Gastrointestinal Ultrasonographic Findings in Cats With Feline Panleukopenia Virus: a Case Series

Feline panleukopenia (FPV) and and and other felids. FPV is particularly widespread among sheltered cats, and is associated with high morbidity and mortality, causing severe gastroenteritis characterized by anorexia, lethargy, fever, dehydration, hemorrhagic diarrhea, and vomiting. There is currently no data on the ultrasonographic features of cats affected with FPV. This case series describes abdominal ultrasonographic ndings in shelter cats with naturally-occurring FPV, and assesses whether are associated with clinical and laboratory ndings. Cats affected by FPV were enrolled in the study if an abdominal ultrasound was performed within 12 hours of diagnosis. Clinical, laboratory and survival data were collected from medical records. Ultrasonographic examinations were reviewed for gastrointestinal abnormalities and their associations with the above data were explored.


Background
Feline panleukopenia virus (FPV) is a single-stranded, non-enveloped DNA virus that infects domestic cats and other felids as well as mink, raccoons, and foxes [1]. Replication of FPV occurs within mitotically active cells, such as intestinal crypt epithelial cells, damaging the intestinal villi, increasing permeability of the intestinal wall, and resulting in malabsorption [1,2]. The most common clinical signs are anorexia, lethargy, fever, dehydration, hemorrhagic diarrhea, and vomiting [3]. Diagnosis is made by combining history, clinical ndings, and hematologic changes together with detection of FPV or canine parvovirus (CPV) antigen in feces or viral DNA in blood or in feces [1,4]. A recent study on survival and prognostic factors of FPV [5,6] showed that shelter cats without signs of lethargy, with a higher body weight, or higher rectal temperature at admission were more likely to survive. Leukopenia on or after the third day of hospitalization was associated with poorer outcome. Treatment with amoxicillin-clavulanic acid, antiparasitics, or maropitant was associated with improved survival.
To the best of the authors' knowledge, there is currently no information on the ultrasonographic features of the gastrointestinal tract of cats affected by FPV, especially regarding acute lesions. In a recent case report of an 8-month old kitten with FPV, a multi-layered appearance of the intestine was described representing superimposed wall layers and therefore resembling intussusceptions,. However, a few hours after the ultrasonographic examination, the cat defecated a 15 cm-long piece of tissue and the histological examination revealed necrotic and cellular debris, indicating a brinonecrotic colonic cast [7,8,9].
The most frequent ultrasonographic abnormalities in dogs with CPV gastroenteritis include uid and gaslled gastrointestinal tract associated with generalized atony, decreased thickness of the mucosal layer, hyperechoic mucosal speckles, and irregularity of the luminal surface of the duodenum and jejunum [10,11].
Gastrointestinal motility disorders are frequent in dogs and cats with acute vomiting or diarrhea. In cats with diffuse functional ileus associated with pancreatitis or abdominal effusion, corrugation of small bowel loops have been observed [9].
The aims of this retrospective study were therefore to: i) describe abdominal ultrasonographic abnormalities in shelter cats with naturally-occurring panleukopenia; ii) determine whether they are associated with clinical and laboratory ndings; and iii) investigate their impact on outcome.

Cats and inclusion criteria
An outbreak of FPV infection occurred in a large cat shelter located in northwestern Italy at the end of autumn 2010 which persisted for more than three consecutive years, thus becoming endemic.
Retrospectively, medical records of cats with FPV infection admitted to the authors' institution between January 2011 and December 2013 were reviewed and information pertaining to signalment, history, clinical signs, and laboratory ndings at presentation, as well as survival at discharge from the hospital were collected. Vaccination status, fecal examination, and whether cats had positive or negative results of anti-FIV antibody and FeLV antigen testing (SNAP FIV/FeLV combo plus test, IDEXX Laboratories, Milan, Italy) were also recorded [22].
In our study, cats were included if diagnosed with FPV based on compatible clinical ndings (i.e., lethargy, diarrhea or vomiting), laboratory abnormalities (i.e., anemia, leukopenia, thrombocytopenia, hypoproteinemia, hypoalbuminemia, hypokalemia or hypoglycemia) and positive fecal antigen test (SNAP Parvo, IDEXX Laboratories, Milan, Italy), and if an abdominal ultrasound had been performed within 12 hours of admission. Fasting longer than eight hours was not possible because the sample included kittens and debilitated cats.

Abdominal ultrasonography
Abdominal B-mode examination was performed in conscious cats placed in dorsal recumbency using a GE-LogiQ S8 (GE Healthcare, Milan, Italy) with a high-frequency transducer (11)(12)(13)(14)(15)(16)(17)(18). Prior to ultrasound, hair was clipped on the abdominal area, and coupling gel was used. A board-certi ed radiologist (S.S.) along with a diagnostic imaging intern (R.I.) reviewed all ultrasound documentation. Cats were included if the quality of images was judged as adequate.
Ultrasound videos, static images and reports were reviewed to assess abdominal organs, with emphasis on the gastrointestinal tract. The ultrasonographic features collected from the gastrointestinal tract were: overall wall and layer thickness (e.g., muscular layer thickening), both measured in a transverse and sagittal axis from the inner hyperechoic interface of the mucosal surface to the outer hyperechoic layer of the serosa; echogenicity of the various layers (e.g., hyperechogenicity of the mucosa); luminal patterns (e.g., uid-lled); morphologic description, location and extension of any intestinal lesion.
Stratigraphy of the intestinal wall was considered abnormal if layers had changes in echogenicity or in thickness, as described by previous authors (Table 1) [23,26]. Peristalsis was reduced if peristaltic acts were ≤3/min and increased if >5/min [29].
In addition, size, echogenicity, echotexture and morphology of jejunal lymph nodes and spleen, and the presence of peritoneal free uid were evaluated. Any other abdominal abnormalities were recorded if present.

Statistical analysis
Statistical analysis was performed using SAS v. 9.3 (SAS Institute Inc, Cary, NC). The median and range were calculated for the overall wall thickness and for individual layers of the gastrointestinal segments (e.g., stomach, duodenum, jejunum).
Several parameters were considered for this exploratory analysis. In particular, i) signalment and clinical variables included age, presence of vomiting, diarrhea, lethargy, and survival; ii) laboratory ndings included erythrocytes, leukocytes, platelet count, serum concentration of total proteins, albumin, glucose, and potassium; and iii) ultrasonography included duodenal wall thickness, duodenal muscularis thickness, jejunal wall thickness, jejunal muscularis thickness, duodenal mucosa hyperechogenicity, jejunal mucosa hyperechogenicity, jejunal hyperechoic mucosal band paralleling the submucosa, and peritoneal free uid.
These clinical and laboratory variables were used to determine whether they were associated with ultrasonographic ndings. Associations between ultrasonographic abnormalities and ultrasonographic, signalment, and clinical and laboratory data were explored using Chi-square and Fisher's exact tests. Due to the numerous analyses, signi cance was corrected with Bonferroni, and set at P<0.001 for associations with ultrasonographic and laboratory data and set at P<0.01 for associations with signalment and clinical data including survival [24].
To explore the above associations, all ndings were categorized into: present vs. absent or abnormal vs. normal. Laboratory parameters, including erythrocytes, leukocytes, platelet count, total proteins, albumin, glucose, and potassium, were considered abnormal if lower than the reference interval. In addition, age was divided into <1 year vs. >1 year. Cats were considered alive if they survived up to discharge from the hospital.

Results
Of the initial 265 cats with suspected FPV infection, 21 (7.9%) met the inclusion criteria and were enrolled in the study. All cats were diagnosed with FPV based on clinical ndings, laboratory abnormalities, and positive fecal antigen test (SNAP Parvo, IDEXX Laboratories, Milan, Italy). Four cats (19%) had been vaccinated between 11 and 20 months before disease onset. All cats were domestic shorthair. A total of 11 cats were intact females (52.4%) and 10 were intact males (47.6%) with a median age of 3 months (range: 1.5-24). Twelve cats (57.1%) survived and 9 (42.9%) died. The 12 cats that survived were discharged on average ve days (median: 5, range: 4-7) after admission; according to the shelter veterinarian all the surviving cats fully recovered. The nine cats that did not survive died on average after two days (median: 2; range: 1-3). None of the cats were euthanized.
In three cats, information on clinical status was not available. For the remaining 18 cats (85.7%), major clinical signs on admission were lethargy in 14 (77.7%), diarrhea in 10 (55.5%), and vomiting in four (22.2%). Clinical signs had been observed on average two days (median: 2, range: 1-4) before admission.
Serological testing for FIV antibody and FeLV antigen were performed in each cat and all were negative.
Fecal examination was performed in four cats (19%) that had not been treated with antiparasitics: three were negative and one was positive for Capillaria, Uncinaria, Giardia, and Toxocara. The remaining cats had been treated with antiparasitics during the week before admission and fecal examination was not performed. Table 2 summarizes the main abnormalities in the hematological and biochemical pro les of the 21 cats.
Information on the evaluation of the stomach was reported in 18 of 21 cats (85.7%); in 6 (33.3%) the stomach was moderately-to-severely distended by food and/or liquid uid, and in 12 (66.6%) cats it was normally distended. The median of the overall wall thickness was 2.3 mm (range: 0.7-6.3).
Thickening of the muscular layer was present in the duodenum (median: 0.4 mm, range: 0.2-0.8) and jejunum (median: 0.6 mm, range: 0.1-1.5) in nine cats (52.9%) and in 12 cats (57.1%), respectively (Insert Image 1). Increased echogenicity of the mucosa was detected in seven cats (41.2%) in the duodenum and in seven (33.3%) in the jejunum (Insert Image 2 and Image 3). Hyperechoic mucosal band paralleling the submucosa was present only in the jejunum of seven cats (33.3%) (Insert Image 4). The duodenum and jejunum were distended by uid and/or echogenic material in two cats (11.8%) and in nine cats (42.8%), respectively. The luminal surface appeared irregular in two cats (11.8%) in the duodenum, and in seven (33.3%) in the jejunum; in these cases the nding was generalized.
The colon was evaluated in 15 of the 21 cats (71.4%), and the wall was considered thickened (thickness: >1.5 mm) in three cats (20%); one cat showed loss of layering.
Jejunal lymph nodes were described in 12 cats (57.1%), and the thickness was deemed normal (median: 4.6 mm, range: 3-6.2). Images of the spleen were available in 18 cats (85.7%) and only in one cat was it considered enlarged (thickness: >9 mm); in seven cats (38.8%) the spleen had a "moth-eaten" parenchyma, and in three (16.6%) it was hypoechoic.
Five cats (23.8%) presented moderate anaechoic abdominal effusion. Other ndings recorded were hyperechogenicity of the mesentery in two cats with no abdominal effusion, multiple bilateral renal cysts in one, and hyperechogenicity of the liver parenchyma with hypoechoic multiple nodules, cystitis and peripancreatic steatitis in another.
Among the parameters assessed with ultrasonography, no signi cant association was identi ed. However, a trend towards signi cance was observed for a positive association between jejunal mucosa hyperechogenicity and duodenal muscularis thickening (P=0.010) or jejunal hyperechoic mucosal band (P=0.007).
When associations were explored between ultrasonographic abnormalities and signalment, clinical ndings, laboratory abnormalities, and survival, positive associations were observed between jejunal wall thickening and age (P<0.001), as well as the presence of peritoneal effusion and vomiting (P=0.002). A trend towards signi cance was observed for the presence of peritoneal effusion and low platelet count (P=0.005). A positive association was observed between survival and increased echogenicity in the jejunal mucosa (P=0.003), and presence of hyperechoic mucosal band in the jejunum (P=0.003).

Discussion
Our study highlights that in cats with acute FPV, the gastrointestinal tract shows several ultrasonographic changes . The most frequent ndings were diffuse small intestine mucosal layer thinning, muscular layer thickening and mucosal hyperechogenicity. Hyperechoic mucosal band paralleling the submucosa and irregular luminal surface were frequent in the jejunum.
Although described mostly in chronic enteropathies [12,13,14,15,16], our study identi ed diffuse small intestine muscular layer thickening, and hyperechoic mucosal band paralleling the submucosa, along with increased mucosal echogenicity in cats with acute gastroenteritis induced by FPV.
Thickening of the muscular layer was observed in the duodenum and jejunum in approximately 50% and 60% of cats, respectively, which has previously been associated with chronic enteropathies (e.g., eosinophilic enteritis) and lymphoma; potential causes are in ltrative diseases and smooth muscle hypertrophy or hyperplasia [12,13,15,16]. No histopathological analysis was performed in our cats, and occult chronic enteropathy (e.g., food intolerance) or lymphoma cannot be rule out in our cases.
However, it seems unlikely that 60% of these young cats had an underlying disease. We assume that muscular layer thickening in cats with acute panleukopenia may be due to transient in ammatory in ltration. Enteritis and hypoproteinemia have been shown to cause diffuse intestinal wall edema (Nyland Matton, 2015), potentially also including the muscular layer.
In our study, the hyperechoic mucosal band paralleling the submucosa was present in the jejunum in onethird of the cats, while but not in the duodenum. Using ultrasound, Penninck and coworkers (2010) described the presence of a mucosal hyperechoic band paralleling the submucosa in cats presenting with gastrointestinal signs. Based on histology, the hyperechoic mucosal band represented an ultrasonographic interface due to the presence of mucosal brosis [14]. In another investigation [13], cats showing a visible hyperechoic mucosal band on ultrasound were prospectively examined, without performing histopathology. Most cats had vomiting or diarrhea, but one-third had no clinical signs or other ultrasonographic intestinal abnormalities. It was concluded that this band can also be observed in apparently healthy cats [13].
We observed increased mucosal echogenicity in approximately 40% of cats in the duodenum, and 30% in the jejunum. In addition, irregularity of the luminal surface was found in 10% of cats in the duodenum, and in 30% of cats in the jejunum. Mucosal changes, such as increased mucosal echogenicity, hyperechoic speckles, and striations have been reported in several conditions in chronic enteropathies in dogs and cats. Increased mucosal echogenicity has been described in lymphoplasmacytic enteritis and eosinophilic enteritis and in cats with mucosal brosis [14,15]. Hyperechoic speckles and hyperechoic striations have been reported in dogs with chronic enteropathies [18]. Anecdotally, a hyperechoic mucosal border on the luminal margin was observed in dogs with acute enteritis [19].
In our cats with FPV, it is possible that these ndings were due to mucus, cellular debris, gas entrapped in the mucosal crypts, and protein accumulation caused by necrosis and in ammation associated with the acute form of the disease, as reported in puppies with CPV [20].
In our study, mucosal thinning of the duodenum and jejunum was observed in two-thirds of cases, along with an irregular luminal surface in two cats (11.8%) in the duodenum, and in seven (33.3%) cats in the jejunum. Villous atrophy secondary to viral-induced crypt cell destruction, necrosis, and sloughing of epithelial cells associated with FPV are reasonable explanations for the reduced thickness. This nding is similar to previous observations in 40 puppies with CPV where the duodenal and jejunal mucosa thickness was signi cantly decreased and the luminal surface of the duodenal and jejunal mucosa was irregular [20]. The thinning of the mucosal layer due to the aforementioned causes, could explain the severe reduction in the jejunal wall that we observed in one cat in which the thickness was 0.6 mm.
In cats with ultrasonographic information on the jejunal lymph nodes, echogenicity, echotexture, and size were normal. This nding is similar to previous observations in puppies with CPV [20], in which parvovirus infection was not associated with jejunal lymphadenopathy by abdominal ultrasound. Stander and coworkers (2010) assumed that cortical atrophy was the result of the tendency of CPV to rapidly divide cells of the lymphoid tissue. It is also possible that the same explanation accounts for the sonographically normal lymph nodes in our study, although histology would have been required for con rmation.
In almost half of the cats with available images of the spleen, a "moth-eaten" parenchyma was observed, and in only one was this nding associated with splenomegaly. A "moth-eaten" parenchyma is characterized by numerous small hypoechoic nodules spread throughout the organ causing a spotted echotexture. However, this nding is non-speci c, and in a previous study was observed in cats with lymphoid hyperplasia, extramedullary hematopoiesis, passive congestion, lymphoma, carcinoma, and histiocytosis, as well as in healthy animals [21]. The use of very high-frequency transducers (11)(12)(13)(14)(15)(16)(17)(18), which improve the quality of images, may also be partly responsible for the high rate of this particular pattern in our cats.
Regarding the associations between ultrasonographic features and clinical ndings, vomiting was positively associated with free peritoneal uid. It is possible that cats with more severe in ammation of the gastrointestinal tract, in addition to vomiting, have a greater permeability of all bowel layers including the serosa, leading to abdominal effusion. Mild abdominal effusion has been described in puppies with CPV, although associations with clinical signs were not investigated [10]. Age was also positively associated with jejunal wall thickening. However, the reason for this nding is not clear, hence a casual association rather than a cause-and-effect relationship cannot be excluded.
Interestingly, based on ultrasonography, the abdominal effusion of the present cats was anechoic. Feline infectious peritonitis seemed unlikely because in this disease, the effusion is usually echogenic and with mobile particles [30] -it is still nevertheless possible. The uid was not collected to clarify its pathogenesis.
With regard to survival and abdominal ultrasonography, a positive outcome was more likely in FPV cats with higher jejunal mucosal echogenicity and hyperechoic mucosal band. The hyperechoic mucosal band may represent an ultrasonographic interface due to the presence of mucosal brosis [14]. Unfortunately, histopathology was not performed in our cats with FPV to verify whether brous tissue was present in the jejunum. Given that FPV has an acute onset in most cats [3], the presence of brous tissue would be unexpected. However, in some cats FPV may be more subclinical, longer lasting and with a better outcome [1]. It is also possible that intestinal brosis develops in some of these cases. We hypothesize that cats with a hyperechoic mucosal band were more likely to survive because the infection with FPV was less severe and relatively chronic. In the another study, the hyperechoic mucosal band was found in association with increased echogenicity of the mucosa [14]. In line with this, in our study there was a trend towards an association between the two ultrasonographic ndings.
The main limitations of the present study are the relatively small number of cats included and the retrospective nature of the investigation. Some medical records were incomplete, in particular those pertaining to images and videos of the ultrasound. Hence, it was not possible to measure all segments of the gastrointestinal tract, although the attending radiologist reported them as normal.
Moreover, follow-up abdominal ultrasound and histological examinations were not performed, which would have characterized the underlying causes of some of the ultrasonographic features. Finally, no control group of shelter cats with other gastrointestinal diseases was available for comparison. However, shelter cats with vomiting or diarrhea but otherwise in good conditions are treated symptomatically by the shelter veterinarian without performing further diagnostics, such as abdominal ultrasound, thus precluding the possibility of a control group.

Conclusions
In brief, cats with naturally-occurring FPV had ultrasonographic features compatible with gastroenteropathy. Diffuse small intestine mucosal layer thinning, muscular layer thickening and mucosal hyperechogenicity, and jejunal hyperechoic mucosal band paralleling the submucosa and irregular luminal surface were frequently observed in the cats examined. Some ultrasonographic abnormalities were associated with clinical ndings and outcome, although the underlying reasons have not been clari ed.

Declarations
Ethics Approval and consent to participate: This work involved the use of non-experimental animals only (both owned and cats in a shelter), and followed established internationally recognised high standards ('best practice') of individual veterinary clinical patient care. Ethical approval from a committee was not necessary. Informed consent (either verbal or written) was obtained from the owner or legal custodian of all animals described in this work for the procedures undertaken.
Consent for publication: For all the animals or humans individually identi able within this publication, informed consent for their use in the publication (verbal or written) was obtained from the people involved.
Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request.
Competing interests: The authors declare that they have no competing interests.
Funding: The author received no nancial support for the research, authorship, and/or publication of this article.
Author's contributions: The idea was conceived by IR, SS, ZE. The study was designed by ZE, SS, AE, IR.
The cases were managed by DE, PF, FF, PM. The data were analyzed by RI, CB, CLM. The paper was written by IR, CS and ZE. All of the authors read and approved the nal manuscript.    Figure 1 Thickening of the muscular layer in the jejunum Figure 2 Increased echogenicity of the mucosa in the duodenum Figure 4 Hyperechoic linear mucosal band paralleling the submucosa in the jejunum