The VPM 201 Course Objective

Transcription

1 The Professor versus Student Lecture Dilemma What is important for students to know? Professor says: This is really important for you to know Students hear: Blah blah blah blah The VPM 201 Course Objective (as I see it): A basic understanding of bacterial and fungal disease relevant to clinical veterinary practice. 1

2 I think this is important for you to know. For example: An organism can be considered significant if it is isolated in large numbers and pure culture from a disease condition. Large numbers, pure culture.. The Microbial Universe Peering into the unknown microbial universe 2

3 The Microbial Universe Do we really understand what we are dealing with? (just a little bit) They are not simple bugs. Their world is completely different from ours. We must change our perspective! The Microbial Universe How old is microbial life? (3.8 billion years) How old is human life? (5 million years) Bacteria have been in existence for billions of years and remain one of the most successful life forms 3

4 Peering into the Enterobacteriaceae Universe Course: VPM 201 Fall, 2011 Lecturer: C. Anne Muckle Enterobacteriaceae What is the BUG? 4

5 The Family Enterobacteriaceae A LARGE family of gram-negative bacilli, ferment glucose with acid & gas production, oxidase-negative (except newcomer Plesiomonas), don t form spores, can be motile or no motile Several genera in the family and new species are being identified in each genera (how many? could be hundreds or thousands) Most that we deal with clinically belong to a few of the many species known Are environmental and normal gut flora, so it is hard to decide if they are important when you isolate them in culture from a sample. Diagnostic dilemma - significant? or contaminant? Remember: Significant if isolated in pure culture from a normally sterile site, ex. from organs (septicemia) or bladder by cystocentesis (UTI) The Family Enterobacteriaceae Identified by classical tube biochemical reactions or commercial identification systems (ex. - API 20E) Grow readily on simple media & selective/differential media for gram-negative enteric bacteria, such as MacConkey, Eosin Methylene Blue, XLD, and Salmonella-Shigella agar 5

6 Enterobacteriaceae on MacConkey Agar Lactose Fermenters (LF): Escherichia, Enterobacter, and Klebsiella, Citrobacter, & Serratia (coliforms) Grow as PINK colonies on MacConkey Enterobacteriaceae on MacConkey Agar Non Lactose Fermenters (NLF): Salmonella, Shigella (E. coli Shigella), Proteus, and Yersinia Grow as PALE, COLOURLESS colonies on MacConkey 6

7 Evolution of Virulence in Enterobacteriaceae (Remember they are not just simple bugs ) Common ancestral bacterium was a commensal adapted to the intestine of animals Escherichia coli (E. coli) and Salmonella are closely related but diverged a long time ago and acquired virulence factors by different genetic mechanisms (Salmonella acquired SPI-1 & SPI-2) The former genus Shigella species are a group of E. coli clones that acquired an invasion plasmid (pinv) and became host-adapted to primates Escherichia coli: E. coli is the major facultative anaerobic bacteria in the intestinal tract (large intestine/ colon) of most animal species (10 9 organisms/ gram feces), except pet birds E. coli is normal enteric flora E. coli can be an opportunistic or primary pathogen Certain serotypes are associated with disease. ex - E. coli O:157:H7 in humans, and O:147:K88:H2 in pigs 7

8 E. coli - Not just a simple bug E. coli has plasmids, bacteriophages, and mobile genetic elements encoding virulence characteristics (fimbriae = pili, toxins, OMPs, & antimicrobial drug resistance) Harmless strains become pathogenic by acquiring virulence factors through horizontal gene transfer (conjugation, transduction, transformation) (CBC News Aug. 11, 2010) British scientists have identified NDM-1, an enzyme that turns bacteria into superbugs resistant to antibiotics, in 180 patients in the U.K., India and Pakistan. NDM-1 has been seen largely in E. coli bacteria (also Klebsiella pneumoniae). The gene for the enzyme called NDM-1, or New Delhi metallobeta-lactamase 1 is found on DNA structures known as plasmids that can be easily copied and passed on to other types of bacteria. Identifying the Bug - E. coli Serotyping O antigen = cell wall LPS, determines serogroup H antigen = flagella, determines serotype K antigen = capsule *Serotyping has traditionally been used to identify pathogenic versus nonpathogenic E. coli strains For intestinal samples routine diagnostic labs can do limited agglutination tests using antiserum for specific serotypes or fimbriae (see in Lab Manual exercises for diagnosing pig and calf diarrhea ETEC serotypes) Serotyping of E. coli isolates is usually performed at Reference Laboratories Serotyping is based on antibody-antigen agglutination reaction 8

9 Identifying and Understanding the Bug E. coli Pathotyping E. coli Pathotypes: Pathogenic E. coli are also grouped into classes, or pathotypes, based on the mechanisms by which they cause disease and further classified as virotypes based on the virulence genes they possess. E. coli Virotypes: An E. coli virotype is a particular combination of virulence genes such as genes for fimbriae and toxins The E. coli Laboratory The EcL, University of Montreal, St. Hyacinthe, Quebec Diagnosis of pathogenic E. coli in animals by: Serotyping and Virotyping (multiplex PCR/ colony hybridization, PFGE) Virotyping is replacing serotyping Dr. John Fairbrother, OIE Expert for E. coli Q. What does this Bug (E. coli) do? (What diseases does E. coli cause?) E. coli causes enteric (intestinal) and extraintestinal disease in many domestic animal species (poultry, pigs, ruminants, dogs, cats, horses, rabbits) Examples are: enteritis, septicemia, mastitis, pyometra, urinary tract infections, omphalitis, yolk sac infection, cellulitis, air sacculitis 9

10 Q - How are virulent/pathogenic strains of E. coli spread in the farm environment? The spread of virulent strains of E. coli in the farm environment is presumed to occur through feed, aerosols, fomites, and carrier animals. Q. What are the most important E. coli pathotypes in animals? The important E. coli pathotypes in animals are: ExPEC = Extraintestinal pathogenic E. coli (includes UPEC = uropathogenic E. coli) ETEC = Enterotoxigenic E. coli EPEC = Enteropathogenic E. coli STEC = Shiga toxin-producing E. coli 10

11 Non-enteric localised disease by extraintestinal E. coli (The ExPEC, which includes UPEC) Invasive strains Strains are usually from the animal s own normal intestinal flora = endogenous source (Remember - Keep your friends close, but your enemies closer. ) Predisposed by crowding, poor ventilation, poor sanitation ex. - in poultry houses, pens Serum-resistant E. coli strains resist complement & phagocytosis by having K1 capsule, plasmidencoded outer membrane proteins (OMP) and can scavenge host iron sources Systemic Disease ExPEC - Non-enteric localised disease and extraintestinal disease Diagram Source: 11

12 EXPEC - Non-enteric localised & extraintestinal disease Septicemia of poultry (called colibacillosis or colisepticemia): ingested or inhaled E. coli in large # causes bacteremia or air-sacculitis & polyserositis omphalitis in chicks from infected yolk sac ( mushy chick disease ) Septicemia in neonates due to lack of immunity from colostrum, poor sanitation Umbilical stump is a common entry point ( navel ill ) EXPEC - Non-enteric localised disease & extraintestinal disease Mastitis in cows & sows (coliform mastitis): post calving; heavily manure-contaminated environment; peracute illness with endotoxic shock & death commercial vaccine contains rough E. coli strain J5 Pyometra in dogs and cats (luteal phase of oestrous cycle favours adherence by E. coli to endometrial cells) Metritis in horses and cattle 12

13 ExPEC: Pyometra in dogs and cats Pyometra = an accumulation of pus in the uterus. Canine pyometra is a distinct disease syndrome associated with cystic endometrial hyperplasia and usually infection by a variety of bacteria, especially Escherichia coli, occurring during diestrus. Photo courtesy of Dr. R. Lofstedt, AVC Clinical signs may include abdominal enlargement, purulent vaginal discharge if the cervix is open, polyuria, polydipsia, and a systemic response, which is more severe if the cervix is closed. An immune-mediated glomerulonephritis may also occur. ExPEC: Metritis in horses Photo courtesy of Dr. R. Lofstedt, AVC 13

14 Q. How does the J5 vaccine for coliform mastitis in dairy cows work? Stimulates antibodies that protect against the systemic effects of endotoxin (endotoxemia) EXPEC - Non-enteric localised disease & extraintestinal disease Urinary tract infections (UTI) caused by UPEC (UPEC are a type of ExPEC): E. coli (UPEC) is the most frequently isolated bacterial cause of UTI in dogs & cats Virulence factors of UPEC: Fimbriae (P) to colonize urinary tract epithelium, endotoxin, cytotoxic necrotizing factors (CNF1 & 2), & alpha haemolysin (RTX toxin) that damage cells, iron-binding proteins (aerobactin), capsule, which blocks complement & biofilms 14

15 Let s talk about diarrhea Diarrhea AKA Gastrointestinal disease, enteric disease, enteritis, scours, colibacillosis E. coli and Enteric Disease Gastroenteric strains cause enteritis Enteritis ( colibacillosis ) in poultry, pigs, ruminants, dogs, cats, horses, and rabbits Diarrhea Strains are usually from an exogenous source (external to animal) 15

16 E. coli pathotypes associated with enteric disease in animals ETEC, EPEC, and STEC are the most important enteric disease-causing E. coli pathotypes in animals: ETEC Enterotoxigenic E. coli Colonisation by fimbriae & fluid loss by enterotoxins, but with little tissue damage Diagram Source: 16

17 How do ETEC cause disease? Two key steps in ETEC Pathogenesis: 1. Attachment: ETEC strains produce fimbriae (pili) for attachment to receptors on microvilli of enterocytes in small intestine pigs: F4 (previously called K88) & F6 (previously 987P) calves: F5 (previously K99) Attachment is animal species-specific, requires specific cell membrane receptors 2. Enterotoxin production electrolyte & water hypersecretion: Enterotoxins (Sta, STb, and LT) have cell binding and enzyme activity resulting in secretion of Cl and H 2 O by enterocytes Turned on hypersecretion by enterocytes There is little damage to enterocytes, little inflammatory response Attachment Enterotoxin production ETEC and neonatal diarrhea Ingestion of ETEC serotypes results in severe watery diarrhea in neonates ( white scours ) death from dehydration, metabolic acidosis, endotoxic shock, and can have terminal bacteremia (yes, ETEC can sometimes capture the castle! see next lecture notes) Predisposing causes: No colostrum = no maternal antibodies; poor hygiene; stresses; low gastric acidity in newborns allows bacteria to reach intestine Why is a calf with ETEC similar to a child with cholera? LT is similar to cholera toxin 17

18 Q. What is the most common cause of E. coli diarrhea in neonatal cattle, pigs, dogs, and rabbits? *ETEC are the most common cause of E. coli diarrhea in farm animals, particularly, neonatal diarrhea in calves, lambs, and pigs. ETEC are a major cause of postweaning diarrhea in pigs. The stress of weaning is critical for the disease to occur. Q. How do you prevent ETEC neonatal diarrhea? By blocking fimbrial attachment and colonization of ETEC (ex. F4 = K88, F5 = K99) How? By stimulating protective anti-k99 pilus immunity in colostrum by vaccinating pregnant dams. By giving anti-pilus antibodies given orally to calves (passive immunity). A new treatment approach?- phage therapy (lytic E. coli T4 phage) to prevent and treat ETEC infections in neonatal farm animals. 18

19 EPEC - cause intimate attachment and damage to enterocyte microvilli = attaching and effacing lesions (AE) AE lesions: APEC induce the formation of pedestals on the host cell surface Diagram Source: How do EPEC cause disease? EPEC hijack host cells differently than ETEC. EPEC do not produce toxins causing fluid production. Instead, they tell the enterocyte to produce a pedestal for them to sit on. This intimate attachment causes effacement (shortening) of the microvilli and cell death. How do EPEC do this? They attach and inject a protein (Tir) that induces cells to produce a pedestal for them to sit on; Tir is the receptor for intimin. Remember Pedestal What is the Result? *Microvilli are damaged, diarrhea results from malabsorption of Na and Cl and PMN influx Diarrhea is mucoid, chronic, causing failure to thrive 19

20 STEC = Shiga toxin-producing E. coli STEC and edema disease in pigs an enterotoxemia due to Shiga-toxin Diagram Source: How do STEC cause edema disease in pigs? Shiga toxin damages vascular endothelium systemically These bacteria adhere by fimbriae (F18) to specific receptors on the intestinal epithelial cells and produce Shiga toxin (a cytotoxin that inhibits protein synthesis) which is transported across the epithelial cells to the circulation (toxin absorption mechanism is still unknown) Shiga toxin damages the endothelial cells of blood vessels, resulting in edema in target tissues, such as the kidneys. What is the result? Ataxia, paddling, confusion, death, see edema of CNS, eyelids, stomach, larynx (pigs have a peculiar squeal) Remember edema disease is an enterotoxemia; the toxin acts systemically, not locally on the enterocytes CNS signs ataxia, paddling Edema of stomach wall 20

21 STEC in animals and humans: Calves and lambs: diarrhoea and dysentery (blood and mucous) Humans: hemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS), caused by E. coli O157:H7, O145, & other STEC serotypes HUS = hemolytic anemia, thrombocytopenia, and renal failure Dogs: HUS in young dogs, with cutaneous & renal glomerular vasculopathy (CRGV) in racing greyhounds fed poor-quality ground beef, 1997 Diagram Source: STEC are an enormous public health issue; they cause an important foodborne zoonosis STEC are normal flora of cattle and ruminants Cattle are the major reservoir of STEC pathogenic for humans Fecal contamination of undercooked meat causes human illness ( hamburger disease ); cross-contamination of raw food; vegetables washed with contaminated water Outbreaks occur commonly due to global food distribution (ex Jack in the Box restaurant; 2006 fresh spinach outbreak) This hamburger could contain meat from some 500 cattle. 21

22 Zoonotic STEC from ruminants to humans Diagram Source: Holy Cow! In 1994 in the USA, O157 E. coli O157:H7 was labelled an adulterant; it is illegal to sell raw ground beef containing it. Starting in 2012 in the USA, it will be illegal to sell raw ground beef containing six other strains of STEC. These big-six non-o157 STEC strains are O26, O11, O103, O45 and O

23 Cows, STEC, Water, & People What happens when you mix cow manure and drinking water, and don t treat the water with chlorine? May, 2000, Walkerton, Ontario: Cattle manure containing E. coli bacteria was washed by heavy rainfall into a town well. The town s water supply was not properly chlorinated, maintained, or tested. Seven people died, 3000 were made ill. 23

24 E. coli O104:H4 June 2011 hemolytic uremic syndrome outbreak in Europe A new hybrid SUPER BUG Deadly E. coli bacteria continues to run rampant throughout Europe > 3, 700 cases, with >800 cases HUS and > 40 deaths, affecting primarily healthy adult females Not a typical Shiga-toxin-producing E. coli (STEC) strain It is a hybrid pathotype: Enteroaggregative (EAggEC) Shiga-toxin producing E. coli (STEC) with aerobactin, which is a virulence factor of EPEC. EAggEC/STEC/EPEC chimera all in one! Also is multi-drug resistant, with an ESBL gene. Raw Sprouts - the food vehicle of transmission? Is this the End? 24