Glanders: A ZooanthroponosisGlanders: A ZooanthroponosisGlanders = Farcy = Equinia = Malleus = Droes

Caused byBurkholderia mallei = Malleomyces mallei = Pseudomonas mallei = Burcholderia mallei

Dr. Bhoj R Singh, Principal Scientist (VM)I/C Epidemiology; Centre for Animal Disease Research and Diagnosis

Indian Veterinary Research Institute, Izatnagar-243122, Bareilly, UP, India. TeleFax  +91-581-2302188

Overview

• Organism• History• Epidemiology• Transmission• Disease in Humans• Disease in Animals• Prevention and Control• Actions to Take

The Organism

• Burkholderia mallei– Gram negative bacillus– Exists primarily in infected host– Prolonged survival in favorable environments– Inactivated by heat and sunlight

• Related to Burkholderia pseudomallei – Cause of meliodosis

History• 3rd Century BC

– Described by Aristotle

• 1664: Contagious nature recognized• 1830: Zoonotic nature suspected• 1891: Mallein test developed• 1900: Control programs implemented

History

• World War I– Suspected use as

biological agent to infect Russian horses and mules

– Large number of human cases in Russia during and after WWI

History

• World War II– Japanese infected horses,

civilians, and POWs– U.S. and Russia investigated

use as biological weapon

History

• 1934– Eliminated from animals in the U.S.

• 1945– Six lab acquired cases at Camp Detrick• 2000• Human case in laboratory worker at USAMRIID• 2007-8• Outbreak in many parts of India• 2011• Outbreak in horses in Allahabad

Transmission: Animals• Ingestion

– Contaminated food and water– Skin exudates, respiratory secretions

• Inhalation• Direct contact, fomites

Transmission FactorsTransmission Factors

•Animal density and proximity favour spread as well as stress-related host factors• Subclinical carries often prove to be more important in transmission of disease than clinical cases

Transmission in Humans

• Direct contact with infected animals– Abraded skin– Mucous membranes

• Fomites• Inhalation• Ingestion• Person-to-person (rare)

Epidemiology• Glanders continues to be reported from Brazil, China,

India, Iran, Iraq, Mongolia, Pakistan, Turkey, and the United

Arab Emirates and is thought to be endemic in various areas of the Middle East, Asia, Africa and South America.

• Endemic– Parts of Africa, the Middle East, Asia, and South America

• Possible occurrence– Balkan states, former Soviet republics

• Sporadic cases– Central America

• Once widespread, has been eradicated from many countries

Host Range• Affects solipeds

– Donkeys : Acute form– Horses: Chronic form

• Equidae, humans, occasionally Felidae, and other species are susceptible; infections are usually fatal• Donkeys are most susceptible, mules somewhat

less and horses demonstrate some resistance manifested in chronic forms of the disease, especially in endemic areas

• Susceptibility to glanders has been demonstrated in camels, bears, wolves and dogs• Carnivores may become infected by eating infected meat (14 lion died of Glanders in Iran Zoo,

Jan, 2011; cattle and pigs are resistant• Small ruminants may also be infected if kept in close contact to glanderous horses

Disease in HumansWho Is At Risk?

• Veterinarians• Grooms• Horsemen• Butchers• Lab workers

Disease in Humans

• Four forms of infection– Localized cutaneous– Pulmonary– Septicemic– Chronic form

• Generalized symptoms– Fever, malaise, muscle ache, chest pain

• Case-fatality rate: 95% (untreated)

Clinical Signs: Cutaneous• Incubation period: 1 to 5 days

– Erythema and ulceration of skin– Lymphadenopathy– Nodules

• Along lymph vessels• Highly infectious exudate

• Case fatality rate: 20% when treated

Clinical Signs: Pulmonary• Incubation period: 10 to 14

days– Inhalation of aerosolized

bacteria– Hematogenous spread to

lungs– Pneumonia, pulmonary

abscesses, pleural effusion●Case-fatality rate– 90 to 95% if untreated– 40% if treated

Clinical Signs: Septicemia

• Incubation period: 1 to 5 days – Any site of infection can lead to sepsis– Fever, chills, myalgia, chest pain, rash– Tachycardia, jaundice, photophobia, lacrimation

• Case-fatality rate−≥95% untreated; >50% treated

• Rapidly fatal

Clinical Signs: Chronic

• “Farcy”– Multiple abscesses

• Muscles, joints, spleen, liver

−Weight loss−Lymphadenopathy

• Case-fatality rate: 50% (treated)• Relapses common• Disease can last up to 25 years

Differential Diagnosis in Humans• Typhoid fever• Tuberculosis• Syphilis• Erysipelas• Lymphangitis• Pyemia• Yaws• Melioidosis• Anthrax

Diagnosis: Humans

• Culture and Gram stain– Sputum, urine, skin lesions, blood– Gram negative bacilli– Safety pin appearance

• Agglutination tests– May be positive after 7 to 10 days– High background titer in normal sera makes

interpretation difficult

Diagnosis: Humans

• Complement fixation – More specific – Positive if titer is equal to or greater than 1:20

• Chest radiograph for the pulmonary form of disease

• PCR

Treatment

• Limited information on treatment• Long term antibiotic treatment necessary (1 to

12 months)• Multiple drug therapy• Drain abscesses

Disease in EquidsDisease in EquidsThree Forms of Disease

NasalPulmonaryCutaneous

Course of DiseaseAcute

ChronicLatent

Disease in Animals• Forms of disease not clearly distinct

– May occur simultaneously– Incubation period: 2 to 6 weeks is typical

• Nasal form– Fever, cough, dyspnea, thick nasal discharge,

ulcers– Lymph node and vessel involvement– Death

• Begins clinically with a high fever, loss of appetite and laboured breathing with coughing.• A highly infectious, viscous, yellowish-green, mucopurulent discharge is present and this may crust around the nares.• A purulent ocular discharge has also been described• Nodules in the nasal mucosa may produce ulcers

Disease in Animals

Pulmonary Form− Nodules and abscesses in lungs− Dyspnea− Coughing− Fever− Progressive debilitation

• Usually requires several months to develop; first manifests itself through fever, dyspnoea, paroxysmal coughing or a persistent dry cough accompanied by laboured breathing

• Diarrhoea and polyuria may also occur; all leading to a progressive loss of condition

Cutaneous Form− Nodules and ulcers on skin− Lymphadenopathy− Swollen joints and edema of legs− Glanderous orchitis in males

• Develops insidiously over an extended period; begins with coughing and dyspnoea usually associated with periods of exacerbation leading to progressive debilitation

• Initial signs may include fever, dyspnoea, coughing and enlargement of the lymph nodes

Disease in Animals

• Acute Disease−More common in donkeys− Nasal and pulmonary signs

Coughing, dyspnea Nodules and ulcers on nasal

mucosa Enlarged submaxillary lymph

nodes− Neurological signs in

experimentally infected horses− Death

Disease in Animals

• Chronic form– Coughing, malaise, fever, weight loss– Nasal discharge and ulcers, skin

nodules– Lymph node and vessel involvement– Swelling of joints and leg edema

• Latent form– May be few symptoms– Nasal discharge– Glanderous orchitis common

Lesions-Necropsy

• Ulcers, nodules, Stellate scars in upper respiratory tract.• Pneumonia; Firm rounded miliary nodules• Swollen lymph nodes and vessels• Histologically B. mallei lesions comprise focal acute

necrotizing hepatitis (F), enlarged hepatocyte nuclei (arrows) and foci of hepatocyte vacuolation (arrowheads)

LesionsNasal form (nasal glanders)• Ulceration in nasal glanders may spread within upper

respiratory passages; perforation of the nasal septum has been observed

• Ulcers of the nasal area, trachea, pharynx and larynx may resolve in the form of star-shaped cicatrices (“stellate scars”)

• Regional lymph nodes (e.g. submaxillary) are enlargePulmonary form (pulmonary glanders)• Lung lesions in pulmonary glanders commence as small light-

coloured nodules surrounded by a haemorrhagic zone or as a consolidation of pulmonary tissue and a diffuse pneumonia

• Pulmonary nodules progress to caseous or calcified state; these eventually discharge their contents thus spreading disease to the upper respiratory tract

• Nodules can also be found in the liver, spleen and kidney

Lesions• Cutaneous form (cutaneous glanders)• Nodules begin to appear in subcutaneous tissue along the

course of lymphatics of the legs, costal areas and ventral abdomen and upon rupturing excrete an infectious purulent, yellow exudate.

• Ulcers result from rupturing of these nodules and these may heal or extend to surrounding tissue

• Infected lymphatics may result in swollen, thickened, cord-like lesions.

• coalescence of lymphatic lesions resemble a string of beads and are sometimes referred to as “farcy pipes.’’

• Nodular lesions can also be found in the liver and spleen• Orchitis has been associated with glanders.• Latent glanders may only demonstrate minor lesions of the

lung.

Differential diagnosis• Melioidosis• Strangles (Streptococcus equi)• Ulcerative lymphangitis (Corynebacterium

pseudotuberculosis)• Botryomycosis• Sporotrichosis (Sprortrix schenkii)• Gutteral pouch empyema• Dermatophilosis• Dermatomycoses• Pseudotuberculosis (Yersinia pseudotuberculosis)• Epizootic lymphangitis (Histoplasma farciminosum)• Horsepox• Tuberculosis (Mycobacterium tuberculosis)• Trauma and allergy

Sampling• Before collecting or sending any samples, the proper

authorities should be contacted• Samples should only be sent under secure conditions and

to authorized laboratories to prevent .• For Identification of the agent: Whole lesions or sections

of lesions, respiratory exudates smears from fresh lesions.• Samples should be kept cool and shipped on wet ice as

soon as possible• Sections of lesions in 10% buffered formalin and air dried

smears of exudate on glass slides should be submitted for microscopic examination

• For Serological tests: Serum sample should be collected aseptically the spread of the disease

Center for Food Security and Public Health, Iowa State University, 2011

Diagnosis: Animals

• Isolation of Burkholderia mallei – Blood, sputum, urine or skin lesions

• Mallein test– Intrapalpebral or conjunctival injection– Swelling 1 to 2 days later

• Serology• Compliment fixation and ELISA

– Most reliable in horses

Identification of the agentMorphology of Burkholderia mallei• Straight pleumorphic, aerobic bacilli, 0.3-0.5 µm x 0.7-5 µm• Methylene blue or Gram-stained organisms from fresh lesions for gram-negative non-

sporulating, non-encapsulated rods and presence of a capsule-like cover has been demonstrated by electron microscopy

Cultural characteristics• Bacteria grow aerobically and prefer media that contain glycerol.• Burkholderia mallei is non-motile, IMViC negative, grow at 42oC, nitratase positive,

hydrolyse arginine, not grow on McConkey agar, give variable results for urease & are positive for catalase, oxidase and gelatinase.

Laboratory animal inoculation• Male guinea-pig inoculated intraperitoneally and observation for severe localised

peritonitis and orchitis (the Strauss reaction); sensitivity of only 20%, hamsters are also susceptible

• Confirmation through bacteriological examination of infected testes

Culture Confirmation• PCR• PCR-restriction fragment length polymorphism• pulsed field gel electrophoresis• ribotyping using restriction enzymes in combination with rDNA probes• VNTR and MLST Confirmation by polymerase chain reaction and real-time PCR.

The Mallein test (a prescribed test for international trade)

• Mallein, purified protein derivative(PPD) is available commercially.• Intradermo-palpebral test - most sensitive, reliable and specific test.• Ophthalmic test - less reliable than the intradermo-palpebral test.• Cuti-reaction (subcutaneous test) - interferes with subsequent serological diagnosis

and so the other two mallein tests are preferred.

1. From a diagnostic standpoint the mallein reaction can only be considered positive when it produces a typical reaction.

2. A reaction is typical when the temperature rises at least 40 to a temperature of over 1040, and during the first day the fever line should show a plain or two summits, and on the second day, and sometimes even on the third day, a higher elevation is reached. The rise in temperature is accompanied with a local or general reaction.

3. The elevations below 1040, and those without a typical reaction, demand a re-examination.4. The slow rising to a stationary high temperature proves glanders, even when it otherwise deviates

from a typical reaction.5. The typical local swelling at the place of injection, if accompanied by clinical symptoms, is a positive

sign of glanders, even when the rise in temperature, and also the general organic reaction, is absent.

6. All contact animals submitted to the mallein test, whether they give a doubtful reaction or not, should always be tested the second time in from 10 to 30 days.

7. The production of mallein should be carried out exclusively in state or endorsed scientific institutions, or in those under municipal, state, or government supervision.

Serological testsComplement fixation test (a prescribed test for international trade)• not as sensitive as the mallein test.• reported to be 90–95% accurate; serum being positive within 1 week of

infection and• remaining positive in the case of exacerbation of the chronic process.• specificity of CF testing has been questioned.Enzyme-linked immunosorbent assays - plate and membrane (blot) ELISA• neither procedure has been shown to differentiate serologically between

B. mallei and B. pseudomallei.• validation pending.• recombinant 0375H and 0375TH proteins for ELISA has been identified at

DRDE Gwalior (V. Pal et al., 2012), exhibited 100% sensitivity and specificity for glanders diagnosis.

Other serological tests• avidin–biotin dot ELISA - not validated• Western blot – not validated• Rose Bengal plate agglutination test (RBT) - validated in Russia only (If titre

is <400 it is negative, 400 to <800 suspicious, ≥800 positive)

Conclusions about diagnostic tests

1. That mallein is the most reliable practical method that we have of diagnosing glanders.

2. That the agglutination test is a very valuable aid in diagnosing glanders, and, in some cases, can be employed where conditions prevent the application of other tests.

3. Straus' method is reliable in clinical cases where a positive reaction is obtained in the pig and Bact. mallei recovered in pure cultures from the lesions.

4. It is advisable to make cultures in suitable culture media, such as glycerine potato, from the suspected material when Straus' method is employed.

5. Complement fixation is very reliable, but is probably too tedious and complicated for routine procedure.

6. Cuti-reaction and ophthalmo reactions with mallein have shown very poor results.

7. ELISA still needs to be validated but too can not be a field test.

Treatment

• Antibiotics effective– Endemic areas

• Treatment controversial– Asymptomatic carriers may result.Ciprofloxacin and doxycycline are recommended

but none is 100% effective to cure an active case however, in horses doxycycline has been found more effective but relapse has been reported after 4-5 weeks of cure.

Both are effective to prevent symptomatic infections.

Prevention and Control Prevention and Control

IMMEDIATELY notify authoritiesCentral

Animal Husbandry Commissioner, DAHD State

Director of Animal HusbandryQuarantine

Recommended ActionsSanitary prophylaxis• Prevention and control of glanders epizootics depends

on a program of early detection and the humane elimination of test positive animals in conjunction with strict animal movement controls, effective premise quarantines and thorough cleaning and disinfection outbreak areas.

• Affected animal carcasses should be burned and buried.

• All disposable materials on positive premises (feed and bedding) should be burned or buried and conveyances and equipment should be carefully disinfected.

Medical prophylaxis• Antibiotic treatments have been used in endemic

zones.• should be noted that this may lead to subclinical

carrier animals which can infect humans and other animals

• Experimentally effective treatments include: doxycycline, ceftrazidime, gentamicin, streptomycin, and combinations of sulfazine or sulfamonomethoxine with trimethoprim

• Case fatality rates can reach 95% if no treatment is administered

Prevention: Humans

• Elimination of disease in animals• Biosafety level 3 required in labs• Protective clothing during exams and

necropsy -Gloves and mask, disposable shoes etc..

Prevention

• Horses– Early detection and

quarantine with disinfection– Test and slaughter

• Reportable to state veterinarian• Vaccine not available for humans or animals

Glanders as a Biological Weapon

• History• Very few organisms required to cause disease• Easily produced• Pulmonary form has high mortality• Limited experience with disease can slow

diagnosis and treatment

Additional Resources• World Organization for Animal Health (OIE)

– www.oie.int• U.S. Department of Agriculture (USDA)

– www.aphis.usda.gov• Center for Food Security and Public Health

– www.cfsph.iastate.edu• USAHA Foreign Animal Diseases

(“The Gray Book”)– www.usaha.org/pubs/fad.pdf