In this article we will discuss about Vibro Cholera:- 1. Morphology and Staining of Vibrio Cholera 2. Cultural Characteristics 3. Transport and Enrichment Media 4. Biochemical Reaction 5. Laboratory Diagnosis 6. Treatment 7. Prevention and Control.
Contents:
- Morphology and Staining of Vibrio Cholera
- Cultural Characteristics of Vibrio Cholera
- Transport and Enrichment Media of Vibrio Cholera
- Biochemical Reaction of Vibrio Cholera
- Laboratory Diagnosis of Vibrio Cholera
- Treatment of Vibrio Cholera
- Prevention and Control of Vibrio Cholera
1. Morphology and Staining of Vibrio Cholera:
Vibrio cholera are short, curved, comma shaped gram-negative bacilli which can be readily stained by aniline dyes. They measure about 1.5 µ 0.2-0.4 µ in size, have rounded or slightly pointed ends. On subculture, the comma shape is lost.
In stained films of fresh mucus from acute cholera case, they appear as “fish in stream” (Fig. 34.1). Because of single polar flagellum, they are actively motile. This type of motility is called as “darting motility” and suggests a “swarm of gnats“. They do not form spores and capsules.
2. Cultural Characteristics of Vibrio Cholera:
Vibrio cholera are predominantly aerobic and their growth is best at an optimum pH 8.6 and temperature 37°C and is inhibited by 7 percent sodium chloride. They grow very well on ordinary alkaline media.
On alkaline nutrient agar, the colonies are moist, translucent, round disks (1-2 mm in diameter) with a bluish tinge in transmitted light and a distinctive odour, On MacConkey’s agar, their colonies become reddish on prolonged incubation as V. cholera are late lactose fermenters.
On blood agar, the greenish zone initially appears around the colonies and later becomes clear due to haemodigestion. In gelatin stab culture, napi-form or funnel shaped liquefaction occurs in 3 days at 22 C. In alkaline peptone water, a fine surface pellicle appears within 6 hours and breaks on shaking.
On alkaline bile salt agar (BSA), Colonies are similar to those on nutrient agar. On Monsur’s gelatin taurocholate trypticase tellurite agar (GTTA) medium the colonies are small translucent with a greyish black centre (due to tellurite reduction) and a turbid halo due to gelatin liquefaction.
Thiosulphate citrate bile and sucrose (TCBS) medium can be used though it is not cost effective. On this medium, colonies convex yellow; because of the acid, from fermented sucrose, which turns the indicator, bromothymol blue of this medium into yellow. These yellow colonies may become green on prolonged incubation.
“String” test can be used to identify V. cholera colonies. A loopful of the growth is mixed with a drop of 0.5% sodium desoxycholate on a slide. If the mixture looses its turbidity, becomes mucoid and forms a string when the loop is drawn away slowly, the test is positive.
3. Transport and Enrichment Media of Vibrio Cholera:
Venkatraman and Ramakrishnan (VR) medium (crude sea salt 20 g; peptone 5 g in 1000 ml distilled water, pH 8.6); Cary Blair medium (sodium chloride, sodium thioglycollate, disodium phosphate and calcium chloride) are suitable transport media whereas alkaline peptone water and Monsur’s taurocholate tellurite peptone water are good transport and enrichment media.
4. Biochemical Reaction of Vibrio Cholera:
V. cholera ferment glucose, mannitol, maltose, mannose and sucrose, but lactose is fermented late. Indole is formed and nitrate is reduced to nitrite. These two properties can be demonstrated by “Cholera Red Reaction” which is tested by adding a few drops of sulphuric acid to a 24 hours peptone water culture.
A reddish pink colour develops due to the formation of nitroso-indole. This reaction is not specific as other nonpathogenic vibrio’s share this reaction.
A positive oxidase test is a key step in the preliminary identification of V. cholera. A small strip of filter paper, soaked in the oxidase reagent (freshly prepared 1 per cent tetramethyl-p-phenyl-enedi-amine hydrochloride solution), is placed on a microscopic slide.
The colony to be tested is fished out with a platinum loop and is smeared on this paper. In a positive reaction, the smeared area turns purple in 10-60 seconds.
The classical V. cholera are Voges-Proskauer (VP) negative and non-haemolytic while el tor vibrio is VP positive haemolytic. Equal volume (0.5 ml) of 24 hours heart infusion broth culture and 1 per cent sheep erythrocyte suspension are mixed and incubated at 37°C for 24 hours and examined for haemolysis after holding at 4°C overnight.
The classical V. cholera are agglutinated by O serogroup 1 antiserum but other vibrio’s, similar to V. cholera, which do not agglutinate with O serogroup 1 antiserum are called as non-agglutinating (NAG) Vibrio or non-cholera vibrio (NCV) which are not of much medical importance.
The main serotypes are Ogawa, Inaba and Hikojima. Recent pulsed field get electrophoresis (PFGE) is more discriminating serotyping of Vibrio cholera.
Chick Red Cell Agglutination Test:
A loopful culture is emulsified in a drop of saline on a slide and a drop of 2.5% chick erythrocyte suspension is added, erythrocytes will clump if test is positive. All el’ tor V. cholera are positive, but classical V. cholera are negative.
Sensitivity to Polymyxin B is done by disk diffusion method using antibiotic disks. All classical V. cholera are sensitive to Polymyxin but el’ tor V. cholera are not. Sensitivity to Cholera phage group IV is a now available test to differentiate between classical and el’ tor V. cholera. Only classical V. cholerae lysed Mukherjee’s group IV phage at routine test solution.
Cholera enterotoxin (exotoxin) is a protein which is composed of two subunits—A (active) B (binding). Single subunit is not toxic; but mixture of these 2 subunits is toxic. Subunit B binds to the receptor of the intestinal mucosa. Subunit A can be dissociated into A1 and A2. A1 subunit activates adenyl cyclase in the intestinal mucosa.
Pathophysiology of Cholera:
Human infection is by ingestion of food or water containing 10–5 to 10–10 V. cholera, in contrast to Salmonella Shigella in which ingestion of 10–2 to 10–5 organisms can induce the infection. Those V. cholera, which escape gastric acidity, multiply in the alkaline small intestinal content. They do not invade into the gut wall and blood stream, hence there is no damage of the intestinal mucosa.
As a result of activation of adenyl cyclase cAMP levels are raised in the gut epithelium, hence there is outpouring of large volume of fluid into the lumen of the intestine causing dehydration, rice water diarrhoea, loss of electrolytes.
The death may ensure due to hypovolemic shock within 24 hours, el’ tor V. cholera, though highly infectious, causes mild and asymptomatic infection than those with classical V. cholera. In the seventh pandemic classical V. cholera has been replaced by el’ tor V. cholera; the previous pandemics were due to classical V. cholera, V. cholera 0139 is a new serotype which first appeared during 1992 in Southern India.
An outbreak of cholera due to V. cholera 0139 in Bangladesh was reported in December 1992. The same serotype was isolated in April 1993 from Cholera cases in Thailand indicating that it has the potential for extensive geographical spread.
5. Laboratory Diagnosis of Vibrio Cholera:
Stool can be collected in the acute stage by rubber catheter, rectal swab; on a clean paper of leaf; it should never be collected from the bed-pan which may still contain disinfectants, used for disinfection, that may destroy V. cholera. For rapid diagnosis, the darting motility of V. cholera is inhibited by the addition of the specific antiserum. This immobilisation test can be done under dark ground illumination.
Specimens, media, colonies suggestive of V. cholera can be tested by slide agglutination with V. cholera subgroup 01 antiserum; this agglutination test can be repeated by using mono-specific Ogawa and Inaba antisera for serotyping.
If positive, the isolates should be differentiated from el’ tor V. cholera. A novel rapid, colorimetric immunodiagnostic kit, Cholera SMART can detect directly V. cholera in clinical specimens similar to recent co-agglutination test and PCR.
6. Treatment of Vibrio Cholera:
The dehydration should be compensated immediately by Oral Rehydration Therapy (ORT) (Fig. 34.3). In resting healthy gut (left) absorption roughly equals secretion. As a result of action of cholera endotoxin (centre) secretion greatly exceeds the absorption with net loss of a large quantity of isotonic fluid.
When glucose electrolyte solution is administrated orally (right) glucose facilitates fluid absorption and counter-balances toxin induced fluid secretion and fluid balance is maintained. Antibacterial therapy is of secondary importance.
(i) Prophylaxis-Killed Vaccine:
The initial trials were with parenteral, killed whole cell vaccines. These vaccines showed protective efficiency ranging 30-80% lasting several months to one year.
(ii) Oral Vaccine:
As only A subunit is responsible for toxin mediated pathophysiology, A subunit gene has been deleted from a classical Inaba strain retaining B subunit. This modified strain provides important protective efficiency with minimal side-effects.
Whole cell/B subunit (WC/BS) Oral vaccine is a mixture of killed classical and el’ tor biotypes and purified B subunit and has shown in Bangladesh a high level of protective efficiency with few side effects, although protection only lasts for six months.
(iii) Live Vaccine:
Recombinant DNA vaccine with expression of V. cholera O1 in attenuated strain of Salmonella typhi Ty21 has been developed. Ty21 a as a carrier bacteria does cause significant side effects. It colonizes Peyer’s patch induces Ig response by local immune system.
Chemoprophylaxis:
It is useful when attendants in endemic areas are administrated with antibiotics.
7. Prevention and Control of Vibrio Cholera:
(A) General Measures:
For the prevention of cholera, like enteric fever, it is necessary to introduce:
(a) Large-scale purification of water supplies;
(b) Better sanitary methods for the disposal of sewage;
(c) An isolation ward for all infected patients with the facility for the disinfection of their excreta; besides, the carriers and contacts should be followed up.
(B) Specific Measures:
Active Artificial Immunization:
(i) For active immunization, a killed vaccine, containing 8,000 million V. cholera per ml with equal numbers of Inaba and Ogawa strains, is widely used. Two injections are given at an interval of one month each. 50-60 per cent inoculated persons are protected against clinical attack and the immunity lasts for 3-6 months.
In mass immunization programme only one injection is possible and protects adults due to its action as a booster on top of prior natural immunization, but in children below 5 years of age, it is not effective.
The aluminium hydroxide and phosphate adjuvant vaccine induces a high degree of immunity in young children. The use of cholera toxoid as vaccine was not successful.
(ii) A non-living oral B. subunit-whole cell (BS-WC) vaccine has undergone field trial in Bangladesh, conducted by the International Centre for Diarrhoeal Diseases, Dacca, in 1985.
This vaccine was prepared by Institute Merienix, France, and National Bacteriological Laboratory, Sweden and contains:
(1) Oral cholera toxin B subunit
(2) Heat killed classical vibrio: Ogawa – 2.5 x 1010
(3) Heat killed classical vibrio: Inaba – 2.5 x 1010
(4) Formalin killed el’ tor vibrio: Ogawa – 2.5 x 1010
(5) Formalin killed el’ tor vibrio: Inaba – 2.5 x 1010 cells
Protection was 85% for 1 year and 60% for 2 years. BS-WC vaccine conferred significant protection against diarrhoea due to E. coli (LT-ETEC).
