routes of infection in the negative control
This investigation describes the effect of route groups (Fig. 3).and heat stress on the pathological changes in S. IHC test: IHC test detected antigen in the brain, eye and agalactiae infection in Red hybrid tilapia.The clinical kidney from 4 hpc with slight immunostaining within features, pathology and detection of antigens were  earlier and more pronounced in heat stressed Red The fact that IP route had more lesion than IC and hybrid tilapia.The Red hybrid tilapia challenged with IM in this investigation showed that the pathogenicity S. agalactiae showed various clinical signs and lesions of the bacteria depends on level of bacteraemia, with such as skin, fin and visceral organ haemorrhage/ the brain being the primary target organ for S. congestion that are consistent with the disease while agalactiae irrespective of the routes employed.The histopathological changes observed in the brain, bacteria tropism at 4 hpc was uniform with wider kidney and eyes are highly suggestive of septicaemia distribution in IP routes as bacteria were found in the [2,15,16,17] with detection of bacteria much earlier at brain, eye and kidney.The wider coverage observed in 4 hpc in the brain and eye when heat stress was IP showed that IP provides a direct route of infection employed.This could explain the higher susceptibility into the body when compared to the IC which had cut as accompanied by higher mortality and bacterial means of entry into the body system while IM has the isolation usually observed during the hot season [10] natural skin defensive mechanisms to contend with.and when the environmental temperature increases.
The high temperature tends to be associated with This might aid the breaking of the skin natural more pronounced clinical signs, macroscopic and defensive mechanisms to enhanced penetration and microscopic lesions in the fish challenged with S. detection observed in IC and IM groups.The detection agalactiae and this further confirm the epidemioof this bacterium in the tissues examined except the logical reports of higher mortality due to this infection gills soon after infection especially when heat stressed in the hot season [10].Additionally, the result showed further suggested the three organs as the target organs that infection could be enhanced by abrasions especially of the organism in the order of the brain, eye and kidney when the water temperature is higher.In this study, [4].
there is a significant difference between different routes The gross pathological changes seen in this study of infection (IP, IC and IM) in most of the organs were typical of septicaemia associated with S. agalactiae especially in heat stressed group.This shows that, the infection.The meningitis and blood vessels congestion infection via IC can be enhanced by heat stress implying were associated with loss of orientation and abnormathat heat stress may play a major role in development of lities often observed in the disease [4,13].The significant streptococcosis.This possible mechanism of this role difference observed between the routes of infection in needs further investigation especially as regards the the brain, eye and kidney further lend credence to the effect of stress hormones and factors on the immunefact that stress may increase the bacterial virulence [4].competence of fish.The pathological changes observed in the blood Conclusion vessel and the subsequent detection of antigens in blood vessels, capillaries in the kidney and meningeal In conclusion, S. agalactiae being recognized as capillaries further strengthen the observations of [3] pathogenic to fish especially tilapia, causes septicaemia that vasculitis and thrombosis are some of the features and severe pathological changes in all different routes of streptococcosis in tilapia.The 50% mortality recorded of infection (24 hpc) used in this study.The lesions in the stressed IP group was in consonance with the observed are more in the brain, eye and kidney than reports of some workers that mortality in fish challenged gills and are more severe in the IP route, followed by IC 8 with 10 CFU using the IM route are often observed and lastly IM especially when heat stress was employed.when the water temperature is above 26C [18,19].The The higher temperature could have contributed to the observed mortality is very high and alarming especially higher percentage of mortality observed in IP route of when compared to the negative control where there infection however the possible mechanism that lead to was no mortality within 24 h despite pathological this observation still needed to be verified.changes observed within this period.The mortality

Figure- 1 .
Figure-1.Lesions of Red hybrid tilapia challenged with S. agalactiae.A: Haemorrhage at the eyes and pectoral fin, B: Haemorrhage at the anal fin, dorsal fin, caudal fin and body.C: Congestion at the gills, D: Exophthalmia, E: Soft brain, F: Congestion and kidney enlargement.

Figure- 4 .
Figure-4.Immunostaining were found in A and D (IM20): the meninges and the meningeal blood vessel lumen and wall (arrow).B and E (IC16): blood vessels and capillaries in the choroid body containing eosinophilic granular cells and neutrophils (arrow).C and F (IP8): kidney; the immunostaining was observed in the blood vessels lumens (arrow); a thrombus in F (arrow).

Table - 1
. Bacteria culture from the organs X: No growth, /:Growth

Table - 2
. Results of IHC examination of different groups and organs of red tilapia for S. agalactiae