X National Congress of Italian Association of Veterinary Pathology (AIPVET), University of Teramo, 29-31 May 2013

Pathology survey on a captive-bred colony of the Mexican Goodeid, extinct in the wild, Zoogoneticus tequila (Webb & Miller, 1998)
Arbuatti Alessio1, Della Salda Leonardo2, Romanucci Mariarita2
practitioner, Mozzagrogna (Chieti, Italy) 2Faculty of Veterinary Medicine, University of Teramo, Italy
1Veterinary

Introduction
The Goodeid family includes 26 viviparous fish species exclusively distributed in central Mexico freshwaters: the varied geological history of this region (Mesa Central) led to the emergence of fish species characterized by extremely limited original diffusion areas and, thus, more susceptible to the consequences of human actions. Zoogoneticus tequila (Fig. 1,3) is considered extinct in the wild (Dominguez-Dominguez et al., 2005a,b; Magurran, 2009), and is maintained under controlled conditions in Europe, USA and Mexico in the international conservation project Goodeid Working Group. The present study analyzes the pathologies encountered in the unique captive-bred Italian colony in order to provide information regarding the proper management of this species aimed at its conservation and possible reintroduction in the natural habitat (Fig.2).

Materials and Methods

The unique Italian colony was founded in 2007. Fish were bred in different structures: two tropical freshwater planted aquariums (180 L and 250 L) (Fig. 4), an outdoor pond (360 L) for warm season (Fig. 5) and a multi-tank aquarium system (180 L) for fry growth (Fig. 6) (Arbuatti et al., 2011). Routine management provided for a weekly water change (20%), using properly bioconditioned water, enriched with filter bacteria, and a daily administration of high quality dry (SHG Microgranules, SHG Spirulina, SHG Artemia salina flakes, Sera FD Tubifex, Sera Daphnia, Sera Artemia shrimps), frozen (Artemia salina, Tubifex sp. and mosquito larvae) and live (Enchytraeus buchholzi) foods. Pathologies observed during breeding management were recorded and some fish showing macroscopically evident lesions were submitted for necropsy, immediately after spontaneous death, and subsequently fixed in 10% neutral buffered formalin for histopathological examination.

Fig. 1 Adult male Z. tequila showing the typical orange-yellow band in the caudal fin and the andropodium (arrow), a copulatory organ allowing the transfer of spermatozeugmata (sperm bundles) in the female genital apparatus. Fig. 2 Rio Teuchitlan sources (Central Mexico), unique original biotope of Z. tequila. Irrigation and zootechnical water use, as well as the building of an aquatic park led to the disappearance of this species. Fig. 3 Adult female Z. tequila. Females give birth to free-swimming fry after an intraovarian gestation.

Spinal deformities are commonly observed in both wild and farmed fish, and recognize a multifactorial etiology (Arbuatti et al., 2013). A combination of genetic defects, traumatic injuries and nutritional imbalances could play a role in the pathogenesis of the curvatures observed in our study, although their incidence was very low. 4 Fig. 4 Tropical freshwater aquarium (250 L), housing the main colony. 5 Fig. 5 External pond for outdoor breeding and reproduction. 6 Fig. 6 Multi-tank aquarium system used for fry and young fish growth. Recurrent abdominal distention due to segmental dilation of distal intestine may be compared to intestinal pseudo-obstruction syndromes, rarely described in humans and various animal species, but not in fish. Although histological lesions may be variable (Huang et al., 1996; Moore et al., 2002; Vandenberge et al., 2009), a severe villous atrophy represents a well-known event in these syndromes, which could be due to bacterial overgrowth with production of toxic metabolites for intestinal epithelium (Schuffler et al., 1978). Histological examination of sub-ocular fluid-filled sac revealed a lymphatic origin (lymphatic cyst), comparable to fluid-filled sacs typical of a Goldfish variety (Carassius auratus) called bubble-eye, which are known to contain lymph (Sawatari et al., 2009). From this pathology survey, revealing an absence of infectious or parasitic diseases, as well as a low incidence of diseases potentially related to nutritional imbalances, it is possible to deduce the suitability of the breeding management of Z. tequila for the conservation of this species and its possible reintroduction in the natural habitat in the next future. 8

Discussion

Results
Out of a total of about 180 fish, the following pathologies were recorded: 7 cases of spinal deformities (scoliosis in 2 males and 5 females) (Fig.7A); 1 case of female fish with congenital deviation of ocular axis (Fig. 8A); 1 case of female fish with recurrent abdominal distention, showing marked dilation and thinning of distal intestinal tract at post-mortem exam (Fig. 9B). Histologically, dilated tract showed complete mucosal and submucosal atrophy, with disappearance of intestinal folds, and moderate atrophy of the outer longitudinal layer of tunica muscularis (Fig. 10A-B). Another case concerned a female fish with a right, large sub-ocular fluid-filled sac (Fig. 11A), associated with progressive cataract formation in the ipsilateral eye. This sac accidentally ruptured, with disappearance of dilation, but without further consequences for the fish, except for a slight cranium asymmetry. Histological examination revealed a large dermal cavity lined by flattened endothelial-like cells, multifocally hardly recognizable for the presence of a moderate lymphocyte infiltration in the cystic wall and surrounding dermis, and in continuity with the overlying hyperplastic epidermis at the rupture site of the cystic wall (Fig. 11B). On the opposite cranium side, the presence of a vascular space lined by endothelial cells, referable to a lymphatic vessel, surrounded by loose dermal tissue, was revealed (Fig. 11C). No infectious or parasitic diseases were observed, except for 1 case of an adult male with left corneal opacity, presumably resulting from traumatic keratitis with secondary bacterial infection, which also darkened in colour, probably as a consequence of partial blindness (Fig. 8B). 9 7

A A B Fig. 7 Lateral (A) and dorsal (B) view of an adult male showing scoliosis. A B 11 e

B Fig. 8 (A) Adult female with congenital deviation of ocular axis (arrow); (B) adult male with left corneal opacity (arrow) and diffuse dark colour.
References Arbuatti A et al. (2011) AACL Bioflux Vol. 4 issue 5:670-683. Arbuatti A et al. (2013) Asian Pac J Trop Biomed 3(3):186-190. Dominguez-Dominguez O et al. (2005a) New Life publication, Homestead, Florida, 515-523. Dominguez-Dominguez O et al. (2005b) New Life publication, Homestead, Florida, 525569. Huang SF et al. (1996) J Pediatr Surg 31:721-725. Magurran AE (2009) Science 325: 1215. Moore SW et al. (2002) Pediatr Surg Int 18:13-20. Sawatari E et al. (2009) Zoolog Sci 26:254-258. Schuffler MD et al. (1978) Dig Dis Sci 23: 821-8. Vandenberge V et al. (2009) Vlaams Diergen Tijds 78: 117-120. Webb SA, Miller RR (1998) Occasional papers of the Museum of Zoology 725:1-23. Ed. The University of Michigan.

Fig. 9 (A) Normal distal intestine of a control fish; (B) dilated distal intestine (arrows) of an adult female with recurrent abdominal distention. 10

A A B C D

Fig. 10 Z. tequila with segmental intestinal dilation: (A-B) dilated intestinal tract showing complete mucosal and submucosal atrophy with fold disappearance; (C-D) adjacent normal tract (E-E, A-C 10X, B-D 40X).

Fig. 11 (A) Adult female with a sub-ocular fluid-filled sac (arrow); (B) cystic cavity in continuity with overlying hyperplastic epidermis (arrow) at the rupture site of the cyst wall; inset: flap of cyst wall showing continuity between epidermis (e) and endothelial lining; (C) lymphatic vessel at the opposite cranium side; inset: endothelial lining of lymphatic vessel (E-E, B 10X, C 20X, insets 40x).