Histological confirmation of epizootic ulcerative syndrome in two cyprinid species from Lake Liambezi, Zambezi Region, Namibia

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African Zoology

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Histological confirmation of epizootic ulcerative

syndrome in two cyprinid species from Lake

Liambezi, Zambezi Region, Namibia

Kyle J. McHugh, Kevin W. Christison, Olaf L.F. Weyl & Nico J. Smit

To cite this article: Kyle J. McHugh, Kevin W. Christison, Olaf L.F. Weyl & Nico J. Smit (2014)

Histological confirmation of epizootic ulcerative syndrome in two cyprinid species from Lake

Liambezi, Zambezi Region, Namibia, African Zoology, 49:2, 311-316

To link to this article: http://dx.doi.org/10.1080/15627020.2014.11407649

Published online: 20 Apr 2015.

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Histological confirmation of epizootic ulcerative

syndrome in two cyprinid species from Lake

Liambezi, Zambezi Region, Namibia

Kyle J. McHugh

_{, Kevin W. Christison}

_{, Olaf L.F. Weyl}

_{& Nico J. Smit}

1

Water Research Group(Ecology), Unit for Environmental Sciences and Management, Potchefstroom Campus,

North-West University, Private Bag X6001, Potchefstroom, 2520 South Africa 2

Department of Agriculture Forestry and Fisheries, Private Bag X2, Roggebaai, 8012 South Africa 3

South African Institute for Aquatic Biodiversity, Private Bag 1015, Grahamstown, 6140 South Africa

Received 3 March 2014. Accepted 7 July 2014

Epizootic ulcerative syndrome (EUS) is a fish disease caused by an infection of the oomycete, Aphanomyces invadans. During a fish biodiversity assessment of Lake Liambezi, Zambezi Region, Namibia, in August 2011, two Barbus haasianus and three Barbus unitaeniatus with circular ulcerative skin lesions were collected. Lake Liambezi receives inflow from the Zambezi, Chobe and Linyanti rivers. The presence of EUS in the two species was confirmed histologically by: a loss of epidermis at the site of the lesion; hyphae visible deep into the muscle layer with an associated granulomatous inflammatory reaction; and muscle fibre degeneration visible with associated penetrating hyphae. This paper provides further histological confirmation of EUS from Lake Liambezi and the first record of the disease in B. haasianus.

Key words: Barbus haasianus, Barbus unitaeniatus, Chobe River, fungal pathogen, histology.

Epizootic ulcerative syndrome (EUS) is a fish

disease, which results in high biodiversity and

economic losses (FAO 2009; Oidtmann 2012). The

disease is caused by the oomycete Aphanomyces

invadans David & Kirk 1997, as listed in the Index of

Fungi (IMI 1997), which causes epidermal lesions

that penetrate into the underlying muscle tissue,

ultimately resulting in skin erosion and exposure

of underlying musculature and ulceration (Lilley

& Roberts 1997). Globally, the first reports of EUS

outbreaks were from South East Asia, when EUS

caused high mortalities of wild and cultured fish

(Lilley & Roberts 1997). In sub-Saharan Africa the

first report of A. invadans and an associated EUS

outbreak was from Botswana in 2007, followed

by Zambia in 2008 (Andrew et al. 2008).

Subse-quently 27 fish species from the region have been

shown to be susceptible to EUS (FAO 2009; Choongo

et al. 2009; Songe et al. 2012; Huchzermeyer & Van

der Waal 2012). Lesions, suspected to be associated

with EUS have previously been reported from

Lake Liambezi by Van der Waal (2008; 2012).

The present study reports the findings of an EUS

assessment of 91 fish, representing 16 species in

seven families, that were sampled from Lake

Liambezi, Zambezi Region, Namibia, as part of a

biodiversity assessment in August 2011 (Fig. 1)

(17°52.942’S, 24°23.706’E). As noted by Van der

Waal (2012), this is highly relevant because the

300 km

_{Lake Liambezi is used extensively by}

artisanal fishermen and EUS-associated fish

mortalities could impact on both food security and

on local economies. Van der Waal (2012) further

emphasized the importance for continued targeted

surveillance for the prevalence of EUS in the lake.

The lake also receives inflow from the Zambezi,

Chobe and Linyanti Rivers which may facilitate

spread of the pathogen into the currently

unaf-fected Kwando River via the Linyanti swamps.

The objective of this paper was therefore to provide

insight into the current extent of EUS in Lake

Liambezi. This was done by means of a histological

assessment on fish that macroscopically exhibited

signs of EUS.

Fish were sampled by means of gill nets,

electro-fishing and angling. Live fish were transferred to a

field laboratory, identified using keys provided

by Skelton (2001), measured, and a presumptive

EUS identification, based on the presence of

typical clinical signs, including skin damage and

haemorrhagic ulcerative skin lesions, was

con-ducted in accordance with FAO (2009).

Of the 91 fish inspected, only five individuals

showed macroscopic alterations to the skin

(Ta-ble 1). The clinically affected fish species were

Barbus haasianus David, 1936 and B. unitaeniatus

Günther, 1866. Individuals of both these species

exhibited varying degrees of severity of skin

ulcer-ations. The lesions were mostly focal and varied

from a red inflamed area to an open ulcerated

wound. In the more severely affected samples,

scales were absent from the lesion. Affected tissues

from these five fish were collected, fixed in 10%

neutral buffered formalin and prepared for

histol-ogy using the methods described by McHugh et al.

(2011, 2013). Sections were stained using

haema-toxylin and eosin and examined using an Olympus

BX 51 compound light microscope to determine

the presence of oomycete hyphae and associated

312 African Zoology Vol. 49, No. 2, October 2014

Fig. 1. Map of Lake Liambezi and its location in the Zambezi Region of northern Namibia.

Table 1. Fish species, number and mean lengths of fish species captured in Lake Liambezi and presence of lesions

confirmed by histology as epizootic ulcerative syndrome (EUS) infections. Abbreviations:n, number; ML, mean length; S.D., standard deviation.

Fish species n ML ± S.D. Clinically Histological (range) in mm affected confirmation

Cichlidae

Coptodon rendalli(Boulenger, 1896) 4 148.3 ± 63.6 (84–211) 0 0

Oreochromis andersonii(Castelnau, 1861) 5 182.8 ± 72.3 (100–279) 0 0

Oreochromis macrochir(Boulenger, 1912) 4 217.8 ± 95.4 (80–291) 0 0

Sargochromis codringtonii(Boulenger, 1908) 7 139.4 ± 10.8 (125–154) 0 0

Serranochromis macrocephalus(Boulenger, 1899) 8 106.4 ± 6.1 (101–115) 0 0

Serranochromis robustus(Günther, 1864) 3 239.7 ± 31.9 (211–274) 0 0

Tilapia sparmaniiSmith, 1840 3 98.3 ± 6.0 (90–104) 0 0

Clariidae

Clarias gariepinus(Burchell, 1822) 1 325 0 0

Clarias ngamensisBoulenger, 1915 5 437.6 ± 11.6 (425–455) 0 0

Clarias theodoraeWeber, 1897 1 239 0 0

pathology in tissue sections. The demonstration of

mycotic granulomas in histological sections of

affected tissues and organs is recognized as one of

three diagnostic tests recommended for the

confir-matory diagnosis of EUS (OIE 2012; FAO 2009).

Epizootic ulcerative syndrome infection could

only be histologically confirmed in one B. haasianus

and two B. unitaeniatus (Table 2). One sample of

the B. haasianus had damage to the skin that

appeared to have been caused by the gill net from

which it was collected. Pathology in the fish tissue

was consistent between both Barbus species and

comprised deeply penetrating, oomycete

infec-tion through the epidermis into the sub-dermal

muscle layers eliciting a pronounced host response

(Fig. 2). Numerous refractile hyphal cell walls,

were visible in cross-section within the muscle

tissue (Fig. 2b,c,d). Muscular degeneration or

necrosis, in association with oomycete

infiltra-tion, was characterized by loss of muscular

striation and in some instances the muscle fibres

were almost entirely replaced by fibrous tissue

and oomycete hyphae surrounded by a

granulo-matous inflammatory tissue reaction (Fig. 2b,c).

Fish species n ML ± S.D. Clinically Histological (range) in mm affected confirmation

Cyprinidae

Barbus haasianusDavid, 1936 2 27.5 ± 3.5 (25–30) 2 1

Barbus unitaeniatusGünther, 1866 3 128.3 ± 7.6 (120–135) 3 2

Hepsetidae

Hepsetus odoe(Bloch, 1794) 6 245.0 ± 52.4 (194–345) 0 0

Mochokidae

Synodontis nigromaculatusBoulenger, 1905 15 156.5 ± 17.0 (125–185) 0 0

Mormyridae

Marcusenius altisambesiKramer, Skelton, 10 182.1 ± 17.6 (154–208) 0 0

van der Bank & Wink, 2007

Schilbeidae

Schilbe intermediusRüppell, 1832 14 214.7 ± 24.4 (195–251) 0 0

Total 91 5 3

Table 2. Clinical and histological observations onBarbus unitaeniatusandBarbus haasianuscollected from Lake Liambezi, Zambezi District, Namibia.

Host species Gross pathology Histology

Barbus unitaeniatus Skin lesion with small red spots Loss of epidermis at site of lesion. Hyphae visible deep into muscle layer with associated granulo-matous inflammatory reaction. Muscle fibre degeneration visible with associated penetrating hyphae. Areas of erythrocytic extravasation visible.

Barbus unitaeniatus Skin lesions with inflamed muscle tissue

No EUS-associated pathology observed.

Barbus unitaeniatus Skin ulceration with inflamed muscle tissue

Lesions were extensive. Total loss of epidermis at site of lesion. Oomycete hyphae visible throughout lesion penetrating deep into muscle layer. Muscle fibres mostly replaced by inflammatory tissue at centre of lesion. Muscle degeneration visible immediately adja-cent to the lesion. Multiple areas of erythrocytic extravasation visible throughout the lesion. Numerous multinucleated giant cells visible throughout lesion often associated with encapsulated hyphae.

The inflammatory cellular infiltration of the

dermal and underlying muscle layers consisted

mostly of cells with relatively large nuclei and very

little or no cytoplasm (typical of lymphocytes) and

cells with large nuclei and eosinophilic cytoplasm

(typical of macrophages). Numerous

multi-nucleated giant cells infiltrating the muscle

layer were also observed (Fig. 2d). According to

Ferguson (2006), multinucleated giant cells are

frequently associated with fungal and metazoan

parasitic infections. Multiple oomycete

granu-lomas were formed from the aggregation of

in-flammatory cells, including numerous

multi-nucleated giant cells often associated with

encap-314 African Zoology Vol. 49, No. 2, October 2014

Fig. 2. Micrographs of muscle tissue sections from the twoBarbusspecies stained with H&E. a, Deeply penetrating, oomycete infection of the epidermis and sub-dermal muscle layers eliciting a pronounced host inflammatory response. b, c, d, Numerous refractile hyphal cell walls (arrows), were visible in cross-section within the muscle tissue: b, c, necrosis of muscle tissue in association with oomycete infiltration surrounded by granulomatous inflam-matory tissue reaction; d, numerous multinucleated giant cells infiltrating the muscle layer (mgc as indicated by arrows). Scale bars = 100 µm.

Table 2 (continued)

Host species Gross pathology Histology

Barbus haasianus Skin damage and tearing

ap-peared to be gill net damage

Focal lesion, loss of epidermis at site of lesion. Oomycete hyphae visible throughout lesion and penetrating deep into the muscle. Refractile hyphae in cross section visible throughout the lesion. Exten-sive, granulomatous inflammatory reaction replacing muscle fibres in centre of the lesion. Muscle degener-ation visible in surrounding tissue.

Barbus haasianus Small skin lesion with raised scales

sulated oomycete hyphae. Multiple areas of

erythrocytes forced from the blood vessels into the

surrounding tissue were also visible within the

lesions.

These findings are consistent with the pathology

described for EUS infections (Chinabut et al. 1995;

Vishwanath et al. 1998; Werner et al. 2002; Kiryu

et al. 2003; Johnson et al. 2004; Oidtmann et al. 2008)

and satisfy the OIE’s requirements for a confirmed

case of EUS (OIE 2012). The current study

there-fore provides histological evidence for EUS in

Lake Liambezi, the infection of B. unitaeniatus and

the first histological-evidence supported record

of EUS in B. haasianus. Further investigations are

needed to assess the impact of EUS on other fish

species in Lake Liambezi and to determine its

spread and population level impact.

Samples were obtained during a biodiversity survey of Lake Liambezi that was financially and logistically supported by a National Research Foundation of South Africa (NRF) grant (UID: 7415) awarded under the Inter-national Science and Technology Agreements between South Africa and Namibia, the Namibia Ministry of Fish-eries and Marine Resources/Namibia Nature Foundation Integrated Co-Management of Zambezi/Chobe River Fisheries Resources Project, the Nedbank Namibia Go Green Fund and the Norwegian Agency for Develop-ment Cooperation (NORAD). Richard Peel is thanked for facilitating this research and providing field support. We acknowledge the assistance of the facilitators at a work-shop on scientific writing, arranged by Young Water Professionals (YWP) and hosted by University of Johan-nesburg in January 2014.

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Responsible Subject Editor: A. Oldewage 316 African Zoology Vol. 49, No. 2, October 2014