Veterinary · Para-clinical · also MSVE VMC-211, General Bacteriology and Mycology
How bacteria and fungi are built, grown, identified and killed, and how bacteria become resistant to antimicrobials.
In the exams: ICAR AICE-JRF core group 06; ICAR AIEEA-PG 13 (Microbiology); PRC Microbiology & Public Health 14%; China microbiology and immunology 35 (its largest single component); TNPSC Micro/VPH/Preventive Medicine 30
General veterinary microbiology is the study of how bacteria and fungi are built, how they grow, how they are cultured and identified in the laboratory, how they are killed, how they exchange genes, and how they resist antimicrobial drugs. It is the grammar that systematic bacteriology, infectious diseases, public health and pharmacology are written in.
The subject matters in practice because almost every clinical decision about a bacterial or fungal infection passes through the diagnostic laboratory. The veterinarian chooses the specimen, keeps it alive or keeps it from overgrowing on the way, asks for the right culture, reads the identification and the susceptibility report, and decides whether the isolate is a cause, a contaminant or a commensal. The same veterinarian sterilises instruments, sets disinfection protocols for a milking parlour or a hatchery, and is increasingly asked by regulators to justify antimicrobial use. Antimicrobial resistance moves between animals, people and the environment, so this subject sits at the centre of One Health.
Worldwide the subject is examined early and often. In India it is taught as General Bacteriology and Mycology (MSVE course VMC-211) and it carries weight in the ICAR postgraduate and PhD entrance tests and in state veterinary officer examinations. It is part of the largest single component (microbiology and immunology) of China's national practising veterinarian examination, and it shares the Microbiology and Public Health paper in the Philippine licensure examination. This manual keeps to the diagnostic and clinical-laboratory level taught to veterinary students. Where practice or standards differ between countries or between standard-setting bodies, the text says so rather than choosing one.
Bacteria are prokaryotes: they have no nucleus bounded by a membrane, no mitochondria, no endoplasmic reticulum and no Golgi apparatus. Their DNA lies in a region of the cytoplasm called the nucleoid, usually as a single circular chromosome, although some bacteria (for example Borrelia) have linear chromosomes and some have more than one chromosome. Most bacteria of veterinary importance measure roughly 0.5 to 1 micrometre across and 1 to 5 micrometres long, so they are visible with the oil-immersion objective of a light microscope but their fine structure needs electron microscopy. Shapes are described as cocci (spheres), bacilli or rods, coccobacilli (short rods such as Pasteurella and Brucella), curved or comma-shaped rods (Vibrio, Campylobacter), spirilla and spirochaetes (Leptospira, Borrelia, Brachyspira), and pleomorphic forms. Arrangement follows the plane of division: cocci in clusters (staphylococci), chains (streptococci), pairs (diplococci) or packets of four or eight (Micrococcus, Sarcina). The cytoplasmic membrane is a phospholipid bilayer without sterols (Mycoplasma is the exception, taking sterols from the host or medium).
The cell wall gives shape and resists osmotic lysis. It is built of peptidoglycan: chains of alternating N-acetylglucosamine and N-acetylmuramic acid joined by short peptide cross-links. Transpeptidases, also called penicillin-binding proteins, make the cross-links; beta-lactam antibiotics bind these enzymes, and glycopeptides such as vancomycin bind the D-alanyl-D-alanine end of the peptide. Gram-positive bacteria have a thick peptidoglycan layer, many layers deep, threaded with teichoic and lipoteichoic acids, and no outer membrane. Gram-negative bacteria have a thin peptidoglycan layer in a periplasmic space, outside which lies an outer membrane. The inner leaflet of the outer membrane is phospholipid; the outer leaflet is largely lipopolysaccharide (LPS). LPS has three parts: lipid A, which is embedded in the membrane and is the endotoxin; a core oligosaccharide; and the O-specific polysaccharide side chain, which is the O antigen used in serotyping Salmonella and Escherichia coli. Mycobacteria, and to a lesser degree Nocardia and Rhodococcus, have a wall rich in mycolic acids (long-chain branched fatty acids) linked to arabinogalactan. This waxy wall resists the Gram stain, resists drying and many disinfectants, and holds carbol fuchsin against acid-alcohol, the basis of acid-fast staining. Mycoplasmas have no cell wall at all: they are pleomorphic, pass through filters that hold back ordinary bacteria, are not stained usefully by Gram's method and are intrinsically resistant to beta-lactams.
Many bacteria secrete a capsule, a well-organised layer outside the wall, or a looser slime layer. Most capsules are polysaccharide; the capsule of Bacillus anthracis is unusual in being a polypeptide of D-glutamic acid. Capsules resist phagocytosis and complement, and they are a major virulence factor and vaccine antigen; the capsular types of Pasteurella multocida (A, B, D, E, F) are used in epidemiology. Capsules are shown by negative staining with India ink or nigrosin, and the B. anthracis capsule appears as pink material around blue square-ended rods in M'Fadyean's polychrome methylene blue stain of blood smears. Flagella are protein (flagellin) filaments driven by a rotary motor in the membrane. Their arrangement is monotrichous (one polar flagellum, as in Vibrio), lophotrichous (a polar tuft), amphitrichous (one at each pole, as in Campylobacter) or peritrichous (all around, as in Salmonella and most motile Enterobacterales). Flagellar protein is the H antigen. Spirochaetes move by periplasmic flagella (axial filaments) lying under an outer sheath. Pili, or fimbriae, are shorter, thinner protein appendages. Common (attachment) fimbriae are adhesins: the F4 (K88) and F5 (K99) fimbriae of enterotoxigenic E. coli bind the intestinal epithelium of piglets and calves. Sex pili connect donor and recipient cells in conjugation. Endospores are dormant, highly resistant structures formed inside the cell by Bacillus and Clostridium when nutrients run out. The spore core is dehydrated and contains calcium dipicolinate and small acid-soluble proteins that protect DNA; around it lie a cortex of special peptidoglycan and tough protein coats. Spores survive boiling, drying and many disinfectants, which is why sterilisation is defined by killing them. Spore position (central, subterminal or terminal) and whether it swells the cell help identification; Clostridium tetani has a round terminal spore giving a drumstick shape. Spores are stained by the Schaeffer-Fulton method with malachite green.
Taxonomy has three parts. Classification arranges organisms into groups by their relationships; nomenclature gives the groups names under agreed rules; identification decides which named group a new isolate belongs to. Bacterial names follow the International Code of Nomenclature of Prokaryotes. A species has a two-part (binomial) Latin name, the genus written with a capital letter and the species epithet in lower case, both in italics in print: Staphylococcus aureus, later abbreviated S. aureus. The ranks above species are genus, family, order, class and phylum. Salmonella is the classic example: the genus has two species, S. enterica and S. bongori, and S. enterica subspecies enterica contains more than two thousand serovars defined by O and H antigens in the White-Kauffmann-Le Minor scheme, so the full name is Salmonella enterica subspecies enterica serovar Typhimurium, usually written Salmonella Typhimurium with the serovar not in italics. Names change as knowledge improves. Several veterinary organisms have been renamed in the last two decades, for example Haemophilus parasuis to Glaesserella parasuis and Haemophilus paragallinarum to Avibacterium paragallinarum, and in 2021 phylum names were brought into line with the code, so Firmicutes became Bacillota and Proteobacteria became Pseudomonadota. Bergey's Manual of Systematic Bacteriology, and its online successor, is the standard reference for classification, while Bergey's Manual of Determinative Bacteriology is organised for identification.
Classical classification used phenotype: shape, Gram reaction, motility, spore formation, oxygen requirement, biochemical reactions and antigenic structure. Molecular methods now anchor classification. The gene for 16S ribosomal RNA is present in all bacteria, has conserved regions for primers and variable regions that differ between taxa, and is the usual first tool for placing an unknown organism in a genus. Its limitation is that closely related species may have almost identical 16S sequences. The traditional species definition was DNA-DNA hybridisation of about 70 per cent or more between strains; genome sequencing has largely replaced this with average nucleotide identity, where a value of roughly 95 to 96 per cent is used as the species boundary. In the diagnostic laboratory, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identifies colonies by their protein fingerprint against a reference library and has replaced many biochemical panels where it is available; its accuracy depends on the library containing the veterinary species concerned.
Veterinary textbooks group bacteria in a way that helps diagnosis rather than strict phylogeny. Gram-positive cocci include Staphylococcus (catalase positive) and Streptococcus and Enterococcus (catalase negative). Gram-positive rods are divided into spore-formers (Bacillus, aerobic; Clostridium, anaerobic) and non-spore-formers (Listeria, Erysipelothrix, Corynebacterium, Trueperella, Rhodococcus). Branching or filamentous Gram-positives include Actinomyces, Nocardia and Dermatophilus; mycobacteria form their own acid-fast group. Gram-negative rods include the Enterobacterales (E. coli, Salmonella, Klebsiella, Proteus, Yersinia), the non-fermenters (Pseudomonas, Burkholderia), the Pasteurellaceae (Pasteurella, Mannheimia, Bibersteinia, Glaesserella, Actinobacillus, Histophilus, Avibacterium), Bordetella, Brucella, Francisella, Moraxella and Taylorella. Curved Gram-negatives include Campylobacter, Helicobacter and Lawsonia. Spirochaetes are Leptospira, Borrelia and Brachyspira. Gram-negative anaerobes include Fusobacterium, Dichelobacter and Bacteroides. Wall-less bacteria are the mycoplasmas (class Mollicutes). Obligate intracellular bacteria, which cannot be grown on ordinary cell-free media, include Chlamydia, Rickettsia, Ehrlichia, Anaplasma and Lawsonia intracellularis; Coxiella burnetii was long placed here but can now be grown in special axenic media. These groupings matter because they predict the first steps in the laboratory: which media, which atmosphere, which staining method and which biochemical tests to set.
Bacteria need a source of carbon, energy, nitrogen, phosphorus, sulphur, minerals and, for many, growth factors they cannot make. Organisms of veterinary importance are nearly all chemoheterotrophs, taking both carbon and energy from organic compounds. Fastidious organisms need enriched media: Haemophilus-group bacteria need haemin (X factor), nicotinamide adenine dinucleotide (V factor) or both; Glaesserella parasuis and Avibacterium paragallinarum are classically V-factor dependent and grow as satellite colonies next to a streak of Staphylococcus, which releases NAD. Temperature groups are psychrophiles (optimum below about 15 degrees C), mesophiles (optimum about 20 to 45 degrees C, including almost all animal pathogens) and thermophiles (optimum above about 45 degrees C). Listeria monocytogenes and Yersinia enterocolitica multiply slowly at refrigerator temperature, which matters for food safety and allows cold enrichment. Thermotolerant Campylobacter species are cultured at about 42 degrees C. Oxygen divides bacteria into obligate aerobes (Pseudomonas, Mycobacterium, Nocardia), facultative anaerobes (Enterobacterales, staphylococci), microaerophiles that need reduced oxygen (Campylobacter, usually cultured in about 5 per cent oxygen with raised carbon dioxide), aerotolerant anaerobes, and obligate anaerobes (Clostridium, Fusobacterium, Bacteroides, Dichelobacter). Obligate anaerobes lack or have little catalase and superoxide dismutase, so toxic oxygen species kill them. Capnophiles need extra carbon dioxide, commonly 5 to 10 per cent; Brucella abortus biovars often need it on primary isolation.
Bacteria reproduce by binary fission: the cell enlarges, copies its chromosome and divides into two identical daughter cells. The interval between divisions under given conditions is the generation time (doubling time). It is short for fast-growing organisms such as E. coli under ideal laboratory conditions (often quoted as about 20 minutes) and very long for Mycobacterium tuberculosis and M. bovis (in the order of 15 to 20 hours), which is why their cultures take weeks. When bacteria are put into a fixed volume of fresh medium (a batch culture) and viable cells are counted over time, four phases appear. In the lag phase the number of cells hardly changes while they adapt, repair damage and make enzymes; its length depends on the state of the inoculum and the change of medium. In the exponential (log) phase cells divide at a constant, maximal rate for the conditions, the logarithm of the count rises in a straight line, and cells are most uniform and most sensitive to antimicrobials that act on growing cells, such as beta-lactams. In the stationary phase nutrients run out and waste products accumulate, so the viable count stays roughly constant as division balances death. In the death (decline) phase viable cells fall, usually exponentially. Biofilms are a different way of life: communities of cells attached to a surface and embedded in a self-made matrix of polysaccharide, protein and DNA. Biofilms on catheters, teat liners, water lines and implants tolerate antimicrobials and disinfectants far better than free-floating (planktonic) cells of the same strain.
During exponential growth the number of cells after n generations is N = N0 x 2 to the power n, where N0 is the starting number. Taking logarithms to base 10, n = (log N minus log N0) divided by log 2, and because log 2 is 0.301, n is about 3.32 times (log N minus log N0). The generation time g is the time elapsed t divided by n. For example, if a culture rises from 2 x 10 to the power 3 to 6.4 x 10 to the power 4 cells per millilitre in 100 minutes, the count has increased 32-fold, which is five doublings, so g = 100 / 5 = 20 minutes. Counting methods fall into two groups. Total counts include dead cells: direct microscopic counts in a counting chamber, and turbidity measured as optical density or compared with McFarland standards, where a 0.5 McFarland suspension corresponds to about 1.5 x 10 to the power 8 bacteria per millilitre. Viable counts include only cells able to form colonies: the spread-plate and pour-plate methods, membrane filtration, and the most probable number (MPN) method based on growth in replicate tubes of serial dilutions. Because a colony may arise from a clump or chain rather than one cell, viable counts are reported as colony-forming units (CFU). The calculation is CFU per millilitre = number of colonies / (volume plated in millilitres x dilution). If 156 colonies grow from 0.1 millilitre of a 10 to the power minus 5 dilution, the count is 156 / (0.1 x 10 to the power minus 5) = 1.56 x 10 to the power 8 CFU per millilitre. Only plates within a countable range are used, commonly 30 to 300 colonies, though some food and dairy standards use 25 to 250; plates with too few colonies are statistically unreliable and plates with too many are crowded.
The best laboratory cannot rescue a bad specimen. Collect the specimen from the lesion or site most likely to contain the agent, at the right stage of disease, and where possible before antimicrobial treatment starts, because even one dose can prevent culture. Use aseptic technique: clean the teat end with alcohol before a milk sample, take urine by cystocentesis rather than free catch when culture matters, and sample the edge of an active lesion rather than a surface crust. Send enough material; a tissue piece or aspirated pus is better than a swab, and a swab should be placed in a transport medium such as Amies or Stuart medium, which keeps organisms alive without letting them multiply. Most specimens for bacterial culture are kept chilled (about 4 degrees C) and delivered quickly, within the time limit the receiving laboratory sets. Specimens for anaerobic culture need air-free containers or anaerobic transport media and should not be exposed to air. Specimens from suspected zoonotic or notifiable diseases, such as anthrax, brucellosis or glanders, must be handled under national rules and at the appropriate biosafety level; in particular, a carcass suspected of anthrax must not be opened, and the national procedure for sampling must be followed.
Culture media are classified by consistency, composition and purpose. Liquid (broth) media allow growth from small numbers of organisms; solid media contain agar, a seaweed polysaccharide that melts near 100 degrees C, sets near 40 degrees C and is not digested by most bacteria, so single colonies can be isolated. Enriched media add blood, serum or egg: blood agar (commonly 5 to 10 per cent sheep or ox blood) supports most pathogens and shows haemolysis; chocolate agar, in which blood is heated to release X and V factors, supports fastidious Pasteurellaceae. Selective media contain inhibitors that suppress unwanted organisms: bile salts and crystal violet in MacConkey agar suppress Gram-positives; high salt in mannitol salt agar selects staphylococci; antibiotics in Campylobacter and Brucella selective media suppress competitors; malachite green in Loewenstein-Jensen medium suppresses contaminants of mycobacterial cultures. Differential media show a property that separates organisms growing on them: MacConkey agar contains lactose and neutral red, so lactose fermenters such as E. coli form pink colonies and non-fermenters such as Salmonella form pale colonies. Enrichment broths favour one organism in a mixed specimen, for example selenite or tetrathionate broth and Rappaport-Vassiliadis broth for Salmonella from faeces. Transport media keep organisms viable without growth. Anaerobes are grown in reduced media such as cooked meat broth or thioglycollate broth, or on blood agar in an anaerobic jar or cabinet.
The streak-plate method dilutes the inoculum across the surface of a plate in successive sectors with a sterile loop, so that single, well-separated colonies grow in the last sectors. A pure culture is grown from one such colony. Plates are usually incubated at 35 to 37 degrees C for 24 to 48 hours; many laboratories hold plates longer for slow growers, and mycobacterial cultures are held for weeks. The atmosphere is chosen for the suspected organism: air; air with 5 to 10 per cent carbon dioxide for capnophiles and many fastidious organisms; microaerobic gas mixtures for Campylobacter; or anaerobic conditions, confirmed with an indicator, for obligate anaerobes. On blood agar, beta-haemolysis is complete clearing around the colony, alpha-haemolysis is partial lysis with greening, and gamma means no haemolysis. Staphylococcus aureus often shows a double (target) zone on sheep or ox blood agar because of two haemolysins. Mucoid colonies suggest capsule, swarming growth over the whole plate suggests Proteus, and a medusa-head colony edge is classical for Bacillus anthracis, which, unlike most other Bacillus species, is non-haemolytic and non-motile. The laboratory also judges significance: heavy, pure growth of a known pathogen from a properly collected specimen is meaningful; light mixed growth from a swab of a contaminated site often is not.
Staining is the quickest step in identification and often guides treatment before culture results arrive. In the Gram stain a heat-fixed smear is flooded with crystal violet, treated with iodine (the mordant, which forms a crystal violet-iodine complex), decolourised with alcohol or acetone and counterstained with safranin or dilute carbol fuchsin. Gram-positive cells keep the violet complex in their thick peptidoglycan and appear purple; Gram-negative cells lose it through the outer membrane and thin wall and appear pink or red. Over-decolourising makes Gram-positives look negative. The Ziehl-Neelsen stain uses hot carbol fuchsin, decolourisation with acid-alcohol and a methylene blue counterstain: acid-fast organisms (mycobacteria) stay red on a blue background. A modified Ziehl-Neelsen method with a weaker decolourising acid shows organisms that are only weakly acid-fast, such as Nocardia, and it is widely used on smears from abortions to show Brucella, Coxiella and Chlamydia as red organisms (often called the Stamp or modified Ziehl-Neelsen stain). Other useful stains are M'Fadyean's polychrome methylene blue for the capsule of B. anthracis, Albert's stain for metachromatic granules, the spore stain, and Giemsa or Leishman stains for blood parasites and for Anaplasma or Ehrlichia inclusions. Dark-field microscopy shows live leptospires, which are too thin to see well by ordinary bright-field staining.
Primary tests separate the main groups. The catalase test (bubbles when a colony is mixed with hydrogen peroxide) separates staphylococci (positive) from streptococci and enterococci (negative); do not take the colony from blood agar with the medium, because red cells contain catalase. The oxidase test detects cytochrome c oxidase: Pseudomonas, Pasteurella, Aeromonas, Campylobacter, Brucella and Neisseria are usually positive, whereas the Enterobacterales are negative, which is a first split among Gram-negative rods. The coagulase test separates coagulase-positive staphylococci (S. aureus, S. pseudintermedius of dogs, some S. hyicus) from coagulase-negative species. Carbohydrate fermentation, often read with a pH indicator, and triple sugar iron (TSI) agar show whether glucose, lactose and sucrose are fermented and whether hydrogen sulphide is produced; Salmonella typically gives an alkaline slant, acid butt and black hydrogen sulphide. The IMViC set (indole, methyl red, Voges-Proskauer, citrate) separates E. coli (positive, positive, negative, negative) from Klebsiella and Enterobacter (usually negative, negative, positive, positive). Urease is produced rapidly by Proteus and by most Brucella species. Other tests used in veterinary work include nitrate reduction, gelatin liquefaction, aesculin hydrolysis, the CAMP test (Streptococcus agalactiae enhances the beta-toxin haemolysis of S. aureus in an arrowhead shape), satellitism for V-factor-dependent organisms, and motility at different temperatures (Listeria is motile at 20 to 25 degrees C but much less so at 37 degrees C).
Serological identification uses known antibodies to detect antigens on the isolate: slide agglutination for Salmonella O and H antigens and E. coli fimbrial antigens, Lancefield grouping of beta-haemolytic streptococci by their cell-wall carbohydrate, and latex agglutination kits. Molecular methods detect nucleic acid: conventional and real-time polymerase chain reaction (PCR) for specific genes such as toxin genes, 16S rRNA gene sequencing for identification, and whole-genome sequencing for typing and outbreak tracing. A positive PCR shows that DNA is present, not that the organism is alive or causing disease. Koch's postulates (the organism is present in every case, is isolated in pure culture, reproduces the disease in a susceptible host and is re-isolated from that host) remain the classical standard for proving cause, but they fail for organisms that cannot be cultured, for carrier states and for multifactorial diseases such as bovine respiratory disease. In daily practice the question is simpler but harder: is this isolate the cause? The laboratory and the clinician consider the site (a normally sterile site versus one with resident flora), the purity and quantity of growth, the organism (a primary pathogen such as Brucella versus an opportunist such as a coagulase-negative staphylococcus), agreement with cytology (bacteria inside neutrophils suggest infection), the clinical picture and repeat isolation. A report of 'no growth' from a specimen taken after treatment, or left warm for two days, proves little.
Pathogenicity is the ability of an organism to cause disease; virulence is the degree of that ability, often measured experimentally as the dose that kills (LD50) or infects (ID50) half of the test animals. A primary pathogen causes disease in healthy hosts; an opportunist causes disease only when defences are weakened by stress, injury, another infection, immunosuppression or an indwelling device. Many important veterinary pathogens live as commensals: Pasteurella multocida and Mannheimia haemolytica in the upper respiratory tract of cattle, E. coli in the gut, staphylococci on skin. Normal flora also protect the host, by competing for space and nutrients and producing inhibitory substances; antimicrobial treatment that disturbs them can allow overgrowth, for example of Clostridioides difficile or yeasts. Infection proceeds in steps: entry through a portal (skin wound, mucosa, umbilicus, teat canal); adherence; colonisation and multiplication; evasion of innate and adaptive defences; spread locally, through lymph or blood, or inside cells; damage to the host; and shedding and transmission.
Adhesins let bacteria resist being washed away: fimbriae such as F4 and F5 of enterotoxigenic E. coli, and non-fimbrial surface proteins. Invasins let bacteria enter non-phagocytic cells; Salmonella uses a type III secretion system, a needle-like apparatus that injects effector proteins into the host cell and makes it take the bacterium in. Many virulence genes lie together on pathogenicity islands, blocks of DNA with a different G+C content from the rest of the chromosome, which suggests they were acquired by horizontal transfer. Antiphagocytic factors include capsules, the M protein of streptococci and protein A of S. aureus, which binds the Fc part of IgG. Some bacteria survive inside phagocytes instead of avoiding them: mycobacteria, Brucella, Listeria, Salmonella, Rhodococcus equi and Francisella are facultative intracellular pathogens that block phagosome-lysosome fusion, escape into the cytoplasm or resist killing, which is why cell-mediated immunity is central to protection and why many antimicrobials that do not enter cells fail against them. Iron is scarce in host tissues because it is bound to transferrin, lactoferrin and haem; bacteria compete with siderophores that seize iron, and with receptors for host iron proteins. Biofilm formation, regulated by quorum sensing (cell-to-cell signalling that switches genes on when the population is dense), protects bacteria on surfaces.
Exotoxins are proteins secreted by, or released from, bacteria, both Gram-positive and Gram-negative. They are usually heat-labile, highly potent and specific in action, strongly antigenic, and can be converted by formaldehyde into toxoids, which are non-toxic but still immunogenic and form the basis of tetanus and clostridial vaccines. Many are A-B toxins: the B (binding) part attaches to a host-cell receptor and the A (active) part is the enzyme. Examples are the neurotoxins of Clostridium tetani, which block release of inhibitory neurotransmitters in the spinal cord and cause spastic paralysis, and of C. botulinum, which block acetylcholine release at the neuromuscular junction and cause flaccid paralysis; the enterotoxins of enterotoxigenic E. coli, where heat-labile toxin raises cyclic AMP and heat-stable toxin raises cyclic GMP, both causing secretory diarrhoea; the membrane-damaging alpha toxin of C. perfringens; and the epsilon toxin of C. perfringens type D in enterotoxaemia of sheep. Superantigens, such as some staphylococcal enterotoxins and toxic shock syndrome toxin, bind MHC class II and T-cell receptors outside the normal antigen groove and activate large numbers of T cells at once. Endotoxin is the lipid A part of the LPS of Gram-negative bacteria. It is part of the outer membrane, released mainly when cells die or divide, is heat-stable, is less potent weight for weight than most exotoxins, is poorly immunogenic and cannot be made into a toxoid. Lipid A binds Toll-like receptor 4 on macrophages and triggers release of tumour necrosis factor, interleukin-1 and other mediators, leading to fever, leucopenia followed by leucocytosis, hypotension, disseminated intravascular coagulation and endotoxic shock, as seen in coliform mastitis and equine colic with gut damage. Endotoxin survives autoclaving, so parenteral fluids and implants must be free of pyrogens as well as sterile.
Sterilisation is the destruction or removal of all living microorganisms, including bacterial spores. Disinfection kills or removes pathogens from inanimate objects but not necessarily spores. For medical devices the usual sterility assurance level (SAL) target is a probability of no more than one in a million (10 to the power minus 6) that a single viable organism survives on an item. Killing by heat, radiation or chemicals follows first-order kinetics under ideal conditions: in each equal interval of time the same fraction of survivors dies. The decimal reduction time, or D-value, is the time at a stated temperature needed to kill 90 per cent of the population, that is, to reduce it by one logarithm. If N0 organisms are exposed for t minutes, the survivors are N = N0 x 10 to the power (minus t / D). For example, 10 to the power 6 spores with a D-value of 1.5 minutes at 121 degrees C need 12 decimal reductions to reach 10 to the power minus 6, so 12 x 1.5 = 18 minutes. The z-value is the rise in temperature that reduces the D-value tenfold; for spores in moist heat it is commonly about 10 degrees C. The F-value expresses the lethality of a process as equivalent minutes at a reference temperature; F0 is the equivalent minutes at 121.1 degrees C with a z-value of 10 degrees C, calculated as the time at temperature T multiplied by 10 to the power ((T minus 121.1) / 10).
The autoclave uses saturated steam under pressure: the standard cycle is 121 degrees C, about 103 kilopascals (15 pounds per square inch) above atmospheric pressure, for at least 15 minutes of holding time once the whole load has reached temperature; higher-temperature cycles such as 134 degrees C for about 3 minutes are used for wrapped instruments in many hospitals. Boiling at 100 degrees C for 10 minutes or more kills vegetative bacteria and most viruses but not reliably spores, so it is disinfection, not sterilisation. Tyndallisation (intermittent steaming at 100 degrees C, often 20 to 30 minutes on three successive days, with incubation between so that spores germinate and are killed next day) sterilises media that would be damaged by autoclaving. Inspissation (about 80 to 85 degrees C on three successive days) coagulates and disinfects egg and serum media such as Loewenstein-Jensen. Pasteurisation of milk (the holder method at about 63 degrees C for 30 minutes, or high-temperature short-time at about 72 degrees C for 15 seconds, with details set by national food law) kills vegetative pathogens such as Brucella, Mycobacterium bovis, Salmonella and Listeria but is not sterilisation. Dry heat kills by oxidation and needs higher temperatures for longer: the hot-air oven is commonly run at 160 degrees C for 2 hours or 170 to 180 degrees C for shorter times, for glassware, metal instruments, powders and oils that steam cannot penetrate.
Filtration removes rather than kills microbes and is used for heat-sensitive liquids such as sera, antibiotic and vitamin solutions and some culture-medium supplements. Membrane filters of 0.22 micrometre (often written 0.2 micrometre) pore size retain bacteria and are the usual sterilising grade; they do not retain most viruses or reliably retain mycoplasmas. Ionising radiation (gamma rays, usually from cobalt-60, or electron beams) penetrates packaging and kills by damaging DNA; it sterilises single-use plastic syringes, catheters, gloves and sutures without heat, which is why it is called cold sterilisation. Ultraviolet light, most germicidal near 260 nanometres where DNA absorbs strongly, forms thymine dimers but penetrates poorly, so it is used only to reduce contamination on exposed surfaces and in air, for example inside safety cabinets; it is not a sterilising method for solid loads. Ethylene oxide gas alkylates proteins and nucleic acids, penetrates packs and sterilises heat-sensitive instruments such as endoscopes and plastics, but it is toxic, flammable and possibly carcinogenic, and items need aeration to remove residues. Physical indicators are the time, temperature and pressure records of each cycle. Chemical indicators change colour when conditions are reached; autoclave tape shows only that a package has been exposed to heat, not that it is sterile, and the Bowie-Dick test checks air removal in pre-vacuum autoclaves. Biological indicators are the definitive check: spores of Geobacillus stearothermophilus for steam and hydrogen peroxide, and spores of Bacillus atrophaeus for dry heat and ethylene oxide, are processed with the load and then incubated to show whether any survive. Prions, the agents of scrapie and bovine spongiform encephalopathy, resist ordinary sterilisation; special extended cycles and chemical treatments are laid down in national guidance.
Microorganisms differ in resistance to chemical disinfectants in a fairly predictable order, from most to least resistant: prions; bacterial spores; protozoal oocysts such as Cryptosporidium and coccidia; mycobacteria; small non-enveloped viruses (for example parvoviruses and picornaviruses); fungi; vegetative bacteria; and enveloped viruses, which are the easiest to kill because their lipid envelope is readily destroyed. Canine parvovirus and Clostridium spores in a kennel or a calving pen need stronger products and longer contact than an enveloped virus such as avian influenza or Newcastle disease virus on a clean surface. The Spaulding classification ranks items by infection risk. Critical items enter sterile tissue or the bloodstream (surgical instruments, needles, implants) and must be sterile. Semi-critical items touch mucous membranes or non-intact skin (endoscopes, laryngoscope blades, some thermometers) and need at least high-level disinfection, which kills everything except large numbers of spores. Non-critical items touch only intact skin or nothing (stethoscopes, tables, floors) and need low- or intermediate-level disinfection.
Alcohols (ethanol or isopropanol at about 60 to 90 per cent in water, commonly 70 per cent) denature proteins and dissolve lipids; water is needed for their action, they evaporate fast, they do not kill spores, and they are poor on dirty surfaces. Halogens: chlorine compounds such as sodium hypochlorite are cheap, broad-spectrum and sporicidal at high concentrations but are inactivated by organic matter, corrode metal and lose strength in storage; iodine and iodophors (iodine complexed with a carrier) are used as skin antiseptics and teat dips. Phenolics act on membranes and remain active in organic matter better than many classes, but some are toxic to cats. Quaternary ammonium compounds are cationic detergents that disrupt membranes; they are good cleaners with low toxicity but are inactivated by soap, hard water and organic matter, are weak against Gram-negatives such as Pseudomonas (which can even grow in diluted solutions) and do not kill spores, mycobacteria or non-enveloped viruses. Biguanides such as chlorhexidine are widely used as skin and surgical-scrub antiseptics, are persistent on skin, and are not sporicidal. Aldehydes (glutaraldehyde, formaldehyde) alkylate proteins and nucleic acids and are high-level disinfectants or chemical sterilants given long contact times; formaldehyde is used for fumigating empty poultry houses and hatcheries under strict safety controls. Oxidising agents (hydrogen peroxide, peracetic acid, potassium peroxymonosulphate products) are broad-spectrum, and peracetic acid remains active with some organic matter. Alkalis such as sodium hydroxide and calcium hydroxide (lime) are used on floors and in foot dips and are effective against many viruses; sodium carbonate and sodium hydroxide are traditional agents for foot-and-mouth disease virus, which is acid- and alkali-labile, but the agents and strengths approved for notifiable diseases are set by national authorities and differ between countries.
Efficacy depends on the concentration of the product, the contact time, temperature, pH, the number and type of organisms present, and above all on organic matter such as faeces, blood, milk and bedding, which inactivates many disinfectants and shields organisms. The rule on a farm or in a clinic is therefore: remove gross dirt, wash with detergent, rinse, let dry, then apply disinfectant at the label dilution for the label contact time, then let dry again. Working dilutions are calculated from C1 x V1 = C2 x V2, where C1 is the concentration of the stock, V1 the volume of stock needed, C2 the concentration wanted and V2 the final volume. To make 1 litre of a 1000 parts per million (0.1 per cent) available-chlorine solution from a stock labelled 5 per cent, V1 = 0.1 x 1000 / 5 = 20 millilitres of stock made up to 1 litre. A dilution of '1 in 200' means 1 part product in a total of 200 parts, so 5 litres needs 25 millilitres of product. The historical phenol coefficient compared a product with phenol against a test organism: the highest dilution of the test product that killed in a set time divided by the corresponding dilution of phenol. It is of limited practical value because it ignores organic matter and surfaces. Modern standards use suspension tests, carrier or surface tests and field-simulation tests with organic load, and express efficacy as a logarithmic reduction; a 5-log reduction means 99.999 per cent of organisms are killed.
The bacterial genome consists of the chromosome plus accessory elements. Plasmids are extrachromosomal DNA molecules, usually circular, that replicate independently. They are not essential for growth under ordinary conditions but often carry genes that help under stress: antimicrobial resistance (R plasmids), toxins, adhesins, capsule synthesis and metabolic abilities. Conjugative plasmids carry the genes for their own transfer; mobilisable plasmids use another plasmid's machinery. Veterinary examples include the two virulence plasmids of Bacillus anthracis, pXO1 carrying the toxin genes and pXO2 carrying the capsule genes; loss of pXO2 is the basis of the non-capsulated live spore vaccine strain. Mutation is a heritable change in the DNA sequence. Point mutations substitute one base, and may be silent, missense (changing one amino acid) or nonsense (creating a stop codon); insertions and deletions not in multiples of three cause frameshifts. Mutations arise spontaneously at low rates, independently of the drug, and are selected when a drug is present; the Luria-Delbrueck fluctuation test showed this. Resistance to fluoroquinolones through mutations in gyrA and parC, and to rifampicin through mutations in rpoB, are chromosomal examples. Bacteria regulate genes to save energy; the lac operon of E. coli is the classic model, in which a repressor blocks transcription of the lactose genes until lactose (as allolactose) is present, and glucose lowers expression further by catabolite repression.
Bacteria acquire genes from other bacteria by three main routes. Transformation is uptake of free DNA from the environment by a competent cell, first shown by Griffith in 1928 with pneumococci and identified as DNA by Avery, MacLeod and McCarty in 1944; naturally competent genera include Streptococcus, Haemophilus-group organisms and Bacillus. Transduction is transfer of bacterial DNA by a bacteriophage. In generalised transduction a phage accidentally packages any fragment of host DNA; in specialised transduction a temperate phage that integrates at a fixed site carries neighbouring genes when it excises imprecisely. Lysogenic conversion is a related phenomenon: an integrated prophage gives its host new properties. Examples in veterinary organisms include the Shiga toxin genes of Shiga toxin-producing E. coli and the botulinum neurotoxin genes of Clostridium botulinum types C and D, which are phage-borne. Conjugation is transfer of DNA from a donor to a recipient through direct contact, usually through a sex pilus. The F (fertility) plasmid of E. coli was the first model, described by Lederberg and Tatum: F-positive cells transfer a copy of the F plasmid to F-negative cells; in Hfr (high-frequency recombination) strains the F factor is integrated in the chromosome and can drag chromosomal genes across. Conjugation is the most important route for spreading resistance genes among Gram-negative bacteria, and it can cross species and genus boundaries, for example between E. coli and Salmonella in an animal's gut.
Transposable elements move within and between DNA molecules in the same cell. Insertion sequences are the simplest, carrying only the transposase gene flanked by inverted repeats. Transposons carry extra genes, often resistance genes, between flanking insertion sequences or repeats; they can jump from a chromosome to a plasmid and then travel by conjugation. Integrons are gene-capture systems: they contain an integrase gene, a recombination site and a promoter, and they collect gene cassettes, each usually carrying one resistance gene, in an array expressed from the shared promoter. Class 1 integrons, often carried on transposons and plasmids, are widespread in Gram-negative bacteria from animals and people and are a marker of resistance gene traffic. In the diagnostic laboratory, genetic knowledge is used through PCR for virulence and resistance genes, plasmid profiling, pulsed-field gel electrophoresis (now largely replaced), multilocus sequence typing and whole-genome sequencing, which can show whether isolates from animals, food and people belong to the same clone and whether a resistance gene is on the chromosome or on a mobile element.
Selective toxicity depends on targets that bacteria have and animal cells lack or have in a different form. Cell-wall synthesis inhibitors are the beta-lactams (penicillins, cephalosporins, carbapenems), which bind penicillin-binding proteins and block cross-linking of peptidoglycan, and the glycopeptides, which bind the D-alanyl-D-alanine terminus. Protein synthesis inhibitors act on the 30S subunit (aminoglycosides, which cause misreading and are bactericidal; tetracyclines, which block aminoacyl-tRNA binding) or on the 50S subunit (macrolides, lincosamides, phenicols such as florfenicol, and pleuromutilins such as tiamulin). Nucleic acid inhibitors include the fluoroquinolones, which inhibit DNA gyrase and topoisomerase IV, and rifampicin, which inhibits RNA polymerase. Folate pathway inhibitors are the sulfonamides, which compete with para-aminobenzoic acid at dihydropteroate synthase, and trimethoprim, which inhibits dihydrofolate reductase; together they block two steps and are often synergistic. Polymyxins (colistin) bind lipid A and disrupt the outer and cytoplasmic membranes of Gram-negatives. Intrinsic resistance is a property of a whole species: Gram-negatives resist vancomycin because it cannot cross the outer membrane, mycoplasmas resist beta-lactams because they lack a wall, anaerobes resist aminoglycosides because uptake needs oxygen-dependent transport, and enterococci resist cephalosporins.
Susceptibility testing measures how an isolate responds to drugs in vitro under standard conditions. The minimum inhibitory concentration (MIC) is the lowest concentration that prevents visible growth after overnight incubation. In broth microdilution, the drug is diluted in twofold steps (for example 64, 32, 16, 8, 4, 2, 1, 0.5 micrograms per millilitre) in cation-adjusted Mueller-Hinton broth, and a standard inoculum (a 0.5 McFarland suspension, about 1.5 x 10 to the power 8 CFU per millilitre, diluted to give about 5 x 10 to the power 5 CFU per millilitre in each well) is added; growth and sterility controls are included. The minimum bactericidal concentration (MBC) is the lowest concentration that kills at least 99.9 per cent of the inoculum, found by subculturing clear wells. In disk diffusion (the Kirby-Bauer method), a lawn of the standard inoculum is spread on Mueller-Hinton agar of standard depth (about 4 millimetres), paper disks with a fixed drug content are applied, and after incubation at about 35 degrees C for 16 to 20 hours the diameter of each inhibition zone is measured. The zone is inversely related to the MIC but it is not an MIC. Results are interpreted against clinical breakpoints to give categories: susceptible (S, high likelihood of success at the standard dose), intermediate or 'susceptible, increased exposure' (the two main standard-setters define this middle category differently) and resistant (R). Breakpoints come from standard-setting bodies, mainly the Clinical and Laboratory Standards Institute (CLSI, whose veterinary standards are VET01 and VET01S) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST, with its veterinary subcommittee VetCAST). Veterinary-specific breakpoints exist only for some drug, species and disease combinations; where they are missing, laboratories may apply human breakpoints, which may not fit the drug's behaviour in the animal. Epidemiological cut-off values (ECOFFs) separate wild-type organisms from those with acquired resistance mechanisms; they are used in surveillance, not to predict clinical outcome. Two practical rules: a lower MIC for one drug than for another does not by itself mean the first drug is better, because each drug has its own breakpoint; and 'S' in the report does not guarantee cure if the drug cannot reach the site (for example an intracellular organism, an abscess, or a biofilm), whereas 'R' strongly predicts failure.
Acquired resistance arises by mutation or by acquiring genes, and works by four main mechanisms. First, enzymatic inactivation: beta-lactamases hydrolyse the beta-lactam ring. Extended-spectrum beta-lactamases (ESBLs), of which the CTX-M family is now the commonest worldwide, hydrolyse third-generation cephalosporins and are inhibited by clavulanic acid; AmpC beta-lactamases hydrolyse cephamycins as well and are not well inhibited by clavulanate; carbapenemases hydrolyse carbapenems and are of the greatest concern for human medicine. Aminoglycoside-modifying enzymes acetylate, phosphorylate or adenylate aminoglycosides. Second, target alteration: methicillin resistance in staphylococci is due to the mecA gene (or the related mecC), which encodes penicillin-binding protein 2a with low affinity for almost all beta-lactams; it underlies methicillin-resistant S. aureus (MRSA) and methicillin-resistant S. pseudintermedius (MRSP) in dogs. Erm methylases alter 23S rRNA and give cross-resistance to macrolides, lincosamides and streptogramin B (the MLSB phenotype). Van genes change the peptidoglycan terminus from D-alanyl-D-alanine to D-alanyl-D-lactate, lowering glycopeptide binding; avoparcin use as a growth promoter in Europe was associated with vancomycin-resistant enterococci in animals before it was banned. Mutations in gyrA and parC alter the fluoroquinolone targets, and ribosomal protection proteins such as Tet(M) protect the ribosome from tetracyclines. Plasmid-borne mcr genes, first reported in 2015 in E. coli from pigs and people in China, encode an enzyme that adds phosphoethanolamine to lipid A and lowers colistin binding. Third, reduced permeability through loss of porins. Fourth, active efflux, for example Tet(A) efflux pumps for tetracyclines and multidrug efflux systems in Pseudomonas. Resistance genes are often linked on the same plasmid or integron, so using one drug can select resistance to unrelated drugs (co-selection), and heavy metals and some biocides can co-select as well. Control rests on prudent use: treat only when bacterial infection is likely, sample for culture and susceptibility when possible, choose narrow-spectrum drugs, use the correct dose and duration, respect national restrictions on critically important antimicrobials, and prevent infection through vaccination, hygiene and biosecurity. WOAH, WHO and FAO lists of antimicrobials important for animal and human health guide these decisions, and national rules differ.
Fungi are eukaryotes: they have a nucleus, mitochondria and 80S ribosomes, which is why antibacterial drugs do not act on them. Their cell wall is made of chitin, glucans (including beta-1,3-glucan) and mannoproteins, and their cell membrane contains ergosterol instead of the cholesterol of animal cells. They are heterotrophs that absorb nutrients and most are saprophytes in soil and plant matter. Two growth forms occur. Yeasts are single cells that reproduce by budding (blastoconidia); some, such as Candida, can form pseudohyphae, and Candida albicans forms true germ tubes in serum within a few hours at 37 degrees C, which is a quick identification test. Moulds grow as tubular filaments called hyphae, which may be septate (Aspergillus, dermatophytes) or broad and sparsely septate (the Mucorales); a mass of hyphae is a mycelium. Dimorphic fungi grow as moulds in the environment at about 25 degrees C and as yeasts or spherules in tissue at body temperature; examples are Blastomyces dermatitidis, Histoplasma capsulatum (including the variety farciminosum, the cause of epizootic lymphangitis of horses and other equids), Coccidioides immitis (which forms spherules in tissue) and Sporothrix schenckii. Asexual spores include conidia borne on conidiophores (Aspergillus), sporangiospores inside a sporangium (Mucorales), arthroconidia formed by fragmentation of hyphae (dermatophytes, Coccidioides), chlamydospores (thick-walled resting cells) and blastoconidia.
Fungi cause disease in three ways. Infections (mycoses) are classified by site: superficial and cutaneous mycoses, chiefly dermatophytosis (ringworm), which affects keratin of skin, hair and claws; subcutaneous mycoses such as sporotrichosis and eumycotic mycetoma, entering through wounds; and systemic mycoses such as blastomycosis, histoplasmosis, coccidioidomycosis and cryptococcosis, usually acquired by inhaling spores, with geographical distributions that follow soil and climate. Opportunistic mycoses occur when defences fail: candidiasis of the crop in young birds or after prolonged antibiotic use, aspergillosis of the lungs and air sacs of poultry (brooder pneumonia) and other birds, guttural pouch mycosis in horses, sino-nasal aspergillosis in dogs, and mycotic abortion in cattle. Malassezia pachydermatis, a lipophilic yeast that, unlike most other Malassezia species, does not need added lipid to grow, is a normal inhabitant of canine skin and ears that overgrows in otitis externa and dermatitis. Cryptococcus neoformans and C. gattii are capsulated yeasts associated with bird droppings and eucalyptus respectively, affecting especially cats. Dermatophytes include Microsporum canis (cats and dogs, commonly zoonotic), Trichophyton verrucosum (cattle ringworm, zoonotic), Trichophyton mentagrophytes complex (rodents and many species) and the geophilic species formerly called Microsporum gypseum, now Nannizzia gypsea. Second, mycotoxicoses are poisonings by fungal toxins produced in feed rather than infections, for example aflatoxins from Aspergillus flavus and A. parasiticus, which damage the liver and appear in milk as aflatoxin M1, and zearalenone from Fusarium, which is oestrogenic in pigs.
Direct examination is fast and often decisive. Hairs or skin scrapings are cleared in 10 to 20 per cent potassium hydroxide to show hyphae and arthroconidia; dermatophytes invade hair and form spores on its surface (ectothrix) or inside it (endothrix). A Wood's lamp (ultraviolet light of about 365 nanometres) makes some, but not all, Microsporum canis infections fluoresce apple-green; many infections and most other dermatophytes do not fluoresce, so a negative result does not rule ringworm out. India ink or nigrosin preparations of cerebrospinal fluid or nasal exudate show the wide capsule of Cryptococcus. Cytology of ear swabs shows the peanut- or footprint-shaped buds of Malassezia. Culture uses Sabouraud dextrose agar, which is slightly acidic (about pH 5.6) and often contains chloramphenicol to inhibit bacteria and cycloheximide to inhibit saprophytic moulds; note that cycloheximide also inhibits some pathogens such as Cryptococcus and Aspergillus, so a plate without it is set when these are suspected. Dermatophyte test medium contains phenol red, which turns red as dermatophytes release alkaline metabolites early in growth; colour change with many contaminants later in incubation causes false positives, so microscopic identification of the colony is still needed. Most fungal cultures are incubated at about 25 to 30 degrees C and held for up to two to four weeks. Antifungal drugs exploit fungal targets. Polyenes (amphotericin B, nystatin) bind ergosterol and make pores in the membrane. Azoles (ketoconazole, itraconazole, fluconazole, voriconazole and topical azoles) inhibit lanosterol 14-alpha-demethylase and so ergosterol synthesis. Allylamines such as terbinafine inhibit squalene epoxidase. Echinocandins inhibit beta-1,3-glucan synthase in the wall. Flucytosine is converted inside fungal cells to 5-fluorouracil and blocks nucleic acid synthesis. Griseofulvin binds fungal microtubules, is used against dermatophytes, and is teratogenic, so it must not be given to pregnant animals. Legal availability and use in food animals differ widely between countries.
Build the subject on a few ideas that explain many facts: the Gram-negative outer membrane, the endospore, the growth curve and first-order killing, the three routes of gene transfer and the four mechanisms of resistance. In practical classes, learn to make and read a Gram and a Ziehl-Neelsen stain, streak for single colonies, read haemolysis and MacConkey plates, and carry out catalase, oxidase and coagulase tests correctly. Practise the numericals until they are automatic: generation time from two counts, CFU per millilitre from a plate, survivors from a D-value, a disinfectant dilution, and an MIC read against a breakpoint. Learn older and newer names side by side, because examiners use both.
In practice this subject turns into daily decisions. You choose whether a case needs culture before treatment, collect a clean specimen (cystocentesis urine, aseptic milk, deep tissue rather than a surface swab), keep it cold and send it with a history. You read the laboratory report critically: is the growth pure and heavy from a sterile site, or light and mixed from a contaminated swab? You interpret MICs against each drug's own breakpoint, choose a narrow-spectrum drug that reaches the site, and recognise MRSP, ESBL and other resistant organisms and their infection-control needs. You also check the clinic autoclave with biological indicators, write disinfection protocols that start with cleaning, and pick products that kill the organism at hand, for example parvovirus in a kennel.
After twenty-five years the subject becomes a matter of systems. A senior clinician, herd veterinarian or laboratory manager designs sampling plans for herds and flocks, sets and audits sterilisation and disinfection procedures for hospitals, hatcheries and processing plants, and leads antimicrobial stewardship: practice formularies, restrictions on critically important antimicrobials, and use of local susceptibility data (antibiograms and MIC90 values) for empirical therapy. The same person talks with regulators about national residue and resistance surveillance, handles biosecurity during outbreaks of notifiable disease, and trains younger colleagues to distinguish colonisation from infection. Accreditation of a diagnostic laboratory, quality control of susceptibility testing, and choosing between CLSI and EUCAST methods are decisions made at this level.
At research level the subject is about mechanisms and evidence. A senior researcher uses whole-genome sequencing to trace the movement of resistance genes on plasmids, transposons and integrons between animal, human and environmental bacteria; studies pathogenicity islands, secretion systems and regulatory networks; and develops veterinary-specific clinical breakpoints by combining MIC distributions, pharmacokinetic and pharmacodynamic data and clinical outcomes. Other work includes validating rapid diagnostics against culture, modelling sterilisation and disinfection kinetics under farm conditions with organic load, and testing interventions that reduce antimicrobial use without harming welfare or production. Such a researcher also advises standard-setting bodies and must judge carefully which findings are established and which are still contested.
This manual covers general principles only. Individual bacterial and fungal pathogens, their diseases and their specific laboratory identification belong to systematic bacteriology and mycology and are mentioned here only as examples. Viruses, immunology and serological tests, and mycotoxicoses are covered in their own subjects. The manual gives no clinical breakpoints, disk contents or zone diameters, because these are set by CLSI and EUCAST, differ between them and between editions, and must be read from the current tables; the breakpoints in the practice questions are invented for calculation only. Approved disinfectants and their concentrations for notifiable diseases, sterilisation parameters for prions, packaging rules for transport of specimens, and pasteurisation standards are set nationally and are given only in outline. Bacterial nomenclature changes often; where a newer name is in use the manual gives both, but it does not list every recent reclassification, some of which are still debated. Antimicrobial doses are deliberately not given.
Original practice questions written to the syllabus and pattern of the exams named in each question's examTags. They are not past papers and do not reproduce any board's questions. Every numerical answer (generation time, plate counts, D, z and F0 values, disinfectant dilutions, MIC and MBC reading) was computed by script and the working is shown. Breakpoints in MIC questions are stated in the question or marked hypothetical; real breakpoints are drug-, species- and site-specific and must be taken from current CLSI or EUCAST/VetCAST tables.
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