Clinical Diagnosis of Sheep

Clinical Diagnosis of  Sheep Diseases

Introduction

Sheep are domesticated ruminant that are widely distributed throughout the world. More than 16 sheep breed are known. In Saudi Arabia many local breeds were found and total population of the sheep is approaching 8 millions. In Saudi sheep are considered as the main source of animal protein and mutton is preferable meat to most of   people.

Many problems are facing sheep rearing but diseases are top in the list facing sheep industry.

                          

Types of diseases in sheep

1-Infectious

2-Non infectious

Infectious diseases

ُExamples	Infectious Agent
Clostridial diseases, foot rot, some types of pneumonia (Pasteurllosis), some abortion diseases, (brucellosis), bacterial mastitis	Bacteria
Foot and Mouth, sore mouth, rabies, pox	Virus
coccidiosis, worms, mange	Parasite
prions are proteins normally found within the body’s nervous tissue (nerves, spinal column, brain) ·for unknown reasons these prions at times will change to a form that resists the normal mechanisms for turn over and break down ·prions continue to buildup on the nerve tissue eventually causing nervous disorders (Scrapie)	Prions

Noninfectious diseases

Type	Information	Examples
Nutritional	·deficiency or excess of particular nutrients in the diet ·can be acute (occur suddenly), but most often is a gradual depletion or buildup of nutrient	White muscle disease, photosensitivity, copper toxicity
Metabolic	·closely tied with nutritional disorders as they are caused by an imbalance in the nutrients supplied in diet with production demands ·Accumulation of metabolites ·animal’s metabolism can not meet the production demands and nutrients are extracted from the animal’s system at a greater rate than they can be replenished ·typically rapid onset of signs ·occurs at times of sudden increases production requirements (e.g. when a ewe begins lactating) or with sudden changes in diet (e.g.hay diet directly to lush pasture)	Pregnancy toxemia, hypocalcaemia, grass staggers
Digestive	·also linked with nutrition and changes in diet ·generally caused by a disruption in rumen function	Bloat, Lactic acidosis
Genetic	·defects which are inherited from parents ·intensive line breeding or inbreeding programs generally cause an increase in these disorders ·keeping good breeding and lambing records will greatly aid in culling the problem out of your flock	overshot jaw

What is diagnosis?

Identification of the disease or condition that affecting the animal at present time.

Diagnosis of sheep diseases is important due to the followings:

1) Accurate and early diagnosis is particularly important for the clinical treatment of diseases

2) Diagnosis is the cornerstone of any control program

3) Diagnosis is important as it gives insight into the extent of public health problems (zoonotic disease transmitted from sheep).

Types of diagnosis

i) Tentative

ii) Confirmatory

iii) Differential

Tentative

Could be achieved by:

1-Full clinical history

 Including the age, sex species and any related information

Age (different diseases will be more likely to occur in certain age)

Gender and reproductive state (e.g. ewes which are very heavily pregnant or that have just lambed)   showed by the animal at time

Number of animals affected and pattern of affection. (e.g. problems such as a lack of water or toxins in feed may acutely (suddenly) affect all animals in the pen, while infectious diseases may affect a small percentage of animals initially, and gradually move through the pen and/or barn.)

 2- Lesions

Some sheep diseases have characteristics (pathognomic lesions) eg: pox

3- Clinical Examination

   Could be visual or physical

Visual:

Check animal for

 Rrumination

Looked ‘depressed’: head down, droopy ears, dull eyes, hunched stance (back arched with forefeet and hind  feet placed close together under the animal)

Look hollow (abdomen/flank is excessively concave and hook bones are prominent)

Show signs of diarrhea (excessive tag or wetness on hindquarters are key signs)

Show signs of bloat (distension of abdomen, particularly high on the left side where the rumen is located)

Show signs of respiratory distress (laboured breathing, nostrils distended, coughing, copious amounts of nasal discharge)

Show signs of neurological disorders (uncoordinated, moving in circles, abnormal gait, posture or head carriage)

Show signs of lameness or stiffness

Physical examination

Body temperature (normal for an adult is 39-40 c⁰)

Notes: Some physiological factors will affect body Tem, (Age, lambs are normally higher than adults, day time, environmental temp.)

A high body temperature indicates that the animal is stressed or the body is staging an immune response to an infection.

A normal body temperature indicates that the problem is due to a noninfectious cause, such as a metabolic disorder

A low body temperature in very young lambs indicates starvation, and in adults may indicate internal bleeding.

Body temperature can be taken by gently inserting a thermometer into the sheep’s rectum. Using a bit of mineral oil or other nontoxic lubricant will make the process easier. Be sure to hold the thermometer while you are taking the temperature, to prevent it from become lost or broken. If you are using a glass thermometer it should remain in the animal for at least 60 to 90 seconds to ensure an accurate result. Digital thermometers signal when the temperature has stabilized

Auscultation to check

1-Heart rate (normal for a sheep is 70-90/ minute). Heart rate will increase under the same circumstances as the respiration rate.

2-To hear respiratory sounds and rumen sounds. Respiration rate (normal for a sheep is 25-35 (respirations/minute) (this also can be observed visually)

An abnormally high rate is an indication of distress caused by diseases that attack the respiratory tract (such as pneumonia), or could be a sign of severe pain due to injury, etc. It is best to observe respiration rate before disturbing the animal, as the stress of being caught will naturally increase the count. The easiest way to determine the respiration rate is to watch the animal’s abdomen and count each complete breath cycle.

Respiration rate will be high in healthy animals that have been running, are stressed, or exposed to high ambient temperatures.

Palpation to check

1- Different organs and tissues

2- Colour of mucus membranes (tissue around eyes and gums). Pale or bluish membranes indicate internal bleeding or poisoning.

Confirmatory Diagnosis

This mainly lab diagnosis:

Tentative diagnosis will let you to choose the appropriate lab. method for confirmatory diagnosis;

Laboratory techniques used for diagnosis

Mainly depends on samples send to the lab. So appropriate sample selection, collection and processing will be the key for accurate diagnosis and it will influence lab efficiency.

General sample selection and collection guidelines should include the followings:

1- Select the correct sample and collect the specimen by the proper technique and the proper supplies.

2- Collect adequate volumes. Insufficient material may yield false results.

3- Place the specimen in the container design to promote the efficiency of the sample and to eliminate leakage and potential safety hazards.

4-label each container with full information (date, site, other observation could be with accompanied sheet)

5- Appropriate device and method of collection.

6-Avoid chemical or microbial contamination when ever is possible to ensure the sample representatives of the condition.

(A) Techniques used for diagnosis of non infectious  diseases

Sample mainly body fluids (blood, urine, milk, CSF)

(B) Techniques used for diagnosis of infectious diseases

1-Microscopic examination

could by :

(i) Direct smear (wet)

(ii) Stained smear

*What is usefulness of direct smear? QUICK

*Can be done at the site of sample collection

 2-Isolation and identification of the causative agent

 Methods of isolation depend on the type of organism, bacteria , virus, fungus,  etc..

The isolation and identification is considered as one of the most confirmatory diagnostic method

       It take time and need sophistication, lab facilities                                               

                                                                 

3- Immunoassay

Analytical methods that incorporate Ags or Abs  to determine the presence of their corresponding (Ags or Abs) in a variety of media (eg body fluids, tissues)

It determinate Abs that produced in response to some nonself protein or Ags ( constituent of organism that have antigenic properties).

The term serology (serologic or serodiagnosis)  is detection of  Abs in the serum. Some times the term could be used when the media is not serum eg saliva or milk as long as the Abs or Ags are detected.

Hence the term serologic assay can be considered essentially synonymous with the term immunoassay.

Purposes and uses of serological tests

1-Particularly valuable in diagnosis infection when it is difficult to culture and dangerous to handle.

2-The most common uses is to identify infection and differentiate between acute from past infection (types of Ab with clinical data).

3-Assessment of diseases progress and understand kinetic of immune response.

Specific immunoassays for the detection and diagnosis of infectious diseases

1-Agglutination

2-Precipitation

3-CF

4-Radioimmunoassay

ELISA

Most popular and revolutionized immunoassay. Easy to perform and  adapted to test large number on samples and do need the use or radioactive substances and also determine the class of Ab.

4-Molecular diagnosis

Three terms,  genetics (genetic engineering),  molecular biology and biotechnology are used in field of diagnosis (what these terms mean)

Molecular biology now commonly used in the diagnosis of infectious diseases and non infectious diseases (eg. genetic disorders) (how)

Many molecular techniques are used in diagnosis of infectious diseases

Molecular techniques   

     1- Polymerase Chain Reaction (PCR)

Polymerase chain reaction is the test tube system for DNA replication that allows a “target” DNA sequence to be selectively amplified many folds in just a few hours

During PCR, high temperature is used to separate the DNA molecules into single strand, and synthetic sequence of single-stranded DNA (20-30 nucleotides) serve as primers.

Two different primer sequences are used to bracket the target region to be amplified. One primer is complementary to DNA strand at the beginning of the target region; a second primer is complementary to a sequence on the opposite DNA strand at the end of the target region.

To perform a PCR reaction, a small quantity of the target DNA is added to a test tube with a buffer solution containing DNA polymerase, oligonucleotide primers, the four deoxynucleotide building blocks of DNA, and the cofactor MgCl₂.

The PCR mixture is taken through replication cycles consisting of temperatures and time periods.

It is necessary to standardize the PCR steps (time and temperatures). This standardization depends mainly on the purpose and nature of the test.

The temperatures and duration of the PCR cycles are controlled by automated thermocycler.

The reaction tubes used in PCR are thin walled straight sides, which ease the heat transfer from the thermocycler to the reaction mixture.

 Strategies are required to standardize reaction reagents and to avoid cross contamination.

The PCR is a sensitive, specific and rapid technique, which has been successfully applied to diagnose disease and study genetic disorders.

The assay could detect several microorganisms in a variety of specimens, including sputum, serum, cerebral fluid, urine, feces, and various tissues

Types of PCR

  1-Random Amplified Polymorphic DNA (RAPD) PCR

2-RT-PCR

Reverse transcriptase was developed to amplify RNA target (RNA). So first DNA is produced form RNA by reverse transcription and the DNA is amplified. Using  RT enzyme.

3-Nested PC

It was developed to increase the sensitivity and the specificity of PCR

It uses two pairs of amplification primers and two round of PCR.

One primer is used the first round for 15-30 cycles and the product of the first cycle was then subjected to the second round of amplification with second set of primers that anneal to sequence internal to the sequence of first primer.

4-Multiplex PCR

Two or more primer sets designed for amplification of different targets are included in the same reaction mixture

By this technique, more than one target sequence in a clinical sample can be co amplified in single tube.

The primers used must be carefully selected due to difference in annealing temps. And should lack complementary.

 it is less sensitive

  Restriction Endonuclease Analysis (REA)

This method consists of extraction of DNA from homogenous population of organisms, digestion of the DNA with a restriction endonuclease enzyme, and electrophoresis of the digested DNA in an agarose gel.

The restriction endonuclease recognizes and cleaves double-stranded DNA at specific 4 or 6 base-pair sequences and a set of fragments is generated. The migration of these fragments in agarose gel is related to molecular weight; a pattern of bands is produced that can be seen by ultraviolet light if stained with ethidium bromide. This  pattern   constitutes a    characteristic   “fingerprint” for a  particular    DNA.

Types of it RFLP

  DNA Hybridization

Hybridization allows specific DNA sequences to be analyzed against the complex background of eukaryotic genome.

To focus on an individual genome, DNA from the target organism is isolated, fragmented with restriction enzymes, and separated by gel electrophoresis.

 The DNA fragments are denatured to render them single strand and exposed to a solution containing radioactive DNA “probe.”

The probe consisted of single-stranded nucleic acid (either DNA or RNA) with a sequence chosen to a base pair with the gene of interest.

Under appropriate conditions of temperature, salt, and pH, called “stringency,” the probe will bind to its corresponding sequence in the target DNA.

The presence of a radioactive signal indicates position of the probe   binding.

Anthrax- a 'Zoonotic Infection'

Anthrax is an acute disease caused by the bacteria Bacillus anthracis. Most forms of the disease are lethal, and it affects both humans and other animals. There are effective vaccines against anthrax, and some forms of the disease respond well to antibiotic treatment.

Like many other members of the genus Bacillus, Bacillus anthracis can form dormant spores that are able to survive in harsh conditions for extremely long periods of time—even decades or centuries. Such spores can be found on all continents, even Antarctica. When spores are inhaled, ingested, or come into contact with a skin lesion on a host they may reactivate and multiply rapidly.

Anthrax commonly infects wild and domesticated herbivorous mammals which ingest or inhale the spores while grazing. Ingestion is thought to be the most common route by which herbivores contract anthrax. Carnivores living in the same environment may become infected by consuming infected animals. Diseased animals can spread anthrax to humans, either by direct contact (e.g. inoculation of infected blood to broken skin) or consumption of a diseased animal's flesh.

Anthrax spores can be produced in vitro and used as a biological weapon. Anthrax does not spread directly from one infected animal or person to another; it is spread by spores. These spores can be transported by clothing or shoes. The dead body of an animal that died of anthrax can also be a source of anthrax spores.

Overview

Color-enhanced scanning electron micrograph shows splenic tissue from a monkey with inhalational anthrax; featured are rod-shaped bacilli (yellow) and an erythrocyte (red).

Anthrax is one of the oldest diseases of grazing animals such as sheep and cattle and is believed to be the Fifth Plague mentioned in the Book of Exodus in the Bible. Anthrax is also mentioned by Homer (in The Iliad), Virgil (Georgics), and Hippocrates. Infection of humans can result from contact with infected animal hides, fur, wool ("Woolsorter's disease"), leather or contaminated soil. Anthrax is now fairly rare in humans, although it still regularly occurs in ruminants, such as cattle, sheep, goats, camels, wild buffalo, and antelopes, in hind-gut fermenters such as zebras and rhinos, and in other wildlife such as elephants and lions in certain endemic areas of the world.

Bacillus anthracis bacteria spores are soil-borne and because of their long lifetime, they are still present globally and at animal burial sites of anthrax-killed animals for many decades; spores have been known to have reinfected animals over 70 years after burial sites of anthrax-infected animals were disturbed.

Until the twentieth century, anthrax infections killed hundreds and thousands of animals and people each year in Europe, Asia, Africa, Australia, and Southern Vietnam, specifically in the concentration camps during WWII, and North America. French scientist Louis Pasteur developed the first effective vaccine for anthrax in 1881. Thanks to over a century of animal vaccination programs, sterilization of raw animal waste materials and anthrax eradication programs in North America, Australia, New Zealand, Russia, Europe and parts of Africa and Asia, anthrax infection is now relatively rare in domestic animals with normally only a few dozen cases reported every year. Anthrax is even rarer in dogs and cats: there had only ever been one documented case in dogs in the USA by 2001, although the disease affects livestock. Anthrax typically does not cause disease in carnivores and scavengers, even when these animals consume anthrax-infected carcasses. Anthrax outbreaks do occur in some wild animal populations with some regularity. The disease is more common in developing countries without widespread veterinary or human public health programs.

There are 89 known strains of anthrax. The virulent Ames strain, which had been used in the 2001 anthrax attacks in the United States, has received the most news coverage of any anthrax outbreak. However, the Vollum strain, developed but never used as a biological weapon during the Second World War, is much more dangerous. The Vollum (also incorrectly referred to as Vellum) strain was isolated in 1935 from a cow in Oxfordshire, UK. This is the same strain that was used during the Gruinard bioweapons trials. A variation of Vollum known as "Vollum 1B" was used during the 1960s in the US and UK bioweapon programs. Vollum 1B is widely believed to have been isolated from William A. Boyles, a 46-year-old scientist at the U.S. Army Biological Warfare Laboratories at Camp (later Fort) Detrick (precursor to USAMRIID) who died in 1951 after being accidentally infected with the Vollum strain. The Sterne strain, named after the Trieste-born immunologist Max Sterne, is an attenuated strain used as a vaccine.

Cause

Bacteria

Gram-positive anthrax bacteria (purple rods) in cerebrospinal fluid sample. If present, a Gram-negative bacterial species would appear pink. (The other cells are white blood cells).

Bacillus anthracis is a rod-shaped, Gram-positive, aerobic bacterium that is about 1 by 9 micrometers in length. It was shown to cause disease by Robert Koch in 1876. The bacterium normally rests in endospore form in the soil, and can survive for decades in this state. Herbivores are often infected whilst grazing or browsing, especially when eating rough, irritant or spiky vegetation: the vegetation has been hypothesized to cause wounds within the gastrointestinal tract permitting entry of the bacterial endo-spores into the tissues, though this has not been proven. Once ingested or placed in an open cut, the bacterium begins multiplying inside the animal or human and typically kills the host within a few days or weeks. The endo-spores germinate at the site of entry into the tissues and then spread via the circulation to the lymphatics, where the bacteria multiply.

It is the production of two powerful exo-toxins and lethal toxin by the bacteria that causes death. Veterinarians can often tell a possible anthrax-induced death by its sudden occurrence, and by the dark, non-clotting blood that oozes from the body orifices. Most anthrax bacteria inside the body after death are out-competed and destroyed by anaerobic bacteria within minutes to hours post-mortem. However, anthrax vegetative bacteria that escape the body via oozing blood or through the opening of the carcass may form hardy spores. One spore forms per one vegetative bacterium. The triggers for spore formation are not yet known, though oxygen tension and lack of nutrients may play roles. Once formed, these spores are very hard to eradicate.

The infection of herbivores (and occasionally humans) via the inhalational route normally proceeds as follows: once the spores are inhaled, they are transported through the air passages into the tiny air particles sacs (alveoli) in the lungs. The spores are then picked up by scavenger cells (macrophages) in the lungs and are transported through small vessels (lymphatics) to the lymph nodes in the central chest cavity (mediastinum). Damage caused by the anthrax spores and bacilli to the central chest cavity can cause chest pain and difficulty breathing. Once in the lymph nodes, the spores germinate into active bacilli which multiply and eventually burst the macrophages, releasing many more bacilli into the bloodstream to be transferred to the entire body. Once in the blood stream these bacilli release three substances: lethal factor, edema factor and protective antigen. Protective antigen combines with these other two factors to form lethal toxin and edema toxin, respectively. These toxins are the primary agents of tissue destruction, bleeding, and death of the host. If antibiotics are administered too late, even if the antibiotics eradicate the bacteria, some hosts will still die. This is because the toxins produced by the bacilli remain in their system at lethal dose levels.

In order to enter the cells, the edema and lethal factors use another protein produced by B. anthracis called protective antigen. Edema factor inactivates neutrophils (a type of phagocytic cell) so that they cannot phagocytose bacteria. Historically, it was believed that lethal factor caused macrophages to make TNF-alpha and interleukin 1, beta (IL1B), both normal components of the immune system used to induce an inflammatory reaction, ultimately leading to septic shock and death. However, recent evidence indicates that anthrax also targets endothelial cells (cells that lines serous cavities, lymph vessels, and blood vessels), causing vascular leakage of fluid and cells, and ultimately hypovolemic shock (low blood volume), and septic shock.

The lethality of the anthrax disease owes itself to the bacterium's two principle virulence factors: (i) the poly-D-glutamic acid capsule, which protects the bacterium from phagocytosis by host neutrophils, and (ii) the tripartite protein toxin, called anthrax toxin. Anthrax toxin is a mixture of three protein components: (i) protective antigen (PA), (ii) edema factor (EF), and (iii) lethal factor (LF). PA plus LF produces lethal toxin, and PA plus EF produces edema toxin. These toxins cause death and tissue swelling (edema), respectively.

Exposure

Occupational exposure to infected animals or their products (such as skin, wool, and meat) is the usual pathway of exposure for humans. Workers who are exposed to dead animals and animal products are at the highest risk, especially in countries where anthrax is more common. Anthrax in livestock grazing on open range where they mix with wild animals still occasionally occurs in the United States and elsewhere. Many workers who deal with wool and animal hides are routinely exposed to low levels of anthrax spores but most exposures are not sufficient to develop anthrax infections. Presumably, the body's natural defenses can destroy low levels of exposure. These people usually contract cutaneous anthrax if they catch anything. Historically, the most dangerous form of inhalational anthrax was called Woolsorters' disease because it was an occupational hazard for people who sorted wool. Today this form of infection is extremely rare, as almost no infected animals remain. The last fatal case of natural inhalational anthrax in the United States occurred in California in 1976, when a home weaver died after working with infected wool imported from Pakistan. The autopsy was done at UCLA hospital. To minimize the chance of spreading the disease, the deceased was transported to UCLA in a sealed plastic body bag within a sealed metal container.

In November 2008, a drum maker in the United Kingdom who worked with untreated animal skins died from anthrax. In December 2009 The New Hampshire Department of Health and Human Services confirmed a case of gastrointestinal anthrax in an adult female. The CDC is currently investigating the source and the possibility that it was contracted from an African drum recently used by the woman. Gastrointestinal anthrax is exceedingly rare in the United States, with only one case on record, reported in 1942, according to the Centers for Disease Control and Prevention. In December 2009 an outbreak of anthrax occurred amongst heroin addicts in Glasgow, Scotland, resulting in ten deaths. The source of the anthrax is believed to be dilution of the heroin with bone meal in Afghanistan.

Also during December 2009, The New Hampshire Department of Health and Human Services confirmed a case of gastrointestinal anthrax in an adult female. The CDC investigated the source and the possibility that it was contracted from an African drum recently used by the woman taking part in a drumming circle. Gastrointestinal anthrax is exceedingly rare in the United States, with only one case on record, reported in 1942, according to the Centers for Disease Control and Prevention. The woman apparently inhaled anthrax [in spore form] from the hide of the drum. She became critically ill, but with gastrointestinal anthrax rather than inhaled anthrax, which made her unique in American medical history. The building where the infection took place was cleaned and reopened to the public and the woman recovered. Jodie Dionne-Odom, New Hampshire state epidemiologist, states, "It is a mystery. We really don't know why it happened."

Mode of infection

Anthrax can enter the human body through the intestines (ingestion), lungs (inhalation), or skin (cutaneous) and causes distinct clinical symptoms based on its site of entry. An infected human will generally be quarantined. However, anthrax does not usually spread from an infected human to a noninfected human. But if the disease is fatal to the person's body, its mass of anthrax bacilli becomes a potential source of infection to others and special precautions should be used to prevent further contamination. Inhalational anthrax, if left untreated until obvious symptoms occur, may be fatal.

Anthrax can be contracted in laboratory accidents or by handling infected animals or their wool or hides. It has also been used in biological warfare agents and by terrorists to intentionally infect as exemplified by the 2001 anthrax attacks.

Pulmonary

Inhalational anthrax, mediastinal widening

Respiratory infection in humans initially presents with cold or flu-like symptoms for several days, followed by severe (and often fatal) respiratory collapse. Historical mortality was 92%, but when treated early (seen in the 2001 anthrax attacks) observed mortality was 45%. Distinguishing pulmonary anthrax from more common causes of respiratory illness is essential to avoiding delays in diagnosis and thereby improving outcomes. An algorithm for this purpose has been developed. Illness progressing to the fulminant phase has a 97% mortality regardless of treatment.

A lethal infection is reported to result from inhalation of about 10,000–20,000 spores, though this dose varies amongst host species. Like all diseases there is probably a wide variation to susceptibility with evidence that some people may die from much lower exposures; there is little documented evidence to verify the exact or average number of spores needed for infection. Inhalational anthrax is also known as woolsorters' or ragpickers' disease as these professions were more susceptible to the disease due to their exposure to infected animal products. Other practices associated with exposure include the slicing up of animal horns for the manufacture of buttons, the handling of hair bristles used for the manufacturing of brushes, and the handling of animal skins. Whether these animal skins came from animals that died of the disease or from animals that had simply laid on ground that had spores on it is unknown. This mode of infection is used as a bioweapon.

Gastrointestinal

Gastrointestinal infection in humans is most often caused by eating anthrax-infected meat and is characterized by serious gastrointestinal difficulty, vomiting of blood, severe diarrhea, acute inflammation of the intestinal tract, and loss of appetite. Some lesions have been found in the intestines and in the mouth and throat. After the bacteria invades the bowel system, it spreads through the bloodstream throughout the body, making even more toxins on the way. Gastrointestinal infections can be treated but usually result in fatality rates of 25% to 60%, depending upon how soon treatment commences.

Cutaneous

Anthrax skin lesion

Cutaneous (on the skin) anthrax infection in humans shows up as a boil-like skin lesion that eventually forms an ulcer with a black center (eschar). The black eschar often shows up as a large, painless necrotic ulcer (beginning as an irritating and itchy skin lesion or blister that is dark and usually concentrated as a black dot, somewhat resembling bread mold) at the site of infection. Cutaneous infections generally form within the site of spore penetration between 2 and 5 days after exposure. Unlike bruises or most other lesions, cutaneous anthrax infections normally do not cause pain.

Cutaneous anthrax is rarely fatal if treated, but without treatment about 20% of cutaneous skin infection cases progress to toxemia and death.

Treatment typically includes antibiotic therapy. Specific guidelines are available for adults, children, pregnant women, and immunocompromised persons. The differential diagnosis includes multiple entities and thus accurate diagnosis is imperative. Clinical examination coupled with culture and cutaneous biopsy can aid in accurate diagnosis.

Diagnosis

Other than Gram Stain of specimens, there are no specific direct identification techniques for identification of Bacillus sp. in clinical material. These organisms are Gram positive but with age can be Gram variable to Gram negative. A specific feature of Bacillus sp. that makes it unique from other aerobic microorganisms is its ability to produce spores. Although, spores are not always evident on a Gram stain of this organism, the presence of spores confirms that the organism is of the genus Bacillus.

All Bacillus sp. grow well on 5% Sheep blood agar and other routine culture media. PLET (polymyxin-lysozyme-EDTA-thallous acetate) can be used to isolate B.anthracis from contaminated specimens and bicarbonate agar is used as an identification method to induce capsule formation.

Bacillus sp. will usually grow within 24 hours of incubation at 35 degrees C, in ambient air (room temperature) or in 5% CO₂. If bicarbonate agar is used for identification then the media must be incubated in 5% CO₂.

B.anthracis appears as medium-large, gray, flat, irregular with swirling projections, often referred to as "medusa head" appearance, and is non-hemolytic on 5% sheep blood agar. It is non-motile, is susceptible to penicillin and produces a wide zone of lecithinase on egg yolk agar. Confirmatory testing to identify B.anthracis includes gamma bacteriophage testing, indirect hemagglutination and enzyme linked immunosorbent assay to detect antibodies.

Prevention

Vaccines

An anthrax vaccine licensed by the U.S. Food and Drug Administration (FDA) and produced from one non-virulent strain of the anthrax bacterium, is manufactured by BioPort Corporation, subsidiary of Emergent BioSolutions. The trade name is BioThrax, although it is commonly called Anthrax Vaccine Adsorbed (AVA). It is administered in a six-dose primary series at 0, 2, 4 weeks and 6, 12, 18 months; annual booster injections are required thereafter to maintain immunity.

Unlike NATO countries, the Soviets developed and used a live spore anthrax vaccine, known as the STI vaccine, produced in Tbilisi, Georgia. Its serious side effects restrict use to healthy adults.

Treatment

Anthrax cannot be spread directly from person to person, but a patient's clothing and body may be contaminated with anthrax spores. Effective decontamination of people can be accomplished by a thorough wash down with antimicrobial effective soap and water. Waste water should be treated with bleach or other anti-microbial agent. Effective decontamination of articles can be accomplished by boiling contaminated articles in water for 30 minutes or longer. Chlorine bleach is ineffective in destroying spores and vegetative cells on surfaces, though formaldehyde is effective. Burning clothing is very effective in destroying spores. After decontamination, there is no need to immunize, treat or isolate contacts of persons ill with anthrax unless they were also exposed to the same source of infection. Early antibiotic treatment of anthrax is essential—delay significantly lessens chances for survival. Treatment for anthrax infection and other bacterial infections includes large doses of intravenous and oral antibiotics, such as fluoroquinolones, like ciprofloxacin (cipro), doxycycline, erythromycin, vancomycin or penicillin. In possible cases of inhalation anthrax, early antibiotic prophylaxis treatment is crucial to prevent possible death. In May 2009, Human Genome Sciences submitted a Biologic License Application (BLA, permission to market) for its new drug, raxibacumab (brand name ABthrax) intended for emergency treatment of inhaled anthrax. If death occurs from anthrax the body should be isolated to prevent possible spread of anthrax germs. Burial does not kill anthrax spores.

If a person is suspected as having died from anthrax, every precaution should be taken to avoid skin contact with the potentially contaminated body and fluids exuded through natural body openings. The body should be put in strict quarantine. A blood sample taken in a sealed container and analyzed in an approved laboratory should be used to ascertain if anthrax is the cause of death. Microscopic visualization of the encapsulated bacilli, usually in very large numbers, in a blood smear stained with polychrome methylene blue (McFadyean stain) is fully diagnostic, though culture of the organism is still the gold standard for diagnosis. Full isolation of the body is important to prevent possible contamination of others. Protective, impermeable clothing and equipment such as rubber gloves, rubber apron, and rubber boots with no perforations should be used when handling the body. No skin, especially if it has any wounds or scratches, should be exposed. Disposable personal protective equipment is preferable, but if not available, decontamination can be achieved by autoclaving. Disposable personal protective equipment and filters should be autoclaved, and/or burned and buried. Bacillus anthracis bacillii range from 0.5–5.0 μm in size. Anyone working with anthrax in a suspected or confirmed victim should wear respiratory equipment capable of filtering this size of particle or smaller. The US National Institute for Occupational Safety and Health (NIOSH) and Mine Safety and Health Administration (MSHA) approved high efficiency-respirator, such as a half-face disposable respirator with a high-efficiency particulate air (HEPA) filter, is recommended. All possibly contaminated bedding or clothing should be isolated in double plastic bags and treated as possible bio-hazard waste. The victim should be sealed in an airtight body bag. Dead victims that are opened and not burned provide an ideal source of anthrax spores. Cremating victims is the preferred way of handling body disposal. No embalming or autopsy should be attempted without a fully equipped biohazard laboratory and trained and knowledgeable personnel.

Delays of only a few days may make the disease untreatable and treatment should be started even without symptoms if possible contamination or exposure is suspected. Animals with anthrax often just die without any apparent symptoms. Initial symptoms may resemble a common cold—sore throat, mild fever, muscle aches and malaise. After a few days, the symptoms may progress to severe breathing problems and shock and ultimately death. Death can occur from about two days to a month after exposure with deaths apparently peaking at about 8 days after exposure. Antibiotic-resistant strains of anthrax are known.

In recent years there have been many attempts to develop new drugs against anthrax, but existing drugs are effective if treatment is started soon enough.

Early detection of sources of anthrax infection can allow preventive measures to be taken. In response to the anthrax attacks of October 2001 the United States Postal Service (USPS) installed BioDetection Systems (BDS) in their large scale mail cancellation facilities. BDS response plans were formulated by the USPS in conjunction with local responders including fire, police, hospitals and public health. Employees of these facilities have been educated about anthrax, response actions and prophylactic medication. Because of the time delay inherent in getting final verification that anthrax has been used, prophylactic antibiotic treatment of possibly exposed personnel must be started as soon as possible.

History

Etymology

The name 'anthrax' comes from anthrax , the Greek word for 'coal', because of the black skin lesions developed by victims with a cutaneous anthrax infection.

Discovery

Robert Koch, a German physician and scientist, first identified the bacteria which caused the anthrax disease in 1875. His pioneering work in the late nineteenth century was one of the first demonstrations that diseases could be caused by microbes. In a groundbreaking series of experiments he uncovered the life cycle and means of transmission of anthrax. His experiments not only helped create an understanding of anthrax, but also helped elucidate the role of microbes in causing illness at a time when debates were still held over spontaneous generation versus cell theory. Koch went on to study the mechanisms of other diseases and was awarded the 1905 Nobel Prize in Physiology or Medicine for his discovery of the bacteria causing tuberculosis. Koch is today recognized as one of history's most important biologists and a founder of modern bacteriology.

First vaccination

In May 1881 Louis Pasteur performed a public experiment to demonstrate his concept of vaccination. He prepared two groups of 25 sheep, one goat and several cows. The animals of one group were injected with an anti-anthrax vaccine prepared by Pasteur twice, at an interval of 15 days; the control group was left unvaccinated. Thirty days after the first injection both groups were injected with a culture of live anthrax bacteria. All the animals in the non-vaccinated group died, while all of the animals in the vaccinated group survived. The human vaccine for anthrax became available in 1954. This was a cell-free vaccine instead of the live-cell Pasteur-style vaccine used for veterinary purposes. An improved cell-free vaccine became available in 1970.

Society and culture

Site cleanup

Anthrax spores can survive for long periods of time in the environment after release. Methods for cleaning anthrax-contaminated sites commonly use oxidizing agents such as peroxides, ethylene oxide, Sandia Foam, chlorine dioxide (used in Hart Senate office building), and liquid bleach products containing sodium hypochlorite. These agents slowly destroy bacterial spores. A bleach solution for treating hard surfaces has been approved by the EPA. Bleach and vinegar must not be combined together directly, as doing so could produce chlorine gas. Rather some water must first be added to the bleach (e.g., two cups water to one cup of bleach), then vinegar (e.g., one cup), and then the rest of the water (e.g., six cups). The pH of the solution should be tested with a paper test strip; and treated surfaces must remain in contact with the bleach solution for 60 minutes (repeated applications will be necessary to keep the surfaces wet).

Chlorine dioxide has emerged as the preferred biocide against anthrax-contaminated sites, having been employed in the treatment of numerous government buildings over the past decade. Its chief drawback is the need for in situ processes to have the reactant on demand.

To speed the process, trace amounts of a non-toxic catalyst composed of iron and tetro-amido macrocyclic ligands are combined with sodium carbonate and bicarbonate and converted into a spray. The spray formula is applied to an infested area and is followed by another spray containing tert-Butyl hydroperoxide.

Using the catalyst method, a complete destruction of all anthrax spores can be achieved in under 30 minutes. A standard catalyst-free spray destroys fewer than half the spores in the same amount of time. They can be heated, exposed to the harshest chemicals, and they do not easily die.

Cleanups at a Senate office building, several contaminated postal facilities and other U.S. government and private office buildings showed that decontamination is possible, but it is time-consuming and costly. Clearing the Senate office building of anthrax spores cost $27 million, according to the Government Accountability Office. Cleaning the Brentwood postal facility outside Washington cost $130 million and took 26 months. Since then newer and less costly methods have been developed.

Clean up of anthrax-contaminated areas on ranches and in the wild is much more problematic. Carcasses may be burned, though it often takes up to three days to burn a large carcass and this is not feasible in areas with little wood. Carcasses may also be buried, though the burying of large animals deeply enough to prevent resurfacing of spores requires much manpower and expensive tools. Carcasses have been soaked in formaldehyde to kill spores, though this has environmental contamination issues. Block burning of vegetation in large areas enclosing an anthrax outbreak has been tried; this, while environmentally destructive, causes healthy animals to move away from an area with carcasses in search of fresh graze and browse. Some wildlife workers have experimented with covering fresh anthrax carcasses with shadecloth and heavy objects. This prevents some scavengers from opening the carcasses, thus allowing the putrefactive bacteria within the carcass to kill the vegetative B. anthracis cells and preventing sporulation. This method also has drawbacks, as scavengers such as hyenas are capable of infiltrating almost any exclosure. The occurrence of previously dormant anthrax, stirred up from below the ground surface by wind movement in a drought-stricken region with depleted grazing and browsing, may be seen as a form of natural culling and a first step in rehabilitation of the area.

Biological warfare

Anthrax was first tested as a biological warfare agent by Unit 731 of the Japanese Kwantung Army in Manchuria during the 1930s; some of this testing involved intentional infection of prisoners of war, thousands of whom died. Anthrax, designated at the time as Agent N, was also investigated by the allies in the 1940s. Weaponized anthrax was part of the U.S. stockpile prior to 1972, when the United States signed the Biological Weapons Convention.

Colin Powell holding a model vial of anthrax while giving a presentation to the United Nations Security Council

Anthrax spores can and have been used as a biological warfare weapon. Its first modern incidence occurred when Scandinavian "freedom fighters" (the rebel groups) supplied by the German General Staff used anthrax with unknown results against the Imperial Russian Army in Finland in 1916. There is a long history of practical bioweapons research in this area. For example, in 1942 British bioweapons trials severely contaminated Gruinard Island in Scotland with anthrax spores of the Vollum-14578 strain, making it a no-go area until it was decontaminated in 1990. The Gruinard trials involved testing the effectiveness of a submunition of an "N-bomb"—a biological weapon. Additionally, five million "cattle cakes" impregnated with anthrax were prepared and stored at Porton Down in "Operation Vegetarian"—an anti-livestock weapon intended for attacks on Germany by the Royal Air Force. The infected cattle cakes were to be dropped on Germany in 1944. However neither the cakes nor the bomb were used; the cattle cakes were incinerated in late 1945.

More recently the Rhodesian government used anthrax against cattle and humans in the period 1978–1979 during its war with black nationalists.

American military and British Army personnel are routinely vaccinated against anthrax prior to active service in places where biological attacks are considered a threat. The anthrax vaccine, produced by BioPort Corporation, contains non-living bacteria, and is approximately 93% effective in preventing infection.

Weaponized stocks of anthrax in the US were destroyed in 1971–72 after President Nixon ordered the dismantling of US biowarfare programs in 1969 and the destruction of all existing stockpiles of bioweapons.

The Soviet Union created and stored 100 to 200 tons of anthrax spores at Kantubek on Vozrozhdeniya Island. They were abandoned in 1992 and destroyed in 2002.

Soviet accident

April 2, 1979

Despite signing the 1972 agreement to end bioweapon production the government of the Soviet Union had an active bioweapons program that included the production of hundreds of tons of weapons-grade anthrax after this period. On April 2, 1979 some of the over one million people living in Sverdlovsk (now called Ekaterinburg, Russia), about 850 miles east of Moscow, were exposed to an accidental release of anthrax from a biological weapons complex located near there. At least 94 people were infected, of whom at least 68 died. One victim died four days after the release, ten over an eight-day period at the peak of the deaths, and the last six weeks later. Extensive cleanup, vaccinations and medical interventions managed to save about 30 of the victims. Extensive cover-ups and destruction of records by the KGB continued from 1979 until Russian President Boris Yeltsin admitted this anthrax accident in 1992. Jeanne Guillemin reported in 1999 that a combined Russian and United States team investigated the accident in 1992.

Nearly all of the night shift workers of a ceramics plant directly across the street from the biological facility (compound 19) became infected, and most died. Since most were men, there were suspicions by NATO governments that the Soviet Union had developed a sex-specific weapon. The government blamed the outbreak on the consumption of anthrax-tainted meat and ordered the confiscation of all uninspected meat that entered the city. They also ordered that all stray dogs be shot and that people not have contact with sick animals. There was also a voluntary evacuation and anthrax vaccination program established for people from 18–55.

To support the cover-up story Soviet medical and legal journals published articles about an outbreak in livestock that caused GI anthrax in people who consumed infected meat, and cutaneous anthrax in people who came into contact with the animals. All medical and public health records were confiscated by the KGB. In addition to the medical problems that the outbreak caused, it also prompted Western countries to be more suspicious of a covert Soviet Bioweapons program and to increase their surveillance of suspected sites. In 1986 the US government was allowed to investigate the incident, and concluded that the exposure was from aerosol anthrax from a military weapons facility. In 1992, President Yeltsin admitted that he was "absolutely certain" that "rumors" about the Soviet Union violating the 1972 Bioweapons Treaty were true. The Soviet Union, like the US and UK, had agreed to submit information to the UN about their bioweapons programs but omitted known facilities and never acknowledged their weapons program.

Anthrax bioterrorism

Theoretically anthrax spores can be cultivated with minimal special equipment and a first-year collegiate microbiological education, but in practice the procedure is difficult and dangerous. To make large amounts of an aerosol form of anthrax suitable for biological warfare requires extensive practical knowledge, training, and highly advanced equipment.

Concentrated anthrax spores were used for bioterrorism in the 2001 anthrax attacks in the United States, delivered by mailing postal letters containing the spores. Only a few grams of material were used in these attacks and in August 2008 the US Department of Justice announced they believed that Dr. Bruce Ivins, a senior biodefense researcher employed by the United States government, was responsible. These events also spawned many anthrax hoaxes.

Due to these events, the U.S. Postal Service installed biohazard detection systems at its major distribution centers to actively scan for anthrax being transported through the mail.

Decontaminating mail

In response to the postal anthrax attacks and hoaxes the US Postal Service sterilized some mail using a process of gamma irradiation and treatment with a proprietary enzyme formula supplied by Sipco Industries Ltd.

A scientific experiment performed by a high school student, later published in The Journal of Medical Toxicology, suggested that a domestic electric iron at its hottest setting (at least 400 °F (204 °C)) used for at least 5 minutes should destroy all anthrax spores in a common postal envelope.