Provisional seniority List of lab techcnicians , AHD

http://www.ahd.kerala.gov.in/index.php/orders-and-circulars?start=25

Leptospirosis

Leptospirosis

Leptospirosis is a worldwide zoonotic disease of domestic animals and wildlife. It is caused by a spirochete bacteria classified under the Leptospira , of which there are ~17 species. The taxonomy of the genus is confusing, with a number of species, including interrogans (sensu stricto), kirschneri , and santarosai. Serovars, as defined by agglutination cross-absorption, are the primary grouping below the species designation. This can add even greater confusion, as some serovars are grouped under more than one species. The term Leptospira interrogans (sensu lato) is used to describe the broad group of pathogenic leptospires associated with animal hosts.


Dogs are considered the reservoir host for serovar canicola . Based on studies that focused on urban populations of dogs, serovars canicola and icterohemorrhagiae were considered the most prevalent serovars infecting dogs. Studies that have included more rural and suburban populations of dogs document the predominance of serovars of grippotyphosa and pomona as causative agents of canine leptospirosis. Other serovars, particularly bratislava , have also been implicated. A variety of factors, such as increased exposure to raccoons and opossums in suburban and rural areas, communities encroaching on wildlife habitats, and poor farming practices, may explain the rise of previously low-prevalence serovars. Rodents continue to play an important role as reservoirs in urban settings.

Diagnosis:

Serology is the most useful and frequently used diagnostic test for dogs. Acute and convalescent titers may be necessary to confirm a diagnosis. Other diagnostic tests, such as darkfield microscopy, fluorescent antibody, PCR, culture, and histopathology are less frequently used antemortem. Demonstration of leptospires in tissues with silver staining or fluorescent antibody testing are preferred postmortem.

Laboratory Diagnosis of Anthrax in Animals

I. Introduction

1. Overview

Anthrax is a zoonotic disease that is transmissible to humans through handling or

consumption of contaminated animal products. The etiologic agent of anthrax, Bacillus

anthracis, is a spore forming gram-positive bacillus. Although anthrax can be found globally

in temperate zones, it is more often a risk in countries with less standardized and less

effective public health programs. Areas currently listed as high risk are South and Central

America, Southern and Eastern Europe, Asia, Africa, the Caribbean, and the Middle East. In

these regions, herbivorous wildlife mammals, such as deer, wildebeest, elephants, and

domesticated livestock, such as goats, sheep, cattle, horses, and swine, are at highest risk

for disease. These animals usually become infected while grazing on contaminated land,

eating contaminated feed or drinking from contaminated water holes. B. anthracis spores

can remain viable in soil for many years. 

2. Human infection

Humans can become infected with B. anthracis by handling products or consuming

under cooked meat from infected animals. Infection may also result from inhalation of B.

anthracis spores from contaminated animal products such as wool or the intentional release

of spores during a bioterrorist attack. Human-to-human transmission has not been

reported. Three forms of anthrax occur in humans: cutaneous, gastro-intestinal, and

inhalational.

2.a. Cutaneous anthrax

Cutaneous infections occur when the bacterium or spore enters a cut or abrasion on the

skin, such as when handling contaminated wool, hides, leather or hair products (especially

goat hair) from infected animals. Skin infection begins as a raised itchy bump or papule

that resembles an insect bite. Within 1-2 days, the bump develops into a fluid-filled vesicle,

which ruptures to form a painless ulcer (called an eschar), usually 1-3 cm in diameter, with

a characteristic black necrotic (dying) area in the center. Pronounced edema is often

associated with the lesions because of the release of edema toxin by B. anthracis. Lymph

glands in the adjacent area may also swell. Approximately 20% of untreated cases of

cutaneous anthrax result in death either because of the infection becomes systemic or

because of respiratory distress caused by edema in the cervical and upper thoracic regions.

Deaths are rare following appropriate antibiotic therapy, with lesions becoming sterile within

24 h and resolving within several weeks.

2.b. Gastrointestinal anthrax

The gastrointestinal form of anthrax may follow the consumption of contaminated meat

from infected animals and is characterized by an acute inflammation of the intestinal tract.

Initial signs of nausea, loss of appetite, vomiting, and fever are followed by abdominal pain,

vomiting of blood, and severe diarrhea. The mortality rate is difficult to determine for

gastrointestinal anthrax but is estimated to be 25%-60%.

2.c. Inhalation anthrax

This form of anthrax results from inhaling B. anthracis spores, and is most likely following

an intentional aerosol release of B. anthracis. After an incubation period of 1-6 days

(depending on the number of inhaled spores), disease onset is gradual and nonspecific.

Fever, malaise, and fatigue may be present initially, sometimes in association with a

nonproductive cough and mild chest discomfort. These initial symptoms are often followed

by a short period of improvement (ranging from several hours to days), followed by the

abrupt development of severe respiratory distress with dyspnea (labored breathing),

diaphoresis (perspiration), stridor (high-pitched whistling respiration), and cyanosis (bluish

skin color). Shock and death usually occur within 24-36 h after the onset of respiratory

distress, and in later stages, mortality approaches 100% despite aggressive treatment.

Physical findings are usually nonspecific. The chest X-ray is often pathognomonic (diseasespecific)

revealing a widened mediastinum with pleural effusions, but typically without

infiltrates.

B. anthracis can be detected by Gram stain of the blood and by blood culture with routine

media, but often not until late in the course of the illness. Only vegetative encapsulated

bacilli are present during infection. Spores are not found within the blood, partially because

CO2 levels in the body inhibit sporulation. Studies of inhalation anthrax in non-human

primates (i.e., rhesus monkeys) showed that bacilli and toxins appear in the blood within 2-

3 days of exposure. The appearance of toxins coincides with the appearance of bacilli in the

blood, and tests are available to rapidly detect the toxins.

3. Antibiotic therapy

Most B. anthracis strains are sensitive to a broad range of antibiotics. Penicillin,

ciprofloxacin, or doxycycline are usually recommended for the treatment of anthrax. To be

effective, treatment should be initiated early. If left untreated, the disease is highly fatal.

4. Anthrax vaccine

An anthrax vaccine for animal is licensed for use in the Kerala state produced by IAH &VB palode. 

II. Laboratory Procedures for the identification of Bacillus anthracis

1. General

The procedures described below function to rule out presumptively identified B. anthracis in

clinical specimens or isolates. These procedures should be performed in microbiology

laboratories that utilize Biological Safety Level 2 (BSL 2) practices. Laboratory coats and

gloves shall be worn when processing specimens and performing tests. Safety glasses or

eye shields are recommended. Any activities that bring hands in contact with mucosal

surfaces (for example, eating, drinking, smoking, or applying make-up) are prohibited.

Hands should be washed before leaving the laboratory. Anthrax vaccination is not required.

1.a. Handling of samples

For safety considerations, analysis of samples for biological threat agents is performed

within a certified Class II biological safety cabinet (BSC). Procedures requiring removal of

items from a BSC, such as slides for microscopy, should follow published microbiological

practices and precautions. When using a BSC, assure that the cabinet does not contain

unnecessary items that will interfere with proper airflow and function. As for any procedure

involving infectious materials, standard personal protective gear should be used, such as

latex gloves and laboratory coats, or disposable over garments. Additional respiratory

protection should also be considered with materials or analytical procedures determined to

be potentially hazardous outside the BSC. Once a biological agent has been identified,

modifications in handling of samples can then be considered.

1.b. Decontamination

Commercially available household bleach solutions contain 5.25% hypochlorite and, when

diluted 1:10, are effective in routine decontamination of surfaces and instruments after

working with B. anthracis. Contaminated items such as pipettes, needles, loops, and

microscope slides should be immersed in decontamination solution until autoclaving. Work

surfaces, such as a biological safety cabinet (BSC), should be wiped down before and after

use with decontamination solution. The method of decontamination of a spill depends upon

the nature of the spill. Spills involving fresh cultures or samples known to have low

concentrations of spores should be flooded with decontamination solution and soaked for 5

min before cleanup. Spills that involve samples with high concentrations of spores, involve

organic matter, or occur in areas of lower than room temperature (refrigerators, freezers)

should be exposed to decontamination solution for at least 1 h before cleanup. Personnel

involved in the cleanup of any spill should wear gloves, safety glasses, and a laboratory coat

or gown during the cleanup process. Respiratory protection should be considered for spills

in which a substantial aerosolization is suspected.

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2. Collection of clinical specimens

2.a. Materials

Sporocidal disinfectant

Sputum cup

Sterile cotton swabs

Blood culture collection kit

Stool collection cup

2.b. Cutaneous anthrax

2.b.1. Vesicular stage: The organism is best demonstrated in this stage. Soak two dry

sterile swabs in vesicular fluid from a previously unopened vesicle.

2.b.2. Eschar stage: Rotate two swabs beneath the edge of the eschar without removing the

eschar.

2.c. Gastrointestinal anthrax

2.c.1. If the patient is able to produce a stool specimen, stool cultures should be performed.

Obtain a stool culture.

2.c.2. In later stages of disease, blood cultures will yield the organism, especially if

specimens are obtained prior to antibiotic treatment.

2.d. Inhalation anthrax

2.d.1. If respiratory symptoms are present and sputum is being produced, obtain a

specimen for culture and smear.

2.d.2. In later stages of disease (2-8 days post exposure) blood cultures may yield the

organism, especially if specimens are drawn before antibiotic treatment.

3. Materials needed for processing of clinical specimens

5% Sheep blood agar plates [SBA] (BD Bioscience or Remel, Inc. or equivalent)

MacConkey agar plates (BD Bioscience or Remel, Inc. or equivalent)

Phenyl ethyl alcohol agar (PEA) plates (for stool specimens)(BD Bioscience or Remel, Inc. or

equivalent)

Trypticase soy broth (BD Bioscience or Remel, Inc. or equivalent)

Clean glass microscope slides

Sterile cotton swabs (commercially available specimen transport swabs for aerobic culture

are preferred)

Disposable bacteriologic inoculation loops

Clinical centrifuge with appropriate biocontainment tube holders

Sporicidal disinfectant (0.5% sodium hypochlorite or 0.5% calcium hypochlorite)

4. Isolation from clinical specimens

4.a. Sputum specimens

Inoculate 3 routine media for sputum specimens (i.e. SBA, MacConkey agar, broth

enrichment).

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4.b. Blood specimens

4.b.1. Routine blood culture methods are sufficient.

4.b.2. There may be enough organisms in the blood to see them on direct smears by Gram

stain. B. anthracis appears as short chains of 2-4 cells which are encapsulated as evidenced

by clear zones around the bacilli. The presence of large encapsulated gram-positive rods in

the blood is strongly presumptive for B. anthracis identification.

4.b.3. If blood culture bottle is positive, perform a Gram stain directly and observe for

encapsulated rods. These blood cultures should also be subcultured to SBA and MacConkey

agar plates.

4.c. Swab specimens

4.c.1. Use one swab to inoculate 3 standard media for surface wounds (e.g., SBA,

MacConkey agar, or broth enrichment).

4.c.2. Prepare a smear for Gram staining with the second swab.

4.d. Stool specimens

4.d.1. Routine stool culture methods are sufficient (e.g., SBA, MacConkey agar, or PEA

plates).

4.d.2. Do not use CVA or hectone agar plates.

4.e. CSF specimens

4.e.1. If a clinical centrifuge with appropriate biocontainment tube holders is available,

centrifuge the CSF specimen at 1500 X g for 15 minutes.

4.e.2. Collect the sediment and prepare a smear for Gram staining.

4.e.3. Inoculate the remainder of the sediment onto SBA and broth enrichment media

(tryptic soy broth or thioglycollate).

5. Incubation and examination of cultures

5.a. Cultures should be incubated at 35-37o C under ambient conditions.

5.b. Cultures should be examined within 18-24 h of incubation. Growth of B. anthracis may

be observed as early as 8 h after inoculation.

6. Differential tests for the presumptive identification of B. anthracis

6.a. Colony characteristics of B. anthracis

6.a.1. After incubation of SBA plates for 15-24 h at 35-37o C, well isolated colonies of B.

anthracis are 2-5 mm in diameter. The flat or slightly convex colonies are irregularly round,

with edges that are slightly undulate (irregular, wavy border), and have a ground-glass

appearance. There are often comma-shaped projections from the colony edge, producing

the "Medusa head" colony.

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6.a.2. Colonies on SBA usually have a tenacious consistency. When teased with a loop, the

growth will stand up like beaten egg white. In contrast to colonies of B. cereus and B.

thuringiensis, colonies of B. anthracis are not β-hemolytic. However, weak hemolysis may

be observed under areas of confluent growth in aging cultures and should not be confused

with β-hemolysis.

6.a.3. When examining primary growth media, it is important to compare the extent of

growth on SBA plates with that on MacConkey agar plates. B. anthracis grows well on SBA,

but does not grow on MacConkey agar. B. anthracis grows rapidly; heavily inoculated areas

may show growth within 6-8 h and individual colonies may be detected within 12-15 h. This

trait can be used to isolate B. anthracis from mixed cultures containing slower-growing

organisms.

Figure 1. B. anthracis and B. cereus colony morphology. Overnight cultures of B. cereus

(left side of plate) and B. anthracis (right side) on SBA.

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6.b. Gram stain morphology of B. anthracis

6.b.1. Procedure

Perform Gram stain by usual procedures.

6.b.2. Interpretation of results

B. anthracis is a large gram-positive rod (1-1.5 X 3-5 μm) that forms oval, central-tosubterminal

spores (1 X 1.5 μm) on SBA that do not cause significant swelling of the cell.

Spores are not present in clinical samples unless exposed to atmospheric levels of CO2; CO2

levels within the body inhibit sporulation. Vegetative cells seen on Gram stain of blood and

impression smears are in short chains of 2-4 cells that are encapsulated. However, cells

from growth on SBA under ambient conditions, are not encapsulated and occur as long

chains of bacilli. When grown on nutrient agar in the presence of 5% CO2 or on other basal

media supplemented with 0.8% sodium bicarbonate, virulent strains will yield heavily

encapsulated bacilli. The capsule can be visualized microscopically using India Ink.

Figure 2. Gram stain of B. anthracis from SBA

Magnification 1,000X.

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6.c. India ink staining of clinical samples (blood and CSF) for capsule

6.c.1. Purpose

India ink is useful for improving visualization of encapsulated B. anthracis in clinical samples

such as blood, blood culture bottles, or cerebrospinal fluid (CSF).

6.c.2. Materials

Microscope slides

Cover glasses

India ink (Bactidrop, Remel, Inc., Catalog # 21-518 or equivalent).

Microscope with 100X oil immersion objective

6.c.3. Controls

a) Control strains

(1) Positive control: Klebsiella pneumoniae on SBA or equivalent

(2) Negative control: E. coli ATCC 25922 or equivalent

b) Method controls: Perform the test with suspensions of fresh cultures of the control

strains. Control strains should be assayed on each day of testing.

c) Resolving an out-of-control result: check the purity and identity of the control strains

and repeat the test.

6.c.4. Procedure

a) For the controls, transfer a small amount of growth (1 mm diameter) from each control

SBA plate into 0.5 ml whole EDTA-treated blood or serum. Mix.

b) For the unknowns, take 100 μl of sample (blood, CSF)

c) Transfer 5-10 μl of unknown or control to a slide, place a cover glass on the drop, and

then add 5-10 μl of India ink to the edge of the cover glass.

d) After the ink diffuses across, view the cells using 100X oil immersion objective with oil on

top of the cover glass.

Figure 3. India ink staining of B. anthracis

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6.c.5. Interpretation of results

The capsule will appear as a well-defined clear zone around the cells for the positive control.

No zone should be present in the negative control.

6.d. Motility test: Wet mount or motility medium

6.d.1. Purpose

This test determines the motility of suspect isolates. B. anthracis is a nonmotile species.

This characteristic is unusual among Bacillus species and is therefore useful in the

preliminary identification of B. anthracis isolates. Two methods are given: the wet mount

and the tubed motility test.

6.d.2. Materials

a) For wet mount procedure

Precleaned microscope slides

Cover glasses

Sterile distilled water

Disposable bacteriologic inoculating loop

Light microscope with 40X objective and 10X eyepiece

Sterile glass tube

b) For tubed motility test

Tubed motility media (Remel, Inc., Catalog # 06-1408 or equivalent), 5 ml per tube

Sterile disposable 1 μl inoculating loop or needle

6.d.3. Controls

a) Control strains

(1) Positive control: Pseudomonas aeuroginosa ATCC 35032 or equivalent

(2) Negative control: Acinetobacter spp. ATCC 49139 or equivalent

b) Method controls: Perform the test with fresh cultures of the control strains using the

same method as with unknowns. Control strains should be assayed on each day of testing.

c) Resolving an out-of-control result: Check the purity and identity of the control strains and

repeat the test.

6.d.4. Procedures

Wet mount and motility

a) Wet mount procedure

(1) Deliver 2 drops (approximately 0.1 ml) of sterile distilled water into the sterile glass

tube.

(2) Using the inoculating loop, sample a suspicious colony from a 12-20 h culture and

suspend the growth in the water. (Alternatively, a loopful of medium from a fresh broth

culture can be used).

(3) Transfer 1 drop of the suspension to the microscope slide and overlay with the cover

glass.

(4) Examine the slide under the microscope using the 40X objective (total magnification =

400X).

(5) Discard slides in 0.5% hypochlorite solution.

b) Motility test medium procedure

(1) Using the sterile inoculating needle, remove some growth from an isolated, suspicious

colony of an 18-24 h culture.

(2) Inoculate the motility tube by carefully stabbing the needle 3-4 cm into the medium

and then drawing the needle directly back out so that a single line of inoculum can be

4/18/01 15

observed.

(3) Incubate the tube aerobically at 35-37o C for 18-24 h.

c) Interpretation of results

(1) For wet mount: Motile organisms can be observed moving randomly throughout the

suspension. Nonmotile organisms either fail to move or move with Brownian motion.

(2) For motility test medium: Nonmotile organisms, such as B. anthracis, will form a

single line of growth that does not deviate from the original inoculum stab. Motile

organisms will form a diffuse growth zone around the inoculum stab.

7. Presumptive identification key for B. anthracis

7.a. From clinical samples, such as blood, CSF, or lesion material: encapsulated grampositive

rods.

7.b. Gram-positive, broad rod, spore-positive: Bacillus species.

7.c. Spores are nonswelling and oval shaped; ground glass appearance of colonies: Bacillus

morphology group 1 (includes B. anthracis, B. cereus, B. thuringiensis, and B. cereus var.

mycoides)

7.d. Nonmotile: B. anthracis and B. cereus var. mycoides

7.e. Nonhemolytic: presumptive B. anthracis

Veterinary Laboratory Technician , Kerala

VETERINARY LABORATORY TECHNICIAN, KERALA   

I.          NATURE OF WORK:

This is a para-professional technical laboratory work in the field of veterinary science responsible for at least one laboratory unit.

Employees in this class, under a minimum of supervision, routinely conduct a broad range of tests requiring independent judgment in a variety of scientific disciplines. Employees in this class perform a broader range of tests than a laboratory technician.

Employees in this class will perform the following laboratory tests, as well as others: culture and identification of microorganisms, fecal examinations for parasites, complete blood count, complete urinalysis, blood chemistries, sperm counts and motility, pregnancy testing; will administer animal inoculations; may test for rabies including the selection of appropriate tissues and distinguishing the type of fluorescence that is a positive or negative indication of rabies.  Work in this position requires considerable accuracy and powers of observation.  Employees must be capable of producing acceptable reports that require a minimum of review.

Supervision is received from veterinarians or laboratory scientists.  Work effectiveness is determined through a review of reports and assignments, as well as the reliability of laboratory test results.  Employees in this class may be required to train and supervise lower-level employees.

II.         EXAMPLES OF WORK:  (Examples are illustrative only)

Under minimum supervision, performs a broad range of examinations or tests in various fields of veterinary diagnostic laboratory technology such as bacteriology, serology, clinical chemistry, hematology, and parasitology to determine whether or not abnormalities are present.  Some examples of diagnostic tests include, but are not limited to, testing for the following diseases: Bovine, Leukemia, Erlichosis, Blood Cell Parasite determinations, Hog Cholera, Pseudorabies, Transmissible Gastro-Enteritis, Equine Virus Abortion, Botulism (A, B, and C), Hemobartonellosis, Dirofilariosis, Babesiosis, Rabies, Swine Dysentery, Shipping Fever Complex, and Infectious Bovine Rhinotracheitis.  Other tests include, but are not limited to, equine cross match to determine blood incompatibility, biological and chemical pregnancy tests in mares, and spinal fluid protein determination to determine abnormalities;

Makes chemical or microbiological analyses of body fluids;

Examines cultures and smears and conducts tests to identify organisms;

Prepares specialized bacteriological culture media and reagents;

Adapts and modifies test equipment as needed;

Prepares technical reports;

Requisitions laboratory supplies;

Trains and supervises laboratory assistants and technicians;

Keeps detailed records;

Performs other related duties.

III.       REQUIRED KNOWLEDGE, SKILLS AND ABILITIES:

Knowledge of the species variations in non-human and avian enzymes and blood

chemistry;

Knowledge of a wide variety of veterinary laboratory diagnostic procedures and techniques including chemical laboratory practices and veterinary clinical laboratory practices.

Introductory knowledge of the anatomy and physiology of non-human and avian species; of endo and ecto-parasites and the methodology required to isolate ad identify microorganisms from the appropriate specimens; of non-human and avian hematology and the methodology required to conduct tests in the various species; of the methodology of performing non-human and avian blood chemistry and enzyme determinations; of non-human urinalysis and the methodology necessary to perform determinations in the various species;

Ability to properly operate and calibrate for the various non-human and avian species a wide range of scientific instruments including, but not limited to, the accustat blood chemistry analyzer, hematology analyzer, cryostat, fluorescent microscopes and standard microscopes, and hemocytometer; to perform a wide range of bacteriological and chemical tests with a minimum of supervision and to train other employees in these procedures; to supervise technical assistants; to prepare reports and maintain records accurately; to maintain effective working relationships with others.

IV.       MINIMUM EDUCATION AND EXPERIENCE REQUIREMENTS:

Education:

Successful completion of  VHSE in MLT

                                        Diploma in MLT from Directrate of Medical Education , Kerala

                                        BSc degree in MLT from any recognized medical colleges in India

                                        Diploma in MLT from any agriculturel universities in India

Experience:

not necessary for entry level

Note:

Completion of a formal course in medical technology consisting of training experience

V.        LICENSES, REGISTRATIONS AND CERTIFICATES:

NOT APPLICABLE.

 VI. Pay scale

         Lab technician grade 11 :   10480 -18300   ( Entry )

         Lab technician grade 1   :   13210 -22360   ( 1 st time bound )

         Lab supervisor              :   18740-33680   ( 2 nd Time bound)

VII. Laboratory pattern

       Veterinary Poly Clinics

District Veterinary Centers

Chief Disease Investigation Office, Palode

Regional disease diagnostic Labs

Rabies Diagnostic Lab, Kannur ,Kollam, Palode

Avian disease Diagnostic Lab

Brucella lab Wayanad 

Institute of Animal Health & veterinary biologicals , Palode

Invitation

All laboratory Technicians are invited to this blog for technology transfer