Pancreatic and biliary duct Hammondia heydorni infection in a dog fed a raw elk-meat diet

Abstract

We describe a 3-y-old Standard Poodle that was fed a raw elk-meat diet and developed anorexia, icterus, and markedly elevated liver enzyme activities. Histopathology of the pancreatic and liver biopsy specimens revealed lymphohistiocytic pancreatitis and hepatitis with intraepithelial protozoa. Histologically, Hammondia spp. are indistinguishable from Neospora caninum. Therefore, we employed PCR and amplicon sequencing to further investigate our case. Amplification and sequencing of the ITS1 marker matched 100% with Hammondia heydorni. Intrapancreatic involvement and the histopathologic features of hepatic and pancreatic H. heydorni infection have not been reported previously in dogs, to our knowledge. H. heydorni is a coccidian organism with an obligatory 2-host life cycle. The definitive hosts are dogs and coyotes; the intermediate hosts are mostly ruminants, including cervids. Ingesting tissue cysts within the skeletal muscle of the intermediate hosts leads to infection in the definitive hosts. H. heydorni has been considered clinically inconsequential; however, increasing reports note gastrointestinal and hepatic disease in dogs associated with this organism, and H. heydorni is considered an emerging pathogen in dogs. Infection in our case was associated with ingestion of raw cervid meat; cases of H. heydorni infection are invariably linked to ingestion of raw-meat diets.

Keywords

cholangitis dogs pancreatitis raw-meat diet

A 3-y-old, castrated male Standard Poodle dog was presented to a primary care veterinarian in Alberta, Canada because of anorexia. The patient had been ingesting a diet containing hunter-harvested raw elk (Cervus canadensis) meat for more than one year. Three months before the onset of anorexia, the patient had been presented because of lameness and suspected allergic dermatitis. The dermatologic lesions were biopsied and revealed pustular dermatitis with pyogranulomatous furunculosis, consistent with a secondary bacterial infection. The patient was discharged and treated with 375 mg amoxicillin trihydrate and clavulanate potassium (PO q12h; Clavamox, Zoetis), meloxicam (PO q24h; Metacam, Boehringer Ingelheim), and diphenhydramine (PO q12h). One month later, the patient was presented to another veterinary clinic and treated with 0.4 mL of methylprednisolone acetate IM (Depo-Medrol; Pfizer) and 100 mg of cyclosporine (PO q24h; Atopica, Elanco). One month following initiation of treatment, cyclosporine was tapered for ~1 mo. At this time, treatment began with 300 mg of gabapentin (q12h; Teva) for increased limb pain. One week following the termination of cyclosporine treatment and one month before presentation with anorexia, cyclosporine was reinstituted at the initial dose in conjunction with 25 mg of prednisolone (PO q24h; Apotex) due to relapsing dermatologic clinical signs. Weight loss, hyporexia, and lethargy were first noted at this time. One month later, the patient was presented for worsening anorexia, weight loss, and icterus. A review of historical body weight revealed a 5.6 kg decrease in weight over the last 9 mo. Before presentation, all chemistry values were within RIs.

On presentation, the patient was severely icteric with lymphopenia (0.87 × 10⁹/L; RI: 0.9–4.7 × 10/L), severe bilirubinemia (436 µmol/L; RI: 0–4 µmol/L), elevated alkaline phosphatase (ALP) activity (21.8 µkat/L [1,300 U/L]; RI: 0.12–1.92 µkat/L), mild hyponatremia (133 mmol/L; RI: 140–154 mmol/L), and mild hypokalemia (2.9 mmol/L; RI: 3.8–5.4 mmol/L). Alanine transaminase (ALT) activity was above the detectable limit of the chemistry analyzer (Vetscan FUSE; Abaxis). The patient was hospitalized overnight with intravenous fluids and maropitant citrate (Cerenia; Zoetis), resulting in mild improvement of ALP activity (19.2 µkat/L [1,150 U/L]) and bilirubinemia (354 µmol/L). The following day, he was transferred to an emergency veterinary clinic, and a chemistry panel was performed. Activities of ALT (106 µkat/L [6,360 U/L]; RI: 0.28–1.59 µkat/L), aspartate aminotransferase (10.5 µkat/L [627 U/L]; RI: 0.30–0.94 µkat/L), ALP (30.0 µkat/L [1,790 U/L]; RI: 0.12–1.92 µkat/L), and gamma-glutamyl transferase (GGT; 1.60 µkat/L [96 U/L], RI: 0–0.13 µkat/L) were markedly increased, as was the total bilirubin concentration (458 µmol/L; RI: 0–4 µmol/L). All hepatic synthetic function values were within RIs. Neospora caninum ELISA IFA, Toxoplasma IgG and IgM serology, Giardia ELISA, Witness Lepto Rapid Test (Zoetis), and fecal parasite and ova evaluation were all negative.

Abdominal ultrasound revealed changes in both the liver and pancreas, including a diffuse increase of echogenicity of the liver, resulting in isoechogenicity of the spleen and severe gallbladder distension by fluid admixed with a small amount of echogenic debris. Additionally, the common bile duct was distended up to 6.2 mm in diameter. The pancreatic parenchyma adjacent to the duodenal papilla and common bile duct was thickened up to 26 mm and was hypoechoic compared with the remainder of the pancreas; the adjacent mesenteric adipose tissue was hyperechoic.

The following day, the patient underwent a laparotomy to evaluate the liver and biliary tree grossly and to collect biopsy specimens. Gross examination of the liver revealed no significant parenchymal abnormalities; the gallbladder and common bile duct were severely distended. The pancreas was slightly firm diffusely but otherwise was unremarkable. An anti-mesenteric enterotomy performed at the level of the duodenal papilla revealed an enlarged duodenal papilla that was easily catheterized, producing a pale green bile. A pancreatic biopsy was collected from the distal right limb using a guillotine technique. Liver biopsies from the left lateral, left medial, and right lateral liver lobes were collected using a guillotine technique. Biopsy specimens were submitted to the Cornell University Animal Health Diagnostic Center (AHDC; Ithaca, NY, USA) for histologic examination and digital copper quantification.⁷ Liver tissue and bile were combined and submitted for aerobic and anaerobic culture, which were negative for growth (Antech Diagnostics).

Formalin-fixed pancreas and liver biopsy specimens were submitted to the AHDC and processed routinely to produce H&E-stained slides. Histologically, dense streams of fibrous tissue dissected throughout the pancreatic parenchyma. Pancreatic ducts were large, tortuous, filled with cellular debris, and surrounded by dense aggregates of neutrophils, macrophages, lymphocytes, and plasma cells. Ductal epithelial cells were frequently expanded by ~20-µm vacuoles containing protozoa in various stages of development, including schizonts containing myriad ~5 × 1-µm, crescentic organisms (merozoites), and solitary, 20-µm, clear ovoid structures, with central, 5-µm, round basophilic structures (developing oocysts; Fig. 1 ). Multifocally infiltrating the adjacent mesenteric adipose tissue were small aggregates of neutrophils.

Figure 1.

Intraepithelial Hammondia heydorni in the pancreas and liver of a 3-y-old, castrated male Standard Poodle. H&E. A. The pancreatic parenchyma has dense streams of fibrous tissue (asterisk), infiltrated by large numbers of neutrophils, macrophages, lymphocytes, and plasma cells. Pancreatic ducts are tortuous and dilated by cellular debris; ductular epithelium is expanded by vacuolated clear spaces (square). B. From the square in Fig. 1A: pancreatic duct epithelium is expanded by ~20-µm vacuoles with protozoa in various stages of development, including schizonts (arrowhead) and myriad ~5 × 1-µm crescentic organisms (merozoites). Solitary, 20-µm, clear ovoid structures with 5-µm, round basophilic structures are developing oocysts (arrow). C. Biliary duct is encircled by concentric layers of fibrous tissue with moderate numbers of neutrophils and macrophages. Biliary duct is dilated, and the ductular epithelium is expanded by vacuolated clear spaces (square). D. From the square in Fig. 1C: biliary duct epithelium is expanded by ~20-µm vacuoles with protozoa in various stages of development, including schizonts (arrowhead) containing myriad ~5 × 1-µm, crescentic organisms (merozoites). Solitary, 20-µm, clear ovoid structures, with 5-µm, round basophilic structures, are developing oocysts (arrow).

Histologically, all liver lobe samples had expansion of centrilobular hepatocytes by eosinophilic, lacey cytoplasm, interpreted as glycogen. Canalicular cholestasis was prominent ( Fig. 2A ), and bile pigment was frequent within macrophages throughout the liver. Portal tracts were infiltrated by numerous neutrophils, macrophages, lymphocytes, and plasma cells. Biliary ducts were encircled by “onion skin” fibrous tissue, suggesting extrahepatic cholestasis ( Fig. 2B ). Occasionally, bile ducts were barely visible within periductal concentric fibrous tissue ( Fig. 2C ), reminiscent of ductopenia associated with destructive cholangitis). Biliary ducts were prominent and tortuous, and the ductal epithelium was expanded by ~20-µm vacuoles containing protozoa similar to those described in the pancreas (Fig. 1B, 1D). Within periportal parenchyma, occasional foci of hepatocyte loss were infiltrated by a few neutrophils and mild hemorrhage, interpreted as bile infarcts (Fig. 2C). Randomly throughout the lobules, small foci of hepatocellular necrosis were infiltrated by neutrophils, macrophages, lymphocytes, fibrin, and hemorrhage ( Fig. 2D ).

Figure 2.

Histologic features of Hammondia heydorni infection in the liver of a 3-y-old, castrated male Standard Poodle. H&E. A. Many bile canaliculi contain bile plugs (arrowheads). B. Bile ducts are often surrounded by concentric periductal fibrous tissue (arrowhead). C. Occasional bile ducts are barely visible within concentric periductal fibrous tissue (arrowhead), similar to destructive cholangitis with ductopenia. Focal hepatocyte loss with minimal inflammatory cell infiltrates and mild hemorrhage, interpreted as bile infarcts (arrow). D. Parenchyma has random focal hepatocellular loss (arrowheads), with fibrin, hemorrhage, macrophages, neutrophils, and lymphocytes.

A panel of histochemical stains (reticulin, Masson trichrome, picrosirius red, Prussian blue, rhodanine) was applied to a section of the left lateral liver lobe to better characterize liver architecture, fibrosis, and heavy metal accumulation. Tight layers of reticulin surrounded portal areas, interpreted as parenchymal collapse; the reticulin meshwork within the remainder of the liver was within normal limits. Dense streams of fibrillar collagen surrounded portal areas and frequently formed concentric rings. There was no evidence of bridging or sinusoidal fibrosis. A robust population of Kupffer cells contained positive-staining iron granules throughout the entire liver. No stainable copper was found. Digital copper quantification⁷ revealed 273 µg Cu/g (RI: 0–400 µg Cu/g). A Giemsa stain of sections of the pancreas and right lateral liver lobe did not highlight the protozoal organisms noted on H&E; the reason for this unexpected result is unknown and suspected to be related to the Giemsa stain technique used.

PCR was elected to definitively identify the protozoal organisms within the pancreas and liver. DNA was extracted from formalin-fixed, paraffin-embedded tissue scrolls corresponding to the lesions (DNeasy blood and tissue kit; Qiagen). Initial PCR amplification targeted the 18S region of the protozoal ribosomal DNA per the published protocol with slight modifications,¹⁸ including using the Green PCR master mix (Invitrogen; ThermoFisher), staining the amplicons with SYBR Green (Lonza), and visualizing (C200 imaging system; Azure Biosystems). Amplicons were cleaned (E.Z.N.A cycle pure kit; Omega Bio-Tek) and submitted to the Cornell University Institute of Biotechnology for Sanger sequencing. BLAST (blastn, https://blast.ncbi.nlm.nih.gov/Blast.cgi) search of the sequenced DNA revealed 100% agreement with either Hammondia heydorni or Neospora caninum.³ As the 18S DNA marker was of poor resolution to differentiate the protozoal species, we performed PCR and sequencing of the internal transcribed space (ITS1)²⁰ with PCR protocol modifications as noted above; the sequenced amplicons exclusively matched 100% to H. heydorni (GenBank KU253797.1). PCR confirmed the presence of intraepithelial ductal H. heydorni, which was associated with moderate, chronic, lymphohistiocytic and neutrophilic pancreatitis and cholangiohepatitis.

Following histologic diagnosis of protozoal hepatitis and pancreatitis, treatment of the patient began with oral clindamycin. Following discharge, the patient’s hepatic chemistry values slowly improved. One month after initiating treatment, his ALT was 20.1 µkat/L (1,204 U/L), ALP was 71.5 µkat/L (4,281 U/L), GGT was 0.7 µkat/L (41 U/L), and bilirubin was 24 µmol/L. The owners reported that the patient’s appetite and activity level also improved, although never returned to normal. Five weeks following discharge, the patient acutely collapsed with a distended abdomen and marked hypotension. There was no evidence of free fluid within the abdomen. Euthanasia was elected due to the poor prognosis, and no additional testing was performed. It is unclear if the acute decline was related to the protozoal disease or an unrelated issue, as postmortem examination was declined.

H. heydorni is a coccidian parasite, closely related to, and morphologically indistinguishable from, N. caninum and Toxoplasma gondii.⁶ H. heydorni has an obligatory 2-host life cycle.⁸ The life cycle involves intermediate hosts, which include cervids, camels, sheep, goats, buffalo, horses, rabbits, guinea pigs, and dogs.^6,8,15 Intermediate hosts are infected with H. heydorni by ingesting sporulated oocysts.⁸ Once ingested, sporozoites excyst from the oocyst into the intestinal lumen. These sporozoites invade the intestinal epithelium, mesenteric lymph nodes, and other tissues, where they rapidly multiply as tachyzoites. Following this replication, bradyzoites encyst in skeletal muscle where they undergo slow replication.¹⁵ Infectious tissue cysts can develop in the intermediate host as early as 14 d following ingestion of oocysts.⁵ Definitive hosts, such as dogs and wild canids,¹⁵ become infected when they ingest tissue cysts within the skeletal muscle of the intermediate hosts. Once ingested, H. heydorni forms cystozoites within the small intestinal epithelium, which develop into meronts, then gamonts, then oocysts.⁸ Oocysts are shed in the feces of the definitive host 5–8 d after consuming tissue cysts.⁵ Once the oocysts sporulate, which occurs in a minimum of 3 d, they become infective to intermediate hosts when ingested.⁵

H. heydorni oocysts have been reported in the feces of dogs that have consumed raw meat containing H. heydorni tissue cysts. In each of these studies, the type of raw meat included beef, cervid, sheep, chicken, turkey, and guinea pig sources.^4,9,10,14,16 Transmission of H. heydorni to dogs via the consumption of raw cervid meat has been documented⁹; white-tailed deer were naturally infected, suggesting an underlying infection in wild cervid populations.⁹ Infectious tissue cysts have been described in other cervids, including moose and roe deer.¹⁰

H. heydorni has been considered a non-pathogenic parasite in both definitive and intermediate hosts, including dogs.⁵ However, there have been increased reports of dogs with diarrhea that are concurrently shedding H. heydorni oocysts.^19,21 A direct correlation between H. heydorni infection and diarrhea cannot be formed as there are many confounding factors, but, in 1 study, 7 of 12 dogs shedding H. heydorni oocysts had diarrhea.¹⁹ Underlying immunosuppression was suggested to play a role in infection in 1 study; an increased incidence of infection in dogs <6-mo-old was supported by a decrease in incidence once dogs reach adulthood.²

Reports of dogs with acute hepatitis associated with H. heydorni–like protozoa within the bile^1,13 contradict a previous theory that H. heydorni cystozoites only remain in intestinal epithelium until shed as oocysts in the feces. Both reports describe nonspecific clinical signs with increased liver enzyme activity. Cytologic examination of the liver and bile in both cases was consistent with neutrophilic inflammation and intrabiliary coccidian organisms, with features consistent with H. heydorni. In both cases, H. heydorni was confirmed via PCR, but histopathology was not performed.^1,13

In a search of Google and PubMed using the search terms “Hammondia heydorni,” “protozoal cholangiohepatitis,” “protozoal pancreatitis,” and “intraductal protozoal organisms,” we retrieved no cases of pancreatic disease associated with H. heydorni infection, any histologic description of hepatic and pancreatic H. heydorni infection, or development of H. heydorni merogony and gamogony within ductal epithelium of the definitive host, suggesting that this condition has not been reported previously in dogs. The pathogenesis of the biliary and pancreatic ductal infection is not known, but we speculate that the most likely route of infection was retrograde movement of the protozoa through the bile and pancreatic ducts from the duodenum. This contradicts previous theories of solely enterocyte involvement in the definitive host. The periductal biliary fibrosis and ductopenia resemble features of destructive cholangitis, and H. heydorni infection may be an additional differential diagnosis for this uncommon condition in dogs.^{11,12,17,22,23}

Given the case reports of dogs with disease associated with H. heydorni infection,^1,13 there is concern that this protozoon may have become a more pathogenic form. Conversely, as suggested,² clinical disease may be associated with immunosuppression. In our case, the patient had received a potent corticosteroid (Depo-Medrol) and non-steroidal immunomodulator (cyclosporine) on several occasions, which could have caused immunosuppression. Additional studies in dogs infected with H. heydorni will help determine if the increase in clinical disease observed is the result of increased pathogenicity of the protozoan, diminished immune response by the host, or improved detection.

As raw-meat diets gain popularity and there are increased reports of clinical disease associated with H. heydorni infection, this protozoon should be considered an emerging pathogen. The progression of the severity of clinical signs additionally raises concern for safety of raw-meat diets in dogs. H. heydorni should be considered a possible cause of hepatitis or pancreatitis in dogs with a history of consuming raw meat, especially cervid meat.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iDs

Darian Giannino

Mason Jager

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