Mycobacterium bovis in Animals
Mycobacterium bovis in Animals Presented by Kathy Orloski, DVM, MS Epidemiologist Cattle Health Center U.S. Department of Agriculture Animal and Plant Health Inspection Service Veterinary Services January 27, 2015
Acknowledgements Bruce Thomsen, DVM, PhD Nancy Roberts, DVM, MS Suelee Robbe-Austerman, DVM, PhD
2
Official Disease Eradication Program • State and Federal Legal Authority • Federal – Code of Federal Regulations – Title 9, Part 77 – Uniform Methods and Rules (UMR)
• State Laws
3
Etiology • Mycobacterium bovis is a member of the M. tuberculosis complex • Other bacteria within the M. tuberculosis complex are: M. tuberculosis, M. africanum, M. canettii, M. microti, M. pinnipedii, M. caprae, and M. bovis BCG • All are acid-fast, intracellular, bacilli
General characteristics of bovine tuberculosis infection • Slow growing in vivo & in vitro • Chronic disease, long incubation • Can survive (not grow) in environment for variable time • Slowly progressive tubercles • Clinically inapparent • Impact on public health – Meat condemnation – Human infection from unpasteurized milk
Routes of Infection Key: infected animals only shed bacteria intermittently • Inhalation of aerosolized bacteria is the most common route – Requires close contact
Routes of Infection • Ingestion – Infected milk – Contaminated feedstuffs
• Uncommon routes – Congenital, cutaneous (oral cavity), and venereal
• Lesion location frequently does not correlated well with route of inoculation
Disease in domestic species Cattle: – Susceptible to 3 types of TB – M. avium infection rare • Immunocompromised – Reservoir host of M. bovis – M. tuberculosis • Reports of cattle being infected but does not cause progressive disease • Has not been detected in cattle in the US
Disease in domestic species Swine – Susceptible to 3 types of TB – High prevalence M. bovis areas: • M. bovis
– Low prevalence M. bovis areas: • M. avium, other mycobacteria
– Food waste: • M. tuberculosis
Disease in domestic species Goats – Susceptible to 3 types: M. bovis, M. avium, M. tuberculosis – Infection similar to cattle – Not as efficient reservoir for M. bovis? – M. tuberculosis may produce lesions and be generalized
Disease in domestic species Sheep –Rare, but can occur –M. bovis or M. avium –Extensive fibrosis and mineralization
Disease in domestic species Horses –Rarely tuberculous –All types isolated
Disease in domestic species Dogs –75% human and 25% bovine –Lung, renal and mesenteric infections –May maintain M. tuberculosis and M. bovis
Disease in domestic species Cats – In high prevalence countries, 90% M. bovis – Have been implicated as source in M. bovis reinfection of dairy herds – Orally exposed – In eradicated areas, TB is rare • M. tuberculosis mainly • M avium and others occasionally
Free-Ranging and Captive Wildlife Elk, deer, llamas and other camelids – M. bovis, can be reservoir hosts – Gross pathology and lesion location varies • M. bovis lesions often suppurative in elk, deer • Potential source of infection for cattle
– Susceptible to M. avium and other environmental mycobacterium – M. tuberculosis ??
Apparent susceptibility to Mycobacteria M. bovis
M. tuberculosis
M. avium
Cattle
X
X
X
Apes
X
X
Chickens
X
Pigs
X
X
X
Sheep
X
Goats
X
X
X
Cats/dogs
X
X
X
Equine
X
Deer
X
X?
X
X
Outcome of exposure to M. bovis • No infection • Infection – - If infected, most cattle will have subclinical disease, with limited, dormant, lesions
• Lesion and infection may then: – Regress – Progress into disseminated disease with clinical signs dependent upon organ system infected – Common clinical signs of severe tuberculosis would be emaciation, anorexia, depression
Route of Infection and the Initial Lesion • Respiratory tract exposure – Granulomas in the thoracic lymph nodes – Granulomas in the lung – Granulomas in lymph nodes of the head and neck
• Alimentary tract exposure – Granulomas in mesenteric lymph nodes
• Relationship of route of infection to lesion location is complex and not straight forward
Typical Gross Appearance of the Granulomatous Lesions - Cattle • Firm, spherical masses • Range in size from 1mm to >10cm • Maybe solitary or numerous granulomas throughout the body • On cut section: – - Outer fibrous white capsule – - Mineralized and “gritty” center that is white to yellow, typically firm and dry, but can be a thick fluid
Lesion Location • In US cattle, bovine TB lesions are most typically found in the lymph nodes • Therefore, slaughter surveillance focuses on inspection of lymph nodes of the head and chest
21
Lymph node TB lesion anatomic location in cattle with one lesion and found through slaughter surveillance FY 2009-20121 Lesion Location Head Cervical Bronchial Mediastinal Lung Portal Mesenteric Total 1 Source:
No of cases 12 3 15 10 1 0 0 41
Percent 29.3% 7.3% 36.6% 24.4% 2.4% 0.0% 0.0% 100.0%
VS 6-35 Laboratory Submission Form; FY = federal fiscal year
K Orloski, VS
K Orloski, VS
J. Nelson NVSL
J Nelson NVSL
J Nelson NVSL
J Nelson NVSL
Finding TB is not always this easy. The crucial first step is a thorough necropsy.
Jeff Nelson NVSL
J. Nelson NVSL
Post mortem Testing BE THOROUGH • Collect samples for: – - Bacterial culture – - Histopathology
Do a systematic necropsy, palpate and incise tissues, and have a check list •
• •
Head – Medial and lateral retropharyngeal LN – Manibular LN – Parotid LN Cervical LN Thorax – Right and left tracheobronchial LN – Cranial, middle, caudal mediastinal LN – Lung – Pleura
•
Abdomen Cranial and caudal mesenteric LN Hepatic (portal) LN Liver Spleen Kidney Ovaries and uterus
•
Other lymph nodes Deep popliteal LN Subiliac LN Medial iliac LN Superficial inguinal LN Mammary LN Mammary gland
Diagnostic Testing • Histopathology – HE and acid fast stained slides
• PCR – Formalin fixed – Fresh
• Culture • Genotyping – Whole genome sequencing (WGS) (spoligotype derived from WGS)
Key points on PCR • Test rapidly identifies DNA for – M. tuberculosis complex (usually M. bovis) in our samples. IS6110 – M. avium species. 16S rRNA – M. avium subspecies paratuberculosis. IS900
• Negative PCR results are not necessarily definitive – Prolonged formalin fixation (>7 days) reduces sensitivity
Basics of DNA Replication Whole Genome Sequencing • Over 1000 replications, we will have on average 5 with errors – In addition, there are other factors that cause DNA changes, such as lethal mutations
• Whole genome sequencing “determines the complete DNA sequence of an organism's genome at a single time” (Wikipedia) • Identify and evaluate the differences in the DNA between strains – Single nucleotide polymorphisms, SNPs
How does a SNP become established? • SNP = single nucleotide polymorphism • SNP occurs in a single organism that is among many as these organisms are dividing. – That bacteria and its progeny must then out compete its neighbors, or go through a bottleneck.
• An example of a bottleneck is a transmission event
• The vast majority of replication errors (SNPs, insertions, deletions, etc..) fail to dominate in the population
36
Principles of evolution and population genetics • DNA replication errors are rare, but they occur regularly • SNPs that dominate a population are less common…very rare and are driven by population dynamics • Consensus SNPs are a permanent change carried in future generations. This adds temporality. • Genotyping pipeline only calls consensus SNPs
Single Nucleotide Polymorphism (SNP) Examples
A = adenine, G = guanine (purine bases) T = thymine, C = cytosine (pyrimidine bases) 38
WGS work flow • Current technology allows for 250 bp reads. The genome is 4,400,000 bp. • We align those short reads to a previously assembled reference strain. (use a UK strain)
39
Phylogenetic trees Ave SNP change ~0.3/yr 0.1-0.7 range NE herd TX herd MX steer 6
CA herd
5
7 17 SNP
13 15 SNP
2 Common ancestor
EPIDEMIOLOGY OF BOVINE TUBERCULOSIS
Conducting an eradication program at the herd level • Agent factors • Transmission – How agent is Shed Transmitted Acquired • Host reaction: Pathology & Immune response • Other factors – Management – Politics
Transmission of bovine tuberculosis Host
Environment
Agent
Interspecies transmission of Mycobacterium bovis * * Zoo
*
*
* = reservoirs
Epidemiology of Mycobacterium bovis in a herd
Shedding Exposed infected
Clinical
Anergic to test
Visible lesions Exposed non-infected
Breaking the transmission cycle • Depopulate • If depopulation not possible: – cull liberally – test frequently – pasture management – eliminate crowding – prevent exposure via milk
After test and removal of positive cattle
Infected - Anergic to test
Exposed non-infected
Herd to herd transmission of Mycobacterium bovis •Carrier animals •Environmental contamination •Inanimate objects •Human carriers
Summary • Bovine Tuberculosis affects many species of animals and humans • The infection is chronic, subclinical, progresses slowly, and has a poorly defined incubation period • Severity depends upon exposure dose, nutrition, genetics, stress, and others • Transmission usually occurs via aerosols over short distances • When an infected herd is detected – Contain – Determine source(s) – Prevent further spread
Thank You ! Questions or Comments?
Acknowledgements Bruce Thomsen, DVM, PhD Nancy Roberts, DVM, MS Suelee Robbe-Austerman, DVM, PhD
2
Official Disease Eradication Program • State and Federal Legal Authority • Federal – Code of Federal Regulations – Title 9, Part 77 – Uniform Methods and Rules (UMR)
• State Laws
3
Etiology • Mycobacterium bovis is a member of the M. tuberculosis complex • Other bacteria within the M. tuberculosis complex are: M. tuberculosis, M. africanum, M. canettii, M. microti, M. pinnipedii, M. caprae, and M. bovis BCG • All are acid-fast, intracellular, bacilli
General characteristics of bovine tuberculosis infection • Slow growing in vivo & in vitro • Chronic disease, long incubation • Can survive (not grow) in environment for variable time • Slowly progressive tubercles • Clinically inapparent • Impact on public health – Meat condemnation – Human infection from unpasteurized milk
Routes of Infection Key: infected animals only shed bacteria intermittently • Inhalation of aerosolized bacteria is the most common route – Requires close contact
Routes of Infection • Ingestion – Infected milk – Contaminated feedstuffs
• Uncommon routes – Congenital, cutaneous (oral cavity), and venereal
• Lesion location frequently does not correlated well with route of inoculation
Disease in domestic species Cattle: – Susceptible to 3 types of TB – M. avium infection rare • Immunocompromised – Reservoir host of M. bovis – M. tuberculosis • Reports of cattle being infected but does not cause progressive disease • Has not been detected in cattle in the US
Disease in domestic species Swine – Susceptible to 3 types of TB – High prevalence M. bovis areas: • M. bovis
– Low prevalence M. bovis areas: • M. avium, other mycobacteria
– Food waste: • M. tuberculosis
Disease in domestic species Goats – Susceptible to 3 types: M. bovis, M. avium, M. tuberculosis – Infection similar to cattle – Not as efficient reservoir for M. bovis? – M. tuberculosis may produce lesions and be generalized
Disease in domestic species Sheep –Rare, but can occur –M. bovis or M. avium –Extensive fibrosis and mineralization
Disease in domestic species Horses –Rarely tuberculous –All types isolated
Disease in domestic species Dogs –75% human and 25% bovine –Lung, renal and mesenteric infections –May maintain M. tuberculosis and M. bovis
Disease in domestic species Cats – In high prevalence countries, 90% M. bovis – Have been implicated as source in M. bovis reinfection of dairy herds – Orally exposed – In eradicated areas, TB is rare • M. tuberculosis mainly • M avium and others occasionally
Free-Ranging and Captive Wildlife Elk, deer, llamas and other camelids – M. bovis, can be reservoir hosts – Gross pathology and lesion location varies • M. bovis lesions often suppurative in elk, deer • Potential source of infection for cattle
– Susceptible to M. avium and other environmental mycobacterium – M. tuberculosis ??
Apparent susceptibility to Mycobacteria M. bovis
M. tuberculosis
M. avium
Cattle
X
X
X
Apes
X
X
Chickens
X
Pigs
X
X
X
Sheep
X
Goats
X
X
X
Cats/dogs
X
X
X
Equine
X
Deer
X
X?
X
X
Outcome of exposure to M. bovis • No infection • Infection – - If infected, most cattle will have subclinical disease, with limited, dormant, lesions
• Lesion and infection may then: – Regress – Progress into disseminated disease with clinical signs dependent upon organ system infected – Common clinical signs of severe tuberculosis would be emaciation, anorexia, depression
Route of Infection and the Initial Lesion • Respiratory tract exposure – Granulomas in the thoracic lymph nodes – Granulomas in the lung – Granulomas in lymph nodes of the head and neck
• Alimentary tract exposure – Granulomas in mesenteric lymph nodes
• Relationship of route of infection to lesion location is complex and not straight forward
Typical Gross Appearance of the Granulomatous Lesions - Cattle • Firm, spherical masses • Range in size from 1mm to >10cm • Maybe solitary or numerous granulomas throughout the body • On cut section: – - Outer fibrous white capsule – - Mineralized and “gritty” center that is white to yellow, typically firm and dry, but can be a thick fluid
Lesion Location • In US cattle, bovine TB lesions are most typically found in the lymph nodes • Therefore, slaughter surveillance focuses on inspection of lymph nodes of the head and chest
21
Lymph node TB lesion anatomic location in cattle with one lesion and found through slaughter surveillance FY 2009-20121 Lesion Location Head Cervical Bronchial Mediastinal Lung Portal Mesenteric Total 1 Source:
No of cases 12 3 15 10 1 0 0 41
Percent 29.3% 7.3% 36.6% 24.4% 2.4% 0.0% 0.0% 100.0%
VS 6-35 Laboratory Submission Form; FY = federal fiscal year
K Orloski, VS
K Orloski, VS
J. Nelson NVSL
J Nelson NVSL
J Nelson NVSL
J Nelson NVSL
Finding TB is not always this easy. The crucial first step is a thorough necropsy.
Jeff Nelson NVSL
J. Nelson NVSL
Post mortem Testing BE THOROUGH • Collect samples for: – - Bacterial culture – - Histopathology
Do a systematic necropsy, palpate and incise tissues, and have a check list •
• •
Head – Medial and lateral retropharyngeal LN – Manibular LN – Parotid LN Cervical LN Thorax – Right and left tracheobronchial LN – Cranial, middle, caudal mediastinal LN – Lung – Pleura
•
Abdomen Cranial and caudal mesenteric LN Hepatic (portal) LN Liver Spleen Kidney Ovaries and uterus
•
Other lymph nodes Deep popliteal LN Subiliac LN Medial iliac LN Superficial inguinal LN Mammary LN Mammary gland
Diagnostic Testing • Histopathology – HE and acid fast stained slides
• PCR – Formalin fixed – Fresh
• Culture • Genotyping – Whole genome sequencing (WGS) (spoligotype derived from WGS)
Key points on PCR • Test rapidly identifies DNA for – M. tuberculosis complex (usually M. bovis) in our samples. IS6110 – M. avium species. 16S rRNA – M. avium subspecies paratuberculosis. IS900
• Negative PCR results are not necessarily definitive – Prolonged formalin fixation (>7 days) reduces sensitivity
Basics of DNA Replication Whole Genome Sequencing • Over 1000 replications, we will have on average 5 with errors – In addition, there are other factors that cause DNA changes, such as lethal mutations
• Whole genome sequencing “determines the complete DNA sequence of an organism's genome at a single time” (Wikipedia) • Identify and evaluate the differences in the DNA between strains – Single nucleotide polymorphisms, SNPs
How does a SNP become established? • SNP = single nucleotide polymorphism • SNP occurs in a single organism that is among many as these organisms are dividing. – That bacteria and its progeny must then out compete its neighbors, or go through a bottleneck.
• An example of a bottleneck is a transmission event
• The vast majority of replication errors (SNPs, insertions, deletions, etc..) fail to dominate in the population
36
Principles of evolution and population genetics • DNA replication errors are rare, but they occur regularly • SNPs that dominate a population are less common…very rare and are driven by population dynamics • Consensus SNPs are a permanent change carried in future generations. This adds temporality. • Genotyping pipeline only calls consensus SNPs
Single Nucleotide Polymorphism (SNP) Examples
A = adenine, G = guanine (purine bases) T = thymine, C = cytosine (pyrimidine bases) 38
WGS work flow • Current technology allows for 250 bp reads. The genome is 4,400,000 bp. • We align those short reads to a previously assembled reference strain. (use a UK strain)
39
Phylogenetic trees Ave SNP change ~0.3/yr 0.1-0.7 range NE herd TX herd MX steer 6
CA herd
5
7 17 SNP
13 15 SNP
2 Common ancestor
EPIDEMIOLOGY OF BOVINE TUBERCULOSIS
Conducting an eradication program at the herd level • Agent factors • Transmission – How agent is Shed Transmitted Acquired • Host reaction: Pathology & Immune response • Other factors – Management – Politics
Transmission of bovine tuberculosis Host
Environment
Agent
Interspecies transmission of Mycobacterium bovis * * Zoo
*
*
* = reservoirs
Epidemiology of Mycobacterium bovis in a herd
Shedding Exposed infected
Clinical
Anergic to test
Visible lesions Exposed non-infected
Breaking the transmission cycle • Depopulate • If depopulation not possible: – cull liberally – test frequently – pasture management – eliminate crowding – prevent exposure via milk
After test and removal of positive cattle
Infected - Anergic to test
Exposed non-infected
Herd to herd transmission of Mycobacterium bovis •Carrier animals •Environmental contamination •Inanimate objects •Human carriers
Summary • Bovine Tuberculosis affects many species of animals and humans • The infection is chronic, subclinical, progresses slowly, and has a poorly defined incubation period • Severity depends upon exposure dose, nutrition, genetics, stress, and others • Transmission usually occurs via aerosols over short distances • When an infected herd is detected – Contain – Determine source(s) – Prevent further spread
Thank You ! Questions or Comments?
