Serratia marcescens-associated subcutaneous abscess in a dog.

This report describes the clinical presentation and progression of a Serratia marcescens-associated subcutaneous abscess in a dog with hypothyroidism, hyperadrenocorticism and diabetes mellitus. The S. marcescens isolate was resistant to several antibiotics. Treatment with antibiotics and topical antiseptics was not successful.

Healthcare-associated infections caused by chlorhexidine-tolerant Serratia marcescens carrying a promiscuous IncHI2 multi-drug resistance plasmid in a veterinary hospital.

The bacterium Serratia marcescens can cause opportunistic infections in humans and in animals. In veterinary settings, the diversity, reservoirs and modes of transmission of this pathogen are poorly understood. The phenotypes and genotypes of Serratia spp. isolated from dogs, cats, horses, a bird and a rabbit examined at an Australian veterinary hospital between 2008 and 2019 were characterised. The isolates were identified as S. marcescens (n = 15) or S. ureilytica (n = 3) and were placed into four distinct phylogenetic groups. Nine quasi-clonal isolates associated with post-surgical complications in different patients displayed high levels of resistance to the antimicrobials fluoroquinolones, cephalosporins, aminoglycosides, and to the disinfectant chlorhexidine. A Serratia sp. with a similar resistance profile was also isolated from chlorhexidine solutions used across the Hospital, suggesting that these infections had a nosocomial origin. A genomic island encoding a homolog of the Pseudomonas MexCD-OprJ biocide efflux system was detected in the chlorhexidine-tolerant Serratia. The nine multi-drug resistant Serratia isolates also possessed a Ser-83-Ile mutation in GyrA conferring fluoroquinolone resistance, and carried a large IncHI2 conjugative plasmid encoding antimicrobial and heavy metal resistances. This replicon was highly similar to a plasmid previously detected in a strain of Enterobacter hormaechei recovered from the Hospital environment. IncHI2 plasmids are commonly found in Enterobacteriaceae, but are rarely present in Serratia spp., suggesting that this plasmid was acquired from another organism. A chlorhexidine-tolerant Serratia isolate which lacked the IncHI2 plasmid was used in mating experiments to demonstrate the transfer of multi-drug resistance from a E. hormaechei donor. This study illustrates the importance of environmental surveillance of biocide-resistance in veterinary hospitals.

Long-lasting nosocomial persistence of chlorhexidine-resistant Serratia marcescens in a veterinary hospital.

Healthcare-associated infections (HAIs) are often overlooked in veterinary medicine. Serratia marcescens isolates were recovered over a ten-year period from companion animals in a French veterinary hospital. The pets were sampled either for diagnostic purposes or to monitor colonization. A retrospective study showed that 32 S. marcescens isolates were identified as HAI cases and a further 22 cases were associated with colonization of the surgical site. Two S. marcescens lineages were responsible for two different outbreaks during the study period. Chlorhexidine solution (1%) used to impregnate gauze was found to be the source of the second S. marcescens outbreak and all isolates had high MIC values for chlorhexidine (MIC = 128 mg/L). This study reports, for the first time to our knowledge, the nosocomial spread of chlorhexidine-resistant S. marcescens in a veterinary setting and highlights consequences of the improper use of disinfectants.

First case of Serratia liquefaciens isolated from urinary tract infection in sows and associated clinicopathological and pathological findings.

An incident of sudden deaths in the breeding stock was reported from a farrow-to-finish commercial pig farm in Greece. The 8·4% of sows during lactation and gestation period presented anorexia, fever, haematuria, return-to-oestrus and sudden deaths (mortality rate: 2·3%). Blood and urine samples were collected from four diseased sows. Furthermore, swabs from urine bladders were collected from two dead sows and four culled sows at the slaughterhouse. Blood testing demonstrated mild leucocytosis and absence of azotaemia. Urinalysis revealed haematuria, proteinuria, bilirubinuria and active urine sediment with bacilli, epithelial cells and leucocytes, crystals and granular casts. Histopathological evaluation of the bladder demonstrated chronic active polypoid cystitis. The bacterial culture revealed the presence of Serratia liquefaciens. The antibiotic susceptibility testing showed high resistance to the most common antibiotics, with the highest sensitivity of the isolate towards quinolones. After the administration of a single dose of 7·5 mg kg-1 body weight enrofloxacin intramuscularly, the mortality rate decreased to less than 0·5% along with a remarkable reduction in the severity of clinical signs. Based on our findings, S. liquefaciens induced severe clinical signs and deaths in sows, mainly due to urinary infection. Inadequate water sanitation might have been responsible for increased exposure to S. liquefaciens. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, the isolation of Serratia liquefaciens from the urinary tract of pigs associated with clinical signs and increased mortality was described for the first time. Serratia liquefaciens is an important cause of hospital-acquired human infections. The isolate in this study was resistant to the most common antibiotics. Therefore, the use of quinolones which are drugs of last resort for treatment of infections was the only therapeutic option. The presence of the resistant bacterium in the urinary tract raises concerns for its zoonotic potential.

Description of two Serratia marcescens associated mastitis outbreaks in Finnish dairy farms and a review of literature.

BACKGROUND: Infection with Serratia spp. have been associated with mastitis outbreaks in dairy cattle herds. Environmental contamination or a point source, like a teat dip product, have often been observed to be potential sources of such outbreaks. We describe two Serratia marcescens associated mastitis outbreaks associated with a contaminated teat dip containing a tertiary alkyl amine, n,n-bis (3-aminopropyl) dodecylamine in two dairy cattle farms in Finland. S. marcescens strains isolated from milk and environmental samples were identified by the MALDI-TOF method. RESULTS: Six specimens (n = 19) on Herd 1 and all specimens (n = 9) on Herd 2 were positive for S. marcescens. Positive specimens were from mastitis milk and teat dip liquid and equipment. Bacteria were not isolated from the unopened teat dip canister. The same clone of S. marcescens was isolated from milk samples and teat dip samples within the farms. Pulsed field gel electrophoresis results to the S. marcescens isolates from these two different herds were tested with unweighted pair-group method using arithmetic average clustering analysis. The isolates were not same clone in both herds, because similarity in that test was only 75% when cut-off value to similarity is 85%. CONCLUSIONS: Our investigation showed that the post milking teat dip and/or temporary containers were contaminated with S. marcescens and these were most likely the sources for new mastitis cases. The negative result from the unopened teat dip canister and positive results from refillable containers demonstrated that the product itself was not contaminated with S. marcescens at the production unit, but became contaminated at the farm level.

Protective effect of neglected plant Diplocyclos palmatus on quorum sensing mediated infection of Serratia marcescens and UV-A induced photoaging in model Caenorhabditis elegans.

Plants are considered to be a leading source for possible human therapeutic agents. This holistic study has investigated the anti-quorum sensing (anti-QS), anti-infection, antioxidant and anti-photoaging properties of neglected plant Diplocyclos palmatus. The results showed that D. palmatus methanolic leaf extract (DPME) effectively inhibited the quorum sensing (QS) regulated virulence factor production as well as biofilm formation in Serratia marcescens. The transcriptomic analysis revealed that DPME significantly downed the expression of QS-regulated genes such as fimA, fimC, flhC, bsmB, pigP and shlA in S. marcescens, which supports the outcome of in vitro bioassays. Further, the docking study revealed that the presence of active compounds, namely tocopherols and phytol, DPME exhibited its anti-QS activity against S. marcescens. In addition, DPME treatment extended the lifespan of S. marcescens infected C. elegans by the action of dropping the internal accumulation. Further, qPCR analysis clearly revealed that DPME treatment significantly up-regulated the expression of the lifespan-related gene (daf-16) and immune-related genes (clec-60, clec-87, lys-7 and bec-1) in S. marcescens infected C.elegans. On the other hand, DPME extensively reduced the UV-A induced ROS stress, thereby, extended the lifespan in UV-A photoaged C. elegans. Further, the qPCR analysis also confirmed the up-regulation of daf-16, clec-60, clec-87 and col-19 genes which advocated the improvement of the lifespan, healthspan and collagen production in UV-A photoaged C. elegans. Further bioassays evidenced that that the lifespan extension of photoaged C. elegans was accomplished by the actions of antioxidants such as tocopherols and phytol in DPME.

Dauer life stage of Caenorhabditis elegans induces elevated levels of defense against the parasite Serratia marcescens.

Host-parasite research often focuses on a single host life stage, yet different life stages may exhibit different defenses. The nematode Caenorhabditis elegans has an alternate dispersal life stage, dauer. Despite dauer's importance in nature, we know little of how it responds to parasites. Previous research indicates that non-dauer C. elegans prefer to consume the virulent bacterial parasite, Serratia marcescens, when given a choice between the parasite and benign Escherichia coli. Here, we compared the preferences of dauer individuals from six strains of C. elegans to the preferences of other life stages. We found that dauer individuals exhibited reduced preference for S. marcescens, and dauers from some strains preferred E. coli to S. marcescens. In addition to testing food preference, a mechanism of parasite avoidance, we also measured host mortality rates after direct parasite exposure to determine if life stage also altered host survival. Overall, dauer individuals exhibited reduced mortality rates. However, dauer versus non-dauer larvae mortality rates also varied significantly by host strain. Collectively, we found evidence of dauer-induced parasite avoidance and reduced mortality in the presence of a parasite, but these effects were strain-specific. These results demonstrate the importance of host life stage and genotype when assessing infection dynamics.

Supposed endogenous endophthalmitis caused by Serratia marcescens in a cat.

An 8-year-old male neutered domestic shorthair cat was presented for evaluation of acute respiratory distress. Respiratory auscultation revealed a diffuse and symmetric increase in bronchovesicular sounds. Thoracic radiographs showed a diffuse unstructured interstitial pulmonary pattern with multifocal alveolar foci. Despite an aggressive treatment with supportive care, including oxygenotherapy and systemic antibiotics, progressive respiratory distress increased. Three days after the presentation, acute anterior uveitis was noticed on left eye. Ophthalmic examination and ocular ultrasonography revealed unilateral panuveitis with ocular hypertension. The right eye examination was unremarkable. Cytological examination of aqueous humor revealed a suppurative inflammation. Serratia marcescens was identified from aqueous humor culture. Primary pulmonary infection was suspected but was not confirmed as owners declined bronchoalveolar lavage. Active uveitis resolved and cat's pulmonary status improved after appropriate systemic antibacterial therapy. Vision loss was permanent due to secondary mature cataract. To the best of authors' knowledge, this is the first report of endogenous bacterial endophthalmitis secondary to S. marcescens infection in a cat.

Imidacloprid Decreases Honey Bee Survival Rates but Does Not Affect the Gut Microbiome.

Accumulating evidence suggests that pesticides have played a role in the increased rate of honey bee colony loss. One of the most commonly used pesticides in the United States is the neonicotinoid imidacloprid. Although the primary mode of action of imidacloprid is on the insect nervous system, it has also been shown to cause changes in insects' digestive physiology and alter the microbiota of Drosophila melanogaster larvae. The honey bee gut microbiome plays a major role in bee health. Although many studies have shown that imidacloprid affects honey bee behavior, its impact on the microbiome has not been fully elucidated. Here, we investigated the impact of imidacloprid on the gut microbiome composition, survivorship, and susceptibility to pathogens of honey bees. Consistent with other studies, we show that imidacloprid exposure results in an elevated mortality of honey bees in the hive and increases the susceptibility to infection by pathogens. However, we did not find evidence that imidacloprid affects the gut bacterial community of honey bees. Our in vitro experiments demonstrated that honey bee gut bacteria can grow in the presence of imidacloprid, and we found some evidence that imidacloprid can be metabolized in the bee gut environment. However, none of the individual bee gut bacterial species tested could metabolize imidacloprid, suggesting that the observed metabolism of imidacloprid within in vitro bee gut cultures is not caused by the gut bacteria. Overall, our results indicate that imidacloprid causes increased mortality in honey bees, but this mortality does not appear to be linked to the microbiome.IMPORTANCE Growing evidence suggests that the extensive use of pesticides has played a large role in the increased rate of honey bee colony loss. Despite extensive research on the effects of imidacloprid on honey bees, it is still unknown whether it impacts the community structure of the gut microbiome. Here, we investigated the impact of imidacloprid on the gut microbiome composition, survivorship, and susceptibility to pathogens of honey bees. We found that the exposure to imidacloprid resulted in an elevated mortality of honey bees and increased the susceptibility to infection by opportunistic pathogens. However, we did not find evidence that imidacloprid affects the gut microbiome of honey bees. We found some evidence that imidacloprid can be metabolized in the bee gut environment in vitro, but because it is quickly eliminated from the bee, it is unlikely that this metabolism occurs in nature. Thus, imidacloprid causes increased mortality in honey bees, but this does not appear to be linked to the microbiome.

Characterization of a Polymicrobial Dermal Infection in a Peninsular Pronghorn ( Antilocapra americana peninsularis) in Baja California, Mexico.

In situ conservation efforts are assisting the recovery of free-ranging populations of the endangered peninsular pronghorns ( Antilocapra americana peninsularis) at the Vizcaino Biosphere Reserve, Baja California Sur, Mexico. We detected a polymicrobial dermal infection. Etiologic agents were identified as a keratinophilic dermatomycete and opportunistic pathogenic bacteria.

Sudden death of an Indian peafowl (Pavo cristatus) at a zoo due to non-pigmented Serratia marcescens infection.

A 16-year-old female Indian peafowl (Pavo cristatus) died two days after recognition of conjunctivitis in the right eye, anorexia and depression. Gross necropsy revealed a thick pseudomembrane under the eyelid and hydropericardium. Histopathological examination revealed hepatocellular necrosis, sinusoidal and vascular congestion and infiltrated inflammatory cells. Infiltration by inflammatory cells was noted in the epicardium. The lungs had mild interstitial pneumonia with the extensive congestion within the capillaries of the air sacs. Tubular interstitial congestion and necrosis was noted in the kidneys. Bacterial culture and nucleotide sequencing of the inflammatory specimens identified the causative agent as Serratia marcescens, an uncommon bacterium in birds. In summary, this study describes the sudden death of an Indian peafowl due to S. marcescens infection, which is rarely seen in animals.

Characterization of putative virulence factors of Serratia marcescens strain SEN for pathogenesis in Spodoptera litura.

Two Serratia marcescens strains, SEN and ICC-4, isolated from diseased insect cadavers were observed to differ considerably in their virulence towards Spodoptera litura. The present study was aimed to characterize the possible virulence factors present in the virulent Serratia marcescens strain SEN. Both the S. marcescens strains were evaluated for the presence of various lytic enzymes such as chitinase, lipase, protease and phospholipase. The virulent S. marcescens strain SEN was observed to possess considerably higher activity of chitinase and protease enzymes; activity of phospholipase enzyme was also higher. Although, all the three toxin genes shlA, phlA and swr could be detected in both the S. marcescens strains, there was a higher expression of these genes in the virulent strain SEN. S. marcescens strain ICC-4 showed greater reduction in overall growth yield in the post-exponential phase in the presence of midgut juice and hemolymph of S. litura larvae, as compared to S. marcescens strain SEN. Proliferation of the S. marcescens strain SEN was also considerably higher in foregut, midgut and hemolymph of S. litura larvae, as compared to strain ICC-4. Peritrophic membrane treated with broth culture of the S. marcescens strain SEN showed higher damage as compared to strain ICC-4. The peritrophic membrane of larvae fed on diet treated with the virulent strain showed considerable damage while the peritrophic membrane of larvae fed on diet treated with the non-virulent strain showed no damage. This is the first report documenting the fate of ingested S. marcescens in S. litura gut and the relative expression of toxin genes from two S. marcescens strains differing in their virulence towards S. litura.

Sepsis and Hemocyte Loss in Honey Bees (Apis mellifera) Infected with Serratia marcescens Strain Sicaria.

Global loss of honey bee colonies is threatening the human food supply. Diverse pathogens reduce honey bee hardiness needed to sustain colonies, especially in winter. We isolated a free-living Gram negative bacillus from hemolymph of worker honey bees (Apis mellifera) found separated from winter clusters. In some hives, greater than 90% of the dying bees detached from the winter cluster were found to contain this bacterium in their hemolymph. Throughout the year, the same organism was rarely found in bees engaged in normal hive activities, but was detected in about half of Varroa destructor mites obtained from colonies that housed the septic bees. Flow cytometry of hemolymph from septic bees showed a significant reduction of plasmatocytes and other types of hemocytes. Interpretation of the16S rRNA sequence of the bacterium indicated that it belongs to the Serratia genus of Gram-negative Gammaproteobacteria, which has not previously been implicated as a pathogen of adult honey bees. Complete genome sequence analysis of the bacterium supported its classification as a novel strain of Serratia marcescens, which was designated as S. marcescens strain sicaria (Ss1). When compared with other strains of S. marcescens, Ss1 demonstrated several phenotypic and genetic differences, including 65 genes not previously found in other Serratia genomes. Some of the unique genes we identified in Ss1 were related to those from bacterial insect pathogens and commensals. Recovery of this organism extends a complex pathosphere of agents which may contribute to failure of honey bee colonies.

In vitro synergistic effects of fisetin and norfloxacin against aquatic isolates of Serratia marcescens.

Serratia marcescens is a common pathogenic bacterium that can cause infections in both humans and animals. It can cause a range of diseases, from slight wound infections to life-threatening bacteraemia and pneumonia. The emergence of antimicrobial resistance has limited the treatment of the diseases caused by the bacterium to a great extent. Consequently, there is an urgent need to develop novel antimicrobial strategies against this pathogen. Synergistic strategy is a new approach to treat the infections caused by drug-resistant bacteria. In this paper, we isolated and identified the first multi-resistant pathogenic Serratia marcescens strain from diseased soft-shelled turtles (Pelodiscus sinensis) in China. We then performed a checkerboard assay; the results showed that out of 10 tested natural products fisetin had synergistic effects against S. marcescens when combined with norfloxacin. The time-kill curve assay further confirmed the results of the checkerboard assay. We found that this novel synergistic effect could significantly reduce the dosage of norfloxacin against S. marcescens.

Identification by real-time PCR with SYBR Green of Leishmania spp. and Serratia marcescens in canine 'sterile' cutaneous nodular lesions.

BACKGROUND: Noninfectious, non-neoplastic, nodular to diffuse, so-called 'sterile' granulomatous/pyogranulomatous skin lesions (SGPSLs) are infrequently identified in dogs and may represent a diagnostic challenge. Their correct identification is based on history, histopathology and absence of intralesional foreign bodies and micro-organisms. HYPOTHESIS/OBJECTIVES: The aim of this study was to investigate the presence of Leishmania spp., Mycobacterium spp., Serratia marcescens and Nocardia spp. by real-time PCR in canine nodular skin lesions histologically diagnosed as putatively sterile. METHODS: Formalin-fixed skin biopsies were collected from 40 dogs. All samples were associated with an SGPSL diagnosis characterized by multifocal, nodular to diffuse, periadnexal and perifollicular pyogranulomas/granulomas. Neither micro-organisms nor foreign bodies were detected with haematoxylin and eosin staining, under polarized light. Further analyses included periodic acid Schiff, Ziehl-Neelsen, Fite Faraco, Giemsa and Gram histochemical stains; anti-Bacillus Calmette-Guérin (BCG) and Leishmania spp. immunohistochemistry; and real-time PCR analysis for Leishmania spp., Mycobacterium spp., S. marcescens and Nocardia spp. RESULTS: Special stains and BCG/immunohistochemistry were negative in all samples. Real-time PCR was positive for Leishmania spp. in four of 40 biopsies and for S. marcescens in two of 40 samples. Real-time PCR for Mycobacterium spp. and Nocardia spp. was negative. No correlation between real-time PCR positivity and a specific histological pattern was identified. CONCLUSIONS AND CLINICAL IMPORTANCE: Leishmania spp. have been previously identified as possible agents of certain SGPSLs, while the involvement of S. marcescens has not been investigated previously. According to our findings, Serratia spp. should be included in the list of agents possibly associated with a subgroup of granulomatous/pyogranulomatous skin lesions in dogs.

The "other" gram-negative bacteria in mastitis: Klebsiella, serratia, and more.

Mastitis caused by gram-negative infections is of increasing importance on modern and well-managed dairy farms. Without a doubt, E coli tends to be the most important cause of these gram-negative infections when the data are tallied across farms.1 However, more precise investigation of individual farms often reveals a farm-specific infection pattern where a single gram-negative bacterial species predominates. Several farms with a predominance of "other" gram-negative IMIs may be observed. We have shown the presence of outbreaks on individual dairy farms with K pneumoniae, S marcescens, and Enterobacter cloacae. On farms with a predominance of these "other" gram-negative infections, a detailed epidemiologic investigation may reveal the source of these infections. It is quite surprising to identify the difference in host immune response pattern and the associated clinical and subclinical presentations of IMIs due to the different gram-negative organisms. Experimental and field observations would suggest that among the gram-negative bacterial causes of mastitis, Klebsiella spp are causing the most severe cases, closely followed by E coli and then much less clinical severity is observed in Serratia spp and Enterobacter spp cases. The precise mechanisms that would explain the difference in clinical severity are not known, but the most likely explanation appears to be the structure of the lipid A fraction of the LPS of the bacterial species. Important differences in the lipid A fraction of LPS between and within bacterial species are observed. The prevention of IMIs with gram-negative bacteria has components that are generic across species and components that are species specific. Generic prevention may be obtained by improving hygiene and reducing exposure of teat ends to environmental contamination. Also the use of a J5 bacterin is expected to provide some reduction in severity of gram-negative IMIs across bacterial species. Specific prevention programs will depend on the actual transmission behavior of the dominant species causing IMIs in the herd. Several clonal outbreaks of gram-negative bacterial species have been described. In such situations, optimal milking procedures, segregation and culling of infected animals, and targeted treatment would be advisable. Even more specific are the prevention procedures associated with S marcescens outbreaks, where resistance against specific biocides will lead to transmission of infection through teat disinfectants. Removal of these biocides from the cow environment is than essential. Antimicrobial treatment of gram-negative bacteria has often considered to be of limited value and treatment should be more targeted toward cow survival and reduction of clinical symptoms. More recently, extended treatment with a third-generation cephalosporin was reported to be efficacious in the treatment of E coli and Klebsiella spp but not of E cloacae. Further investigations in effective treatment protocols for gram-negative IMIs are warranted.

Antimicrobial susceptibilities and bacteriological characteristics of bovine Pseudomonas aeruginosa and Serratia marcescens isolates from mastitis.

The presence of metallo-ß-lactamase (MBL)-producing and multidrug-resistant Pseudomonas aeruginosa (MDRP) strains among bovine isolates of Gram-negative bacilli, and O-serotypes of bovine Serratia marcescens and P. aeruginosa isolates have been reported rarely. The aims of this study were to (1) elucidate antimicrobial susceptibilities and O-serotypes of P. aeruginosa and S. marcescens isolates from bovine mastitis and the presence of MBL-producers and MDRP strains among them and (2) evaluate their relationships to human isolates. We investigated the MICs of 24 antimicrobials and O-serotypes for 116 P. aeruginosa and 55 S. marcescens isolates in Japan, primarily in 2006. A total of 171 isolates exhibited high antimicrobial susceptibilities with the exception of a partial drug. P. aeruginosa isolates exhibited high susceptibilities of ≥ 95.7% to ciprofloxacin, imipenem, meropenem, piperacillin, ceftazidime, cefepime, cefoperazone/sulbactam, amikacin, tobramycin, and gentamicin; however, they exhibited a susceptibility of only 69.8% to aztreonam. They exhibited substantial resistances to ceftriaxone, enrofloxacin, cefotaxime, and moxalactam. S. marcescens isolates exhibited high susceptibilities of ≥ 90.9% to kanamycin, ceftiofur, sulfamethoxazole-trimethoprim, and the 15 aforementioned drugs, but exhibited resistance to minocycline. Neither MBL-producers nor MDRP strains were detected among the 171 strains. The dominant serotypes of P. aeruginosa isolates were OG, OA, OB, OI, OF, OE, and OK; those of S. marcescens isolates were O6 and O5. Every S. marcescens isolate was pigmented. These findings suggest that bovine P. aeruginosa and S. marcescens isolates differ from human isolates from both antibiogram and phenotypic perspectives, and could help to evaluate differences in bacteriological characteristics between bovine and human isolates.

Fatal aortic endocarditis associated with community-acquired Serratia marcescens infection in a dog.

A 12 yr old Dalmatian was referred for evaluation of acute lethargy, fever, neurologic signs, and a recently ausculted heart murmur. Echocardiography in combination with blood cultures resulted in a diagnosis of nonhospital-acquired Serratia marcescens bacteremia and aortic valve endocarditis. Despite early diagnosis and aggressive therapy, the dog failed to respond to antimicrobials and died within 6 hr after admission. Necropsy findings included aortic valve endocarditis, septicemia, and diffuse thromboembolic disease. There was no history of pre-existing underlying disease or immunosuppressive therapy, and the dog had not been hospitalized before referral.