The book is a comprehensive guide that delves into the essential concepts and laboratory practices of microbiology as applied to veterinary science. Designed for undergraduate and postgraduate veterinary students, this textbook presents a balanced blend of theoretical knowledge and practical applications, fostering diagnostic proficiency and critical understanding of microbial diseases in animals.
The book begins with an introduction to the clinical microbiological laboratory and progresses through the principles of specimen collection, bacterial isolation, Gram staining, and biochemical identification techniques. It emphasizes the role of drug sensitivity testing and molecular diagnostics in effective therapeutic planning.
An extensive portion of the book covers the classification, morphology, and pathogenic characteristics of significant bacterial genera such as Staphylococcus, Streptococcus, E. coli, Salmonella, Brucella, Mycobacterium, and others. It also explores advanced immunological diagnostic methods including ELISA, agglutination, and haemagglutination-inhibition tests.
Special sections are dedicated to emerging and re-emerging diseases, transboundary animal diseases, and their implications for global trade. The book concludes with appendices highlighting viral and bacterial diseases of major domestic animals and birds.
With clear explanations, updated content, and a structured approach aligned with current curricula, this textbook is an indispensable resource for students, educators, and professionals in the field of veterinary microbiology.
The book titled “Diagnostic Veterinary Microbiology – An update” has been completed and edited especially for the veterinary undergraduates, JRF, Veterinary Assistant Surgeon and aspirants for public service commission examinations. The emergence and re- emergence of zoonotic diseases has redefined the role of veterinary researchers in infectious diseases. The animal models of diseases are of paramount importance to understand the origin, evolution, epidemiology and underlying molecular mechanisms involved in various diseases of animals and humans. No break through in science is possible in watertight close compartment. Therefore, in present scenario, climate and environmental changes share in emergence of various infectious diseases which lead to early diagnosis and an one health concept emphasizing a multidisciplinary approach to devise efficacious diagnostics, therapeutics and vaccines. The use of Artificial intelligence and Bioinformatics for designing POCT and synthetic vaccines and mathematical model for disease forecasting are classical examples. This book not only contains the most commonly address problems of paraclinical subject, the present book entitled “ Diagnostic Veterinary Microbiology-An Update” would be helpful to scholars to design their research problems and would motivate them to cross – disciplinary boundaries as per the nature of their research problem. The book will also appraise field veterinarians of recent updates. We express our sincere thanks with gratitude to our beloved teachers, friends and my dearest colleagues for their timely help, inspiration and guidance.
Location and Layout • As the laboratory will be handling materials hazardous to human and animal health, it must be isolated as much as possible from livestock yards, poultry farms and quarantine stations and should not function in or near establishments handling healthy animals, food products for human or animal consumption or near vaccine production laboratories. • Animal houses, garages, workshops, stores, water tanks, residential accommodation, administrative blocks have to be constructed as per the need of ideal microbiological laboratory. Expansion possibilities should also be kept in mind. The building can be arranged in 5 main groups: • Administrative / laboratory rooms • Decontamination and disposal area • Animal house facilities • Workshops / stores/garages and • Residential accommodation for the staff, hospital, schools, playgrounds, parks, shops etc. • The laboratory building may be rectangular in blocks, arranged in L, T or U shapes and may be of one or several stories. The temperature, wind velocity, rainfall,
On the face of an outbreak of a disease, collection of right clinical specimens in proper preservative, their transportation to the laboratory under ideal conditions and quick processing of specimens in the laboratory to identify the causative agent are crucial to successful control of the disease. This attains more importance when a highly contagious disease appears. Collection of specimens from a virus-infected host is the first step for demonstration or isolation of a virus. Specimens for virus isolation should be collected at the time when the maximum amount of virus can be expected to be present. i.e. collect the specimens as early as possible after the onset of clinical symptoms. In many viral diseases maximum viral production occurs before the patient becomes ill so that it is ideal to collect the specimen at an even early time. The nature of the specimen taken in any particular instance is determined by the patient’s clinical syndrome and previous history. They are best considered under three main headings. 1. Specimen for virus isolation/antigen detection/ nucleic acid detection 2. Specimen for serological investigation 3. Specimen for direct examination
The chief functions of Clinical Microbiological laboratory are to examine and culture specimens for microorganisms, to make accurate species identification of important isolates and to perform antibiotic susceptibility tests when indicated. These tasks will assist physicians in the diagnosis and treatment of infectious diseases. Once the specimen is received in the laboratory, the specimens are examined visually depending on the physicians order and the nature of the specimen. Wet mounts and smears may be prepared and stained for microscopic examination. Observations may or may not be immediately reported to the physicians depending on the definitiveness of the results. Timely information may often be used to establish a presumptive diagnosis and institute a specific course of therapy. Specimens that require definitive identification of potentially pathogenic microbes are processed further. All agar plates are streaked for colony isolation; then plates and broths. All cultures are placed in an incubator with appropriate temperature and environmental conditions to maximize the growth and replication of microbes. Often a presumptive microbial identificationcan be made. A final report should be delayed while subcultures and additional test procedures are performed to identify the organisms definitively. Preliminary Identification of Unknown Bacteria A number of techniques may be used in the direct microscopic examination of clinical specimens to demonstrate the presence of microbes The examination of wet mounts of unstained materials by phase contrast or dark field microscopy is useful for demonstrating motility, spirochaetes and endospores. Giemsa, Wright or acridine orange stains may be helpful in observing bacterial forms that stain poorly or that have little contrast from back ground material.
Special Staining A. Spore stain Spores may be spherical or oval and may be central subterminal or terminal in position within the cell. Shape, size and position of the spore in the cell are useful for the identification of sporulated organisms. Spores are difficult to stain and when once stained, they are difficult to decolourize. With Gram’s stain, mature spores remain unstained bodies. Modified Ziehl-Neelsen’s method of staining will be used for the staining of spores. Composition a. Concentrated carbol fuchsin b. Sulphuric acid 0.5 % solution. c. Loeffler’s alkaline methylene blue Materials Old cultures of B. subtilis and Cl. Chauvoei and staining solutions. Procedure 1. Make smears and fix over flame. 2. Stain with carbol fuchsin and steam for 3 to 4 minutes. 3. Wash with tap water. 4. Decolourize with 0.5% sulphuric acid. 5. Wash with water. 6. Counter stain with Loeffler’s methylene blue for 2 minutes.
Antimicrobial Susceptibility Test The routine use of the modified Kirby-Bauer method is recommended. Material • Sterile cotton swabs • Soyabean – Casein digest broth or Tryptone soya broth • Non selective agar such as nutrient agar. • 0.5 Mc Farland turbidity standard: Solution A Bacl2, 2H2O. 1.75G Make upto 100 ml with distilled water. Solution B H2SO4 1.0 ml Make upto 100 ml with distilled water. Stock Solution 0.5 ml of solution A + 99.5 ml of solution B. Dispense in 5 ml amounts. Seal and store in dark. • Glass vials or tubes 10 ml capacity • Muller – Hinton agar • Antimicrobial discs • Sterile forceps • Ruler or calipers
Viruses can be cultivated only in living cells. Laboratory animals, embryonated avian eggs and cell cultures are three systems available for this purpose. These three systems are used for the basic procedures of isolation and identification of viruses, maintenance of stock cultures and production of vaccines. Cultivation of Viruses in Laboratory Animals Animal inoculation helps to confirm the infectious nature of the disease. Laboratory animals used for many kinds of virological works such as clinical studies, pathogenicity of viruses, epizootiology, potency tests on vaccines and production of diagnostic and prophylactic antisera. An ideal experimental animal is one that is 1. Uniformly and fully susceptible to the virus under study 2. Free from intercurrent infection and adventitious lesions and 3. Inexpensive purchase and maintain If possible it is better to use germ free, specific pathogen free or colostrums deprived animals for experimental purpose. The same species of animals or atleast closely related species of the natural host is selected for inoculation, wherever possible. For example in case of swine fever, which shows a high degree of host specificity, the same species should be used for inoculation. in others such as in case of rabies with low host specificity, the causative virus will grow in a wide range of animals.
Molecular biology involves the study of DNA, RNA and proteins in cells. It emphasizes the biomolecules that carry out biological processes. One should understand the biomolecules participating in the process to understand the cellular function. In diagnostic microbiology, biomolecules are the analytes for testing and characterization. The analytes for molecular testing are the genomes (DNA), transcriptomes (RNA) and proteomes (Proteins) of the microorganism. Traditional methods of microorganism characterization depend on the culturing of the organisms, which is time-consuming and sometimes challenging to perform. Molecular techniques involve the characterization of DNA, RNA and protein to identify microorganisms. Nucleic acid sequence details are used to classify bacterial species, and mass spectrometry is used to identify microorganisms based on peptide profiles. DNA (Deoxy ribonucleic acid) DNA contains the genetic information required for the development and functioning of the living organism. It is the central informational molecule comprising a sequence of A, T, G and C nucelotides linked together by a phosphodiester bond. DNA is double helical in structure, composed of two complementary strands of nucleotides held together by hydrogen bonds between nitrogenous bases.
Precipitation and agglutination reactions are similar except in that for precipitation reaction antigen must be in soluble form. The precipitation reaction is highly specific and very sensitive for the detection of antigen being capable of demonstrating antigens in dilutions of 1:100,000 to 1: 1,000,000 or more. It is important to bear in mind that antigens used in precipitation reactions are of molecular size not cellular. A soluble antigen reacted with its homologous antibody at the optimum proportions produce an insoluble precipitate. The essential ingredients for precipitation test are soluble antigen (precipitinogen), antibody (precipitin) and an electrolyte (normal saline). a) Precipitation ring test The antigen is carefully layered over the antiserum, without mixing so that an interface is formed. Diffusion of each reagent will then occur into the other. If the system is homologous, precipitation will occur at the point in the tube, where the proper ratio of antigen to antibody is reached. Eg: Ascoli’s test for anthrax diagnosis. In advanced decomposition of animals when cultures would be almost impossible, the precipitation method of Ascoli is used for diagnosis of Anthrax. This method is a mixing of immunized rabbit serum with an extract of organs under examination for the production of a precipitate. Procedure 1. Grind up organs or blood of suspected animals. 2. Suspend in saline. 3. Boil for five minutes 4. Filter through filter paper and allow to cool 5. Carefully stratify 0.5 ml of this extract on 0.5 ml immune serum in small test tubes.
Among various immunodiagnostic tools enzyme immunoassay becomes increasingly popular for the detection of humoral immune response (seroconversion) or microbial antigens for the purpose of diagnosis. Enzyme Linked Immunosorbent Assay (ELISA) is widely used in studies of infectious diseases.Despite several merits of ELISA, it has some limitations such as requirement of ELISA reader to obtain accurate measurements. To overcome this, the dot immunobinding assay was developed .In Dot Immunoassay nitrocellulose membrane is used as an adsorbent matrix instead of plastic surface.The antigen is dotted onto the nitrocellulose membrane and the membrane then incubated with test antibody, and secondly with enzyme conjugated antibody directed against the first antibody.In case of positve reaction, (specific antigen –antibody reaction) color dot is developed against the white background at the site of antigen deposition.In case of negative reaction, no color development takes place at the site of antigen deposition . Principle • In direct ELISA antibody is directly attached to solid phase and targeted by added antigen (detecting antigen). • These added antigens are targeted by antispecies antibodies linked to enzyme termed as conjugates. • The bound enzyme is developed by the addition of substrate /chromogen, then stopped and finally read using a spectrophotometer
Principle The principle of the test is that antibodies possess two antigen-binding sites that will attach specifically with the antigenic determinants on the surfaces of the bacteria, cells or particles. Under suitable conditions one antibody molecule will combine with the determinant group on the surfaces of two bacteria and in this way a lattice is formed. These lattices are generally visible with the naked eye as clumps and they sediment readily due to the large size of the clump. Pre-requisites for Agglutination Reaction a) The antigen (agglutinogen) should be in particulate form, which will remain in suspension for sufficiently long time. b) The antibody (agglutinin) should be directed to the target surface epitope. c) Antigen – antibody should be present in optimal proportions. d) An electrolyte is necessary for the reaction to take place and usually the reaction is carried out in physiological saline solutions (0.85% NaCl). Because of the overall negative charge on red cells or bacteria at neutral pH it is difficult for them to come close enough, so that for molecule such as IgG antibodies are able to form a bridge between epitope of two different cells or bacteria. The presence of salt tends to neutralize the charge effects to allow agglutination. Plate Agglutination Test This is employed to screen sera within a short period for preliminary testing. Materials • Brucella abortus coloured antigen • suspected serum • glass plate.
Principle Some viruses like Orthomyxo, Paramyxoviruses etc. have the property to clump the erythrocytes of certain species of birds or animals. This is not an antigen-antibody reaction but a network of erythrocytes formed by the bridging virions. These virions on their envelop have peplomers that binds with the receptors on the erythrocytes. If large quantities of virus are used for HA and allowed to act on red blood cells for a long period of time, the agglutinated red cells shed the virus particles which then float freely in the suspending fluid and neither attach to the same red cells nor agglutinate them. This process of dissociation of virus from the red cells is known as elution, and is due to the destruction of receptors on red cells by the virion associated receptor destroying enzyme - neuraminidase. The red cells freed from the virus particles are known as stabilized red cells. The elution will occur most rapidly if the temperature is raised to 37°C as the enzyme neuraminidase will be more active at this temperature. Practical Application of Haemagglutination a) Spot test: For rapid detection of the presence of a haemagglutinating virus. This can be done by mixing two or three drops of suspected sample (infected chick embryo fluids or cell culture fluids) with equal quantity of 5% chicken red cells in normal saline. The appearance of the clumps of agglutinated red cells, indicate the presence of a haemagglutinating virus in the sample. b) Titration of the virus using the haemagglutinating property.
Microbiology is the study of microorganisms or microbes. They are very small in size and hence cannot be seen with naked eyes. Microorganisms are excellent models for understanding cell function in higher organisms, including humans. Hence the science of microbiology is the foundation of all the biological sciences and microorganisms consist of bacteria, fungi, algae, protozoa and viruses. The fields according to the organisms studied are henceforth termed as: Bacteriology - Bacteria (singular: bacterium) Phycology (phyco, seaweed) - Algae (singular: alga) Mycology (myco, a fungus) - Fungi (singular: fungus) Protozoology (proto, first; zoo, animal) - Protozoa (singular: protozoan) Virology - Viruses Microbiology began when people learned to make lenses from glass and combine them to produce magnifications great enough to see microbes. The importance of bacteria in the lives of people, animals and the environment has been unfolded in the past 100 years. It was also believed that the incidence of the disease was due to divine reasons since man had no idea of microbes at this point of time. In the earlier days, leprosy was considered as a dangerous infectious disease and spread of the disease was attributed to direct contact with the affected persons. Hence, certain procedures were specified for segregation of the affected persons. At this point of time various theories were also attributed to the genesis of disease. Some of the important theories are as follows: 1. Theurgical theory – Disease was attributed to the wrath of divine spirits as a punishment of individual sins. 2. Miasmatic theory – This theory was proposed by Hippocrates and further developed by his student Galen. It states that the disease was dueto the emanations from the
Classification of Organisms Over the years, scientists have developed several systems for the classification of organisms. Greek phlosopher Aristotle (384-322 BC) grouped life forms as either plants or animals. Microscopic organisms were unknown at that time. Fungi were included in plants. Aristotle’s system distinguished only between plants and animals on the basis of movement, feeding mechanism, and growth patterns. In 1735, Carolus Linnaeus formalized the use of two Latin names to identify each organism, a system called Binomial nomenclature. He grouped closely related organisms and introduced the modern classification groups: kingdom, phylum, class, order, family, genus, and species. At that time singlecelled organisms were observed but not classified. This classification had two kingdoms. Plantae: containing Plants and Fungi Animalia: containing Animals In 1866, Ernst Haeckel proposed a third kingdom- Protista, to include all single-celled organisms like amoebas and diatoms. Some taxonomists also placed simple multicellular organisms, such as seaweeds, in Kingdom Protista. In 1938, Herbert Copeland proposed a fourth kingdom, Monera, to include only bacteria. This was the first classification proposal to separate prokaryotes from eukaryotes, at the kingdom level. In 1957, Robert H. Whittaker proposed a fifth kingdom –Fungi.Fungi do not ingest food as animals do, nor do they make their own food, as plants do. They secrete digestive enzymes around their food, breaking it down before absorbing it into their cells.
1. Morphology of Bacteria Structure of Bacteria Bacteria are very small with a diameter of 0.5 – 1 μm in size. Due to this small size the surface area / volume ratio is very high compared to that of large organisms of similar shape. The large surface area compared to a small volume facilitates easy nourishment of all areas of the cell. So there is no circulatory mechanism to distribute the nutrients and there is little or no cytoplasmic movement within the cell. i) Shape Bacteria exhibit three types of morphology; • Spherical - cocci (coccus) - Greek word kokkos, meaning a berry • Straight rods - bacilli (bacillus) - Latin word bacillus, meaning a stick • Helically curved rods -spirilla (spirillum) Variations in these basic forms are also seen. Eg: cocobacillary, ovoid and filamentous.
First discovered by Scottish surgeon sir Alexander ogston (1880) in infected tissues. He named it as staphylococcus” (Greek staphyle, bunch of grapes; KOKKAS, berry). Systematics Domain : Bacteria Phylum : Firmicutes Class : Bacilli Order : Bacillales Family : Staphylococcaceae Genus : Staphylococcus Species : Staph. aureus Staph. Intermedius Staph. hyicus Habitat and Ecology Staphylococcus occurs worldwide in mammals although the spread of staphylococcal strains between different animal species is limited. They colonise the nasal cavity, skin and mucous membranes and can be transient in the intestinal tract. It is an opportunist type organism.
Systematics Domain : Bacteria Phylum : Firmicutes Class : Bacilli Order : Lactobacillales Family : Streptococcaceae Genus : Streptococcus Species : Str. agalactiae, Str.dysgalactiae, Str. equi subsp. zooepidemicus, Str. uberis, Str. equi subsp equi, Str.suis, Str. canis, Str. pyogenes (human) History Rivolta (1873) described chain forming organisms in pus from a case of strangles in horses. In 1878-79, Pasteur recognized this organism as a pusforming agent. In 1903, Hugo Schottmuller introduced blood to differentiate various types of hemolysis. In 1928, Rebecca Lancefield reported a serological method of grouping Streptococci. Habitat Streptococci are worldwide in distribution. Most of the Streptococci of Veterinary interest live as commensals in the mucosa of the upper respiratory and lower urogenital tracts. They do not survive for long away from the animalhosts.
B.anthracis causes anthrax in animals and Wool sorter’s disease, hide porter’s disease, Malignant Pustule in humans. B.cerus causes food poisoning in humans. Other bacillus in this group is non-pathogenic and they are called as anthracoids. B.licheniformis is an emerging pathogen and it is implicated in sporadic abortions in cattle and sheep. Systematics Domain : Bacteria Phylum : Firmicutes Class : Bacilli Order : Bacillales Family : Bacillaceae Genus : Bacillus Species : B. anthracis, B. cereus, B. subtilis, B. mycoides, B. megaterium, B.mesentricus Family Characters They are gram +ve large rods, aerobic (facultative anaerobic), endospore forming, capsulated, mostly catalase positive and fermentative organisms. They are motile by peritrichous flagella. Bacillus Anthracis History • Discovery of the anthrax bacillus is credited to Davaine and Rayer (1863 –1868). • Considerable historic interest attached to anthrax bacilli;
The genus Clostridium consists of gram positive, spore forming anaerobic bacilli. The spores are wider than the bacillary bodies, giving the bacillus a swollen appearance, resembling a spindle. Hence, the name clostridium (Kloster- meaning spindle) was given. Systematics Domain : Bacteria Phylum : Firmicutes Class : Clostridia Order : Clostridiales Family : Clostridiaceae Genus : Clostridium The clostridia can be divided into four major groups according to the kind of disease they produce. They are as follows.
Listeria has been divided into seven species with two distinct groups. Among which the Listeria monocyotgenes and Listeria ivanovii are haemolytic and pathogenic for animals. The Listeria murrayi and Listeria grayi are nonhaemolytic, rarely isolated and considered to be non pathogenic. Among which the genus L. monocytogens, being the cause of septicaemia, abortion and CNS infections in a wide range of animal species including humans. History L.monocytogenes first described by Murray (1926) who named it as bacterium monocytogenes because of characteristic monocytosis infection in laboratory animals. It was renamed Listerella hepatolytica by Pirie (1927) and the present name given by him in 1940. The Listeria momnocytogenes was first isolated by Gill (1929) from sheep. Natural Habitat Listeria species are widely distributed in the environment and can be isolated from soil, faeces, plants, decaying vegetation and silage (pH 5.5) in which the bacteria can multiply. Silage is commonly implicated in outbreaks of listeriosis in cattle and sheep. In poor quality silage the listerial numbers may reach 107 cfu/kg of silage. Asymptomatic faecal carriers occur in man and many animal species. L.monocytogens can be excreted in bovine milk. Human foods associated with listeriosis in man include soft cheeses, milk and poultry meat.
Natural Habitat The bactrerium is widespread in nature and has been recovered from a wide variety of wild and domestic animals including mammals, fish (both fresh and salt water), birds, reptiles and amphibians. It is present in the soil and can survive for 20 days or longer in alkaline soil. The major source of infection for swine and turkeys is carrier animals of the same species. It is reported that 30-50% of pigs carry the bacterium in their tonsils, other lymphoid tissues. It is present in slurry of piglets and can be recovered from the faeces of carrier pigs. Morpholgy Erysipelothrix rhusiopathiae (previously named Erysipelothrix insidiosa) from S (Smooth) -form colonies and usually from acute syndromes is a grampositive rod, the R (rough) form colonies usually from chronic disease is a gram-positive filament. The organism is non-motile, non-spore forming, nonacid fast, capsulated occur either in singly, in groups or in chains. Cultural Characters It is a facultative anarobe, but growth is enhanced by 10% CO2. It is able to grow in a temperature range of 50C to 420C, within a pH range 6.7 to 9.2 and 8% NaCl2. Growth occurs on nutrient agar but is improved by the addition of serum or blood. It will not grow on Mac Conkey agar. Media contain either sodium azide (0.1%) or crystal violet (0.001%) may be used as selective media. On blood agar, non-haemolytic pinpoint colonies (0.5 mm) appear at 24hrs incubation. Colonial variation becomes obvious at 48hrs incubation when azone of greenish haemolysis often develops under and just around the colonies.
Mycobacteria are slender rods of varying lengths that sometimes show branching filamentous form resembling ‘fungal mycelium’. Hence, the name mycobacteria, meaning fungus like bacteria. Although cytochemically gram positiive, the Mycobacteria do not take up the dyes of the gram stain because the cell walls are rich in lipids – Mycolic acid. Once a dye has been taken up by the cells they are not easily decolourised, even by acid-alcohol. Mycobacteria are therefore called as acid-fast bacilli. The genus includes animal and human pathogens as well as saprophytic members often referred to as atypical, anonymous, opportunistic, tuberculoid and MOTT (Mycobacteria other than typical tubercle) bacilli. Clasification of Mycobacteria (Tubercle Bacilli) I. Slowly growing Mycobacteria • Mycobacterium tuberculosis causes human tuberculosis in human and dogs. • Mycobacterium bovis causes bovine tuberculosis in many animal species and also cause tuberculosis in human • Mycobacterium africanum causes human tuberculosis. • The human type (Mycobacterium tuberculosis) is primarily a pathogen for man. But can cause disease in cattle, pigs, dogs, monkeys, parrots and other species. The bovine type (Mycobacterium bovis) is a common cause of disease in domestic animal particularly cattle, pigs, cat, dogs and horse. The avian type (Mycobacterium avium) is primarily a pathogen for birds. But can cause disease in cattle, sheep, goat and pigs.
Corynebacteria are gram positive, non-acidfast, non-motile, non-capsulated, small pleomorphic rods. They frequently occur in rods, coccoid, club and filamentous shape. The term coryneform which refers to the pleomorphic club shape of these gram-positive bacteria. (From Coryne – meaning club). The major pathogen is Corynebacterium diptheria. Which causes diptheria in children. Corynebacteria associated with animals are called diphtheroids. Morphology They are gram-positive slender rod with a tendancy to clubbing at one or both ends; they are non-sporing, non-motile, non-capsulated and non-acidfast. They have granules composed of (high energy phosphate stores) – polymetaphosphate. The granules are more strongly gram positive than the rest of the bacterial cell. Stained with Loeffler’s methylene blue, the granules take up a reddish purple color and hence they are called metachromatic granules. They are called as volutinor Babes Ernst Granules. They are often situated at the poles of the bacilli and are called polar bodies. Special stains, such as Albert’s, Neisser’s and Ponder’s have been devised for demonstrating the granules clearly. Stained smears from animal tissues often reveal groups of cells in parallel (Palisades) or cells at sharp angles to each other (Chinese letter or Cuneiform arrangement). This is due to the incomplete separation of the daughter cells after binary fission. Rhodococcus equi can appear as a gram-positive coccus or a rod or club shaped form arranged in clusters. It is capsulated and sometimes weakly acid fast.
The actinomycetes comprise a heterologous group of prokaryotes that have the ability to form gram positive, branching filaments of less than 1μm in d.m. The main animal pathogens in the actinomycetes are in the genera Actinomyces, Arcanobacterium, Actinobaculum, Nocardia and Dermatophilus. Nonpathogenic, prolific producers of antimicrobial substances – streptomyces are also included in Actinomycetes. Actinomyces Natural Habitat The actinomyces species are present on mucous membrane of the host animal, often in the oral cavity, tonsils, and nasopharynx. The soil is the natural habitat of many actinomyces species. History The generic name Actinomyces was first used by Harz (1879). Boestrom (1891) isolated Actinomyces bovis. Cummins (1962) clearly demonstrated Actinomyces were bacteria and they are distinct from other branching genera. Morphology The organisms show considerable pleomorphism. Actinomyces species are usually long and fialmentousalthough short V, Y, and T configuration also occur. In lesions of actinomycosis, the pus contains small pale yellow granules referred as sulfur granules. The sulphur granule is composed of bacterial filaments and mineralized calcium phosphate of host origin. When the granules are crushed and gram stained, a mass of gram-positive branching filaments about 1μm in width, short rods, and cocci are evident. Around this mass, a circle of club shaped bodies with their narrow ends pointing towards the centre-staining gram negative. Hence, called ray fungus. They are nonacid fast, non-spore forming, nonmotile, non-capsulated and do not form endospores or conidia.
Nocardia has the ability to form gram-positive, branching filaments of less than 1um in diameter. It is closely related to Corynebacterium, Mycobacterium and Rhodococcus species History Nocard described this organism in 1888, following its isolation from a case of bovine ‘farcy’, hence the name of the type species: Nocardia farcinica. Natural Habitat Nocardia species are soil borne saprophytes Morphology They are gram-positive, obligate aerobes ability to form branching filaments. Some produce true mycelia and some strains are acid-fast. All species are nonmotile. Gram stained smears from lesions revealed gram-positive branching filaments that often show fragmentation into coccobacillary elements. The modified ZN stained smears exhibit a similar morphology but most of the filaments retain the carbol-fuchsin dye and stain red. Cultural Characters The Nocardia species grow very well in blood agar incubated aerobically at 370C for upto 7 days. Inoculate the suspected colonies from blood agar into Sabouraud dextrose agar (SDA) and incubated at 370C for upto 10 days.
Dermatophylaceae is a group of bacteria with mycelial filaments which divide transversely and in at least two longitudinal planes to form masses of coccoid or cuboidal cells, which characteristically become motile. They are gram positive, non acid fast, aerobic and produce aerial mycelium when their growths are stimulated by 10% CO2. Dermatophilus congolensis, Dermatophilus dermatonomus and Dermatophilus pedis are the pathogens causing variety of skin lesions in mammals, including man. Dermatophilus congolensis causes very severe clinical disease and its infection is most common in tropical and subtropical regions. Diseases caused by Dermatophilus species Dermatophilus congolensis mainly affects Cattle, horses, sheep and goats, but many animal species and man can be infected.The disease has many names; Cattle : Streptothricosis or Dermatophilosis Horse : Skin Funk (Rain Rot, Rain Scald, Dew Poisoning), Grease heal Sheep: Mycotic dermatitis (general infection), lumpy wool (wool- covered skin) and strawberry foot rot (skin of lower leg and coronet Natural Habitat Dermatophilus congolensis is the only species in the genus is thought to maintain itself in small foci of infection on a carrier animal or within scab particles in dust. It can survive in scab material for period’s upto 3 years. History Bovine disease was first described by Van Saceghem in 1915 in the Congo now known as Zaire in Africa.
Introduction Escherichia coli (commonly abbreviated as E. coli) is a Gram-negative, facultative anaerobic, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Escherichia coli are common inhabitants of the terminal small intestine and large intestine of mammals. They are often the most abundant facultative anaerobes in this environment. They can occasionally be isolated in association with the intestinal tract of non-mammalian animals and insects. The presence of E.coli in the environment is usually considered to reflect faecal contamination and not the ability to replicate freely outside the intestine. Morphology E.coli is Gram-negative, facultative anaerobic and non-sporulating. Cells are typically rod-shaped, and are about 2.0 micrometers (μm) long and 0.25- 1.0 μm in diameter, with a cell volume of 0.6–0.7 μm. It can live on a wide variety of substrates. Strains that possess flagella are motile. The flagella have a peritrichous arrangement. It is motile by peritrichous flagellae, though some strains are non-motile.Spores are not formed. Capsules and fimbriae are found in some strains. Cultural Characteristics Escherichia coli or E.coli cells may grow on a solid or in a liquid growth medium under a laboratory condition. Solid and liquid media may have exactly the same composition except that the solid medium contains an extra 1.5% agar. Different E.coli clones may have different properties. Colonies growing on solid media represent different clones. It is an aerobe and a facultative anaerobe. Optimal growth of E. coli occurs at 37°C (98.6°F) but some laboratory strains can multiply at temperatures of up to 49°C (120°F).
Klebsiella pneumoniae (also known as Friedlander’s bacillus) is a Gramnegative, non-motile, encapsulated, lactose fermenting, facultative anaerobic, rod shaped bacterium found in the normal flora of the mouth, skin, and intestines. Klebsiella spp. are Gram-negative, nonmotile, usually encapsulated rod-shaped bacteria, belonging to the family Enterobacteriaceae. These bacteria produce lysine decarboxylase but not ornithine decarboxylase and are generally positive in the Voges-Proskauer test. Members of the Enterobacteriaceae family are generally facultative anaerobic, and range from 0.3 to 1.0 μm in width and 0.6 to 6.0 μm in length. Klebsiella spp. often occurs in mucoid colonies. The genus consists of 77 capsular antigens (K antigens), leading to different serogroups. Morphology of Klebsiella pneumoniae (K. pneumoniae) • Shape – Klebsiella pneumoniae is a short, plump, straight rod shape (bacillus) bacterium. • Size – The size of Klebsiella pneumoniae is about 1–2 μm × 0.5–0.8 μm (micrometer). • Arrangement Of Cells – K. pneumoniae is arranged singly, in pairs, or in short chains and sometimes in clusters. • Motility – Klebsiella pneumoniae is a non-motile bacterium. • Flagella – K. pneumoniae is a non-flagellated bacterium. • Spores – The Klebsiella pneumoniae is a non–sporing bacterium. • Capsule – Capsules are present in Klebsiella pneumoniae which can easily be demonstrated using India ink preparation, appear as a clear halo in a dark background. • Gram Staining Reaction – Klebsiella pneumoniae is a Gram -ve (Negative) bacterium.
Salmonella consists of bacilli leading to Enteric fever, Gastroenteritis, Speticaemia etc. The important member of the genus is Salmonella typhi, which causes Typhoid fever. Salmonella are of two groups; (i) Enteric fever group consisting of typhoid & Paratyphoid bacilli exclusively or primary human parasites (ii) Food poisoning group, which are animal parasite but may infect humans causing gastrointestinal infections Salmonella is oxidase negative, catalase positive, indole and Voges Proskauer (VP) negative, methyl red and Simmons citrate positive, H2S producing andurea negative. Characteristics Cells are rod-shaped, non-spore-forming, and predominantly motile by means of peritrichous flagella with diameters of around 0.7-1.5μm and lengths of 2-5μm with a few exceptions. On blood agar, colonies are 2-3mm in diameter. Colonies are generally lactose non-fermenters. They obtain their energy from oxidation and reduction reactions using organic sources, and are facultative anaerobes. They produce acid from glucose usually with the production of gas, and are oxidase negative. Most produce hydrogen sulphide except Salmonella paratyphi A and Salmonella typhi, which is a weak producer. They are identified with a combination of serological and biochemical tests. Salmonella species are classified and identified into serotypes according to the White-Kauffmann-Le Minor scheme; there are more than 2,500 Salmonella serotypes that have been described and reported.
Yersinia species are non-lactose fermenters and, with the exception of Y. pestis are motile. Although there are more than 10 Yersinia species, only Y. pestis, Y. enterocolitica and Y. pseudotuberculosis are pathogenic for animals and man. Yersinia ruckeri causes perioral haemorrhagic inflammation in some species of fish. Growth of yersiniae tends to be less rapid than other members of the Enferobacteriaceae. They characteristically demonstrate bipolar staining in Giemsa-stained smears from animal tissues Natural Habitat Y. pestis, the cause of bubonic plague in man and a sylvatic cycle in animals, is transmitted mainly by fleas from tolerant rodents. Human infections through cuts, bites, scratches and aerosols can also occur. Cats are susceptible to Y.pestis and naturally infected cats can pose a health hazard for humans in endemic areas. Y. pseudotuberculosis persists in wild rodents and birds as well as in the environment. The intestinal tract of wild and domestic animals appears to be the reservoir for Y. enterocolitica. Pigs, particularly, are carriers of Y. enterocolitica strains pathogenic for humans. Isolation Yersinia species grow on nutrient, blood and MacConkey agars but the colonies, after 24 hours incubation, tend to be smaller than those of the other members of the Enterobacteriaceae. Y. pestis grows poorly on agars containing desoxycholate whereas Y. enterocolitica and Y. pseudotuberculosis grow well on these media. Yersinia selective medium (CIN agar) containing the antibiotic supplement cefsulodin (15mg/litre), irgasin (4 mg/litre) and novobiocin (2.5 mg/litre) is designed for the isolation of Y. enrerocolitica from faeces.
The genera Proteus and Providencia belong to the tribe Proteae of the family Enterobacteriaeceae. The members of both these genera are gram negative, motile bacilli, aerobes and facultative anaerobes and can grow on basic media. A characteristic feature which distinguishes tribe Proteae from other members of Enterobactereaceae is the presence of the enzyme phenylalanine-deaminase which converts phenylalanine to phenylpyruvic acid (PPA reaction).They also produces a powerful urease enzyme which rapidly hydrolyses urea to ammonia. Genus Proteus They are gram-negative bacilli, 1-3 μm long and 0.6 μm wide. They are noncapsulated and are actively motile by peritrichous flagella. The name ‘Proteus’ refers to their pleomorphism, after the Greek God Proteus who could assume any shape. Four species: Proteus mirabilis, P.vulgaris, P.penneri and P.myxofaciens are recognized. Proteus mirabilis, P.vulgaris are widely recognised as human pathogens. Culture Characteristics These can grow on ordinary media like nutrient agar with a characteristic fishy or seminal odour. On MacConkey and Teepol lactose agar, lactose nonfermenting pale colonies, around 2-3 mm in size are formed. On non-inhibitory solid media such as blood and nutrient agar Proteus mirabilis and P vulgaris show characteristic swarming growth in the form of a uniform film, which spreads over the whole surface of the plate. In young swarming cultures, many of the bacteria are long, curved and filamentous, sometimes reaching upto 80 μm in length. When two different strains of swarming proteus mirabilis encounter one another on an agar plate, swarming ceases and a visibile line of demarcation forms. This is known as the Dienes phenomenon. In liquid medium (peptone water, nutrient broth), Proteus produces uniform turbidity with a slight powdery deposit and an ammoniacal odour.
History Pseudomonas is comprised of two Greek words-Pseudo meaning false and Monas meaning unit, so literally the term means “false unit”. However Pseudomonas is a real bacteria so we really do not know the basis of the nomenclature by Walter Migula. A scientist by the name of Migula gave the Genus name of Pseudomonas in 1894 to these bacteria. Classification The classification of Pseudomonas is given below: • Class : Gamma Proteobacteria • Order : Pseudomonadales • Family : Pseudomonadaceae • Genus : Pseudomonas • Eight groups : P. aeruginosa ; P. chlororaphis; P. fluorescens; P.pertucinogena; P.putida; P. stutzeri; P. syringae; P. incertae sedis. The family has 191 valid species. These include bacteria which are saprophytic, free living, and human, animal and plant pathogens. The type species is Pseudomonas aeruginosa. Usual Habitat Pseudomonas species are environmental organisms which occur worldwide in water and soil, and on plants. Pseudomonas aeruginasa is also found on the skin, on mucous membranes and in faeces. Burkholderia pseudomallei, which is found in soils, occasionally infectsanimals and man. Wild rodents can act as reservoirs of this organism. It is widely distributed in some tropical and subtropical regions of Southeast Asia and Australia.Although B.mallei can survive in the environment for upto 6 weeks its reservoir is infected Equidae.
Pasteurella and Mannheimia species are small (0.2 x 1-2 μm) non-motile, Gramnegative rods or coccobacilli. They are oxidase-positive facultative anaerobes, and most species are catalase-positive. Although non-enriched media will support their growth, these organisms grow best on media supplemented with blood or serum. They usually remain viable for only a few days on culture plates. Some species, such as Mannheimia haemolytica, Pasteurella trehalosi and P. aerogenes can tolerate the bile salts in MacConkey agar. In smears from infected tissues stained by the Giemsa method, pasteurellae exhibit bipolar staining. The family Pasteurellaceae comprises five genera, Actinobacillus, Haemophilus, Mannheimia, Pasteurella and Lonepinella. These genera share a number of common features and some organisms have been reclassified within these genera following deoxyribonucleic acid hybridization studies and 16s rRNA sequencing. The pasteurellae are small (0. 2 μm by up to 2.0 μm), Gram-negative rods or coccobacilli. They are nonmotile,non-sporing, facultatively anaerobic, fermentative(except for P. anatipesrijer), oxidase-positive andcatalase - positive (except for P. caballi). Althoughunenriched media support their growth, they grow beston media supplemented with serum or blood. Recent Changes in Nomenclature Previous name Present name Pasteurella ureae Actinobacillus ureae Haemophilus avium Pasteurella avium Pasteurella pneumotropica Pasteurella dogmatis (Henriksen biotype)
Actinobacillus species are non-motile, Gram-negative rods (0.3 to 0.5 x 0.6 to 1.4 μm) which occasionally have a coccobacillary appearance. They are non-motile, non-sporeformingand non-acid-fast. A surface slime is presentin the three major species (A. ligllieresii. A. equuli andA. suis) and may be related to the stickiness of theircolonies on agar. Surface cultures have low viabilityand die in 5-7 days. The actinobacilli ferment carbohydrates, without the production of gas, within 24 hours and most species produce urease and grow on MacConkey agar. Most species are urease and oxidase positive. The reactions in the oxidase andcatalase tests are variable. The genus is still in a stateof flux, mainly because of the close similaritiesbetween actinobacilli and species in the genera Pasteurella and Haemophilus. These facultative anaerobes ferment carbohydrates producing acid but not gas. Actinobacilli exhibit some host specificity and are mainly pathogens of farm animals. Usual Habitat Actinobacilli are commensals on mucous membranes of animals particularly in the upper respiratory tract and oral cavity. As actinobacilli cannot survive for long in the environment, carrier animals play a major role in transmission. Colonial Appearance • A. lignieresii: small, glistening colonies develop in 24 hours. They are usually slightly sticky (viscid) on primary isolation but lose th is characteristic on subculture. The colonies are non-haemolytic and develop to about 2 mm in diameter in 48 hours. The organism grows well on MacConkey agar, the colonies are at first pale but become pinkish as A. lignieresii is a late lactose-fermemer. • A. equuli: some strains are haemolytic and the colonies are sticky with this feature remaining on subculture. I t is a lactose-fermenter onMacConkey agar.
Introduction The genus Haemophilus contains small, nonmotile, nonsporing, oxidase positive, pleomorphic, gram negative bacilli that are parasitic on human beings or animals. Haemophilus means blood loving organisms. Haemophilus species are small (less than 1 pm x 1 to 3 μm), Gram-negative rods, which often appear coccobacillary and may occasionally form short filaments. These motile organisms, which are facultative anaerobes with variable reactions in catalase and oxidase tests, do not grow on MacConkey agar. They are fastidious bacteria requiring one or both of the growth factors X (haemin) and V (nicotinamide adenine dinucleotide, NAD). Optimal growth occurs in an atmosphere of 5-10% CO2 on chocolate agar which supplies both X and V factors. Small, transparent, dewdrop-like colonies are formed by most Haemophilus species after incubation for 48 hours. Colonies of H.somnus have a yellowish hue and some isolates are haemolytic on sheep blood agar. The main pathogens in the genus are H.somnus in cattle and sheep, H.parasuis in pigs and H.paragallinarum which is responsible for infectious coryza of chickens. The haemophili are motile, facultative anaerobes, produce acid from glucose, reduce nitrates and are variable in the oxidase and catalase tests. They are nutritionally fastidious, will not grow on MacConkey agar and grow best on chocolate agar (supplying the X and V factors) under 5- 10 per cent CO2 at 37°C. The growth of many of the Haemaphilus species is enhanced by 10 per cent CO2, As this is not inhibitory for any of them, CO2 should be used for routine isolation.The inoculated chocolate agar plates are incubated under 10 per cent CO2 at 35-37°C for 3-4 days, although some growth may be seen after 24 hours.
Brucella species are small (0.6 x 0.6 to 1.5 pm), nonmotile, coccobacillary, Gram-negative bacteria. As they are not decolourized by 0.5% acetic acid in the modified Ziehl-Neelsen (MZN) staining technique, they are classed as MZN-positive. In MZN-stained smears of body fluids or tissues, they characteristically appear as clusters of red coccobacilli. For taxonomic purposes, all Brucella species should be classified as Brucella melitensis as DNA hybridization studies have shown that the genus contains only one species. Brucella species are aerobic, capnophilic and catalase-positive. Apart from B.ovis and B.neotomae, they are oxidase-positive. All Brucella species are urease-positive except B.ovis. Brucella ovis and some biotypes of B.abortus require 5 to 10% CO2 for primary isolation. Moreover, the growth of other Brucella species is enhanced in an atmosphere of CO2. Media enriched with blood or serum is required for culturing B.abortus biotype 2 and B.ovis. Recently, brucellae have been detected in sea-mammals. Smears are made from specimens and stained by the modified Ziehl- Neelsen (MZN) stain. Brucellae appear as small, red-staining coccobacilli in clumps because of their intracellular growth. Isolation The brucellae grow well on 5-10 per cent blood agar. However, other than foetal abomasal contents and colostrum, the specimens are likely to contain many contaminating bacteria and fungi, so selective media are usually required. The selective media contain a nutritive blood agar base with 5 per cent sterile seronegative equine or bovine serum and an antibiotic supplement. The antibiotic supplement used in selective media for B.ovis usually differs from that for B. abortus. Skirrow agar is a satisfactorymedium for both the Campylobacter fetus subspecies and for brucellae, including the most fastidious species such as B. abortus biotype 2, B.canis and B.ovis.
Introduction Vibrios are Gram-negative, rigid, curved rods that are actively motile by means of a polar flagellum. The name ‘Vibrio’ is derived from the characteristic vibratory motility (from vibrare, meaning to vibrate). They are asporogenous and noncapsulated. Vibrios are present in marine environments and surface waters worldwide. The most important member of the genus is Vibrio cholerae, the causative agent of cholera. It was first isolated by Koch (1883) from cholera patients in Egypt, though it has been observed earlier by Pacini (1884) and others. Natural Habitat Vibrio spp. can be present in both freshwater and seawater as well as in the alimentary tracts of animals and man. At least five Vibrio spp. are human pathogens including V. cholerae, the cholera bacillus, and V.parahaemolyticus which causes food poisoning. Only V. metschnikovii is associated with disease in domestic animals. It causes a cholera-like disease in chickens and other birds but its geographical distribution is very limited. V. anguillarum causes infections in many species of fish especially in salt or brackish water. It causes high mortality in salt water eels. Vibrio Cholerae Morphology The cholera vibrio is a short, curved, cylindrical rod, about 1.5 × 0.2-0.4 mm in size, with rounded or slightly pointed ends. The cell is typically comma shaped (hence the old name V comma) but the curvature is often lost on subculture. S-shaped or spiral form may be seen due to two or more cells lying end to end. Pleomorphism is frequent in old cultures. In stained films of mucus flakes from acute cholera cases, the vibrios are seen arranged in parallel rows,
Campylobacter species are slender, curved motile Gram negative rods (0.2 to 0.5 pm wide) with polar flagella. Daughter cells which remain joined have a characteristic gull-winged appearance and long spirals formed by joined cells also occur. These microaerophilic organisms grow best on enriched media in an atmosphere of increased CO2 and decreased oxygen tension. Many Campylobacter species grow on MacConkey agar. They are non-fermentative and oxidase-positive and have variable catalase reactions. Usual Habitat Many Campylobacter species are commensals in the intestinal tracts of warm-blooded animals. Campylobacter jejuni and C. lari (formerly C. laridis) colonize the intestines of birds, which can result in faecal contamination of water courses and stored food. A number of Campylobacter species are excreted in the faeces of pigs. Campylobacter fetus subspecies venerealis appears to be adapted principally to bovine preputial mucosa. Differentiation of Campylobacter Species Campylobacter species are strictly microaerophilic, requiring an atmosphere of 5 to 10% oxygen and 1 to 10% CO2 for growth. A selective enriched medium such as Skirrow agar is usually used for primary isolation. Differentiation of isolates is based on colonial morphology and certain cultural, biochemical and antibiotic-susceptibility characteristics. Colonial Morphology • Campylobacter fetus subspecies venerealis and C.fetus subspecies fetus have small, round, smooth, translucent colonies with a dewdrop appearance. • Campylobacter jejuni produces small, flat, grey colonies with a spreading, watery appearance.
to be coccobacillary.They are strict aerobes and do not attack carbohydratesbut derive energy by the oxidation of amino acids. B. avium and B. bronchiseptica are motile by peritricholls flagella but B. pertussis and B. parapertussis are non-motile. All are catalase-positive and oxidase-positive. B. bronchiseptica and B. aium willgrow on MacConkey agar. The genus Bordetella contains four species, B.pertussis, B.parapertussis, B. bronchiseptica and B. avium. Bordetella pertussis, the type species, and B.parapertussis are human pathogens associated with whooping cough in children. Bordetella bronchiseptica infects a wide range of animal species including man, while B.avium is a pathogen of avian species. The bordetellae are occasional pathogens which have an affinity for ciliated respiratory epithelium. Bordetella bronchiseptica and B.avium are small (0.2 to 0.5 x 0.5 to 1.5 pm), Gramnegative rods with a coccobacillary appearance. They are catalasepositive, oxidase-positive aerobes and are motile peritrichous bacteria. Because they cannot utilize carbohydrates, they derive their energy mainly from the oxidation of amino acids and have no special growth requirements. They grow on MacConkey agar. Natural Habitat The bordetellae are inhabitants primarily of the upper respiratory tract of healthy and diseased humans, animals and birds. B. pertussis and B. parapertussis are human pathogens causing whooping cough and a mild form of whooping cough, respectively. B. bronchiseptica can be present in the upper respiratory tract of pigs, dogs, cats, rabbits, guinea-pigs, rats, horses and possibly other animals. B. avium inhabits the respiratory tract of infected poultry, principally turkeys. Mammalian infections are mainly transmitted by aerosols but in turkeys indirect spread can occur via water and litter.
Moraxella bovis occurs as short (1.0 to 1.5 x 1.5 to 2.5 μm), plump Gramnegative rods or, occasionally, cocci which typically occur in pairs. This organism is non-motile, aerobic and usually catalase-positive and oxidasepositive. Although proteolytic, it is unable to utilize sugars. Growth, which is enhanced by the addition of blood or serum to media, does not occur on MacConkey agar. The optimal temperature for growth is 33-35°C. Most M.phenylpyruvica strains will grow on MacConkey agar. But M.bovis and M.lacunata are unable to do so. Virulent strains, when isolated from cases of infectious bovine keratoconjunctivitis, are fimbriate, haemolytic and grow into the agar. Species, other than M.bovis, which are periodically isolated from clinical specimens, are generally regarded as non-pathogenic. Usual Habitat Moraxella bovis is found on mucous membranes of carrier cattle. The organism is susceptible to desiccation and is short-lived in the environment. It can survive for up to 72 hours in the salivary organs and on the body surface of flies, which can act as vectors. Colonial Appearance On blood agar after 48 hours’ incubation the colonies of M.bovis are flat, round, small (1 mm diameter), greyish-white and friable, surrounded by a narrow zone of complete haemolysis. The appearance is not unlike that of a beta-haemolytic streptococcus. New isolates are often piliated and erode the agar, sinking into it. Colonial growths will autoagglutinate when suspended in saline. On subculture, colonial variation is common, pili are no longer formed and the colonies are butyrous and less likely to autoagglutinate. Some colonies can become non-haemolytic. The strains of M. hovis (M.equi) isolated from horses are non-haemolytic even on primary isolation. M.lacunata and M. phenylpyruvica are non-haemolytic on blood agar and some strains of M. phenylpyruvica will grow on MacConkey agar.
Bacteroides spp are non-spore forming gram-negative bacilli that are part of the human resident flora. Microbiologically, they are distinguished from other genera by growth in 20% bile. At present, the Bacteroides fragilis group consists of ten species: B. fragilis (the most frequent isolate), B. distasonis, B. thetaiotaomicron, B. vulgatus, B. ovatus, B. eggerrthii, B. merdae, B. stercoris, B. uniformis, and B. caccae. Since 1990, many organisms previously designated as Bacteroides have been reclassified (see chapter on Anaerobes other than Bacteroides). Bacteroides, the predominant genus in the human intestine, are important in numerous metabolic activities and may provide some level of protection from invasive pathogens. All 10 species are usually isolated from the colon, although infections caused by or associated with them can include virtually any organ. Characteristics The gram-negative Bacteroides spp. or closely related genera are capsulated obligatory anaerobic bacilli that are non-spore forming, pale-staining, and some are motile by peritrichous flagella, while other taxa are non-motile. Bacteroides, Parabacteroides, Odoribacter are generally bile resistant, distinguished from genera which are bile sensitive. They are normally commensal, found in the intestinal tract of humans (mouth, colon, urogenital tract) and other animals. Many cultures of Bacteroides strains display brown to black pigmentation on blood agar media caused by esculin hydrolysis. Morphology In general, all strains were stained poorly with safranin, but well with dilute carbolfuchsin. Motility was not observed in any strains. The morphology of Bacteroides fragilis in fresh cultures and in pus is not especially characteristic. In this form, the bacillus measures 0.5 by 2.0 to 2.5 micra. Older cultures tend to develop larger forms (0.6 to 0.8 by 2.0 to 9.0 micra) but even in this state the organism cannot be distinguished from aerobic Gram-negative bacilli, by its morphology.
Many non-spore-forming, anaerobic, Gram-negative bacteria cause opportunistic mixed infections, often in association with facultative anaerobes. Synergistic interactions between the organisms in these mixed infections are common. Fusobacterium species and bacteria formerly referred to as Bacteroides species account for more than 50%of the anaerobic organisms isolated from these infections. Usual Habitat Nan-spore-forming, Gram-negative anaerobes are often found on muwus membranes, particularly in the digestive tract, of animals and man. They are excreted in the faeces and they can survive for short periods in the environment. Dichelobacter nodosus, a primary pathogen of the epidermal tissues of the hoof region of ruminants, survives for less than 4 days in mud. Diagnostic Procedures In order to ensure that isolates of anaerobes are aetiologically significant, specimens for isolation procedures should be obtained by direct sampling from discharges or lesions and by supra pubic puncture in urinary infections. Specimens should be processed promptly after collection. Commercial kits and transport media are available for specimens from suspected anaerobic infections. In the core of a tissue specimen over 2-3 cm, an anaerobic microenvironment is usually maintained. Samples of fluid in a syringe remain suitable for anaerobic culture if air is expelled from the syringe and the needle is plugged. Anaerobic jars with an atmosphere of hydrogen and 10%CO2 are used for incubating cultures at 37°C for up to 7 days.
The order Spirochaetales contains two families, Leptospiraceae and Spirochaetaceae. It comprises spiral or helical bacteria (spirochaetes) which share some unique morphological and functional features. Members of the order are motile by means of endoflagella which are located within the periplasm. The spirochactes are slender, motile, flexuous, unicellular, helically coiled bacteria ranging rrom 0.1 -3.0 μm in width. The outer sheath, the outermost layer of a spirochaete cell, is a multi layered membrane that completely surrounds the peri plasmic flagella (axial filament) and the helical protoplasmic cylinder. The cylinder consists or the nuclear material, cytoplasm, cytoplasmic membraneand the peptidoglycan portion of the cell wall. The periplasmic flagella are wrapped around the cylinder and are in the peri plasmic space of these Gram-negative bacteria. One end of each flagellum is inserted near a pole of the protoplasmic cylinder and attached by platelike structures called insertion discs. The distal end of each flagellum is not inserted and extends to the centre of the cell and may overlap the flagellum from the opposite end. The periplasmic flagella facilitate the motility of the bacteria in viscid environments. Leptospira species Members of this species (leptospires) are motile helical bacteria (0.1 x 6 to 12 μm) with hook-shaped ends. Although cytochemically Gram-negative, they do not stain well with conventional bacteriological dyes and are usually visualized using dark-field microscopy. Silver impregnation and immunological staining techniques are used to demonstrate leptospires in tissues. Leptospirosis, which can affect all domestic animals and humans, ranges in severity from mild infections of the urinary or genital systems to serious systemic disease.
Emerging Infectious Diseases Emerging infectious diseases are those due to newly identified and previously unknown infections which cause public health problems either locally or internationally. Re-emerging Infectious Diseases Re-emerging infectious diseases are those due to the reappearance and increase of infections which are known, but had formerly fallen to levels so low that they were no longer considered a public health problem. What Causes Emergence or Re-emergence of Infectious Diseases? Several factors contribute to the emergence and re-emergence of infectious diseases, but most can be linked with the increasing number of people living and moving on earth: rapid and intense international travel; overcrowding in cities with poor sanitation; changes in handling and processing of large quantities of food; and increased exposure of humans to disease vectors and reservoirs in nature. Other factors include a deteriorating public health infrastructure which is unable to cope with population demands, and the emergence of resistance to antibiotics linked to their increased misuse. Avian Influenza Avian influenza is a zoonotic, globally important disease of birds that can be categorised as either low pathogenic (LPAI) or highly pathogenic (HPAI) according to the virulence of the virus in animals. Outbreaks of LPAI are common around the world but LPAI typically causes no clinical signs or only minor illness in infected birds. LPAI strains are generally less of a threat to human health; in addition, LPAI can have significant economic repercussions as a result of export restrictions and culling of birds, particularly in developing countries where the use of vaccines and other veterinary medicines is difficult due to weak veterinarian services and small farming units. Compared with LPAI, HPAI is more readily detected in poultry due to very high levels of mortality in infected birds, and can cause potentially catastrophic economic consequences, from significantly reduced livestock populations to lost export markets. Though relatively rare, sporadic human infections of HPAI have occurred in cases of close contact with infected birds and caused serious illness and even death.
Members of the genus Serpulina (Treponema) are host-associated spirochaetes found in the oral cavity, intestinal tract and genital region of animals and humans. The cells are wider, are not as tightly coiled as th e leptospires and can be stained by aniline dyes. The species of veterinary significance are S. Hyodysenleriae (swine dysentery) and T. paraluiscunicllii (ventdisease of rabbits). S. innocens, present in the faecesof pigs and dogs, is thought to be non- athogenic,although some workers regard S. innocens as a strainof S. hyodysenteriae of lower virulence. S. hyodysenteriae and S. innocens are s imilarmorphologically, culturally as well as biochemicallyand can be grown on laboratory media. T. Paraluiscuniculi has not been cultured in vitro. Normal Habitat T. paraluiscliniculi produces a benign venereal disease of rabbits and is present in lesions in the genito-perineal area of rabbits. It causes latentin fection in mice, guinea- pigs and hamsters. The treponemes can be found in the lymph nodes of these animals. The reservoir of S. hyodysenteriae is the intestinal tract of pigs, wild rats and mice. Recovered, asymptomatic pigs can excrete these organisms in faeces for 3 months or more. Survival of S. hyodysenteriae in soil or voided pig faeces is short, about 24 48 hours. Infection is by the faecal-oral route.
Borreliae, which are longer and wider than other spirochaetes, have a similar helical shape. In addition to a linear chromosome, which is unique among bacteria, borreliae possess linear and circular plasmids. Although these spirochaetes can cause disease in animals and humans, subclinical infections are also common. Borreliae are transmitted by arthropod vectors. Usual Habitat Borreliae are obligate parasites in a variety of vertebrate hosts. Although these organisms persist in the environment for short periods, they depend on vertebrate reservoir hosts and arthropod vectors for long-term survival. Differentiation of Borrelia Species Borreliae can be differentiated from other spirochaetes by their morphology, by the low guanine and cytosine content of their genomic DNA and by ecological, cultural and biochemical characteristics. Identification of Borrelia species depends mainly on genetic analysis. At least nine genospecies or genomic groups of B.burgdorferi sensulato, have been identified using DNA-DNA hybridization, 16s rRNA sequencing and other molecular techniques. Clinical Infections The species of particular veterinary importance are B. burgdorferi sensu lato, the cause of Lyme disease in animals and humans, and B. anserina which causes avian borreliosis. The significance of two other species, B.theileri andB.coriaceae, as animal pathogens, is uncertain. Lyme Disease This condition, also known as Lyme borreliosis, was first identified in 1975 following investigation of a cluster of arthritis cases in children near the town of Old Lyme, Connecticut. The causative agent, a spirochaete, was named Borrelia burgdorferi.
Five genospecies of intestinal spirochaetes have been isolated from pigs namely Brachyspira hyodysenteriae,B. pilosicoli, B. innocens, Serpulina intermedia and S. murdochii. The genera Serpulina and Brachyspira were recently combined. These anaerobic spirochaetes have six to fourteen spirals and are 0.1 to 0.5 μm in width. Usual Habitat Pathogenic Brachyspira species are found in the intestinal tract of both clinically affected and normal pigs. Carrier pigs can shed B.hyodysenteriae for up to three months and are the principal source of infection for healthy pigs. Differentiation of Brachyspira Species The differentiation of B.hyodysenteriae from other intestinal spirochaetes is based on its pattern of haemolysis on blood agar. Tests for detecting indole production or the hydrolysis of hippurate are also useful diagnostically. Restriction endonnclease analysis, restriction fragment length polymorphism, ribotyping using 16s rRNA analysis, PCR-based assays and multilocus enzyme electrophoresis have been developed both for differentiating species and for distinguishing strains of organisms within species. Brachyspira hyodysenteriae isolates can also he allocated to several serogroups and serotypes. Pathogenesis Most information on the pathogenesis of Brachyspira species derives from studies of B.hyodysenteriae. Motility in mucus is an essential virulence factor of thisorganism; mutant strains with altered motility are lesscapable of colonizing the pig intestine. Colonization may be enhanced by factors in mucuswith chemotactic activity for the organisms. Factors with such chemotactic activities have been demonstrated in vitro. Haemolytic activity,demonstrated in vitro, correlates with pathogenicity andthree genes encoding haemolytic and cytotoxic activityhave been cloned and sequenced.
The mycoplasmas are microorganisms in the class Mollicutes. Of the nine genera in this class, five contain species of veterinary interest. The genus Mycoplasma, in which there are about 100 species, contains most of the animal pathogens. The first mycoplasma identified in 1890 was Mycoplasma mycoides subspecies mycoides, the cause of contagious bovine pleuropneumonia. Similar types of mycoplasmas which were subsequently identified were called pleuropneumonia- like organisms (PPLO). Mycoplasmas, the smallest prokaryotic cells capable of self-replication, are pleomorphic organisms ranging from spherical (0.3 to 0.9 pm in diameter) to filamentous (up to 1.0 μm long). Because they cannot synthesize peptidoglycan or its precursors, they do not possess rigid cell walls but have flexible, triplelayged outer membranes. Their flexibility allows them to pass through bacterial membrane filters of pore size 0.22 to 0.45 μm. Mycoplasmas are susceptible to desiccation, heat, detergents and disinfectants. However, they are resistant to antibiotics such as penicillin which interfere with the synthesis of bacterial cell walls. Based on 5s rRNA sequence analyses, the mycoplasmas have been shown to be linked phylogenetically to Gram-positive bacteria such as Clostridium species which have low guanine-cytosine content in their DNA. They require enriched media for growth, characteristically forming umbonate micro-colonies when illuminated obliquely and microcolonies with a ‘fried egg’ dppearance in transmitted light. The dense central zone is due to extension of the microcolony into the agar. Mycoplasmas, which have relatively small genomes (approximately 800 genes) are fastidious in their growth requirements. Most mycoplasmas are facultative anaerobes and some grow optimally in an atmosphere of 5 to 10% CO2. Nonpathogenic anaerobic mycoplasmas are found in the rumens of sheep and cattle. The genera Mycoplasma and Ureaplasma contain animal pathogens.
Introduction Q fever is a zoonotic bacterial infection caused by Coxiella burnetii, an obligate intracellular parasite, classified within the family Rickettsiaccae. Q fever (Q for “query”) was first used in 1937 to describe a mysterious febrile illness of packing house workers in Brisbane, Australia. The causative agent was isolated from infected workers and later was identified as Rickettsiae. Almost simultaneously the same organism was identified wood tick collected in Montana. Cox, an American, and Burnet, an Australian, where honored for their early work with this organism, hence the name “Coxiella burnetii”. Host and Susceptibility Coxiella burnetii is a well-established infectious agent that has reached a state of balanced pathogenicity in a plethora of host. Human are unnatural and usually dead-end-host. The microorganism generally maintained in a less pathogenic form in separate cycle existing independently among wild mammals and their haematophagus arthropods, principally tick and in domestic animals. Morphology It is an intracellular organism which is a polymorphic bacillus (0.2-0.4mm width, 0.4-1.0 mm length), which has a cell membrane, like Gram negative bacteria. However, it stains poorly with pigmented Gram stain, but gimenez staining is traditionally used to stain the Coxiella burnetii pathogen from pathological materials and crop. Coxiella burnetii has several distinctive characteristics, including a sporulationlike process that protects the organism against the external environment, where it can survive for long periods. In mammals, the usual host cell of C. burnetii is the macrophage, which is unable to kill the bacterium. The other an important characteristic of C. burnetii is its antigenic variations, those called phase variation. This antigenic shift can be measured and is valuable for differentiating acute from chronic Q fever. Thus, it displays two antigenic phases those are phase-I and phase- II that are liable to the Lipopolysaccharide (LPS) of the membrane.
Aetiology Classification of the causative agent • Ehrlichia ruminantium (formerly Cowdria ruminantium) Order Rickettsiales, Family Anaplasmataceae • Small, Gram negative, pleomorphic coccus, and obligate intracellular parasite. • Strains of E. ruminantium are very diverse and vary in virulence: while some strains are highly virulent, others appear to be less-pathogenic. • E. ruminantium has a high level of genomic plasticity. Several different genotypes can co-exist in a geographical area, and may recombine to form new strains. • E. ruminantium multiplies in vascular endothelial cells throughout the body to cause severe vascular compromise. • It usually occurs in clumps of from less than five to several thousand organisms within the cytoplasm of infected capillary endothelial cells, and can be detected in brain smears by light microscopy. Resistance to Physical and Chemical Action Temperature Heat labile and loses its viability within 12–38 hours at room temperature. Infective stabilities can be cryopreserved in DMSO (dimethyl sulphide) or better yet in sucrosepotassium phosphate-glutamate medium (SPG). Infective half-life of thawed stabilate kept on ice is only 20–30 minutes. pH: Not applicable. Disinfectants: Not applicable. Survival:
Bacteria from the genus Anaplasma belong to the Procaryota kingdom, the family Anaplasmataceae. Anaplasma belong to obligate intracellular microorganisms, Gram-negative bacteria, living in the blood cells of mammals. Vertebrates can be their reservoir, i.e. an environment where the pathogen can live and proliferate for many years. However, in many cases bacteria from the genus Anaplasma cause diseases in domestic animals and people. Taxonomical Position of Bacteria from the Genus Anaplasma The genera Anaplasma, Ehrlichia, Neorickettsia and Wolbachia include obligate intracellular bacteria, parasitizing in the vacuoles of cells in eukarytic hosts. Animal pathogens were attributed to the genus Anaplasma, such as A. centrale, A. marginale, A. platys, A. ovis and A. bovis, and also the aetiological factor of human anaplasmosis, A. phagocytophilum. Anaplasma marginale A. marginale are obligate intracellular bacteria parasitizing in erythrocytes of higher vertebrates, mostly ruminants. Anaplasma Centrale A. centrale is a parasite in the erythrocytes of ruminants, mainly cattle. As opposed to A. marginale, it creates concentrations in the central part of the cell. A. centrale is less pathogenic to cattle than A. marginale but, most importantly, occasionally gives resistance against the latter. Hence it is used for the preparation of live vaccine strains, assuring immunological protection against bovine anaplasmosis. Anaplasma Bovis A. bovis is a bacterium detected mainly in cattle, but also observed in small mammals which are probably a reservoir of this bacterium. It occurs in monocytes, and the disease it causes is called monocytic anaplasmosis. The symptoms of the disease are most visible in calves, but also in adult animals; symptoms include weakening of the body, marked reduction in weight, elevated temperature, enlargement of prescapular lymph nodes, paling of the mucous membranes, and in many cases an elevated amount of secreted mucus.
Organisms in the order Rickettsiales form a diverse group of small (0.3 to 0.5 x 0.8 to 2.0 pm), non-motile, pleomorphic Gram-negative bacteria which replicate only in host cells. They can be cultured in the yolk sac of embryonated eggs or in selected tissue culture cell lines. Because they stain poorly with aniline dyes, these organisms should be stained by Romanowsky methods such as Giemsa or Leishman. In addition to host-cell dependence and poor affinity for basic dyes, a requirement for an invertebrate vector distinguishes them from conventional bacteria and the Chlamydiales. Epidemiology Animal hosts and arthropod vectors are the reservoirs for most rickettsiae. A number of rickettsia1 organisms, including Ehrlichia canis, Anaplasma marginale and Haemobartonella felis produce latent infections. In arthropods, rickettsiae replicate in the epithelial cells of the gut before spreading to other organs, including the salivary glands and ovaries where further replication may occur. Organisms are transmitted when the arthropod feeds on the animal host. Some organisms such as Rickettsia rickettsii are maintained in a tick population by transovarial transmission. Trans-stadial but not transovarial transmission of E. canis and E. phagocytophila occurs in ticks. With the exception of Coxiella burnetii, which produces endospore-like forms and can remain viable in dust for up to 50 days, most rickettsiae are labile outside host cells. Aerosol transmission of C.burnetii commonly occurs in domestic animals and humans. In addition, a silent cycle involving ticks and small wild mammals may constitute a possible source of infection for some domestic species. Pathogenesis and Pathogenicity Many Rickettsia species including the causal agents of typhus (R. prowuzekii), murine typhus (R. typhi) and scrub typhus (R. tsutsugamushi) are primarily human pathogens. Rocky Mountain spotted fever, caused by Rickettsia rickettsii, which is a common rickettsia1 disease of humans, also affects dogs.
Chlamydiae are obligate intracellular bacteria with an unusual developmental cycle during which unique infectious forms are produced. They replicate within cytoplasmic vacuoles in host cells. On account of their apparent inability to generate ATP, with resultant dependence on host cell metabolism, they have been termed ‘energy parasites’. The family Chlamydiaceae belongs to the order Chlamydiales. Currently two genera, Chlamydia and Chlamydophila, and nine species are described. Formerly a single genus and four species, Chlamydia trachomatis, C. psittaci, C. pneumoniae and C. pecorum, were recognised. This classification was based on phenotypic characteristics such as host preference, inclusion morphology, iodine staining for the presence of glycogen, and sulphonamide susceptibility. However, recent nucleic acid sequencing studies of the 16s and 23s rRNA genes confirm two distinct lineages. In the developmental cycle of chlamydiae, infectious and reproductive forms are morphologically distinct. Infectious extracellular forms, called elementary bodies (EBs) are small (200 to 300 nm), metabolically inert and osmotically stable. Each EB is surrounded by a conventional bacterial cytoplasmic membrane, a periplasmic space and an outer envelope containing lipopolysaccharide. The periplasmic space does not contain a detectable peptidoglycan layer and the EB relies on disulphide cross-linked envelope proteins for osmotic stability. Elementary bodies enter host cells by receptor-mediated endocytosis. Acidification of the endosome and fusion with lysosomes are prevented by mechanisms which are not fully understood. A process of structural reorganization within the pathogen, of several hours duration, results in the conversion of an EB into a reticulate body (RB). The RB, about 1 μm in diameter, is metabolically active, osmotically fragile and replicates by binary fission within the endosome. The endosome and its contents, when stained, is called an inclusion. When a number of inclusions containing RBs of C.trachomatis are formed in an infected cell, fusion of these structures may occur.
Emerging Infectious Diseases Emerging infectious diseases are those due to newly identified and previously unknown infections which cause public health problems either locally or internationally. Re-emerging Infectious Diseases Re-emerging infectious diseases are those due to the reappearance and increase of infections which are known, but had formerly fallen to levels so low that they were no longer considered a public health problem. What Causes Emergence or Re-emergence of Infectious Diseases? Several factors contribute to the emergence and re-emergence of infectious diseases, but most can be linked with the increasing number of people living and moving on earth: rapid and intense international travel; overcrowding in cities with poor sanitation; changes in handling and processing of large quantities of food; and increased exposure of humans to disease vectors and reservoirs in nature. Other factors include a deteriorating public health infrastructure which is unable to cope with population demands, and the emergence of resistance to antibiotics linked to their increased misuse.
Trans-boundary Animal Diseases (TADs) are highly contagious diseases of livestock in the world and transmissible diseases which have the potential for very serious and rapid spread, irrespective of national borders, which has serious socio-economic or public health consequence and they are importance in the international trade of animals and animal products. With rapidly increasing globalization, an associated risk of movement of trans-boundary diseases is emerging. Trans-boundary animal diseases represent a serious threat. They reduce production and productivity, disrupt local and national economies and also threaten human health (zoonotic). Trans-boundary animal disease is a concern globally; cumulative effort is needed at international level to minimize the spread of infectious diseases across the borders. Considering that livestock rearing constitutes a significant share in the national economy of a developing country, it is imperative to take up disease control initiatives. Measures are required to safeguard the livestock industry from epidemics of infectious diseases and to uphold safe international trade of livestock and their products. In this regard, it is essential to develop scientific and risk-based standards that facilitate the international trade in animal commodities. Introduction Trans- boundary Animal Diseases (TADs) are highly contagious diseases of livestock in the world. Moreover, their economic importance is a major constraint in international trade. Their implication on human Health and National food security cannot be over emphasized. Zoonotic diseases among TAD’s include diseases like West Nile Virus (WNV), Rift Valley Fever (RVF), Mad Cow disease (BSE), Bovine Tuberculosis and Highly Pathogenic Avian Influenza (HPAI). Other important TADs are Foot and Mouth Disease (FMD), Contagious Bovine Pleuropneumonia (CBPP), Lumpy Skin Disease, African Swine Fever (ASF) and Newcastle Disease (ND). They have the potential for very rapid spread, irrespective of national borders and these diseases can cause serious socio-economic and possibly public health consequences.
