key: cord-0737509-z6ytcj47
authors: Gary, J.M.; Langohr, I.M.; Lim, A.; Bolin, S.; Bolin, C.; Moore, I.; Kiupel, M.
title: Enteric Colonization by Staphylococcus delphini in Four Ferret Kits with Diarrhoea
date: 2014-09-22
journal: J Comp Pathol
DOI: 10.1016/j.jcpa.2014.08.004
sha: b62576b1566e272b3f29f8a16ae06c1ec363707f
doc_id: 737509
cord_uid: z6ytcj47

Four, 1-to 4-week-old ferret kits were submitted to the Diagnostic Center for Population and Animal Health at Michigan State University for post-mortem examination. Grossly, multiple bowel loops in all ferret kits were distended by mucoid faecal material. Microscopically, there was no evidence of inflammation or notable alteration to the normal mucosal morphology. Gram-positive coccoid bacteria colonized variable segments of the small intestine. These bacteria were identified as Staphylococcus delphini by phenotypic and molecular analyses. Enzyme-linked immunosorbent assay for detection of Staphylococcus enterotoxins was positive and polymerase chain reaction detected the gene for Staphylococcus enterotoxin E in the isolates. The hypersecretory diarrhoea in these ferret kits may have been associated with colonization of the small intestine by S. delphini, cultures of which were shown in vitro to be potentially capable of producing enterotoxin E. The condition described in these ferrets is similar to ‘sticky’ kit syndrome in mink.

'Sticky' kit syndrome is an enteric disease that affects mink kits between 10 and 14 days of age. Clinical signs include hypersecretion of cervical apocrine glands and mucoid diarrhoea, which causes severe dehydration that can rapidly proceed to death. Large outbreaks of sticky kit syndrome at mink farms are not uncommon, causing death in up to 30e40% of affected kits (Englund et al., 2002) . During one such outbreak, post-mortem examinations conducted at the Michigan State University Diagnostic Center for Population and Animal Health (DCPAH), in Lansing, MI, revealed that the intestinal mucosa of affected kits was colonized by gram-positive coccoid bacteria. Bacterial culture and typing indicated that they were a strain of Staphylococcus delphini that was capable of producing enterotoxins A and E (Sledge et al., 2010) .

Staphylococcus delphini is coagulase-positive and is part of the Staphylococcus intermedius group. Mustelids are reported to be natural hosts of S. delphini group A, which has been isolated from their skin, urinary tract, nasal swabs and faeces (Guardabassi et al., 2012) , and from urinary tract lesions (Guardabassi et al., 2012) and enteric disease in mink (Sledge et al., 2010) . The purpose of this report is to describe enteric colonization by S. delphini in four ferret kits with diarrhoea, which is similar to 'sticky' kit syndrome previously described in mink.

Four ferret kits, comprising a <1-week-old female (kit 1), a 2-week-old female (kit 2), a 3-week-old male (kit 3) and a 4-week-old male (kit 4), all from the same facility, had a 1-day history of pasty, yellow (kits 1, 2 and 4) to green (kit 3) diarrhoea. They were submitted dead to the Michigan State University DCPAH. On gross examination, the coats of kits 1, 2 and 4 appeared normal except for mild faecal staining in the perineal region, while kit 3 had a rough and greasy hair coat. The stomachs of all kits contained milk. The small and large intestines of kits 1, 2 and 4 contained moderate amounts of yelloweorange, mucoid to liquid faecal material (Fig. 1) . The jejunal and ileal contents of the third kit were yellowewhite and mucoid, while the colonic contents were bright green and of mucoid to liquid consistency. Brain, heart, lung, liver, spleen, thymus, kidney, pancreas and small and large intestine were collected from the four kits and were fixed in 10% neutral buffered formalin for at least 24 h. Representative sections were processed routinely and embedded in paraffin wax. Sections (5 mm) were stained with haematoxylin and eosin (HE). Selected sections of the small intestine were stained by the Gram's method (Hucker-Twort stain). Immunohistochemistry (IHC) was performed for coronavirus as previously described (Garner et al., 2008) .

Microscopical examination of small intestine revealed numerous 1e2 mm diameter, gram-positive coccoid bacteria along the apical and lateral surfaces of the intestinal villi (Figs. 2 and 3). In some segments, the cocci were also present in large numbers in the lumen of intestinal crypts. Rarely, sections of small intestine from kit 3 were characterized by mild degeneration of the mucosal epithelium associated with minimal neutrophilic inflammation. The small intestine was negative by IHC for ferret enteric coronavirus in all the kits. For bacterial culture, representative sections of fresh frozen small intestine were pooled based on the age of the affected kits. For each pooled sample, intestinal contents were cultured as described by Sledge et al. (2010) . Similarly, pooled samples of faeces were collected for parasitological evaluation and sections of small intestine were tested for the presence of ferret enteric coronavirus and group C rotavirus using polymerase chain reaction (PCR) assays (Wise et al., 2006 (Wise et al., , 2009 . Small intestinal samples were negative by PCR for ferret enteric coronavirus and group C rotavirus and by qualitative faecal examination for intestinal parasites in all the kits.

Pooled small intestinal culture from the two younger kits grew gram-positive cocci that were identified as Staphylococcus spp. by colony morphology, production of catalase, haemolysis on enriched sheep blood agar, the presence of coagulase enzyme and positive Gram stain. The bacteria did not grow on P agar and were VogeseProskauer test negative, which characterized them as member of the S. intermedius group. Few Escherichia coli and few Enterococcus spp. were also cultured from the small intestine. There was no growth of Salmonella in any culture.

Samples of the bacterial colonies phenotypically consistent with Staphylococcus spp. were submitted for extraction of DNA and subsequent PCR assays for 

characterization of the bacteria. These assays targeted the 16S ribosomal DNA (16S rDNA), the superoxide dismutase (SodA) gene and the heat shock protein 60 (Hsp60) gene (Sledge et al., 2010) . Phylogenetic analysis using the nucleic acid sequence of the three selected genes identified the bacteria as S. delphini (Fig. 4) . The amplified nucleic acid sequences of the 16S rDNA and Hsp60 genes were identical among the bacterial isolates obtained in the current study and the S. delphini isolated previously from mink with sticky kit syndrome (unpublished sequence) (Sledge et al., 2010) . Further, the 1,052 base pair sequence derived from 16S rDNA in the current study was 100% identical to multiple corresponding sequences from S. delphini in the GenBank database and the 485 base pair sequence of the Hsp60 gene had 95e99% similarity with corresponding sequences for S. delphini in GenBank. The 348 base pair pair segment of the SodA gene of the S. delphini from sticky kit disease in mink was identical to that of the bacterial isolates from the current study except for a single A to G transition that did not result in a change of the predicted amino acid sequence.

To determine if the S. delphini isolated in the current study contained toxin genes, overnight cultures of bacteria in tryptic soy broth were centrifuged individually at 1,730 g for 10 min at 10 C. The clarified culture supernatants were injected through low protein binding 0.22 mm syringe filters. The filtered culture supernatants were then screened for enterotoxin production using a commercial enzyme-linked immuo-sorbent assay (ELISA) kit that detects the presence of Staphylococcus enterotoxins (SETs) A, B, C, D and E following procedures recommended by the manufacturer (RIDASCREEN Ò SET Total, R-Biopharm AG, Darmstadt, Germany). The clarified bacterial culture supernatants were positive for enterotoxin(s) by ELISA. The presence of staphylococcal enterotoxin genes A to E was assessed by PCR assays, using PCR primers designed to amplify fragments of the Staphylococcus aureus enterotoxin genes (sea, seb, sec, sed and see) (Fischer et al., 2009) . Nucleic acid sequence analysis of the amplicons confirmed the detection of an enterotoxin gene fragment that was 92% similar to a corresponding fragment of the enterotoxin E gene of S. aureus.

In this study, small intestinal colonization with S. delphini was seen in four ferret kits with diarrhoea. Minimal degeneration of intestinal epithelium with rare associated neutrophils, likely representing a mild reaction to bacterial toxins, was present in one ferret kit. In previous reports, bacterial colonization of the intestine by S. delphini with associated diarrhoea and excessive cervical apocrine gland secretion was described in mink kits ranging from 10 to 14 days of age (Sledge et al., 2010) . The four ferret kits described in this report showed similar clinical signs to those 'sticky' mink kits: the ferret kits had pasty, yellow diarrhoea and one kit had also a sticky coat. However, ferret kits were affected at a wider age range (1e4 weeks) than mink kits. Many of the features of the diarrhoeal disease described in these ferret kits, including the intestinal colonization by grampositive cocci, the isolation of S. delphini and the partial nucleic acid sequences of three genes of the isolates, are consistent with the findings previously described in the mink kits with sticky kit syndrome (Sledge et al., 2010) . The similar clinical and histological features and the comparable bacterial isolates suggest that ferret kits can experience a 'sticky kit syndrome' similar to the syndrome described in mink. gene (448 base pairs) and SodA gene (313 base pairs). The concatenated sequence for the HSP60 and SodA genes was trimmed to match the length of sequences for both genes that were available in GenBank. The tree was constructed using the neighbour-joining method (Saitou and Nei, 1987) with 1,000 bootstrap replicates (Felsenstein, 1987) . The percentage of replicate trees in which the associated taxa clustered together is shown above the branches. The phylogenetic analyses were conducted in MEGA4 (Tamura et al., 2007) .

Astrovirus epidemiologically linked to pre-weaning diarrhoea in mink

Clinicopathologic features of a systemic coronavirus-associated disease resembling feline infectious peritonitis in the domestic ferret

Mustelidae are natural hosts of Staphylococcus delphini group A

Outbreak of neonatal diarrhoea in farmed mink kits (Mustella vison) associated with enterotoxigenic Staphylococcus delphini

MEGA4: molecular evolutionary genetic analysis (MEGA) software version 4.0

Molecular characterization of a novel coronavirus associated with epizootic catarrhal enteritis (ECE) in ferrets

Detection of group C rotavirus in juvenile ferrets (Mustela putorius furo) with diarrhoea by reverse transcription polymerase chain reaction: sequencing and analysis of the complete coding region of the VP6 gene

We would like to acknowledge L. Moon and the DCPAH Bacteriology Laboratory and the DCPAH Histology Laboratory for their help in this study.

The authors declare no potential conflicts of interest with respect to the research, authorship and/or publication of this article. The authors received no financial support for the research, authorship and/or publication of this article.