Overview on Campylobacter Situation in Pets

Overview on Campylobacter situation in pets

        El-Gedawy,A .A*Rasha A. Mohsen** and Abeer Elrefaie**Yasser Hana*

*Bacteriology Dept., Animal Health Research Institute, Dokki. **Bacteriology Dep., AHRI, Mansoura.

Abstract

Bacteriological examination of 200 collected fecal swap from both human (80) and pet animal (120) show that the rate of campylobacter positive sample was 49 sample(24.5%) with (20%)  in pet animal and (31.25%) in human, the result of isolation  was (21.5%).Animal was grouped to healthy and symptomatic with nearly equal rate of isolation, convential PCR used for campylobacter spp. Detection using  C. coli ceuE and  C. jejuni mapA  where all the samples was positive for c.jejuni, campylobacter pathogensity was examined using the following virulence genes (flaA , wlaN,  VirB11), gene sequencing was performed to detect identify between both human and pet samples.

Introduction 

Campylobacter is Gram-negative, non-spore forming, curved S-shaped rods, microaerobic, catalase and oxidase positive bacteria.

Campylobacter is one of the most common causes of zoonotic gastroenteritis in developed and developing countries (ECDC, 2015).

Pet animals can be colonized with numerous Campylobacter spp including mainly C. jejuni, C. upsaliensis, and C. helveticus.  Pets are known as a carrier since about 50% of the healthy dogs can shed the campylobacter spp. in feces without any clinical signs with similar rate of isolation between symptomatic and asymptomatic dogs ( Smith and Taylor 2019).

FlaA sequence typing is a genotyping method that is increasingly used in epidemiological investigations of Campylobacter species  FlaA sequencing is another commonly used, cost-effective, and well-accepted sequence based typing method for Campylobacter with low levels of non-typeability ( Kittl et al., 2013).

Campylobacter infection in pets and humans from a common source is possible. However, the importance of pets as a source of Campylobacter infections to human remains unclear as genetic studies have shown that human and canine strains are distinct, suggesting that host-specific genotypes may exist among strains ( Damborg et al., 2008).

Dogs feeding diet containing raw meat especially poultry are considered to be one of the most important risk factor (LeJeune and Hancock 2001). Feeding of poultry slaughter-house offal is the main source of pet animals feeding in Egypt.

In addition, there are no studies undertaken to highlight the campylobacter infection in pet animals in Egypt. So, the aim of this work was :

Isolation of campylobacter species from pet animals and human.Prevalence of Campylobacter in fecal samples in healthy and diarrheic pet animals and human.Biochemical and molecular identification of isolated strains.Molecular detection of some virulence genes of isolated stains.Gene sequencing for determination of similarity between isolated strains in pet animals and human to identify the possibility of transmission between them.

Material and Methods

sampling: (OIE 2008)

The total number and classification of samples was illustrated in table (1).

Table (1): The total number and classification of samples

. Isolation and Identification: according to (ISO 2006):

.Molecular Identification:

DNA extraction : using QIAamp DNA Mini Kit ( Catalogue no.51304)

Oligonucleotide primers sequences used in cPCR

Table (2): Oligonucleotide primers sequences used for PCR assay:

Table (3): Preparation of PCR Master Mix for cPCR

 -  Cycling conditions of the primers during cPCR 

Temperature and time conditions of the two primers during PCR are shown in Table (4).  

Table (4): Cycling conditions of the primers during cPCR.

purification of the PCR Products:

Using QIAquik PCR product purification protocol, Using QIAquick PCR Product extraction kit. (Qiagen Inc. Valencia CA).

Sequencing reaction:

  A purified RT-PCR product was sequenced in the forward and/ or reverse directions on an Applied Biosystems 3130 automated DNA Sequencer (ABI, 3130, USA). Using a ready reaction Bigdye Terminator V3.1 cycle sequencing kit. (Perkin-Elmer/Applied Biosystems, Foster City, CA), with  Cat. No. 4336817.

A BLAST® analysis (Basic Local Alignment Search Tool) (Altschul et al., 1990) was initially performed to establish sequence identity to GenBank accessions. The sequence reaction was done according to the instruction of the manufacture as following:

Table (5): Preparation of master mix using Big dye Terminator V3.1 cycle sequencing

 Phylogenetic analysis:

A comparative analysis of sequences was performed using the CLUSTAL W multiple sequence alignment program, version 1.83 of MegAlign module of Lasergene DNAStar software Pairwise, which was designed by ( Bacon et al.,2002) and  Phylogenetic analyses were done using maximum likelihood, neighbour joining and maximum parsimony in MEGA6 (Tamura et al., 2013).

Results:

 Table (6): Prevelance of campylobacter species in fecal samples of pet animals and human:

Table (7): Prevelance of campylobacter species in fecal samples of pet animals and human according to Health status:

Table (8): Molecular characterization of Campylobacter spp. in pet animals and human.

Fig (1): Agarose gel electrophoresis of conventional PCR for detection of C. jejuni mapA gene in 10 examined samples from pet animals and human showing amplification of 589 bp. fragment.

L (lader): 100 bp plus DNA ladder (100-3000 bp).

(Pos): positive control. (Campylobacter jejuni strain identified by RLQP). 

(Neg): negative control. (Master Mix without DNA).

Lanes (3, 4, 5,6, 7, 8, 9 and 10): positive samples. Lane (6): negative sample.

Fig (2): Agarose gel electrophoresis of conventional PCR for detection of C. coli ceuE spp. in 10 examined samples from pet animals and human showing negative resluts.

L (lader): 100 bp plus DNA ladder (100-3000 bp). (Pos): positive control. (C. coli strain identified by RLQP).  (Neg): negative control. (Master Mix without DNA).

Moleculer detection of C. jejuni virulence genes:

Molecular charaterization of 7 different virulence genes (23S rRNA, wlaN, VirB11,     flaA) were illustrated in table (9).

Table (9): Moleculer detection of C. jejuni virulence genes.

Nd: not detected

 23s RNA gene

All the ten representive samples were examined for the presence of 7 virulance genes. Result showed that 8 samples (no. 3,4,5,6,7,8,9,10) was positive for 23s rRNA (80%) at 6500 bp.

Fig (3): Agarose gel electrophoresis of conventional PCR for detection of C. jejuni 23s rRNA gene in 10 examined samples from pet animals and human showing amplification of 6500 bp. fragment. L (lader): 100 bp plus DNA ladder (100-3000 bp).

(Pos): positive control. (Campylobacter jejuni strain identified by RLQP). 

(Neg): negative control. (Master Mix without DNA). Lanes (3, 4, 5, 6, 7, 8, 9 and 10): positive samples. Lane (1,2): negative sample.

wlaN gene

All the eight samples show negative result to all of wlaN.

Fig (4): Agarose gel electrophoresis of conventional PCR for detection of C. jejuni WlaN gene in 10 examined samples from pet animals and human showing amplification of 672 bp. fragment. L (lader): 100 bp plus DNA ladder (100-3000 bp).

(Pos): positive control. (Campylobacter jejuni strain identified by RLQP). 

(Neg): negative control. (Master Mix without DNA). Lanes (1 to 8): negative samples.  Virb11 gene.

All the eight samples show negative result to Virb11 gene.

Fig (5): Agarose gel electrophoresis of conventional PCR for detection of C. jejuni Vibr 11 gene in 10 examined samples from pet animals and human showing amplification of 449 bp. fragment. L (lader): 100 bp plus DNA ladder (100-3000 bp).

(Pos): positive control. (Campylobacter jejuni strain identified by RLQP). 

(Neg): negative control. (Master Mix without DNA). Lanes (1 to 8): negative samples.

FlaA gene

The result confirmed that only three samples out of the eight samples were positive for flaA gene (37.5%). Human samples (3, 7) and animal sample (5).

Fig (6): Agarose gel electrophoresis of conventional PCR for detection of C. jejuni flaA gene in 8 examined samples from pet animals and human showing amplification of 855 bp. fragment. L (lader): 100 bp plus DNA ladder (100-3000 bp).

(Pos): positive control. (Campylobacter jejuni strain identified by RLQP). 

(Neg): negative control. (Master Mix without DNA). Lanes (3, 5, 7): positive samples.

Lane (4, 6, 8, 9, 10): negative samp

FlaA gene sequencing:

A representative positive isolates from pet animal (L5) and human (L7) were were selected for C. jejuni characterization by FlaA gene sequencing and were submitted to NLQP for sequencing of the PCR product. The data of the chosen isolates was illustrated in table (14). It was conducted in forward directions and the nucleotide sequence of the FlaA gene from nucleotide positions 0 to 830 were determined for each isolate and used for nucleotide analysis and deduced amino acid analysis.

Table (10): Data of the chosen isolates for sequencing:

FlaA nucleotide sequencing

 The sequence analysis of the FlaA gene of the two isolates strains were used for comparison in this study. The result indicated that the the identiy percentage between the two representative isolates was 95%.

The flaA sequences of the two representive isolates were determined where BLAST analysis revealed significant homology with reported Campylobacter species at the NCBI GenBank database. The data of these referenced campylobacter species showed in table (15). the the result showed that the two chosen isolates were more closly related to isolates from different sources including human, chicken and turkey (Tab. 15 &Fig.18).

Phylogenetic tree was constructed from the nucleotide sequences of the flaA gene to assess the genetic relationship among the C.jejuni isolates (Fig. 7)

Phylogenic tree was separated into two main distinct branches, one branch include only C.jejuni strain CS0052 (JQ991582.1) and the other branch include the rest of isolates

 The tree shows that our representive isolate (L7 human) was closely related to C. jejuni strain B30 KF846024.1 (isolated from human in Tanzania) and Campylobacter jejuni strain B24 KF846024.1 (isolated from human in Tanzania), Campylobacter jejuni strain Cjst25 KM396359.1 (isolated from human in Iran)

The tree also shows that our representive isolate (L5 dog) was closely related to C. jejuni strain L17 AF050193.1 (isolated from poultry in USA) and C. jejuni D2290 AF050189.1 (isolated from poultry in USA).

Table (11): Nucleotides identity of the chosen C. jujeni isolates from pet animal (L5) and human (L7) in comparison to commonly used reference isolates obtained from GenBank.

Fig (7): Phylogenitic tree of FlaA gene sequence from C. jujeni isolated from pet animals (L5_C.Jejuni_dog) and human (L7_ C.Jejuni_human) and closely related isolates obtained fom Gene Bank

 Discussion

Campylobacter is one of the most common causes of zoonotic gastroenteritis in developed and developing countries (ECDC 2015). Consumption of contaminated meat especially poultry, drinking contaminated water, contact with pets, also during traveling are the most likely sources of Campylobacter contamination (Kittl et al., 2012).

Result indicated that the overall prevalence of Campylobater spp was 24.5% (49/200). The prevalence in human samples was 31.25% (25/80).

There is variation among campylobacter isolation in human population. Lower percentage of Campylobacter isolation was reported by Rozynek and Dzierzanowska (1994) who found that the mean frequency of C. jejuni infections did not exceed (9.1%) and (Pazzaglia et al. 1995) who reported 17.2% in hospitalized diarrheic children in Alexandria, Egypt.

On the other hand, higher prevalence of C. jejuni was approximately 46% among annual bacterial foodborne illnesses in the USA ( Frost et al., 2002).

Concerning pet animals, our investigation revealed that the overall prevalence of Campylobater spp was 20% (24/120). The prevalence was 20.8% (15/72) in dog and 18.57% (9/48) in cat.

The rate of isolation of campylobacter in dog (20.8%) is little higher than that of the cat (18.75%) and this was in agreement with the previously reported results of ( Frost et al., 2002). who reported higher prevalence in dogs than cat.

The study  detected  the rate of isolation in both healthy and diseased animal to confirm the ability of pets to be a carrier without any clinical signs and possibility of transmission to human. .Some Previous studies showed that approximately 50% of the healthy dogs can shed campylobacter spp. in their feces without any clinical signs which indicates similar rate of isolation between symptomatic and asymptomatic dogs. The reason for equal rate of isolation between symptomatic and asymptomatic pets  may be due to the ability of the pet animal to shed the bacteria in their feces for long period of time in contrast to human(Romich, 2008).

Molecular characterization of C. jejuni was carried out. In our study, molecular identification of 8 out of 10 reprehensive samples was confirmed to be C. jejuni through investigation of its characteristic mapA gene which indicates that the recovery of C.jejuni in the chosen isolates was 80%.  In contrast, investigation of C. coli ceuE spp. gene in the examined samples from pet animals and human showing negative results .This was in agreement with  Couturier et al. (2012) who state that C. jejuni is considered to be the most commonly isolated species out of the 17 species within the genus Campylobacter as it cause  90% of the infections .

The conventional PCR used for detection of campylobacter strain virulence genes revealed that all the 8 representive samples was positive for 23s rRNA (100%).wlaN gene was not detected in any sample where (Bacon et al. 2002) found that the detection rate of the wlaN gene from human clinical samples (25.0 %) was similar to that from poultry meat (23.8 %).All the 8 representive samples show negative result for VirB11. This was in accordance with Bacon et al. (2002) .

Three samples out of the eight representative samples showed positive results for the flaA gene (37.5%)  and the positive samples was obtained from human (no. 3,7) and animal (no. 5).

   FlaA sequence typing was performed. FlaA sequence is a genotyping method that is increasingly used in epidemiological investigations of thermo-tolerant Campylobacter species (Kittl et al., 2012).

Nucleotide sequence of the flaA gene showed 2 highly variable regions; one at positions 539 to 689 designated the flaA and a large variable region ( flaA-LVR), at approximately positions 700 to 1,600 where The sequence data of the LVR showed similar results to those of the SVR; owing to the length of the sequence and the complexity of LVR data analysis, this region is not suitable for molecular epidemiological studies of Campylobacter infections ( Singh and Kwon 2013).

The flaA sequences of the two representive isolates were determined  and undergo BLAST analysis which revealed significant homology with reported Campylobacter species at the NCBI GenBank database, the result showed that the two chosen isolates were more closely related to isolates from different sources including human, chicken and turkey.

There is a shortage in the literature reviewing the molecular typing and gene sequencing of C. jejuni in pet animals. On the other hand, many studies were undertaken in especially in poultry. 

Phylogenetic tree was constructed from the nucleotide sequences of the flaA gene to assess the genetic relationship among the c.jejuni isolates whereas  the tree shows that our representive isolate (L7 human) was closely related to C. jejuni strain B30 KF846024 (isolated from human in Tanzania) and C. jejuni strain B24 KF846024 (isolated from human in Tanzania), C. jejuni strain Cjst25 KM396359 (isolated from human in Iran)  the high identity rate between our isolate (L7 human, Egypt) and human isolates from other countries indicates that campylobacter can be worldwide.

The tree also shows that our representative isolate (L5, dog) was closely related to C. jejuni strain L17 AF050193 (isolated from poultry in USA) and C. jejuni D2290 AF050189 (isolated from poultry in USA).

These high degree of identity between our isolate (L5 dog) and poultry show that dogs can acquire the infection from poultry as mentioned before by LeJeune and Hancock (2001) who state that dogs feeding diet containing raw meat especially poultry are considered to be one of the most important risk factor of campylobacter infection.

Conclusion

Consumption of contaminated meat, especially poultry considered to be one of the major source of Campylobacter infections in both human and pets so Veterinarians should advise owners of the potential dangers of using raw or undercooked protein sources for their pets.

human can acquire infection from their pets through the fecal oral route there for owners should practice appropriate hygienic measures.

The high identity between the C. jejuni isolates from pet animal and human potentiate the hypothesis of zoonotic possibility of campylobacter transmission between pet animal and human.