Experimental Bacterial Co-infection in Nile Tilapia Shows High Pathogenicity from Lake Kariba Isolates

Experimental Bacterial Co-infection in Nile Tilapia Shows High Pathogenicity from Lake Kariba Isolates | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Experimental Bacterial Co-infection in Nile Tilapia Shows High Pathogenicity from Lake Kariba Isolates Frederick Chitonga Zulu , View ORCID Profile Kunda Ndashe , View ORCID Profile Katendi Changula , Mazuba Siamujompa , Chanda Chitala , Mwansa M. Songe , Ladslav Moonga , View ORCID Profile Stephen R. Reichley , View ORCID Profile Bernard Mudenda Hang’ombe doi: https://doi.org/10.1101/2025.03.30.646194 Frederick Chitonga Zulu 1 The University of Zambia, School of Veterinary Medicine, Department of Paraclinical Studies , Lusaka, Zambia BVM Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kunda Ndashe 1 The University of Zambia, School of Veterinary Medicine, Department of Paraclinical Studies , Lusaka, Zambia PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kunda Ndashe For correspondence: ndashe.kunda{at}gmail.com Katendi Changula 1 The University of Zambia, School of Veterinary Medicine, Department of Paraclinical Studies , Lusaka, Zambia PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Katendi Changula Mazuba Siamujompa 1 The University of Zambia, School of Veterinary Medicine, Department of Paraclinical Studies , Lusaka, Zambia 2 Copperbelt University, Faculty of Natural Resources, Department of Zoology and Aquatic Sciences , Kitwe, Zambia MVSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chanda Chitala 1 The University of Zambia, School of Veterinary Medicine, Department of Paraclinical Studies , Lusaka, Zambia 3 Ministry of Fisheries and Livestock, Department of Veterinary, Central Veterinary Research Institute , Lusaka, Zambia MSc Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mwansa M. Songe 3 Ministry of Fisheries and Livestock, Department of Veterinary, Central Veterinary Research Institute , Lusaka, Zambia PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ladslav Moonga 1 The University of Zambia, School of Veterinary Medicine, Department of Paraclinical Studies , Lusaka, Zambia BAEd Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stephen R. Reichley 4 Mississippi State University, Global Center for Aquatic Health and Food Security ,, Mississippi State, USA 5 Mississippi State University, College of Veterinary Medicine, Department of Pathobiology and Population Medicine , Mississippi, USA PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Stephen R. Reichley Bernard Mudenda Hang’ombe 1 The University of Zambia, School of Veterinary Medicine, Department of Paraclinical Studies , Lusaka, Zambia 4 Mississippi State University, Global Center for Aquatic Health and Food Security ,, Mississippi State, USA PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bernard Mudenda Hang’ombe Abstract Full Text Info/History Metrics Preview PDF Abstract Objective This study aimed to determine the pathogenicity of bacteria ( Aeromonas spp., Lactococcus garvieae, Acinetobacter spp., and Klebsiella spp.) isolated from Nile tilapia ( Oreochromis niloticus ) in small-scale aquaculture establishments on Lake Kariba through experimental infections. Materials and methods Healthy fish (50g ± 5g) were distributed among six transparent fish tanks labeled A to F. Fish in tanks A, B, C, and D were exposed to Aeromonas spp., Lactococcus garvieae, Acinetobacter spp., and Klebsiella spp., respectively, through intraperitoneal inoculation. Tank E received a co-infection of all isolates, while tank F served as the control group with an injection of normal saline. Clinical signs, mortalities and post-mortem lesions were recorded in all experimental groups, with histopathological examinations performed on liver, kidney, and spleen tissues. Results The findings indicate that Acinetobacter spp. and Klebsiella spp. exhibit low pathogenicity, evidenced by few clinical signs such as lethargy, pale skin, and fin erosion. The study further highlighted Aeromonas spp. and Lactococcus garvieae as the bacterial isolates causing significant clinical symptoms in Nile tilapia compared to Acinetobacter spp. and Klebsiella spp. following experimental infection. Conclusion Co-infection with all bacterial pathogens demonstrated very high pathogenicity, with 100% mortality reported within seven days post-infection. The study proposes the development of a polyvalent vaccine to control fish disease outbreaks in small-scale aquaculture operations on Lake Kariba, thereby sustaining aquaculture production in Zambia. 1. Introduction Globally, aquaculture production growth is driven by both large commercial operations and small-scale producers ( 1 ). Over the past two decades, global aquaculture production has increased at an annual rate of 6.7%, making it the fastest-growing sector in animal production ( 1 ). However, this growth declined to 3.5% for the period 2016 to 2021 ( 2 ). In some parts of the world the significant factor contributing to this decline is disease outbreaks in production facilities ( 3 , 4 ). Globally, the production of Nile tilapia ( Oreochromis niloticus ) is particularly affected by disease outbreaks, predominantly bacterial in nature ( 5 , 6 ). Reported diseases in farmed Nile tilapia include francisellosis ( Francisella noatunensis subsp. orientalis), lactococcosis ( Lactococcus garvieae ), streptococcosis ( Streptococcus agalactiae, S. iniae , and S. dysagalactiae ), edwardsiellosis ( Edwardsiella tarda and E. ictaluri ), and motile hemorrhagic septicemia ( Aeromonas hydrophila, A. sobria , and A. veronii ) ( 7 – 11 ). These bacterial infections often emerge following exposure to stressors such as overcrowding, poor water quality, and suboptimal nutrition, which suppress fish immunity and increase susceptibility to environmental bacteria. Consequently, it is common to isolate multiple bacterial pathogens during an outbreak in farmed tilapia ( 12 ). In Zambia, aquaculture production has recently increased, with significant participation from small-scale producers over the past five years ( 13 ). The Government of the Republic of Zambia, through various projects and funding agencies, has promoted financial investment in small-scale fish farming ( 14 ). Substantial investment has been directed toward increasing the number of small-scale producers on Lake Kariba, the largest fish production region in Zambia ( 15 ). Siamunjopa et al . (2023) reported significant mortality losses of fish among small-scale producers on Lake Kariba due to Aeromonas spp., Pseudomonas spp., Micrococcus spp., Klebsiella spp., Lactococcus spp., Streptococcus spp., and Acinetobacter spp ( 16 ). Despite the isolation of bacteria from diseased Nile tilapia in small-scale fish farms on Lake Kariba, the pathogenicity of these isolates remains unknown. It is well documented that immunosuppressed fish, due to poor husbandry practices, are susceptible to multiple bacterial pathogens ( 12 , 17 , 18 ). Therefore, this study aimed to determine the pathogenicity of the bacterial isolates from diseased Nile tilapia from small-scale producers to elucidate the role of these bacterial pathogens in the disease process. 2. Materials and Methods This study was undertaken in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Health Research Ethics Committee of Zambia. The protocol of this study was approved by the Excellence in Research Ethics and Science (ERES) Converge, (Protocol Number: 2019/AUG/024). All efforts were made to minimize suffering and stress of the fish, both during handling and sampling. As infection was one of the humane endpoints, subjects were withdrawn from the experiment (euthanized and sampled) after clinical signs appeared. The decision criteria to euthanize animals included two or more clinical signs (e.g. poor body condition, severe skin erosion and hemorrhage, loss of balance, extensive abdominal swelling, scale protrusion, and exophthalmia). Where signs of disease or abnormal behaviour were observed, the fish were euthanised by stunning with a blow to the head followed by dislocation of the cervical vertebra. 2.1. Animals and environmental conditions Healthy Oreochromis niloticus (Nile tilapia) juveniles with average 40g ± 12.5g body weight were obtained from Palabana Fisheries, a commercial farm without history of disease outbreak, located in Chirundu district, South-East of Zambia. The fish population in this farm had been observed for clinical signs and sampled for evidence of disease over a period of 6 months. The fish were maintained in 500-L tanks for at least 2 weeks before conducting the experiments. The animals were maintained on commercial feed which was supplied twice a day to satiety. Feeding was withdrawn 24 h before experiments, and the animals were anesthetized in 10 mg L of tricaine methanesulfonate (MS222) before bacterial administration. Water parameters such as dissolved oxygen (DO), temperature, pH, ammonia, total dissolved solids and electrical conductivity were continuously measured. 2.2. Bacterial Pathogens Bacterial pathogens used in this study were Aeromonas spp ., Lactococcus garvieae ., Acinetobacter spp., and Klebsiella spp. e previously isolated from a natural outbreak of disease in Nile tilapia in small cage cultured fish on Lake Kariba, Siavonga, Zambia ( 16 ). The bacteria were cultured in brain–heart infusion (BHI) agar enriched with 5% defibrinated ovine blood and incubated at 28 °C for 24 h. Individual isolates of each bacteria were grown in BHI broth, and stocks were made with 15% glycerol and kept at −80 °C until use. 2.3. Determination of Lethal Dose (LD 50 ) of the bacteria An experimental challenge study was conducted to find out the lethal dose - 50% end point (LD50) in Nile tilapia. The bacteria were subcultured in 10 ml of Brain Heart Infusion (BHI) broth and were incubated at 37 ºC for 24h. After 24 h incubation, the stock solutions were prepared by dissolving colonies of each bacterial isolate in saline solution to match the turbidity of four McFarland standards (Riga, Latvia) [ Table 1 ]. View this table: View inline View popup Download powerpoint Table 1: McFarland standards The test was conducted with batches of 10 fish per dose by intraperitoneal injection in Nile tilapia with 24 hrs of bacterial suspension. The fishes were injected with 0.1 ml of bacterial suspension intraperitoneally with final concentrations of 1.5 × 10 8 , 3.0 × 10 8 , 6.0 × 10 8 , 9.0 × 10 8 and 12.0 × 10 8 CFU per ml, respectively. The control fishes were injected with 0.2 ml of normal saline. The challenge study was carried out in triplicates. A total number of 20 fishes were kept in each tank (40L). Fish mortality was recorded in every 24 h interval for 10 days. LD 50 was calculated using the Reed and Muench (1938) method. 2.4. The Challenge Study The study had six treatment groups: Aeromonas spp ., Lactococcus garvieae, Acinetobacter spp., Klebsiella spp, co-infection of all bacteria, and negative control. Twenty( 20 ) fish per treatment group were inoculated intraperitoneally with 100 μl bacterial suspension at the determined lethal doses according to the treatment groups [ Table 2 ]. View this table: View inline View popup Download powerpoint Table 2: Treatment groups for the trial experiments Clinical signs after bacterial inoculation were monitored daily for 21 days and the gross pathology and mortality rate were recorded daily. Necropsy, bacteriological, and histological examination was conducted on moribund fish r. At 21 days post-infection, all surviving fish were euthanized using an overdose of eugenol solution. 2.5. Histopathological examination of challenged fish Tissue samples (Anterior kidney, liver, and spleen) from the control and infected groups (three per group) were collected for histological study. These samples were preserved in 10% (vol/vol) neutral buffered formalin. After 24 h, the formalin was replaced with a fresh 10% formalin solution. Tissue sections were embedded with paraffin and stained with hematoxylin and eosin (H&E) using standard histological procedures. 2.6. Bacteriological examination of challenged fish To satisfy Koch’s postulates the bacteria was reisolated and identified from the moribund fishes. The brain, liver, and kidney were sampled using sterile inoculation loops and streaked on MacConkey agar (HiMedia, India), nutrient agar (HiMedia, India), and blood agar (HiMedia, India) using a strictly aseptic technique. The inoculated Petri dishes were stored at 28 °C for 24 h. Pure cultures were obtained by carrying out subculturing procedures and subjecting them to a second round of incubation at room temperature for another 48 hours, ensuring the isolation of uncontaminated bacterial strains. The isolates were identified by determining colony morphology and afterward the isolates were grouped accordingly. Two to three representative isolates from each group were subjected to Gram-staining. Conventional biochemical tests to characterize the bacteria was done according Siamunjompa et al ., (2023) ( 16 ). 2.7. Data Analysis Data were entered into an Excel spreadsheet (Microsoft Excel 2010 version, Redmond, WA, USA) and then exported to DATA Tab™ (Styria, Austria), a Web-App for statistical data analysis, where descriptive statistics (frequencies and proportions) were computed and presented using tables for categorical parameters ( 19 ). The summary tables and graphs were prepared in accordance with the objective of the study. 3. Results 3.1. Observation of clinical signs The clinical signs observed in the days following infection indicated that in fish infected with Klebsiella and Acinetobacter , the initial signs appeared 2 days post-infection [ Figure 1 ]. Klebsiella -infected fish exhibited lethargy, skin ulcerations, and mortality, while Acinetobacter -infected fish showed pale skin, loss of appetite, and mortality [ Figure 1 ]. In contrast, the first clinical signs in fish infected with Aeromonas spp and Lactococcus garvieae were recorded 1 day post-infection [ Figure 1 ]. Aeromonas -infected fish displayed hemorrhages on the body and trunk, skin ulcerations, ascites, and loss of appetite and mortality? [ Figure 1 ]. Fish infected with Lactococcus garvieae exhibited erratic swimming, fin erosion, lethargy, corneal opacity, skin ulcerations, and mortality [ Figure 1 ]. For the co-infected group, significant clinical signs included loss of appetite, body hemorrhages, skin ulcerations, fin hemorrhages, and high mortality [ Figure 1 ]. Download figure Open in new tab Figure 1: Clinical signs observed on fish infected with the various bacteria in infection experiments 3.2. External pathological lesions The external pathological lesions observed in the experimental groups are detailed in Figure 2 . In the Aeromonas group, there were slight erosions, minor skin ulcerations, and hemorrhaging on the operculum and eyes ( Figure 2A ). The Klebsiella group exhibited hemorrhages on the trunk and fins ( Figure 2B ). Fish in the Acinetobacter group appeared pale with slight hemorrhages on the operculum ( Figure 2C ). The Lactococcus garvieae group showed corneal opacity, missing scales, fin erosion, and hemorrhages on the operculum ( Figure 2D ). The co-infection group had skin ulcerations, fin erosions, hemorrhages on the operculum, and wounds around the mouth ( Figure 2E ). Download figure Open in new tab Figure 2: The external pathological lesions observed in the experimental groups were as follows: In the Aeromonas group (A), there were slight erosions, skin ulcerations, and hemorrhaging on the operculum and eyes. The Klebsiella group (B) exhibited minor skin ulceration, and hemorrhages in the eyes, on the trunk, and fins. Fish in the Acinetobacter group (C) appeared pale with slight hemorrhages in the eyes and on the operculum. The Lactococcus garvieae group (D) showed corneal opacity, missing scales, fin erosion, and hemorrhages on the operculum. The co-infection group (E) had skin ulcerations, fin erosions, hemorrhages on the operculum, and wounds around the mouth. 3.3. Histopathological assessments The liver of fish infected with Klebsiella spp exhibited congestion and hemorrhages ( Figure 3A, B, C ), parenchymal degeneration ( Figure 3B ), and lymphocyte infiltration ( Figure 3D ). In fish infected with Acinetobacter spp, the liver showed congestion ( Figure 4A ), parenchymal degeneration ( Figure 4B ), and hepatocyte vacuolation ( Figure 4B ), while the spleen revealed eosinophil infiltration ( Figure 4C ) and melanomacrophage centers ( Figure 4C ). Fish infected with Aeromonas spp presented main lesions of parenchymal degeneration ( Figure 5A ), hepatocyte vacuolation, and lymphocyte infiltration ( Figure 5A ). In fish infected with Lactococcus garvieae , significant liver lesions included congestion ( Figure 6A ), lymphocyte infiltration ( Figure 6B, D ), and parenchymal degeneration ( Figure 6D ), with inflammatory cell infiltration observed in the epidermis ( Figure 6C, F ). Co-infected fish displayed significant lesions across the liver, kidney, skin, and spleen. In the kidney, there was excessive lymphocyte infiltration ( Figure 7A, B ), parenchymal necrosis and degeneration ( Figure 7B ). The liver showed lymphocyte infiltration ( Figure 7C, D ), parenchymal degeneration ( Figure 7D ), and hepatocyte vacuolation ( Figure 7D ). In the skin, inflammatory cell infiltration was noted, while the spleen exhibited eosinophil infiltration ( Figure 7F ) and parenchymal degeneration ( Figure 7E ). Download figure Open in new tab Figure 3: Liver of fish infected with Klebsiella spp showing congestion and haemorrhages (A, B and C), degeneration of parenchyma (B) and lymphocyte infiltration (D). Download figure Open in new tab Figure 4: In the liver of Fish infected with Acinetobacter spp showed congestion (A) and degeneration of parenchyma (B), and vacuolation of hepatocytes (B). In the spleen, eosinophil infiltration (C) and centres of melanomacrophages were seen. Download figure Open in new tab Figure 5: in the fish infected with Aeromonas spp, the main lesions were degeneration of the parenchyma (A), vacuolation of hepatocytes, and lymphocyte infiltration. Download figure Open in new tab Figure 6: In the fish infected with Lactococcus garvieae in the liver the significant lesions were congestion (A), infiltrations of lymphocytes (B and D), and degeneration of parenchyma (D). in the epidermis, the significant lesions were infiltration of inflammatory cells (C and F). Download figure Open in new tab Figure 7: the co-infected fish has significant lesions seen in liver, kidney, skin and spleen. In the kidney excessive infiltration of lymphocytes (A and B), and necrosis and degeneration of parenchyma (B). In the liver, infiltration of lymphocytes (C and D), degeneration of parenchyma (D), and vacuolation of hepatocytes (D). In the skin the significant lesion was infiltration of inflammatory cells. In the spleen, eosinophil infiltration (F) and degeneration of parenchyma (E) were observed. 3.4. Mortality trends in the different experimental groups Comparative analysis of cumulative mortality revealed the most rapid demise in the co-infection group, with 100% (20/20) mortality by 7 days post-infection (dpi) [ Figure 8 ]. In the Lactococcus garvieae -infected group, initial mortality was observed at 2 dpi, and all succumbed by day 12 dpi [ Figure 8 ]. Aeromonas spp. infection also exhibited initial mortality at 2 dpi, with complete mortality by day 16 dpi [ Figure 8 ]. Conversely, the Klebsiella and Acinetobacter groups displayed the lowest mortality rates with 7 and 5 mortalities out of twenty recorded respectively at the study’s conclusion [ Figure 8 ]. Download figure Open in new tab Figure 8: Number of mortalities of fish challenged with indicated bacteria isolated in this study. 3.5. Bacteriological examination of challenged fish Bacterial isolation from the experimental fish revealed that in groups A and B, the experimental bacteria Klebsiella spp and Acinetobacter spp were isolated exclusively from the liver [ Table 3 ]. In groups C and D, the experimental bacteria Aeromonas spp and Lactococcus garvieae were isolated from the spleen, liver, and kidney [ Table 3 ]. Additionally, Lactococcus garvieae was isolated from brain tissue. In the co-infection group, Aeromonas spp and Lactococcus garvieae were isolated from the spleen, kidney, and brain, while all the bacteria were isolated from the liver [ Table 3 ]. View this table: View inline View popup Table 3: Isolation of bacteria from organs of fish in the infection experiment. 4. Discussion Bacterial pathogens affecting farmed Nile tilapia in both large and small-scale operations on Lake Kariba have been documented ( 11 , 16 ). Fish reared in floating cages are naturally exposed to the microbial communities in the aquatic environment, inevitably leading to co-infections during disease outbreaks. Our study demonstrate that co-infection with four species of bacteria resulted in extremely high pathogenicity among the experimental fish. Aeromonas spp and Lactococcus garvieae , well-documented fish pathogens ( 9 , 10 , 20 ), exhibited high pathogenicity, whereas Klebsiella spp and Acinetobacter spp, which are not well-characterized as fish pathogens, demonstrated reletively low pathogenicity. Challenge fish receiving Acinetobacter spp. exhibited clinical signs of pale skin, loss of appetite, and mortality. These pathogens have not previously been reported to cause significant disease outbreaks in Nile tilapia production globally. However, Acinetobacter spp. have been documented to cause disease outbreaks in other fish species, such as rohu ( Labeo rohita ) ( 21 ). The clinical signs observed in Acinetobacter outbreaks in rohu included ulcerative lesions, hemorrhages on the skin and tail, and fin rot, which differ from the observations in this study where Nile tilapia presented with pale skin and loss of appetite. Histopathological examination in the present study revealed liver lesions, including parenchymal degeneration and hepatocyte vacuolation, and in the spleen, eosinophil infiltration and melanomacrophage centers. These findings were consistent with those reported by Laltlanmawia et al . (2023) in diseased rohu ( 21 ). The mortality rate at the end of the present experiment, 21 days post-infection, was 50%, in contrast to the infection experiment in rohu, where 100% mortality was recorded within the first 3 days post-infection ( 21 ). In the present study, fish challenged with Klebsiella spp. exhibited clinical signs of skin ulcerations, lethargy, and mortality. This pathogen has previously been isolated from diseased tilapia, where significant mortality was recorded ( 16 , 22 , 23 ). Reported clinical signs in these studies included lethargy, anorexia, subcutaneous hemorrhages, urogenital bleeding, and ascites, some of which were consistent with the present findings ( 16 , 22 , 23 ). Vaneci-Silva et al. (2022) reported significant histopathological lesions such as multifocal edema and fatty degeneration in the liver, as well as melanomacrophage centers in the spleen and kidney ( 23 ). The present study also identified significant histopathological lesions in the liver, including congestion and hemorrhages, parenchymal degeneration, and lymphocyte infiltration. Additionally, the cumulative mortality rate in this study was 70% at 21 days post-challenge, whereas an outbreak in juvenile Nile tilapia at a farm in Brazil resulted in 100% mortality within 4 days ( 23 ). Aeromonas spp have been reported globally as significant pathogens in aquaculture, often causing outbreaks secondary to stressors in production systems ( 9 , 20 , 24 , 25 ). Clinical signs of Aeromonas infections include hemorrhagic patches on the skin, at the base of the pectoral fin, and around the anal opening, as well as scale desquamation, skin ulcerations and fin erosions ( 20 , 24 ). These signs were consistent with those observed in the present study. Histopathological examination of liver tissues in this study revealed parenchymal degeneration, hepatocyte vacuolation, and lymphocyte infiltration, consistent with findings by Dong et al. (2017), who additionally reported hemosiderin accumulation around vessels and hepatocytes, and hyperemia and hemorrhage in the spleen ( 9 ). The cumulative mortality rate in the present study was 100% at 16 days post-challenge, contrasting with the 70% cumulative mortality rate at 21 days post-challenge reported by Dong et al ., (2017). Since the initial isolation of Lactococcus garvieae from Nile tilapia in Zambia in 2018, this pathogen has continued to be reported as a cause significant losses due to disease outbreaks among both small and large commercial producers ( 11 , 16 , 22 ). The main clinical signs observed in the present study, such as erratic swimming, corneal opacity, fin erosion, and loss of scales, are consistent with those reported in outbreaks among farmed tilapia in Zambia ( 11 , 16 ). Histopathological lesions recorded in this study included congestion, lymphocyte infiltration, parenchymal degeneration, and inflammatory cell infiltration in the skin epidermis. These lesions are similar to those reported in cage-cultured cobia ( Rachycentron canadum ) ( 26 ). The present study recorded a cumulative mortality rate of 100% by 12 days post-infection which highlights the pathogenic nature of the bacterial causing significant mortality in many tilapia production farms, both small and large-scale, in Zambia ( 11 , 16 ). Previous experimental studies on co-infection in Nile tilapia primarily focused on bacteria, viruses, or parasites ( 9 , 27 , 28 ). This study demonstrated the co-infection of four bacterial pathogens, previously isolated from farmed Nile tilapia on Lake Kariba among small-scale producers ( 16 ). To the best of our knowledge, this is the first study to elucidate the co-infection of four bacterial pathogens in Nile tilapia. Clinical signs were observed within a day post-infection, earlier than for any of the single pathogens included in the study. These signs included skin ulcerations, mouth lesions, fin erosions, corneal opacity, and hemorrhages, with the first three lesions being more severe in the co-infection group than in the mono-infection experiments. Dong et al. (2017) reported heavy mortalities in Nile tilapia infected with Aeromonas jandaei and Aeromonas veronii ( 9 ). In the present study, co-infection with four bacterial pathogens resulted in very high mortality, with cumulative mortality reaching 100% by the seventh day post-infection. Histopathological examination further demonstrated the pathogenicity of the co-infection, showing a combination of lesions seen in the mono-infections. These lesions included excessive lymphocyte infiltration, necrosis, and parenchymal degeneration in the kidney; lymphocyte infiltration, parenchymal degeneration, and hepatocyte vacuolation in the liver; inflammatory cell infiltration in the skin; and eosinophil infiltration and parenchymal degeneration in the spleen. The results of this study indicate that the presence of Klebsiella spp., Acinetobacter spp., Aeromonas spp., and Lactococcus garvieae in the cage environment, coupled with stressors, will lead to severe mortalities in farmed fish. This study demonstrated that Nile tilapia infected with Klebsiella spp. and Acinetobacter spp. exhibited low pathogenicity. Furthermore, we reported for the first time the experimental co-infection of Nile tilapia with four bacterial pathogens ( Klebsiella spp., Acinetobacter spp., Aeromonas spp., and Lactococcus garvieae ). The high pathogenicity observed in these bacterial co-infections suggests a potential threat to the aquaculture industry in Zambia. Therefore, future research should focus on developing strategies to control these bacterial co-infections, including the development of polyvalent vaccines. 6. Statements and Declarations Competing interests The authors declare that they have no financial or personal relationships that may have inappropriately influenced them in writing this article. Authors’ contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Kunda Ndashe, Frederick Chitongo Zulu, Ladslav Moonga and Katendi Changula. The first draft of the manuscript was written by Kunda Ndashe, and Frederick Chitongo Zulu and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript Data Availability The data that support the findings of this study are available from the corresponding author, Kunda Ndashe, upon reasonable request. Ethics approval This study was undertaken in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Health Research Ethics Committee of Zambia. The protocol of this study was approved by the Excellence in Research Ethics and Science (ERES) Converge, (Protocol Number: 2019/AUG/024). Statement of Animal Rights All procedures involving animals were conducted in compliance with ethical guidelines and approved by the relevant institutional ethics committee. The welfare of the animals was ensured throughout the study, adhering to the principles of humane treatment, minimizing distress, and following the 3Rs (Replacement, Reduction, and Refinement) wherever applicable. Conflict of Interest Statement The authors declare no conflicts of interest related to this study. 5. Acknowledgements The authors would like to thank the technical and administrative staff of the University of Zambia, School of Veterinary Medicine, Bacteriology and Pathology Units who supported and faciliated the study. References 1. ↵ FAO . The State of World Fisheries and Aquaculture 2022: Towards Blue Transformation [Internet] . 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