Dr. Ronald Ross received the Nobel Prize for Physiology or Medicine in 1902 for his outstanding achievement in unlocking the mystery of the malaria fever. He did his research on malaria in Secunderabad-Deccan (the twin city of Hyderabad-Deccan, South India). His research and the details published by him offered a very large picture of the population of India and the primitive methods he used to conduct his research and studies which ultimately unlocked the mystery of malaria.
Dr. Ross entered the medical service of India in 1881. He found malaria to be a more limited and localised disease; its outbreak appeared to occur among troops merely as a result of chill and fatigue. He noted in his research that the population of India in 1900 was nearly 300 million. The statistical data maintained by the government of India at the beginning of the 20th century showed that deaths in 1900 from fever alone were 4,919,591 – roughly about five million deaths yearly. That same year, out of the total of 305,927 persons from the army and jailed prisoners, 102,640 were admitted into hospital for malarial fever.
His medical curriculum made him aware of many facts, hypotheses, observations and postulates about the malarial fever and showed that malarial fever inflicted misery on mankind during the 17th, 18th and 19th centuries. Malaria, essentially an endemic and local fever, was found in the fertile, well-watered and luxuriant areas. Dr Ross learned that towards the middle of the 17th century physicians discovered that cinchona bark could be used as a drug to cure a certain class of fevers. The therapeutical benefits of this medicine and the pathological advances made later suggest that the physicians could cure malarial fever with the cinchona bark.
Physicians and researchers studying the causes of malarial fever at the beginning of the 19th century suggested that it is produced by an unknown poison, present in the stagnant water on the ground, which enters the human body. In the middle of the 19th century, physicians discovered a peculiar black granular substance in the blood and some tissues of those suffering from the malarial fever. This was identified as the malarial pigment or melanin.
It was later discovered that the melanin is produced within minute amoeboid parasites which live in the blood corpuscles of a malaria patient. This discovery led to the establishment of a new department of parasitic pathology. Later, minute clinical and pathological studies showed that many vertebrates have similar parasites in their blood. Additional research led to the development of a method for staining the parasites. A parallel research conducted at that time involved inoculating the blood of patients into healthy persons resulting in producing infection in healthy persons.
One of the clinical pathological studies conducted by physician Laveran clearly showed that a certain type of parasite was the cause of malarial fever. Many physicians of his time, including Marchiafava, Celli, Mannaberg, Metchnifoo, Canalis, Antolisei and others added important details to the malarial research. Their conclusions were used to establish several important laws concerning the whole group of organisms that caused malarial fever. The laws stated that Laveran’s (in honour of Laveran) parasite is the cause of malarial fever and that three varieties of human parasites caused quartan, tetrain and irregular fevers. This allowed for the treatment of the patients but did not help in the prevention of the disease on a large scale.
It was determined that the parasites occur in the human blood. The scientific research conducted to find the path that allowed the parasites to arrive in the blood was done very extensively by recognising the fact that malarial fever was essentially an endemic disease – a disease that does not easily spread from human being to human being like small-pox or plague. One of the hypotheses indicated that malarial fever was caused due to miasmata exhaled from the stagnant water.
Another theory was put forward that said that the soil possessed a poisonous effluent so powerful at certain spots that it can produce fever in human beings. The hypothesis, constructed to explain this, said that the effluent escapes like a gas when the surface of the ground is disturbed and infects those in the vicinity. This hypothesis could not be verified experimentally.
Using the stagnant water as the focal point and assuming that the parasites in the water have taken the saprophytic (a bacterium that obtains food from dead or decaying organic matter) life and could enter the human body by inhaling water vapours or by drinking dirty water. Many physicians conducted experiments by making healthy persons drink water brought from unhealthy sources. The experiments produced entirely negative results and no malarial fever was detected among those who drank the water.
Equipped with this knowledge, Dr. Ross was especially attracted to the study of malarial problems in 1889. He found that outbreak of malaria occurred among troops merely as the result of chill or fatigue. He also observed that the symptoms were not lining up with what was previously determined. He became very dissatisfied with the accepted theories and decided to review the whole question of malaria by close analysis. At first he thought that the disease was caused due to intestinal auto-intoxication. Later he conducted studies by examining samples of malarial blood and was unable to find the large and more obvious parasites.
He returned to England in 1894 and sought advice of many doctors and professors about malaria. He also collected information on malaria from essays, findings, theories and hypotheses. In November 1894, Dr. Sir Patrick Manson informed him that he has developed a hypothesis on the basis of monographs of physicians like Mannaberg, Marchiafava and Bignami, and epidemiological findings and work of King, and has arrived at the conclusion that the mosquito is the carrier of the malaria parasite.
Convinced by Dr. Mason’s arguments, Dr. Ross decided to give this theory a close experimental examination on his return to India. In 1895, after his arrival in India, he was appointed medical officer of a regiment of soldiers stationed at Secunderabad (Deccan), South India. Many of the soldiers were suffering from malarial fever. He examined the mosquitoes caught at random in the barracks and the hospital. Based on the limited information from encyclopaedias and other valuable literature on Indian mosquitoes, he was still able to separate mosquitoes into three groups: brindled mosquitoes, grey mosquitoes and spotted-winged mosquitoes. Then he caught and bred mosquitoes in large quantities, collected their larvae and kept them in jars until they hatched. These hatched mosquitoes were released from the breeding-jars into a mosquito-net which was already occupied by a patient. The mosquitoes fed on human blood were caught in bottles and later dissected for microscopic examination.
Dr. Ross prepared slides of the blood sample taken from the mosquitoes to study the process by which the motile filaments and strands of cell escape from the parent cell within the stomach of the mosquitoes. He also developed a technique which involved dissecting a mosquito, from one minute to several hours, after feeding on human blood, and examining the stomach contents. After several weeks of examining the mosquitoes and studying the results, he concluded that the presence of motile filaments in the blood samples was only 5 per cent but in the mosquito’s stomach it was around 60 per cent. It showed that a mosquito’s stomach was a more favourable locus for the presence of motile filaments.
His next task was to locate the destination of the motile filaments in mosquitoes. This was a very difficult task because the microscope that were available at that time were not very powerful, which made it impossible to follow the movement of motile filaments. During the study he found that the filaments constantly disappeared. He analysed the data and concluded that filaments neither migrate nor enter the tissues of mosquitoes.
On September 9, 1895, he was sent to Bangalore, to combat a serious outbreak of cholera. The population of Bangalore at that time was 80,000. He was stationed in Bangalore from 1895 to 1897. In the meantime Physician Mason, who had done work on mosquitoes, came up with a more advanced hypothesis and stated that the motile filaments after living in the mosquito would later slip into the water, lay eggs and die. This contaminated water when drank or just inhaled as a vapor would produce infection in human being.
Dr. Ross decided to test this speculation by conducting an experiment. He caused a number of mosquitoes to feed on a selected patient and kept them in a jar half filled with water and continued to add different batches of fed mosquitoes until they died one by one. The water from this jar was given to three persons. One of the men developed mild fever and after a thorough examination, Ross found parasites in his blood.
Subsequent tests that were conducted later ended up in failure, forcing Dr. Ross to reject the hypothesis developed by Mason. Yet, in March 1896, Mason delivered three lectures at the Royal College of Physicians in London and supported his own hypothesis on the basis of the test conducted by Dr. Ross and urged Dr. Ross to continue his tests and experiments. Dr. Ross conducted 22 experiments. In these experiments he made various persons drink infected water in which mosquitoes fed on cases of malaria and those persons died.
In phase two of the experiments he made persons drink water that contained gregarines parasites of mosquitoes. The majority of the results were negative and only in three cases a slight reaction did occur. On the basis of these experiments no definite conclusions could be reached. Dr. Ross documented his finding that there may be other routes for infection. One of the possibilities he considered was that the mosquitoes, previously infected from persons suffering from malaria or possibly from other mosquitoes, might inoculate the parasites into healthy person(s) during bite or might deposit them on the skin.
A new series of experiments and observations were made to test the theory of inoculation by allowing the mosquitoes (specimens of brindled and grey mosquitoes) bred from larvae in captivity to feed upon several patients who had suffered from malaria fever earlier. The patients bitten by these mosquitoes did not develop any malaria fever. The results were negative indicating that mosquitoes did not carry the infection. Dr. Ross was also aware of the fact that most of the mosquito’s species were bridled and grey group of mosquitoes and not a single species was from the dappled-winged group.
In 1896 new theories were presented by Italian physicians King and Bignami. In his theory Bignami made criticism of Mason’s hypothesis and speculated that the motile filaments are the result of the death of parasites. Both Bignami and King continued to speculate that mosquitoes would carry the poison from marsh and swamp lands and transfer them to human beings during their bites.
Dr. Ross conducted experiments and the results showed that the escape of the filaments depends on certain conditions and not on the death of the parasites. These conditions were the changes in the specific gravity of the blood by removal of water by partial evaporation or through the addition of a little water. Dr. Ross contemplated that mosquitoes became infected from human beings and other mosquitoes and then transfer the parasites to the healthy persons. Dr. Ross continued to see failure after failure and was forced to reconsider the whole basis of his work, yet he was convinced that there is no flaw in his fundamental reasoning.
Dr. Ross stayed in India for 15 years in Madras, Bangalore, the Andamans, Secunderabad, Burma, Bombay, Poona, Calcutta, Karachi, Quetta and Nilgri Hills and found malaria in all these places. His study and experiments suggested a connection between the disease and the mosquitoes. In April 1896 he went on a two-month vacation to Ootacamund, Nilgiri Hills, and South India. Here he found that all employees working for the coffee and tea plantations at the foot of Sigur Ghats were suffering from severe malaria, anaemia, and emaciation and enlarged spleen. He conducted tests and the test results showed presence of parasite in the blood stream. He discovered to his astonishment that there were no mosquitoes in the houses of employees. He was informed by some of the employees of the plantations that they were often bitten at night by mosquitoes.
At this point, Dr. Ross developed three hypotheses about mosquitoes and malaria. His first hypothesis was that while sucking blood the mosquitoes deposited minute drops of excreta containing gregarines on the skin that might contain the spores of the parasites of malaria, which might end up in the blood of the bitten victim.
The second hypothesis was that the malaria spores might be voided by mosquitoes upon rotting vegetations or damp earth and may develop into extra corporal form capable of infecting human beings by airborne spores.
The third hypothesis was that infected mosquito could introduce the parasite directly into the blood during biting.
During his research Dr. Ross discovered that the grey and brindled mosquitoes were domesticated insects and could be found in corners of the dwellings by day in large numbers, but the dappled-winged mosquitoes would fly away out of the houses at day break. Some of the results of the experiments made him arrive at the conclusion that the probability of infection by drinking water was very small. At the end of his vacation he resumed his duties as a Medical Officer at Secunderabad-Deccan.
In July 1897, he noticed that malaria had continued unabated during the two years (1895-1896) in his regiment at Secunderabad, while he was stationed in Bangalore. These were not merely relapses and he believed that some cause of infection was actually at work among the troops. He decided to test experimentally all the varieties of mosquitoes found near the barracks. He collected larvae from the areas around barracks and kept them in separate bottles, and the developed mosquitoes were released in mosquito nets where patients were placed.
The gorged mosquitoes were delicately dissected, and the contents of the intestine searched and examined under the worn-out microscope that had rusted screws from the sweat of hands and forehead. The results were absolutely negative on these grey and brindled mosquitoes. It immediately dawned on him that he has not tested the dappled-winged mosquitoes.
On August 15, 1897 one of his assistants brought a bottle of larvae and he found dappled-winged insects among the hatched one. He fed them on a patient suffering from malaria and dissected them and found nothing. On August 20, 1897, while examining one of the dissected mosquitos’ stomach, he observed granules of black substance like the pigment of the parasite of malaria.
In a nutshell, the process of finding the malaria-bearing species was long that involved infecting men by the bites of successive species of mosquitoes and in Dr. Ross’ own words, “a multitude of costly and dangerous experiments.” The next process was to conduct experiments to observe the development of the pigmented cells in mosquitoes to their probable relation to the parasite of malaria.
Dr. Ross spent the next month examining the stomachs of all the mosquitoes caught for the pigmented cells. Finally, on September 21, 1897, on dissecting the stomach of mosquitoes that was caught after it had fed on a patient, he found pigmented cells in considerable numbers. The question was as to where this mosquito became infected, because it had not been bred from the larva. Since the patient who was bitten by this mosquito was in a bed alone in a largely empty ward, Dr. Ross concluded that the mosquito obtained the pigmented cells from the patient.
In the meantime an isolated pool of rain water was found on the top of a hillock near the hospital that had swarms of small grey larvae. This swarm of larvae was brought to the laboratory and placed in breeding bottles and after hatching proved to be the larvae of the long sought small dappled-winged mosquitoes. They were released in large numbers within the mosquitoes-net of a patient. The next day two of the mosquitoes who had fed themselves were dissected and in the stomach of one of them large number of small pigmented cells were found. His experiment proved that the dappled-winged mosquitoes that belonged to the genus Anopheles mosquitoes transmitted malaria parasite to humans.
[Mohammad Yacoob is a retired industrial engineer and an engineering proposals analyst who lives in Los Angeles, California]