Date Published: August 15, 2018
Publisher: BioMed Central
Author(s): Ronja Mathiesen, Julie Melsted Birch, Mariann Chriél, Henrik Elvang Jensen, Jens Frederik Agger, Peter Mikael Helweg Heegaard, Tina Struve.
Pre-weaning diarrhea (PWD) is a syndrome affecting farm-raised neonatal mink kits. Apart from diarrhea it causes greasy skin exudation, dehydration, and distressed behavior and can ultimately lead to death. No specific causative agents have been identified and the syndrome is regarded as multifactorial. The aim of the present study was to investigate a possible inflammatory state in mink kits with PWD, as indicated by raised serum concentrations of the acute phase protein serum amyloid A (SAA) and by changes in intestinal pathomorphology and intestinal contents of bacteria. Samples collected from 20 diarrheic mink kits with PWD and 20 age-matched non-diarrheic control mink kits from two commercial Danish farms during the pre-weaning period (April–May) in 2016 were analyzed.
Concentrations of SAA in serum samples from mink kits with PWD were significantly higher (up to 1000-fold) compared to non-diarrheic control mink kits. Significant features of enterocytic vacuolization, atrophy and fusion of villi in jejunum and mucosal atrophy of the colon of kits with PWD were found. Moreover, attachment of coccoid bacteria to enterocytes was more often found in kits suffering from PWD, while intra-cytoplasmic eosinophil bodies were more frequently observed in control kits. Cellular infiltrations with mononuclear and neutrophil leukocytes were not associated with disease status. Bacteria from the Staphylococcus intermedius group, such as Staphylococcus delphini, were more frequently cultivated from control mink kits, whereas Enterococcus spp. dominated in mink kits with PWD. Escherichia coli was cultivated from both control and mink kits with PWD, but with a higher frequency from mink kits with PWD.
A significant increase in circulating concentrations of SAA was found in PWD affected mink kits from 6 to 23 days old compared to controls. The histopathological changes in PWD mink kits suggest that the type of diarrhea is secretory. Attachment of coccoid bacteria, therefore, might be responsible for an enterotoxic effect causing a loss of balance in movements of ions and water leading to the vacuolization and swelling of the enterocytes. The slight to moderate infiltrations of neutrophils irrespectively of diarrheic status and the attachment of coccoid bacteria to enterocytes are comparable to observations found in piglets suffering from New Neonatal Porcine Diarrhea Syndrome. Mechanisms behind the correlation between increased SAA levels and the observed pathological intestinal features remain obscure.
The pre-weaning diarrhea syndrome (PWD) in mink (Neovison vison) kits is a major cause of concern in the mink industry due to both economic losses and decreased animal welfare. It may affect more than 30% of the litters [1, 2] and has been observed in farm-raised neonatal mink for several decades worldwide . Mink kits affected by PWD display diarrhea with concomitant excessive secretions from the cervical apocrine glands, and exudate on the skin surface, the tail, and the claws. Moreover, dehydration may ultimately lead to the death of the affected kits [4, 5]. The onset of clinical signs generally occurs in the whole litter during the pre-weaning period, at 5–20 days of age, with a morbidity rate varying from 0 to more than 30% of the litters, and a mortality of typically one or two kits per litter [6, own findings, 2017]. The PWD syndrome is considered multifactorial, and there is a lack of consistency in isolated bacteria and viruses in kits with PWD. Studies have aimed to define the causality of the syndrome and mink astrovirus (MiAstV) isolated from mink kits with PWD indicate involvement in the syndrome [7–11]. However, all of the proposed putative pathogens including also Campylobacter jejuni, rota-, calici-, and mink coronavirus, Escherichia coli, and Staphylococcus delphini have also been isolated from clinically healthy kits, so their role in PWD remains elusive [10–13]. Apart from having a multifactorial infectious origin, other factors including management factors have been associated with an increased risk of developing PWD. For example, litters from 1-year old females and in females with low energy supply in the late gestation period are at increased risk of being affected by PWD [2, 14]. Moreover, the presence of dogs on the farm area as well as the size of the farm (total number of females) have also been associated with high morbidity of PWD . Regarding the intestinal pathomorphology accompanying PWD, only a few studies have been published [15, 16]. Moreover, intestinal lesions in mink kits suffering from PWD have not been classified, according to the standard pathomorphological paradigm, as either non-inflammatory/secretory, inflammatory or invasive . A possible biomarker to assess infection or inflammation in mink kits suffering from PWD is serum amyloid A (SAA). SAA is an acute phase protein found in low concentrations in healthy animals and is released following inflammation, infection, or tissue injury in both mink [18–20] and many other species, including humans [21, 22]. It is synthesized predominantly by the liver in response to the cytokine interleukin 1, however, other organs such as the intestine, have also been shown to produce it . The aim of the present study was to examine if the levels of SAA could be a biomarker for PWD in mink kits and to characterize and compare the intestinal pathomorphology, and the bacterial intestinal contents between healthy controls and mink kits suffering from PWD.
We report for the first time that PWD in mink kits between 6 and 23 days old is associated with a significant increase in circulating concentrations of SAA. Two of the control kits showed elevated concentrations of SAA in serum, however only one of them (1315 µg/mL; Fig. 1) showed signs of cholangitis, which could explain the elevated concentration of SAA (data not shown). SAA have been shown by Bruun et al.  to be elevated in mink after subcutaneous injection with lipopolysaccharide from E. coli. The gastrointestinal tract of mink is very short and the passage of feed through the entire gastrointestinal tract takes approximately 2–3 h . Previous studies have shown the difficulty in culturing bacteria from the small intestine in mink  and therefore we decided to use swabs from the colon, where the density of bacteria is relatively high. Furthermore, the decision to only cultivate the bacteria aerobically was based on the fact that only aerobic bacteria have so far been isolated from PWD-affected mink, whereas anaerobic bacteria have not [11, 28, 29]. We isolated E. coli most frequently from kits suffering from PWD, which corresponds well with previous findings that E. coli is commonly isolated from PWD mink kits [11, 16, 30, 31]. A quantitative study of healthy mink kits showed that the intestinal counts of E. coli were highest when kits were around 4 weeks of age , but in the present study E. coli was isolated from younger kits suffering from PWD. However, E. coli has also been identified as part of the normal mink kit intestinal microflora [11, 16, 30, 32]. Moreover, analysis of virulence factors of E. coli has revealed that the population of E. coli in mink consists of several serogroups with no apparent association to outbreaks of PWD [11, 32]. In general, enterococci are not regarded as pathogenic in mink, but Enterococcus hirae has been associated with diarrhea in 4–7 week old mink kits submitted for diagnostic testing (Chriél, pers. comm., 2017), as well as in other neonatal animals such as suckling rats, kittens and piglets [33–35]. Gut sections from PWD mink kits showed clear disease associated changes, including vacuolization of enterocytes and pronounced attachment of coccoid bacteria, both of which have been observed in other studies [7, 15, 16]. The intracytoplasmic eosinophilic bodies (PAS positive vacuoles) within the enterocytes that most frequently were identified in the healthy mink kits have also been described previously [15, 16]. Although their role has not been clarified in mink, their staining properties and localization is comparable to the absorptive, intra-cytoplasmic vacuoles found in neonates of pigs and ruminants . Therefore, the presence of these vacuoles in enterocytes of healthy mink kits is not surprising and may be related to the intestinal uptake of maternal milk antibodies , taking place up to 4–5 weeks after birth in mink kits . Although more pronounced changes of the gut architecture like atrophy and fusion of villi were present in the PWD kits, no significant difference in the degree of neutrophil and mononuclear leucocyte infiltration were observed between controls and PWD mink kits. This lack of histopathological signs of inflammation indicates that PWD in the mink kits represents a secretory type of diarrhea. The observed attachment of coccoid bacteria may be responsible for an enterotoxic effect causing a loss of balance of movements of ions and water leading to the vacuolization and swelling of the enterocytes. Interestingly, the attachment of enterococci and E. coli to enterocytes and the slight to moderate infiltrations of neutrophils irrespective of diarrheic status has recently been found in piglets suffering from New Neonatal Porcine Diarrhea Syndrome (NNPDS) [38–40], suggesting similarities in mechanisms between diarrhea in the pre-weaning period of mink kits and piglets. Elevated levels of SAA and adhesion of bacteria to the intestinal wall has been seen for segmented filamentous bacteria (SFB), which adhere to the enterocytes, inducing epithelial SAA production [41, 42]. Atarashi et al.  colonized rats and germ-free mice with SFB from 20 strains of bacteria isolated from feces from patients suffering from ulcerating colitis and from E. coli and found that they all promote the induction of Th17 cells, which in turn could lead to an increased SAA production in enterocytes. Research on local expression of SAA in the intestine is needed to elucidate if circulating SAA levels are increased as a consequence of local production of SAA by epithelial intestinal cells in mink kits affected by PWD. In rodent models, a local induction of SAA in enteric epithelial cells in response to an altered microbiota in the absence of inflammation has indeed been demonstrated , however the impact on circulating SAA concentrations has not been reported. Although more pronounced changes of the gut architecture, like atrophy and fusion of villi were present in the PWD kits, no significant difference in the degree of neutrophil and mononuclear leucocyte infiltration were observed between controls and PWD mink kits. Thus, the possibility of an association between increased SAA levels and an unidentified inflammatory state in mink kits suffering from PWD should be investigated in more detail.
We identified a significant increase in circulating concentrations of SAA and attachment of coccoid bacteria in kits affected by PWD. The slight to moderate infiltrations of neutrophils irrespectively of diarrheic status and the attachment of coccoid bacteria to enterocytes show similarities with observations found in piglets suffering from NNPDS, and suggest that PWD in mink is a secretory type of diarrhea.