TITLE OF THE PROJECT:
VULTURE DROPPINGS DO NOT SPREAD ANTHRAX

Introduction:

Vultures are most important scavenging birds. Although it is well known that due to scavenging action vultures prevents spread of anthrax, but according to some; they may spread anthrax through their droppings. Thus this experiment was done to find out whether vulture droppings contain anthrax bacilli.

In India increase of anthrax cases may be related to a fast declining vultures that are excellent scavengers. Wildlife scientists issued an alert that an unprecedented decline in the population of vultures across India might lead to the spread of zoonotic diseases from livestock to people.

India has lost nearly 90% of its vultures to an illness that wildlife research suspect is caused by an avian virus and massive expansion of the Indian poultry industry in recent years is a potential source of novel avian viruses for vultures. India lacks facilities for incineration and for sophisticated carcass processing. The efficient and rapid disposal of anthrax carcasses is a vital issue for anthrax control and if one cannot burn or bury the animals in this condition vultures are preferred scavengers than dogs, wolves, foxes etc.

Sometime human scavenging is a public health problem. Non-consumption of Parse corpses by vultures may have an impact. Parse Zoroastrians believe burning or burying the dead pollutes the air or land, and so they place corpses on towers for vultures to consume. At Mumbai’s biggest Parse Dhokmas many corpses lay unconsummated for months till nature acted on them.

Experiment and observations:

This study was done on vulture droppings around different roosts, which were present in Botanical Gardens, Kolkata. Droppings were present on dead leaves as well as on herbs, which were present, surrounding their roosts. The size of their droppings was big and in this way droppings of other birds could be differentiated. During our observation, we could not justify the previous statements by other workers that due to acid nature, droppings usually destroy the trees. Except that few top branches were affected by the weight of the vultures, no harm was done of the plants. Even herbs totally smeared with the droppings did not die, and they were as good as the other similar plants in that locality.

We have studied pH and bacteria in our laboratory, which are given below.

pH: Droppings were mildly acidic; pH ranges between 6.0 to 6.8.

Cultures: Only aerobic bacteriological cultures were made on nutrient agar medium with droppings and swabs taken from feathers. Following findings were noted. Opaque, 2-4 mm colonies, Gram’s stain showed gram negative bacilli ; translucent confluent colonies, Gram’s stain showed gram positive slender bacilli ; small opaque colonies, Gram’s stain showed gram negative filamentous bacilli ; very small opaque colonies, Gram’s stain showed gram positive bacilli ; big opaque colonies, Gram’s stain showed gram positive bacilli with spores . There was no colony similar to anthrax bacilli and they mainly consist of colibacilli, Bacillus sp. Other than anthrax bacilli, few micrococci and corynebacteria.

The most interesting finding was that there were plenty bacteriophages in the cultures creating plaque like gaps here and there in the colonies. Thus it may happen that these bacteriophages (viruses which infect bacteria) may kill pathogenic bacteria which may enter in the intestine of the vultures during feeding and thus they really act as a natural incinerator as stated by previous workers.

Conclusion as per the completed work so far:

Vulture droppings did not contain anthrax bacilli; the main reason is the bacteriophages, which were present in abundant number in the droppings. Thus it is unlikely that vultures can spread anthrax through their droppings.

ONGOING PROJECT TITLE:
SODIUM METASILICATE AS MOSQUITO LARVICIDE

Mosquitoes are controlled by public health agencies to prevent spread of mosquito borne diseases. There are different methods to control mosquitoes. Among them the larvicidal agents are an important group widely used for mosquito control. The two most important aspects of an ideal mosquito larvicidal agent are biodegradable nature and safe for human beings as well as for other non-target organisms in the environment.

Many safe larvicidal agents have been described recently (Perich et al, 1990; Prasad and Kalyanasundaram, 1991;Schafer and Mulligan, 1991; Floore et al, 1998;Su and Mulla, 1998; Murugan and Jeyabalan, 1999; el-Shazly and el-Sharnoubi, 2000; Hallmon et al, 2000; Massoud and Labib, 2000). However, recent problems regarding the use of malathion and opinions against use of all pesticide spraying creates a scope for search of other mosquito control agents which are somewhat related to nature.

After study of physical and chemical characters of sodium metasilicate it appears to us to be a probable good larvicidal agent particularly for an urban set up. It increases pH of the medium and it has got a detergent/surface tension lowering action; but within a short span of time, particularly at lower concentrations, it is automatically degraded to silica in an aqueous solution and can be incorporated in nature, as silica is abundantly present in nature. Therefore, it appears to us that these physico-chemical properties of sodium metasilicate can be exploited to kill mosquito larvae.

Conversion of sodium metasilicate to silica can also be done easily by addition of dilute (0.01%) hydrochloric acid at the final outlet of the urban sewer system or in water accumulated places. With these ideas we started experiments to prove this hypothesis.

Laboratory colonies of Culex quinquefasciatus (Diptera: Culicidae) was used in our studies. They were maintained at 27+/-2 degree Celsius and at 75% relative humidity under a 10:14 light dark photoperiod cycle. The required quantities of sodium metasilicate (Fluka, Switzerland) were mixed thoroughly with 200 ml of rearing water in 500 ml plastic troughs to prepare different concentrated solutions (0.5%, 1%, 2% and 4%). For control experiment one plastic trough containing only 200 ml of rearing water was taken .One hundred fourth-instar mosquito larvae were released into each trough.

Finely ground dog biscuit was added in each trough (1 g per day per trough) which acted as larval food. For pupation dried coconut midribs were placed over water as the substratum. At 24-h intervals dead larvae and pupae were removed and counted. Observations on percentages of larval mortality, percentages of pupation and adult emergence were recorded. The experiment was replicated five times.

Observations are summarized in the Table.

Table: Effect of sodium metasilicate on the larval mortality, pupation and adult emergence of Culex quinquefasciatus.

SM: Sodium metasilicate. ** 58% died after emergence. *** All died after emergence.

Thus in order to develop and utilize a new safe chemical agent – sodium metasilicate in mosquito control program as a potential larvicide four different concentrations (see Table) were investigated against larvae of Culex quinquefasciatus under laboratory conditions.

The data presented in the Table clearly indicates a significant larvicidal action of this chemical. Sodium metasilicate appears to be a good larvicidal agent because it is spontaneously degraded to silica in aqueous solution and silica is present in huge quantities in earth crust.

Thus presently we can use it in storm drains, roof gutters, unused swimming pools and in old tires. If any residual activity of it remains after the desired time schedule, it can be easily converted to silica after addition of a dilute (0.01%) hydrochloric acid. Thus our observations suggest that sodium metasilicate is a safe naturally occurring chemical larvicide which can be used in an urban set up very easily.

Progress report of another ongoing research project

Rapid Urease Tests(RUT): Specimens were placed one each in CLO slide, Paper strip urease test(RUTP) and silica gel slide(RUTG) for comparative studies.

CLO test is the commonly used RUT outside India. Thus comparative studies were done with this test.

Paper strip urease test(RUTP): In this test RUT reagent was made mixing 10ml of 10% urea broth in deionised water (pH 6.8), 10 mg KH2PO4 and 500 ul of 1% phenol red. The test filter paper strips were soaked with the solution. It is then dried in incubator at 37 C and kept at room temperature. The strips showed a shelf life of more than six months. The indicator in the strip showed mild yellowish colour but changed to pink when reagent became alkaline due to urease activity.

Silica gel slide(RUTG): The idea of silica gel was taken from one of our previous paper(29). In a plastic pouch 59 mg silicate(Na2SiO3), 25 mg urea, 0.1 mg phenol red is kept in a sealed cover, it can be kept at room temperature, just before endoscopy one should add 0.5 ml deionised water followed by 5 drops (250 ul) of 16% V/V aqueous solution of phosphoric acid (H3PO4). Its shelf life is about 18 months.

Experiments to find out optimum concentration of urea in these new media:
Media were prepared with different concentrations of urea ( 0.1%, 0.5%, 1.0%, 5.0%, 10.0%), pH and temperature were maintained at 6.5 and 37C respectively, Endoscopy biopsy materials of 66 patients with RUT positive tests were applied on these media and the time required for the change of colour was observed (Graph1).

Experiments to find out optimum pH for reaction in these new media:
Media were prepared with different pH levels (4.5, 5, 5.5, 6, 6.5, 7.0), urea and temperature were maintained at 5% and 37C respectively, Endoscopy biopsy materials of 28 patients with RUT positive tests were applied on these media and the time required for the change of colour was observed (Graph 2).

Experiments to find out influence of different ions, stabilizing agents, catalyzing agents on these new media:
Fe+++, Cu++, Co++, Zn++ salts (all at a concentration of 1mM,10 patients), Fe++, Mn++, Ni++ salts ( all at a concentration of 10 Mm, 10 patients), glycerol(1%, 5 patients) , 2 mercaptoethanol(5 patients), alpha keto glutarate ( both at a concentration of 0.05%)(5 patients) were used in these media and time required for colour change were compared with control media.

The rapid urease test (RUT) results:

The rapid urease test (RUT) results read at 1 hour compared to histology done outside in 83 patients are given in Table 1.

The sensitivity, specificity, positive and negative predictive values and accuracy of RUTP were 91%, 100%, 100%, 100%, 95% and of RUTG they were all 100% which appeared very significant. RUTP and RUTG were both stable for more than 6 months at room temperature.

Regarding standardization of RUTP and RUTG it was found that 5-10% urea concentration in the medium showed very good result. Regarding optimum pH we did not find a clear picture. However, it is better to keep the pH between 6.5 to 6.8, because this will help to observe rapid colour change from acidic to alkaline side.

Fe+++(8/10), Cu++(7/10), Co++(8/10), Zn++(8/10) ion inhibited urease activities in about 80% cases in test samples in comparison to controls and Fe++(3/10), Mn++(2/10), Ni(3/10) ions inhibited urease activities in about 30% cases in test samples. Glycerol(average –4 minutes), 2 mercaptoethanol(average –5 minutes) and alpha ketoglutarate (average –10 minutes) showed rapid enzymatic activities. All these changes were statistically significant ( P value significant at 0.01 level).

Discussion:

Helicobacter pylori, a gram-negative, microaerophilic, spiral-shaped bacterium, is an etiologic agent of human gastritis and peptic ulceration and is highly restricted to the gastric mucosa of humans. Urease, synthesized at up to 6% of the soluble cell protein, hydrolyzes urea, thereby releasing ammonia, which may neutralize acid, allowing survival of the bacterium and initial colonization of the gastric mucosa. Urease is an enzyme found in plants and bacteria but not in mammals.

The nickel metalloenzyme urease catalyses the hydrolysis of urea to ammonia and carbamate, and thus generates the preferred nitrogen source of many organisms. When produced by bacterial pathogens in the gastroduodenal region, urease acts as a virulence factor. At the sites of infection urease is known to enhance the survival of the infecting bacteria. Ammonia resulting from the action of urease is believed to increase the pH of the environment to one more favourable for growth, and to injure the surrounding epithelial cells. Bacterial urease gene clusters contain from seven to nine genes depending upon the species. These genes encode the urease structural subunits and accessory polypeptides involved in the biosynthesis of the nickel metallocentre.

Much work regarding the structure and function of urease enzyme has been done recently by Prof. Robert P. Hausinger of University of Minnesota. So far, three distinct mechanisms of urease gene expression have been described for ureolytic bacteria. Some species constitutively produce urease; some species produce urease only if urea is present in the growth medium; and some species produce urease only during nitrogen-limiting growth conditions. For either the urea-inducible genes or the nitrogen-regulated genes transcription appears to be positively regulated. In the nitrogen-regulated systems, urease gene expression requires Nac (nitrogen assimilation control), a member of the transcriptional activators. Urea dependent expression of urease requires UreR (urease regulator), also a member of the transcriptional activators. An evolutionary tree for urease genes of eight bacterial species is proposed (33).

Invasive and non-invasive methods are currently available for the detection of Helicobacter pylori . Invasive methods involve obtaining biopsy at endoscopy for histology, culture or urease tests. Histology of gastric antral biopsies for Helicobacter pylori remains the gold standard for the diagnosis of Helicobacter pylori infection, although the commercially available CLO test outside India and Helicochek, H-P test and Pylorichek in India are being used increasingly because of their simplicity and convenience.

To assess the current attitude to Helicobacter pylori among doctors Binek et al (34) made an elaborate study. The initial diagnostic methods, the indications for eradication therapy, the therapeutic regimen and its duration, together with eradication control, were indicated in questionnaires sent out to the members of the Swiss Society for Gastroenterology and Hepatology at the beginning of 1997.Helicobacter pylori was diagnosed mainly with a rapid urease test and/or histology. Peptic ulcer disease (100%), mucosa associated lymphoid tissue (MALT) lymphoma (94.5%) and therapy-resistant dyspepsia (78.7%) were clear indications for Helicobacter pylori eradication.

Histology, culture and CLO test have a sensitivity of 91%, 70% and 77-98% respectively and specificity of 100%, 100% and 96-97% respectively as observed by previous workers( 35). In this study RUTP and RUTG also showed a sensitivity of 91% ,100% and specificity of 100%, 100% respectively. Thus RUTP and RUTG when read at 1 hr, gave a comparable sensitivity and specificity levels with those of other tests. RUTG showed even better sensitivity and specificity levels in comparison to the widely used CLO test. The result shows that RUTP and RUTG are suitable for use as diagnostic test for Helicobacter pylori.

False negative RUTP may occur when very low numbers of Helicobacter pylori are present, or when the bacterium has a patchy distribution. Biopsy from both the antral and body mucosa may improve the sensitivity. False positive RUTs are very rare , in this series it is absent, and they usually not react before 3 hours because other urease producing bacteria produce much less urease than Helicobacter pylori (35). In this series, although we excluded patients with previous Helicobacter pylori eradication, recent antibiotics therapy or proton pump inhibitor therapy; however, a few patients may not given proper information and this may contribute to some of the false negative results.

The advantage of RUTP and RUTG is that they are inexpensive and easy to make. Moreover RUTP and RUTG can give quick result in comparison to other conventional tests. This is extremely useful in an outpatient setting where Helicobacter pylori eradication therapy can be started at once.

The most interesting point regarding RUTP and RUTG is that they can be used at room temperature and no cold chain is required for their storage and transport. Thus the endoscopist even in remote places can easily use them and thus it appears to be ideal for all developing countries. The rapid colour change in RUTG is due to the fact that there are wide molecular gaps in silica gel in comparison to the agar medium and silica may induce increased enzymatic activities ( 36).

Histology, although it is very good, can give us information on morphological features like the presence or absence of gastritis etc. It is more expensive and gives slower results. Culture is more demanding in terms of transport and more time-consuming, but may be worthwhile for resistant strains.

Non-invasive methods for detecting Helicobacter pylori include serological tests and urea breath test. Generally, both have good sensitivity and specificity: serology, 98% and 88% respectively, and urea breath test, 100% and 100% respectively ( 35). The lower specificity of serological tests is largely due to previous treatment of Helicobacter pylori and therefore it is not suitable within 6 – 12 months of eradication therapy, but is expensive and not commonly available in general practice.

In conclusion, RUTP and RUTG are inexpensive tests for Helicobacter pylori infection with good sensitivity and specificity and stable at room temperature. Sensitivity and specificity of RUTG is comparable only to urea breath test. They are useful in an outpatient setting because they give quick result and no cold chain is required for their storage. Their routine use can be recommended.