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______________________________ Cause and Effect Association DOES NOT EQUAL Causation Kochs Postulates o Agent grown in pure culture

e o Agent isolated from diseased organism o Cultured agent should induce disease in healthy o Agent must be re-inoculated and tested Hills Criteria: o Strength of association- stronger = more likely connected o Temporality- cause must precede event o Consistency- multiple data points o Plausibility- rational/theoretical basis o Coherence- agrees w/ known data o Specificity- effect has only one cause o Dose-response relationship- direct relationship between risk and effect o Experiment- data based on tests o Analogy- similar relationship can be applied in different fields

Main Criteria for causation: o Exposure must precede outcome o Dose response (most cases) o Reasonably big effect (OR or RR)- statistically significant/clinically important o Sensible connection between cause and effect o Results consistent with other work o Statistical association IS NOT causation o One study IS NOT causation o Usually a complicated COMBO OF FACTORS is needed for an outcome Component cause- needed in some cases Necessary cause- needed in all cases Sufficient cause- a group of causes makes disease inevitable Agent-host-environment triad represents the interaction and interdependence of factors and time as used in investigation of diseases and epidemics Agent virulence, transmission, dose, environmental survival, life cycle, carrier/subclinical dz, incubation pd, prevalence Host genetics, immune status, age/sex/breed/species, vax, body condition, nutrition, stress, repro status, concurrent dz Environment weather, season, geography, pop density, cleanliness, food/water sources, ventilation, housing, travel hx, other animals, air quality, pecking order Study designs and causation Randomized clinical trial? Cohort study? Case-control study? Cross-sections study?

Case report/case series?

______________________________ Bias and Confounding Bias SYSTEMATIC DEVIATION of results or inferences from the truth- towards/away from null, all study types, from PI/observers, occurs in design/conduct of a study Chance = imprecise results Bias = inaccurate results 3 types: 1. Selection bias selection differences between groups changes exposure/outcome; nonrep pops, etc. Occurs if controls/cases more/less likely to be included if they have been exposed retrospective cohort study Ex: self-selection, response, loss to follow-up 2. information bias misclassification bias; info gathering leads to systemic errors; distorts true strength of association; differential bias is worse than non-differential bias control by blinding, questionnaire, accuracy, use multiple controls ex: recall (exposed = greater sensitivity), reporting, interview 3. confounding third factor that relates to both exposure and outcome and which accounts for some/all of the observed relationship between the two; NOT result of exposure; to be a confounder: a. be associated with the exposure w/o having the consequence of the exposure b. be associated with the outcome, but be independent of the exposure (not intermediary) To control: study design randomization, matching, restriction; analysis stratification, multi variable analysis ______________________________ Measures of Association II All measures can be calculated for cohort and experimental trials; RD and AF also calculated for cohort and experimental trials Risk Ratios and Odds Ratios -- measure of the strength of association between exposure and outcome RR < 1 exposure is protective of disease RR = 1 exposure has no effect on disease RR > 1 exposure is positively associated with the disease RR is significant if CI does not include 1 Be able to calculate and interpret Absolute Risk difference AR = (RE+ - RE-) Difference in the incidence of disease in exposed animals and the incidence in unexposed animals; excess risk attributed to exposure; shows magnitude of RR on a given population AR < 0 exposure is protective AR = 0 exposure has no effect AR > 0 exposure is positively associated with disease AR is significant if the confidence interval does not include 0 + AR = Absolute risk increase; - AR = Absolute risk reduction Be able to calculate and interpret Attributable Fraction AF = (RE+ - RE-)/ RE+ OR AF = (RR-1)/RR The fraction of outcomes attributable to exposure; MUST have cause/effect relationship

Be able to calculate and interpret Population Absolute Risk difference PAR = (R - RE-) Like AR, shows simple difference in risk between two groups BUT is the increase in risk in the entire population that is due to the exposure; PAR = (Incidence in total pop) - (incidence in unexposed) Be able to calculate and interpret Population Attributable Fraction PAF = (R - RE-)/R = (PAR / R) Like AF, but disease in the entire population rather than the exposed group; shows proportion of disease in the whole population that is attributed to the exposure and would be avoided if the exposure was removed from the pop PAF = [(Incidence in total population)-(incidence in unexposed group)] / (Incidence in total population) Be able to calculate and interpret NNT and NNH NNT = 1/0.1 = 10 if the expose has a protective effect (negative association) NNH = 1/0.1 = 10 if the exposure has a harmful effect (positive association) ___________________ Poultry Lecture ___________________ Definitions, Concepts & Veterinarians Be able to define and explain major Public Health activities Environmental health - Lifestyle and behavior, Environment, Human biology, Organization of health programs and systems Zoonoses diagnosis, surveillance, prevention, control, eradication Occupational hazards and diseases associated with livestock and their products Biologics development and production Prevention and control of foodborne diseases Meat inspection Outbreak investigation Vector, water, wildlife and animal monitoring Biomedical research Emergency actions in natural and man-made disasters Social aspects of natural and man-made disasters Social aspect of service animals and human-animal bonding Be able to define and discuss food defense, security, and safety Food defense- The protection of food products from intentional adulteration by biological, chemical, or physical or radiological agents Food security- Access of all people all the time to sufficient, safe, nutritious food to maintain a healthy and active life (WHO) Food safety- Development and implementation of control and mitigation strategies to prevent or reduce microbial, chemical and physical contamination of food; Agencies: Be able to define and discuss foodborne pathogen surveillance in the U.S. Pre-harvest- reduce residues/pathogens; HACCP Hazard Analysis and Critical Control Points Post-harvest- food inspection, sanitization, storage/handling, HACCP Know major U.S. agencies involved in protecting U.S. food USDA, APHIS, FDA, FAS, CDC

Also: EPA, Homeland Security, local agencies, NCIMS (Dairy Products) Foodnet- CDC's Emerging Infections Program (EIP); collaboration w/ CDC, USDA, FSIS, FDA o Laboratory-based surveillance with active data collection, clinical laboratory/physician/population surveys, epidemiologic studies o Salmonella, Shigella, Campylobacter, E coli O157, Listeria monocytogenes, Yersinia, Vibrio, Cryptosporidium, Cyclospora

__________________ Antimicrobial Resistance Discuss susceptibility testing in populations Minimum Inhibitory Concentration- conc drug required to inhibit growth of a bacterial isolate under standardized conditions; S/I/R based on breakpoints (clinical prediction based on data) Susceptible does not mean that the drug will be effective in all cases Phenotypic resistance: MIC, Disk-diffusion (Kirby-Bauer), MIC, E-strip, Agar dilution Antibiograms (multiple resistance) MICs for several agents Genetic- gene sequences coding for resistance; expression is not assured, even if sequences present Discuss development/emergence, dissemination, accumulation and transmission of AMR within bacterial populations, within and among animal populations, and from animals to humans Cross-resistance- Resistance to one drug is associated with resistance to another drug with same MOA Co-resistance (or associated resistance)- coexistence of genes or mutations in the same strain each conferring resistance to a different drug or class of drugs For surveillance: describe changes in AMR in space and time, emergence of new resistant strains, describe/ compare AMR in different populations For epidemiological studies: differences in AMR between groups of animals in different conditions/ interventions; identify risk factors for AMR levels Spread thru direct or indirect transfer; greatest human risk is ingestions Discuss why AMR/MICs change over time in populations of bacteria and populations of animals and humans Bacteria change based on inherent resistance, acquired resistance (vert/horz), and mutations due to competition, environment, etc. Usually more virulent = less fit for long term survival Changes due to differences of exposure and interactions with hosts Discuss potential implication of AMR on veterinary medicine AMR surveillance program in Denmark- DANMAP FDA interest in vet drugs Not conclusive data; use drugs cautiously __________________ HACCP - Hazard Analysis Critical Control Points Be able to discuss the basic principles and concepts of a HACCP plan Mgmt system for prevention of problems to produce food products that are safe for consumption; methodical/systematic applications in process control as well Developed in 1960s by Pillsbury and the Army for NASA Most focus is on microbial contamination Prevention of risks Focus on POINTS in a production process not EVERY stage/carcass/kitchen

Identify hazards, critical points and target control points to reduce risk

HACCP Principles 1. Conduct an analysis of potential hazards (Hazard analysis) 2. Determine the critical control points (CCP) for the target hazard or hazards (ie food safety) 3. Establish critical (preventive) limits that trigger measures; done at each CCP 4. Establish monitoring requirements for the CCPs- continuous or sampling 5. Establish corrective measures when CCPs are not under control; ID problems, keep records 6. Monitor system- monitor procedures/documentation 7. Establish documentation concerning all procedures and records The 5 steps for HACCP implementation 1. Assemble a multidisciplinary, facility based HACCP team 2. Describe the final product and the method of distribution 3. Describe the intended use of the product and target consumer 4. Develop a flow diagram that describes the production and distribution process 5. Verify the flow diagram Be able to discuss how HACCP can be used to identify hazards and CCPs on farms and ranches Hazard = agent that causes the event to occur Risk = probability that an event will occur Advantages: o Focuses on identifying, PREVENTING hazards o Objective is scientifically-based o More efficient: record-keeping allows long-term assessment of performance o Manufacturers and distributors assume responsibility o Increases international competitiveness and safety/value of commodity o Reduces barriers to international trade HACCP advantages over solely using Good Management Practices o Establishment of critical limits o Systematic monitoring of CCPs o Predetermined corrective measures o Monitoring of system performance _______________ Herd health and biosecurity Herd Health- A planned animal health and production management program with regularly scheduled veterinary activities and good herd management designed to achieve and maintain optimum animal health and production o Objective: The maintenance of animal health and production at the most efficient level that provides economic returns to the animal owner while maintaining animal well-being and minimizing pollution Be able to determine major herd health components A good herd health program integrates disease biology and production management The different components of the triad present opportunities to reduce disease at multiple steps in the transmission cycle Dont get stuck in the agent factor! Be able to discuss principles for biosecurity measures on livestock operations

Biosecurity- action taken to prevent transmission of disease onto and within an animal operation o Sometimes prevention ONTO an operation = biosecurity and prevention WITHIN = biocontainment o Implemented between groups of animals and within groups of animals Four principles: o Resist- Increase the animals ability to resist disease- stress, immunity, vaxs o Isolate- Prevent contact between animals within a controlled environment o Traffic- Control traffic onto and within an operation- fomites, animals, feedstuff o Sanitation- Eliminate sources of the infectious agents Clean is not just the use of disinfectants Main purpose to prevent fecal material entering the oral cavity of animals USE HACCP!!!!! (dairy farm flowchart example)

________________ Foodborne Zoonoses The Food and Drug Administration - inspects imported foods, milk pasteurization plants, promotes better food preparation techniques in restaurants and food processing plants, and sale of turtles The US Department of Agriculture - monitors the health of food animals, inspects egg pasteurization plants, and is responsible for the quality of slaughtered and processed meat The US Environmental Protection Agency- regulates and monitors the safety of drinking water

Salmonella Gram (-), facultatively anaerobic rods In the intestinal tract of humans and animals; 1 transmitted via fecal-oral Can survive for long periods in the environment (esp warm/wet) Pathogenesis: Enterotoxin activates adenylate cyclase increased secretion of water and electrolytes diarrhea Most virulent isolates are Salmonella enterica; rarely S. bongori o Salmonella Typhimurium most common subtype Designated based on virulence factors lipopolysaccharide (O), flagellar protein (H), and capsular (Vi) antigens Worldwide, but seems to be most common where intensive animal husbandry is practiced Vertical transmission possible Most clinical cases are seen in young birds- anorexia, lethargy, diarrhea, polydipsia, CNS signs Transmission- raw/ undercooked meat and eggs, unpasteurized milk products, cross-contamination, contamination from an infected animal (or person) !!WASH HANDS!! o Diarrhea, fever, and abdominal cramps 12 to 72 hours after infection; illness usually lasts 4 to 7 days, and most persons recover without treatment o Illness is usually worse in the very young, the very old, and the immunocompromised (e.g. septicemia) Campylobacter Worldwide distribution- one of most common foodborne illnesses in the world Common species in animals: o C. jejuni and C. coli basically everything (fecal/oral; cause enteritis) o C. fetus subsp. fetus - cattle, sheep and goats (can cause infertility and abortion) o C. fetus subsp. venerealis cattle (can cause infertility and abortion) Transmission to humans:

o Ingesting undercooked poultry and other meats, raw milk, raw clams, contaminated foodstuffs or unchlorinated water; raw chicken is the most common o Contact with infected pets or livestock o Person-to-person transmission is unusual, but can occur; humans do not usually become carriers Disease in humans: o Infectious dose is relatively small o Incubation period for gastroenteritis is 1-10 days (most often 2 to 5 days) o The vast majority (99%) of human illness is caused by C. jejuni; usually mild Needs moist environments Prevention: o Common sense for humans o All in/all out flocks/herds, avoid new animals o Prevent fecal contamination of water with cows o Chlorination of drinking water

Listeria Needs moist environments Low incidence, high mortality Reservoirs soil, GI of asymptomatic animals- wild and feral mammals, birds, fish and crustaceans Infected animals can shed L. monocytogenes in the feces, milk and uterine discharges Poor quality silage with a high pH has been involved in many outbreaks because the bacteria can multiply in this environment Disease in animals: o In ruminants, listeriosis can cause encephalitis, abortion or septicemia, depression, loss of appetite, fever, lack of coordination, salivation, facial paralysis, and circling o Disease is more common in younger animals (1 to 3 years old) o Infection can also cause mastitis in cows o Some animals can be carriers without getting sick Disease in humans: o Most infections are caused by eating the bacteria in food, but the bacteria can also be spread by inhalation or direct contact (e.g., during calving, lambing, or necropsy) o Contaminated food sources include raw meat and fish, unpasteurized dairy products and uncooked vegetables or processed foods that have become contaminated after processing, particularly soft cheeses, deli cold cuts, sliced or grated cheese, and ice cream o Gastroenteritis in healthy people has an incubation period of approximately 1 to 2 days; susceptible is ~3 weeks o Possible vertical transmission - infection in utero or during delivery; abortions in last trimester o Cutaneous eruption is common o Febrile gastroenteritis usually self-limiting Diagnosis must be isolation from blood, CSF, the placenta or aborted fetus, or other normally sterile location Treatment with antibiotics; may take up to six weeks or more L. monocytogenes is relatively resistant to freezing, drying, heat and basic pH conditions ________________ Antemortem and Postmortem Inspection for Livestock Food Safety and Inspection Service Public Health Regulatory Agency

Regulates meat, poultry, and processed egg industries Ensures products are safe, wholesome, and accurately labeled

Meat Inspection Act of 1906 began system as we know it today and provided for mandatory PM inspection Wholesome Meat Act of 1967 state programs must be equal to Inspection is NOT Grading! (grading is optional) FSIS Areas of Responsibility Sanitation Antemortem inspection- dispositions by DVM only: Pass for Regular Slaughter, Pass for Slaughter as a U.S. Suspect, U.S. Condemned o Can do on-site or lab based testing Product Reinspection- Processed Products, Imported Products, Export Products FSIS Verification of Food Safety System HACCP SSOP Generic E. coli and other testing programs Risk Inspection based ______________ Foreign Animal Disease Outbreak Response FAD Important transmissible livestock or poultry disease believed to be absent from a country Exotic Animal Disease animal diseases which are unusual, including FADs Transboundary disease a new term used by many international documents to describe FADs

Animal costs- Intro of new diseases into a completely niave pop can have devastating effects on animal health High morbidity, high mortality, chronic losses in productivity, ecological damage, animal welfare/well being Direct/indirect economic costs Social and political costs Every outbreak is unique and usually relies on local vet reporting! Affected zone Surveillance zone Tracing There is no zero risk!!! _______________ Diagnostic Testing Continued Understand that different biological factors may affect Se and Sp Usually assume that Se and Sp are constant terms when talking about the performance of tests- dont change depending on population, but not always true Factors affecting Se: o Immune status o The stage of disease / severity of disease o Duration of infection

o Can be an informal relationship between prevalence and factors mentioned above if they differ in e.g. high and low prevalence populations o The relationship between predictive values and prevalence is formal Factors affecting Sp: o Most test do not have perfect Sp - some cross-reaction due to non-target components; may change over time/regions o Maternal antibodies & Vaccine antibodies T/F positives?; diagnose disease vs. immune status o Sp may improve over time in control programs- e.g. bovine tuberculosis eradication

Understand that Se and Sp can be estimated without the use of a reference test (Gold Standard) Maximum likelihood or Bayesian approaches -- results from two or more tests applied to the same individuals Understand why different testing scenarios may require different cutoffs for a positive/negative test, i.e. Se / Sp Selection of appropriate cutoff value: o Purpose of testing o Relative cost of false positive and negative results- economic, phyco-social, or political However, test with the largest overall accuracy = (Se+Sp)/2 For Receiver-operating characteristic curves o The cutoff that gives the best accuracy is found by selecting the point closest to the left uppermost (Northwestern) corner BUT may not always be the cutoff selected o Selection of the cutoff depends on the intended use of the test Understand that new and old tests can be compared without the use of a reference test Use Receiver-operating characteristic curves- Se on the y-axis against (1-Sp) on the x-axis o The area under the curve (AUC) is a measure of the overall accuracy (performance) of a test o If the AUC=1.0, the test has perfect Se and Sp o The diagonal line from (0,0) to (1,1) is the reference line equivalent to Se=Sp=0.5 o AUC can be used to compare the overall performance of two or more tests o The larger area under the curve (AUC) the more accurate the test is Use Kappa - a measure of agreement o Can be used for pair-wise comparison of two or more observers or tests o Assumptions The observers (tests) must be independent conditional on disease status They should be blinded to each others results o When kappa is calculated on populations consisting of both true positive and negative subject, the statistic is dependent on the prevalence o Can have values between -1 and ; value of 0 = only agreement by chance; 1 = perfect agreement Understand the relationship between true and apparent prevalence Prevalence measured by the applied test o TP = AP + Sp -1 / Se + Sp -1 Use sero-prevalence if Se and Sp are unknown Understand that tests often are used in combination, and how that affects the Se and Sp In practice, most diagnoses are based on the use of multiple tests o Often starting with a test with high Se and followed by a test with high Sp o Surveillance, Screening, Bovine tuberculosis, Johnes disease Assumptions: The tests are independent conditional on disease status

Rationale: Increase either Se or Sp Effect on the predictive values? o Depends on the application of the tests (parallel or series) and disease prevalence Conditional independence o Two tests are conditionally independent when the SENSITIVITY of the second test does not depend on whether the results of the first test are positive or negative among INFECTED individuals o Two tests are conditionally independent when the SPECIFICITY of the second test does not depend on whether the results of the first test are positive or negative among NON-INFECTED individuals Parallel interpretation of test results o The animal is considered positive if it is positive on either or both tests o The animal is considered negative if negative on both tests o Increases sensitivity of the combined tests o Decreases specificity of the combined tests o Additional tests can be used to verify positive test results on one or both tests (to clarify true disease status) Serial interpretation of test results o An animal must be positive on both tests to be considered positive o The animal is considered negative if it is negative on either or both tests o Increases the specificity of the combined tests o Decreases the sensitivity of the combined tests o Additional tests can be used to verify negative test results

High/low Se or High/low Sp? Under different circumstances, you may desire a test with very low probability of FP or FN results o Low FN Tradeoff with FPs Import situation (protection) o Low FP Tradeoff with FNs Export situation Being able to sell Understand which factors should be included in a good study for determining Se and Sp Experimental studies o Control over infections status Disease-free animals are infected experimentally (disease positives) Disease-free animals not infected (disease negatives) o Overestimate Se artificially high infection doses No variability in infective dose and immune response o Overestimate Sp Using Specific pathogen-free animals as controls No exposure to potential cross-reacting organisms Cross-sectional studies o Random or other systematic sampling o Verification of all samples (D+, D-, T+, T-) o Representative of the spectrum of disease o Stratify on factors that affect the test Se and Sp

Age/ other factors affecting spectrum of disease Observational studies o Slow or chronic diseases Convenient samples o Allows for selection based on spectrum of disease Hospital and diagnostic laboratory samples o Submissions change over time

Checklist for validation of diagnostic tests General o Test purpose and analytical unit described o Test protocol sufficiently described Reference test (Gold Standard) o The choice of reference test described More accurate than the new is necessary Selection of reference populations o The reference population is sufficiently described - time, location, age, breed, gender o The reference population should reflect the target population, i.e. appropriate spectrum disease (other conditions) o Selection criteria must be presented Sampling of the reference population o Sampling procedure described in detail o Exclusion criteria included (pos neg results) o Sample size estimation Performance of test and reference test o Testing protocols sufficiently described Def. of negative and positive results o Results of all tests evaluated blindly Presentation of results o Point estimates and confidence intervals o ROC curves Discussion of results o In relation to study design o In relation to reference test ________________ Herd-level testing and bovine tuberculosis Understand how the U.S. bovine Tb eradication program works Michigan example States that are bovine Tb positive: Michigan, Minnesota, California, New Mexico Focus on herd-level testing o Used to identify infected herds in control and eradication programs Movement restrictions in place that require all cattle to be tested before movement, i.e. individual animal testing TESTING: o Decisions on infection status made at the herd level o The caudal fold tuberculin (CFT) test used as a screening test

o Usually all animals older than 12 months of age were tested o Animal positive on the CFT test were tested with the comparative cervical tuberculin (CCT) test OR gamma-interferon test o If animals reacted on the CCT, they were submitted for necropsy, histological exam (and PCR) and culture for M. bovis o If one or more animals were positive for M. bovis on culture and/or PCR, the herd was diagnosed positive for bovine tuberculosis o Herd depopulation or Test & Remove o Export ban/Movement ban Understand how herd-level testing results are interpreted Correct classification of herd status is important for: o Specific-pathogen-free (SPF) programs/ other health certification programs o Disease eradication programs (cull whole herds as compared to individual animals) For some diseases, herd-level status easier to establish than individuals status o Test Se for individuals is low but the test has high or perfect Sp o IF the Se of a test is low, then by testing many animals the probability of detecting a disease is increased o Can be complicated by the number of FP results if Sp is not perfect Decision rules that are applied to interpretation of herd-level test results will: o Usually be applied to individual animal first Selecting a cutoff value for individual test => positive or negative animal o then aggregated across results for all sampled animals in the herd Selecting a cutoff for the herd-level interpretation I.e. Number (or proportion) of positive individual-animal results that is sufficient to designate the herd as positive If Sp is perfect, then cutoff of one test positive animal Herd-level sensitivity (HSe): probability that a positive herd yields a positive herd-test results Herd-level specificity HSp): probability that a negative herd yields a negative herd-test results Herd-level PPV (HPPV): Probability that a test positive herd is truly a disease positive herd Herd-level NPV (HNPV): Probability that a test negative herd is truly a disease negative herd o The number of animals tested sample size (some times entire herd) o The true within herd prevalence o The herd cutoff value used to declare a herd positive o Se o Sp Herd-level predictive values are dependent on: o Herd-level sensitivity o Herd-level specificity o The true prevalence of positive herds (herd-level prevalence) o WHY IS PPV RELATIVELY LOW AND NPV VERY HIGH? Understand how individual animal test performance affect herd-level test performance Common for all these situations is that interventions are determined by the status of the herd rather than by the status of the individual animals within the herd