CHAPTER ONE
1.0 GENERAL INTRODUCTION
Campylobacteriosis, caused by thermophilic bacteria of the genus Campylobacter, is a
significant zoonotic gastrointestinal disease affecting humans and animals, including dogs,
globally (Coker et al., 2002; Kaakoush et al., 2015; Carron et al., 2018; EFSA and ECDC, 2018;
Elmali and Can, 2019; Igwaran and Okoh, 2019). In humans, the disease is characterized by
fever, diarrhea, and pain. It is caused by Campylobacter jejuni and Campylobacter coli and
rarely, by other emerging Campylobacter species including Campylobacter lari, C. concisus, and
C. upsaliensis (Nachamkin and Blaser, 2000; Vandamme, 2000; Prasad et al., 2001; Sahin et al.,
2002; Moore et al., 2005; Man, 2011; Selwet and Galbas, 2012; Kaakoush et al., 2015). Majority
of the human infections are attributed to consumption of contaminated poultry (Wingstrand et
al., 2006; Sahin et al., 2015; Tresse et al., 2017; Carron et al., 2018), raw milk (Peterson, 2003)
and water (Diergaardt et al., 2004; Galanis, 2007; Abe et al., 2008).
Dogs were first associated with human campylobacteriosis in 1960 (Wheeler and Borchers,
1961) and Campylobacter jejuni was the first species isolated from dogs in 1977 (Skirrow,
1977). Since then, several studies have reported C. jejuni isolation from healthy and diarrheic
dogs worldwide with pathogenic involvement occurring most frequently in puppies (less than
one year) and is precipitated by factors such as crowding, stress, and concurrent diseases
(Engvall et al., 2003; Hald et al., 2004; Marks et al., 2011).
Most studies have found that the most frequently isolated species from dog feces is C.
upsaliensis, followed by C. jejuni and less frequently C. coli, C. lari, and C. hyointestinalis
1
,(Engvall et al., 2003; Hald et al., 2004; Workman et al., 2005; Rossi et al., 2008; Acke et al.,
2009; Koene et al., 2009; Parsons et al., 2011; Goni et al., 2017). Most dogs are asymptomatic
and shed these pathogens in their feces ultimately infecting humans and other animals by
contaminating the environment (Fox, 1990; Hald and Madsen, 1997). The prevalence of
Campylobacter species in dogs varies widely (Acke et al., 2006; Parsons et al., 2010; Kumar et
al., 2012; Verma et al., 2014; Holmberg et al., 2015; Lazou et al., 2016; Torkan et al., 2018;
Thépault et al., 2020). This variation depends on the age, the method of diagnosis, study design,
geographic region, housing type, the presence of infection or concomitant disease, and diarrheic
versus healthy dogs (Acke et al., 2009; Marks et al., 2011; Iannino et al., 2017).
The isolation of Campylobacter species using the culture method is generally used for the
diagnosis of campylobacteriosis; however, it is time-consuming and labor-intensive due to the
fastidious nature of Campylobacters (Li et al., 2014). In addition, this method is biased towards
the recovery of C. jejuni and C. coli (Lastovica, 2006), thereby potentially underestimating the
emerging species. Due to the close phylogenetic relationship between C. jejuni and C. coli,
biochemical assays cannot reliably distinguish between these two species (Miller et al., 2010).
This is however overcome by molecular-based assays such as polymerase chain reaction (PCR)
and sequencing which allow for rapid and specific detection, thus enhancing epidemiological
studies (Kaakoush et al., 2015; Vinueza- Burgos et al., 2017; Ricke et al., 2019).
The pathogenesis of Campylobacter infection is complex and poorly understood. However,
different studies have indicated that different virulence markers play a role in the motility and
adherence of these bacteria to the intestinal mucosa, enterocyte invasion, and toxin production
(Humphrey et al., 2007; Dasti et al., 2010; Wieczorek and Osek, 2013; Lapierre et al., 2016;
Younis et al., 2018). This contributes to their increased occurrence and epidemiology in
2
,comparison to other enteric bacteria (Bolton, 2015; Otigbu et al., 2018). The flaA gene is
involved in motility, colonization, and biofilm formation (Park, 2002; Guerry, 2007) whereas the
cadF gene is involved in the adherence of these bacteria to the intestinal mucosa (Carvalho et al.,
2001; Eucker and Konkel, 2012; Pillay et al., 2020). The pldA, ciaB, and iam genes are
responsible for the expression of invasion of enterocytes (Carvalho et al., 2001; Dasti et al.,
2010; Hamidian et al., 2011; Eucker and Konkel, 2012; Shams et al., 2016; Pillay et al., 2020).
Campylobacters also excrete several cytotoxins (encoded by cdtA, cdtB, and cdtC genes) that
contribute to disease development (Hickey et al., 2000; Bolton, 2015; Tresse et al., 2017) .
Although majority of dogs may be subclinically infected, others especially puppies less than 6
months of age or those from stressful environments may develop mild to moderate enteritis
presenting as mild to watery diarrhea or as bloody or mucoid diarrhea with tenesmus (Brown et
al., 1999; Chaban et al., 2010; Weese, 2011; Acke, 2018). Additional clinical signs include
anorexia, dehydration, lethargy, and rarely fever, vomiting, and abdominal pain (Brown et al.,
1999; Sykes and Marks, 2013; Marks et al., 2011). Antimicrobial treatment is usually
unnecessary (Kim et al., 2019), however, in severe cases where antimicrobial treatment is
needed, macrolides (erythromycin), fluoroquinolones (ciprofloxacin) and tetracyclines are
recommended (Guerrant et al., 2001; Iovine, 2013; Lübbert, 2016; Yang et al., 2019).
Antimicrobials such as amoxicillin-clavulanic acid and gentamicin could be used to treat
systemic Campylobacter infections (Dai et al., 2020).
A significant use of antimicrobials in humans and animals has led to an increase in antimicrobial
resistant Campylobacter spp. (Marks, 2003; Humphrey et al., 2007; Luangtougkum et al., 2009;
Agnes et al., 2013; Abay et al., 2014; Abdollahpour et al., 2015; Aslantaş, 2017; Issa et al.,
2018) which has led the World Health Organization (WHO) to classify Campylobacters as
3
, antibiotic-resistant ‗high‘ priority zoonotic pathogens (WHO, 2017). There are reports of wide
ranging prevalence of Campylobacter strains resistant to: macrolides, fluoroquinolones,
tetracyclines, aminoglycosides, betalactams, cephalosporins and sulphonamides (Ishihara et al.,
2004; Perez-Boto et al., 2010; Pollett et al., 2012; Karikari et al., 2017; Agunos et al., 2018;
Ewers et al., 2018; Zachariah et al., 2021). Transmission of resistant Campylobacter species or
their resistance genes is possible between dogs and humans via direct or indirect contact, through
the environment (Iannino et al., 2019). Therefore, monitoring of Campylobacter resistance is
important to public health and great attention should be paid in choosing the most appropriate
antimicrobial therapy (Wieczorek et al., 2018).
In Kenya, Campylobacter species have been reported in several studies amongst several
domestic animal spp. including poultry (Conan et al., 2017; Mageto et al., 2018; Carron et al.,
2018), cattle (Osano and Arimi, 1999), pigs, ducks, sheep, and adult dogs (Turkson et al., 1988).
However, despite the close proximity to humans, data indicating the presence and extent of
Campylobacter infection in puppies is limited in Kenya.
The aim of this study was to determine the prevalence, risk factors, virulence genes,
antimicrobial resistance profiles, and molecular epidemiology of thermophilic Campylobacter
species in puppies in the Nairobi Metropolitan Region, Kenya.
1.1 Objectives of the study
1.1.1 General objective
To determine the prevalence, risk factors, molecular characterization, virulence genes, and
antimicrobial resistance profiles of thermophilic Campylobacter species in puppies
in the Nairobi Metropolitan Region, Kenya.
4
1.0 GENERAL INTRODUCTION
Campylobacteriosis, caused by thermophilic bacteria of the genus Campylobacter, is a
significant zoonotic gastrointestinal disease affecting humans and animals, including dogs,
globally (Coker et al., 2002; Kaakoush et al., 2015; Carron et al., 2018; EFSA and ECDC, 2018;
Elmali and Can, 2019; Igwaran and Okoh, 2019). In humans, the disease is characterized by
fever, diarrhea, and pain. It is caused by Campylobacter jejuni and Campylobacter coli and
rarely, by other emerging Campylobacter species including Campylobacter lari, C. concisus, and
C. upsaliensis (Nachamkin and Blaser, 2000; Vandamme, 2000; Prasad et al., 2001; Sahin et al.,
2002; Moore et al., 2005; Man, 2011; Selwet and Galbas, 2012; Kaakoush et al., 2015). Majority
of the human infections are attributed to consumption of contaminated poultry (Wingstrand et
al., 2006; Sahin et al., 2015; Tresse et al., 2017; Carron et al., 2018), raw milk (Peterson, 2003)
and water (Diergaardt et al., 2004; Galanis, 2007; Abe et al., 2008).
Dogs were first associated with human campylobacteriosis in 1960 (Wheeler and Borchers,
1961) and Campylobacter jejuni was the first species isolated from dogs in 1977 (Skirrow,
1977). Since then, several studies have reported C. jejuni isolation from healthy and diarrheic
dogs worldwide with pathogenic involvement occurring most frequently in puppies (less than
one year) and is precipitated by factors such as crowding, stress, and concurrent diseases
(Engvall et al., 2003; Hald et al., 2004; Marks et al., 2011).
Most studies have found that the most frequently isolated species from dog feces is C.
upsaliensis, followed by C. jejuni and less frequently C. coli, C. lari, and C. hyointestinalis
1
,(Engvall et al., 2003; Hald et al., 2004; Workman et al., 2005; Rossi et al., 2008; Acke et al.,
2009; Koene et al., 2009; Parsons et al., 2011; Goni et al., 2017). Most dogs are asymptomatic
and shed these pathogens in their feces ultimately infecting humans and other animals by
contaminating the environment (Fox, 1990; Hald and Madsen, 1997). The prevalence of
Campylobacter species in dogs varies widely (Acke et al., 2006; Parsons et al., 2010; Kumar et
al., 2012; Verma et al., 2014; Holmberg et al., 2015; Lazou et al., 2016; Torkan et al., 2018;
Thépault et al., 2020). This variation depends on the age, the method of diagnosis, study design,
geographic region, housing type, the presence of infection or concomitant disease, and diarrheic
versus healthy dogs (Acke et al., 2009; Marks et al., 2011; Iannino et al., 2017).
The isolation of Campylobacter species using the culture method is generally used for the
diagnosis of campylobacteriosis; however, it is time-consuming and labor-intensive due to the
fastidious nature of Campylobacters (Li et al., 2014). In addition, this method is biased towards
the recovery of C. jejuni and C. coli (Lastovica, 2006), thereby potentially underestimating the
emerging species. Due to the close phylogenetic relationship between C. jejuni and C. coli,
biochemical assays cannot reliably distinguish between these two species (Miller et al., 2010).
This is however overcome by molecular-based assays such as polymerase chain reaction (PCR)
and sequencing which allow for rapid and specific detection, thus enhancing epidemiological
studies (Kaakoush et al., 2015; Vinueza- Burgos et al., 2017; Ricke et al., 2019).
The pathogenesis of Campylobacter infection is complex and poorly understood. However,
different studies have indicated that different virulence markers play a role in the motility and
adherence of these bacteria to the intestinal mucosa, enterocyte invasion, and toxin production
(Humphrey et al., 2007; Dasti et al., 2010; Wieczorek and Osek, 2013; Lapierre et al., 2016;
Younis et al., 2018). This contributes to their increased occurrence and epidemiology in
2
,comparison to other enteric bacteria (Bolton, 2015; Otigbu et al., 2018). The flaA gene is
involved in motility, colonization, and biofilm formation (Park, 2002; Guerry, 2007) whereas the
cadF gene is involved in the adherence of these bacteria to the intestinal mucosa (Carvalho et al.,
2001; Eucker and Konkel, 2012; Pillay et al., 2020). The pldA, ciaB, and iam genes are
responsible for the expression of invasion of enterocytes (Carvalho et al., 2001; Dasti et al.,
2010; Hamidian et al., 2011; Eucker and Konkel, 2012; Shams et al., 2016; Pillay et al., 2020).
Campylobacters also excrete several cytotoxins (encoded by cdtA, cdtB, and cdtC genes) that
contribute to disease development (Hickey et al., 2000; Bolton, 2015; Tresse et al., 2017) .
Although majority of dogs may be subclinically infected, others especially puppies less than 6
months of age or those from stressful environments may develop mild to moderate enteritis
presenting as mild to watery diarrhea or as bloody or mucoid diarrhea with tenesmus (Brown et
al., 1999; Chaban et al., 2010; Weese, 2011; Acke, 2018). Additional clinical signs include
anorexia, dehydration, lethargy, and rarely fever, vomiting, and abdominal pain (Brown et al.,
1999; Sykes and Marks, 2013; Marks et al., 2011). Antimicrobial treatment is usually
unnecessary (Kim et al., 2019), however, in severe cases where antimicrobial treatment is
needed, macrolides (erythromycin), fluoroquinolones (ciprofloxacin) and tetracyclines are
recommended (Guerrant et al., 2001; Iovine, 2013; Lübbert, 2016; Yang et al., 2019).
Antimicrobials such as amoxicillin-clavulanic acid and gentamicin could be used to treat
systemic Campylobacter infections (Dai et al., 2020).
A significant use of antimicrobials in humans and animals has led to an increase in antimicrobial
resistant Campylobacter spp. (Marks, 2003; Humphrey et al., 2007; Luangtougkum et al., 2009;
Agnes et al., 2013; Abay et al., 2014; Abdollahpour et al., 2015; Aslantaş, 2017; Issa et al.,
2018) which has led the World Health Organization (WHO) to classify Campylobacters as
3
, antibiotic-resistant ‗high‘ priority zoonotic pathogens (WHO, 2017). There are reports of wide
ranging prevalence of Campylobacter strains resistant to: macrolides, fluoroquinolones,
tetracyclines, aminoglycosides, betalactams, cephalosporins and sulphonamides (Ishihara et al.,
2004; Perez-Boto et al., 2010; Pollett et al., 2012; Karikari et al., 2017; Agunos et al., 2018;
Ewers et al., 2018; Zachariah et al., 2021). Transmission of resistant Campylobacter species or
their resistance genes is possible between dogs and humans via direct or indirect contact, through
the environment (Iannino et al., 2019). Therefore, monitoring of Campylobacter resistance is
important to public health and great attention should be paid in choosing the most appropriate
antimicrobial therapy (Wieczorek et al., 2018).
In Kenya, Campylobacter species have been reported in several studies amongst several
domestic animal spp. including poultry (Conan et al., 2017; Mageto et al., 2018; Carron et al.,
2018), cattle (Osano and Arimi, 1999), pigs, ducks, sheep, and adult dogs (Turkson et al., 1988).
However, despite the close proximity to humans, data indicating the presence and extent of
Campylobacter infection in puppies is limited in Kenya.
The aim of this study was to determine the prevalence, risk factors, virulence genes,
antimicrobial resistance profiles, and molecular epidemiology of thermophilic Campylobacter
species in puppies in the Nairobi Metropolitan Region, Kenya.
1.1 Objectives of the study
1.1.1 General objective
To determine the prevalence, risk factors, molecular characterization, virulence genes, and
antimicrobial resistance profiles of thermophilic Campylobacter species in puppies
in the Nairobi Metropolitan Region, Kenya.
4
