Campylobacteriosis in Sub-Saharan Africa

Abstract

Research and clinical works have documented various species of campylobacter in Africa. Thermophilic campylobacter has been shown to be endemic in the sub-Saharan Africa (SSA) region, and the prevalence is suspected to be increasing. To define the geographic boundaries of SSA, the United Nations macrogeographic definition of Africa has been used in several studies https://unstats.un.org/unsd/methodology/m49/. According to this UN definition, sub-Saharan Africa is divided into East Africa, Central Africa, Southern Africa, and West Africa. The zoonotic potential of campylobacter has been extensively studied and documented in the SSA region. Children are the most affected by campylobacter infections, and the infections exhibit seasonal patterns. Research has shown varied sources of infection such as foods of animal and plant origins, as well as unpasteurized milk and water, but animal meat is the most common source of infection. This chapter will delve into finding more recent information on campylobacter in the region such as the species, their prevalence, virulence, and risk factors. It will also explore the options in management such as vaccines and recommended diagnosis therapeutic protocols in humans and animals.

1. Introduction

Campylobacter is a gram-negative, nonspore-forming, curved or spiral bacilli, which are oxygen-sensitive and prefer to grow under micro-aerobic conditions. Certain species are relatively thermotolerant and therefore are considered thermophilic. Such species include Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), which are of critical importance to food safety, grow optimally at 42°C [1]. Campylobacter pathogen is common and endemic in sub-Saharan Africa (SSA) and causes gastroenteritis in animals and humans. The bacteria is highly infectious zoonotic pathogens and a major cause for the global human gastroenteritis infections with over 400 million cases reported annually in developing nations [2].

Assessment and quantification of the true burden of campylobacteriosis in the African context is hampered by the under-reporting of symptomatic diarrhea as well as inadequate surveillance programs of foodborne illnesses, as well as the minimal attention to Campylobacter as a pathogen. During diagnosis and laboratory testing, Campylobacter is rarely considered among the top suspects of diarrhea therefore not investigated, diagnosed, and reported [3]. Owing to the thermotolerant nature of various species of Campylobacter, they can survive various temperatures of cooking and cause cross-contamination of foods and food products leading to human and animal infections.

Most campylobacter infections do not need to be treated with antimicrobial agents, since there is evidence of spontaneous recovery. However, in a subset of patients especially pediatric and geriatric patients, campylobacter may cause severe complications and increased risk for death and therefore requires treatment. Other groups who are vulnerable include especially in immune-deficient or immune-suppressed individuals [4]. The most common drugs of choice in the treatment of such infections including fluoroquinolones such as ciprofloxacin or macrolides such as erythromycin are currently used because of their large spectra activity on enteric pathogens [5].

The disease is endemic in all sub-Saharan countries with varying prevalence rates across the region. The main sources of campylobacter infections include meats and milk with the most common source being poultry meat and eggs. A review publication by Gahamanyi et al. [4] identified the highest prevalence in Nigeria among all age groups with the most prevalent species being C. coli.

2. Campylobacter virulence

Virulence refers to the propensity of an infectious agent to cause a disease. The proteins and the genes which have an important function in disease development are known as virulent factors or determinants. Virulence factors of campylobacter include toxins, adhesins, invasion factors, flagellum proteins for motility, iron acquisition factors chemotaxis, lipooligosaccharide (LOS) secretion systems and campylobacter polysaccharide (CPS), antigens, genes, and response to environmental and oxidative stress [6].

2.1 Motility

The Campylobacter motility system needs flagella and a chemotaxis-based system that regulates the movement based on conditions of the environmental. Chemotaxis is the capability to move toward environments which are favorable that contain higher nutrients concentration or lower concentration of toxicity [7]. Chemotaxis has implication in the virulence of various pathogenic bacteria, which relies on this process to invade hosts. The movement of motile bacteria can be controlled by different extracellular chemical gradients detected by transducer-like proteins (Tlps) also known as methyl-accepting chemotaxis proteins (MCPs). These external stimuli, which bind to, relay a signal to chemotaxis proteins in the cytoplasm, which initiate a signal transduction cascade resulting in directed flagellar movement. Motility is significant for survival under the various gastrointestinal tract conditions and for small intestine colonization [8]. Campylobacter has uncommon movement more so in viscous substances. This is because of the presence of one or two polar flagella and the helical cell shape. The former provides propulsive cell movement, while the helical shape ensures the corkscrew rotation [9].

2.2 Adhesins

Campylobacter adhesion to the host intestinal epithelium is important for colonization. C. jejuni has a large number of different adhesins that individually or together mediate bacterial attachment to different cellular structures and various hosts [10]. Flagellum, outer membrane proteins (OMPs), and lipopolysaccharides (LPSs) are among the presumed adhesins. Campylobacter adhesion protein to fibronectin (CadF) attaches to epithelial cells fibronectin as the ligand. This adhesion stimulates a β-integrin receptor which triggers phosphorylation of the epidermal growth factor receptor. Erk1/2 signaling pathway is the activated one, and the GTPases Rac1 and Cdc42 are recruited and stimulated by Cia proteins, which begin the engulfing of Campylobacter via cytoskeleton reagent and membrane ruffling [10].

2.3 Invasion

After the bacterial adhesion to host cells intestinal membranes, C. jejuni invades the cells via endocytosis. Invasion process requires the Campylobacter-stimulated rearrangement of the cytoskeleton through microtubules and microfilaments [6]. Flagella are thought to have a second function in addition to that of motility designed to function as an export device type III secretion system (T3SS) in secretion of non-flagellar proteins during host invasion. It is also known that C. jejuni invasiveness in vitro is associated with de novo synthesis of entry-enhancing proteins and requires host cell signal transduction. Variants of the flagellin proteins such as flaA, flaB, flgB, and flgE genes have reduced invasiveness, while flaC and Campylobacter invasion antigens (Cia) gene products are important in colonization and invasion and are taken into the host cell’s cytoplasm using this flagellar secretion system. Full invasion of INT-407 cells requires CiaC, while CiaI has a function in intracellular survival [9].

2.4 Toxin production

The bacterial invasion process does not appear to be solely responsible for the cytopathic effects associated with C. jejuni infection. Toxins are likely involved in the disease process. In Campylobacter only, one toxin cytolethal distending toxin (CDT) is a known toxin produced in Campylobacter and has DNAse activities which lead to damage of DNA. Cytolethal distending toxin functions in the host cell invasion and results to extended period of symptoms and persistence of infection. Formation of CDT is activated by many factors including quorum sensing and is synthesized after C. jejuni has invaded the intestinal epithelial membrane. The toxin consists of three subunits encoded by cdtA, cdtB, and cdtC gene, and gene products are needed for the toxin to be functionally active [9]. Once the toxin is inside the cell, cdtB results in DNA double-strand break and probably cell death [6].

2.5 Carbohydrate structures

Lipooligosaccharides (LOS) majorly O- and N-linked glycans and a capsule on the cell surface of the Campylobacter facilitate colonization and associated genes. The lipooligosaccharide molecule contains an oligosaccharide core and lipid A which have various roles, including, host cell adhesion, immune evasion, and invasion. Sialylation of the LOS increases invasive potency and lowers immunogenicity [11]. Polysaccharides have a central role in the host-bacteria interaction and are essential for virulence and antigenicity. Cell surfaces of C. jejuni are covered by a polysaccharide capsule that enables survival, adhesion, and evasion of host immune system. Capsule mutants show decreased evasion process [7].

C. jejuni contains an N-linked glycosylation system controlling posttranslational changes of periplasmic proteins. Flagellin subunits are the only ones modified by O-linked glycosylation. N-linked glycosylation regulates evasion as the glycosyl molecules are immunodominant leading to slow generation of antibodies against the protein fraction [11]. N-glycans also protect C. jejuni surface proteins against protease enzymes of the gut. This explains why Campylobacter lari (PglB) mutants with a deficiency in the expression of oligosaccharyltransferase reveal slow growth in media supplemented with cecal contents [12].

3. Campylobacter prevalence in SSA

A wide variety of animals, including poultry, wild birds, sheep, cows, pigs, cats, and dogs, serve as natural reservoirs and source of transmission for Campylobacter [4]. As a result, Campylobacter colonization in various reservoirs creates a significant danger for human health due to the pathogen’s release into animal waste, contaminated water sources, the environment, and food. In Africa, a sizeable share of the population keeps poultry or livestock and most cases both. Unfortunately, these animals are frequently kept and slaughtered in unhygienic and unsanitary settings; hence, the high rates of campylobacter reported in animal husbandry [13]. Information on prevalence of campylobacter is scanty in SSA region because of the cumbersome and expensive procedures involved in its isolation, although some studies have attempted to determine both human and animal prevalence.

According to results of a recent systematic review and meta-analysis, the species C. jejuni is the most common in sub-Saharan Africa [14]. These results are consistent with another systematic study done in West Africa in 2022 [13] in which it was shown that C. jejuni was the most frequently detected species compared to C. coli, with a prevalence rate of 52% and 30%, respectively. Likewise, it is the most prevalent campylobacter species found in food, and the one commonly associated with human campylobacteriosis is C. jejuni [15].

A review carried out by [4] reported prevalence of thermophilic Campylobacter in humans ranging from 9.6 to 62.7% on average in sub-Saharan Africa. Nigeria reported the highest prevalence of 62.7% in humans followed by Malawi (21%) then South Africa (20.3%). For Campylobacter infections in children under 5 years of age, Kenya reported 16.4% of her cases, followed by Rwanda (15.5%) and Ethiopia (14.5%). The mean prevalence among all age groups and the children under 5 years of age at the country level was 18.6% and 9.4%, respectively. The prevalence is within the ranges found in other low- and middle-income countries (LMICs) as shown by Coker et al. [16] study. The prevalence was, however, higher and lower than that reported from Korea and the USA, respectively [17, 18]. This difference could be explained by the fact that campylobacteriosis is hyperendemic in LMICs, perhaps as a result of poor sanitation and close contact between people and domestic animals [4].

According to a systematic review carried out by Hlashwayo et al. [14] in SSA, prevalence rate ranged from 0–100%. According to the review, species identified in various regions were C. jejuni, C. coli, C. fetus subsp. venerealis, C. hyointestinalis, C. upsaliensis, C. fetus, C. fetus subsp. fetus, C. troglodytis sp. nov, C. sputorum subsp. Sputorum, C. lari and C. f. venerealis biovar intermedius. Like most other studies, the studies showed that C. jejuni and C. coli were the most prevalent species; while C. hyointestinalis, C. sputorum, and C. troglodytis were the least prevalent. Hlashwayo et al. [14] also reported that Western Africa recorded higher prevalence of Campylobacter species compared to other SSA regions. The high prevalence could be explained by transportation of unchecked poultry and other animals due to the presence of large market for live animals in the region [19].

Campylobacter subgroup analysis studies carried out in the same region by [13] recorded a pooled prevalence in poultry (39%, 95% CI: 27–52) higher than in other livestock (26%, 95% CI: 17–38). In poultry, the individual prevalence was estimated between 4–88% and 11–93% in livestock. Also, the same study recorded a pooled estimate of 10% (95% CI: 6–17) in humans with a lot high level of heterogeneity (I² = 98%).

Eastern Africa comes second in terms of percentage Campylobacter prevalence from studies [14]. Different animals have been screened for the presence of campylobacter prioritizing poultry and cattle. Isolates reported were C. jejuni, C. coli, C. lari, C. upsaliensis, C. fetus, and C. troglodytis. A 100% campylobacter prevalence was found in fecal materials from wild monkeys [14]. C. fetus subsp. venerealis has also been identified as the source of enzootic infertility in smallholder herds in this area.

In a systemic and meta-analysis review carried out by Zenebe et al. [20] in Ethiopia, the overall Campylobacter species prevalence was 10.2% (95% CI 3.79, 16.51) and heterogeneity was not observed across the included studies (I² 0.01%; Q = 3.23, p = 1.00). Also, 75% of the studies reported C. jejuni and C. coli at the species level.

Middle Africa has the least data on campylobacter in SSA. C. jejuni and C. coli are the only species reported. A prevalence of 92.7% was reported from slaughtered chicken, highlighting the role of food animals in the epidemiology of campylobacter in the region just like other regions. This suggests that they may not be epidemiologically delinked or varied from other SSA regions.

In Southern African region, poultry and cattle have been studied more, while pigs, sheep, goats, and dogs have also been studied to a limited extent. C. jejuni, C. coli, C. upsaliensis, and C. fetus were the common isolates in the region. A higher prevalence of C. jejuni and C. coli was reported in diarrheic chicken and goats [21].

In overall, data on Campylobacter prevalence are limited due to the expensive procedures and capacity required for such studies compared to other bacteria.

4. Risk factors associated with campylobacteriosis

Generally, campylobacter presents itself in asymptomatic nature, forming natural commensals in the intestinal tracts of majority of animals but is more pronounced in birds. These animals act as natural reservoirs for the bacteria and more often a source of human infection by contamination through pathogen shedding in fecal matter [22]. Studies by Ogden et al. [23] faulted contaminated food products of animal origin as the source of human gastroenteritis.

4.1 Risk factors

There are numerous factors that predispose humans and animals to campylobacteriosis infections in SSA, and case-control studies have been done to quantify on their impacts to provide strategic and targeted control measures. These factors include:

4.1.1 Contaminated animal products

Contaminated animal products, especially poultry meat and unhygienic handling of food items, pose a human risk factor for sporadic campylobacter infections. Animal meat production patterns in SSA ranging from home slaughtering to unhygienic meat preparations in open fires and barbecues are channels for campylobacter transmission. Unhygienic slaughtering procedures and poor handling of meat and its products may provide an avenue for human contamination in abattoirs. The enteric nature of campylobacter species constitutes the risk of cross-contamination from the fecal matter of same or different animal during flaying and evisceration. Contamination can also occur through cross-contamination between hide and carcass or in situations where contaminated water is used to clean the animal carcasses [24].

A study in Tanzania that targeted screening of cattle in abattoirs for Campylobacter reported a 5.6% prevalence rate of thermophilic campylobacter with C. jejuni as the predominant strain [25]. Different studies have reported a prevalence range of between 5% and 89% of campylobacter in cattle [24, 26]. In 1999, Osano and Arimi [27] reported a 2% contamination level of campylobacter on carcasses in Kenya.

High levels of campylobacter carcass contamination in pigs have been reported in SSA. In Tanzania, pork carcasses have been recorded to have 10.16% levels of contamination Komba et al. [28]. This is similar to a study outside the SSA in Brazil Aquino et al. [29]. Other studies in other developing nations have shown high rates of pork contamination between 34 and 63.6% [30] and Malakauskas et al. [31].

Developed nations have recorded low levels of campylobacter contamination on pig carcasses, and this has been attributed to low levels of enteric campylobacter due to animal feeds, abattoir levels of hygiene, and carcass handling procedures [32, 33]. Unpasteurized milk and raw milk products are associated with campylobacter illness in developing nations. Contamination of milk often occurs during milking through fecal contamination of the animal’s udders or udder infection. Poor personnel hygiene also contributes substantially in contamination of the udders [4]. In SSA, there are no strict legislations with regard to production and sale of milk to consumers so as to curb on unpasteurized milk contamination which is associated with campylobacter infections.

4.1.2 Domestic animal vectors

Animal contact is a major risk factor for acquiring human campylobacteriosis, and this is due to occupational exposure to animals in a slaughterhouse, pet shop, farm, or zoo. Animal contact also occurs during food handling/preparation or animal husbandry [34]. Most of the population living in rural areas and small farmers make up the largest population of animal keepers. Farm animals are mainly kept in free range systems, where there is close interaction between animals and humans, and thus an exposure to zoonotic pathogens is made possible [35]. Occupational exposure poses a potential risk factor, especially when biosecurity measures such as limited restriction to the animals or poultry housing and personnel security and hygiene are not followed. Cleaning and disinfection of animal housing prior to restocking and presence of a medicated footbath at the entrance plays a key role in the prevention of transfer campylobacter to the personnel house [36]. Children are found to be more susceptible to acquiring campylobacteriosis through animal contact during play than the general population. A study carried out by [34] reported a significant relationship between animal contact and acquiring campylobacteriosis.

4.1.3 Wild animal vectors

Wild birds are considered notable reservoirs of Campylobacter and often contaminate the environment through fecal droppings [37]. Children are at risk of ingesting campylobacter in the open playgrounds because of exposure to contaminated fecal matter. Open fields and playgrounds often act as natural habitats for wild birds and stray dogs in SSA and present a potential reservoir for campylobacter [35].

A case study by [38, 39] in New Zealand found a 12.5% positivity for C. jejuni in avian fecal samples from a children’s playing field indicating the possibility of a high-risk factor in SSA [40]. Livestock manure and other uncovered waste are also very prominent sources of human and animal infections. Therefore, handling livestock manure and drinking untreated water pose a risk of health risk associated with campylobacter [41].

There is a wide range of natural reservoirs for campylobacter, including chickens and other poultry, wild birds, pigs, dogs, cats, sheep, and cows. Consequently, colonization of various reservoirs by campylobacter poses a significant risk to humans as the pathogen becomes contaminated in livestock waste and the environment [4]. Manure gets contaminated when the reservoirs shed the pathogens.

4.1.4 Contaminated water

Water bodies such as lakes and rivers have been associated with campylobacteriosis in SSA because of contamination with animal feces, draining of sewage effluent, discharge from slaughterhouses, and slurry that is used in agricultural farms. Campylobacter can remain infective in water for over 120 days [42]. In developing countries in SSA, water bodies act as sources of drinking water both for the animals and humans, providing platforms for bathing, swimming, and other water sports that can all act as routes for sporadic campylobacter contamination. Studies have also shown that rainwater can act as sources of campylobacteriosis through avian fecal contamination [43].

4.1.5 Age

Campylobacteriosis in children under the age of 5 years is common is SSA, and it is attributed to undesirable hygienic conditions and poor water sanitation systems. Poor maternal hygiene also predisposes young children to diarrhea infections through feeding, cleaning, and other routine childcare practices. Children are also at risk during outdoor play as they come closer to animal wastes on the environment and are often less keen to hand and body hygiene. Exposures to different environmental conditions influence children’s risk to diarrhea [34].

A review article by [4] based on 33 articles showed that a young age is a high-risk factor for campylobacter infections in SSA. Kenya recorded the highest prevalence of campylobacteriosis at 16.4% in children under the age of 5 years. In Rwanda, the prevalence rate was 15.5%, while Ethiopia reported 14.5% on the same observation group.

4.1.6 Underlying diseases

Underlying diseases have been shown to act as predisposing factors for campylobacteriosis. Chronic conditions such as chronic gastrointestinal disease, gastric ulcers, celiac disease, liver disease, asthma, or diabetes have been associated with campylobacteriosis in the different populations [4]. People with underlying diseases get immunocompromised, hence easily predisposed to campylobacteriosis [44]. For instance, human immunodeficiency virus (HIV)-infected patients with diarrhea are more likely to be infected with Campylobacter than uninfected individuals with diarrhea. In addition, the incidence of Campylobacter-related diseases is higher in HIV-infected patients than in the general population. Also, studies by [45] reported a high incidence of campylobacter-associated illness.

4.2 Campylobacteriosis diagnosis and species identification methods

4.2.1 Specimen collection

Appropriate clinical specimen types for Campylobacter testing are liquid or semi-soft stool and rectal or stool swabs. Though rare, Campylobacter spp. may also be recovered from specimens such as blood and tissue majorly liver for C. lari, and fetal uterine content in abortion caused Campylobacter spp. Specimens should be collected during the acute phase of the diarrheal illness before antibiotic treatment is initiated. Urine-free stool and swabs should be collected in a sterile, airtight container containing modified Cary-Blair (CB) transport medium [46]. Stools with evidence of blood, mucus, or pus are optimal. Rectal swabs are acceptable in infants and young children when feces are otherwise difficult to obtain; however, these are not acceptable specimen types for many culture-independent diagnostic tests (CIDTs)-based test platforms. Modified CB-moistened swabs provide good recovery of Campylobacter, though other swabs, including Amies, have also shown good recovery for campylobacter.

Typically, a single specimen is sufficient, particularly for the recovery of C. jejuni and C. coli. In cases of persistent diarrhea with a negative culture or any other time when initial testing does not provide a definitive pathogen, collecting a second specimen may be appropriate. Specimen rejection may be appropriate upon receipt of solid or formed stool, stool mixed with urine, dry swab or swab lacking visible evidence of stool, evidence of barium, leakage from the container, a frozen specimen, or a specimen submitted in expired or parasitic transport medium [47].

4.2.2 Transport and storage of isolates presumptive and confirmed

Campylobacter isolates may be submitted to Public Health Labs for confirmation and/or characterization. Post-culture, and for proper transportation, fresh campylobacter isolates (24 hours old) should be swabbed from a plate, placed in transport media (modified CB or Amies Transport Medium) and shipped on ice overnight or as a frozen bacterial culture in trypticase soy broth with 20% glycerol on dry ice. Isolates that are not preserved in glycerol should not be frozen or come into direct contact with ice packs, as this will reduce recovery. If isolates are to be submitted on solid media, Columbia agar with blood, Brain Heart Infusion (BHI) or Wang’s should be used. Shipment on Trypticase Soy Agar is not preferred.

Prior to further testing, Campylobacter isolates should be held as frozen stocks (−60°C) in glycerol or maintained on fresh culture media with routine passage. It may be useful to store antibiotic resistant and outbreak-associated isolates for later reference and characterization, as per the laboratory’s isolate retention policy. Frozen stocks should never be completely thawed. Instead, a small amount of the stock should be partially thawed, removed, and the stock returned to the freezer as soon as possible. Repeated freeze-thaw cycles should be avoided [48].

4.2.3 Direct diagnosis

4.2.3.1 Culture-dependent diagnostic tests (CDTS) for the detection of Campylobacter

Campylobacter usually grows on most nonselective culture media, especially when enriched with blood. The majority of these media have been developed for isolation of C. jejuni and are rarely suitable for other species, hence limited application to veterinary samples. Five percent of blood agar is suitable for culturing C. fetus and C. jejuni of aborted ewe’s samples. Pre-contamination filtration membrane is recommended to minimize contamination with other bacteria. Optimum atmospheric growth conditions are 55 oxygen, 10% CO₂, and 85% hydrogen and nitrogen and are artificially generated by commercial gas generating kits in conjunction with standard anaerobic jars use of tri-gas incubator [49].

4.2.3.2 Materials and reagents

• 5% sheep or horse blood agar plates

• 0.65 u millipore membrane filters

• Gas jars

• Gas packs—oxide gas generating packs for Campylobacter.

4.2.3.3 Procedure

Centrifuge macerated tissue/fecal/fetal stomach contents samples at 100 g for 10 minutes. Aseptically remove supernatant and incubate at 37°C for 1 hour. Place 0.65 um membrane filter on a surface of each isolating agar plate. Place between 5 and 10 drops of incubated supernatant fluid onto the center of each filter and incubate at 37°C for 1 hour. Remove and discard the filters and spread the filtrate over the agar surface. Allow the plates to dry. Incubate the plates on atmospheres of 5% O₂, 10% CO₂, and 85% H₂ or N₂ and examine by plate microscopy for typical colony of campylobacter species after 48–72 hours.

4.2.3.4 Results and interpretation

Following culturing on media containing blood at 37°C colonies will vary in appearance from small round and complex one. Campylobacter fetus are large and mucoid colonies. C. jejuni and C. coli will produce large and small colonies coexisting on plate which can be sub cultured for single colony isolation. Stains of C. jejuni can develop a metallic sheen over the culture surface. Generally, coccoid forms of Campylobacter are invariably due to long incubation periods and may be considered degenerative and nonviable; however, C. jejuni cultures produce coccoid forms before 48 hours. Hemolysis is not observed in blood agar.

4.3 Identification of campylobacter species

Campylobacter colonies on plate agar plates can be confirmed by gram stain, oxidase reaction, and catalase reaction. C. fetus and C. jejuni can be distinguished from other species by growth temperature studies antibiotic sensitivity, production of hydrogen sulphide, and hippurate hydrolysis. In gram stain, campylobacter cells are short gram-negative rods and have a distinctive curved or spiral appearance.

In oxidase test, 1% tetramethyl-p-phenalene diamine hydrochloride aqua solution is used, and it forms dark purple color within 10 seconds. In catalase test, C. fetus, C. jejuni, and C. laridis all possess catalase enzymes which catalyze release of oxygen from hydrogen peroxide. Campylobacter fetus will grow best between 25° and 37 but not at 42°. C. jejuni, C. coli, and C. laridis will grow at between 37 and 42 but not at 25°C.

4.4 Indirect diagnosis

4.4.1 Polymerase chain reaction

4.4.1.1 IQ-check campylobacter PCR technology

Polymerase chain reaction has advantage over the standard gold test of culturing, since culture diagnostic test for Campylobacter spp. is lengthy protocol [50]. The test is based on gene application and detection by real-time PCR [51]. The kit is ready to use PCR reagent containing oligonucleotides (primers and probes) for specific C. jejuni, C. coli, and C. laridis as well as DNA polymerase and nucleotides (IQ-check campylobacter PCR technology kit manual, BioRad). Detection and data analysis is optimized by Bio-Rad real-time PCR instrument called CFX 96 Touch Deep Well System. The test is used for qualitative detection of Campylobacter species in food products, environmental samples, fecal matters, and animal tissues.

The protocol involves sample enrichment, free DNA treatment, DNA extraction, real-time PCR, and data analysis. The sample enrichment step is a key in subculturing the contaminated samples to increase bacterial growth of Campylobacter. This protocol step is specific on type of sample diagnostics for food sample contamination, and n/10 g of sample is added into 9n/10 ml of supplemented Bolton broth in a stomacher bag with incorporated filter then incubated without shaking for 4 hours at 37 ± 1°C under micro-aerobic condition and transferred to 41.5 ± 1°C for additional 24 hours under microaerobic condition. For carcass rinsed sample, the carcass is rinsed in 40 ml of buffered peptone water for 1 minute, and the rinse of 30 ml is added to 30 ml of double-strength blood-free Bolton enrichment broth (2XBF-BEB), mixed gently, and then incubated for 24 hours at 42 ± 1°C under microaerobic conditions. For carcass swab after sponging carcass, 25 ml of 2XBF-BEB is added into it, mixed gently, and then incubated at 42 ± 1°C for 24 hours at microaerobic condition.

Fecal matters are homogenized into supplemented Bolton broth in stomacher bag with incorporated filter then allowed to decant at room temperature for 10 minutes. The next step is free DNA removal treatment. For DNA extraction, lysis reagent is aliquoted into wells of deep plates, enriched media sample added to the mixture and mixed by pipetting up and down until homogenized. The deep well is crossed by pre-pierced sealing. It is then heat-blocked at 95°C for 15 minutes and thereafter incubated under agitation at 1300 rpm at 95°C for 25 minutes. After that, it is vortexed at high speed for 2 minutes. The supernatant is then extracted and stored at 20°C and always allowed to thaw,homogenize and then centrifuged at 12,000 g for 5 minutes before reusing.

The next step is real-time PCR. PCR involves preparing the mix containing application solution and the fluorescent probes. The volume of PCR mix needed depends on number of samples and controls to be analyzed, and at least one positive and negative control must be included in each PCR run. The application solution and fluorescent probes must be used within 1 hour after storage at 2–8°C. 45 ul PCR mix is aliquoted to each well of the plate, then 5 ul of DNA extract, negative control, and positive control are added to the corresponding wells. The wells of the PCR plates are sealed, centrifuged/ quick-spun to eliminate any bubble, then PCR plates are placed in thermocycler. To run PCR, the iQ check kits instructions should be followed in real-time PCR system guide. The PCR data analysis is done by CFX manager IDE software. Then data interpretation is done when the parameters have been set and the Cq values of each sample are interpreted. Positive and negative control sample results should always be verified before interpreting. Positive campylobacter samples have Cq values more than 10 FAM fluorophore (IQ-check campylobacter PCR technology kit manual, BioRad).

4.4.1.2 Real-time PCR

Different protocols of real-time PCR are applied for the detection of different species. In the detection of Campylobacter jejuni, primers and corresponding probes targeting hip O genes are used, while Campylobacter coli quantitative protocol of [52, 53] is used and adapted to the fast real-time PCR method by minor adaption (omitting ‘G’s at 3′-end) of forward primers. For other species, C. lari and C. hyointestinalis genus-specific 16S rRNA encoding DNA [54] region is targeted. To this purpose, the method by Lund et al. [55] is adapted to the fast real-time PCR method. To this end, the forward primer is elongated with three bases, a new reverse primer is designed, and the TaqMan probe is redesigned to contain the minor groove binding (MGB) quencher dye. Real-time PCR is performed on an Applied Biosystems 7500 thermal cycler, using the TaqMan® Fast Universal PCR Master Mix. For the real-time PCR, 5 ml of DNA, 10 ml of TaqMan® FastUniversal PCR Master Mix, 1 ml (10 pmol) of forward and reverse primers 1 ml (5 pmol) TaqMan probe are mixed, and 2 ml of DNase free water is added to a final volume of 20 ml. The cycling conditions consisted of 3 min at 95°C, followed by 40 cycles of 3 s at 95°C and 30 s at 60°C. Real-time data were analyzed with Applied Biosystems 7500 software (version 1.4). Upon completion of the run, a cycle threshold (Ct) is calculated and plotted against the log input DNA to provide standard curves for the quantification of unknown samples [56].

5. Conclusion

Campylobacter infection, or campylobacteriosis, is a challenge in the SSA, and the prevalence seems to be increasing with increasing surveillance and diagnosis. The disease seems to be more prevalent among the farming households and those living under lacking hygienic standards. Despite the disease being self-limiting, its impact, zoonotic potential, and cost cannot be ignored. With increased demand of animal protein for nutrition and this being a key source of infection, more work needs to be done with a focus of SSA in mind due to financial constraints and poverty. The work should be geared toward development of rabid diagnostics for this disease to enable early diagnosis and limit its effects on populations and economies.