Table of contentsIntroductionClinical signsPostmortem lesionDetection method by conventional techniqueMorphology and cultural characteristicsBiochemical characteristics, pathogenicity testSerological identificationDNA-based techniquesRandomly amplified polymorphic DNA PCRExtragenic extragenic based PCR on a palindromic sequenceRestriction endonuclease analysisPulsed-field gel electrophoresis (PFGE)Status of avian cholera in Ethiopia and diagnostic techniques usedConclusion and recommendationsIntroductionThe diagnosis of avian cholera depends on the identification of the responsible bacteria, P. multocida, after isolation of birds showing signs and lesions consistent with this disease. Presumptive diagnosis may be based on the observation of typical signs and lesions and/or microscopic demonstration of bacteria exhibiting bipolar staining in tissue smears, such as blood, liver or spleen. Mild forms of the disease can occur (OIE, 2015). Confirmatory diagnosis is carried out by isolation and identification of the causative agent. Various laboratory diagnostic techniques have been developed over the years for pasteurellosis and used regularly in the laboratory. Among these techniques, molecular diagnostic techniques are the most important. This technique not only gives a 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 Should Not Be Banned”? Get the original essay Clinical Signs The clinical signs of acute avian cholera are unfitness, fever, ruffled feathers, discharge of mucus from the mouth, dyspnea, and watery or yellowish diarrhea (Rhoades and Rimler, 1990). Birds suffering from a chronic form of the disease may exhibit depression, conjunctivitis, dyspnea, lameness, stiff neck, swelling of the wattles, sinuses, limb joints, foot pads and sternal bursae (Christensen and Bisgaard, 2000). In cases of significant lung involvement, there will be loud wheezing and coughing as the disease progresses. Depending on the particular strain of P. multocida involved, morbidity and mortality can be high or very high. With less virulent strains, some affected birds may recover slowly, after a variable period of depression. With more virulent strains, death usually occurs quickly after a brief period of prostration, accompanied by convulsive wing flapping and paddling. Birds that survive acute illness may recover completely or develop exudative arthritis of the leg or wing joints. Arthritis can occur without signs of acute systemic disease, particularly in very young or old birds (Wilkie, et al., 2012). Postmortem lesions 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 site of chronic infections (OIE, 2008). The most common gross 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 by conventional techniqueIdentification and characterization of P. multocida is based on the ability to cultivate or purify the organism inlaboratory. The purified organism is then classified according to phenotypic characteristics such as morphology, carbohydrate fermentation patterns, and serological properties. However, culture conditions can influence the expression of these attributes, thereby decreasing the stability and reliability of phenotypic methods of 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 from birds that succumb to the acute form of the disease, as well as exudative lesions from birds with the chronic form of the disease, are generally easy to achieve. Isolation of chronically ill birds that show no signs of illness other than emaciation and lethargy is often difficult. Under these conditions or when host decay has occurred, bone marrow is the tissue of choice for isolation attempts. The surface of the tissue to be cultured is grasped with a hot spatula and a sample is obtained by inserting a sterile cotton swab, thread 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). Morphology and cultural characteristics Identification of P. multocida can be done based on morphological study and coloring properties. , cultural and biochemical characteristics, as described by Cheesbrough (2006) and cultural and morphological examinations can be carried out as described by Cowan and Steel (2004). Accordingly, samples suspected of avian cholera are first grasped with a spatula and incised with a small sterile scalpel blade and forceps. The sample is inoculated directly into tryptose broth medium, incubated for 2 to 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 their ability to produce any changes in the medium such as hemolysis on blood agar can be examined. If our sample is a swab from these organs, it is inoculated directly onto a selective medium, such as casein sucrose yeast (CSY) agar, blood agar, and incubated aerobically at 370°C for 24 hours. Then, suspected colonies are subjected to Gram staining and methylene blue for cell morphology. The Gram stain result showing Gram negative, with characteristics of bipolar coccobacilli, was considered P. multocida. Biochemical characteristics, pathogenicity test Phenotypic characterization of Pasteurella multocida by biochemical reaction from various samples based on the sugar fermentation reaction (Cowan and Steel, 2004). Pasteurella multocida does not cause hemolysis, it is not mobile and 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. Nitrates are reduced; indole and hydrogen sulfide are produced, and methyl red and Voges-Proskauer tests are negative (Glisson, et al., 2008). The pathogenicity test of P. multocida strains can be carried out using a pure colony grown for 18 h in a shaker. incubator at 37°C in Brain Heart Infusion (BHI) broth. Approximately 0.2 ml of each culture containing approximately 2.4 x 108 colony forming units/ml can be inoculated into three mice tested per route.intraperitoneal and observed for 72 h to examine the mortality profile. If the organism is re-isolated from heart blood collected from dead mice on a blood agar plate and a liver print smear reveals the agent by the Giemsa staining method and again, if the Re-isolated colonies showed characteristics similar to those of P. multocida and the impression smears revealed a bipolarity typical of the organism, P. multocida is considered pathogenic (Shivachandra et al., 2005).Serological identificationSerological tests , such as enzyme-linked immunosorbent assays (ELISA), agglutination and indirect hemagglutination (IHA) tests, have been used to identify antibodies against Pasteurella multocida in poultry sera (Marshall et al., 1981). An 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 success in attempts to monitor seroconversion in vaccinated poultry. The ELISA test has been used for decades to screen for antibodies against avian cholera in avian species (Marshall et al., 1981; Solano et al., 1983). Commercial ELISA kits are available for chickens and turkeys. ELISA is a rapid, very sensitive and specific serological test (Poolperma et al., 2017). Among the ELISA types, the indirect ELISA test is most commonly applied. This test is designed to measure the relative level of antibodies against P. multocida (Pm) in bird serum. The antigen is placed on 96-well plates. Upon incubation of the test sample in the coated well, P. multocida (Pm)-specific antibodies form a complex with the coated antigen. After removing unbound material from the wells, a conjugate is added that binds to any bird antibodies attached in the wells. Unbound conjugate is washed away and an enzyme substrate is added. Further development of color is directly related to the amount of antibodies against P. multocida (Pm) present in the tested sample (Aydin, 2015).DNA-based techniquesPhenotypic 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. Thus, in recent years, phenotypic differentiation tools have been frequently replaced by genotypic methods (Taylor et al., 2010). Unlike conventional methods, PCR-based typing techniques have been shown to be rapid and highly sensitive in identifying and differentiating strains. Pulsed-field gel electrophoresis (PFGE) is known to be the standard for epidemiological typing of P. multocida strains, although one study indicated that repetitive palindromic sequence-based extragenic PCR (REP-PCR) compares favorably. Additionally, randomly amplified polymorphic DNA (RAPD) is a suitable technique to study 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 performed based on a PCR targeting the capsular gene cap specific to P. multocida, 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 quality are checked by running a 5 μL 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 aamplicon 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 grade water, and DNA template 2 μL. The thermal profiles 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 seconds, annealing at 55°C for 30 seconds and elongation at 72°C for 90 seconds. dry, and a final extension at 72°C for 5 min. 5 μL of PCR product can be loaded into a 1% (w/v) agarose gel with 1 μL of 6X loading dye for electrophoresis in 1X TBE buffer at 100 V for 30 min. A standard 100 bp DNA ladder can also be loaded into the same gel to compare the size of amplified PCR products. Before running the gel, ethidium bromide (0.5 μg/mL) can be added to the gel. PCR products were visualized under UV light in an image documentation system (Mahmuda, 2016). Randomly amplified polymorphic DNA PCR The randomly amplified polymorphic DNA technique relies on polymorphic DNA which can be amplified by one or more short oligonucleotide primers of arbitrary sequences with 8 to 12 nucleotides (ziva et al., 2008). As RAPD is a simple, rapid 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 effective in discovering genetic variations due to its simplicity and arbitrary sequence of primers (Mohamed and Mageed, 2014). Additionally, this technique does not require sequence information to establish genetic relatedness or variation among field isolates (Welsch and McClelland, 1990). Extragenic Repetitive Palindromic Sequence Based PCRSpecific primers for 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 rapid epidemiological analysis and characterization of bacteria and has been used in several studies (Blackall and Miflin, 2000). Additionally, 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 provided information on the genomic characteristics of bacteria (Jabbari and Esmaelizadeh, 2005). Except for the time required for digestion and electrophoresis, PCR-RFLP is a novel and rapid method for classification of P. multocida (Tsai et al., 2011). DNA fingerprinting of P. multocida by restriction endonuclease analysis (REA) has proven valuable in epidemiology. investigations of avian cholera in poultry flocks. P. multocida isolates sharing both a capsular serogroup and a somatic serotype can be distinguished by REA. Agarose gels stained with ethidium bromide are analyzed after electrophoresis of the DNA digested with Hhal or Hpall endonuclease (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 additional hybridization with a labeled DNA probe facilitating reading of the banding pattern and giving the necessary interpretation. The probe can be marked with radioactive or non-radioactive materials. The rRNA probe is widely accepted for hybridization and subsequent interpretation (Blackall, 2000). Pulsed-field gel electrophoresis.