Foundations MT2 LOs.pdf

VIROLOGY LECTURE 4: HOST DEFENSE, SUSCEPTIBILITY, IMMUNE RESPONSE Define window of susceptibility. Time at which antibody level is no longer sufficient to protect from infection, yet high enough to prevent vaccination. initial vaccine series - increasing coverage, not ne...

VIROLOGY LECTURE 4: HOST DEFENSE, SUSCEPTIBILITY, IMMUNE RESPONSE Define window of susceptibility. Time at which antibody level is no longer sufficient to protect from infection, yet high enough to prevent vaccination. initial vaccine series - increasing coverage, not necessarily boosting ◦ Puppies that are successfully immunized at 6 weeks may not get added protection out of the next vaccine ‣ the Ab developed from the first will block the next vaccine from having an effect ‣ another round of vaccines is given because you do not know which puppies were protected from the first vaccine it is cheaper to re-vaccinate than to test titers Define innate immune response. the innate response is the first line of defense and is essential to activate the adaptive immune response ◦ innate response alone may not be sufficient to clear a viral infection ◦ acquired immune response is essential for clearing the viral infection AND providing immunological memory involves: ◦ cells: infected target, NK cells, macrophages, dendritic cells, granulocytes ◦ soluble mediators: cytokines, chemokines, defensins, pentraxins, complement, etc Define adaptive immune response. faster and stronger than the innate immune response is more specific than innate immunity - cells have memory essential for clearing viral infection involves:  ◦ cells: T lymphocytes, B lymphocytes ◦ soluble products: cytokines, chemokines, antibodies Define Interferon. Interferons are proteins produced by cells in response to viral infections and other immune challenges. play a crucial role in the immune response by interfering with viral replication, activating immune cells, and enhancing the body's defenses against pathogens. regulate activity of other immune cells Compare and contrast the innate and adaptive immune response. both the innate and adaptive immune systems are needed for survival against a viral infection - even in immunocompetent individuals the innate response is the first line of defense - essential to activate the adaptive immune response you are BORN with INNATE IMMUNITY, you DEVELOP ADAPTIVE IMMUNITY List viral products that trigger the innate immune system and what that trigger induces. Toll-Like Receptors (TLR) recognize viral PAMPs ◦ TLR3 - recognizes dsRNA ◦ TLR4 - some viral glycoproteins ◦ TLR7 - recognizes 5'ppp (triphosphate) ssRNA ◦ TLR9 - recognizes unmethylated CpG motifs in viral DNA (eg. from herpesviridae)  RNA helicases in the cytoplasm of host cells ◦ recognize ssRNA or dsRNA intermediates ◦ Retinoic acid Inducible Gene-1 (RIG-1) ◦ Melanoma differentiation antigen (MDA5) Host response to viral PAMPs isn't the same to that of bacterial PAMPs like LPS ◦ Viremia - some fever ◦ Bacteremia - fever, hypotension, disseminated intravascular clotting Viral activation of PRRs leads to type 1 interferon production ◦ Virus infected cells produce type I interferon to protect neighboring cells ‣ A viral infected cell prodices type I interferon (alpha and beta) produced for a brief period after viral infection (~10h) it interferes with viral replication ‣ released in a paracrine manner to induce an antiviral state in uninfected neighboring cells they become refractory to viral replication ◦ virus infected cells spread the news via type I interferons ‣ Interferons can induce ISGs in infected neighboring cells, increase antigen presentation and chemokine production in innate cells, induce antibody production, and increase effector T cell responses Describe how NK cells interact with other cells and decide when to release toxic granules to kill virally infected cells. NK cells are the first cell that responds to a viral infection NK cells become activated within hours of a viral infection ◦ antiviral activity of NK cells is brief; peaks 2-3 days post infection activated NK cells produce large amounts of IFNy (interferon gamma) ◦ IFNy activates macrophages to more efficiently kill intracellular pathogens How does a NK cell identify a viral infected cell? ◦ NK cells recognize the total amount of MHC class I molecules on the cell's surface ‣ viral infected cells commonly express less MHC I on their surface and are targeted for killing by MK cells NK cells are regulated by the balance between activating inhibitory receptors ◦ NK cells express both activating and inhibitory receptors ‣ a fine balance of these receptors controls the release of toxic granules from NK cells, which leads to killing of a viral infected cell  ‣ If inhibitory receptors on NK cells are not activated (by MHC I on a host cell), activating receptors lead to NK cell degranulation and killing of a viral infected cell ‣ some viruses encode MHC I homologues to avoid killing by NK cells Describe how macrophages respond to viruses. Resident macrophages are strategically placed throughout the body (eg. liver - kupffer cells; lung - alveolar macrophages) to search for invading microbes, such as viruses a macrophage that phagocytoses a virus may cause the production of cytokines that limit viral replication and awaken other cells in the innate and acquired immune system (eg NK and CD8+ T cells, respectively) to kill the virus macrophages also restrict virus spread by inactivating extracellular virions ◦ Generation of reactive oxygen species (free radicals), nitric oxide (NO) and others Neutrophils play a very small role, if any, in inactivating viruses Describe the concept of memory as it relates to adaptive immunity Memory is the hallmark of adaptive immunity ◦ cells that have memory ‣ B cell - mediators of humoral immunity ‣ CD8+ T cell - mediators of cell mediated killing; CTLs ‣ CD4+ T cell - helper T cells, assist CD8 and B cells ‣ all 3 have specific antigen receptors ◦ some become memory T cells and memory B cells. ‣ persist in the body long-term ‣ allow the immune system to respond more rapidly and effectively if the same pathogen is encountered again Describe how antibodies can target free virus and virus infected cells and how some viruses evade antibodies. Functions of CD4 T cells in viral immunity ◦ B cell help: produces cytokines to help differentiate B cells into plasma cells and undergo isotype switching ◦ T cell help: Produces IL-2 to help activate CD8 T cells to kill viral infected cells ◦ Antibodies can neutralize viral infectivity in a number of ways ‣ interfere with virion binding to receptors ‣ block uptake into cells  ‣ prevent uncoating of the genomes in endosomes ‣ cause aggregation of virus particles ‣ many enveloped viruses are lysed when antiviral antibodies and serum complement disrupt membranes ◦ Opsonization: providing specificity to phagocytosis and enhanced signaling ‣ process: opsonization of microbe by IgG binding of opsonized microbes to Fc receptors (FcyRI) Fc receptor signals activate phagocyte phagocytosis of microbe killing of ingested microbe ◦ Antibody-dependent cellular toxicity ‣ viral antigens are expressed on the surface of infected cells ‣ IgG binds to antigens on the surface of the target cell ‣ NK cells recognize bound antibodies through their Fc receptors which specifically bind to the Fc region of the antibodies FcyRIII specifically binds bound IgG - does NOT bind free IgG ‣ binding of Fc receptors triggers the activation of NK cells ‣ activated NK cells release cytotoxic molecules (such as perforin and granzymes) that induce apoptosis in the target cell ‣ target cell is destroyed viruses have developed mechanisms to evade antibodies: ◦ enveloped viruses ‣ bud from internal membrane (no viral proteins on plasma membrane) ‣ cell-to-cell spread/syncytia formation never have to leave cell - intracellular travel ex: respiratory syncytia virus ‣ heavily glycosylated receptors evade immunity ‣ rapidly mutating receptors ◦ persistent viruses ‣ latency very few viral proteins -> cell will not look infected Functions of CD8 T cells in viral immunity ◦ also known as cytotoxic T cell or CTL ◦ endogenous antigens (eg viral proteins) are loaded onto MHC I molecules  ◦ If the T cell receptor (TCR) of a CD8 T cell recognizes the viral peptide presented on MHC I molecule -> infected cell will be killed and halt viral replication ‣ perforin/granzyme killing ‣ Fas/FasL killing ‣ CD8 T cells do not kill extracellular virions ◦ virus specific CD8 T cells are generated 4-5 days post infection and proliferate to sufficient numbers to kill viral infected cells ‣ faster than antibodies viruses have evolved multiple strategies to evade lysis by CD8+ T cells ◦ some viruses escape presentation of their antigens on MHC class I molecules ‣ epstein barr virus encodes some proteins that are resistant to proteosome-mediated lysis -> viral peptides cannot get loaded onto MHC I ‣ Herpes simplex 1 virus encodes a protein that blocks TAP-mediated transport of peptides from the cytosol to the endoplasmic reticulum -> viral peptides are not loaded onto MHC I ‣ many viruses down regulate the movement MHC class I to the cell surface -> poor recognition by CD8 T cell function of CD8 T cells in persistent viral infections ◦ the antiviral function of CD8 (and CD4) T cells may decline or become dysfunctional during some chronic viral diseases (ex hepatitis C virus in humans and FIV or FeLV in cats) ‣ CD8 T cells lose their cytotoxic activity ‣ CD8 T cell may lessen their cytokine production ‣ Memory CD8 T cells may be lost from the host - slow or no recall to antigen Explain how maternal antibodies, young immune systems, and the window of susceptibility interact to produce current vaccine schedules that suggest several vaccinations. effect of age on host resistance ◦ young and elderly animals are more susceptible to most viruses and produce more severe disease ‣ a few exceptions: Spanish flu (1918) was harsh on 18-30 year olds, H1N1 in 2009, chicken pox, mumps, and polio viruses cause less severe disease in the young, worsen in teen and middle age ◦ waning immune system in elderly - slower to respond ◦ immature immune system in young ‣ B and T cells may not function optimally in young animals  ‣ young animals may lack protective titers of antibodies immune-interference may develop from an interaction of vaccine and maternal antibodies in young animals failure of passive transfer ◦ cells may lose susceptibility to some viruses with age ‣ rotaviruses replicate in piglet enterocytes during the first days of life, but become refractory to infection > 1 month of age. vaccination challenges ◦ infant immune system not fully developed ‣ solution: vaccinate the mother -> ingestion of antibodies via colostrum protects young while they build their own active immunity ◦ But interference by maternal antibody ‣ Ab in colostrum reduces vaccine efficacy window of susceptibility ◦ the time at which antibody level is no longer sufficient to protect from infection, yet high enough to prevent vaccination initial vaccine series - increasing coverage, not necessarily boosting ◦ Puppies that are successfully immunized at 6 weeks may not get added protection out of the next vaccine ‣ the Ab developed from the first will block the next vaccine from having an effect ‣ another round of vaccines is given because you do not know which puppies were protected from the first vaccine it is cheaper to re-vaccinate than to test titers Discuss host factors that could alter the course of a disease. Hormonal factors ◦ corticosteroids are immunosuppressive ‣ impair the function of T and B cells and neutrophil migration to tissue ‣ the stress of long distance transport, overcrowding, and others may cause the production of endogenous corticosteroids in cattle, and make them more susceptible to primary respiratory viral infections and secondary bacterial infections ◦ periparturient or pregnant animals may be immunosuppressed due to pregnancy hormones ‣ pregnant sheep are more susceptible to Rift Valley fever virus other determinants of host resistance ◦ nutrition - effect on the integrity of the mucous membranes, skin, phagocytes, and the immune response ◦ obesity - can have pro-inflammatory or suppressive effects ◦ polymicrobial infection - depletion of immune resources, complementary pathology, skewing of immune response to non-protective outcome (co-infection) Discuss the effect of age on host resistance. effect of age on host resistance ◦ young and elderly animals are more susceptible to most viruses and produce more severe disease ‣ a few exceptions: Spanish flu (1918) was harsh on 18-30 year olds, H1N1 in 2009, chicken pox, mumps, and polio viruses cause less severe disease in the young, worsen in teen and middle age ◦ waning immune system in elderly - slower to respond ◦ immature immune system in young ‣ B and T cells may not function optimally in young animals ‣ young animals may lack protective titers of antibodies immune-interference may develop from an interaction of vaccine and maternal antibodies in young animals failure of passive transfer ◦ cells may lose susceptibility to some viruses with age ‣ rotaviruses replicate in piglet enterocytes during the first days of life, but become refractory to infection > 1 month of age. LECTURE 5: PREVENTING, DIAGNOSING, AND TREATING VIRAL INFECTIONS Define passive vaccine Passive vaccines deliver the product of an immune respones to a recipient ◦ short term protection ◦ no memory response ◦ maternal protection Define active vaccine Active vaccines vaccinate with a modified form of the pathogen or part of a pathogen to induce the recipient to make antibodies ◦ long term production ◦ induce a memory response Define inactivated vaccine Chemically inactivate the virus using... ◦ formalin ◦ Beta-propriolactone ◦ nonionic detergents infectivity is eliminated antigenicity is not compromised "looks just like the real thing" safer than live vaccines for people who are immunocompromised requires multiple doses to maintain immunity Define subunit vaccine break virus into components; clone specific viral proteins and express in bacteria, yeast, insect cells, or cell culture, purify proteins and use for vaccination ◦ antigens are usually capsids or membrane proteins can be used in people with weak immune systems and generally have fewer side effects may require multiple doses to achieve and maintain immunity Define replication competent/attenuated/modified live vaccine termed "modified live vaccines" viral replication occurs, stimulates immune response  infection induces mild or inapparent disease long lasting immunity with just 1 or 2 doses not suitable for people with a weakened immune system; could also revert to virulent form (or become more virulent) Define antigen An antigen is a molecule or molecular fragment that is recognized by the immune system as foreign and capable of triggering an immune response. Antigens are typically proteins, peptides, polysaccharides, or lipids found on the surface of pathogens (such as viruses, bacteria, or fungi), as well as on infected or abnormal cells, like cancer cells. When an antigen is detected, it binds to specific receptors on immune cells, such as T cells or B cells, initiating processes that aim to neutralize or eliminate the source of the antigen. Define antibody An antibody is a Y-shaped protein produced by B cells as part of the immune response. Antibodies specifically recognize and bind to antigens. Once bound to an antigen, antibodies help neutralize the threat by marking it for destruction by other immune cells, neutralizing toxins, or directly preventing pathogens from infecting cells. Each antibody is highly specific to a particular antigen, allowing the immune system to target a wide range of pathogens effectively. Explain the goal of vaccination and how vaccines work. the goal of vaccination is to prevent symptoms of disease really good vaccines also: ◦ prevent transmission ◦ have long lasting immunity vaccines educate the immune system about a pathogen ◦ when pathogen is encountered, yields a quick memory response that produces antibodies to the antigen which prevents disease symptoms vaccines do not prevent infection, but they prevent disease vaccines are our best defense against viruses vaccines work by inducing a critical level of immunity in individuals that will prevent them from getting sick - the best vaccines will prevent infection altogether when enough people in a population have the critical level of immunity, the infectious agent will be unable to spread! (HERD IMMUNITY) ◦ each virus has a different threshold to prevent its spread  ‣ smallpox: 80-85% ‣ measles: 93-95% vaccines are not perfect and not everyone will be protected, but everyone needs to get vaccinated to protect those who cannot receive the vaccine Explain the pros and cons of using inactivated vaccines versus attenuated vaccines Inactivated vaccines: contain pathogens that have been killed or inactivated to not cause disease; still trigger an immune response ◦ pro: safer than live vaccines for immunocompromised people ◦ con: requires multiple doses to maintain immunity Live attenuated vaccines: weakend form of a live pathogen, modified to still stimulate an immune response but not cause illness ◦ pro: long lasting immunity after 1 or 2 doses ◦ con: not suitable for immunocompromised individuals, and can revert to more virulent form Explain what it means when an antigen test is positive versus when an antibody test is positive If you are testing for viral antigen ◦ a positive test allows you to conclude the animal contains the virus at the time of the test ◦ *remember every test will have a sensitivity/specificity which should be considered when evaluating results if you are testing for host antibodies against a viral antigen, ◦ a positive test allows you to conclude the animal has been exposed to the pathogen sometime in the past and has generated an immune response to the pathogen ◦ *remember every test will have a sensitivity/specificity which should be considered when evaluating results Understand what viral product is being detected in PCR tests PCR amplifies specific sequences of DNA ◦ can use to detect RNA viruses, but must first convert the RNA to DNA using reverse transcriptase ◦ tests will have specific primers to amplify specific viruses - ie you must have an idea of what virus you are testing for Explain why antivirals are not used often in veterinary medicine antivirals are not common in veterinary medicine because... ◦ expensive and not cost effective for food animals ‣ slaughter withdrawal issies in food animals ◦ unsure whether they will really help animals ‣ need to administer early in disease when virus is actively replicating and being shed ‣ adverse effects ‣ few studies are done to prove efficacy in pet/food animals ◦ difficult to develop for animal use ‣ there are no broad spectrum antiviral drugs, in contrast to penicillin which kills most gram positive bacteria ◦ resistance is a possibility ‣ especially for ssRNA viruses ◦ only 50 antiviral drugs have been approved for human use, cpmpared to many more antibiotics ‣ mostly developed for HIV, HCV, and Herpesviridae ‣ these antiviral drugs can be used extra-label in companion veterinary medicine, but never in food production animals vaccines do a good job of preventing infection, but cannot do anything for individuals that are already infected most antivirals are for viruses that cause persistent infections ◦ most drugs block viral nucleic acid replication (polymerase inhibitors) ◦ other viral specific unctoins are "good" targets for antivirals ‣ proteases ‣ integrases ‣ fusion ◦ because targeting the virus typically induces fewer side effects, most drugs target viral enzymes why dont we have more antivirals? ◦ drugs need to block the virus without causing damage to the host ‣ viral replication is linked to host cells -> difficult to find the right compounds ◦ some viruses do not have good cell culture models -> antiviral testing difficult ◦ compounds need to be very potent ‣ to stop a virus that replicates at a logarithmic rate, an extremely effective compound is needed, but are rare findings  antivirals need to stop virus replication completely ◦ in contrast to other drugs taken for things like high cholesterol which need to slightly increase or decrease host enzyme activity ‣ viral replication extremely rapid -> so many viral particles produced -> antivirals need to block the virus to make an impact ◦ resistance is also a concern acute viral infections are difficult to treat ◦ by the time acute infections show symptoms, the virus may already be jumping to a new host or viral replication is slowing ‣ hit and run viruses (common cold, flu, GI bugs) ◦ most antivirals need to stop peak viral replication to show a therapeutic effect ‣ but most patients to not feel sick until after a peak in viral replication Explain how AXT and acyclovir stop viral replication. AZT and nucleoside analogs ◦ AZT is a deoxythymidine analog that is converted to the triphosphate form (pppAZT) in host cells by the cellular enzyme thymidine kinase ◦ pppAZT is incorporated by reverse transcriptase into inactively growing viral DNA, and blocks the ability of RT to incorporate more nucleotides ◦ 3' N3 group is present instead of OH -> DNA chain termination ‣ toxic to host because it affects DNA synthesis in uninfected host cells too since the development of AZT, many nucleoside analogs have been developed ◦ newer generation non-nucleoside reverse transcriptase inhibitors bind and block RT enzyme of Retroviridae with fewer side effects than nucleoside analogues Acyclovir ◦ only acts in herpesviridae infected cells -> viral encoded thymidine kinase (TK) phosphorylates acyclovir and then is processed by viral DNA polymerase ‣ fewer side effects than AZT because the drug is only activated in virally infected cells ◦ nucleoside analogue of guanosine -> no 3' OH in deoxyribose molecule so it terminates DNA replication ◦ resistant herpesviridae fail to phosphorylate acyclovir or viral DNA polymerase wont incorporate phosphorylated drug into DNA chain Explain how NA inhibitors decrease influenza spread Neuraminidase (NA) is required to cleave sialic acid moieties from infected cells so the bidding  virions can escape and infect additional cells NA inhibitors block activity and viral particles do not spread well Oseltamivir (Tamiflu) and Zanamivir (Relenza) ◦ inhibitors designed to mimic sialic acid, the ligand for viral neuraminidase ◦ interfere with release of progeny influenza virus from infected host cells -> limit infection of new host cells ◦ only effective if administered early in infection - no effect if given too late ◦ resistance to oseltavimir has rapidly emerged in human H1N1 viruses ‣ zanamibir is closest mimic of sialic acid - no resistance detected ◦ some veterinarians use oseltamivir to treat CPV2 infections ‣ has no effect on CPV2 replication Describe mechanisms to prevent disease in the clinic and the field. hygeine - wash your hands often when handling animals, and do it correctly sanitation - clean all areas of your practice using appropriate disinfectants; shower between visiting different farms and animals; change clothes use PPE ◦ be ethical and change PPE between patients - PPE can become a fomite  nRBCs: usually metarubricytes; correct WBC count if >5/100 WBCs ◦ appropriate: regenerative anemias ◦ inappropriate: nRBCs without reticulocytes, nRBCs are disproportionate to the regenerative response, related to splenic dysfunction/disease/splenectomy, and bone marrow (neoplasia; damage (trauma hypoxia heat stroke) lead..) Basophilic stippling ◦ risidual aggregates of RNA ◦ small, fine basophilic dots ◦ regenerative anemias ‣ typical of regeneration in ruminants often more prominent than polychromasia ‣ dogs and cots, only with marked regeneration ◦ other causes ‣ without appropriate regeneration; lead toxicity, dyserythropoiesis END EXAM 1 MATERIAL Parasitic RBC inclusions usually need to be at 100x oil magnification both epicellular (on the surface) and intracellular (within the cell) must not confuse with other inclusions (HJ bodies, basophilic stippling) Hemotropic Mycoplasma spp. ◦ epicellular: small bacteria on the surface of RBCs ◦ present in many species ‣ pathogenicity varies with host and species ‣ most pathogenic in cats and pigs ◦ parasitemia will wax and wane ‣ diagnosis by microscopic blood smear exam has low sensitivity and specificity ‣ test of choice: PCR ‣ species specific infections: dog: M. haemocanis cat: M haemofelis  Sheep, goats: M ovis Pigs: M. Haemosuis Cattle: M. wenyonii Llama, alpaca: M. haemolamae ◦ can be difficult to distinguish from stain precipitate - look for organisms at outer edge of cells, basophilic dots (cocci), small rings, linear chains ◦ can fall off RBCs in stored blood - we will see organisms in the background of the smear ◦ stain precipitate: ‣ much more common. clean stain is imperative! if you focus up and down using the fine focus of the microscope, you woll see stain precipitate in a slightly different plane than the RBCs Cytauxzoon felis ◦ tick transmitted, intracellular protozoal organism, closely related to Babesia ‣ regional to SE USA ◦ reservoir species: bobcats ◦ domestic species: only in cats ‣ often fatal disease, but pathogenicity can vary  ‣ has a 1) tissue storage stage (schizonts) and a 2) RBC stage (piroplasms) ◦ Tissue storage (schizonts) ‣ primary cause of clinical disease ‣ before organisms can be seen on a blood smear ‣ Schizonts (parasites in macrophages) line and occlude vessels of most organs ‣ sometimes schizonts can be seen on the feathered edge of blood smears (more common in tissue samples) ◦ RBC stage (piroplasms) ‣ second phase of infection ‣ variable parasitemia, can be low ‣ very small, 0.5-2.5um ‣ round to oval ‣ dot-like nucleus ‣ resembles Babesia gibsoni ◦ initial signs are non-specific, but there is rapid progression within few days ‣ severe, non-regenerative anemia likely extravascular hemolysis (parasites within RBCs) pre-regenerative? bone marrow damage? ‣ sepsis-like signs leukopenia with toxic change thrombocytopenia shock-like signs ‣ history: cats that have access to outside; more common in outdoor only cats ◦ early diagnosis is critical due to rapid progression ◦ check for organisms on the blood smear ‣ primary method of diagnosis ‣ organisms can be very low in numbers ‣ in suspect cases with negative blood smear, look for tissue phase (fine needle aspirate) ◦ PCR is also available (confirmatory)  Anaplasma spp. ◦ small, 0.5-1 um, round, basophilic structures at the periphery of the cell ◦ must differentiate from HJ bodies: smaller than HJ bodoes, may see multiple per cell, more marginated Babesia spp ◦ tick transmitted, intraerythrocytic protozoan parasites ◦ only has an RBC stage (merozoite) ◦ > 100 species described ◦ can be described as large form or small form species based on size ◦ primarily in dogs ◦ also in cattle, horses, and sheep; some of them to Theileria ◦ large-form babesia ‣ B. canis low virulence in the US, but strains outside the US are more virulent other large form species appear similar (B. coco) often subclinical clinical disease ◦ more likely in puppies (the sick or the immunosuppressed) ◦ mild anemia to moderate thrombocytopenia large: 2-4 um Piriform dot-like red nucleus  may be paired ◦ small-form Babesia ‣ B. gibsoni world-wide distribution, moderately virulent increased incidence in pit bulls dog fighting or dog bites increases infection risk moderate to marked regenerative anemia, intra and extravascular hemolytic anemia, +/- spherocytes - secondary IMHA, moderate to parked thrombocytopenia small: < 2um round to oval dot-like red nucleus thin cytoplasmic membrane ◦ diagnosis ‣ blood smear exmaination: critical for virulent species (B. gibsoni) low sensitivity when present in low numbers using capillary blood (ear vein, nail clip) may concentrate organisms ‣ serology (titers) sensitive screening test can only document exposure but cannot confirm acute infection ‣ PCR sensitive and specific longer turnaround time Microfilaria ◦ Dirofilaria immitits (heartworm), Dipetalonemia reconditum ◦ these are big! ‣ sometimes you will only see them on the feathered edge or in the deep body of the blood smear ◦ difficult to distinguish types of microfilaria on stained blood smear ‣ might need Knott's test, or heartworm antigen test LECTURE 3: WBCs I - TERMINOLOGY, IMPORTANT LEUKOGRAM PATTERNS, LEFT SHIFT WBC categories Neutrophils - predominant in most (but not all) species; antimicrobial activity, inflammation, phagocytosis ◦ granulocyte Eosinophils - hypersensitivity responses, parasites ◦ granulocyte basophils - rare; function poorly understood, hypersensitivity responses ◦ granulocyte lymphocytes - usually second most common cell type, acquired immune response ◦ mononuclear cell monocytes - form of a histiocyte (in tissue: macrophage); phagocytosis, antigen presentation, cytokine secretion ◦ mononuclear cell Limitations of hematology analyzers will provide falsely decreased PLT count when PLTs are large (impedance) or clumped cannot recognize important morphologic changes ◦ RBCs - agglutination, spherocytes, schistocytes ◦ presence of nRBCs (often falsely counted as lymphocytes) ◦ band neutrophils, toxic changes ◦ neoplastic cells and other cells usually not present on CBC (ie mast cells) ◦ cannot detect hemoparasites (heartworm filariae, babesia spp, anaplasma spp) whenever analyzer flags a value, verify results with a microscopic blood smear exam The manual WBC differential can be performed on 40x dry, 50x oil, or 100x oil in the monolayer spend time deciding who is who before starting to actually count move in an orderly manner and count 100 consecutive white blood cells, avoid double counts stay in the monolayer! both the manual WBC differential and correcting automated WBC counts will prevent cells from being counted more than once and should keep you from counting cells too close to the feathered edge Major patters of leukocyte responses physiologic leukocytosis ◦ AKA epinephrine response, fight or flight response ◦ neutrophilia and lymphocytosis corticosteroid response ◦ AKA stress response ◦ neutrophilia, lymphopenia, monocytosis, eosinopenia inflammatory leukogram ◦ variable ◦ classic: neutrophilia with a left shift and monocytosis Physiologic Leukocytosis due to excitement, fear, acute pain, exercise ELSI = Epinephrine causes Lymphocytes and Segmented neutrophils to Increase Neutrophilia - due to redistribution of neutrophils in the marginated pool lymphocytosis - likely due to splenic contraction young animals, especially cats and horses short-lasting: only as long as epinephrine surge occurs common in young, healthy cats ◦ Neutrophilia may exeed 39,000/uL ◦ lymphocytosis may exceed 35,000/uL, can predominate common in healthy foals ◦ neutrophils and lymphocytes up to 14,000/uL each pigs: 2-3x upper reference interval for neutrophils and lymphocytes uncommon in dogs ◦ more likely in puppies or excitable breeds (toy breeds) ◦ neutrophil counts up to 20,000/uL ◦ lymphocytosis of up to 12,000/uL cattle: parturition and exercise induced Corticosteroid (stress) response caused by endogenous or exogenous corticosteroids ◦ steroid treatment, hyperadrenocorticism (Cushings) ◦ excessive release with pain, trauma, fear, chronic/severe illness  SSMILED ◦ stress/steroids cause segmented neutrophils and monocytes to increase, lymphocytes and eosinophils decrease Neutrophilia ◦ 2-3x the upper reference interval in dogs ◦ No significant left shift, no toxic changes ◦ increased release from bone marrow storage pool ◦ redistribution from marginated pool to circulating pool ◦ prolonged circulating pool lymphopenia ◦ lymphocyto-lysis and redistribution of lymphocytes Monocytosis ◦ usually only seen in dogs Eosinopenia typically seen in sick animals (stress due to underlying disease) ◦ if ill patient lacks a stress response, suspect Hypoadrenocorticism (Addisons) often seen in animals with inflammatory disease ◦ combined stress and inflammatory leukogram Neutrophil production, tracking, and kinetics Production of neutrophils ◦ production in the bone marrow, orderly maturatino from plast to segmented neutrophil ‣ beginning to end: about 5-6 days ‣ bovines: 7 days (held in BM for one additional day) trafficking and kinetics of neutrophils ◦ Blood pools: total neutrophil pool ‣ circulating neutrophil pool + marginating neutrophil pool ◦ circulating neutrophil pool: free flowing in the blood stream ◦ marginating pool: stuck to the endothelial cells, not sampled during blood draw ◦ species variation: ‣ horses, dogs, calves: 50/50 split ‣ cats: marginating 3x circulating ◦ Storage pools vary by species ‣ dogs = largest storage pool 5 day supply -> tend to not develop neutropenia  ◦ seen sometimes with pyometra and other large pus formations ‣ cats, horses = intermediate storage pool may run out faster than dogs ‣ ruminants = small storage pool frequently develop neutropenia with increased demand (mild inflammation) - not overly concerning on its own ◦ travel from blood to tissue is one way ‣ blood transit time is 10-14 hours ◦ number of neutrophils in peripheral blood affected by: ‣ shifts between circulating pool and marginating pool epi and glucocorticoids cause shift from marginating pool to circulating pool bacterial endotoxins can cause a shift from circulating to marginating pool (increase adhesion) ‣ rate of production in bone marrow ‣ rate of release from bone marrow in the blood ‣ rate of migration into the tissue - indicates demand Neutrophil left shifts ◦ immature neutrophils in peripheral blood ◦ typically consists of band neutrophils ◦ should be orderly ‣ most immature stages present in the lowest numbers ‣ disorderly release suggests a bone marrow disorder like leukemia ◦ band neutrophils in left shifts ‣ horse-shoe shaped nucleus ‣ parallel sides ‣ smooth outline ‣ no or minimal constrictions no indentations grater than half the width of the nucleus no folds (note: if the nucleus is long enough to fold = segmented neutrophil) ‣ coarse chromatin ‣ slightly larger, bluer cytoplasm compared to segmented neutrophils ‣ subjective - always check lab's reference interval  ‣ monocytes vs. band neutrophils: Regenerative left shift ◦ orderly ◦ normal or increased segmented neutrophils and lower number of immature cells (typically band neutrophils) ◦ Mature cells >> immature cells ◦ segmented neutrophils >> band neutrophils >> metamyelocytes etc Degenerative left shift ◦ immatures >> matures (ie bands >> segmented neutrophils) ◦ OR can be characterized by Neutropenia + any left shift ◦ indicates overwhelming inflammation - things may go downhill Neutrophil toxic changes ◦ morphologic abnormalities of the neutrophil cytoplasm that occur during maturation, seen with: ‣ infections/sepsis ‣ severe inflammation ‣ excessive tissue damage ◦ mild changes ‣ potentially any condition with inflammation, infectious or non-infectious (eg IMHA) ◦ moderate to marked changes ‣ bacterial infections, other infections, sepsis ◦ do not confuse toxic change with degenerative changes ‣ toxic change: cytoplasm, only evaluated in blood ‣ degenerative change: nucleus, usually happens in tissue ◦ Döhle bodies ‣ blue-grey aggregates of retained ER ‣ rare amount is normal in cats ‣ mildest ◦ Basophilic cytoplasm ‣ increased basophilia ‣ more pronounced ◦ vacuolization ‣ note: vacuoles (foaminess) in neutrophils without other toxic changes (basophilia, Döhle bodies) is likely and artifact artifact could be caused by prolonged storage/sample being old ◦ Toxic granulation ‣ purple red granules ‣ rare in cats, dogs ‣ uncommon in LA can be more common in horses than cattle ‣ common in birds, reptiles amphibians - best clue into infection for exotics ‣ highest degree of toxic change ◦ grading is subjective, based on: ‣ changes seen Döhle bodies -> basophilia -> vacuolization -> toxic granulation ‣ number of affected cells ◦ reporting ‣ on a scale as "total toxic change" slight, mild, moderate, marked 1+, 2+, 3+, 4+ ‣ by individual feature few döhle bodies, mild basophilia, etc LECTURE 4: WBC II - INFLAMMATION, DISORDERS, AND INCLUSIONS Inflammation Neutrophils ◦ changes on CBC will depend on... ‣ intensity of the stimulus ‣ acute vs. chronic ‣ location of the inflammation ‣ species ◦ Neutrophil count on a CBC reflects the balance between production and demand ‣ increased, normal, or decreased ◦ Neutrophilia ‣ the classic response to inflammation ‣ you may also see a left shift and toxic changes ‣ with chronicity, left shift may go away ‣ bone marrow outpaces the demand/consumption in tissue enough time for bone marrow to respond to the peripheral demand tissue demand is milder ◦ dog can get really pronounced response ‣ degree of neutrophilia depends on the intensity of the stimulus and the species' bone marrow reserve/responsiveness ‣ ranges of neutrophilia by species: dog: 20,000-100,000/uL cats: 20,000 - 60,000/uL horses: 15,000 - 30,000/uL cattle: 10,000 - 25,000/uL ◦ how to tell stress neutrophilia from inflammatory response ‣ often present together ->. clinically important to identify inflammation ‣ inflammation is more likely if: neutrophilia is greater than 2-3x the upper reference interval left shift and/or toxic changes are present other evidence of inflammatory disease (eg fever, increased acute phase proteins) ◦ extreme neutrophilia ‣ high end of the ranges or even higher ‣ AKA leukemoid response ‣ localized, pirulent responses - "pus pockets"  big bone marrow stimulus + localized lesion pyothorax pyoabdomen pyometra walled off abscesses endocarditis, pyelonephritis ‣ less common causes Hepatozoon americanum infection ◦ tick-borne protozoal disease ◦ gulf coast, occasionally in the southeast US ◦ pyogranulomatous mystitis and periostial bone reactions leukocyte adhesion deficiency neoplastic proliferation of neutrophils ◦ chronic myelogenous leukemia (CML) - rare ◦ Neutropenia ‣ neutropenia due to inflammation occurs when tissue/demand/consumption exceeds production in the bone marrow usually an acute, severe, overwhelming demand (clinically important) ‣ other mechanisms endotoxemia -> margination of neutrophils (circulating pool -> marginal pool) ◦ transient (1-3 hours) and early on some infectious agents can directly destory neutrophil precursors in BM ◦ canine and feline parvovirus ‣ typically associated with left shift + toxic changes ‣ varies by species based on bone marrow storage pool of neutrophils dogs - largest storage pool of all species ◦ neutropenia -> severe, overwhelming inflammation (bad!) cats, horses -> moderate storage pool ◦ neutropenia -> normal response to significant acute inflammation ◦ calves -> higher storage pool, respond more like dogs and cats ‣ ex: gram negative bacterial infection/septicemia; salmonellosis (horses) ◦ Normal neutrophil counts ‣ can be seen with  mild inflammation recovery from neutropenia (regenerating from acute, severe inflammation) - Good! progression towards neutropenia (degenerating) - Bad ‣ how to detect inflammation? left shift and/or toxic changes other indications of underlying inflammation (eg fever) changes in acute phase proteins (fibrinogen, albumin, Crp, serum amyloid A) ◦ species differences ‣ species with less responsive bone marrow/lower BM storage pool ruminants! horses to some degree neutropenia develops more rapidly with acute inflammation neutrophil changes not as prominent as in other species (dogs, cats) ‣ fibrinogen routinely reported on UGA LA CBCs increases with inflammation and dehydration ‣ other acute phase proteins horses: SAA (serum amyloid A) ‣ ruminants: neutrophil to lymphocyte ratio healthy adult ruminants are lymphocyte predominant (N:L = 1:2) ◦ inversion of the ratio is suggestive of inflammation ◦ summary of neutrophil patterns ◦ Non-inflammatory neutropenia ‣ chemotherapy, cytotoxic drugs ‣ pancytopenia (decrease in all 3 cell lines): aplastic anemia myelophthisis (diseases that 'crowd' out regular bone marrow cells) ‣ immune-mediated neutropenia (rare) ‣ breeds border collies - trapped neutrophil syndrome grey collies - cyclic hematopoiesis  ‣ idiopathic ◦ Acquired disorders of neutrophil function ‣ Glucocorticoids decreased adhesion molecules ‣ systemic diseases: diabetes mellitus, renal failure, liver cirrhosis ‣ neutritional deficiencies - copper, cobalt, selenium, molybdenum, thiamine, sulfur, zinc ‣ some viral diseases: FeLV, BVD, others ‣ burn patients ‣ periparturient neutrophil dysfunction in diary cattle poorly understood increased incidence of mastitis ◦ Important neutrophil inclusions ‣ granulocytic ehrlichiosis and anaplasmosis Ehrlichica ewingii Anaplasma phagocytophilum primarily in dogs, rarely in cats, large animals thrombocytopenia anemia polyarthritis Morulae in neutrophils (rarely eosinophils) ◦ pale blue lavender, round, stippled to clumped structure ('raspberry') ◦ identification of morulaeis an insensitive method of detection serology, IFA, PCR more sensitive blood smear ID useful in acute disease ‣ Monocytic ehrlichiosis E. canis & E. chaffeensis morulae in monocytes, not neutrophils ◦ rarely seen in blood; only present during acute infection and in very low numbers diagnosis: serology, PCR acute disease: mild anemia, thromocytopenia, hyperglobulinemia, lymphocytosis chronic infection: thrombocytopenia and lymphocytosis may persist; some progress  to bi- or pancytopenia due to marrow hypoplasia or aplasia ‣ Distemper inclusions aggregates of viral particles, transiently visibile in blood of dogs with acute infection magenta, purple, lavendar, pale blue, variabily sized round structures - stain best with diff quick the virus infects hematopoietic precursor cells in the bone marrow; therefore, inclusions can be seen within all types of leukocytes and erythrocytes ‣ Bacteria? usually not seen in peripheral blood, located in tissue LECTURE 3: WBC III OTHER WBC Lymphocytes Predominant WBC type in adult ruminants, rats, pigs, mice, and many birds Stress response ◦ lymphopenia ◦ SSMILED : increased segmented neutrophils, decreased lymphocytes, increased monocytes, decreased eosinophils Epinephrine (physiologic) leukocytosis ◦ Lymphocytosis ◦ ELSI: increased segmented neutrophils, increased lymphocytes Lymphocytosis ◦ antigenic stimulation ‣ mild increase post vaccination ‣ Infectious diseases Chronic E. canis infection: up to 30,000/uL - differential: chronic lymphocytic leukemia? ‣ Autoimmune diseases IMHA in cats (up to 20,000/uL) ◦ Endocrine disease ‣ Hypoadrenocorticism: up to 13,000/uL (opposite of a stress response) ‣ Hyperthyroidism: up to 9,000/uL  ◦ Lymphoid neoplasia: lymphoma or lymphocytic leukemia ◦ Persistent Lymphocytosis of cattle ‣ BLV induced non-neoplastic proliferation ◦ Primary response to diseases in some birds nRBC or lymphocyte? Compared with lymphocytes, nRBCs have: ◦ grayish blue cytoplasm ◦ Greater amount of cytoplasm ◦ Darker, coarser chromatin Lymphopenia Acute viral infection - trapping in lymphoid tissue Stress Leukogram - Lymphocytosis and trapping in lymphoid tissue Destruction ◦ Of lymphocytes ‣ Some viruses: distemper, parvovirus, FIV ‣ Chemotherapy, radiation ◦ Of lymph node architecture ‣ Lymphoma Loss of lymphocyte rich lymph fluid ◦ Chylous effusion, protein losing enteropathies Immunodeficiency viruses: FIV, BIV Severe, combined immunodeficiency (SCID) Monocytes Rapid maturation in the bone marrow: 1-2 days ◦ Immediately released upon production, no bone marrow storage pool ‣ Increasing monocyte counts usually precedes recovery from neutropenia ◦ Blood transit time: 12-24 hours  Develop into ◦ Macrophages, antigen presenting cells ‣ Phagocytosis of cellular debris, foreign materials, infectious agents ‣ Secrete inflammatory cytokines Monocytosis Stress response - only consistent in the dog Chronic inflammation, esp with ◦ Tissue necrosis ◦ Hemolysis and hemorrhage ◦ Pyogranulomatous inflammation Monocytopenia - not clinically relevant Eosinophils Eosinophilia ◦ Worms, wheezes, and weird sneezes ◦ Worms ‣ Parasitic infection, especially those with tissue stages ◦ Wheezes ‣ Hypersensitivity reactions Athsma, eosinophilic bronchopneumopathy, atopic dermatitis, flea bite allergy, etc ◦ Weird diseases ‣ Infection Fungal infection Pythiosis ‣ Associated with neoplasia (paraneoplastic) Mast cell tumor (dogs, horses, not cats) T-cell lymphoma Some carcinomas ‣ Hypereosinophilic syndrome Rottweilers, other dog breeds, rarely cats Presumed to be dysregulation of eosinophils ◦ Hypoadrenocortism - opposite of a stress response (20% of patients)  ◦ Hyperthyroidism in cats Basophils Production similar to neutrophils Functions appear to be similar to mast cells ◦ Hypersensitivity reactions ◦ Participation in hemostasis ◦ Rejection of parasites Rare in most species ◦ High in rabbits (10-15%), turtles and tortoises (30-60%), birds Basophilia ◦ Often parallels eosinophilia, but to a smaller degree ◦ Basophilia without eosinophilia is uncommon ‣ Similar differentials Mast cells bone marrow derived, but not seen in blood in health Morphology ◦ Large cell, round nucleus ◦ Fine, magenta granules ‣ Granules may not stain with quick stains ◦ Check feathered edge (large, heavy cells) increased numbers (mastocytosis) ◦ poor venipuncture ◦ dogs: mostly reactive ‣ GI disease, esp parvo infection ‣ other inflammatory diseases ‣ advanced stages of aggressive mast cell tumor s ◦ cats: mast cell tumor! LECTURE 6: NON-MAMMALIAN HEMATOLOGY State appropriate handling procedures and recommended anticoagulants for non-mammalian blood samples Bird blood collection ◦ right jugular vein ‣ common site in most pet species (psittacines) and smaller birds (passerines) ◦ Basilic "wing" vein ‣ medium to large sized birds ‣ when jugular venipuncture isn't possible ◦ medial metatarsal vein ‣ waterfowl, chickens, flightless birds Reptile blood collection ◦ blind venipuncture ◦ lizards - ventral coccygeal (tail) vein, jugular vein ‣ use caution in species with tail autonomy (ex geckos and skinks) ◦ snakes - ventral coccygeal (tail) vein, cardiocentesis ◦ chelonians - jugular vein ‣ other sites at risk for lymph contamination ◦ what is lymph contamination? ‣ crossly seen by a flash of clear yellow to pink fluid prior to blood during venipuncture ‣ falsely decreaes the PCV and increases lymphocyte counts ‣ any reptile is susceptivel depending on the site of blood collection sample handling ◦ maximum volume of blood (mL) = 0.01 x weight (gm) ◦ volume required: minimum~200uL for CBC ‣ a properly prepared blood smear has priority - microscope evaluation alone an provide relevant diagnostic information ◦ storage: ‣ whole blood has short-term stability at room temperature ‣ refrigerate (~39F or 4C) and use ideally within 24 hours ‣ make a blood smear before storing ◦ EDTA or heparin anticoagulants for CBC ‣ EDTA preferred in most birds except Corvids and Ratites ‣ heparin preferred in most reptiles except green iguanas and chinese water dragons ‣ Use albumin if there are problems with cell preservation  2:1 ratio, two drops blood:one drop albumin ◦ heparin vial for chemistry ‣ using plasma maximizes sample volume compared to a red top ‣ EDTA can cause artificial changes (eg decrease in calcium and potassium) Understand the different ways to obtain a total leukocyte count in non-mammalian species Leukocyte Counting Methods ◦ indirect counting method ‣ uses Pholxine B stain available as solution that is then diluted or as a pre-diluted kit (Leukopette) whole blood is added to the stain and allowed to sit causes lysis of RBCs and stains heterophils and eosinophils bright pink ‣ count phloxine-stained granulocytes (heterophils and eosinophils) in all 9 large squares of the chamber and on both sides of the hemacytometer ‣ determine the differential count on the blood smear ◦ direct counting method ‣ uses Natt & Herrick's solution to count both RBCs and WBCs whole blood is diluted (1:200) with Natt & Herrick's solution stains all leukocytes dark blue ◦ leukocyte estimates using blood smears (always recommended) ‣ performed on a stained blood smear (diff-quick or wright-giemsa) ‣ count number of leukocytes in 40x (get an average over 10 fields) ‣ estimated leukocytes/uL = average number of leukocytes in 10 firlds x 2,000 (use 2500 instead if 50x) ‣ should be considered a rough estimate -> variable due to smear density and/or quality ‣ used together with either the direct or indirect counting methods to validate counts ◦ regardless of the method, you will need to determine the leukocyte differential ‣ leukocyte differentials are performed on a stained blood smear (diff quick or wright- giemsa) ‣ same concept used in mammals - 100 or 200 cell count of leukocytes by cell type ‣ yields a percentage -> used to calculate the absolute counts for each cell type ‣ requires an understanding of species differences and patience ◦ Hemocytometer ‣ used for indirect or direct counting methods ‣ thick glass slide with counting chambers Be able to identify each of the avian and reptilian leukocytes, including species differences. (if they ask us about interpretation of these im sorry) Heterophils ◦ analogous to mammalian neutrophils ◦ predominate leukocyte in birds, chelonians, and crocodilians ◦ exhibit prominent species-specific differences ‣ birds, lizards and most amphibians have segmented nuclei (2-3 lobes) with coarse chromatin ‣ chelonians, crocodilians, and snakes have round, eccentric nuclei ◦ cytoplasm is generally clear with eosinophilic (secondary) granules ‣ birds -> bright pink, oval to spindle-shaped granules ‣ reptiles -> orange to bright pink, round to spindle shaped granules ◦ heterophilia: physiologic (summer (reptiles), gravidity), stress (same as mammals), inflammation ◦ heteropenia - overwhelming inflammation Eosinophils ◦ round to rod-shaped granules that are bright pink in most species ‣ exception -> eosinophil granules stain blue in Psittacines, Tegus, and Iguanas ◦ eosinophils make up a similar % in birds as in mammals ‣ variable in reptiles -> chelonians have the highest % eosinophils, whereas eosinophils are virtually absent in snakes ◦ eosinophilia -> immune hypersensitivity/ allergy, parasitic infections, antigenic stimulation Basophils ◦ different morphology from mammals - more similar to mast cells ◦ round, non-segmented nucleus and contain many deep purple granules ‣ basophils may degranulate or fail to stain making their ID challenging ◦ present in low numbers in health ◦ basophilia -> hypersensitivity/allergy, antigenic stimulation, glucocorticoid induced stress, respiratory disease, tissue damage, certain infectious agents  Monocytes ◦ morphology and function similar to mammals ‣ oval to reniform nucleus with basophilic cytoplasm +/- vacuoles ◦ monocytosis -> chronic inflammation, foreign body reaction, some nutritional deficiencies ◦ in reptiles, monocytes may contain melanin pigment granules Azurophils ◦ unique to reptules and commonly seen in lizards, snakes, and crocodilians ‣ second most common leukocyte in snakes (makes up 35%) ◦ look similar to monocytes but contain many pale, blue-purple, dust like granules giving them a pink-purple tinge ◦ often counted together with monocytes in the leukocyte differential due to their similar function ‣ exception -> azurophils of snakes are more similar to mammalian neutrophils and should be counted separately ◦ azurophilia -> acute inflammation in snakes and chronic inflammation in other reptiles Lymphocytes ◦ predominant leukocyte in some birds and reptiles ‣ waterfowl, lizards, snakes ◦ similar functoin and morphology as mammalian lymphocytes ‣ round cells with round to oval nuclei and coarse chromatin ‣ may contain few red granules ◦ lymphocytosis -> antigenic stimulation, neoplasia ◦ lymphopenia -> glucocortoicoid-induced stress, viral infections Thrombocytes ◦ non-mammalian version of platelets ◦ nucleated with phagocytic activity ◦ round to oval shape with clear, blebbed cytoplasm ◦ may appear activated -> basophilic cytoplasm with few vacuoles and/ or purple granules ◦ difficult to differentiate from lymphocytes Be able to identify the common hemoparasites of non-mammalian species and understand their significance. Inclusion body disease ◦ seen in boas and pythons ◦ results in a mild to marked lymphocytosis with characteristic smooth, basophilic inclusions within lymphocytes, thrombocytes, +/- other leukocytes ◦ cause is unkown but a retrovirus is suspected (Iridovirus) ◦ fatal infection with no effective treatment  constant fraction faster process exception: high doses - saturation ◦ zero order kinetics: constant amount Lecture 3: TOXICOKINETICS/TOXICODYNAMICS Biotransformation Enzyme driven reactions ◦ substrate: toxicant ◦ Phase I, Phase II reactions Sites ◦ Liver, lung, kidney ‣ skin, intestine, testes, placenta outcome ◦ detoxification (inactivation) ◦ bioactivation ‣ aka toxification, lethal synthesis ◦ facilitate excretion ‣ more hydrophilic (water-soluble) / larger Phase I Phase I reactions are simple degradation reactions ◦ ATP independent add or expose a "functional" (ionizable) group ◦ hydroxyl, carboxyl, amino ‣ act as a tag or a handle metabolite excreted or proceed to Phase I Phase I reactions ◦ Oxidation (loss of electrons) ‣ addition of oxygen - first discovered oxygenation (addition of O to replace H) ‣ involvement of oxygen is not required (dehydrogenation) ◦ reduction (gain of electrons, addition of H to replace O) ◦ hydrolysis Phase I: P450 Enzymes Oxidation ◦ Cytochrome P450 (CytP450) mized function oxidases ‣ located in endoplasmic reticulum, sits on ER membrane most important mono-oxygenases / oxidases ‣ multiple isoforms (isozymes) broad range of substrates some polymorphism within species ‣ located on ER membrane microsomal (MFOs) ‣ expression: induced or suppressed CytP450 oxidase reactions ◦ requires: oxidized CytP450, NADPH, CytP450 reductase, 02 ◦ reaction steps" ‣ oxidized CytP450 (Fe3+) binds toxicant ‣ enzyme toxicant complex reduced Fe3+ -> Fe2+ ◦ NADPH, reductase ‣ reduced complex binds O2 ‣ second reduction takes place NADPH reductase ‣ final complex splits into 3 products functionalized toxicant metabolite Oxidized CytP450 (regenerated) H2) Bioactivation by Phase I Enzymes: ◦ Aflatoxin B1 from Fungi ‣ DNA binding, liver tumors, and renal papilla neoplasia ‣ 1ppb carcinogenic to rats in chronic exposures ‣ mind TD in dogs as low as 60 ppb over several weeks -> liver failure Phase II Basics ◦ conjugation reactions  ‣ ATP dependent ‣ polar groups from donor compounds added to functional groups (Phase I) ◦ enzymes ‣ specific transferases ‣ require specific cofactors that act as donors ‣ redominantly cytosolic (1 exception) Glucuronidation reaction - occurs in the endoplasmic reticulum and reacts to steroids ◦ promote excretion ‣ larger, charged, water soluble two different reactions can occur in phase II ◦ glucuronidation ‣ major phase II reaction ‣ sugar conjugation donor: Uridine Diphosphate Glucuronic Acid (UDPGA) enzyme: UDP glucuronsyl transferase ◦ cats are deficient in this enzyme ‣ low affinity, high capacity * ‣ enzyme bound to ER membrane ‣ inducible ◦ sulfation (sulfonation) ‣ similar substrates as glucuronidation cats: important reaction ‣ sulfotransferases ‣ donor/cofactor: 3'phosphoadenosine 5' phosphosulfate (PAPS) atp inorganic sulfate ‣ conjugates SO3- (sulfonate) to toxicant ‣ high affinity, low capacity ‣ not readily inducible ◦ glutathione conjugation ‣ reaction steps formation of glutathione toxicant (metabolite) conjugate ◦ via thiol group ◦ GST  recycling of glutathione peptides ◦ peptidases formation of final conjugate ◦ acetyltransferase ◦ mercapturic acid metabolite ◦ Phase II conjugations ‣ Toxicodynamics mechanisms of toxicitiy ◦ step 1: delivery from the site of exposure to the target ◦ step 2: reaction of the ultimate toxicant with the target molecules ◦ step 3: cellular dysfunction and resultant toxicities Toxicodynamics: Step 2 ◦ general mechanisms of toxicant-target interactions ‣ direct interaction with target non-covalent, reversible covalent, irreversible hydrogen abstraction electron transfer enzymatic ‣ indirect - disrupt intracellular environment ‣ common targets proteins: enzymes, receptors, transporters lipids: cellular, subcellular membranes nucleic acids Toxicodynamics Step 3 ◦ two roles of target molecules and their effects:  ‣ Cell regulation (signaling) dysregulation of gene expression ◦ inappropriate cell division (neoplasia, teratogenesis) ◦ inappropriate apoptosis (tissue involution, teratogenesis) ◦ inappropriate protein synthesis (ex peroxisome proliferation) dysregulation of ongoing cell function ◦ ex: inappropriate neuromuscular activity ‣ tremors, convulsion, spasm, cardiac arrhythmia ‣ necrosis, paralysis, paresthesia ‣ cell maintenance impaired internal maintenance ◦ leading to cell injury or death impaired external maintenance ◦ impaired function of integrated systems ‣ eg: hemostasis -> bleeding Examples of Toxic Mechanisms specific protein interactions ◦ enzyme systems ‣ toxicant is an enzyme (Botulism toxins, tetanus toxin) ‣ toxicant is a substrate active site (Anticoagulant rodenticides) allosteric ‣ toxicant causes enzume induction or suppression (OPs) ◦ receptor systems ‣ agonist (amitraz) ‣ antagonist (strychnine) ‣ receptor types ligand gated ion channels; voltage gated ion channels G- protein coupled receptors cytosolic/nuclear receptors ◦ transport systems ‣ hemoglobin (CO) ‣ Pumps: Na-K ATPases (ouabain [cardiac glycoside]), H+ ATPase ‣  ◦ non-selective, opportunistic actions ‣ nearby proteins, lipids, nucleic acids ◦ toxicant is: ‣ electrophilic or ‣ biotransformation (bioactivation) formation of electrophilic metabolite formation of free radicals (small molecule or an atom with an unpaired electron or electrons formation of reactive intermediate (occurs during CytP450 oxidation-reduction) ◦ redox cycling Reactive Intermediates of Toxic Mechanisms Paracetamol -> NAPQI ◦ GSH depletion required; covalent binding Bromobenzene -> Quinone/Epoxide ◦ GSH depletion required; covalent binding carbon tetrachloride -> Trichloromethyl radical ◦ GSH depletion may occur; covalent binding Furosemide -> Furosemide epoxide ◦ GSH depletion may occur; covalent binding Oxidative damage of toxic mechanisms Oxygen reduction ◦ complete tetravalent: transfer of 4 electrions simultaneously to O2 physiologic: membrane-embedded and soluble protein carriers; low risk of ROS ◦ partial ‣ univalent ‣ one electron transfer -> ROS formed Toxic reactive oxygen species (ROS) ROS: ◦ superoxide anion ‣ proteins metal groups: Fe (Cu, Mn), Fe-S clusters  ◦ hydrogen peroxide ‣ crosses membranes ‣ proteins, free AAs, nucleic acids S-H (thiols) Fe-S clusters ◦ hydroxyl radical ‣ protein thiols, lipids, nucleic acids, carbohydrates protective mechanisms ◦ Non-enzymatic ‣ Vitamin E (tocopherol) located in tissue membranes and extracellular fluid convers O2, OH, and lipid peroxyl radicals to less reactive compounds, chain breaking antioxidant ‣ Vitamin A (beta-carotene) located in tissue membranes scavenges O2-; interacts directly with peroxyl radicals ‣ Vitamin C (ascorbic acid) widely distributed in intra- and extracellular fluid interacts directly with O2-, OH. Neutralizes ROS released from WBCs; can regenerate Vit E from E radical form ‣ Glutathione mainly intracellular interacts direct with O2-, OH and lipid hyperperoxides. Serves as substrate for GSH recycling enzymes ‣ Uric acid widely distributed binds transition metals; interacts with O2-, OH. and peroxyl radicals; spares or prevents oxudation of ascorbic acid ◦ Enzymatic ‣ SOD mitochondria, cytosol converts O2 to H2O2 by dismutation reaction ‣ GSH recycling system GSH peroxidase ◦ cytosol and mitochondria  ◦ reduces H2O2 and other hydroperoxidases, low Km, functions during normal metabolism GSH reductase ◦ cytosol and mitochondria ◦ reduces low molecular weight disulfides using NAD(P)H ‣ Catalase (CAT) peroxisomes reduces H2O2, high Km, functions mainly in disease states LECTURE 3.2: DIAGNOSIS AND CARE OF THE INTOXICATED PATIENT Diagnostic assessment initial assessment ◦ who is your patient? # of animals? What's their status? ◦ suspected exposure to toxicants? ‣ known or suspected source? ‣ bring sample of suspect agent, vomitus, container, or label ‣ use care when collecting agent/materials ◦ DO NOT ASSUME INTOXICATION JUST BECAUSE OWNER THINKS IT! clinical signs: ◦ how acute is it? ‣ time of onset of signs, duration, early signs vs late signs environment ◦ pasture, roaming, food/water source, pesticide management, new plants in the home Medical history Physical Exam ◦ quick, but thorough ◦ update patient status: triage ◦ fever: not a rule out for toxicants ‣ uncouplers of OxPhos ◦ handle with care diagnostic confirmation ◦ lab tests ◦ recovery of toxicant ◦ response to therapy do not make diagnosis on clinical signs alone Patient Care treat the animal, not the toxicant! treatment goals: ◦ emergency support ◦ maintain vital organ function ◦ decontamination to prevent further absorption ◦ antidotes (if available) ◦ enhance elimination treatments: ◦ emergency care: ‣ establish patent airway ‣ assist ventilation ‣ maintain or correct fluid and electrolyte balances ‣ control arrhythmias, seizures ‣ maintain body temp ◦ once stabilized: ‣ estimate dose ‣ prevent further exposure decontamination ◦ GI ‣ emesis apomorphine, ropinirole (dogs) xylazine, dexmedetomide (cats) Hydrogen peroxide 3% (both; mostly dogs; rapidly diminishing use) rules of emetic use: ◦ patient must be able to protect airway! ‣ gag reflex should be intact ‣ do not use if patient is: unconscious, severely CNS depressed, has seizures, or, is in respiratory distress ◦ emetics should not be used... ‣ when caustic, corrosive, or volatile compounds are the suspected/known toxicants ‣ in species that do not vomit effectively horses, ruminants, rodents  ‣ Gastric lavage unconscious or anesthetized animal intubation to protect airway ‣ adsorbents activated charcoal (eg Toxiban) - known as universal antagonist ◦ neutral, large particles absorbed best ◦ does not work for heavy metals, hydrocarbons, alcohols, strong acids and bases! multiple dose activated charcoal (MDAC) ◦ AC may be repeated in the presence of enterohepatic recycling, high dose exposures, or toxicants with long T1/2 other adsorbents: clay-based products (bentonite adsorbs paraquat (herbicide); novacil (mycotoxins) ‣ cathartics to promote GI tract emptying osmotic: sodium sulfate, sorbitol (in Toxiban); should be given only once NOT RECOMMENDED: ◦ bulk cathartics: metamucil - too slow ◦ oily cathartics: mineral and castor oils - irritating ‣ evacuation of stomach with either emesis or gastric lavage now considered of questionable benefit by many in most cases; recommend going directly to adsorbents ‣ use of adsorbents and cathartics: cannot give drugs orally do not give cathartics to animals with diarrhea do not give cathartics more than once ◦ dermal or ocular exposures: bathe with mild soap and water, ocular irrigation use antidotal therapy when appropriate and available ◦ chemical antidotes: chelators (CaNa2EDTA for lead) ◦ functional: depressant vs stimulant (diazepam for seizures) ◦ competitive: antagonist (Vit K for anticoagulent rodenticides) ◦ non-competitive: acts at a different target site (atropine for OPs) ◦ intravenous lipid/fat emulsion (ILE/IFE): lipophilic chemical toxicosis (ex: cholecalciferol) enhance elimination ◦ promote renal excretion ‣ controlled fluid therapy: saline and dextrose ‣ diuretics: furosemide and mannitol ‣ ion trapping: urinary alkalinization: sodium bicarbonate, enhances excretion of weak acids urinary acidification: ammonuim chloride, enhances excretion of weak bases ◦ currently recommended only on a limited basis ‣ complications of forced fluid therapy: cerebral edema pulmonary edema metabolic acidosis or alkalosis electrolyte imbalances Parasitology LECTURE 4: PARASITICIDES Categories of Parasiticides and their uses. Endoparasiticide: kills internal parasites ectoparasiticide: kills external parasites ◦ activity against: ticks, mites, fleas, flies, lice ◦ may be appled to animal or environment ◦ ex: insecticides, acaricides, repellents, insect growth regulators, insect development inhibitors, synergists ◦ many are used in both pet and agricultural (crops) settings ◦ adulticides kill adults! IGRs kill babies! ◦ administration: ‣ animal: topical: spot-on/sprays, dip, shampoo, collar systemic: oral, parenteral, topical ‣ environment: foggers, strips, premise sprays, dusts/powders ‣ choice of which to go with may depend on: lifestyle, exposure to ectoparasites, what is the client willing to give (difficulty to administer, packaging, smell) and cost endectocide: kills both internal and external parasites ◦ new term coined specifically for avermectin/milbemycin (macrocyclic lactone) drugs antiprotozoals: activity against protozoal parasites ◦ ex: clazuril, diclazuril, toltrazuril, ponazuril, nitromezuril, amprolium ‣ benzimidazoles (albendazole, fenbendazole) can also be used, are technically antihelminthics ◦ some antibiotics can be used to treat protozoal infections ‣ metronidazole, sulfadimethoxine anthelminthics: activity against helminths (worms) ◦ categories: nematocides, flukicides, cestocides ◦ administration routes: ‣ oral tablet/chewable; liquid, drench; paste, bolus, SR bolus; mineral block, in water ‣ topical: pour-on, spot-on ‣ injection  ◦ administration frequency: daily, monthly, or PRN ◦ major classes: benzimidazoles, avermectin/milbemycins (macrolide endectocides), membrane depolarizers, isoquinolone (praziquantel and epsiprantel), cyclodepsipeptides (emodepside), arsenicals insecticides/acaricides: activity against arthropods (insects and/or arachnids) Major Classes of Anthelminthics (Lecture 5) Benzimidazoles (fenbendazole, febantel, albendazole, oxibendazole) ◦ MOA: binds to beta-tubulin, disrupts polymerization of tubulin into microtubules ◦ broad spectrum, safe, lots of resistance ◦ impacts the metabolism of the parasite, so will not instantly kill it Macrocyclic Lactones (ivermectin, moxidectin, doramectin, selamectin, milbemycin oxime) ◦ MOA: cause paralysis of pharyngeal and somatic muscles ‣ may cause blockage or rupture in host depending on level of infection ◦ effective against most nematodes and arthropods, safe ‣ BUT ivermectin causes toxicity in dogs ◦ white feet? do not treat! ‣ indicative of genetic gene mutation that makes blood-brain barrier more permeable Membrane depolarizers (levamisole, pyrantel) ◦ MOA: depolarizing neuromuscular blocking agents, spastic paralysis of worms ◦ broad spectrum of nematodes, most very safe ‣ Levamisole can cause neurological damage in small ruminants because of small window of safety Isoquinoline (praziquantel) ◦ MOA: causes paralysis of worms ◦ efficacy against cestodes, some nematodes, safe ◦ SA: tx of choice for cestodes and trematodes ◦ LA: will not treat trematodes Cyclooctadepsipeptide (emodepside) ◦ MOA: unknown ◦ new class of drug Strategies for Parasiticide Use Therapeutic = treatment ◦ see it? treat it. prophylactic = prevention ◦ dont want to see it? treat it to prevent.  strategic = control ◦ treat at strategically timed intervals to provide optimal control. Extra-label drug use Extra-label drug use is a privilege of veterinary profession ◦ only permitted by or on the order of a veterinarian and only when a bona fide veterinarian- client-patient relationship exists and an appropriate medical diagnosis has been made ◦ ensure proper dosage, proper drug labeling, tx records, and withdrawal times ◦ should use labeled product if available and equally efficacious ◦ does not matter if rx or OTC may use extra-label if... ◦ no labeled drug for indication or labeled product failed (drug resistance) ◦ published data supports that application ◦ COST IS NOT CONSIDERED AN ACCEPTABLE FACTOR :/ LECTURE 5: PARASITOLOGY CONTROL PROGRAMS Treatment versus control Anthelminthic treatments kill parasites ◦ may or may not control parasites control implies the prevention of clinically and/or economically important levels of parasites/ disease strategic control of parasites involves judicious use of anthelminthics, together with knowledge of the host, parasite, and environment and the properties of the drug being used ◦ environment more prevalent for production animals variation between small and large animals: ◦ zoonotic concern ‣ more of a SA issue because pets may sleep in the owner's bed or share blankets with the family, which is where some parasites may be spread from pet to parent ◦ production loss control programs are individualized ◦ host-specific (cat vs cow; pets in a home versus a shelter; age, breed, reproductive status) ◦ consider environment, client/owner goals ‣ housing, financial constraints ‣ travel; animal's job ‣ individual versus herd client-owned small animals  ◦ annual testing for major parasites ‣ vector-borne parasite testing annually heartworms (antigen + microfilariae in dogs) tick transmitted pathogens (ex: borrelia, anaplasma, ehrlichia) ‣ internal parasite testing AT LEAST annually fecal diagnostics (flotation, PCR, coproantigen) increase testing frequency in young animals, "risky" lifestyle ◦ ex: arya's behavior at daycare ‣ treatment "every pet, all year long" ◦ also true for indoor cats - parasites like ticks can be brought in from the environment on an owner test after tx as well large animal (equine and production) ◦ parasite eradication in every animal is impossible ‣ goal is to minimize disease ◦ herd-wide control puts heavy selection pressure on worm populations for drug resistance (major concern in LA) ‣ maintain refugia (proportion of the worm population not selected by drug tx) ‣ use drugs in combination ‣ integrate non-chemical modalities ◦ how to keep a balance with a good parasite control program ‣ manage the level of pasture contamination ‣ use anthelminthics properly ‣ monitor and treat the animals selectively ‣ quarantine and treat new introductions ‣ investigate treatment failure Drug resistance Drug resistance is increasing in heartworm, hookworm (Ancylostoma caninum), flea tapeworm (Dipylidium caninum) ◦ might have to start integrating large animal drugs to help in tx of these parasites environmental health concerns, drug residues risk-based recommendations ◦ spectrum of care  some strategies to delay resistance in large animals ◦ targeted selective treatment ‣ FEC (only method for horses) ‣ diarrhea score ‣ BCS ‣ FAMACHA scoring (best method for SR - Haemonchus) ◦ selective non-treatment (best method for cattle) ‣ treat only 80-90% of the herd leave heaviest or best-looking untreated Pharmacology LECTURE 4: NSAIDS AND CORTICOSTEROIDS NSAIDS Nonsteroidal anti-inflammatory drugs (NSAID) inflammation is part of nearly every tissue injury but can be a primary disease entity pain is a significant manifestation of inflammation and NSAIDs are a mainstay in the treatment of pain, most effective when given pre-emptively NSAIDs are also anti-pyretic, anti-endotoxemic, and anti-neoplastic, and one of the fastest growing classes of drugs in veterinary medicine produce anti-inflammatory and analgesic effects by inhibiting cyclooxygenase, COX (enzyme that metabolizes arachidonic acid to prostaglandins (PG)). PG and leukotrienes are key factors in production of peripheral sensitization used to treat mild to moderate inflammatory pain and visceral pain PGs produce a protective gastric barrier to intralumenal acidity, sustain normal gastric secretions, maintain normal gut motility PGs regulate renal blood flow and maintain normal tubular function PG inhibition can lead to altered GI motility, GI ulceration, renal or liver toxicity, impairment of jejunal epithelial restitution, and has the potential to delay clotting Leukotrienes mediate inflammation via inflammatory cell recruitment and activation NSAID Mechanisms of Action NSAIDs inhibit the biosynthesis of prostaglandins by preventing the substrate Arachidonic Acid from binding to the COX enzyme active site Arachidonic Acid: formed through the actions of the enzyme phospholipase on cellular membrane lipids in response to tissue damage or release of inflammatory mediators ◦ interacts with two enzymes: Lipoxygenase (LOX) & Cyclooxygenase (COX) regulation of COX enxzyme is the main mechanism for the therapeutic effects of NSAIDs ◦ first gen NSAIDs nonspecifically decreased activity of COX -> supression of all prostaglandin production (no bueno) COX isoforms: ◦ COX-1: constitutive ◦ COX-2: inducible; particularly with tissue damage and inflammation; can increase by 20 fold over baseline ◦ COX-3 (maybe): a splice variant of the COX-1 enzyme; role in inflammation, pain and fever has yet to be completely elucidated, clinical importance unclear  ‣ AKA COX-1b Anti-nociceptive effects occur both peripherally and centrally ◦ penetrate inflamed tissues -> local effect ◦ central action - at both the spinal and supraspinal levels; contributions from COX-1 and COX-2 ‣ central effect may account for overall well-being and improved appetite seen in patients receiving parenterally administered NSAIDs for relief of acute pain All NSAIDS are weak acids; all share similarities in pharmacokinetic properties ◦ over 90% bound to albumin, influences distribution and drug-drug interaction potential NSAID classifications most popular classification method of NSAIDs has been based on their COX 1 or 2 suppression profile ◦ profile often expressed as a ratio -> [NSAID] needed to inhibit 50% of COX-1 enzyme to [NSAID] needed to inhibit 50% of COX2 ◦ the greater the suppression ratio, the more specific the NSAID is for COX2 ‣ COX-1 selective: ratio 1000 none commercially available Adverse effects and contraindications GI tract ◦ COX1 and 2 are necessary for proper function, maintenance, and repair of GI mucosa ‣ COX1 related prostaglandins help regulate mucosal blood flow, secretion of buffers and  mucous, and turnover of epithelial cells ‣ COX2 plays a role in mucosal protection and repair ◦ the GI tract is by far the most common site of NSAID toxicity ‣ since COX2 is necessary for mucosal healing, the more selective a drug is for COX2, the more likely it is to cause GI ulceration/prevent healing of pre-existing lesions ◦ NSAIDs should be used with caution in dehydrated, debilitated, very young, or very old animals Renal ◦ prostaglandins regulate renal blood flow and glomerular filtration, esp during periods of systemic hypotension ◦ both COX1 and 2 are required to maintain adequate renal perfusion ◦ renal prostaglandins work with catecholamines to autoregualte renal blood flow & maintain renal perfusion when MAP is between 60 and 150 mmHg ◦ in stress, prostaglandins are upregulated in the kidneys to maintain adequate blood flow ◦ NSAID suppression of renal prostaglandin can disrupt autoregulation and result in renal ischemia -> acute renal papillary necrosis ◦ cats may be uniquely sensitive to NSAID toxicity -> possess approx 1/2 the number of nephrons at birth compared to most species Hepatic ◦ NSAIDs have been implicated in hepatocellular damage and hepatic failure in several species ◦ acetaminophen toxicity is one of the leading causes of acute liver failure in humans ◦ NSAIDs ar metabolized in the liver and excessive dosing can also lead to hepatotoxicity ‣ idiosyncratic hepatocellular toxicosis with NSAIDs can also occur ◦ excessive dosing of phenylbutazone can produce hepatotoxicity in horses ‣ used in chronic lameness in lowest dose possible ◦ liver enzyme activity should be periodically in patients prescribed NSAIDs on a long term bases ‣ 3-5 fold increase in activity above baseline could indicate hepatotoxicity esp if values return to normal after tx is discontinued NSAIDs and Cats! ◦ cats have trouble metabolizing NSAIDs reliant on glucuronidation ‣ lack gluconyl transferase ◦ goal: dose as infrequently as possible ◦ meloxicam, piroxicam, robenacoxib are metabolized via oxidation, so they can be used for cats  ◦ NSAIDs currently labeled for cats in the US: ‣ meloxicam: one time only injection ‣ Robenacoxib: three days Platelets ◦ thromboxane is necessary for proper platelet function and is produced via COX1 ◦ NSAIDs that strongly suppress COX1 could have significant effects on platelets and clot formation ◦ Aspirin can inhibit platelet aggregation - why it is used ◦ Ketoprofen has been shown to clinically affect hemostasis - should be avoided in cases where surgical bleeding may be difficult to control ◦ the coxib-type NSAIDs do not affect platelet function - older NSAIDs Contraindications ◦ should not be used in patients with: acute renal insufficiency, hepatic insufficiency, dehydration, hypotension, conditions associated with low effective circulating volume (CHF or ascites), coagulopathies, evidence of gastric ulceration, shock/hemorrhage, patients with spinal injury (potential for hemorrhage and neurological deterioration & excessive bleeding at the surgical site) ◦ Aspirin may exacerbate asthma (common in cats) ◦ NSAIDs should not be administered during pregnancy ‣ may lead to cessation of labor, premature closing of the DA, and disruption of fetal circulation ◦ avoid in breeding females ‣ COX2 induction is necessary for ovulation and subsequent implantation of the embryo ◦ avoid in lactating mothers ‣ COX2 is required for maturation of the embryological kidney ‣ HOWEVER one dose of carprofen post c-section before mom leaves hospital for pain - amount passed in milk insignificant -> no impact on kidneys Clinical Use NSAIDs are the most commonly used class of analgesics in vet met - used to reduce effects of primary disease such as acute and chronic pain, inflammation, fever, endotoxemia, and hypercoagulability as well as some neoplastic processes should be reserved for patients without renal, GI, or hepatic dysfunction ◦ exception for horses with colic should be avoided in immature animals in which organ maturation is not complete (less than 6 weeks)  most anesthetic agents can affect CO, blood pressure, and tissue perfusion - adverse affects of NSAIDs may be magnified when given prior to anesthesia During NSAID therapy, all patients should be monitored for hematochezia or melena, vomiting, increased water consumption, and nonspecific changes in demeanor Acetaminophen (NSAID) Acetaminophen has been used in dogs for analgesia and can be found in commercially available combinations with opioids in dogs with ventricular arrhythmias, acetaminophen reduced the number of etopic beats that developed during ischmia and reperfusion acetaminophen has demonstrated anti-oxidant effects on low-density lipoproteins -> cardioprotection acetaminophen is not approved for use in animals and under no circumstances should be given to a cat ◦ cats have deficiencies in hepatic glucuronidation -> cats cannot metabolize acetaminophen in the liver with glucuronic acid ◦ N-acetyl-p-benzoquinone forms and binds covalently to cellular molecules ultimately resulting in hepatic necrosis other adverse effects that occur in cats and dogs include methemoglobinemia and Heinz-body formation Aspirin mainly inhibits COX1, irreversibly inactivates COX enzymes via acetylation ◦ distinct property of Aspirin - duration of aspirin's effect is related to the turnover rate of the COX enzymes ◦ other NSAIDs work via competition with arachidonic acid for binding sites - duration of action is related to drug concentration and disposition aspirin is associated with chondrodestruction, irreversible platelet dysfunction, and GI bleeding and ulceration has been used specifically for its effects on platelets in horses ◦ aspirin is the most effective for antiplatelet therapy ◦ suppression of COX1 via asprin also inhibits thromboxane A2 -> necessary for platelet aggregation ◦ in the equine, platelet thromboxane A2 plays a minor role in aggregation ◦ antiplatelet effects of aspirin have been used for tx of laminitis, DIC, and equine verminous  arteritis in cattle aspirin has been used as an antipyretic and for inflammation associated with lower respiratory tract infections Carprofen - most commonly prescribed NSAID the only NSAID licensed in the US for dogs as both oral and injectible, making it practical for post- op use carprofen considered more potent than aspirin or phenylbutazone for tx of pain and inflammation, may also be safer than older NSAIDs antithromboxane activity of carprofen appears to be minimal carprofen-associated hepatic toxicosis is idiosyncratic, and, when ID'd early, usually resolves after discontinuation of carprofen and administration of supportive care in HEALTHY cats, A SINGLE DOSE of carprofen does not appear to cause GI or renal lesions ◦ toxicity more likely with prolonged administration in cats with concurrent systemic disease carprofen is effective for treating visceral pain in horses and the duration of action is about 12 hours ◦ banamine is still cheaper though Deracoxib One of the fist coxib tybe drugs approved for veterinary use in the US ◦ approved as an oral formulation for control of postoperative and osteoarthitis pain in dogs effective in decreasing lameness and pain associated with synovitis has been associated with GI perforation ◦ recommended that deracoxib should only be used at approved dosages and never co- administered with corticosteroids or other NSAIDs Meloxicam the only injectable NSAID approved for use in cats US available as an injectable forumulation approved for use in dogs and for a single injection in cats; also as an oral formulation approved for use in dogs only FDA warning: "repeated use of meloxicam in cats has been associated with acute renal failure and death. Do not administer additional injectable or oral meloxicam to cats" long-term oral administration in dogs has been investigated and meloxicam was second only to carprofen in the lowest frequency of adverse GI effects, followed by Etodolac, flunixin, and ketoprofen  meloxicam minimally affects hemostasis and is considered safe for perioperative use in dogs may also have an antineoplastic agent Ketoprofen COX1 selective NSAID that is effective in dogs for treatment of orthopedic pain only licensed for injection in horses in the US has increased incidence of adverse effects ◦ prolonged bleeding times, gastric lesions in horses, ketoprofen has potent anti-inflammatory effects and accumulates in inflammatory exudates and inflamed joints Diclofenac (Surpass) approved for use in horses in the US as a topically applied 1% liposomal cream ◦ developed for topical application to a localized area of inflammation -> reduces frequency and severity of toxicity ◦ there is evidence of systemic absorption of the drug and potential for systemic toxicity exists Dipyrone thought to have a mechanism of action similar to acetaminophen best used as antipyretic ◦ used in bacteremic neonatal foals has been shown to induce blood dyscrasias in human patients, and should not be used in any animal that may enter the human food chain Etodolac labeled for use in dogs in the US and is effective for tx of orthopedic conditions adverse effects of this drug appear to occur primarily in the GI tract not commonly rx anymore, but some older practitioners still use it (good to be aware of) Firocoxib only coxib NSAID approved for use in horses in the US ('Equinoxx') the most COX1 sparing NSAID available in the US for dogs approved for OA in dogs!!!! ◦ effective in decreasing urate-induced synovitis, pain, inflammation, and lameness ◦ librella may reduce the need for Firocoxib - still too new to know  clinical impression is that it is not that effective for visceral, eye, or laminitic pain comparable to phenylbutazone in horses for horses with SI colic, firocoxib may be preferred over flunixin meglumine -> inhibits recovery of ischemia-injured mucosa to a lesser degree Flunixin meglumine considered moderately effective of the control of both soft tissue and orthopedic pain ◦ also decreases intraocular inflammation after opthalmic sx systemic use of flunixin in dogs results in severe adverse effects including gastric ulceration, perforation, and peritonitis, increased plasma ALT activity and creatinine, and renal failure ◦ long story short, dont ever use banamine in a dog more commonly used NSAID in horses ◦ can be administered orally, IV, or IM (can cause myonecrosis) ◦ oral or IV route preferable ◦ myonecrosis also seen in cattle efficacious for tx of OA pain, comparable to phenylbutazone for tx of navicular syndrome mainly used for