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Ulcerative disease emergence in grass carp (Ctenopharyngodon idellus) aquaculture in China: Possible impact of temperature abnormality
Aquaculture 517 (2020) 734811
Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aquaculture
Ulcerative disease emergence in grass carp (Ctenopharyngodon idellus) aquaculture in China: Possible impact of temperature abnormality
T
⁎
Xiaoli Huanga,1, , Guanqing Xionga,1, Yang Fenga,1, Kaiyu Wangb, Yufeng Liua, Liang Zhonga, Sha Liua, Yi Gengb, Ping Ouyangb, Defang Chena, Shiyong Yanga a b
Department of Aquaculture, College of Animal Science & Technology, Sichuan Agricultural University, WenJiang 611130, Sichuan, China College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Grass carp Ctenopharyngodon idellus Epidemic ulcerative disease Genus Aeromonas Temperature abnormality
In 2019, an epidemic of ulcerative lesions emerged in grass carp (Ctenopharyngodon idellus) in the Yangtze River basin of China. Diseased fish displayed hemorrhagic ulcers of the tail muscle and reach 70% cumulative mortality, causing serious economic losses. The objective of the present study is to describe disease characteristics, analyze disease factors, and elucidate the etiology. Diseased fish from Qingbaijiang and Deyang, Sichuan, were dissected for the necropsy, pathology, and microbiology investigation. The histopathological examination revealed that the target organs of grass carp were muscle, spleen, and kidney, all showing severe necrosis and varying degrees of inflammation and edema. There were slight changes in the intestine, liver, gill, and heart. Nine strains of bacteria were isolated from diseased grass carp and were identified as belonging to four Aeromonas species. Subsequently, symptoms of tail muscle hemorrhage similar to the clinical symptoms were duplicated through re-infection. There was also a period of low temperatures in the affected regions in the early spring of 2019. Based on the results, we concluded that the genus Aeromonas was the pathogen causing the grass carp epidemic ulcerative disease, which perhaps links to the temperature abnormality.
1. Introduction For most parts of the world, climate changes regularly throughout the year. If this law is broken, certain diseases and even epidemics in animals can occur (Chretien et al., 2015). Freshwater environments and their aquatic organisms are particularly vulnerable to climate change because the persistence and quality of aquatic habitat depend heavily on climatic and hydrologic regimes (Mohammed et al., 2017; Seiller and Anctil, 2013). Changes in weather patterns have increased susceptibility to fish diseases. For example, seasonal increase in sea temperature triggers pancreas disease outbreaks in salmon (SchmidtPosthaus et al., 2012; Stene et al., 2013). Additionally, Batrachochytrium dendrobatidis infection results in a higher mortality rate in frogs when they are subjected to unstable temperatures than a constant one (Raffel et al., 2013). Aquatic organisms are able to dive into different layers of water or migrate to cope with the climate abnormality in natural ecology. For aquaculture, however, climate anomalies can lead to catastrophic damage. Aquatic organisms are more susceptible in early spring due to the anemia and decrease in serum proteins that results from periods of
dormancy and starvation that have occurred during the winter (Woo and Bruno, 2011). Some psychrophilic pathogens such as Flavobacterium, Yersinia and Aeromomas are able to survive and begin to proliferate in low-temperature environments (Marcos Lopez et al., 2010), which results in outbreaks of the pathogen in aquatic organisms in the spring. Grass carp (Ctenopharyngodon idellus) is naturally distributed in the lower reaches of the Heilongjiang River and Yellow River, and the middle and lower reaches of the Yangtze River, China, and accounts for 20% of the total yield of freshwater fish in China (Xianliang et al., 2018). Grass carp frequently suffer various diseases such as reovirus caused hemorrhage (Rao and Su, 2015), A. hydrophila caused septicemia (Song et al., 2014) and Acinetobacter lwoffii caused hemorrhage (Lu et al., 2017). In 2019, an epidemic ulcerative disease emerged in grass carp aquaculture of the Yangtze River basin in the early spring, causing great mortality and economic loss. The main objective of the present study is to describe disease characteristics, analyze disease factors, and elucidate the etiology, with the aim of providing evidence of the effects of climate abnormality on disease emergence in freshwater aquatic animals.
⁎
Corresponding author. E-mail address: [email protected] (X. Huang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.aquaculture.2019.734811 Received 20 July 2019; Received in revised form 25 November 2019; Accepted 1 December 2019 Available online 02 December 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
Aquaculture 517 (2020) 734811
X. Huang, et al.
2. Materials and methods
and Use Committee of Sichuan Agricultural University, under permit number FYS2017530302. Healthy grass carp (75) with bright body color and responsive were selected after random verifying the absence of bacteria in the liver, spleen, and kidney. The carps were divided into 5 groups (1 control group and 4 experimental groups) of 15 fish each. The control group received 0.1 mL of saline (0.85%) intraperitoneal (IP) injection, while the experimental group received a 0.1 mL IP injections of 1 × 105 CFU/ mL purified bacteria. All fish were checked daily for clinical signs and mortality. Re-isolation and identification of the bacteria from the liver, kidney, and spleen of moribund fish were performed.
2.1. Necropsy Eight diseased grass carp with typical clinical signs of disease were collected from affected farms in Deyang and Qingbaijiang, Sichuan, and submitted for necropsy in March 2019. The fish were euthanized by using MS-222. All fish were examined for parasites of the gill and skin. The examinations included gross external, internal lesions and content of the alimentary tract. 2.2. Histopathology
2.6. Data The tissues, including muscle, skin, gill, brain, eye, trunk-kidney, head-kidney, spleen, heart, liver, and intestine, were collected and fixed immediately in 10% neutral buffered formalin. After decalcification, tissues were trimmed into cassettes, dehydrated in graded ethanol solutions, cleared in xylene, and embedded in paraffin wax. Sections (5 μm) were prepared for hematoxylin and eosin (H&E) staining before microscopic analysis. The degree of congestion/hemorrhage, degeneration, hyperplasia, infiltration, necrosis, edema, and hypertrophy in organs was evaluated by reference to the scoring system designed by Bernet and colleagues (Bernet et al., 2010). Every measure was assessed using a score (S) ranging from 0 to 6, depending on the degree and extent of the alteration: (0) unchanged; (2) mild; (4) moderate; and (6) severe (diffuse lesion).
The temperature records were obtained from the National Meteorological Information Center (http://data.cma.cn/) from 11 grass carp epidemic regions (Chizhou, Jingzhou, Changde, Yueyang, Wuxi, Suzhou, Yangzhou, Chengdu, Deyang, Honghu, and Changshu) and 23 neighboring regions (Hefei, Tongling, Anqing, Huangshan, Jingmen, Xiaogan, Yichang, Xianning, Changsha, Xiangtan, Zhuzhou, Yiyang, Nanjing, Changzhou, Zhenjiang, Taizhou, Jiaxing, Huzhou, Mianyang, Suining, Meishan, Ya'an, and Chongqing). 3. Results 3.1. Disease prevalence
The muscles and spleen of diseased grass carp were sampled, immediately fixed in 2.5% glutaraldehyde, and postfixed in 2% veronalacetate-buffered osmium tetroxide. After dehydration in graded alcohol, the samples were embedded in Araldite. The blocks were sectioned on a microtome with a glass knife. The sections (6 μm) were placed on uncoated copper grids, stained with uranyl acetate, and poststained with 0.2% lead citrate. The subcellular structures of the samples were examined with a Hitachi H-600 Transmission Electron Microscope (Hitachi, Tokyo, Japan).
In 2019, grass carp demonstrated a wide range of morbidity in China. The epidemic included 11 cities in Sichuan, Hubei, Hunan, Anhui and Jiangsu provinces, which are located in Minjiang River system, Dongting Lake system, and Taihu Lake system in the Yangtze River Basin, with a horizontal span of 1550 km (Fig. 1). The disease began to appear in early Dec. 2018, and concentrated outbreaks occurred from mid-February to mid-March 2019. The incidence of grass carp infection varies from region to region, with the most effected regions showing 100% mortality and over 50% morbidity (Fig. 2A). Specifically, the disease regions located at the junction of the distribution belts of 12–16 °C and 16–20 °C (Huang et al., 2011), which is supportive of the connection between epidemic and temperature.
2.4. Bacteriology
3.2. Necropsy findings
The spleen, muscle, liver, and kidney of each fish was aseptically streaked on brain–heart infusion (Becton Drive, USA) agar plates and incubated at 18 °C for 18–24 h for routine bacteriology. The isolates from diseased fish were sub-cultured under the same conditions to check the purity of the isolations. Pure cultures of selected isolates were preservation using glycerin and stored at −80 °C. The bacterial species were identified with 16S rRNA sequencing (primers: F: 5’-AGAGTTTGATCCTGGCTCAG-3′ and R: 5’-GGCTACCTT GTTACGACTT-3′). The amplified sequences were compared with the GenBank database (https://www.ncbi.nlm.nih.gov/) with a BLAST analysis. Homology analysis was performed with MEGA 7.0 and a phylogenetic analysis with the neighbor-joining method.
At the time of disease occurrence, the grass carp gathered and emerged to the surface with weak swimming, and died within only a few days. The diseased fish presented with hemorrhaging around the head and gill cover (Fig. 2B1) and muscle ulcers where the skin remained relatively intact (Fig. 2B2). Severe muscle hemorrhage could be observed after the skin was dissected Fig. 2B2’). The swim bladder was overinflated and congested, the spleen was swollen (Fig. 2B3), and occasionally fatty liver was observed (Fig. 2B3’). Similar gross lesions also occurred in silver carp and crucian carp in some polyculture farms (Fig. 2C-D).
2.5. Animal infection assays
Under the microscope, muscle demonstrated marked necrotizing hemorrhagic myositis (Fig. 3A-B). In the spleen, the hematopoietic cells were severely reduced and reticular fibroblasts were severely necrotic (Fig. 3C-D). The trunk-kidney showed severe edema, a severe reduction in renal interstitial hemopoietic tissue, and severe degeneration and necrosis of the renal tubule (Fig. 3E-F). Additionally, severe dilation of the capillary and moderate necrosis of hematopoietic cells was seen in the head-kidney (Fig. 3G-H). Also, there were moderate changes in the intestine and gill, and mild change in liver, eyes, brain and heart, which showed different degrees of degeneration, inflammation, and necrosis (Fig. 3I-L). The results of the pathological score demonstrated that the
2.3. Ultrastructural examination
3.3. Histopathological diagnoses
Healthy grass carp (100; 22.6 ± 2.5 cm, 104 ± 7.6 g) were purchased from a fish farm in Mianyang, Sichuan. The fishes were acclimatized in the laboratory for 2 weeks before experimentation. The carp were exposed to uninterrupted oxygen supply to ensure more than 5 mg/L dissolved oxygen, a pH of 6.5–8 and a temperature of about 15 °C. The water in the tank was pretreated with an aeration process and 30% of the culture water was renewed every day. Fish were fed twice daily with a commercial diet. All animal infections were conducted following protocols approved by the Institutional Animal Care 2
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Fig. 1. Records of grass carp ulcerative disease in the Yangtze River basin of China from Feb. to Apr. 2019.
Fig. 2. Gross lesions in ulcerative diseased carps. A: Dead fish from the diseased farm. B: ulcerative lesions of grass carp: hemorrhage of the head and gill cover (B1), ulcer of the tail muscles, detachment of scales (B2), severe muscle hemorrhage (B2’) after dissection of the skin; swollen spleen, swim bladder congestion (B3), and occasionally fatty liver symptoms (B3’). C-D: Similar ulcerative lesions of silver carp and crucian carp in polyculture pond. ☆: Ulcer site similar to grass carp. 3
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Fig. 3. Histopathologic lesions in ulcerative diseased grass carp. A-B: severe muscle bleeding (arrow), severe necrosis of muscle fibers (★), and moderate inflammatory cell infiltration (arrowhead); C-D: sharp decrease in hematopoietic cells in the spleen, severe necrosis of reticular fibers (★) and hematopoietic cells (arrow), and moderate inflammatory cell infiltration (arrowhead); E-F: moderate renal edema (★), renal interstitial cell sharp decrease, necrosis of renal tubular and red blood cell (arrow), vacuolar degeneration of renal tubular cell (*), and mild inflammatory cell infiltration (arrowhead); G-H: head kidney edema (★), moderate necrosis of hematopoietic cells (arrow); I: severe vacuoles denaturation of intestinal mucosa (*), a large reduction of mucosal cells (arrow); J: hepatic capillary edema (★), mild to moderate hepatocyte necrosis (arrow); K: submucosal cell proliferation (arrow); L: epicardial mild inflammatory cell infiltration (arrow). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
spleen, muscle, middle kidney, and head kidney were the main pathological target organs in the diseased fish, and infiltration and necrosis were the main pathological symptoms (Fig. 4). The histopathological study indicated that there was a strong correlation between the disease and microorganism. 3.4. Ultrastructural pathology Ultrastructural pathology was performed for further analysis. In muscle, the myofibrils were breaking and the cytoplasm of the myocytes was filled with abundant vacuoles (Fig. 5A-C). In the hematopoietic cells, there was marked swelling of the mitochondria and the endoplasmic reticulum (Fig. 5D). Additionally, a small number of autophagosomes were observed in muscle cells and hematopoietic cells (Fig. 5A-D). Moreover, mild swelling occurred in both mitochondria and endoplasmic reticulum in the endothelial cells of the spleen (Fig. 5E). The results showed the disease caused serious cell damage, which would lead to organ disorder. 3.5. Isolation and identification of pathogens Nine strains were originally isolated from the spleen and muscles of diseased grass carp. The 16S rRNA gene of the isolates was amplified, identified with a BLAST search, and deposited in GenBank (Accession Numbers: MN173487, MN173487, MN173489, MN173492, MN173493, MN173502, MN173512, MN173513, and MN173514). Based on the phylogenetic analysis, three strains shared homology with A. salmonicida, three strains shared homology with A. sobria, two strains shared
Fig. 4. Histopathological scores of different organs of diseased grass carp. The size of the circle represents the number of fish with corresponding symptoms (n = 8), and the different colors represent the degree of symptoms.
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Fig. 5. Ultrastructural lesions in ulcerative diseased grass carp. A-C: Ultrastructural changes under the TEM of muscle fibers; D: Ultrastructural changes under TEM in hematopoietic cells; E: Ultrastructural changes under TEM of blood cells. N: nucleus; Mt.: mitochondria; M: myofibrils; ER: endoplasmic reticulum; L: lipid droplets; R: glycogen; arrow: vacuoles; arrowhead: mitochondrial swelling; *: endoplasmic reticulum swelling; ★: autophagosome.
3.6. Pathogenicity of isolates
homology with A. veronii, and one strain shared homology with A. hydrophila (Fig. 6). The results showed all isolates belonged to the genus Aeromonas, which has been reported to infect carps (Byadgi et al., 2018; Chen et al., 2018; Wang et al., 2017; Yamin et al., 2017).
To confirm the pathogenicity, four Aeromonas species were used to experimentally infect the grass carp. There were no aberrant results or mortality observed in the control group of fish during the experiment
Fig. 6. Phylogenetic tree was generated with MEGA 7.0 based on an alignment of the 16S rRNA gene sequences of the isolated strains (FYp0313 1-9) and related species. The 16 s gene sequence of related species originate from NCBI (https://www.ncbi.nlm.nih.gov/). 5
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Fig. 7. Artificial infection of 4 Aeromonas strains. A. Survival rate (%) of grass carp infected with genus Aeromonas. B. Number of infected fish with relative gross lesion.
(Fig. 7A). In contrast, all fish died within 60 h after A. sobria injection, and all A. salmonicida infected grass carp also died at 96 h (Fig. 7A). During the 96 h experiment, A. veronii caused 66.7% grass carp death, while A. hydrophila caused 53.3% (Fig. 7A). The gross lesions of infected fish were recorded; some showed the hemorrhage of tail muscle that was similar to the natural disease (Fig. 7B). Moreover, the bacteria were re-isolated and identified from infected fish. The results demonstrate the pathogenicity of four isolates, confirming the pathogenicity of genus Aeromonas on the ulcerative disease.
outbreak, these regions experienced a low-temperature period of 1 month (1.5–10 °C) (Fig. 8). The continuous low temperature would facilitate the proliferation of pathogens and lead to grass carp ulcerative disease. Regrettably, since it is difficult to determine the incidence of disease, we cannot statistically correlate this epidemic with temperature. Either there is no significant difference in the temperature of the city near the affected regions, or whether an epidemic has occurred in these regions has not been reported. 4. Discussion
3.7. Possible impacts of temperature abnormality on the epidemic Currently, common bacteria in aquatic organisms such as Flavobacterium, Yersinia, Aeromomas, Lactococcus, and Renibacterium can withstand low-temperature environments (Marcos Lopez et al., 2010). Among them, Aeromonas can be divided into motile species such as A. hydrophila, A. veronii, and A. sobria, and non-motile species A. salmonicida (Praveen et al., 2016). Aeromonas has a worldwide distribution and frequently causes disease in cultured and feral fish (Beaz-Hidalgo and Figueras, 2013). Motile Aeromonas could lead to dermal ulceration, tail or fin rot, ocular ulcerations, erythrodermatitis, hemorrhagic septicemia, red sore disease, red rot disease, and scale protrusion disease (Cipriano and Austin, 2011). Also, non-motile A. salmonicida species can cause enlarged spleen, sloughing of scales, necrotic dermis, degeneration of the musculature, and septicemia (Cipriano and Austin, 2011). The present study found that the target organs of diseased grass carp were muscle, spleen and kidney, all of which showed significant necrosis, hemorrhage, and varying degrees of inflammation. Moreover, nine strains were isolated from diseased grass carp and identified them as genus Aeromonas, and their pathogenicity was determined by experimental infection. The results conclude that the grass carp ulcerative disease is an epidemic caused by Aeromonas infection. Opportunistic bacteria with a broad host spectrum have a high possibility of involvement in co-infections (Kotob et al., 2016). Genus Aeromonas is the most famous pathogenic bacteria in fish, which often causes outbreaks of disease in aquaculture (Cipriano and Austin, 2011). The outbreak of the ulcerative disease was wider and more serious in 2019, which is suggestive for a role of abnormal temperature in these infections. There was a report that A. salmonicida had elevated virulence at low temperature (Ishiguro et al., 1981). Low immunity of fish combined with high pathogenicity of Aeromonas may contribute to the epidemic in grass carp. At present, the temperature of the world, including China, is experiencing global warming and abnormal changes in specific local areas (Du et al., 2019). This suggests that abnormal temperature not only affects the growth of animals but also leads to changes in the regularity of the disease. Global climate variability shifts the distribution of infectious diseases with potentially adverse consequences for disease control.
We identified an interesting phenomenon: most affected regions were located at the junction of the distribution belts of 12–16 °C and 16–20 °C (Fig. 1). The temperature in the affected region in 2019 was lower than in previous years. More interestingly, before the epidemic
Fig. 8. Temperature change in the diseased area of grass carp. Arrow: Temperature lag compared to previous years. 6
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Data availability
Huang, Y., Feng, G., Dong, W., 2011. Temporal changes in the patterns of extreme air temperature and precipitation in the various regions of China in recent 50 years. Acta Meteorol. Sin. 69, 125–136. Ishiguro, E.E., Kay, W.W., Ainsworth, T., Chamberlain, J.B., Austen, R.A., Buckley, J.T., Trust, T.J., 1981. Loss of virulence during culture of Aeromonas salmonicida at high temperature. J. Bacteriol. 148, 333. Kotob, M.H., Menanteau-Ledouble, S., Kumar, G., Abdelzaher, M., El-Matbouli, M., 2016. The impact of co-infections on fish: a review. Vet. Res. 47, 1–12. Lu, A., Wei, Z., Hu, X., Sun, J., Pei, C., Zhang, C., Li, L., Li, A., 2017. Isolation and characterization of Acinetobacter lwoffii from the intestine of grass carp (Ctenopharyngodon idella). Isr. J. Aquacult Bamid. 69, 1–7. Marcos Lopez, M., Gale, P., Oidtmann, B., Peeler, E., 2010. Assessing the impact of climate change on disease emergence in freshwater fish in the United Kingdom. Transbound. Emerg. Dis. 57, 293–304. Mohammed, R., Scholz, M., Zounemat-Kermani, M., 2017. Temporal hydrologic alterations coupled with climate variability and drought for transboundary river basins. Water Resour. Manag. 31, 1489–1502. Praveen, P.K., Debnath, C., Shekhar, S., Dalai, N., Ganguly, S., 2016. Incidence of Aeromonas spp. infection in fish and chicken meat and its related public health hazards: a review. Vet. World. 9, 6–11. Raffel, T., Romansic, J., Halstead, N., McMahon, T., Venesky, M., Rohr, J., 2013. Disease and thermal acclimation in a more variable and unpredictable climate. Nat. Clim. Chang. 3, 146–151. Rao, Y., Su, J., 2015. Insights into the antiviral immunity against grass carp (Ctenopharyngodon idella) reovirus (GCRV) in grass carp. J Immunol Res 2015, 1–18. Schmidt-Posthaus, H., Bettge, K., Forster, U., Segner, H., Wahli, T., 2012. Kidney pathology and parasite intensity in rainbow trout Oncorhynchus mykiss surviving proliferative kidney disease: time course and influence of temperature. Dis. Aquat. Org. 97, 207–218. Seiller, G., Anctil, F., 2013. Climate change impacts on the hydrologic regime of a Canadian river: comparing uncertainties arising from climate natural variability and lumped hydrological model structures. Hydrol. Earth Syst. Sc. 10, 14189–14227. Song, X., Jie, Z., Bo, Y., Liu, Z., Kang, W., Gong, C., 2014. Aeromonas hydrophila induces intestinal inflammation in grass carp (Ctenopharyngodon idella): an experimental model. Aquaculture. 434, 171–178. Stene, A., Bang Jensen, B., Knutsen, ø., Olsen, A.B., Viljugrein, H., 2013. Seasonal increase in sea temperature triggers pancreas disease outbreaks in Norwegian salmon farms. J. Fish Dis. 37, 739–751. Wang, S.-T., Meng, X.-Z., Li, L.-S., Dang, Y.-F., Fang, Y., Shen, Y., Xu, X.-Y., Wang, R.-Q., Li, J.-L., 2017. Biological parameters, immune enzymes, and histological alterations in the livers of grass carp infected with Aeromonas hydrophila. Fish Shellfish Immunol. 70, 121–128. Woo, P., Bruno, D., 2011. Fish Diseases and Disorders. Sbs Publishers, pp. 1–930. Xianliang, Z., Fang, X., Shumin, L., Xinzhong, L., Xu, H., Kiayong, J., Honglang, H., 2018. China Fisheries Statistical Yearbook 2018. China Agriculture Press, Beijing, pp. 24–35. Yamin, G., Falk, R., R Avtalion, R., Shoshana, N., Ofek, T., Smirnov, R., Rubinstein, G., Rijn, J., 2017. The protective effect of humic-rich substances on atypical Aeromonas salmonicida subsp. salmonicida infection in common carp (Cyprinus carpio L.). J. Fish Dis. 40, 1783–1790.
All data generated or analyzed are included in the article. All materials are available from the corresponding author, on reasonable request. Authors contributions Xiaoli Huang, Guanqing Xiong, and Yang Feng contributed the work equally. All authors read and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This research was supported by grants Sichuan Agricultural University Pathological Test Project (NO. 015H1500). References Beaz-Hidalgo, R., Figueras, M.J., 2013. Aeromonas spp. whole genomes and virulence factors implicated in fish disease. J. Fish Dis. 36, 371–388. Bernet, D., Schmidt, H., Meier, W., Burkhardt-Holm, P., Wahli, T., 2010. Histopathology in fish: proposal for a protocol to assess aquatic pollution. J. Fish Dis. 22, 25–34. Byadgi, O., Chen, Y.-C., Maekawa, S., Wang, P.-C., Chen, S.-C., 2018. Immune-related functional differential gene expression in koi carp (Cyprinus carpio) after challenge with Aeromonas sobria. Int. J. Mol. Sci. 19, 2107. Chen, J.J., Cao, X.N., Liu, L.T., Cao, X.L., 2018. Immunomodulation with probiotics against aeromonas veronii in grass carp (Ctenopharyngodon idellus). Isr. J. Aquacult Bamid. 70, 1–14. Chretien, J.-P., Anyamba, A., Small, J., Britch, S., Sanchez, J., C Halbach, A., Tucker, C., Linthicum, K., 2015. Global climate anomalies and potential infectious disease risks: 2014-2015. PLoS Currents Outbreaks. 1, 1–16. Cipriano, R.C., Austin, B., 2011. Furunculosis and other Aeromonad diseases. In: Fish Diseases and Disorders: Viral, Bacterial and Fungal Infections. Sbs Publishers, pp. 435–494. Du, Q., Zhang, M., Wang, S., Che, C., Ma, R., Ma, Z., 2019. Changes in air temperature over China in response to the recent global warming hiatus. J. Geogr. Sci. 29, 496–516.
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Contents lists available at ScienceDirect
Aquaculture journal homepage: www.elsevier.com/locate/aquaculture
Ulcerative disease emergence in grass carp (Ctenopharyngodon idellus) aquaculture in China: Possible impact of temperature abnormality
T
⁎
Xiaoli Huanga,1, , Guanqing Xionga,1, Yang Fenga,1, Kaiyu Wangb, Yufeng Liua, Liang Zhonga, Sha Liua, Yi Gengb, Ping Ouyangb, Defang Chena, Shiyong Yanga a b
Department of Aquaculture, College of Animal Science & Technology, Sichuan Agricultural University, WenJiang 611130, Sichuan, China College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, Sichuan, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Grass carp Ctenopharyngodon idellus Epidemic ulcerative disease Genus Aeromonas Temperature abnormality
In 2019, an epidemic of ulcerative lesions emerged in grass carp (Ctenopharyngodon idellus) in the Yangtze River basin of China. Diseased fish displayed hemorrhagic ulcers of the tail muscle and reach 70% cumulative mortality, causing serious economic losses. The objective of the present study is to describe disease characteristics, analyze disease factors, and elucidate the etiology. Diseased fish from Qingbaijiang and Deyang, Sichuan, were dissected for the necropsy, pathology, and microbiology investigation. The histopathological examination revealed that the target organs of grass carp were muscle, spleen, and kidney, all showing severe necrosis and varying degrees of inflammation and edema. There were slight changes in the intestine, liver, gill, and heart. Nine strains of bacteria were isolated from diseased grass carp and were identified as belonging to four Aeromonas species. Subsequently, symptoms of tail muscle hemorrhage similar to the clinical symptoms were duplicated through re-infection. There was also a period of low temperatures in the affected regions in the early spring of 2019. Based on the results, we concluded that the genus Aeromonas was the pathogen causing the grass carp epidemic ulcerative disease, which perhaps links to the temperature abnormality.
1. Introduction For most parts of the world, climate changes regularly throughout the year. If this law is broken, certain diseases and even epidemics in animals can occur (Chretien et al., 2015). Freshwater environments and their aquatic organisms are particularly vulnerable to climate change because the persistence and quality of aquatic habitat depend heavily on climatic and hydrologic regimes (Mohammed et al., 2017; Seiller and Anctil, 2013). Changes in weather patterns have increased susceptibility to fish diseases. For example, seasonal increase in sea temperature triggers pancreas disease outbreaks in salmon (SchmidtPosthaus et al., 2012; Stene et al., 2013). Additionally, Batrachochytrium dendrobatidis infection results in a higher mortality rate in frogs when they are subjected to unstable temperatures than a constant one (Raffel et al., 2013). Aquatic organisms are able to dive into different layers of water or migrate to cope with the climate abnormality in natural ecology. For aquaculture, however, climate anomalies can lead to catastrophic damage. Aquatic organisms are more susceptible in early spring due to the anemia and decrease in serum proteins that results from periods of
dormancy and starvation that have occurred during the winter (Woo and Bruno, 2011). Some psychrophilic pathogens such as Flavobacterium, Yersinia and Aeromomas are able to survive and begin to proliferate in low-temperature environments (Marcos Lopez et al., 2010), which results in outbreaks of the pathogen in aquatic organisms in the spring. Grass carp (Ctenopharyngodon idellus) is naturally distributed in the lower reaches of the Heilongjiang River and Yellow River, and the middle and lower reaches of the Yangtze River, China, and accounts for 20% of the total yield of freshwater fish in China (Xianliang et al., 2018). Grass carp frequently suffer various diseases such as reovirus caused hemorrhage (Rao and Su, 2015), A. hydrophila caused septicemia (Song et al., 2014) and Acinetobacter lwoffii caused hemorrhage (Lu et al., 2017). In 2019, an epidemic ulcerative disease emerged in grass carp aquaculture of the Yangtze River basin in the early spring, causing great mortality and economic loss. The main objective of the present study is to describe disease characteristics, analyze disease factors, and elucidate the etiology, with the aim of providing evidence of the effects of climate abnormality on disease emergence in freshwater aquatic animals.
⁎
Corresponding author. E-mail address: [email protected] (X. Huang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.aquaculture.2019.734811 Received 20 July 2019; Received in revised form 25 November 2019; Accepted 1 December 2019 Available online 02 December 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
Aquaculture 517 (2020) 734811
X. Huang, et al.
2. Materials and methods
and Use Committee of Sichuan Agricultural University, under permit number FYS2017530302. Healthy grass carp (75) with bright body color and responsive were selected after random verifying the absence of bacteria in the liver, spleen, and kidney. The carps were divided into 5 groups (1 control group and 4 experimental groups) of 15 fish each. The control group received 0.1 mL of saline (0.85%) intraperitoneal (IP) injection, while the experimental group received a 0.1 mL IP injections of 1 × 105 CFU/ mL purified bacteria. All fish were checked daily for clinical signs and mortality. Re-isolation and identification of the bacteria from the liver, kidney, and spleen of moribund fish were performed.
2.1. Necropsy Eight diseased grass carp with typical clinical signs of disease were collected from affected farms in Deyang and Qingbaijiang, Sichuan, and submitted for necropsy in March 2019. The fish were euthanized by using MS-222. All fish were examined for parasites of the gill and skin. The examinations included gross external, internal lesions and content of the alimentary tract. 2.2. Histopathology
2.6. Data The tissues, including muscle, skin, gill, brain, eye, trunk-kidney, head-kidney, spleen, heart, liver, and intestine, were collected and fixed immediately in 10% neutral buffered formalin. After decalcification, tissues were trimmed into cassettes, dehydrated in graded ethanol solutions, cleared in xylene, and embedded in paraffin wax. Sections (5 μm) were prepared for hematoxylin and eosin (H&E) staining before microscopic analysis. The degree of congestion/hemorrhage, degeneration, hyperplasia, infiltration, necrosis, edema, and hypertrophy in organs was evaluated by reference to the scoring system designed by Bernet and colleagues (Bernet et al., 2010). Every measure was assessed using a score (S) ranging from 0 to 6, depending on the degree and extent of the alteration: (0) unchanged; (2) mild; (4) moderate; and (6) severe (diffuse lesion).
The temperature records were obtained from the National Meteorological Information Center (http://data.cma.cn/) from 11 grass carp epidemic regions (Chizhou, Jingzhou, Changde, Yueyang, Wuxi, Suzhou, Yangzhou, Chengdu, Deyang, Honghu, and Changshu) and 23 neighboring regions (Hefei, Tongling, Anqing, Huangshan, Jingmen, Xiaogan, Yichang, Xianning, Changsha, Xiangtan, Zhuzhou, Yiyang, Nanjing, Changzhou, Zhenjiang, Taizhou, Jiaxing, Huzhou, Mianyang, Suining, Meishan, Ya'an, and Chongqing). 3. Results 3.1. Disease prevalence
The muscles and spleen of diseased grass carp were sampled, immediately fixed in 2.5% glutaraldehyde, and postfixed in 2% veronalacetate-buffered osmium tetroxide. After dehydration in graded alcohol, the samples were embedded in Araldite. The blocks were sectioned on a microtome with a glass knife. The sections (6 μm) were placed on uncoated copper grids, stained with uranyl acetate, and poststained with 0.2% lead citrate. The subcellular structures of the samples were examined with a Hitachi H-600 Transmission Electron Microscope (Hitachi, Tokyo, Japan).
In 2019, grass carp demonstrated a wide range of morbidity in China. The epidemic included 11 cities in Sichuan, Hubei, Hunan, Anhui and Jiangsu provinces, which are located in Minjiang River system, Dongting Lake system, and Taihu Lake system in the Yangtze River Basin, with a horizontal span of 1550 km (Fig. 1). The disease began to appear in early Dec. 2018, and concentrated outbreaks occurred from mid-February to mid-March 2019. The incidence of grass carp infection varies from region to region, with the most effected regions showing 100% mortality and over 50% morbidity (Fig. 2A). Specifically, the disease regions located at the junction of the distribution belts of 12–16 °C and 16–20 °C (Huang et al., 2011), which is supportive of the connection between epidemic and temperature.
2.4. Bacteriology
3.2. Necropsy findings
The spleen, muscle, liver, and kidney of each fish was aseptically streaked on brain–heart infusion (Becton Drive, USA) agar plates and incubated at 18 °C for 18–24 h for routine bacteriology. The isolates from diseased fish were sub-cultured under the same conditions to check the purity of the isolations. Pure cultures of selected isolates were preservation using glycerin and stored at −80 °C. The bacterial species were identified with 16S rRNA sequencing (primers: F: 5’-AGAGTTTGATCCTGGCTCAG-3′ and R: 5’-GGCTACCTT GTTACGACTT-3′). The amplified sequences were compared with the GenBank database (https://www.ncbi.nlm.nih.gov/) with a BLAST analysis. Homology analysis was performed with MEGA 7.0 and a phylogenetic analysis with the neighbor-joining method.
At the time of disease occurrence, the grass carp gathered and emerged to the surface with weak swimming, and died within only a few days. The diseased fish presented with hemorrhaging around the head and gill cover (Fig. 2B1) and muscle ulcers where the skin remained relatively intact (Fig. 2B2). Severe muscle hemorrhage could be observed after the skin was dissected Fig. 2B2’). The swim bladder was overinflated and congested, the spleen was swollen (Fig. 2B3), and occasionally fatty liver was observed (Fig. 2B3’). Similar gross lesions also occurred in silver carp and crucian carp in some polyculture farms (Fig. 2C-D).
2.5. Animal infection assays
Under the microscope, muscle demonstrated marked necrotizing hemorrhagic myositis (Fig. 3A-B). In the spleen, the hematopoietic cells were severely reduced and reticular fibroblasts were severely necrotic (Fig. 3C-D). The trunk-kidney showed severe edema, a severe reduction in renal interstitial hemopoietic tissue, and severe degeneration and necrosis of the renal tubule (Fig. 3E-F). Additionally, severe dilation of the capillary and moderate necrosis of hematopoietic cells was seen in the head-kidney (Fig. 3G-H). Also, there were moderate changes in the intestine and gill, and mild change in liver, eyes, brain and heart, which showed different degrees of degeneration, inflammation, and necrosis (Fig. 3I-L). The results of the pathological score demonstrated that the
2.3. Ultrastructural examination
3.3. Histopathological diagnoses
Healthy grass carp (100; 22.6 ± 2.5 cm, 104 ± 7.6 g) were purchased from a fish farm in Mianyang, Sichuan. The fishes were acclimatized in the laboratory for 2 weeks before experimentation. The carp were exposed to uninterrupted oxygen supply to ensure more than 5 mg/L dissolved oxygen, a pH of 6.5–8 and a temperature of about 15 °C. The water in the tank was pretreated with an aeration process and 30% of the culture water was renewed every day. Fish were fed twice daily with a commercial diet. All animal infections were conducted following protocols approved by the Institutional Animal Care 2
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Fig. 1. Records of grass carp ulcerative disease in the Yangtze River basin of China from Feb. to Apr. 2019.
Fig. 2. Gross lesions in ulcerative diseased carps. A: Dead fish from the diseased farm. B: ulcerative lesions of grass carp: hemorrhage of the head and gill cover (B1), ulcer of the tail muscles, detachment of scales (B2), severe muscle hemorrhage (B2’) after dissection of the skin; swollen spleen, swim bladder congestion (B3), and occasionally fatty liver symptoms (B3’). C-D: Similar ulcerative lesions of silver carp and crucian carp in polyculture pond. ☆: Ulcer site similar to grass carp. 3
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Fig. 3. Histopathologic lesions in ulcerative diseased grass carp. A-B: severe muscle bleeding (arrow), severe necrosis of muscle fibers (★), and moderate inflammatory cell infiltration (arrowhead); C-D: sharp decrease in hematopoietic cells in the spleen, severe necrosis of reticular fibers (★) and hematopoietic cells (arrow), and moderate inflammatory cell infiltration (arrowhead); E-F: moderate renal edema (★), renal interstitial cell sharp decrease, necrosis of renal tubular and red blood cell (arrow), vacuolar degeneration of renal tubular cell (*), and mild inflammatory cell infiltration (arrowhead); G-H: head kidney edema (★), moderate necrosis of hematopoietic cells (arrow); I: severe vacuoles denaturation of intestinal mucosa (*), a large reduction of mucosal cells (arrow); J: hepatic capillary edema (★), mild to moderate hepatocyte necrosis (arrow); K: submucosal cell proliferation (arrow); L: epicardial mild inflammatory cell infiltration (arrow). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
spleen, muscle, middle kidney, and head kidney were the main pathological target organs in the diseased fish, and infiltration and necrosis were the main pathological symptoms (Fig. 4). The histopathological study indicated that there was a strong correlation between the disease and microorganism. 3.4. Ultrastructural pathology Ultrastructural pathology was performed for further analysis. In muscle, the myofibrils were breaking and the cytoplasm of the myocytes was filled with abundant vacuoles (Fig. 5A-C). In the hematopoietic cells, there was marked swelling of the mitochondria and the endoplasmic reticulum (Fig. 5D). Additionally, a small number of autophagosomes were observed in muscle cells and hematopoietic cells (Fig. 5A-D). Moreover, mild swelling occurred in both mitochondria and endoplasmic reticulum in the endothelial cells of the spleen (Fig. 5E). The results showed the disease caused serious cell damage, which would lead to organ disorder. 3.5. Isolation and identification of pathogens Nine strains were originally isolated from the spleen and muscles of diseased grass carp. The 16S rRNA gene of the isolates was amplified, identified with a BLAST search, and deposited in GenBank (Accession Numbers: MN173487, MN173487, MN173489, MN173492, MN173493, MN173502, MN173512, MN173513, and MN173514). Based on the phylogenetic analysis, three strains shared homology with A. salmonicida, three strains shared homology with A. sobria, two strains shared
Fig. 4. Histopathological scores of different organs of diseased grass carp. The size of the circle represents the number of fish with corresponding symptoms (n = 8), and the different colors represent the degree of symptoms.
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Fig. 5. Ultrastructural lesions in ulcerative diseased grass carp. A-C: Ultrastructural changes under the TEM of muscle fibers; D: Ultrastructural changes under TEM in hematopoietic cells; E: Ultrastructural changes under TEM of blood cells. N: nucleus; Mt.: mitochondria; M: myofibrils; ER: endoplasmic reticulum; L: lipid droplets; R: glycogen; arrow: vacuoles; arrowhead: mitochondrial swelling; *: endoplasmic reticulum swelling; ★: autophagosome.
3.6. Pathogenicity of isolates
homology with A. veronii, and one strain shared homology with A. hydrophila (Fig. 6). The results showed all isolates belonged to the genus Aeromonas, which has been reported to infect carps (Byadgi et al., 2018; Chen et al., 2018; Wang et al., 2017; Yamin et al., 2017).
To confirm the pathogenicity, four Aeromonas species were used to experimentally infect the grass carp. There were no aberrant results or mortality observed in the control group of fish during the experiment
Fig. 6. Phylogenetic tree was generated with MEGA 7.0 based on an alignment of the 16S rRNA gene sequences of the isolated strains (FYp0313 1-9) and related species. The 16 s gene sequence of related species originate from NCBI (https://www.ncbi.nlm.nih.gov/). 5
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Fig. 7. Artificial infection of 4 Aeromonas strains. A. Survival rate (%) of grass carp infected with genus Aeromonas. B. Number of infected fish with relative gross lesion.
(Fig. 7A). In contrast, all fish died within 60 h after A. sobria injection, and all A. salmonicida infected grass carp also died at 96 h (Fig. 7A). During the 96 h experiment, A. veronii caused 66.7% grass carp death, while A. hydrophila caused 53.3% (Fig. 7A). The gross lesions of infected fish were recorded; some showed the hemorrhage of tail muscle that was similar to the natural disease (Fig. 7B). Moreover, the bacteria were re-isolated and identified from infected fish. The results demonstrate the pathogenicity of four isolates, confirming the pathogenicity of genus Aeromonas on the ulcerative disease.
outbreak, these regions experienced a low-temperature period of 1 month (1.5–10 °C) (Fig. 8). The continuous low temperature would facilitate the proliferation of pathogens and lead to grass carp ulcerative disease. Regrettably, since it is difficult to determine the incidence of disease, we cannot statistically correlate this epidemic with temperature. Either there is no significant difference in the temperature of the city near the affected regions, or whether an epidemic has occurred in these regions has not been reported. 4. Discussion
3.7. Possible impacts of temperature abnormality on the epidemic Currently, common bacteria in aquatic organisms such as Flavobacterium, Yersinia, Aeromomas, Lactococcus, and Renibacterium can withstand low-temperature environments (Marcos Lopez et al., 2010). Among them, Aeromonas can be divided into motile species such as A. hydrophila, A. veronii, and A. sobria, and non-motile species A. salmonicida (Praveen et al., 2016). Aeromonas has a worldwide distribution and frequently causes disease in cultured and feral fish (Beaz-Hidalgo and Figueras, 2013). Motile Aeromonas could lead to dermal ulceration, tail or fin rot, ocular ulcerations, erythrodermatitis, hemorrhagic septicemia, red sore disease, red rot disease, and scale protrusion disease (Cipriano and Austin, 2011). Also, non-motile A. salmonicida species can cause enlarged spleen, sloughing of scales, necrotic dermis, degeneration of the musculature, and septicemia (Cipriano and Austin, 2011). The present study found that the target organs of diseased grass carp were muscle, spleen and kidney, all of which showed significant necrosis, hemorrhage, and varying degrees of inflammation. Moreover, nine strains were isolated from diseased grass carp and identified them as genus Aeromonas, and their pathogenicity was determined by experimental infection. The results conclude that the grass carp ulcerative disease is an epidemic caused by Aeromonas infection. Opportunistic bacteria with a broad host spectrum have a high possibility of involvement in co-infections (Kotob et al., 2016). Genus Aeromonas is the most famous pathogenic bacteria in fish, which often causes outbreaks of disease in aquaculture (Cipriano and Austin, 2011). The outbreak of the ulcerative disease was wider and more serious in 2019, which is suggestive for a role of abnormal temperature in these infections. There was a report that A. salmonicida had elevated virulence at low temperature (Ishiguro et al., 1981). Low immunity of fish combined with high pathogenicity of Aeromonas may contribute to the epidemic in grass carp. At present, the temperature of the world, including China, is experiencing global warming and abnormal changes in specific local areas (Du et al., 2019). This suggests that abnormal temperature not only affects the growth of animals but also leads to changes in the regularity of the disease. Global climate variability shifts the distribution of infectious diseases with potentially adverse consequences for disease control.
We identified an interesting phenomenon: most affected regions were located at the junction of the distribution belts of 12–16 °C and 16–20 °C (Fig. 1). The temperature in the affected region in 2019 was lower than in previous years. More interestingly, before the epidemic
Fig. 8. Temperature change in the diseased area of grass carp. Arrow: Temperature lag compared to previous years. 6
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Data availability
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All data generated or analyzed are included in the article. All materials are available from the corresponding author, on reasonable request. Authors contributions Xiaoli Huang, Guanqing Xiong, and Yang Feng contributed the work equally. All authors read and approved the final manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This research was supported by grants Sichuan Agricultural University Pathological Test Project (NO. 015H1500). References Beaz-Hidalgo, R., Figueras, M.J., 2013. Aeromonas spp. whole genomes and virulence factors implicated in fish disease. J. Fish Dis. 36, 371–388. Bernet, D., Schmidt, H., Meier, W., Burkhardt-Holm, P., Wahli, T., 2010. Histopathology in fish: proposal for a protocol to assess aquatic pollution. J. Fish Dis. 22, 25–34. Byadgi, O., Chen, Y.-C., Maekawa, S., Wang, P.-C., Chen, S.-C., 2018. Immune-related functional differential gene expression in koi carp (Cyprinus carpio) after challenge with Aeromonas sobria. Int. J. Mol. Sci. 19, 2107. Chen, J.J., Cao, X.N., Liu, L.T., Cao, X.L., 2018. Immunomodulation with probiotics against aeromonas veronii in grass carp (Ctenopharyngodon idellus). Isr. J. Aquacult Bamid. 70, 1–14. Chretien, J.-P., Anyamba, A., Small, J., Britch, S., Sanchez, J., C Halbach, A., Tucker, C., Linthicum, K., 2015. Global climate anomalies and potential infectious disease risks: 2014-2015. PLoS Currents Outbreaks. 1, 1–16. Cipriano, R.C., Austin, B., 2011. Furunculosis and other Aeromonad diseases. In: Fish Diseases and Disorders: Viral, Bacterial and Fungal Infections. Sbs Publishers, pp. 435–494. Du, Q., Zhang, M., Wang, S., Che, C., Ma, R., Ma, Z., 2019. Changes in air temperature over China in response to the recent global warming hiatus. J. Geogr. Sci. 29, 496–516.
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