Index IntroductionClinical signsPost-mortem lesionDetection method using conventional techniqueMorphology and cultural characteristicsBiochemical characteristics, pathogenicity testsSerological identificationDNA-based techniquesRandomly amplified polymorphic DNAPCRrepetitive sequence-based extragenic palindromic PCRRrestriction endonuclease analysisElectrophoresis pulsed field gel (PFGE) Status of avian cholera in Ethiopia and diagnostic techniques used Conclusion and recommendations Introduction The diagnosis of avian cholera depends on the identification of the causative bacterium, P. multocida, following isolation from birds with signs and lesions consistent with this illness. Presumptive diagnosis may be based on observation of typical signs and lesions and/or microscopic demonstration of bacteria showing bipolar staining in tissue smears, such as blood, liver, or spleen. Mild forms of the disease may occur (OIE, 2015). Confirmatory diagnosis is made by isolation and identification of the causative agent. Over the years, several laboratory diagnostic techniques for pasteurellosis have been developed and routinely used in the laboratory. Among these techniques, the molecular diagnostic technique is the most important. This technique not only provides the diagnosis but also provides information on the capsular type of Pasteurella multocida (Rajeev et al., 2011). Say no to plagiarism. Get a tailor-made essay on "Why Violent Video Games Shouldn't Be Banned"? Get an original essay Clinical Signs Clinical signs of acute avian cholera are loss of appetite, fever, ruffled feathers, oral mucus discharge, dyspnea, and watery or yellowish diarrhea (Rhoades and Rimler, 1990). Birds affected by the chronic form of the disease may experience depression, conjunctivitis, dyspnoea, lameness, stiff neck, swelling of the wattles, sinuses, limb joints, legs and sternal bursae (Christensen and Bisgaard, 2000). In cases with significant lung involvement, you will have loud wheezing and coughing as the disease progresses. Depending on the particular strain of P. multocida involved, morbidity and mortality can be high to very high. With less virulent strains, some affected birds may recover slowly, after a variable period of depression. With the more virulent strains, death usually occurs rapidly after a short period of prostration, accompanied by convulsive flapping and paddling. Birds that survive the acute disease may recover completely or may develop exudative arthritis in the leg or wing joints. Arthritis can occur without signs of acute systemic disease, particularly in very young or elderly birds (Wilkie, et al., 2012). Postmortem lesions Chronic infections also occur with clinical signs and localized infection-related lesions. The pulmonary system and tissues associated with the musculoskeletal system are often sites of chronic infections (OIE, 2008). The most common necropsy findings in birds with confirmed avian cholera were acute fibrinous and necrotizing lesions affecting the liver, spleen, air sacs, and pericardium, as well as nonspecific hepatomegaly and splenomegaly (Michelle et al., 2016). Detection method using conventional technique Identification and characterization of P. multocida has relied on the ability to culture or purify the organism in the laboratory. The purified organism is subsequently classified based on phenotypic traits such as morphology, carbohydrate fermentation patterns, and serological properties. However, cultural conditions can influence the expression of these attributes thus decreasing the stability and reliability of the methodsphenotypic tests for strain identification (Matsumoto and Strain, 1993; Jacques et al., 1994). Isolation of the organism from visceral organs, such as the liver, bone marrow, spleen, or cardiac blood of birds succumbing 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 exhibit no signs 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 burned with a hot spatula and a sample is obtained by inserting a sterile cotton swab, thread or plastic pass through the hot 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 external surface (OIE, 2015). Morphology and cultural characteristics The identification of P. multocida can be carried out on the basis of the morphological study and color properties. , cultural and biochemical characteristics, as described by Cheesbrough (2006) and cultural and morphological examinations can be conducted as described by Cowan and Steel (2004). Accordingly, specimens suspected of fowl cholera are first scalded with a spatula and incised with a small sterile scalpel and forceps. The sample 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, colony size, pigmentation, and its ability to produce any changes in the medium, such as hemolysis on blood agar, can be examined. If our sample is taken from a swab of these organs, it is inoculated directly onto a selective medium, such as casein sucrose yeast agar (CSY), blood agar and incubated aerobically at 370°C for 24 hours. Then, the suspicious colonies were subjected to Gram and methylene blue staining for cell morphology. The Gram stain result showing Gram negative, with bipolar coccobacilli characteristics was considered as P. multocida. Biochemical characteristics, pathogenicity tests Phenotypic characterization of Pasteurella multocida by biochemical reaction from various samples based on sugar fermentation reaction (Cowan and Steel, 2004). Pasteurella multocida does not cause haemolysis, is not motile and grows only rarely 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 sulfide are produced and methyl red and Voges-Proskauer tests are negative (Glisson, et al., 2008). Pathogenicity testing of P. multocida strains can be performed from pure colonies grown for 18 hours in a shaker. as an incubator at 37°C in Brain Heart Infusion (BHI) broth. Approximately 0.2 ml of each culture containing approximately 2.4x108 colony forming units/ml can be inoculated into three test mice intraperitoneally and observed for 72 hours to observe the mortality pattern. If the organism is reisolated from cardiac blood collected from dead mice on a blood agar plate and an impression smear of the liver reveals the agent by the Giemsa staining method and again if the reisolated colonies showed similar characteristics of P. multocida and the impression smears revealed the typical bipolarity of the organism, P. multocida is considered pathogenic (Shivachandra et al., 2005). Serological identification Serological tests, such as enzyme-linked immunosorbent assays(ELISA), agglutination and indirect haemagglutination (IHA) tests have been used to identify antibodies against Pasteurella multocida in poultry sera (Marshall et al., 1981). An indirect haemagglutination procedure can be developed for the identification of various 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 test has been used for decades to detect antibodies against avian cholera in avian species (Marshall et al., 1981; Solano et al., 1983). Commercial ELISA kits are available for chicken and turkey. ELISA is a rapid, highly sensitive and specific serological test (Poolperma et al., 2017). Among the ELISA types, the indirect ELISA test is the most commonly applied. This test is designed to measure the relative level of antibodies against P. multocida (Pm) in bird serum. The antigen is coated onto 96-well plates. After incubation of the test sample in the coated well, the P. multocida (Pm)-specific antibody forms a complex with the coated antigen. After washing unbound material from the wells, a conjugate is added that binds to any attached anti-bird antibodies in the wells. The unbound conjugate is washed away and the enzyme substrate is added. The subsequent color development is directly related to the amount of antibodies against P. multocida (Pm) present in the test sample (Aydin, 2015). DNA-based techniques Phenotypic methods, such as serotyping and biotyping, can be used to differentiate strains, but these methods are very difficult, extremely tedious, and often produce unclear results. Therefore, in recent years, phenotypic differentiation tools have often been replaced with genotypic methods (Taylor et al., 2010). In contrast to conventional methods, PCR-based typing techniques have proven to be rapid and highly sensitive for identifying and differentiating strains. Pulsed-field gel electrophoresis (PFGE) is known to be the standard for epidemiological strain typing of P. multocida, although one study has indicated that repetitive extragenic palindromic sequence-based PCR (REP-PCR) holds up favorably to comparison. Furthermore, randomly amplified polymorphic DNA (RAPD) is a suitable technique to study the host adaptation of P. multocida and the epidemiology of avian cholera ( Klaudia et al., 2012 ). Polymerase chain reaction (PCR): Confirmation of the isolated organism as P. multocida can be carried out based on PCR targeting the P. multocida-specific capsular gene cap as described in (OIE, 2008). Bacterial DNA can be extracted using the Wizard genomic DNA Purification Kit according to the manufacturer's instructions. DNA extraction and its quality are checked by running a 5μL suspension 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′) which amplified the amplicon of approximately 511 bp. All PCR can be performed in a final volume of 25 μL containing 12.5 μL of PCR master mix, 1 μL of each primer (10 pmol), 8.5 μL PCR water, and 2 μL DNA template. The thermal profile followed for the PCR is 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 of PCR product can be loaded into 1% (w/v) agarose gel together with 1μL of 6X loading dye for electrophoresis in 1X TBE buffer at 100V for 30 minutes. In the same gel it is also possible to loada standard 100 bp DNA ladder to compare the size of the amplified PCR products. Before casting the gel, ethidium bromide (0.5 μg/mL) can be added to the gel. The PCR products were visualized under UV light in an image documentation system (Mahmuda, 2016). PCR with randomly amplified polymorphic DNA. The randomly amplified polymorphic DNA technique is based on polymorphic DNA that can be amplified by one or more short oligonucleotide primers of arbitrary sequences with 8–12 nucleotides (Ziva et al., 2008). Since RAPD is a simple, fast and sensitive method, it is one of the most promising genotyping techniques, used to differentiate closely related bacterial species and strains (Huber et al., 2002). Characterization of P. multocida by RAPD -PCR is efficient for discovering genetic variations due to its simplicity and arbitrary primer sequence (Mohamed and Mageed, 2014). Furthermore, this technique does not require sequence information to establish genetic relatedness or variation among field isolates (Welsch and McClelland, 1990). Palindromic Sequence-Based PCR The specific primers for palindromic sequence-based extragenic repetitive PCR (REP-PCR ) complement these repetitive sequences and provide the reproducible and unique REP-PCR DNA fingerprinting patterns. Overall, the REP-PCR method is a valuable tool for rapid epidemiological analysis and characterization of bacteria and has been used in several studies (Blackall and Miflin, 2000). Furthermore, it has been used for molecular typing of P. multocida strains (Shivachandra et al., 2008). Restriction endonuclease analysis Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) has indicated information about the genomic characteristics of bacteria (Jabbari and Esmaelizadeh, 2005). Except for the time required for digestion and electrophoresis, PCR-RFLP is a new and rapid method for the classification of P. multocida (Tsai et al., 2011). DNA fingerprinting of P. multocida by restriction endonuclease analysis (REA) has proven valuable in epidemiological settings. investigations on avian cholera in poultry farms. P. multocida isolates that share both capsular serogroup and somatic serotype can be distinguished by REA. Agarose gels stained with ethidium bromide are analyzed after electrophoresis of DNA digested with Hhal or Hpall endonucleases (Wilson et al., 1992). Ribotyping in conjunction with REA has been widely used to characterize and differentiate Pasteurella multocida isolates (Blackall et al., 1995). REA followed by further hybridization with a labeled DNA probe made it easy to read the banding pattern and provide the necessary interpretation. The probe may be labeled with radioactive or non-radioactive materials. The rRNA probe is widely accepted for hybridization and subsequent interpretation (Blackall, 2000). Pulsed-field gel electrophoresis (PFGE) The utility of agarose gel electrophoresis for visualizing 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 bacterial chromosome restriction fragment patterns was an important step towards genome-based epidemiological analysis (Chijioke, 2016). PFGE analysis has consistently shown greater discrimination in identifying bacterial species than ribotyping, but, has limited application in typing Pasteurella multocida isolates (Townsend et al., 1997a). THE.