Laboratory evaluation of the GIT

Question 1

Discuss the ways in which Helicobacter infection can be diagnosed in the stomach of dogs.

Answer 1

Helicobacter can be diagnosed via:

1. Gastric biopsy - histopathology
   
   • Special stain - Warthrin Starry
2. PCR (on biopsy tissue)
3. Brush cytology obtained via endoscopy
4. Fluorescent in situ hybridization
5. Indirectly via urease test on gastric biopsy
6. Culture?

Question 2

Helicobacter

Discuss the evidence surrounding the clinical significance of Helicobacter infection in dogs

Answer 2

• AVJR paper from 2017 (Chile) revealed no correlation between number or density of Helicobacter-like organisms and gastritis scores on endoscopy or biopsy.
• 2007 study by Leib et al documented an improvement in vomiting and gastritis scores after treatment with amoxicllin, metronidazole and bismuth subsalicylate (with or without famotidine) in 24 dogs.
• A 2012 study by Castiglioni (Vet Microbiol) identified Helicobacter in colonic biopsy samples from dogs. There was a significant association between heavy infections and mucosal fibrosis/atrophy. The heavy infections were also more prevalent in younger dogs
• Transmission between infected dogs has been demonstrated in controlled studies: Treated dogs kept in isolation did not show recurrence, where treated dogs allowed contact with positive dogs had recurrence of infection 80% of the time.
• Omeprazole has been shown to reduce numbers of Helicobacter organisms in gastric biopsies
• 4/13 cats in an experimental study had persistence of Helicobacter DNA despite treatment with amoxicillin, metronidazole, clarithromycin and omeprazole for 14 days.

Question 3

What laboratory tests are available for the diagnosis of parvovirus infection?

What is the sensitivity and specificity of each available test?

Answer 3

1. In-house ELISA test is the most commonly used test
   
   • Sensitivity is reduced when compared to PCR
     
     • Sensitivity has been reported between 18% and 82%
     • False negatives can be seen with low faecal virus load and high antibody titres (Ab binding of virus)
   • Highly specific though false positives can be seen with recent vaccination
2. PCR - more sensitive than ELISA, but delay in receiving results compared to bedside test.

Question 4

Note the various bacteria that can be identified via PCR testing

Answer 4

1. Salmonella spp
2. Campylobacter jejuni, Campylobacter coli
3. Canine Distemper virus
4. Canine enteric coronavirus
5. Clostridium spp and the clostridial toxin genes

Question 5

What is FISH?

Describe how FISH contributes to a diagnosis of enteroinvasive E. Coli

Answer 5

• FISH utilises a fluorescently labelled genetic probe specific for a segment of DNA
• The probes specifically identify unique segments of nucleic acid
• Fluorescence microscopy is then used to identify where the fluorescent probe is bound.
• Often utilised in human medicine to identify specific DNA sequences for genetic councelling or to identify RNA sequences in circulating tumour cells
• For enteroinvasive E. coli, FISH is used on colonic biopsy specimens of dogs with ulcerative colitis
• Fluorescent markers specific for a segment of the E coli DNA are utilised.
• Identification of the fluorescent marker in the presence of ulcerative colitis is diagnostic of the disease.

Question 6

Note the various tests available for detection of GIT endoparasites

Answer 6

Available tests include:

1. Faecal smear - Giardia, protozoal and helmith infections may be detected but with a low sensitivity
2. Faecal floatation - good test for the diagnosis of helmithiasis and useful for Giardia. Sensitivity is increased with zinc sulphate floatation and centrifugal floatation
3. Immunofluorescence - excellent test for the diagnosis of Giardia and cryptosporidium
4. ELISA for Giardia - highly sensitive, but less specific than immunofluorescence
5. PCR - numerous organisms and only test with appropriate sensitivity for Tritrichomonas blagburni

In practicality, floatation techniques combined with PCR is the most utilised broad diagnostic test for intestinal parasitism.

Question 7

List the various tests that can be used to assess intestinal function

Answer 7

1. Serum folate
2. Serum cobalamin
3. Faecal alpha₁-Proteinase Inhibitor Concentration
4. Serum C-reactive Protein
5. Gastrointestinal Permeability Testing
6. Serum or Faecal concentrations of inflammatory markers
7. Gastrointestinal endocrinology

Question 8

Discuss the mechanism of folate absorption and the disease processes that can lead to folate malabsorption

When might serum folate be increased?

Answer 8

• Folate is a water soluble B vitamin (Vitamin B₉)
• Most folate within food is presented as folate polyglutamate
  
  • Folate polyglutamate is not readily absorbed by the GIT
• Folate polyglutamate is deconjugated to folate monoglutamate in the proximal small intestine
  
  • Reaction is catalysed by the brush border enzyme folate deconjugase
• Folate monoglutamate is transported by specific folate carriers in the prox. small intestine.
• Proximal small intestinal disease can lead to decrease of both folate deconjugase and the folate carriers
  
  • Leads to decreased folate absorption
  • With chronicity, body folate stores can become depleted
• Serum folate concentrations can be increased in cases of intestinal dysbiosis due to bacterial production of folate.

Question 9

Describe the process of cobalamin absorption in the gastrointestinal tract.

Answer 9

• Normal dietary cobalamin (vitamin B₁₂) is plentiful in most foods and bound to animal-based dietary proteins
• Gastric acid and pepsin digest the protein and release cobalmin
• Free cobalmin is bound to the cobalamine transport protein, R-protein in the stomach
• R-protein is digested by pancreatic proteases and bound by intrinsic factor
  
  • intrinsic factor is almost exclusively produced by the exocrine pancreas
• Complexed cobalamin and intrinsic factor are absorbed in the distal ileum by binding to specific cobalmin receptors
• Cobalamin is then bound to transport proteins (transcobalamin) within the circulation and transported to the cells

Question 10

Which disease processed can cause cobalamin deficiency or excess and why

Answer 10

1. Exocrine pancreatic insufficiency
   
   • Cobalamin-R protein complex is not digested.
   • Lack of intrinsic factor reduces cobalamin binding
   • Intrinsic factor binding is essential for transport from the ileum via the cobalamin receptor
2. Ileal disease
   
   • Inflammatory or other disease within the ileum causes a loss of cobalamin receptors and thus reduced absorption
3. Intestinal dysbiosis may lead to cobalamin deficiency due to microbial competition for abailable cobalamin

Question 11

Faecal Alpha₁-Proteinase Inhibitor

In what circumstances would Alpha₁PI be increased?

When is testing for faecal increases indicated

Answer 11

• Alpha₁-Proteinase Inhibitor is synthesized within the liver and has a similar size to albumin (~60,000 Da)
• Alpha₁-PI is not hydrolysed by GIT and bacterial proteinases
• Alpha₁-PI loss in the faeces can be a surrogate for intestinal protein loss.

Clinical Indications:

• In patients with hypoalbuminaemia without gastrointestinal signs for which another cause for the low albumin cannot be determined
• Dogs with a high incidence of familial protein losing enteropathy such as soft coated wheaten terriers intended for breeding purposes.

Question 12

List the parameters assessed in the CIBDAI

Answer 12

Each parameter is assessed as either: normal (0), mild (1), moderate (2) or severe (3). A score of 0-3 is assigned to each parameter.

• Attitude / activity
• Appetite
• Vomiting
• Stool consistency
• Stool frequency
• Weight loss: None, < 5%, 5-10%, > 10%