https://doi.org/10.52973/rcfcv-e33214

Received: 15/11/2022 Accepted: 20/12/2022 Published: 07/02/2023

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Revista Científica, FCV-LUZ / Vol. XXXIII, rcfcv-e33214, 1 - 7

ABSTRACT

Helicobacter species such as H. heilmannii, H. pylori, H. felis, H.

bizzozeronii and H. salomonis have been identied in cats and dogs,

and research suggesting that these species may be zoonotic agents

and has been studied intensied in recent years. The aim of this study

was to reveal the presence, comparing the histopathological ndings

and Real-time PCR results of H. felis, H. heilmannii, and H.pylori in

the stomach and liver tissues taken during the necropsies of owned,

stray or shelter dogs. The material of the study consisted of stomach

and liver tissues taken from 35 dogs that died for different reasons

and were brought for necropsy. DNA copies of H. heilmannii were

detected by Real-time PCR in the liver samples of 30 dogs using

H.heilmannii-specic primers. In the case of gastric samples, Real-

time PCR detected H. heilmannii in 13 cases, H. pylori in 3 cases, both

H. heilmannii and H. pylori in 13 cases, and H. felis, H. heilmannii and

H. pylori in 3 cases. Microscopically, neutrophil leukocyte inltration,

epithelial degeneration, brosis and oedema in the lamina propia, and

lymphoplasmacytic cell inltration were determined in the stomachs.

In the Hemotoxylin Eosin staining of the sections, 5 cases and 14 cases

in the Warthin–Starry staining were found positive for Helicobacter-like

microorganisms. Microscopically, dissociation of the remark cords

and hydropic degeneration in hepatocytes, and focal mononuclear

cell inltrations in some sections were determined in the livers. In

conclusion, with this study, it was understood that Real-time PCR

analyzes are very useful in the diagnosis of H. felis, H. heilmannii, and

H. pylori. However, histopathological examinations are necessary to

associate the presence of bacteria with the development of the disease.

Key words: Canine; histopathology, real-time PCR; H. felis;

H.heilmannii; H. pylori

RESUMEN

Especies de Helicobacter como H. heilmannii, H. pylori, H. felis, H.

bizzozeronii y H. salomonis han sido identicadas en gatos y perros, y en

los últimos años se han intensicado las investigaciones que sugieren

que estas especies pueden ser agentes zoonóticos. El objetivo de este

estudio fue por medio de la comparación de los hallazgos histopatológicos

y los resultados de PCR en tiempo real, revelar la presencia de H. felis,

H. heilmannii y H. pylori en los tejidos del estómago e hígado tomados

durante las necropsias de perros con dueño, callejeros o procedentes

del refugio. El material del estudio consistió en tejidos de estómago

e hígado tomados de 35 perros fallecidos por diferentes causas y que

fueron traídos para la realización de la necropsia. Se detectó ADN de

H. heilmannii mediante PCR en tiempo real en las muestras de hígado

de 30 perros utilizando primers especícos de H. heilmannii. En el caso

de las muestras gástricas, la PCR en tiempo real detectó H. heilmannii

en 13 casos, H. pylori en 3 casos, tanto H.heilmannii como H. pylori en 13

casos, y H. felis, H. heilmannii y H.pylori en 3 casos. Microscópicamente

en el tejido estomacal se observó inltración de leucocitos neutrólos,

degeneración epitelial, brosis y edema en la lámina propia e inltración

de células linfoplasmocitarias. En la tinción de Hematoxilina y Eosina

de los cortes, 5 casos resultaron positivos para microorganismos tipo

Helicobacter y 14 casos en la tinción Warthin-Starry. Microscópicamente,

en el tejido hepático se observó disociación de las columnas de Remark

y degeneración hidrópica en los hepatocitos, e inltraciones de células

mononucleares focales en algunas secciones. En conclusión, con este

estudio se comprendió que los análisis de PCR en tiempo real son muy

útiles en el diagnóstico de H. felis, H. heilmannii y H. pylori. Sin embargo,

los exámenes histopatológicos son necesarios para asociar la presencia

de dichas bacterias con el desarrollo de la enfermedad.

Palabras clave: Canino, histopatología, Real-Time PCR, H. felis,

H.heilmannii; H. pylori

Investigation with Real-Time PCR and Histopathology on the presence of

H. felis, H. heilmannii and H. pylori in dogs

Investigación con PCR en Tiempo Real e Histopatología sobre la presencia de

H. felis, H. heilmannii y H. pylori en Perros

Gokhan Akcakavak

* , Mehmet Tuzcu

, Nevin Tuzcu

, Zeynep Celik

, Aysenur Tural

and Osman Dagar

Yozgat Bozok University, Faculty of Veterinary Medicine, Department of Pathology Sorgun, Yosgat, Turkey.

Selcuk University, Faculty of Veterinary Medicine, Department of Pathology. Selcuklu, Konya, Turkey.

Selcuk University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology. Selcuklu, Konya, Turkey.

*Email: gokhan.akcakavak@bozok.edu.tr

Real-time PCR for diagnosis of Helicobacter species in dogs / Akcakavak et al. ____________________________________________________

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INTRODUCTION

After the detection of Helicobacter pylori in humans and its

association with gastritis, peptic ulcer and gastric neoplasia, research

on spiral-shaped bacteria in both humans and cats (Felis catus) and

dogs (Canis lupus familiaris) has increased [2, 16, 23]. Helicobacters

are known as gram-negative, comma-shaped, ‘S’ or spiral-shaped,

agellate, sporeless, non-encapsulated, microaerobic and non-acid-

fast microorganisms [12, 18]. Helicobacter species such as H. heilmannii,

H. pylori, H. felis, H. bizzozeronii, H. salomonis were detected in cats and

dogs [4]. In addition, studies have been published stating that these

agents are found in 0.2-6% of human gastric biopsies and that they

may be zoonotic agents [10, 33].

H. pylori is known as a pathogen that infects almost half of the

population of the World [7, 34]. It has been reported that H. heilmannii

is found in the gastric mucosa of many animal species, especially

dogs, cats, pigs (Sus scrofas domesticus) and primates (Macaca spp.),

and its pathogenicity is lower than that of H. pylori [5]. Numerous

researchers have reported that they have identied Helicobacter

species in both healthy dogs and suffering from gastritis [28, 31, 32

]. It was stated that although Helicobacter species were observed

in the histopathology of the stomach of cats and dogs, they were

not related to the severity of the inammation [21]. Using scraping

cytology and histopathological examination methods for diagnosing

Helicobacter spp., it was reported that gastric Helicobacter spp. was

detected in dogs at a rate of 84.4% in scraping cytology and 65.6%

in histopathological examinations [6].

Although basic gastrointestinal findings such as vomiting and

diarrhoea have been reported clinically in Helicobacter infections in

cats and dogs [14, 35] some researchers reported that most cats and

dogs with Helicobacter were asymptomatic [8, 27, 30]. Mild gastritis

and mononuclear cell inltration are commonly observed in dogs with

histopathologically determined Helicobacter spp. in the gastric mucosa

[8, 13, 26]. In a previous study, it was reported that glandular degeneration

was more common in the fundus region in Helicobacter spp-infected cats

and dogs compared to non-infected ones. Additionally, pycnotic parietal

cells and enlarged canaliculi have also been reported [35].

Diagnosis of Helicobacter species is made with tests like rapid urease

test, histopathology, culture, Polymerase Chain Reaction (PCR) from

tissue samples or non-invasive tests such as urea breath test, blood

tests, and serological tests [3, 13, 15, 26]. The aim of this current study

was to reveal the presence of H. felis, H. heilmannii, and H. pylori in the

stomach and liver tissues taken during necropsy from owned, stray

dogs and dogs kept in shelters, which died from different causes in

the rst stage, by Real-time PCR and histopathological examination.

Then, compare the histopathological ndings with Real-time PCR

results to determine the incidence of H. felis, H. heilmannii and H. pylori.

MATERIALS AND METHODS

Animal material

The material of the study consisted in the stomach and liver tissues

of 35 dogs that died between 2019 and 2022 due to different reasons

and were brought to Selcuk University Veterinary Faculty Pathology

Department for necropsy. Of the dogs studied in the study, thirteen

were stray dogs staying in the shelter, and thirteen were brought in by

city ocials and animal lovers. The remaining nine were owned dogs.

The study was approved by SÜVDAMEK (Decision number: 2022/76).

Histopathological examination

Stomach and liver samples taken for histopathological examinations

were xed in 10% buffered formalin solution and underwent routine

tissue follow-up procedures. Afterwards, the tissues were embedded

in paraffin, and paraffin blocks were obtained. 4-5 micron (µm)

sections were taken from paran blocks to slides and stained with

Hematoxylin Eosin (HE) and Warthin Starry (WS) methods [22].

Real-time PCR examination

Deoxyribonucleic acid (DNA) copies of H. felis, H. heilmannii and H.

pylori were investigated by Light Cycler 2.0 Real-time PCR (Roche,

Life Sciences, Germany) device using primer probes prepared by a

private company. Deionized water was used as negative control. Primer

sequences used in Real-time PCR analysis are given in TABLE I. DNA

isolation from stomach samples taken for PCR examinations was

performed in accordance with the commercial DNA isolation Kit (Roche,

MagNA Pure LC DNA, Cat No; 03264785001) procedure. The obtained

DNAs were stored at – 20°C (Frigidaire 20.0 Cu. Ft. Upright Freezer,

Turkey). Reaction mixture; 10 microliters (µL) of enzyme and dNTP

mixture, 0,5 µL of forward primer, 0,5 µL of reverse primer, 0,2 µL of

TaqMan probe (20 µM) and 3.8 µl of nuclease-free water, in total, 15 µl

were prepared. The mixture was spun, and then 15 µL of the reaction

mixture was transferred to the capillaries for each reaction, and 5 µL

of sample or control sample was added to each capillary. Thermal

cycle; It was 10 minutes (min) for denaturation at 95°C, 30 seconds

(seg) for annealing at 55°C, and 1 seg for elongation at 72°C. The Light

Cycler 2.0 software was used for the presence-absence data analysis.

TABLE I

Primary probe sequences used in the study

Genes and

Species

Primer sequences Reference

üreA, üreB Genes

(H. felis)

F: 5’-GTGAAGCGACTAAAGATAAACAAT-3’

R: 5’-GCACCAAATCTAATTCATAAGAGC-3’

[9]

üreB Gen

(

H. heilmannii)

F: 5’-GGGCGATAAAGTGCGCTTG-3’

R: 5’-CTGGTCAATGAGAGCAGG-3’

[24]

glmM

(

H. pylori)

F: 5’-GGATAAGCTTTTAGGGGTGTTAGGGG-3’

R: 5’-GCTTACTTTCTAACACTAACGCGC-3’

[20]

“F, forward; R, reverse”

RESULTS AND DISCUSSION

Macroscopic ndings

It was determined that the pathological diagnoses of the necropsied

dogs were bronchopneumonia in four, hepatitis in four, hydronephrosis

in one, meningoencephalitis in one, myocarditis in one, nephritis in

one, trauma in ten and gastroenteritis in thirteen.

Microscopic ndings

Neutrophil leukocyte inltration, epithelial degeneration, brosis

and oedema in the lamina propria, and lymphoplasmacytic cell

inltration were determined in the microscopic examination of HE

stained preparations prepared from samples taken from the antrum

regions of the stomachs during necropsy (FIGS. 1, A-B-C). In addition,

FIGURE 1. A. Degeneration of gastric mucosal epithelium (arrow) and brosis in the propria (arrowhead), x200, HE. B. Inammatory cell inltration

(small picture) and congestion (arrowhead) in the case with H.pylori, x200, HE. C. Inammatory cell inltration (arrow) and degenerative changes

in gland structures (arrowhead) in which all three agents are present (H. heilmannii, H. pylori, H. felis), x200, HE. D. Hydropic degeneration of the

liver (arrow), x400, HE. E. Focal inammatory cell inltrates in the liver (arrow), x400, HE, F. Helicobacter-like microorganisms (arrow), WS Staining.

(HE; Hematoxylin Eosin, WS; Warthin-Starry)

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5 cases in HE staining and 14 cases in WS staining were positive

for Helicobacter-like microorganisms (FIGS. 1, F). The microscopic

examination of HE stained preparations from liver samples revealed

dissociation in the remark cords and hydropic degeneration in

hepatocytes, and focal mononuclear cell inltrations in some sections

(FIGS. 1, D-E). Helicobacter-like organisms could not be detected in

HE stains and WS stains. Histopathological ndings determined in

the microscopic examination of stomach and liver tissues are given

in TABLE II.

TABLE II

Histopathological ndings determined in the histopathological examination of stomach and liver tissues

Microscopic Results H. heilmannii H. pylori H. felis + H. heilmannii + H. pylori H. heilmannii + H. pylori

Epithelial degeneration 4/13 1/3 1/3 4/13

Lymphoid cell inltration in Lamina propria 6/13 1/3 1/3 3/13

Fibrosis in Lamina propria 4/13 1/3 1/3 4/13

Edema in Lamina propria 4/13 2/3 1/3 3/13

Degeneration of gastric gland epithelium 4/13 0/3 1/3 2/13

Degeneration in hepatocytes 8/13 1/3 1/3 3/13

Remark cords dissociation 8/13 1/3 1/3 3/13

Mnh cell inltration in the liver 5/13 0/3 0/3 2/13

Agent in HE 2/13 0/3 1/3 2/13

Agent in WS 4/13 0/3 2/3 8/13

(Mnh: Mononuclear cell inltration, HE: Hematoxylin Eosin, WS: Warthin-Starry)

FİGURE 2. A. Real-time PCR, H. felis amplication curve. B. Real-time

PCR, H. heilmannii

amplication curve in male

Real-time PCR for diagnosis of Helicobacter species in dogs / Akcakavak et al. ____________________________________________________

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Real-Time PCR (qPCR) ndings

The detection rates of H. felis, H. heilmannii and H. pylori in stomach

samples by Real-time PCR and the rates of detection of H. heilmannii,

H. pylori and H. felis in gastric samples according to age and gender are

given in TABLE III. The relevant amplication curves are given in FİG 2.

The presence in gastric samples of H. heilmannii in 13 cases, H. pylori

in 3 cases, both H. heilmannii and H. pylori in 13 cases, and H. felis, H.

pylori and H. heilmannii in 3 cases was determined by Real-time PCR.

In the study performed with Real-time PCR on liver samples using

H. heilmannii-specic primers, the DNA of H. heilmannii was detected

in 30 dogs, while DNA of H. felis and H. pylori could not be determined.

The detection rates of H. heilmannii in liver samples by Real-Time

PCR and the distribution of these rates according to age and gender

are given in TABLE IV

TABLE III

Distribution of relevant agents by gender and age in stomach samples

N Stomach Male Female < 1 year old 1-3 years old > 3 year old

H. felis - - - - - - -

H. heilmannii 13 13/35 7/35 6/35 4/35 5/35 4/35

H. pylori 3 3/35 2/35 1/35 1/35 1/35 1/35

H. felis + H. pylori

H. heilmannii

3 3/35 2/35 1/35 1/35 1/35 1/35

H. pylori

+ H. heilmannii

13 13/35 6/35 7/35 5/35 4/35 4/35

TABLE IV

Distribution of H. heilmannii by age and gender

Agent N Male Female

< 1 year

old

1-3 years

old

> 3 year

old

H. heilmannii 30 16/35 14/35 10/35 9/35 11/35

It was observed that 16 of 30 cases of H. heilmannii determined by

Real-time PCR from liver samples were male dogs, and 14 of them were

female dogs. It was determined that 10 of the dogs with H. heilmannii

were younger than 1 year old, 9 of them were between 1-3 years old,

and 11 of them were older than 3 years old.

Determination of hepatitis and gastroenteritis in microscopic

examination of stomach and liver samples, entity of Helicobacter-like

organisms in HE and WS staining of samples taken from stomach

and determination of H. pylori, H. felis and H. heilmannii DNA copies

in these tissues by Real-time PCR are shown in TABLE V.

DNA copies of H. felis, H. heilmannii or H. pylori could not be

determined in the liver of 5 dogs and in the stomach of 6 dogs in

the examinations made with Real-time PCR. In 2 of these dogs,

degeneration of gastric mucosal epithelium, degeneration of

hepatocytes and dissociation of remark cords were detected in

the liver. In one dog, in addition to the degeneration found in the

gastric mucosa epithelium, oedema, brosis in the lamina propria

and degeneration in the gland epithelium were observed.

It is seen that the tendency towards adopting pets (cats and dogs)

and meeting their needs such as care and nutrition is increasing day

by day [1, 29]. Helicobacter species such as H. heilmannii, H. pylori, H.

felis, H. bizzozeronii, H. salomonis were detected in cats and dogs [4]. In

order to reveal the pathology and zoonotic importance of Helicobacter

species, studies have been performed in the elds of both human and

veterinary medicine [6, 9, 31]. In studies investigating the presence of

Helicobacter spp. in dogs, gastric Helicobacter was determined at rates

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Table V

Determination of hepatitis and gastroenteritis in microscopic examination of stomach and liver

samples, entity of Helicobacter-like organisms in H-E and WS staining of samples taken from stomach

and determination of H. heilmannii, H. felis and H. pylori in these tissues by Real-time PCR

Histopathology Agent Real Time PCR

Hepatitis Gastroenteritis HE WS

Stomach Liver

H. felis H. heilmannii H. pylori H. felis H. heilmannii H. pylori

1 - + - + - + + - +

2 - - - - - + - - + -

3 - + - - - + + - + -

4 - - - - - + - - + -

5 + + + + - + - - + -

6 - - - - - - - - - -

7 - + - + - + + - + -

8 - + - + + + + - + -

9 - - - - - + - - + -

10 - - - - - - + - - -

11 - + - + - + - - + -

12 - - - - - - + - - -

13 + - - - - + - - + -

14 + - - - - + + - + -

15 - - - + - + + - + -

16 - + - - + + + - + -

17 - - - - - + - - + -

18 - + + + + + + - + -

19 - + - + - + + - + -

20 - - - - - + + - + -

21 + - - - - + - - + -

22 - - - + - + + - + -

23 - + + + - + + - + -

24 - - - - - - - - - -

25 - - - - - + - - + -

26 - - - - - - + - + -

27 - - - + - + + - + -

28 - + + + - + + - + -

29 - - - - - + - - + -

30 - - - - - + - - + -

31 - - - - - + - - + -

32 - + - + - + + - + -

33 - - - - - - - - - -

34 - + + + - + - - +

35 - - - - - + + - + -

4 13 5 14 3 29 19 - 30 -

(HE: Hematoxylin Eosin, WS: Warthin-Starry)

Real-time PCR for diagnosis of Helicobacter species in dogs / Akcakavak et al. ____________________________________________________

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ranging from 67 to 100%. For this reason, studies have focused on dogs

with the suspicion that dogs with whom people share their habitats

may be the source [3, 8, 18, 19]. In this study, H. pylori was determined

in 54.28%, H. heilmannii in 82.85%, and H. felis in 8.57% by Real-time

PCR in dogs, revealing the importance of studies on the prevalence

and diagnosis of Helicobacter-like organisms in dogs.

Happonen et al. [12], in their study on the diagnosis of Helicobacter

spp. in dogs and cats, reported that they determined Helicobacter spp.

in 20 (95%) of 21 dogs with gastric clinical signs and in all (100%) of the

25 dogs without gastric clinical signs. Hwang et al. [17] reported the

presence of Helicobacter in 77.5% of dogs with gastric symptoms and

67.5% of dogs with no gastric symptoms. The researchers interpreted

that the prevalence of Helicobacter was determined at similar rates

in dogs with and without gastric symptoms, as Helicobacter species

may be a part of the normal gastric ora. In this study, DNA copies

of H. felis, H. pylori and H. heilmannii were determined in 100% of the

stomach samples of 13 dogs with gastroenteritis and in 20 (90.90%) of

the stomach samples of 22 dogs without gastroenteritis, supporting

the conviction that Helicobacter-like organisms may be a part of the

stomach ora.

Eaton et al. [8] detected Helicobacter spp. in all (100%) of 31 stray

dogs and in 10 (67%) of 15 owned dogs. In this study, the detection

by Real-time PCR of H. felis, H. pylori and H. heilmannii in 6 (66.66%)

of 9 owned dogs was similar to the ndings of Eaton et al. [8]. Again

in this study, DNA copies of H. felis, H. pylori and H. heilmannii were

determined in 10 (76.92%) of 13 shelter dogs and 10 (76.92%) of 13 stray

dogs. These rates were found to be higher than the rates determined

in owned dogs, which is consistent with the results of Happonen et

al. [12] that the prevalence of Helicobacter spp in stray dogs is higher

than in owned dogs.

Although Hänninen et al. [11] stated that the presence of Helicobacter

spp. is higher in young dogs, Happonen et al. [12] stated that they

found Helicobacter spp. at an equal rate in young and old animals in

their studies on the prevalence of Helicobacter. In this study, of 32

cases identied as H. felis, H. pylori and H. heilmannii by Real-time

PCR, 11 were under 1 year old, 11 were between 1-3 years old and 10 were

over 3 years old. Similarly, in this study, out of 30 liver samples of H.

heilmannii, 10 belonged to dogs under 1 year old, 9 to dogs between

1-3 years old and 11 to dogs older than 3 years. Our ndings were

consistent with those of [12].

Sagnak and Ozgur [32] found Helicobacter spp. DNA in 29 (58%) of

the samples taken from 50 male dogs and 36 (78.3%) of the samples

taken from 46 female dogs in the evaluation made by PCR in dog faeces;

it was reported that the difference between female and male dogs

was statistically signicant (P=0.034). Considering the distribution

of Helicobacter spp. DNA copies in both liver tissues and stomachs by

gender in this study, it is understood that it was higher in males, unlike

Sagnak and Ozgur [32]. However, a statistical comparison could not

be made because the number of samples was insucient.

Husnik et al. [16] reported in their studies using molecular, cytologic and

histopathologic techniques to determine the prevalence of Helicobacter

spp. and its correlation with gastric pathology, that they detected 71.4%

Helicobacter spp. by PCR diagnostic method and H. heilmannii was

the predominant species. Similar to the literature, in this study it was

determined that H. heilmannii was the dominant species detected by

Real-time PCR in both liver and stomach tissues. A positivity rate of

85.7% in liver tissues and 82.85% in stomach tissues was determined,

which is slightly higher than the rate determined by Husnik et al. [16].

Hermanns et al. [14] reported that they detected Helicobacter in

82% of dogs in their study to determine the histopathological changes

in cats and dogs carrying Helicobacter spp.. They reported that in

these dogs, glandular degeneration with neutrophil granulocyte and

lymphocyte inltration, brosis and oedema in the lamina propria,

and lymphoplasmacytic inltrates were detected microscopically.

Husnik et al. [16] reported the absence of a signicant relationship

between Helicobacter spp. infection status and the presence

of inammatory inltration and gastritis, epithelial damage, and

brosis. The histopathological ndings determined in this study

were consistent with the literature.

Diagnostic methods such as histopathology, cytology, culture, urea

breath test and serological tests are constantly used in the diagnosis

of Helicobacter infections in human medicine. However, the only way

to identify agents at the species level is through PCR and culture tests

[25]. When the ndings of this study were evaluated, it strengthened the

opinion that Real-time PCR was a rapid, specic, sensitive diagnostic

test that can be used to detect Helicobacter-like organisms in dogs.

CONCLUSION

In conclusion, with this study, the presence of H. felis, H. heilmannii

and H. pylori in stomach and liver samples taken during necropsy

from dogs who died from different causes, living in shelters and on

the street was revealed by Real-time PCR and histopathologically.

It has been understood that Real-time PCR analyses are very useful

in the diagnosis of H. felis, H. heilmannii and H. pylori, however

histopathological examinations are necessary to correlate the

presence of bacteria with the disease state.

Conict of interest

The authors declare that they have no conict of interest.

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