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Diagnosis of fowl cholera depends on identification of the causative bacterium, P. multocida, following isolation from birds with signs and lesions consistent with this disease. Presumptive diagnosis may be based on the observance of typical signs and lesions and/or on the microscopic demonstration of bacteria showing bipolar staining in smears of tissues, such as blood, liver, or spleen. Mild forms of the disease may occur (OIE, 2015). Confirmatory diagnosis is done by isolation and identification of causative agent. A variety of laboratory diagnostic techniques have been developed over the years for pasteurellosis and used routinely in the laboratory. Among these techniques molecular techniques of diagnosis is most important. This technique not only gives diagnosis but it also provides information regarding capsular type of Pasteurella multocida (Rajeev et al., 2011).
Clinical signs of acute fowl cholera are inaptence, fever, ruffled feathers, oral mucus discharges, dyspnea and watery or yelowish diarrhoea (Rhoades and Rimler, 1990). Birds suffering from chronic form of the disease may show depression, conjunctivitis, dyspnea, lameness, torticollis, swelling of the wattles, sinuses, limbjoints, footpads and sternal bursae (Christensen and Bisgaard, 2000). In cases with significant pulmonary involvement, there will be loud respiratory rales and coughing as the disease progresses. Depending on the particular strain of P. multocida involved, there may be high to very high morbidity and mortality. With less virulent strains, some affected bird may slowly recover, after a variable period of depression. With more virulent strains, death usually occurs swiftly after a brief period of prostration, accompanied by convulsive wing flapping and paddling. Birds which survive the acute disease may recover completely, or may develop an exudative arthritis in leg or wing joints. Arthritis may occur without signs of acute systemic illness, particularly in very young or old birds (Wilkie, et al., 2012).
Chronic infections also occur with clinical signs and lesions related to localized infections. The pulmonary system and tissues associated with the musculoskeletal system are often the seats of chronic infection (OIE, 2008). The most common gross necropsy findings in the birds with confirmed avian cholera were acute fibrinous and necrotizing lesions affecting the liver, spleen, air sacs, and pericardium, and nonspecific hepatomegaly and splenomegaly (Michelle et al., 2016).
Identification and characterization of P. multocida has relied on the ability to cultivate or purify the organism in the laboratory. The purified organism is subsequently classified according to phenotypic traits such as morphology, carbohydrate fermentation patterns and serological properties. However, culture conditions can influence the expression of these attributes thus diminishing the stability and reliability of phenotypic methods for strain identification (Matsumoto and Strain, 1993; Jacques et al., 1994).
Isolation of the organism from visceral organs, such as liver, bone marrow, spleen, or heart blood of birds that yield to the acute form of the disease, and from exudative lesions of birds with the chronic form of the disease, is generally easily accomplished. Isolation from those chronically affected birds that have no evidence of disease other than emaciation and lethargy is often difficult. In this condition or when host decomposition has occurred, bone marrow is the tissue of choice for isolation attempts. The surface of the tissue to be cultured is seared with a hot spatula and a specimen is obtained by inserting a sterile cotton swab, wire or plastic loop through the heat-sterilized surface. Alternatively the sterilized surface can be cut with sterile scissors/scalpel and the swab or loop inserted into the cut without touching the outer surface (OIE, 2015).
Identification of P. multocida can be done based on morphological study, staining properties, cultural and biochemical characteristic, as described by Cheesbrough (2006) and Cultural and morphological examinations can be conducted as described by Cowan and Steel (2004). Accordingly, Samples suspected of fowl cholera are first seared with spatula and incised with a small sterile scalpel blade and forceps. The specimen is inoculated directly into tryptose broth medium, incubated for 2–3 hours, transferred to Casein Sucrose Yeast (CSY) agar, blood agar, nutrient agar, MacConkey agar and citrate agar. Growth of the organism, size of colony, pigmentation and their ability to produce any change in the medium like haemolysis on blood agar can be examined. If our sample is swab from these organs, it is inoculated directly onto selective medium, such as Casein Sucrose Yeast (CSY) agar, blood agar and incubated aerobically at 370C for 24 hours. Then, suspected colonies subjected to Gram’s and methylene blue staining for cellular morphology. Gram stain result showing gram negative, with bipolar coccobacilli characteristics were considered as P. multocida.
Phenotypic characterization of Pasteurella multocida by biochemical reaction from various samples based on the basis of sugar fermentation reaction (Cowan and Steel, 2004). Pasteurella multocida does not cause haemolysis, it is not motile and only rarely grows on MacConkey agar. It produces catalase, oxidase, and ornithine decarboxylase, but does not produce urease, lysine decarboxylase, beta-galactosidase, or arginine dihydrolase. Phosphatase production is variable. Nitrate is reduced; indole and hydrogen sulphide are produced, and methyl red and Voges–Proskauer tests are negative (Glisson, et al., 2008).
Pathogenecity test of strains of P. multocida can be carried out from pure colony grown for 18 h in a shaker-come-incubator at 37oC in Brain Heart Infusion (BHI) broth. About 0.2 ml each culture containing approximately 2.4×108 colony forming units/ml can be inoculated into three test mice by the Intraperitoneal and observe for 72 h to look at the mortality pattern. If the organism is re-isolated from heart blood collected from dead mice on a blood agar plate and an impression smear from the liver reveals the agent by Giemsa method of staining and again if the re-isolated colonies showed similar characteristics of P. multocida, and impression smears revealed typical bipolarity of the organism, P. multocida is said to be pathogenic (Shivachandra et al., 2005).
Serological tests, such as enzyme-linked immunosorbent assays (ELISA), agglutination and indirect hemagglutination assay (IHA) have been used to identify antibodies against Pasteurella multocida in poultry sera (Marshall et al., 1981). Indirect hemagglutination procedure can be developed for the identification of different capsular antigens of Pasteurella multocida (Solano et al., 1983)
ELISA: has been used with varying degrees of success in attempts to monitor seroconversion in vaccinated poultry. The ELISA assay was used for decades for investigation of antibody to fowl cholera in avian species (Marshall et al., 1981; Solano et al., 1983). Commercial ELISA kits are available for chickens and turkey. The ELISA is a rapid, highly sensitive and speciﬁc serological test (Poolperma et al., 2017). From ELISA types the indirect ELISA test is the most commonly applied one. This assay is designed to measure the relative level of antibody to P. multocida (Pm) in bird serum. Antigen is coated on 96-well plates. Upon incubation of the test sample in the coated well, antibody specific to P. multocida (Pm) form a complex with the coated antigen. After washing away unbound material from the wells, a conjugate is added which binds to any attached bird antibody in the wells. Unbound conjugate is washed away and enzyme substrate is added. Subsequent colour development is directly related to the amount of antibody to P. multocida (Pm) present in the test sample (Aydin, 2015).
The phenotypic methods, like serotyping and biotyping, can been used to differentiate the strains, but these methods are so hard, extremely tedious and often produce unclear results. Thus, in the recent years, the phenotypic differentiation tools have been frequently replaced with the genotypic methods (Taylor et al., 2010). Contrary to conventional methods, the PCR-based typing techniques were found to be rapid and highly sensitive for identifying and differentiating the strains. It is known that pulsed field gel electrophoresis (PFGE) is the standard for epidemiologic strain typing of P. multocida, although one study indicated that Repetitive Extragenic Palindromic sequence-based PCR (REP-PCR) compares favorably. In addition, randomly amplified polymorphic DNA (RAPD) is suitable techniques for studying the host adaptation of P. multocida and the epidemiology of fowl cholera (Klaudia et al., 2012).
Polymerase Chain Reaction (PCR): Confirmation of the isolated organism as P. multocida can be done based on PCR targeting capsular gene cap specific for P. multocida as described in (OIE, 2008). Bacterial DNA can be extracted using Wizard genomic DNA Purification Kit according to the instruction of the manufacturer. Extraction of DNA and its quality is checked by running 5μLsuspension of the extracted DNA in a 1% (w/v) agarose-gel (Mahmuda, 2016).
The primers used in the PCR are PMcapEF (5′-TCCGCAGAAAATTATTGACTC-3′) and PMcapER (5′-GCTTGCTGCTTGATTTTGTC-3′) that amplified around 511-bp amplicon. All the PCR can be done in a final 25 μL volume containing 12.5 μL PCR master mix, 1 μL of each primer (10 pmol), PCR grade water 8.5 μL and DNA template 2 μL. The thermal profile followed for PCR are as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 30 sec, annealing at 55°C for 30 sec and elongation at 72°C for 90 sec, and a final extension at 72°C for 5 min. 5μL PCR product can be loaded into 1% agarose gel (w/v) along with 1μL 6X loading dye for electrophoresis in 1X TBE buffer at 100Vfor 30min. A standard 100-bp DNA ladder can also be loaded in the same gel to compare the size of the amplified PCR products. Prior to casting the gel, ethi-dium bromide (0.5μg/mL) can be added to the gel. The PCR products were visualized under UV light in an image documentation system (Mahmuda, 2016).
The Randomly amplified polymorphic DNA technique relies on the polymorphic DNA that can be amplified by one or several short oligonucleotide primers of the arbitrary sequences with 8–12 nucleotides (ziva et al., 2008). Since the RAPD is a simple, fast and sensitive method, it is one of the most promising genotyping techniques, which has been used to differentiate closely related bacterial species and strains (Huber et al., 2002). Characterization of P. multocida by RAPD-PCR is efficient to find out genetic variations due to its simplicity and arbitrary sequence of primers (Mohamed and Mageed, 2014). In addition this technique does not require the sequence information to establish genetic relatedness or variation between fields isolates (Welsch and McClelland, 1990).
The specific primers for the Repetitive Extragenic Palindromic sequence-based PCR (REP-PCR) complement these repetitive sequences and provide the reproducible and unique REP-PCR DNA fingerprint patterns. In general, the REP-PCR method is a valuable tool for the rapid epidemiological analysis and characterization of bacteria and it has been used in several studies (Blackall and Miflin, 2000). Additionally, it was employed for the molecular typing of the P. multocida strains (Shivachandra et al., 2008).
Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) has indicated information about genomic characteristic of bacteria (Jabbari and Esmaelizadeh, 2005). Except for time consuming for digestion and electrophoresis, PCR-RFLP are novel and rapid method for classification of P. multocida (Tsai et al., 2011). DNA fingerprinting of P. multocida by restriction endonuclease analysis (REA) has proved valuable in epidemiological investigations of fowl cholera in poultry flocks. Isolates of P. multocida having both capsular serogroup and somatic serotype in common may be distinguished by REA. Ethidium-bromide stained agarose gels are analysed following electrophoresis of DNA digested with either Hhal or Hpall endonuclease (Wilson et al., 1992).
Ribotyping in conjunction with REA has been widely used to characterize and differentiate the Pasteurella multocida isolates (Blackall et al., 1995). REA followed by additional hybridization with a labeled DNA probe made easy to read the banding pattern and give the necessary interpretation. The probe may be labeled either by radio active or non radioactive materials. rRNA probe is widely accepted for hybridization and subsequent interpretation (Blackall, 2000).
The usefulness of agarose-gel electrophoresis to visualize the intracellular nucleic acid content of bacterial cells (Goering, 2010) was a revolutionary milestone in molecular biology that rapidly found clinical application including molecular epidemiology. The use of agarose-gel electrophoresis to comparatively analyze patterns of bacterial chromosomal restriction fragments was an important step toward genome-based epidemiological analysis (Chijioke, 2016). PFGE analysis has consistently shown the greater discrimination in identification of bacterial species than ribotyping but, it has limited application in the typing of Pasteurella multocida isolates (Townsend et al., 1997a).The major drawbacks of this technique are the requirements of highly purified intact DNA and specialized and expensive electrophoresis equipment, which is generally not available in normal diagnostic laboratories (Dutta et al., 2005).
Ethiopia, even though the in frequent complaints of the state and private poultry farms due to the high morbidity, mortality, loss of production and high treatment cost pertaining this disease to the National Veterinary Institute, the prevalence of the disease has not been quantified (Bitew, 2008).
Most poultry outbreaks, particularly in more remote parts of the country, remain undiagnosed and dead chickens are simply discarded. Therefore, information on the prevalence and significance of infectious poultry diseases can only readily be obtained through indirect serological studies on apparently healthy chickens. It is difficult to design and implement chicken health development programs without an understanding of the diseases present in the backyard poultry production system. One study revealed a high seroprevalence of fowl cholera (65%), and this constitutes the first report of fowl cholera seroprevalence in Ethiopia (Chaka et al., 2012).
Fowl cholera, a septicemic disease, is associated with high morbidity and mortality in poultry especially chicken and ducks. There are different detection approaches of pasturella multocida which includes conventional and advanced molecular methods of diagnosis. Conventional methods for pasturella multocida detection techniques are laborious and time consuming. However, advances in technology have enabled the development of a variety of rapid test methods that is advanced molecular diagnostic approaches such as RAPD-PCR PFGE, Ribotyping, and REA. The main advantage of RAPDs is that they are quick and easy to assay. Because PCR is involved, only low quantities of template DNA are required. Since random primers are commercially available, no sequence data for primer construction are needed. This technique is very effective for identification, characterization and diagnosis of P. multocida.
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