Vibrio. Laboratory diagnosis

Vibrio. Laboratory diagnosis of cholera. Campylobacter, helicobacter and aeromonas infections.

 

The Cholera Vibrio.  

The causative agents of cholera are the classical Vibrio cholera biovars discovered by R. Koch in 1883 and the El Tor vibrio biovar isolated from the cadaver of a pilgrim on the Sinai peninsula by Gotschlich in 1906, RO- and O139 strains.

Vibrio cholerae biovar Proteus (N. Gamaleya, 1888) and Vibrio cholerae biovar albensis were discovered” later. V. cholerae was described by F. Pacini in 1854.

 

 

 

 

Morphology. Cholera vibrios are shaped like a comma or a curved rod measuring 1-5 mcm in length and 0.3 mcm in breadth (Fig.).

 

Figure. Vibrio cholerae: 1-pure culture; 2- flagellate vibrios

 

They are very actively motile, monotrichous, nonsporeforming, noncapsulated, and Gram-negative.

 

 

The cholera vibrio is subject to individual variation when it is exposed to physical and chemical factors. On artificial media and in old cultures it occurs in the form of grains, globes, rods, threads, clubs or spirals. When it is re-inoculated into fresh media, the organism assumes its initial form.

Electron microscopy reveals flagella 25 nm thick and three-layer cell wall and cytoplasmatic membrane. Small vacuoles are seen  between the wall and the cytoplasmatic membrane. The formation of the vacuoles is assumed to be linked with exotoxin synthesis. The nucleoid is in the centre of the cell. No essential difference has beeoted between the structure of the classical cholera vibrion and that of the El  Tor vibrio. The G-C content in DNA ranges between 45 and 49 per cent.                            

Cultivation.

Diagnosis and Detection

EPIDEMIOLOGY

 

Cholera is a vastly underreported disease, with an estimated 3 to 5 million cases and 100,000 deaths annually. Cholera is endemic in the developing countries of Asia and Africa and has caused epidemics in Asia, the Middle East, and South and Central America [1-5]. In 2009, the number of cases of cholera reported to the World Health Organization increased by 16 percent over the previous year (221,226 cases from 45 countries) [6]. Globally, a high incidence in the Americas in the early 1990s has shifted to a high incidence in Africa in the 2000s, with few cases in Asia.

 

Recent cholera epidemics include the 2008-2009 epidemic in Zimbabwe and the 2010 epidemic following the January earthquake in Haiti [7-9]. The V. cholerae strain responsible for the Haiti epidemic is nearly identical to the El Tor O1 strains predominant in southeast Asia; the ancestry is distinct from that of circulating Latin American and East African strains of V. cholerae, suggesting introduction of the strain from Asia [10]. During the first two years of the 2010 Haiti epidemic the cumulative attack rate was 6.1 percent [11]. The same strain was subsequently identified in cholera cases in the Dominican Republic and in Florida [12]. (See ‘Microbiology’ below.)

 

In the United States, approximately 10 laboratory-confirmed cases of cholera are reported to the Centers for Disease Control and Prevention each year [13]. Of these, about half are acquired outside the United States; the rest are acquired via consumption of contaminated seafood. Toxigenic Vibrio cholerae infection was reported in Louisiana in October 2005, related to consumption of contaminated and improperly cooked seafood after Hurricanes Katrina and Rita [14].

 

Vibrio cholerae growing on thiosulphate citrate bile salt sucrose (TCBS) agar plates

 

Electron micrograph of Vibrio cholerae

It is almost impossible to distinguish a single patient with cholera from a patient infected by another pathogen that causes acute watery diarrhea without testing a stool sample. A review of clinical features of multiple patients who are part of a suspected outbreak of acute watery diarrhea can be helpful an identifying cholera because of the rapid spread of the disease.

 

While management of patients with acute watery diarrhea is similar regardless of the illness, it is important to identify cholera because of the potential for a wide spread outbreak.

 

How to Diagnose

Isolation and identification of Vibrio cholerae serogroup O1 or O139 by culture of a stool specimen remains the gold standard for the laboratory diagnosis of cholera.

 

Cary Blair media is ideal for transport, and the selective thiosulfate–citrate–bile salts agar (TCBS) is ideal for isolation and identification. Reagents for serogrouping Vibrio cholerae isolates are available in all state health department laboratories in the U.S. Commercially available rapid test kits are useful in epidemic settings but do not yield an isolate for antimicrobial susceptibility testing and subtyping, and should not be used for routine diagnosis.

Cholera is an infection of the small intestine caused by the bacterium Vibrio cholerae.

 

The main symptoms are watery diarrhea and vomiting. Transmission occurs primarily by drinking water or eating food that has been contaminated by the feces (waste product) of an infected person, including one with no apparent symptoms.

 

The severity of the diarrhea and vomiting can lead to rapid dehydration and electrolyte imbalance, and death in some cases. The primary treatment is oral rehydration therapy, typically with oral rehydration solution, to replace water and electrolytes. If this is not tolerated or does not provide improvement fast enough, intravenous fluids can also be used. Antibacterial drugs are beneficial in those with severe disease to shorten its duration and severity.

 

Worldwide, it affects 3–5 million people and causes 100,000–130,000 deaths a year as of 2010[update]. Cholera was one of the earliest infections to be studied by epidemiological methods.

Signs and symptoms[edit]

A person with severe dehydration due to cholera. Note the sunken eyes and decreased skin turgor which produces wrinkled hands and skin

The primary symptoms of cholera are profuse diarrhea and vomiting of clear fluid.^[1] These symptoms usually start suddenly, half a day to five days after ingestion of the bacteria.^[2] The diarrhea is frequently described as “rice water” iature and may have a fishy odor.^[1] An untreated person with cholera may produce 10 to 20 litres (3 to 5 US gal) of diarrhea a day.^[1] Severe cholera kills about half of affected individuals.^[1] Estimates of the ratio of asymptomatic to symptomatic infections have ranged from 3 to 100.^[3] Cholera has beeicknamed the “blue death” because a victim’s skin turns bluish-gray from extreme loss of fluids.^[4]

 

 

Typical “rice water” diarrhea

If the severe diarrhea is not treated, it can result in life-threatening dehydration and electrolyte imbalances.^[1]

Fever is rare and should raise suspicion for secondary infection. Patients can be lethargic, and might have sunken eyes, dry mouth, cold clammy skin, decreased skin turgor, or wrinkled hands and feet. Kussmaul breathing, a deep and labored breathing pattern, can occur because of acidosis from stool bicarbonate losses and lactic acidosis associated with poor perfusion. Blood pressure drops due to dehydration, peripheral pulse is rapid and thready, and urine output decreases with time. Muscle cramping and weakness, altered consciousness, seizures, or even coma due to electrolyte losses and ion shifts are common, especially in children.^[1]

Susceptibility[edit]

About 100 million bacteria must typically be ingested to cause cholera in a normal healthy adult.^[1] This dose, however, is less in those with lowered gastric acidity (for instance those using proton pump inhibitors).^[1] Children are also more susceptible, with two- to four-year-olds having the highest rates of infection.^[1] Individuals’ susceptibility to cholera is also affected by their blood type, with those with type O blood being the most susceptible.^[1][6] Persons with lowered immunity, such as persons with AIDS or children who are malnourished, are more likely to experience a severe case if they become infected.^[7] Any individual, even a healthy adult in middle age, can experience a severe case, and each person’s case should be measured by the loss of fluids, preferably in consultation with a professional health care provider.^{[medical citatioeeded]}

The cystic fibrosis genetic mutation in humans has been said to maintain a selective advantage: heterozygous carriers of the mutation (who are thus not affected by cystic fibrosis) are more resistant to V. cholerae infections.^[8] In this model, the genetic deficiency in the cystic fibrosis transmembrane conductance regulator channel proteins interferes with bacteria binding to the gastrointestinal epithelium, thus reducing the effects of an infection

 

Rapid Tests

In areas with limited to no laboratory testing, the Crystal VC® dipstick rapid test can provide an early warning to public health officials that an outbreak of cholera is occurring. However, the sensitivity and specificity of this test is not optimal. Therefore, it is recommended that fecal specimens that test positive for V. cholerae O1 and/or O139 by the Crystal VC® dipstick be confirmed using traditional culture-based methods suitable for the isolation and identification of V. cholerae.

Cholera vibrios are facultative (anaerobes). The optimum growth temperature is 37° C, and growth is arrested below 14 °C and above 42° C. The organisms grow readily on alkaline media at pH 6.0-9.0, and on solid media the colonies are transparent with a light-blue hue, forming domes with smooth edges. On gelatin the organisms produce transparent granular colonies which, when examined under a microscope, resemble broken glass. In 48 hours the medium surrounding the colonies becomes liquefied and the colonies sink into this area. Six-hour-old cultures on alkaline meat broth and peptone water produce a pellicle, which consists of cholera vibrios.

The organism is also subjected to cultural changes. It dissociates from the S-form to the R-form, this process being accompanied by profound changes in antigenic structure.

Fermentative properties. The cholera vibrio liquefies coagulated serum and gelatin; it forms indole and ammonia, reduces nitrates to nitrites, breaks down urea, ferments glucose, levulose, galactose, maltose, saccharose, mannose, mannite, starch, and glycerine (slowly) with acid formation but does not ferment lactose in the first 48 hours, and always coagulates milk. The cholera vibrio possesses lysin and ornithine decarboxylases and oxidase activity. B. Heiberg differentiated vibrios into biochemical types according to their property of fermenting mannose, arabinose, and saccharose. Eight groups of vibrios are known to date; the cholera vibrios of the cholerae and El Tor biovar belong to biochemical variant 1.

The haemolytic activity and haemagglutinating properties of the cholera vibrios in relation to different erythrocytes (sheep, goat, chick, and others) as well as the ability for forming acetylmethylcarbinol are not stable characteristics and are taken into account as less important data in differentiating microbes of the genus Vibrio.

Toxin production. The cholera vibrio produces an exotoxin (cholerogen) which is marked by an enterotoxic effect and plays an important role in the pathogenesis of cholera; the endotoxin also exerts a powerful toxic effect. The cholera vibrios produce fibrinolysin, hyaluronidase, collagenase, mucinase, lecithinase, neuraminidase, and proteinases.

V cholerae produces diarrhea as a result of the secretion of an enterotoxin, choleratoxin, which acts identically to E coli LT to stimulate the activity of the enzyme adenylcyclase. This, in turn, converts ATP to cAMP, which stimulates the secretion of Cl^– and inhibits the absorption of NaCl. The copious fluid that is lost also contains large amounts of bicarbonate and K⁺. Thus, the patient has both a severe fluid loss and an electrolyte imbalance.

The enterotoxin has been shown to bind specifically to a membrane ganglioside designated GM₁. Interestingly, V cholerae produces a neuraminidase that is unable to remove the y-acetylneuraminic acid from GM₁, but it is able to convert other gangliosides to GM₁, thus synthesizing even more receptor sites to which its enterotoxin can bind. Like the LT of E coli, choleragen is composed of five B subunits that react with the cell receptor, an Ai-active subunit that enters the cell and, together with a cellular ADP-ribosylating factor, carries out the ADP-ribosylation of the GTP-binding protein, and a small A; subunit that seems to link the Ai subunit to the B subunit. Interestingly, unlike LT, the DNA en-coding choleragen is not plasmid mediated but is on the chromosome of V cholerae.

CT (as well as the LT produced by E coli) can be quantitated by a number of in vivo, cell culture, or immunologic assay units. In one method, a segment of rabbit small intestine is tied to form a loop. Enterotoxin is serially diluted, and an aliquot of each dilution is injected into a loop. The highest dilution that stimulates fluid accumulation in the loop is recorded as the titer of the enterotoxin. A second method takes advantage of the fact that cAMP causes a morphologic response in cultured Chinese hamster ovary cells, and that enterotoxin will induce such cells to produce cAMP. To quantitate enterotoxin using this assay, a standard, curve is established (with purified enterotoxin) that can be used subsequently to assay an unknown enterotoxin from E coli or V. cholerae (Fig.).

 

A standard curve to equate Escherichia coli enterotoxin with purified cholera toxin. The percentage of Chinese hamster ovary cells that have elongated after growing 24 hours in the presence of cholera toxin in 1% fetal calf serum is plotted against the concentration of cholera toxin present in the culture. As shown, heated toxin or toxin preincubated with antitoxin (anti-CT) have no effect on the morphologic features of the cells.

 

As is true with essentially all diarrhea-producing bacteria, V cholerae must specifically colonize the intestinal epithelial cells to produce disease. In this case, however, the pili binding the bacteria to the host cells seem to be under the same regulator as choleragen production and, as a result, are termed toxin-coregulated pili. Mutants unable to bind to intestinal cells are avirulent in spite of their ability to produce choleragen. Moreover, antibody directed to toxin-coregulated pili are protective.

Remember that non-01 and non-0139 strains of V.cholerae also cause a wide spectrum of infections, ranging from mild diarrhea to one indistinguishable from classic cholera. Some of these serotypes are known to produce a choleratoxin that is identical to that of the classic biotypes, whereas other products a heat-stable enterotoxin analogous to the ST of E. coli.

Antigenic structure. The cholera vibrios have thermostable O-antigens (somatic) and thermolabile H-antigens (flagellar). The O-antigen possesses species and type specificity, the H-antigen is common for the genus Vibrio. According to the O-antigen content, the vibrios are separated into subgroups of which there are more than 140. The cholerae vibrios, El Tor biovars and biovars cholera belong to the O-1 subgroup. In the 0-1 subgroup there are three O-antigens (A, B, and C) according to the combination of which three serological variants, Ogawa (AB), Inaba (AC) and an intermediate variant Hikojima (ABC), are distinguished.

Classification. Vibrio cholerae belongs to family Vibrionaceae, genus Vibrio consisting of 5 species. The species Vibrio cholerae is subdivided into four biological variants: biovar cholerae, biovar El Tor, biovar Proteus, and biovar albensis.

Biovar cholerae and biovar El Tor of Vibrio cholerae are the causative agents of human cholera. Biovar Proteus of Vibrio cholerae causes diarrhoea in birds and gastroenteritis in humans; biovar albensis of Vibrio cholerae was revealed in fresh water and in human faeces and bile.

Resistance. The cholera vibrio survives for a long time at low temperatures. It lives in faeces for up to a month, in oysters, crabs, on the surface offish and in their intestines from 1 to 40 days, in water for several days, on foodstuffs from 1 to 10 days, and in the intestines of flies from 4 to 5 days. .

The El Tor vibrio is marked by high resistance. It lives more than four weeks in sea and river water, 1-10 days on foodstuff’s, and 4-5 days in the guts of flies. It is possible that under favourable conditions El Tor vibrio may reproduce in various water reservoirs.

The organism shows a low resistance to sunlight, X-rays, desiccation, and high temperatures. It is destroyed instantly at 100°C, and in 5 minutes at 80° C. Cholera vibrios are highly sensitive to disinfectants, particularly to acids (e. g. a 1 :10000 solution of hydrochloric acid kills them within one minute). The organism is also very sensitive to the action of gastric juice.

Pathogenicity for animals. Iature animals are not attacked by cholera, but an intraperitoneal injection of the culture into rabbits and guinea pigs gives rise to general toxicosis and peritonitis which is followed by death.

In his experiments with rabbit-sucklings E. Metchnikoff produced a disease similar to human cholera by oral infection. R. Koch reproduced the disease in guinea pigs previously alkalizing the gastric juice and introducing opium. An intravenous vibrio injection into rabbits and dogs gives rise to lethal toxaemia.

Pathogenesis and diseases in man.  Cholera is undoubtedly the most dramatic of the water-borne diseases. As far as is known, cholera was confined to India for the almost 2000 years between its first description by Hindu physicians in 400 b c and its spread to Arabia, Persia, Turkey, and Southern Russia in the early1800s. There were six major pandemics of cholera during the 1800s covering the entire world, killing millions wherever it struck. During one such outbreak in London during 1849, the famous physician, John Snow, traced the spread of the disease to a Broad Street pump from which area residents obtained their water. The spread of cholera in this area was stopped when Snow recommended that the handle of the pump be removed. This is particularly remarkable when one remembers that the germ-theory of disease had not yet been formulated.

The cholera vibrios are transmitted from sick persons and carriers by food, water, flies, and contaminated hands. Via the mouth the organisms gain entrance into the small intestine, where the alkaline medium and an abundance of products of protein catabolism furnish favourable conditions for their multiplication. When the cholera vibrios perish, a large amount of toxin is released. This toxin invades the patient’s blood owing to necrosis of the intestinal    epithelium and the resulting condition promotes disturbance of the intestinal vegetative nerve fibres, dehydration of the body, and a development of intoxication.                                                

Cholera is characterized by a short incubation period of several hours to up to 6 days (in a disease caused by the El Tor vibrio it lasts three to five days), and the disease symptoms include  general weakness, vomiting, and a frequent loose stool. The stools resemble rice-water and contain enormous numbers of torn-off intestinal epithelial cells and cholera vibrios. The major symptom of cholera is a severe diarrhea in which a patient may lose as much as 10 to 20 L or more of liquid per day. Death, which may occur in as many as 60% of untreated patients, results from severe dehydration and loss of electrolytes.

Three phases can be distinguished in the development of the disease.  1. Cholera enteritis (choleric diarrhoea) which lasts 1 or 2 days. In some cases the infectious process terminates in this period and the patient recovers. 2. Cholera gastroenteritis is the second phase of the disease. Profuse diarrhoea and continuous vomiting lead to dehydration of the patient’s body and this results in lowering of body temperature, decrease in the amount of urine excreted, drastic decrease in the number of mineral and protein substance, and the appearance of convulsions. The presence of cholera vibrios is revealed guite frequently in the vomit and particularly in the stools which have the appearance of rice water. 3.  Cholera algid which is characterized by severe symptoms. The skin becomes wrinkled due to the loss of water, cyanosis appears, and the voice becomes husky and is sometimes lost completely. The body temperature falls to 35.5-34° C. As a result of blood concentration cardiac activity is drastically weakened and urination is suppressed.

In severe cases the algid period is followed by the asphyctic phase characterized by cyanosis, dyspnoea, uraemia, azotaemia, and unconsciousness (cholera coma), which lead to prostration and death. Effective treatment and proper nursing care may induce a change of the algid period to the reactive phase during which urination becomes normal, intoxication decreases, and the patient recovers. Fulminate forms of cholera (dry cholera or cholera sicca) may occur in a number of cases.These forms are characterized by the absence of diarrhoea and vomiting and result in death due to severe intoxication. Atypical and latent forms of cholera are exhibited quite frequently, particularly in children, resembling mild cases of gastroenteritis.

Non-specific complications in cholera include pneumonia, erysipelas, phlegmons, abscesses, occasionally sepsis, etc. Among the specific complications cholera typhoid is the most menacing. It is accompanied by a rise in body temperature to 38-39° C, eruptions on the skin, vomiting and fetid loose stools. This condition causes a mortality rate of 80-90 per cent.

Erased and mild forms are observed in 80 to 90 per cent of cases caused by El Tor vibrio. Severe forms with a fatal outcome are encountered in individuals whose condition is aggravated by various somatic diseases which reduce the general body resistance, in those with hypoacidic gastric function, and in elderly persons.

Post-mortem examination of cholera cases reveals distinct hyperaemia of the peritoneum and serosa of the small intestine, which are covered with a sticky exudate. The mucous membrane of the small intestine is congested, peach-coloured, the intestinal epithelium is frequently desquamated, and there are haemorrhages in the submucosa. The vibrios are present in great abundance in the intestinal wall, particularly in Lieberkuhn’s glands, and, not infrequently, in the gall-bladder.

Cholera mortality was quite high in the past (50 to 60 per cent), but has markedly decreased with the application of aetiotropic and pathogenetic therapy. According to WHO, in 1969-1971 it was 17.7 per cent.

Immunity acquired after cholera is high-grade but of short duration and is of an anti-infectious (antibacterial and antitoxic) character. It is associated mainly with the presence of antibodies (lysins, agglutinins, and opsonins). The cholera vibrios rapidly undergo lysis under the influence of immune sera which contain bacteriolysins.

E. Metchnikoff attributed definite significance to phagocytosis following immunity. The normal activity of the stomach, whose contents are bactericidal to the cholera vibrio, plays an essential role in the natural defence mechanism.

Laboratory diagnosis. A strict regimen is established in the laboratory. Examinations are carried out in accordance with the general rules observed for particularly hazardous diseases.

Test specimens are collected from stools, vomit, organs obtained at autopsy, water, objects contaminated by patient’s stools, and, in some cases, from foodstuffs. Certain rules are observed when the material is collected and transported to the laboratory, and examination is made in the following stages.

 

 

 

 

 

 

 

1. Stool smears stained by a water solution of fuchsin are examined microscopically. In the smears, the cholera vibrios occur in groups similar to shoals of fish (Fig.).

 

2. A stool sample is inoculated into 1 per cent peptone water and alkaline agar. After 6 hours incubation at 37°C the cholera vibrios form a thin pellicle in the peptone water, which adheres to the glass. The pellicle smears are Gram stained, and the culture is examined for motility. A slide agglutination reaction is performed with specific agglutinating 0-serum diluted in a ratio of 1 in  100.

The organisms are then transferred from the peptone water onto alkaline agar for isolation of the pure culture. If the first generation of the vibrios in peptone water is not visible, a drop taken from the surface layer is re-inoculated into another tube of peptone water. In some cases with such re-inoculations, an increase in the number of vibrios is achieved.

The vibrio culture grown on solid media is examined for motility and agglutinable properties. Then it is subcultured on an agar slant to obtain the pure culture.

3. The organism is identified finally by its agglutination reaction with specific 0-serum, determination of its fermentative properties (fermentation of mannose, saccharose, and arabinose), and its susceptibility to phagolysis (Table ).

Table

Differentiation of Biovars of Cholera vibrio

 

Note   “A” – carbohydrate fermentation with acid production; “+””– positive result; “–” – negative result; “+” – negative or positive result is not always observed.

 

The following procedures are undertaken for rapid diagnosis: (1) dark field microscopy of the stool; (2) stool culture by the method of  tampons incubated for 16-18 hours in an enrichment medium with repeated dark field microscopy; (3) agglutination reaction by the method of fluorescent antibodies;

 

 (4) bacterial diagnosis by isolation of cholera vibrios (the faecal mass is seeded as a thin layer into a dish containing non-inhibiting nutrient agar and grown for 4-5 hours, the vibrio colonies are detected with a stereoscopic microscope, and the culture is tested by the agglutination reaction with O-serum on glass; (5) since neuraminidase is discharged by the cholera vibrios and enters the intestine, a test for this enzyme is considered expedient as a means of early diagnosis (it is demonstrated in 66-76 per cent of patients, in 50-68 per cent of vibrio carriers, and occasionally in healthy individuals).

Treatment. The mortality rate of cholera can be reduced to less than1% by the adequate replacement of fluids and electrolytes. Antibiotics of the tetracycline group (tetracycline, sigmamycin), amphenicol, and streptomycin are prescribed at first intravenously and then by mouth.

Pathogenetic therapy is very important: control of dehydration, hypoproteinaemia, metabolic disorders, and the consequences of toxicosis, acidosis in particular, by infusion of saline (sodium and potassium) solutions, infusion of plasma or dry serum, glucose, the use of warm bath, administration of drugs which improve the tone of the heart and vessels.

Prophylaxis. Cholera patients and vibrio carriers are the source of the disease. Individuals remain carriers of the El Tor vibrio for a lengthy period of time, for several years. Vibrios of this biotype are widely distributed in countries with a low sanitary level. They survive in water reservoirs for a long time and have been found in the bodies of frogs and oysters. Infection may occur from bathing in contaminated water and fishing for and eating shrimps, oysters, and fish infected with El Tor vibrio.The following measures are applied in a cholera focus:

 

(1) detection of the first cases with cholera, careful registration of all sick individuals, immediate information of health protection organs;

(2) isolation and hospitalization, according to special rules, of all sick individuals and carriers, observation and laboratory testing of all contacts;

(3) concurrent and final disinfection in departments for cholera patients and in the focus;

(4) protection of sources of water supply, stricter sanitary control over catering establishments, control of flies; in view of the possibility of El Tor vibrio reproducing in water reservoirs under favourable conditions (temperature, the presence of nutrient substrates) systematic bacteriological control over water reservoirs has become necessary, especially in places of mass rest and recreation of the population in the summer;

(5) strict observance of individual hygiene; boiling or proper chlorination of water, decontamination of dishes, hand washing;

(6) specific prophylaxis: immunization with the cholera monovaccine containing 8 thousand million microbial bodies per 1 ml or with the cholera anatoxin. Chemoprophylaxis with oral tetracycline is conducted for persons who were in contact with the sick individual or for patients with suspected cholera.

 

Immunization with heat-killed cholera organisms seems to give some protection, and recovery from the disease imparts immunity of an unknown degree or duration. Killed whole cells of V cholerae given orally along with purified B subunits of the toxin induced immunity in about 85% of persons who received it. Another experimental engineered oral vaccine consists of a live attenuated V. cholerae El Tor Ogawa strain. This mutant no longer expresses the A subunit of the toxin but does produce B subunits. It seemed to provide good immunity in volunteers but it has not been used in large-scale field trials.

A experimental vaccine that induces toxin-neutralizing antibodies in mice uses. an ingenious technique in which a 45-base-pair oligonucleotide encoding an epitope of the B subunit of CT is inserted into the flagellin gene of an avirulent Salmonella. This 15-amino acid insert was expressed at the flagellar surface without abolishing flagellar function. The concept of placing an immunogen in a prominently displayed position on the bacterial surface could be used as a cholera vaccine as well as for inserting a number of other epitopes from both bacteria and viruses.

Remember, however, that none of these vaccines offer any protection against the newly described 0139 strain of V. cholera, and it is necessary to develop new vaccines for these organisms.

General epidemic measures play the principal role in cholera prophylaxis, whereas immunization is regarded as an auxiliary measure.

Cholera dates back to the most ancient times. Its endemic focus is India (Lower Bengal, and the deltas of the Ganges and Brahmaputra rivers).

There were six cholera pandemics between 1817 and 1926: in 1817-1823, 1826-1837, 1846-1862, 1864-1875, 1883-1896, and in 1900-1926. In 1961-1963 the seventh outbreak of cholera pandemic occurred, it was caused by the El Tor vibrio.

According to WHO, 668650 cholera cases were recorded between 1953 and 1961 in the countries of Asia and Africa and 348752 cases between 1961 and 1966.

Beginning with 1966, over 50 per cent of cholera cases in the countries of Asia were caused by the El Tor vibrio. According to WHO, however, the incidence of cholera induced by the classical Vibrio cholerae has doubled. There were cases with cholera in the southern regions of the Soviet Union (Astrakhan, Odessa, Kerch) in 1970. More than 464307 people sick with cholera were recorded in all countries in the period between 1970 and 1976. Cholera epidemic with high morbidity and
mortality rates occurred in the second half of 1977 in the Near East (Syria, Saudi Arabia, and other countries).

Vibrio Parahaemolyticus. Vibrio parahaemolyticus was discovered in 1963 by R. Sakazaki and colleagues. It was isolated from sea water, sea animals (fish) and the stool of humans sick with acute enteritis. Two biovars have been identified, biovar 1 (parahaemolyticus) and biovar 2 (alginolyticus). According to the 0-antigen, the Vibrio haemolyticus contains 12 serovars.

Vibrio parahaemolyticus is the causative agent of toxinfections. It produces haemolysin which has an enterotoxic effect. The ocean water along the coast of Japan is the natural reservoir of Vibrio parahaemolyticus. The sea fish and crustaceans are seeded with the organism but it reproduces within them only after they have been caught.

Vibrio parahaemolyticus strains isolated from humans cause lysis of erythrocytes and a cytopathic effect in human tissue cell cultures, whereas strains isolated from food and sea water are devoid of these properties. Fermentation of saccharose and arabinose is not a constant property.

Vibrio anguilarum is isolated from sea and fresh water and from sick fish. Vibrio fischeri from sea water and sea animals, and Vibrio costicola from canned meat and pickles.

The principles of therapy and prophylaxis are the same as those in other toxinfections.

Vibrio vulnificus is a halophilic organism that characteristically produces an overwhelming primary sepsis without an obvious source of infection, or an infection of a preexisting wound followed by a secondary sepsis. Theprimary sepsis seems to follow the ingestion of undercooked or raw seafood, particularly raw oysters. The number of V. vulnificus infections totals fewer than 100 per year in the United States, but the mortality rate is 45 % to 60 % particularly in individuals with liver disease, or thoses with diabetes, kidney disease and and other ailments affecting immune system. As a result, the CDC have strongly recommended: “Don’t eat raw oysters if you suffer from any kind of liver disease.” Secondary sepsis may also occur after the exposure of wounds to salt water or infectes shellfish.

Vibrio fluvialis is another halophilc that has been isolated from the diarrheal stools of many patients in Bangladesh. It has also been found in coastal waters shellfish on the east and west coasts of the United States. This organism has been reported to produce bott enterotoxin-like substances and an extracellular cytotoxin that kills tissue cells.

Vibrio mimicus, an organism similar to certain non-O1 V. choleras strains, also produces a cholera-like disease and reports indicate that it produces an entcrotoxin thatis indistinguishable from choleragen.

Campylobacter. Members of the genus Campyhbacter are gram-negative, curved, spiral rods possessing a single polar flagellum. Four acknowledged species of Campylobacter exist, and several additional species have been termed Campylobacter-like organisms. All seem to be inhabitants of the gastrointestinal tract of wild and domestic animals, including household pets. Transmission to humans occursby a fecal-oral route, originating from farm animals, birds, cats, dogs, and particularly processed poultry. Fifty percent to 70% of all human infections result from handlingor consuming improperly prepared chicken. Because the organisms often are found in unpasteurized milk, many epidemics of campylobacteriosis have been spread via milk. Some epidemics have occurred in school children who were given unpasteurized milk during field trips to dairies. The Food and Drug Administration has, therefore, specifically recommended that childreot be permitted to sample raw milk during such visits.

Campylobacter jejuni ranks along with rotaviruses and ETEC as the major cause of diarrheal disease in the world, particularly in developing countries. Clinical isolates of this organism have been shown to produce a heat-labile enterotoxin that raises intracellular levels of cAMP. Furthermore, the activity of this enterotoxin is partially neutralized by antiserum against E. coli LT and CT, demonstrating that Campylobacter enterotoxin belongs to this same group of adenylate cyclase-activating toxins. The production of this cholera-like toxin does not, however, explain the mechanism by which C. jejuni causes an inflammatory dysentery or bloody diarrhea. Analysis ofstrains producing such infections have revealed the presence of an additional cytotoxin that is biologically distinct from Shiga-like and Clostridium difficile toxins. The role of this toxin as a cause of inflammatory colitis, however, remains unknown. As is true for most intestinal pathogens, C jejuni has been shown to possess an adhesin for intestinal mucosa.

A number of reports have also indicated a close association between certain serotypes of C. jejuni and Guillain-Barre syndrome, but the nature of this relationship is completely unknown. In one study of 46 patients with Guillain-Barre syndrome, C. jejuni was isolated from 30% of patients compared with 1% of controls. Of these, 83% were serotype 19 and 17%  were serotype 2.

Campylobacter fetus also causes human diarrheal disease, but this species is more likely to progress to a systemic infection resulting in vascular necrosis.

The incubation period for the diarrheal disease usually is 2 to 4 days. The organisms can be grown readily on an enriched medium under microaerophilic conditions (6% O₂ and 10% CO₂). Gentamicin, erythromycin, and a number of other antibiotics may be used successfully for the treatment of Campylobacter infections.

Helicobacter. A. new species of gram-negative curved rods, named Helicobacter pylori, was first described in 1983. This organism was found growing in gastric epithelium, and it is accepted by most investigators that H. pylori is the primary etiologic agent of chronic gastritis and duodenal ulcers in humans. Symptoms of chronic gastritis include abdominal pain, burping, gastric distention, and halitosis. The disease can be reproduced in gnotobiotic piglets and in human volunteers after the ingestion of H. pylori. The observation that their eradication by antibacterial treatment results innormalization of the gastric histology and prevents there currence of peptic ulcers strongly supports the role of a this agent in chronic gastritis and peptic ulcer disease. Notice that Helicobacter mustelae can be routinely isolated from both normal and inflamed gastric mucosa of ferrets, and H. felis routinely colonizes the gastric mucosa of cats.

 

Surprisingly, H. pylori infection is widespread, particularly in developing countries where it occurs at a younger age than in developed countries. For example, the prevalence of H. pylori infection in Guangdong Province in China was 52.4%, and it has been suggested that earlya cquisition and, hence, long-term infection may be animportant factor predisposing to gastric cancer.

Adhesins, proteases, and cytotoxins all have been reported as virulence factors for H. pylori. One adhesin that has definitely been characterized is the blood group antigen, Lewis^b, (Le^b) which, if present, is found on the surface of gastric epithelial cells in the stomach. Gastric tissue lacking Le^b antigen or antibodies to the Le^b antigen inhibited bacterial binding. Thus, because Le^b is part of the antigen that determines blood group A, individuals with blood group O run a greater risk for developing gastric ulcers. A second adhesin reported to occur on the surface of H. pylori binds specifically to the monosacchande sialic acid, also found on glycoproteins on the surface of gastricepithelial cells.

The production of a cytotoxin that induces vacuolation of eucaryotic cells has been reported to occur inabout 50% of all isolates. Interestingly, one small study suggested that infection with toxin-producing strains was associated with increased antral inflammation.

All wild-type strains of H. pylori do produce the enzyme urease, and a number of reports have indicated that urease may protect the organisms from the acidic environment of the stomach by the release of ammonia from urea. Urease may also function as a cytotoxin, dc- stroying gastric cells that are susceptible to its activity.

 

Notice that over-the-counter medications containing bismuth salts have been used for years to treat gastritis (Pepto-Bismol, Procter & Gamble Pharm., Norwich, NY) and the fact that H. pylori is sensitive to bismuth may explain its efficacy for the relief of gastric symptoms.

Plesiomonas shigelloides. Plesiomonas shigelloides has been implicated as a cause of diarrhea in the United States as well as in tropical and subtropical countries. The mechanism by which this organism causes diarrhea is unknown, but a report indicated that sterile nitrates of growth medium obtained from 24 different strains of P. shigelloides induced the synthesis of cAMP in Chinese hamster ovary cells. Moreover, this effect was eliminated by either heating the filtrates or by preincubation of them with cholera antitoxin, suggesting that the diarrhea produced by P. shigelloides results from the formation of a cholera-like toxin.

These organisms have been isolated from surface waters, the intestines of fresh water fish, pet shop aquariums, and many animals, particularly dogs and cats. It is more common in tropical and subtropical areas, and isolations from Europe and the United States have been rare and usually associated with foreign travel or consumption of raw oysters.

Aeromonas. Aeromonas species are gram-negative, facultatively anaerobic bacteria that are found in soil, fresh and brackish water, and as pathogens of fish, amphibians, and mammals; symptoms range from diarrhea in piglets to fatal septiccmia in fish and dogs and abortion in cattle. Human infections are most commonly seen as a gastroenteritis but Aeromonas organisms have also been recovered from wounds and soft tissue abscesses that have been contaminated with soil or aquatic environments.

In 1988, California became the first state to make infections by Aeromonas a reportable condition and during that year 280 infections were reported, of which 81% were gastroenteritis and 9% were wound infections. Others were isolated from blood, bile, sputum, and urine, occurring mostly in persons with chronic underlying diseases.

Virulence factors that have been reported for Aeromonas include cholera-like and heat stable entcrotoxins andat least two hemolysins, one or both of which may be cytotoxic or enterotoxic. Aeromonas hydrophila and Aeromonas sobria probably are the only clinically important species.

 Transmission[edit]Cholera is typically transmitted by either contaminated food or water. In the developed world, seafood is the usual cause, while in the developing world it is more often water.[1] Most cholera cases in developed countries are a result of transmission by food. This occurs when people harvest oysters in waters infected with sewage, as Vibrio cholerae accumulates in zooplankton and the oysters eat the zooplankton.[9] Cholera has been found in two animal populations: shellfish and plankton.[1]

 

People infected with cholera often have diarrhea, and if this highly liquid stool, colloquially referred to as “rice-water”, contaminates water used by others, disease transmission may occur.[10] The source of the contamination is typically other cholera sufferers when their untreated diarrheal discharge is allowed to get into waterways, groundwater or drinking water supplies. Drinking any infected water and eating any foods washed in the water, as well as shellfish living in the affected waterway, can cause a person to contract an infection. Cholera is rarely spread directly from person to person. Both toxic and nontoxic strains exist. Nontoxic strains can acquire toxicity through a temperate bacteriophage.[11] Coastal cholera outbreaks typically follow zooplankton blooms, thus making cholera a zoonotic disease.[medical citatioeeded]

When consumed, most bacteria do not survive the acidic conditions of the human stomach.[12] The few surviving bacteria conserve their energy and stored nutrients during the passage through the stomach by shutting down much protein production. When the surviving bacteria exit the stomach and reach the small intestine, they need to propel themselves through the thick mucus that lines the small intestine to get to the intestinal walls where they can thrive. V. cholerae bacteria start up production of the hollow cylindrical protein flagellin to make flagella, the cork-screw helical fibers they rotate to propel themselves through the mucus of the small intestine.[medical citatioeeded]

 

Once the cholera bacteria reach the intestinal wall they no longer need the flagella to move. The bacteria stop producing the protein flagellin to conserve energy and nutrients by changing the mix of proteins which they express in response to the changed chemical surroundings. On reaching the intestinal wall, V. cholerae start producing the toxic proteins that give the infected person a watery diarrhea. This carries the multiplying new generations of V. cholerae bacteria out into the drinking water of the next host if proper sanitation measures are not in place.[medical citatioeeded]

 

The cholera toxin (CTX or CT) is an oligomeric complex made up of six protein subunits: a single copy of the A subunit (part A), and five copies of the B subunit (part B), connected by a disulfide bond. The five B subunits form a five-membered ring that binds to GM1 gangliosides on the surface of the intestinal epithelium cells. The A1 portion of the A subunit is an enzyme that ADP-ribosylates G proteins, while the A2 chain fits into the central pore of the B subunit ring. Upon binding, the complex is taken into the cell via receptor-mediated endocytosis. Once inside the cell, the disulfide bond is reduced, and the A1 subunit is freed to bind with a human partner protein called ADP-ribosylation factor 6 (Arf6).[13] Binding exposes its active site, allowing it to permanently ribosylate the Gs alpha subunit of the heterotrimeric G protein. This results in constitutive cAMP production, which in turn leads to secretion of H2O, Na+, K+, Cl−, and HCO3− into the lumen of the small intestine and rapid dehydration. The gene encoding the cholera toxin was introduced into V. cholerae by horizontal gene transfer. Virulent strains of V. cholerae carry a variant of temperate bacteriophage called CTXf or CTXφ.

 

Microbiologists have studied the genetic mechanisms by which the V. cholerae bacteria turn off the production of some proteins and turn on the production of other proteins as they respond to the series of chemical environments they encounter, passing through the stomach, through the mucous layer of the small intestine, and on to the intestinal wall.[14] Of particular interest have been the genetic mechanisms by which cholera bacteria turn on the protein production of the toxins that interact with host cell mechanisms to pump chloride ions into the small intestine, creating an ionic pressure which prevents sodium ions from entering the cell. The chloride and sodium ions create a salt-water environment in the small intestines, which through osmosis can pull up to six litres of water per day through the intestinal cells, creating the massive amounts of diarrhea. The host can become rapidly dehydrated if an appropriate mixture of dilute salt water and sugar is not taken to replace the blood’s water and salts lost in the diarrhea.[medical citatioeeded]

 

By inserting separate, successive sections of V. cholerae DNA into the DNA of other bacteria, such as E. coli that would not naturally produce the protein toxins, researchers have investigated the mechanisms by which V. cholerae responds to the changing chemical environments of the stomach, mucous layers, and intestinal wall. Researchers have discovered a complex cascade of regulatory proteins controls expression of V. cholerae virulence determinants. In responding to the chemical environment at the intestinal wall, the V. cholerae bacteria produce the TcpP/TcpH proteins, which, together with the ToxR/ToxS proteins, activate the expression of the ToxT regulatory protein. ToxT then directly activates expression of virulence genes that produce the toxins, causing diarrhea in the infected person and allowing the bacteria to colonize the intestine.[14] Current research aims at discovering “the signal that makes the cholera bacteria stop swimming and start to colonize (that is, adhere to the cells of) the small intestine.”[14]

Diagnosis[edit]A rapid dipstick test is available to determine the presence of V. cholerae.[16] In those samples that test positive, further testing should be done to determine antibiotic resistance.[16] In epidemic situations, a clinical diagnosis may be made by taking a patient history and doing a brief examination. Treatment is usually started without or before confirmation by laboratory analysis.

 

Stool and swab samples collected in the acute stage of the disease, before antibiotics have been administered, are the most useful specimens for laboratory diagnosis. If an epidemic of cholera is suspected, the most common causative agent is V. cholerae O1. If V. cholerae serogroup O1 is not isolated, the laboratory should test for V. cholerae O139. However, if neither of these organisms is isolated, it is necessary to send stool specimens to a reference laboratory.

 

Infection with V. cholerae O139 should be reported and handled in the same manner as that caused by V. cholerae O1. The associated diarrheal illness should be referred to as cholera and must be reported in the United States.

 

Prevention

Cholera hospital in Dhaka, showing typical “cholera beds”The World Health Organization recommends focusing on prevention, preparedness, and response to combat the spread of cholera.They also stress the importance of an effective surveillance system.[18] Governments can play a role in all of these areas, and in preventing cholera or indirectly facilitating its spread.

 

Although cholera may be life-threatening, prevention of the disease is normally straightforward if proper sanitation practices are followed. In developed countries, due to nearly universal advanced water treatment and sanitation practices, cholera is no longer a major health threat. The last major outbreak of cholera in the United States occurred in 1910–1911 Effective sanitation practices, if instituted and adhered to in time, are usually sufficient to stop an epidemic. There are several points along the cholera transmission path at which its spread may be halted:[medical citatioeeded

 

Sterilization: Proper disposal and treatment of infected fecal waste water produced by cholera victims and all contaminated materials (e.g. clothing, bedding, etc.) are essential. All materials that come in contact with cholera patients should be sanitized by washing in hot water, using chlorine bleach if possible. Hands that touch cholera patients or their clothing, bedding, etc., should be thoroughly cleaned and disinfected with chlorinated water or other effective antimicrobial agents.

Sewage: antibacterial treatment of general sewage by chlorine, ozone, ultraviolet light or other effective treatment before it enters the waterways or underground water supplies helps prevent undiagnosed patients from inadvertently spreading the disease.

Sources: Warnings about possible cholera contamination should be posted around contaminated water sources with directions on how to decontaminate the water (boiling, chlorination etc.) for possible use.

Water purification: All water used for drinking, washing, or cooking should be sterilized by either boiling, chlorination, ozone water treatment, ultraviolet light sterilization (e.g. by solar water disinfection), or antimicrobial filtration in any area where cholera may be present. Chlorination and boiling are often the least expensive and most effective means of halting transmission. Cloth filters or sari filtration, though very basic, have significantly reduced the occurrence of cholera when used in poor villages in Bangladesh that rely on untreated surface water. Better antimicrobial filters, like those present in advanced individual water treatment hiking kits, are most effective. Public health education and adherence to appropriate sanitation practices are of primary importance to help prevent and control transmission of cholera and other diseases.

Surveillance

Surveillance and prompt reporting allow for containing cholera epidemics rapidly. Cholera exists as a seasonal disease in many endemic countries, occurring annually mostly during rainy seasons. Surveillance systems can provide early alerts to outbreaks, therefore leading to coordinated response and assist in preparation of preparedness plans. Efficient surveillance systems can also improve the risk assessment for potential cholera outbreaks. Understanding the seasonality and location of outbreaks provides guidance for improving cholera control activities for the most vulnerable.For prevention to be effective, it is important that cases be reported to national health authorities.

 

Cholera vaccine

 

Preventive inoculation against cholera in 1966A number of safe and effective oral vaccines for cholera are available.Dukoral, an orally administered, inactivated whole cell vaccine, has an overall efficacy of about 52% during the first year after being given and 62% in the second year, with minimal side effects. It is available in over 60 countries. However, it is not currently recommended by the Centers for Disease Control and Prevention (CDC) for most people traveling from the United States to endemic countries.One injectable vaccine was found to be effective for two to three years. The protective efficacy was 28% lower in children less than 5 years old. However, as of 2010, it has limited availability. Work is under way to investigate the role of mass vaccination.The World Health Organization (WHO) recommends immunization of high-risk groups, such as children and people with HIV, in countries where this disease is endemic.[ If people are immunized broadly, herd immunity results, with a decrease in the amount of contamination in the environment.

 

Sari filtration[edit]An effective and relatively cheap method to prevent the transmission of cholera is the use of a folded sari (a long cloth garment) to filter drinking water. In Bangladesh this practice was found to decrease rates of cholera by nearly half. It involves folding a sari four to eight times. Between uses the cloth should be rinsed in clean water and dried in the sun to kill any bacteria on it. A nylon cloth appears to work as well.

Antibiotics[edit]

Antibiotic treatments for one to three days shorten the course of the disease and reduce the severity of the symptoms.^[1] Use of antibiotics also reduces fluid requirements.People will recover without them, however, if sufficient hydration is maintained. The World Health Organization only recommends antibiotics in those with severe dehydration.

Doxycycline is typically used first line, although some strains of V. cholerae have shown resistance.Testing for resistance during an outbreak can help determine appropriate future choices. Other antibiotics proven to be effective include cotrimoxazole, erythromycin, tetracycline, chloramphenicol, and furazolidone.^[36] Fluoroquinolones, such as norfloxacin, also may be used, but resistance has been reported.

In many areas of the world, antibiotic resistance is increasing. In Bangladesh, for example, most cases are resistant to tetracycline, trimethoprim-sulfamethoxazole, and erythromycin. Rapid diagnostic assay methods are available for the identification of multiple drug-resistant cases.New generation antimicrobials have been discovered which are effective against in in vitro studies.

Zinc supplementation[edit]

In Bangladesh zinc supplementation reduced the duration and severity of diarrhea in children with cholera when given with antibiotics and rehydration therapy as needed. It reduced the length of disease by eight hours and the amount of diarrheal stool by 10%.Supplementation appears to be also effective in both treating and preventing infectious diarrhea due to other causes among children in the developing world.^[40][41]

Prognosis

If people with cholera are treated quickly and properly, the mortality rate is less than 1%; however, with untreated cholera, the mortality rate rises to 50–60%. For certain genetic strains of cholera, such as the one present during the 2010 epidemic in Haiti and the 2004 outbreak in India, death can occur within two hours of becoming ill.

Epidemiology[edit]

See also: Cholera outbreaks and pandemics

 

Hand bill from the New York City Board of Health, 1832—the outdated public health advice demonstrates the lack of understanding of the disease and its actual causative factors.

Cholera affects an estimated 3–5 million people worldwide, and causes 58,000–130,000 deaths a year as of 2010. This occurs mainly in the developing world. In the early 1980s, death rates are believed to have been greater than 3 million a year. It is difficult to calculate exact numbers of cases, as many go unreported due to concerns that an outbreak may have a negative impact on the tourism of a country.Cholera remains both epidemic and endemic in many areas of the world.

Although much is known about the mechanisms behind the spread of cholera, this has not led to a full understanding of what makes cholera outbreaks happen some places and not others. Lack of treatment of human feces and lack of treatment of drinking water greatly facilitate its spread, but bodies of water can serve as a reservoir, and seafood shipped long distances can spread the disease. Cholera was not known in the Americas for most of the 20th century, but it reappeared towards the end of that century.

History[edit]

The word cholera is from Greek: χολέρα kholera from χολή kholē “bile”. Cholera likely has its origins in the Indian Subcontinent; it has been prevalent in the Ganges delta since ancient times. Choleras first origins, within the Indian Subcontinent, are believed to have occurred due to the result of poor living conditions as well as the presence of pools of still water; both of which are ideal living conditions for cholera to thrive.The disease first spread by trade routes (land and sea) to Russia in 1817, then, through technological advancements, to the rest of Europe, and from Europe to North America and the rest of the world.^[1] Seven cholera pandemics have occurred in the past 200 years, with the seventh originating in Indonesia in 1961.

 

Map of the 2008–2009 cholera outbreak in sub-Saharan Africa showing the statistics as of 12 February 2009.

The first cholera pandemic occurred in the Bengal region of India starting in 1817 through 1824. The disease dispersed from India to Southeast Asia, China, Japan, the Middle East, and southern Russia. The second pandemic lasted from 1827 to 1835 and affected the United States and Europe particularly due to the result of advancements in transportation and global trade, and increased human migration, including soldiers.^[49] The third pandemic erupted in 1839, persisted until 1856, extended to North Africa, and reached South America, for the first time specifically infringing upon Brazil. Cholera hit the sub-Saharan African region during the fourth pandemic from 1863 to 1875. The fifth and sixth pandemics raged from 1881–1896 and 1899-1923. These epidemics were less fatal due to a greater understanding of the cholera bacteria. Egypt, the Arabian peninsula, Persia, India, and the Philippines were hit hardest during these epidemics, while other areas, like Germany in 1892 and Naples from 1910–1911, experienced severe outbreaks. The final pandemic originated in 1961 in Indonesia and is marked by the emergence of a new strain, nicknamed El Tor, which still persists today in developing countries.

From a local disease, cholera became one of the most widespread and deadly diseases of the 19th century, killing an estimated tens of millions of people.^[51] In Russia alone, between 1847 and 1851, more than one million people perished of the disease. It killed 150,000 Americans during the second pandemic.^[53] Between 1900 and 1920, perhaps eight million people died of cholera in India.^[54] Cholera became the first reportable disease in the United States due to the significant effects it had on health.John Snow, in England, was the first to identify the importance of contaminated water in its cause in 1854.^[1] Cholera is now no longer considered a pressing health threat in Europe and North America due to filtering and chlorination of water supplies, but still heavily affects populations in developing countries.

In the past, vessels flew a yellow quarantine flag if any crew members or passengers were suffering from cholera. No one aboard a vessel flying a yellow flag would be allowed ashore for an extended period, typically 30 to 40 days.^[55] In modern sets of international maritime signal flags, the quarantine flag is yellow and black.

 

Signal flag “Lima” called the “Yellow Jack” which when flown in harbor means ship is under quarantine. A simple yellow flag (also called the “Yellow Jack”) had historically been used to signal quarantine (it stands for Q among signal flags), but now indicates the opposite, as a signal of a ship free of disease that requests boarding and inspection.

Historically many different claimed remedies have existed in folklore. In the 1854–1855 outbreak in Naples homeopathic Camphor was used according to Hahnemann.^[56] While T. J. Ritter’s “Mother’s Remedies” book lists tomato syrup as a home remedy from northern America. While elecampagne was recommended in the United Kingdom according to William Thomas Fernie

Cholera cases are much less frequent in developed countries where governments have helped to establish water sanitation practices and effective medical treatments. The United States, for example, used to have a severe cholera problem similar to those in some developing countries. There were three large cholera outbreaks in the 1800s, which can be attributed to Vibrio cholerae’s spread through interior waterways like the Erie Canal and routes along the Eastern Seaboard. The island of Manhattan in New York City touched the Atlantic Ocean, where cholera collected just off the coast. At this time, New York City did not have as effective a sanitation system as it does today, so cholera was able to spread.

Research

The bacterium was isolated in 1855 by Italian anatomist Filippo Pacini, but its exact nature and his results were not widely known.

Spanish physician Jaume Ferran i Clua developed a cholera vaccine in 1885, the first to immunize humans against a bacterial disease.

Ukrainian bacteriologist Waldemar Haffkine developed a cholera vaccine in July 1892.

One of the major contributions to fighting cholera was made by the physician and pioneer medical scientist John Snow (1813–1858), who in 1854 found a link between cholera and contaminated drinking water.^[ Dr. Snow proposed a microbial origin for epidemic cholera in 1849. In his major “state of the art” review of 1855, he proposed a substantially complete and correct model for the etiology of the disease. In two pioneering epidemiological field studies, he was able to demonstrate human sewage contamination was the most probable disease vector in two major epidemics in London in 1854. His model was not immediately accepted, but it was seen to be the more plausible, as medical microbiology developed over the next 30 years or so.

 

Robert Koch (third from the right) on a cholera research expedition in Egypt in 1884, one year after he identified V. cholerae.

Cities in developed nations made massive investment in clean water supply and well-separated sewage treatment infrastructures between the mid-1850s and the 1900s. This eliminated the threat of cholera epidemics from the major developed cities in the world. In 1883, Robert Koch identified V. cholerae with a microscope as the bacillus causing the disease.

Robert Allan Phillips, working at the US Naval Medical Research Unit Two in Southeast Asia, evaluated the pathophysiology of the disease using modern laboratory chemistry techniques and developed a protocol for rehydration. His research led the Lasker Foundation to award him its prize in 1967.

Cholera has been a laboratory for the study of evolution of virulence. The province of Bengal in British India was partitioned into West Bengal and East Pakistan in 1947. Prior to partition, both regions had cholera pathogens with similar characteristics. After 1947, India made more progress on public health than East Pakistan (now Bangladesh). As a consequence,the strains of the pathogen that succeeded in India had a greater incentive in the longevity of the host. They have become less virulent than the strains prevailing in Bangladesh. These draw upon the resources of the host population and rapidly kill many victims.

More recently, in 2002, Alam, et al., studied stool samples from patients at the International Centre for Diarrhoeal Disease in Dhaka, Bangladesh. From the various experiments they conducted, the researchers found a correlation between the passage of V. cholerae through the human digestive system and an increased infectivity state. Furthermore, the researchers found the bacterium creates a hyperinfected state where genes that control biosynthesis of amino acids, iron uptake systems, and formation of periplasmic nitrate reductase complexes were induced just before defecation. These induced characteristics allow the cholera vibrios to survive in the “rice water” stools, an environment of limited oxygen and iron, of patients with a cholera infection.

Society and culture[edit]

In many developing countries, cholera still reaches its victims through contaminated water sources, and countries without proper sanitation techniques have greater incidence of the disease.^[67] Governments can play a role in this. In 2008, for example, the Zimbabwean cholera outbreak was due partly to the government’s role, according to a report from the James Baker Institute. The Haitian government’s inability to provide safe drinking water after the 2010 earthquake led to an increase in cholera cases as well. Similarly, South Africa’s cholera outbreak was exacerbated by the government’s policy of privatizing water programs. The wealthy elite of the country were able to afford safe water while others had to use water from cholera-infected rivers.

According to Rita R. Colwell of the James Baker Institute, if cholera does begin to spread, government preparedness is crucial. A government’s ability to contain the disease before it extends to other areas can prevent a high death toll and the development of an epidemic or even pandemic. Effective disease surveillance can ensure that cholera outbreaks are recognized as soon as possible and dealt with appropriately. Oftentimes, this will allow public health programs to determine and control the cause of the cases, whether it is unsanitary water or seafood that have accumulated a lot of Vibrio cholerae specimens. Having an effective surveillance program contributes to a government’s ability to prevent cholera from spreading. In the year 2000 in the state of Kerala in India, the Kottayam district was determined to be “cholera-affected”; this pronouncement led to task forces that concentrated on educating citizens with 13,670 information sessions about human health. These task forces promoted the boiling of water to obtain safe water, and provided chlorine and oral rehydration salts. Ultimately, this helped to control the spread of the disease to other areas and minimize deaths. On the other hand, researchers have shown that most of the citizens infected during the 1991 cholera outbreak in Bangladesh lived in rural areas, and were not recognized by the government’s surveillance program. This inhibited physicians’ abilities to detect cholera cases early.

According to Colwell, the quality and inclusiveness of a country’s health care system affects the control of cholera, as it did in the Zimbabwean cholera outbreak.While sanitation practices are important, when governments respond quickly and have readily available vaccines, the country will have a lower cholera death toll. Affordability of vaccines can be a problem; if the governments do not provide vaccinations, only the wealthy may be able to afford them and there will be a greater toll on the country’s poor.The speed with which government leaders respond to cholera outbreaks is important.

Besides contributing to an effective or declining public health care system and water sanitation treatments, government can have indirect effects on cholera control and the effectiveness of a response to cholera.A country’s government can impact its ability to prevent disease and control its spread. A speedy government response backed by a fully functioning health

 

Additional materials for diagnosis

CHOLERA

Cholera is a particularly dangerous infectious disease, caused by Vibrio cholerae and Vibrio El Tor biovars, which runs as gastroenteritis associated with dehydration.

The main method in the laboratory diagnosis of cholera is bacterio­logical examination. Some 10-20 ml of faeces and vomited matter from patients with suspected cholera are collected with a sterile metallic or wooden spoon, transferred into a sterile wide-mouthed vessel, and tightly stoppered with a-glass or cork plug.

The second portion of faeces and vomit (1-2 ml) is inoculated into 1 per cent peptone water (50 ml) at the patient’s bedside. Both vessels are sealed and immediately sent to the laboratory.

If the patient has no bowel movements at the moment of material collection, cut off soiled samples of the bed linen or underclothes and collect the contents of the rectum with a sterile wire loop inserted 5-8 cm deep. Following removal, put the loop with a faecal sample into a flask with a nutrient medium. At autopsy the material to be tested is obtained in the following manners mark off three sites in the area of the upper, middle and lower portions of the small in­testine and a site of the rectum some 10-15 cm long; then from each end of the marked section express the contents of the intestine side-wise, apply two ligatures, and cut an intestine between them. The gallbladder is removed with a part of the liver. The water (1 L) and foodstuffs (no less than 200 g) should also be examined.

In examining convalescents, individuals who have contacted with patients or carriers, it is recommended that a purgative or a cholagogue (25-30 g of magnesium sulphate, etc.) be preliminary given to them to obtain liquid faeces from the upper part of the intestines and the contents of the gallbladder.

The material to be studied is collected, packed, and sent to the laboratory with special measures of precaution. The glassware should not contain any traces of disinfectants, particularly of acids; it is sterilized or boiled for 15 min.

Jars and test tubes should be closed with glass or rubber stoppers. When cork plugs are used, cellulosic film is placed under them. After the  material has been collected, the plugs are sealed with paraffin and wrapped with double cel­lulosic film.

On each vessel stick on a slip of paper with the name and age of the patient, his or her home and office address, diagnosis, the dates of the onset of the disease and hospitalization, as well as the date and exact time of material collection, and also the name of the person who has sent in the analysis.

The material should be brought to the laboratory no later than six hours after its collection. If the delivery within this period is impossible, the samples are inoculated into 1 per cent peptone water with potassium tellurite and onto plates with alkaline agar. If the laboratory is a long way off, jars and test tubes with the specimens to be tested are put, packing them with saw dust, into a metallic container which, in turn, is packed into a wooden box. The latter is wrapped, sealed, signed “Top, fragile”, and is sent with a cou­rier.

The material should be exam­ined in a special laboratory. Yet, if no such laboratory is available, the samples are sent to any bacte­riological laboratory which may provide an isolated room with a separate entrance and exit. No other analyses are taken in this case and stricter measures of precau­tion are introduced. Personnel with special training only is allowed to do this kind of investigation. No operators on a fasting stomach should be allowed in the laboratory. The examination is carried out around the clock since the results should be available no later than 30-36 hrs later.

Bacteriological examination. Stage I. Using the material col­lected, prepare smears, dry them in the air, fix with alcohol or Nikiforov’s mixture, stain by the Gram technique, and examine under the microscope. Later on, if laboratory findings confirm the diagnosis of cholera in at least one case, stain the smears with Pfeiffer’s fuchsine only. Cholera vibrios appear as thin curved Gram-negative rods (Fig. 15). Because of great polymorphism the smear may, along with typical cells, contain coccal, rod-shaped, and spiral forms, which diminishes the value of this method.

The first preliminary answer is given after the microscopic exam­ination of the smear. It refers to the presence of vibrios and the nature of their Gram-staining.

At this stage of bacteriological examination, one can also perform the immunoftuorescence test, using specific labelled 0-cholera sera. Moreover, the cholera vibrio may be recovered by the immune Indian ink method. In the latter case smears fixed on a glass slide are treated for 2 min in a humid chamber with Indian ink mixed with immune serum, then washed with water and examined with a microscope. The vibrio is stained black by Indian ink: the walls of the cell are black-brown, the centre is slightly greyish. If the bacteria are few, they are preliminarily cultivated for 3-5 hrs in peptone water.

Simultaneously with bacterioscopy, the material tested is inoculat­ed onto liquid and solid nutrient media. Enrichment liquid media that are usually recommended for use include alkaline 1 per cent peptone water, 1 per cent peptone water with potassium tellurite in a ratio of 1 to 100 000, and alkaline taurocholate-tellurite-peptone medium (Monsur’s liquid medium), etc.

Solid nutrient media usually employed are alkaline meat-peptone agar and one of the selective nutrient media: Aronson’s medium, Monsur’s  alkaline taurocholate-lellurite-gelatine-agar medium, TCBS, etc.

To isolate the vibrio from carriers or patients with subclinical forms of cholera, use media which improve the growth of vibrios and suppress the attendant flora (predominantly E. coli). All inoculated cultures are placed in an incubator at  37 °C.

Aronson’s medium consists of 2-3 per cent of meat-peptone agar to which sucrose and destained fuchsine are added.

Monsur’s alkaline taurocholate-tellurite-gelatine-agar medium contains 10 g of trypticase, l0 g of sodium chloride, 50 g of sodium taurocholate, 30g of sodium carbonate, 1 g of gelatin, 15 g of agar-agar, and 1 L of distilled water.

TCBS (thiosulphate-citrate-bromthymol sucrose) is manufactured in the form ready for use; 69 g of the dry medium is taken per 1 L of distilled water.

Stage II. Some 5-6 hours after inoculation examine the film on the peptone water. To do it, tilt the test tube or the vial so that a delicate bluish film is attached to the wall. Prepare smears from the  film or the surface of the medium, stain them by the Gram method, evaluate motility, and conduct presumptive slide agglutination test with 0-cholera (0-1) serum diluted 1:100 or the reaction of cholera vibrio immobilization with 0-cholera serum. The results of the latter are estimated by phase-contrast microscopy. Inhibition of vibrios motility and the formation of agglutinate occur within 1-2 min.

On the basis of the results obtained give a second preliminary result referring to the motility of the vibrio and its relation to the agglutinating serum.

Subculture the material from the film onto plates with alkaline agar or selective medium and simultaneously onto the second peptone water and look for changes in 5-6 hrs.

Stage III. Some 10-16 hrs after inoculation, examine the growth in the second enrichment medium (peptone water) and on the plates with the culture of the native material. The film formed on the peptone water is examined as described above.

On an alkaline agar the cholera vibrio grows with the formation of round, smooth, flat, bluish, homogeneous colonies which are 1–2 mm in diameter, transparent in the transmitted light and have smooth edges. They are oily in consistence, are readily removed and emulsified. Examination of the material from convalescents, bac­teria carriers, and individuals treated with antibiotics may reveal atypical colonies.

On Aronson’s medium colonies of cholera vibrios are scarlet in the centre and pale-pink or colourless at the periphery. On Monsur’s medium colonies are transparent or semitransparent, or they may be of a greyish-black colour with turbid edges. On the TCBS medium they appear as flat and yellow against a bluish-grey background.

The selected colonies are introduced into test tubes with Oikenitsky’s medium or onto an agar slant for enrichment of pure culture and placed in an incubator.

Preliminary identification of cholera vibrios grown on plates with solid media is based on the study of cultural and morphological characteristics and on a presumptive slide agglutination test with 0-cholera serum diluted 1:100 and with Ogawa’s and Inaba’s sera in a 1 to 60 dilution, which is carried out to determine the serovar.

If the examination demonstrates signs typical of the cholera vibrio, a third preliminary answer about the positive result of the investigation is issued. Some material from the typical colonies may be transferred to a broth; then, using a 3-4-hour old culture, perform a standard agglutination test, check fermentation of carbohydrates, and determine whether the isolated culture belongs to Group I according to Heiberg and whether it is liable to phagolysis by cholera phages C and El Tor 2. If the results are positive, an answer concerning the isolation of the causative agent is given within 18-24 hrs from the beginning of the study.

Stage IV. After the results of the standard agglutination test and the reaction of phagolysis and fermentation of carbohydrates by 3-4-hour broth culture have been analysed, a preliminary conclusion about the isolation of the cholera vibrio is made. Plates with the inoculated culture on the second peptone water are examined, using the scheme which is employed in examining the plates with the culture of the native material. On Oikenitsky’s medium the vibrio breaks down sucrose without gas formation and does not ferment lactose (reddening of the medium in the column without gas formation).

To distinguish vibrios from homogeneous species of microorganisms (Aeromonas, Pseudomonas, Plesiomonas), a number of tests may be employed: the oxidase test, glucose oxidation-fermentation reac­tion, the “strand” test (Table ).

Differential-Diagnostic Signs of Vibrios and Related Types of Bacteria

The oxidase test consists of placing a solution of paraaminodimethyl–aniline and alpha-naphthol onto the culture in a Petri dish or onto a meat-peptone agar slant.

To carry out the oxidation-fermentation test, medium with the following composition (per 100 ml) is prepared: 2.0 g of peptone; 5.0 g of sodium chloride;   0.3 g of potassium hydrophosphate; 3.0 g of agar-agar; bromthymol blue (1 per- cent aqueous solution).

Dispense the medium in 3-4-ml portions into 13 X 100 mm test tubes and sterilize for 15 min at 120 °C. After that, add to the tubes 10 per cent glucose solution sterilized by nitration to adjust to the final concentration of 1 per cent. Inoculate the test culture into two tubes with the above mentioned medium- Into one of the tubes pour a layer (1.5-2 cm) of sterile petrolatum oil. Incubate the test tubes for four days and note acid and gas formation. Darkening of the medium in the open test tube indicates oxidation and in the tube with the oil, fermentation. Gas formation is sometimes observed.

The “strand” test. Onto a glass slide, place a drop of 0.5 per cent solution of sodium desoxycholate in buffer isotonic saline. Into this drop, introduce a loop-ful of the tested culture of vibrios grown on a solid nutrient medium and mix. If the result is positive, the mixture becomes transparent, acquires mucilaginous consistency, and trails the loop in the form of a strand in the first minutes after its preparation.

To differentiate between the classical cholera vibrio and the El Tor vibrio, utilize tests determining the sensitivity of cholera vibrios toward phage’s and polymixin and the ability of vibrios to agglutinate chick erythrocytes.

Sensitivity to diagnostic phages is determined by streaking onto a plate with a culture of whole cholera phages C and EI Tor 2, with ten-fold dilutions of the above. Phage C is active only toward the classic cholera vibrio, while the El Tor 2 phage is active toward El Tor biovar. The presence of lysis in the form of one “sterile” spot or a group of small spots in the place of phage introduction is assessed as a positive result.

Sensitivity to polymixin is determined by inoculating the isolated culture onto Petri dishes with nutrient agar containing 50 U of polymixin M or B in 1 ml of nutrient medium. El Tor vibrios are insensitive to antibiotics and show good growth on the dishes, un­like the classic cholera vibrios.

Haemagglutination of chicken erythrocytes is performed on a glass slide. In a drop of isotonic sodium chloride solution, comminute a loopful of 18-hour culture of the vibrio and add a drop of 2.5 per cent suspension of chicken erythrocytes. The cholera El Tor vibrio agglutinates the red blood cells within 1-3 min, whereas the classical biovar fails to induce any clumping.

Haemolysis of sheep erythrocytes (Greig’s test) occurs after their 2-hour incubation with broth culture of cholera El Tor vibrios at 37 °C. Yet, this sign is not stable and some strains of the El Tor biovar, similar to the classical cholera vibrio, display no haemolytic effect.

The Voges-Proskauer test is based on the ability of El Tor vibrios to form acetylmethylcarbinol, which is recognized by the fact that Clark’s glucose-phosphate broth becomes pink or ruby-red, following 1-3 day incubation of the inoculated cultures with addition of alpha-naphthol.

The hexamine test is performed with 24-hour broth culture of the vibrio a loopful of which is streaked onto 1 ml of a glucose-hexamine medium. Following incubation at 37 °C for 6-24 hrs, the El Tor vibrio alters the colour of the medium from green to yellow. The classical cholera vibrio induces no changes in the medium colour over this time.

Production of enterotoxin by the cholera vibrio is determined by means of a specific reaction of passive immune haemolysis. To carry out this reaction, re-suspend in 0.08 M phosphate buffer the erythrocytes from defibrinated sheep blood (after their triple washing). Prepare 10 per cent suspension of red blood cells in 0.02 M solution of the buffer. With a micropipette introduce 0.025 ml portions of 0.02 M phosphate buffer into agglutinating plates, then add two­fold dilutions of the antigen and 1 per cent suspension of erythrocytes. Cover the plates and place them into a 37 °C incubator for 30 min, add to each well (1.025 ml of antitoxic serum diluted 1:50 and 0.02 per cent bovine serum al­bumin, reincubate the culture for 30 min, then add 0.025 ml of complement and replace the culture into the incubator for 90 min. .Haemolysis is evaluated after 30 min of keeping the plates at room temperature. Simultaneously, one monitors non-immune haemolysis and the ingredients of the serum, antigen, and complement.

Isolation of non-agglutinating vibrios brings about the  necessity of studying their biochemical properties, namely: liquefaction of gelatine, splitting of 5tarch (Cadamot’s test), formation of indol from triptophane, reduction of nitrates into nitrites, as well as oxidase and decarboxylase activity. It is also necessary to classify the culture with one of the biochemical groups according to Heiberg.

Demonstration of cholera vibrios in water is of great importance for identifying the factors of infection transmission and conducting anti-epidemic measures. Using a saturated solution of sodium hydrocar-bonate, alkalize the water (900 ml) delivered to the laboratory to pH of 7.8-8.0, add 100 ml of basic peptone, pH 8.0 (peptone, 100 g, sodium chloride, 50 g, potassium nitrate, 1 g, sodium hydrocarbon-ate, 20 g, distilled water, 1000 ml), and dispense it into flasks or vials in 100-200-ml portions. Incubate the inoculated cultures at 37 °C for 5-8 hrs and then inspect them in the manner employed for studying other inoculated cultures in peptone water (vomited matter and faeces). The results are more reliable when the water tested is filtered through membrane filters. Large amounts of water (1.5-2.5 1) are examined and the deposit from the filters is transferred to peptone water (pH 8.0) and alkaline agar.

Rapid detection of cholera vibrios in drinking water. If water con­tamination with cholera vibrios is heavy (at least 100 vibrios per 1 ml), the agglutination reaction is utilized for their recovery. To the water to be assayed add weakly alkaline concentrated solution of peptone in a quantity sufficient to produce 1 per cent solution. With this mixture dilute the 0-cholera agglutinating serum from 1:100 to its titre. Use a mixture free of the serum as a control. Place the test tubes into an incubator and read the results of the test in 6 hrs. The reaction is considered positive it flocculation is observed upon serum dilution to half the litre or the litre.

Rapid recovery of the cholera vibrio in water may also be based on increase in the phage titre.

Rapid method of wide-scale screening for carriers. During an out­break of cholera wide-scale screening for carriers of the cholera vibrio is performed. When a large number of analyses is to be made in the laboratory, faeces from ten subjects are examined simultaneously. Faeces are collected with wire loops and placed into one flask con­taining 200 ml of peptone water and 0-cholera agglutinating serum which is diluted to half the titre. The flask is placed into a 37 “C incubator. In 3-4 hrs the multiplied cholera vibrios begin to agglu­tinate and fall to the bottom in the form of flakes. If this is the case, faecal material is taken from each of the ten individuals, and the examination is repeated with each sample.

Serological diagnosis of cholera is supportive and relies on de­tecting agglutinins and vibriocidal antibodies in the patient’s serum. It is recommended that paired sera obtained from the patients at a 6-8 day interval be used for these reactions. Titres of agglutinins and vibriocidal antibodies usually tend to increase simultaneously. The most sensitive test is demonstration of vibriocidal antibodies. The presence of agglutinating antibodies in the titre of 1:80-1:320 and vibriocidal ones in the titre of 1:1000 is considered diagnostically positive.

Campylobacter

Campylobacter (meaning ‘twisted bacteria’) is a genus of bacteria that are Gram-negative, spiral, and microaerophilic. Motile, with either unipolar or bipolar flagella, the organisms have a characteristic spiral/corkscrew appearance (see photo) and are oxidase-positive.Campylobacter jejuni is now recognized as one of the main causes of bacterial foodborne disease in many developed countries.At least a dozen species of Campylobacter have been implicated in human disease, with C. jejuni and C. coli the most common. C. fetus is a cause of spontaneous abortions in cattle and sheep, as well as an opportunistic pathogen in humans.

The genomes of several Campylobacter species have been sequenced, providing insights into their mechanisms of pathogenesis. The first Campylobacter genome to be sequenced was C. jejuni, in 2000.

Campylobacter species contain two flagellin genes in tandem for motility, flaA and flaB. These genes undergo intergenic recombination, further contributing to their virulence.Nonmotile mutants do not colonize.

Campylobacteriosis is an infection by Campylobacter. The common routes of transmission are fecal-oral, ingestion of contaminated food or water, and the eating of raw meat. It produces an inflammatory, sometimes bloody, diarrhea, periodontitis or dysentery syndrome, mostly including cramps, fever and pain. The infection is usually self-limiting and in most cases, symptomatic treatment by liquid and electrolyte replacement is enough in human infections. The use of antibiotics, on the other hand, is controversial. Symptoms typically last for five to seven days.

Cause

The sites of tissue injury include the jejunum, the ileum, and the colon. Most strains of C jejuni produce a toxin (cytolethal distending toxin) that hinders the cells from dividing and activating the immune system. This helps the bacteria to evade the immune system and survive for a limited time in the cells. A cholera-like enterotoxin was once thought to be also made, but this appears not to be the case. The organism produces diffuse, bloody, edematous, and exudative enteritis. Although rarely has the infection been considered a cause of hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, no unequivocal case reports exist. In some cases, a Campylobacter infection can be the underlying cause of Guillain–Barré syndrome. Gastrointestinal perforation is a rare complication of ileal infection.

campylobacter Questions and Answers

 

 

What is Campylobacter? What harm can Campylobacter bacteria cause? Are more people becoming ill from campylobacteriosis? Who is most susceptible? How can Campylobacter be controlled? What is FSIS doing to prevent Campylobacter infections? What is the best way to prevent Campylobacter infections?

“Campylobacter” bacteria are the second most frequently reported cause of foodborne illness. A comprehensive farm-to-table approach to food safety is necessary in order to reduce campylobacteriosis. Farmers, industry, food inspectors, retailers, food service workers, and consumers are each critical links in the food safety chain. This document answers common questions about the bacteria “Campylobacter,” describes how the Food Safety and Inspection Service (FSIS) of the U.S. Department of Agriculture (USDA) is addressing the problems of “Campylobacter” contamination on meat and poultry products, and offers guidelines for safe food handling to prevent bacteria, such as “Campylobacter,” from causing illness. Q. What is Campylobacter? A. Campylobacter [pronounced “kamp-e-lo-back-ter”] is a gram negative, microaerophilic bacterium and is one of the most common bacterial causes of diarrheal illness in the United States. Campylobacter jejuni, the strain associated with most reported human infections, may be present in the body without causing noticeable illness. Campylobacter organisms can be found everywhere and are commonly found in the intestinal tracts of cats, dogs, poultry, cattle, swine, rodents, monkeys, wild birds, and some humans. The bacteria pass through the body in the feces and cycle through the environment. They are also found in untreated water. Q. What harm can Campylobacter bacteria cause? A. Infection caused by Campylobacter bacteria is called campylobacteriosis and is usually caused by consuming unpasteurized milk, raw or undercooked meat or poultry, or other contaminated foods and water, and contact with feces from infected animals. While the bacteria can exist in the intestinal tracts of people and animals without causing any symptoms or illness, studies show that consuming as little as 500 Campylobacter cells can cause the illness. Symptoms of Campylobacter infection, which usually occur within 2 to 10 days after the bacteria are ingested, include fever, abdominal cramps, and diarrhea (often bloody). In some cases, physicians prescribe antibiotics when diarrhea is severe. The illness can last about a week. Complications can include meningitis, urinary tract infections, and possibly reactive arthritis (rare and almost always short-term), and rarely, Guillain-Barre syndrome, an unusual type of paralysis. While most people who contract campylobacteriosis recover completely within 2 to 5 days, some Campylobacter infections can be fatal, resulting in an estimated 124 deaths each year.  Are more people becoming ill from campylobacteriosis? A. The Foodborne Diseases Active Surveillance Network (FoodNet) found a decline, in the rates of infection in 2009 for Campylobacter (30% decrease), in comparison with the previous three years of surveillance (1996 to 1998). Still, according to the Centers for Disease Control and Prevention (CDC), campylobacteriosis causes an incidence of about 13 cases per 100,000 population diagnosed in the United States annually. Food Net is a collaborative project among CDC, the 10 Emerging Infections Program sites (EPIs), USDA, and the U.S. Food and Drug Administration (FDA). One of the objectives of FoodNet is to measure effectiveness of a variety of preventive measures in reducing the incidence of foodborne illness attributable to the consumption of meat, poultry, and other foods. Q. Who is most susceptible? A. Anyone may become ill from Campylobacter. However, infants and young children, pregnant women and their unborn babies, and older adults, are at a higher risk for foodborne illness, as are people with weakened immune systems (such as those with HIV/AIDS, cancer, diabetes, kidney disease, and transplant patients). Q. How can Campylobacter be controlled? A. Campylobacter can be controlled at a number of different points in the food production and marketing chain. On the farm: Good sanitary practices on farms, as recommended by USDA, minimize the opportunity for the bacteria to spread among animals and birds. Pasteurization of milk and treatment of municipal water supplies eliminate another route of transmission for Campylobacter and other bacteria. In the plant: Raw foods are not sterile, and there are no requirements that they be sterile. Food processing companies are accountable for following good, up-to-date manufacturing practices that minimize the opportunity for the spread of Campylobacter and other bacteria. At retail: A food recall is a voluntary action by a manufacturer or distributor to protect the public from products that may cause health problems or possible death. FSIS conducts a sufficient number of effectiveness checks to verify the recalling firm has contacted the distributor or retailer. Individuals: Reporting the problem is another way to control these bacteria and prevent others from becoming exposed to the source of contamination. Any individual that experiences symptoms of campylobacteriosis should contact a physician. Physicians who diagnose campylobacteriosis and clinical laboratories that identify this organism should report their findings to the local health department. What is FSIS doing to prevent Campylobacter infections? A. In its commitment to ensure that the public has a safe, wholesome food supply, FSIS is constantly working to improve the level of safety and reduce contaminants in the meat and poultry supply. In 1998, FSIS began enforcing a combination of Hazard Analysis and Critical Control Points (HACCP) based process control, microbial testing, pathogen reduction performance standards, and sanitation standard operating procedures which significantly reduce contamination of meat and poultry with harmful bacteria and reduce the risk of foodborne illness. Establishments can choose to include Campylobacter in their HACCP analysis. If Campylobacter is identified by the establishment as being reasonably likely to occur or if it becomes evident that it is an emerging problem in their process, FSIS would expect the establishment to have controls in place designed to address this microbial food safety hazard. HACCP clarifies the responsibilities of industry and FSIS in the production of safe meat and poultry products. The role of FSIS is to set appropriate food safety standards and maintain vigorous inspection oversight to ensure that those standards are met. USDA is supporting research to learn more about Campylobacter in food and how to control it. Finally, FSIS maintains extensive safe food handling education programs to help individuals prevent and reduce the risks of foodborne illness. Q. What is the best way to prevent Campylobacter infections? A. Meat and poultry can contain Campylobacter. However, the bacteria can be found in almost all raw poultry because it lives in the intestinal track of healthy birds. Improving safe food handling practices in kitchens will reduce the number of Campylobacter illnesses. Campylobacter bacteria are extremely fragile and are easily destroyed by cooking to a safe minimum internal temperature. They are also destroyed through typical water treatment systems. Freezing cannot be relied on to destroy the bacteria. Home freezers are generally not cold enough to destroy bacteria. To destroy Campylobacter and minimize the risk of foodborne illnesses: CLEAN: Wash Hands and Surfaces Often Wash your hands with warm soapy water for 20 seconds before and after handling food and after using the bathroom, changing diapers, and handling pets. Wash utensils, cutting boards, dishes, and countertops with hot soapy water after preparing each food item and before you go on to the next item. Consider using paper towels to clean kitchen surfaces. If you use cloth towels, wash them often in the hot cycle of your washing machine. SEPARATE: Don’t Cross-contaminate Separate raw meat, poultry, and seafood from other foods in your grocery shopping cart and in your refrigerator. If possible, use one cutting board for fresh produce and a separate one for raw meat, poultry, and seafood. Always wash cutting boards, dishes, countertops, and utensils with hot soapy water after they come in contact with raw meat, poultry, and seafood. Never place cooked food on a plate which previously held raw meat, poultry, or seafood. COOK: Cook to Safe Temperatures Use a clean food thermometer when measuring the internal temperature of meat, poultry, casseroles, and other foods to make sure they have reached a safe minimum internal temperature: Cook all raw beef, pork, lamb and veal steaks, chops, and roasts to a minimum internal temperature of 145 °F (62.8 °C) as measured with a food thermometer before removing meat from the heat source. For safety and quality, allow meat to rest for at least three minutes before carving or consuming. For reasons of personal preference, consumers may choose to cook meat to higher temperatures. Cook all raw ground beef, pork, lamb, and veal to an internal temperature of 160 °F (71.1 °C) as measured with a food thermometer. Cook all poultry to a safe minimum internal temperature of 165 °F (73.9 °C) as measured with a food thermometer. For optimum safety, cook stuffing separately to 165 °F (73.9 °C). Egg dishes, casseroles to 160 °F (71.1 °C). Fish should reach 145 °F (62.8 °C) as measured with a food thermometer. Bring sauces, soups, and gravy to a boil when reheating. Reheat leftovers thoroughly to at least 165 °F (73.9 °C). In addition, do not eat or drink foods containing raw, unpasteurized milk. CHILL: Refrigerate Promptly Keep food safe at home, refrigerate promptly and properly. Refrigerate or freeze perishables, prepared foods, and leftovers within 2 hours — 1 hour if the temperature is above 90 °F (32.2 °C). Freezers should register 0 °F (-17.8 °C) or below and refrigerators 40 °F (4.4 °C) or below. Thaw food in the refrigerator, in cold water, or in the microwave. Foods should not be thawed at room temperature. Foods thawed in the microwave or in cold water must be cooked to a safe minimum internal temperature before refrigerating. Marinate foods in the refrigerator. Divide large amounts of leftovers into shallow containers for quick cooling in the refrigerator. Don’t pack the refrigerator. Cool air must circulate to keep food safe. For more information about Campylobacter, see the Centers for Disease Control and Prevention (CDC) Web site at: http://www.cdc.gov/nczved/divisions/dfbmd/ diseases/campylobacter/

helicobacter

Helicobacter is a genus of Gram-negative bacteria possessing a characteristic helix shape. They were initially considered to be members of the Campylobacter genus, but since 1989 they have been grouped in their own genus. The Helicobacter genus belongs to class Epsilonproteobacteria, order Campylobacterales, family Helicobacteraceae and already involves >35 species.

Some species have been found living in the lining of the upper gastrointestinal tract, as well as the liver of mammals and some birds.The most widely known species of the genus is H. pylori which infects up to 50% of the human population. Some strains of this bacterium are pathogenic to humans as it is strongly associated with peptic ulcers, chronic gastritis, duodenitis, and stomach cancer. It also serves as the type species of the genus.

Helicobacter spp. are able to thrive in the very acidic mammalian stomach by producing large quantities of the enzyme urease, which locally raises the pH from ~2 to a more biocompatible range of 6 to 7. Bacteria belonging to this genus are usually susceptible to antibiotics such as penicillin, are microaerophilic (optimal oxygen concentration between 5 – 14%) capnophiles, and are fast-moving with their flagella.

Comparative genomic analysis has led to the identification of 11 proteins which are uniquely found in members of the family Helicobacteraceae. Of these proteins, 7 are found in all species of the family, while the remaining 4 are not found in any Helicobacter strains and are unique to Wollinella. Additionally, a rare genetic event has led to the fusion of the RpoB and RpoC genes in this family, which is characteristic of this family.

ecently, new gastric (Helicobacter suis and Helicobacter baculiformis) and enterohepatic (Helicobacter equorum) species have been reported. Helicobacter pylori is of primary importance for medicine; however, non-pylori Helicobacter species (NPHS), which naturally inhabit mammals (except humans) and birds, have been detected in human clinical specimens. NPHS encompass two (gastric and enterohepatic) groups, showing different organ specificity. Importantly, some species such as Helicobacter hepaticus, Helicobacter mustelae and, probably, Helicobacter bilis exhibit carcinogenic potential in animals. NPHS harbour many virulence genes and may cause diseases not only in animals but also in humans. Gastric NPHS such as H. suis (most often), Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis have been associated with chronic gastritis and peptic ulcers in humans and, importantly, with higher risk for MALT lymphoma compared to H. pylori. Enterohepatic species e.g., H. hepaticus, H. bilis and Helicobacter ganmani have been detected by PCR in but still not isolated from specimens of patients with hepatobiliary diseases. Moreover, NPHS may be associated with Crohn’s disease, inflammatory bowel disease and ulcerative colitis. The significance of avian helicobacters (Helicobacter pullorum, Helicobacter anseris and Helicobacter brantae) also has been evaluated extensively. NPHS such as Helicobacter cinaedi and Helicobacter canis can cause severe infections, mostly in immunocompromised patients with animal exposure. Briefly, the role of NPHS in veterinary and human medicine is increasingly recognised. However, despite the growing interest in the possible association between NPHS and the chronic hepatobiliary or intestinal diseases in humans, more studies are still required to prove the suggested association. Several other topics such as isolation of still uncultured species, antibiotic resistance and treatment regimens for NPHS infections and, last but not least, NPHS pathogenesis and possible carcinogenesis

ANSWERING YOUR QUESTIONS ABOUT HELICOBACTER

WHAT IS HELICOBACTER?

Helicobacter is a genus of spiral bacteria that amazingly is able to survive the severe acidity of the stomach. We have known of the existence of such bacteria since 1889 but it wasn’t until nearly 100 years later that their significance was realized.

Until the 1980s, stomach ulcers were treated with an assortment of antacids with the idea that excess acid had caused the ulcer. In fact, most stomach and duodenal ulcers of humans stem from infection with Helicobacter bacteria. Currently, this ulcerative infection is treated both with antacids and antibiotics specifically directed against Helicobacter.

HOW DOES HELICOBACTER CAUSE DAMAGE?

Very few organisms can withstand the extreme acidity of the stomach. The tissue of the stomach is protected by a layer of mucus into which bicarbonate is secreted as an acid neutralizer. The integrity of this mucus lining keeps us from being burned by our own stomach acid.

Helicobacter survives by using enzymes to create its own layer of protective bicarbonate. This little safety suit allows the bacteria to burrow into the stomach’s mucus layer. Its presence generates inflammation in the stomach tissue. Many patients are colonized by Helicobacter and do not develop symptoms; however, if Helicobacter penetrates deeply enough, it will bind to the mucus secreting cells of the stomach and disrupt their ability to produce normal mucus. Ultimately, the mucus lining is disrupted, stomach acid gains access to the stomach tissue, and burning results. Ulcers are thus formed. Making matters worse, Helicobacter organisms are able to stimulate extra acid secretion by the stomach tissue. More burning and more ulcers result and soon the patient is experiencing pain, nausea and/or vomiting. It is unclear what constitutes a few Helicobacter bacteria sharing the stomach with its host peacefully and numerous Helicobacter organisms disrupting the stomach lining integrity and causing disease. It is possible that without additional stomach disease (such as inflammatory bowel disease) or other factors (stress, anxiety), Helicobacter causes no trouble. Helicobacter organisms are often found in small numbers in normal stomachs.

Some Helicobacter species are also capable of producing toxins but the role of such toxins in this disease process is not clear.

Helicobacter seems to be one reason why an animal who has been stable with inflammatory bowel disease or some other stomach disease might suddenly get much worse.

DOES HELICOBACTER INFECTION CAUSE CANCER?

In humans, it appears that Helicobacter infection may indeed cause cancer. We know that Helicobacter infection represents a 400% risk increase for the development of stomach cancer for people. Pets, however, get infected with different Helicobacter species and the same association with cancer in these species has not been made.

DOES MY PET HAVE HELICOBACTER OVERGROWTH?

There are many excellent ways to determine if a pet’s chronic gastrointestinal problem is being complicated by Helicobacter infection.

• BIOPSY – While it is possible to miss Helicobacter if only certain areas of the stomach are colonized, biopsy is by far the most accurate test. This method not only detects the infection but also assesses the degree of inflammation and checks for cancer.

small purple “sticks” are Helicobacter organisms

 

• THE RAPID UREASE TEST – Some gastroenterologists will keep a special broth handy during the biopsy procedure. A spare tissue sample can be dropped in the broth and incubated for an hour. The presence of urease, the enzyme that creates Helicobacter’s protective bicarbonate layer, induces a color change in the solution. In this way, Helicobacter can be detected in an hour rather than after the 2 days it takes to obtain biopsy results.

test kit showing negative and positive results for Helicobacter

 

• PCR TESTING – This especially sensitive DNA testing can be used but is only available in a few centers. 

• BLOOD TESTS – antibodies against Helicobacter can be detected but their levels take months to decline even after the Helicobacter organism is long gone. This limits the usefulness of such testing. 

• BREATH TESTING – A radioisotope labeled meal is fed and the patient’s breath is tested for Helicobacter metabolites. This form of testing is easy to use for monitoring the eradication of Helicobacter, plus it is non-invasive. In humans, Helicobacter eradication is usually confirmed 4 to 8 weeks after treatment has been completed. With the breath test, a second biopsy or endoscopy is not needed. Unfortunately, this type of testing is not readily available for pets.

WHAT IS THE TREATMENT?

Treatment protocols generally consist of two antibiotics and an antacid and are referred to as “Triple Therapy.” Confusing matters is that there are many medication combinations referred to as “Triple Therapy” but at least they seem to all be effective. The following is a list of medications that have been combined in Triple Therapy protocols in the treatment of Helicobacter:

• Amoxicillin (an antibiotic) 

• Tetracycline (an antibiotic) 

• Pepto-Bismol (the bismuth actually accumulates in the Helicobacter cell wall and destroys the organism) 

• Flagyl (an antibiotic) 

• Omeprazole (an antacid) 

• Pepsid AC (an antacid) 

• Clarythromycin (an antibiotic) 

• Azithromycin (an antibiotic) 

• Erythromycin (an antibiotic)

CAN MY PET INFECT ME?

We do not currently know the answer to this question. We do know that there is at least one Helicobacter species capable of infecting both humans and cats. We know that cat ownership does not seem to represent an increased risk for Helicobacter infection in humans. Transmission of the disease is felt to be through contact with vomit or fecal matter.

 

Aeromonas infections

The genus Aeromonas consists of gram-negative rods widely distributed in freshwater, estuarine, and marine environments [1,2]. Aeromonas species grow at a range of temperatures, although they are isolated with increasing frequency during warmer months (May through October in the Northern hemisphere). Aeromonas species cause a wide spectrum of disease syndromes among warm- and cold-blooded animals, including fish, reptiles, amphibians, mammals, and humans.

 

 

scanning electron micrographs of A. hydrophila

he genus Aeromonas was re-categorized from the family Vibrionaceae to the family Aeromonadaceae in the mid-1980s, when phylogenetic evidence from molecular studies became available to support this distinction. The genus Aeromonas has been divided into two major groups:

• Motile, mesophilic species, including eight that can cause disease in humans (table 1).

• Non-motile, psychrophilic species that generally cause disease only in fish.

Aeromonas species are oxidase positive and ferment glucose. The organisms grow at a range of temperatures from 0 to 42ºC.

Bull’s-eye-like colonies of A. caviae on CIN agar at 48 h.

Aeromonas infections are caused by bacteria which are present in the water all of the time. Usually, when fish get sick with an Aeromonas infection, something has happened to make them susceptible to bacterial invasion. There are several species of Aeromonas which can infect fish. The first is Aeromonas salmonicida, which causes a disease called furunculosis in salmon and trout. This bacteria is not usually of concern for producers of warmwater fish and will not be discussed further in this publication. The two species of Aeromonas which do cause disease in warmwater fish are Aeromonas hydrophila and Aeromonas sobria. The difference between these two bacteria is of greater interest to scientists than of practical importance to producers; thus, they will be referred to collectively as Aeromonas infections or Motile Aeromonas Septicemia (MAS). Aeromonas infections are probably the most common bacterial disease diagnosed in cultured warm water fish. Usually, mortality rates are low (10% or less) and losses may occur over a period of time (2 to 3 weeks or longer). In these instances, some factor; usually stress, has caused the fish to become more susceptible to the bacteria. Common sources of stress are poor water quality, overcrowding, or rough handling. Some strains of Aeromonas are more virulent, which means that they possess special properties which enable them to cause more serious disease outbreaks. If these more damaging strains become endemic in a population of fish (which means that they are there all of the time and the fish develop an immunity to them), it becomes difficult to introduce new fish into the water body without suffering major losses of newly-stocked fish.  hydrophila in humans is an opportunistic pathogen associated with blood infections, wound infections, and diarrhea. Reports of wound infections have become more common recently and can cause severe damage possibly requiring amputation. Wound infections can be classified into 3 categories: cellulitis, myonecrosis, and ecthyma. Cellulitis is the most frequently encountered type of infection and involves inflammation of skin tissue. Myonecrosis is more serious and less common, involving the formation of lesions that can require ampuation if not treated agressively. Ecthyma can occur after a blood infection becomes septic and is usually fatal. These diseases are rare in humans, occuring mainly in people with weakened immune systems, and can be prevented by taking proper care of wounds, especially by not washing wounds with lake or river water. A. hydrophila is also considered a cause of diarrhea in humans, usually found in young children and people with weakened immune systems. A. hydrophila is resistant to penicillin and penicillin derivatives but several other antibiotics can be used to treat infections.Signs of Aeromonas infection There is no single physical or behavioral sign specific for Aeromonas infections. Infected fish frequently have: small pinpoint hemorrhages at the base of the fins or on the skin, distended abdomens, and protruding eyes. Internal signs include: fluid in the abdomen, swollen liver and spleen, and the intestines are distended and fluid-filled.

 

 

Cellulitis infection MyonecrosisSubmission of suspect fish to a diagnostic laboratory It is important to submit fish suspected of being infected with Aeromonas to a diagnostic laboratory to confirm the disease, and to determine the antibiotic sensitivity of the strain of Aeromonas causing the problem. In addition, because Aeromonas is a stress-mediated disease, it is not unusual to find that infected fish are heavily parasitized or concurrently infected with another systemic disease agent. Contact your county extension agent for assistance and information on where and how to submit samples for diagnostic services. Management of an Aeromonas outbreak When MAS, or any bacterial infection, is suspected in your fish, you should immediately submit a live, sick fish to the nearest diagnostic facility. If Aeromonas is diagnosed, you need to know what legal drug the isolate is sensitive to and whether or not other infectious agents are present. There are two antibiotics legal for the treatment of bacterial diseases of channel catfish. Both of these are administered in the feed. The first, Terramycin, has been available for many years and many strains of Aeromonas are resistant to it. If the bacteria is resistant to the drug then there is no benefit attained by feeding that medication. Terramycin~, an oxytetracycline product, is available in sinking feed only, and is fed for 10 days followed by a 21-day withdrawal time. The other product, Romet-30 , a potentiated sulfonamide, has only been available since 1985. It is available in a floating feed, and is fed for 5 days, followed by a 3-day withdrawal period. The withdrawal period is the time you need to wait after feeding the medicated feed for the last time until the fish can be sold for human consumption.In many cases, it may not be necessary to treat Aeromonas infections with medicated feeds. For example, if fish are heavily parasitized, they may resist the bacterial disease if the parasites are removed. Similarly, if disease susceptibility is attributed to poor water quality, then correction of the basic husbandry problem could result in a resolution of the bacterial disease outbreak. Keep in mind that the purpose of antibiotics is to keep disease-causing bacteria at bay long enough for the fish to heal itself. In addition, if the affected system is an indoor or closed system, good sanitation is essential to decrease the number of bacteria in the system.

There is no single physical or behavioral sign specific for Aeromonas infections. Infected fish frequently have: small pinpoint hemorrhages at the base of the fins or on the skin, distended abdomens, and protruding eyes. Internal signs include: fluid in the abdomen, swollen liver and spleen, and the intestines are distended and fluid-filled.

Submission of suspect fish to a diagnostic laboratory

It is important to submit fish suspected of being infected with Aeromonas to a diagnostic laboratory to confirm the disease, and to determine the antibiotic sensitivity of the strain of Aeromonas causing the problem. In addition, because Aeromonas is a stress-mediated disease, it is not unusual to find that infected fish are heavily parasitized or concurrently infected with another systemic disease agent. Contact your county extension agent for assistance and information on where and how to submit samples for diagnostic services.

 Campylobacter pylori is a newly described, spiral-shaped, gram-negative bacillus that is oxidase positive, catalase positive, and urease positive and grows slowly in culture. Although observed in human tissue at the beginning of the century, it was not cultured until 1982. Because there are significant morphological and genetic differences between this organism and other species of Campylobacter, it will probably be reclassified in a new genus. Current information indicates that the organism primarily resides in the stomach tissue of humans and nonhuman primates and may occasionally spread to the esophagus or other parts of the alimentary tract under appropriate conditions. Significant evidence has accumulated in the last several years to show that it causes gastritis, and there is mounting evidence that it may participate in the development of duodenal ulcers. It may also be associated with gastric ulcers and nonulcer dyspepsia. It can be detected in patients by culture of biopsy specimens or histological staining of biopsy tissue. Indirect evidence for the presence of the organism can be obtained by detection of urease in a tissue biopsy specimen, by urea breath tests, or by detection of specific antibody. It may not be necessary to implement these procedures for routine use, however, until the role of the organism can be defined better. Ultimately, the discovery of this organism may lead to radical changes in the diagnosis and treatment of gastric disease.

Campylobacter Jejuni and other Enteric Campylobacters

Clinical Manifestations

Campylobacter species cause acute gastroenteritis with diarrhea, abdominal pain, fever, nausea, and vomiting. Recently, Campylobacter infections have been identified as the most common antecedent to an acute neurological disease, the Guillain-Barré syndrome.

Structure

Campylobacter species are Gram-negative, microaerophilic, non-fermenting, motile rods with a single polar flagellum; they are oxidase-positive and grow optimally at 37° or 42°C.

Classification and Antigenic Types

Campylobacter species have many serogroups, based on lipopolysaccharide (O) and protein (H) antigens. However, only a few serogroups account for most human isolates in a given geographic region. C jejuni possesses several common surface-exposed antigens, including porin protein and flagellin.

Pathogenesis

The bacteria colonize the small and large intestines, causing inflammatory diarrhea with fever. Stools contain leukocytes and blood. The role of toxins in pathogenesis is unclear. C jejuni antigens that cross-react with one or more neural structures may be responsible for triggering the Guillian-Barre syndrome.

Host Defenses

Nonspecific defenses such as gastric acidity and intestinal transit time are important. Specific immunity, involving intestinal immunoglobulin (IgA) and systemic antibodies, develops. Persons deficient in humoral immunity develop severe and prolonged illnesses.

Epidemiology

C jejuni and C coli infections are endemic worldwide and hyperendemic in developing countries. Infants and young adults are most often infected. Disease incidence peaks in the summer. Domestic and wild animals are the reservoirs for the organisms. Outbreaks are associated with contaminated animal products or water.

Diagnosis

Observation of darting motility in fresh fecal specimens or of vibrio forms on Gram stain permit presumptive diagnosis; definitive diagnosis is established by stool culture, and occasionally by blood culture.

Control

Control depends on measures to prevent transmission from animal reservoirs to humans.

Helicobacter Pylori and other Gastric Helicobacter-like Organisms

Clinical Manifestations

Helicobacter pylori is associated with chronic superficial gastritis (stomach inflammation) and plays a role in the pathogenesis of peptic ulcer disease. Increasing evidence indicates that H pylori infection is important in causing gastric carcinoma and lymphoma. Acute infection may cause vomiting and upper gastrointestinal pain; hypochlorhydria and intense gastritis develop. Chronic infection usually is asymptomatic.

Structure

This Gram-negative curved or spiral rod is distinguished by multiple, sheathed flagellae and abundant urease.

Classification and Antigenic Types

The antigenic structures are not completely defined and no universal typing scheme has been developed; strains may be differentiated by genotypic methods including restriction endonuclease analysis, and polymerase chain reaction (PCR).

Pathogenesis

Helicobacter pylori is sheltered from gastric acidity in the mucus layer and a small proportion of cells adheres to the gastric epithelium. The microorganism does not appear to invade tissue. Production of urease, a vacuolating cytotoxin, and the cagA-encoded protein is associated with injury to the gastric epithelium.

Host Defenses

Local and systemic humoral immune responses are essentially universal, but are not able to clear infection.

Epidemiology

H pylori infection has a worldwide distribution; about 1/3 of the world’s population is infected. The prevalence of infection increases with age. The major, if not exclusive, reservoir is humans but the exact modes of transmission are not known. H pylori has now been isolated from feces and dental plaque.

Diagnosis

Examination of gastric biopsy or stained smears allows presumptive diagnosis; definitive diagnosis is made by culture. Recently, non-invasive techniques such as the urea breath test and serologic tests have been developed to diagnose H pylori infection, with accuracy exceeding 95 percent.

Control

Several indications have emerged for the use of antimicrobial therapies that eradicate H pylori infection. No vaccine is yet available.

Other Pathogenic Camplyobacter and Helicobacter Species

Campylobacter fetus causes bacteremia in compromised hosts and self-limited diarrhea in previously healthy individuals. Helicobacter cinaedi and H fennelliae cause enteric and extraintestinal diseases and are more common in homosexual men and in travelers.

Campylobacter and Helicobacter are Gram-negative microaerophilic bacteria that are widely distributed in the animal kingdom. They have been known as animal pathogens for nearly 100 years. However, because they are fastidious and slow-growing in culture, they have been recognized as human gastrointestinal pathogens only during the last 20 years. They can cause diarrheal illnesses, systemic infection, chronic superficial gastritis, peptic ulcer disease Helicobacter pylori is a Gram-negative organism that has a helical or spiral shape and has 6-8 flagella at one end. The size of the organism measures about 2-4 um x 0.5-1.0 um. H. pylori are found in a very acidic environments, at a pH of 2.0 or less. The bacterium has been cultured in microaerobic (low oxygen conditions) but it adapts to high oxygen at high culture densities. It is commonly found inside the lining of the stomach and the duodenum. H. pylori are a slow growing organisms that can cause peptic ulcers and gastritis that can lead to gastric cancer and gastric MALT (mucosa-associated lymphoid tissue) lymphoma.

It was first observed in 19th century that curved bacteria were living in the lining of the stomach, but growing and isolating the bacteria was neglected. H. pylori was later isolated in Perth, Western Australia by Barry Marshall and Robin Warren in 1983. They discovered that H. pylori were related to peptic ulcers. To prove this hypothesis, the organism was cultured from the stomach, and through conclusive studies was determined that H. pylori was the bacteria that caused peptic ulcers and gastritis [Tomb] In 2005, Marshall and Warren received the Nobel Prize in physiology or medicine for their discovery of the Helicobacter pylori.

Helicobacter pylori was initially named Campylobacter pylordis because it appeared that the organism was similar to other Campylobacters. It shared a similar appearance with Campylobacter jejuni. [Megraud] Using rRNA hybridation and sequencing, H. pylori was shown to be different from the Campylobacter genus. H. pylori was separated into its own genus Helicobacter in 1989. The Helicobacter name reflects the appearances of the organism. However, the bacteria are helical in vivo, but often rod like in vitro. [Marshall]

The genomes of two strains have been completely sequenced: H. pylori 26695 and J99. Both were sequenced using a random shotgun approach from libraries of cloned chromosomal fragments of ~2.5kb. The 26695 genome was 24kb larger than the J99, but both of the genomes had GC% of 39%. Both genomes had similar average lengths of coding sequences, coding density and the bias of initiation codons. The origin of replication of the genome J99 was not clearly identifiable. [Mobley]

The genome of Helicobacter pylori strain “26695” is circular and contains 1,667,867 base pairs, and strain “J99” contains 1,643,831 base pairs. The chromosome of the organism contains genes that encode the urease gene cluster, cytotoxins in the membrane, and the cag pathogenicity island. In 1989, CagA gene was found and identified as the marker strain of the risk of peptic ulcers and gastric cancer. The CagA pathogenicity island recognizes the type IV secretion system, which CagA proteins are moved to the host cells. The DNA content of H. pylori has GC range of 35-38% which categorized itself to the Campylobacter species. However, the comparisons of the 16S ribosomal RNA showed that H. pylori were different from Campylobacter but similar to Wolinella succinogenes which its GC range was 42-49%. H. pylori were placed into its own genus, Helicobacter after the analysis of the ultra structure, fatty acid composition and biochemical tests which proved different for H. pylori and W. succinogenes.

The analysis of H. pylori sequences specifies the diversity and the development of the organism. The genome contains sequences that encode for the membrane proteins. For example, the F1F0 ATP synthase complex, various oxidoreductases such as cytochrome o, and some transporters. Sequences show that H. pylori contians a “well developed systems for motility, for scavenging iron, and for DNA restriction and modification\ Helicobacter pylori are capable to uptake DNA from other H. pylori. Due to the uncertainty of the strain linkages, recombination occurs because of the repetitive DNA sequences, which allows high frequency deletion and duplication and mismatch in-between the strands. Lack of mismatch repairing can increase in frequency of random variation but it can also convert the gene which can bring down the diversity of the organism.

The motility of H. pylori depends on the flagella which is driven by the proton motive force. The motility and the shape of the bacteria is specifically adapted to the gastric mucus. The flagella have a molecular weight of 50,000-62,000. The shape helps the bacteria to move easily in viscous environments. They do not only provide motility but they have bulbs on the ends of the flagella which favors the adhesion. The flagella are 4um in length (average), and the diameter of each flagella is 30nm.

The genome of H. pylori has homologs for all the enzymes required for the assembly of peptidoglycan by the use of precursors of the cytoplasmic synthesis. After the transport through the cytoplasmic membrane, the precursors are imbedded into the peptidoglycan layer by penicillin-binding proteins. The peptidoglycan of H. pylori differs from that of E. coli. H. pylori peptidoglycan has muropeptides with pentapeptide side chains that ends with glycine. Through the genome, F0 proton-channeling complex (a,b,c subunits) and catalytic, peripheral headpiece of the F1 have all been identified.Through the analysis of the genomes of 26695 and J99, H. pylori do not use complex carbohydrates as energy sources. The only carbohydrate used by H. pylori is glucose. It is metabolized via the Entner Douderoff pathway (reaction that catabolize glucose to pyruvate using glycolysis or pentose phosphate pathway). But it is likely used for anabolic biosynthesis rather than catabolic production. The primary sources of pyruvate in H. pylori are lactate, L-alanine, L-serine, D-Amino acids rather than glucose or malate. It has been reported that fermentation of pyruvate produces acetate. J99 contains homologs to the pta (phosphate acetyl transferase) and ackA (acetate kinase) genes. The 26695 pta has a frameshift mutation which inactivates the gene product.

The urease is a potent virulence factor for H. pylori. Urease is central to H. pylori’s metabolism and virulence and helps the microorganism colonize the gastric mucosa. Urease: NH2C=ONH2 + H2O —> NH3 + NH2-COOH and NH2COOH + 2H2O —> NH3 + H2CO3. This reaction increases in pH. H. pylori synthesize a huge amount of urease. They use it to convert urea into ammonia and bicarbonate to counteract the low acidity of the stomach. With high urease activity, H. pylori can protect the bacterium from acid damage by buffering the cell and the environment. The hydrolysis of urea molecules in the gastric juices creates ammmonia which acts as an acceptor for the H+ ions to increase the local pH. H. pylori demonstrate the highest activity of acidity. The enzyme must be consistently available for the organism to survive in the acidic environment. The defense of the body cannot fight H .pylori because killer T cells and white cells cannot easily get through the lining of the stomach. As the defense cells die, the H. pylori feed off the superoxide radicals on the stomach lining of the cells.

Helicobacter pylori is found commonly in the lining of the stomach and the duodenum because they adapt well to the acidic, low pH environment. Urease is the central metabolism of H. pylori. In order to survive, the organism uses urea to produce ammonia and bicarbonate to neutralize the acid in the stomach. The metabolic products from H. pylorican alter the host, and change the acidity of the environment and increase the supplements of nutrients to colonize the stomach.

Helicobacter pylori are known for peptic ulcers and gastritis. The organism weakens the mucous lining of the stomach and allows acid to enter to the sensitive coating of the stomach. The acid and the bacteria irritate the lining and cause an ulcer. It is very common to humans, and animals. Half of the population is infected by H. pylori. Since it is a slow growing bacterium, many people have the organism but are asymptomatic, that is they don’t get ulcers or gastritis. H. pylori are able to adapt and survive in the acidic environment of the stomach because the conversion of urea to bicarbonate and ammonia neutralizes the gastric acid. The motility of the bacteria, the flagella and the shape favors colonization which allows spiral shape to excavate through the lining.

The protein cagA has an increasing host response. The function is unknown but it is part of the pathogenicity island which has a region that contains 40 genes, and appears to affect virulence. This island contains DNA with different bases compared to the rest of the genome. CagA is coexpressed with VacA. VacA is known as a gene that is mosaic. It shows a clear pathway for the secreted toxins that contribute to the virulence and the colonization in many ways. Another gene that is mostly identified with virulence is iceA. iceA shows a clear relationship between expression and clinical outcome of the genes. There are number of membrane inserted proteins involved with the export of proteins within the pathogenicity island. It is sometimes difficult to detect the infection of H. pylori by using its genomic base. In Asia, CagA is not the marker for pathogenesis in contrast to Western countries.

Several spiral shaped bacteria looking like H. pylori have been found in the gastric mucosa in animals like cats, dogs, baboons and ferrets. They are often called gastric Campylobacter like organisms, but they do not cause gastritis or ulcer. Macaca mulatta, M. nemestrina and baboons seem to be the only animals that naturally harbor H. pylori. Using ELISA and immunoperoxidase staining, the antibodies of the H. pylori was found in serum. Most people with H. pylori do not show symptoms, usually they are asymptomatic. But people with the infection are likely to develop peptic ulcers. Symptoms in patients can be pain in the upper abdomen, nausea, vomiting, loss of appetite, and indigestion.

It is found that H. pylori, has a Lys gene that is an autolytic (break down of tissue) enzyme that degrades the walls of Gram-positive and Gram-negative bacteria. It was found in the unrelated clinical strain. The gene is expressed in vitro, and lytic activity is on both Gram-positive and negative cell walls. The hydrolytic action of the protein was confirmed by the clone and the expression of the gene.

Helicobacter pylori flagella: antigenic profile and protective immunity.

Recent research of the vaccine for H. pylori has been tested on mice. The colonization of the organism is hard to express because H. pylori has a “reproducible induction of sterilizing immunity.’’ Since motility is crucial for this organism by using its flagella, researchers hypothesized that the vaccine which targeted the flagella would improve the protection and reduce the colonization of this organism. To prove this hypothesis, the vaccine was tested on mice and it was observed that immunized mice with whole cell lysate cultivated for the flagella sheath proteins had reduced the colonization of the organism. However, it is indicated that the proteins of the flagella are not evident in “whole cell lysate and shows the differences in antigenicity of the whole cell lysate antisera.”

 

Primary antibiotic resistance in Helicobacter pylori strains isolated iorthern and central Italy.

To determine the antibiotic resistance in the strains of H. pylori, researchers used the two strains that were isolated in Italy. The two strains were isolated in two locations, Bologna, Northern Italy and Rome, Central Italy. The strain was isolated from patients who never got treated for the infection. The antibiotic resistance that was tested on the isolated strain was clarithromycin, metronidazole and levofloxacin, and the purpose was to break the inhibitory concentration point of the strain. The 255 strain had a resistance rate of 16.9%, 29.4%, and 19.1% for clarithromycin, metronidazole and levofloxacin. The patients who had non ulcer dyspepsia had a higher resistance rate in clarithomycin. Italian patients had higher resistance in metronidazole and old patients had higher resistance in levofoxacin. The levofoxacin resistance was more likely to appear in a strains with either clarithomycin or metronidazole resistance. The study of the three antibiotic resistances that were tested was in a very high rate.

 

Targeting Helicobacter pylori in gastric carcinogenesis.

Genes associated with virulence located in the pathogenicity island has been identified with being related to the risk of gastric cancer. Recent studies show that H. pylori was recognized with “both bacterial and host factors.” The host’s gastric inflammatory is affected by the virulence and the cytotoxin associated genes which mediate the cytokine receptors that trigger the risk of having gastric cancer. They targeted the organism with antibiotics and indicated that it may prevent the gastric cancer, but only to patients who have not yet developed “preneoplastic lesions”. The best way of preventing gastric cancer is to target the organism with vaccination, and can lead to gastric carcinoma

References:

1. Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002, P. 223-225, 235-241, 

2. Essential of Medical Microbiology /Wesley A. Volk and al. / Lippincott-Raven Publishers, Philadelphia-Ney-York, 1995, 725 p.

3. Hadbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed. by Yu.S. Krivoshein, 1989, P. 96-105.

cholera. Campylobacter, helicobacter and aeromonas infections.