An Interview with Manuel F. Varela and Ann F. Varela:  Anna Wessels Williams and Diphtheria

Sep 6, 2021 by

Courtesy of Schlesinger Library, Harvard Radcliffe Institute.

From my earliest memories, I was one of those who wanted to go places. When I couldn’t go, I would have my dreams about going.”

—Anna Wessels Williams

I was starting on a way that had been practically untrod before by a woman…I believed, therefore, that females should have equal opportunities with males to develop their powers to the utmost.”

—Anna Wessels Williams

I am happy to have the honor of having my name thus associated with Dr. Park.”

—Anna Wessels Williams

Michael F. Shaughnessy

1) This chapter recognizes a female scientist who joins the ranks of “unsung heroes”—those who have never really gotten the recognition they deserve—Anna Wessels Williams—where and when was she born?

Dr. Anna Wessels Williams was a bacteriologist and virologist extraordinaire. Williams would be noted for her groundbreaking scientific work leading to the treatment of diphtheria, diagnosis of rabies, and the study of the causative microbes for scarlet fever, influenza, and trachoma.

On March 17, 1863, Anna Wessels Williams was born in Hackensack, New Jersey. She died on November 20, 1954, of heart failure in Westwood, New Jersey. Her parents were Jane Van Saun and William Williams, a private educator. Her parents homeschooled their children until the age of twelve. She was born into a large religious family, and one of her sisters was named Millie. Although she was interested in science from the young age of twelve after looking into a microscope, Millie’s near-death experience in 1887 during childbirth transformed Williams’ teaching career into a medical career.

After graduating from a public high school, Williams began her teacher training at the New Jersey State Normal School and graduated in 1883. She taught for two years, but later, Williams attended the Women’s Medical College of the New York Infirmary. Two of her most prominent teachers were Elizabeth Blackwell, the first woman to receive a medical degree in the United States, and Mary Putnam Jacobi, the first woman to study medicine at the University of Paris. Williams graduated in 1891, earning her M.D. degree, and began teaching pathology and hygiene at the Women’s Medical College at the New York Infirmary. From 1892 to 1893, she also continued her medical training in Vienna, Heidelberg, Leipzig, and Dresden.

Due to a cholera outbreak, the New York City Department of Heath’s (DOH) diagnostic laboratory sought volunteers to work in their municipal laboratory in the United States. Williams was one of the volunteers who worked with the lab’s director, William H. Park, on a project relating to diphtheria. Within the first year of working in Park’s lab, Williams isolated a strain of the diphtheria bacillus, which proved invaluable for producing the antitoxin for diphtheria. Park was out of the lab on vacation when Williams made her discovery, and due to laboratory work being considered collaborative in nature, the stain was named Park-Williams No. 8 in honor of both researchers. Down the road, the strain was shortened to Park 8. Williams maintained a positive attitude toward this name change stating, “I am happy to have the honor of having my name thus associated with Dr. Park.” Williams remained at the New York City DOH for 39 years.

2) In 1894, she isolated a strain of the very infectious disease—diphtheria—why is this important? What groundwork does it set?

Among the many significant discoveries, Williams is perhaps most famous for isolating an atypical strain, number 8, of the Corynebacterium diphtheriae bacterium. See Figure 1. This history-making bacterial strain was used to isolate the diphtheria toxin, which in turn would be used to develop worldwide antitoxin therapy against diphtheria. Thanks to the work of Williams, her momentous discovery of the diphtheria bacillus strain eight that could efficiently produce the toxin, the antitoxin became available, making its production cost-effective. The antitoxin treatment for diphtheria became a widespread practice in the U.S. and England shortly after 1900.

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Figure l. Corynebacterium diphtheriae.

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At the time, in the 1880s, diphtheria was a strikingly cunning illness. The disease severity ranged wildly in the humans infected with the bacterium. An asymptomatic form occurs in entirely immune individuals, a mild respiratory illness in partially immune patients, and an often fatal disease in susceptible patients who lack an effective immunity.

The most severe form of these assorted illnesses caused by the bacterium is fulminant respiratory diphtheria. In such diphtheric patients, the microbe was fast-acting, often killing the victim within a week after the onset of symptoms. This severe diphtheria begins with a sore throat, mild fever, and production of an exudate that turns into a thickened pseudomembrane structure in the throat and elsewhere in the body, like the tonsils and the larynx. The pseudomembrane formation is pathological and consists of bacteria, fibrin, immune cells like plasma cells and lymphocytes, and host dead cells. The pseudomembrane is tough and leather-like, and this characteristic is the basis of the disease’s name. Coined by French physician Pierre Bretonneau, the moniker diphtheria means hide leather or skin. The patient is killed in one of two ways. First, the pseudomembrane can occlude the airway, and the patient can suffocate to death. Secondly, the patient can suffer severe injury because of the toxin shed from the Corynebacterium diphtheriae bacteria. The toxin is produced at the site where the bacteria take up residence but quickly disseminates through the blood circulation and produces systemic signs of diphtheria as tissues and organs are damaged or destroyed.

The legendary strain number 8 had been isolated by Williams from a clinical specimen containing Corynebacterium diphtheriae. Due to this groundbreaking discovery, i.e., a fast toxin-producing strain (the Park-Williams 8 strain) could be used to produce large quantities for vaccine production, leading to widespread immunizations and antitoxin treatments. Shortly after introducing the then readily available diphtheria vaccine, the incidence and prevalence of the dread disease were significantly reduced in the U.S. For instance, in 1921, several hundred thousand cases of diphtheria were reported, while at the turn of the new 21st century, only a handful of cases have occurred. The success in significantly reducing the cases and deaths was genuinely remarkable. Thanks to widespread immunization programs, the once dreaded diphtheria are rare in the U.S. On a worldwide scale, however, the epidemiological numbers are not encouraging, with roughly 50,000 cases and almost 2,000 deaths annually. Epidemiologists believe that the illness remains a public health concern because of poor, crowded urban conditions and areas with low vaccination rates. The microbe is persistently present in populations where humans live in poor and unvaccinated areas, and asymptomatic individuals are considered carriers via respiratory transmission and contact with their skin.

3) Working with William Park, Anna Williams tried to find a strain of toxin to activate the antitoxin—and, apparently, when Park was on vacation—Williams found something—what was it?

Legend holds that while Park was away on vacation, Dr. Williams had succeeded in isolating the history-making strain eight from a hospital patient with a surprisingly mild case of diphtheria. However, the clinical isolate strain 8 produced massive quantities of the potent toxin. Williams had joined the Department of Health Laboratory in New York in 1894, headed by William H. Park. At that time, Williams volunteered to help the lab with samples of tissues, blood, food, and drink to identify disease-causing bacteria.

Before Williams would purify the history-altering strain 8 of Corynebacterium diphtheriae in the laboratory, all other previous clinical isolates, and cultures of the bacterial species needed weeks or even months to grow and produce enough toxin from which to make a vaccine. When Emile Roux and Andre Yersin prepared the bacterium for toxin production, it was labor-intensive and time-consuming work, and the yield of the toxin was minuscule. Thus, the limited availability of the toxin led to few individuals who could be vaccinated against diphtheria.

After Williams discovered strain 8 of the Corynebacterium diphtheriae bacterium, she and Park found that under specific growth conditions, the isolate could generate enormous amounts of potent toxin within a day or two! That is, the culturing of the toxin-producing bacteria did not require weeks to months. Instead, William’s strain 8 needed only a day or two to make the toxin. It was a remarkable discovery. What is more, the toxin was deadly potent. For example, a few microliters of the secreted toxin from a new culture could kill a 500-gram laboratory guinea pig in just three days! Williams and Park would publish the finding with a 22-page article in the Journal of Experimental Medicine in 1896.

The toxin work led to the production of the antitoxin, which could be used to treat individuals against diphtheria. In contemporary times, the diphtheria vaccination occurs in a combination injection consisting of three ingredients, an inactivated diphtheria toxin called a toxoid, plus antigens of pertussis and tetanus. The triple immunization cocktail of diphtheria-pertussis-tetanus, called DPT or DPaT, is given in five injections to children amid the ages of 2 months and four-to-six years.

The Corynebacterium diphtheriae microbe is named after its club-shaped cell structure. In Greek, “coryne” means club, and “bacterion” means little rod. The diphtheria toxin studied by Williams consists of a two-chain affair: the A-chain and the B-chain. It is sometimes called the Tox, A-B toxin, or diphtherotoxin. See Figure 2. The toxin is actively secreted outside the bacterium’s cell. Thus, the A-B diphtheria toxin is also a member of the exotoxin class of microbial virulence factors. The A-chain is the active part of the toxin, and the B-chain is the binding part.

Interestingly, the diphtheria toxin production is aided by a bacteriophage virus, β-phage, which harbors the gene encoding the toxin. Thus, without a helping virus, the bacterium cannot cause diphtheria.

Mechanistically, the diphtheria A-B toxin works by first attaching to the host cell’s receptor, called heparin-binding epidermal growth factor, via the toxin’s B-chain. The cell receptor is located in many host cell types but prominently on heart and nerve cells. Next, the A-B chains are internalized into the host cell by endocytosis. Inside the cell, the toxin is enclosed in an endosome, sometimes called a vacuole. Then the A-chain escapes from the confines of the endosome, and it enters the host cell’s cytoplasm where it binds to ribosomes, inhibiting protein synthesis and killing the cell. The A-B toxin is so potent that one molecule can kill an entire cell!

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Figure 2. Diphtheria A-B toxin.

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In general, diphtheria exotoxin action is involved in inducing inflammation and toxemia. A complication of the disease is the pseudomembrane that is produced during the inflammatory process. Toxemia is also a complication. Usually, when the diphtheria toxin is absorbed from the throat, the bacterial toxin protein can go into the blood and onto the heart and nervous system, causing severe symptoms. Without prompt treatment, the patient can die from suffocation, heart damage, or respiratory involvement.

Treatment can be given with the antitoxin to reverse the pathological effects of the toxin. Diphtheria can be prevented with the DPaT vaccine.

4) Over time, the Park-Williams strain No. 8 simply became Park 8—I assume during this period, women were not provided the recognition they deserved. Your thoughts?

Initially, Park and Williams named the famous Corynebacterium diphtheriae bacterial clinical isolate “strain number eight.” Later, it became known as the Park-Williams strain of diphtheria. Then, it was simply denoted as Park 8, a moniker that has lasted into the present time. The genuine motivation for the omission of Williams’ name from the historically famous bacterium is a mystery.

Perhaps the omission was a blunder. Park’s first name was William, and readers may have confused his first name, William, with her last name, Williams. The term Park-Williams tantalizingly resembles William Park. Therefore, one could logically surmise that a mix-up in nomenclature occurred in which an unknown putative investigator had attempted to “set the record straight” or perhaps even clarify the strain’s name, thinking that Williams was a misspelling of Park’s first name. After all, names of microbes or other elements of discovery are rarely named after the first names of scientific investigators. Why keep the first name “William?” Better to call it “Park 8.” We think, however, that such a misstep in scientific nomenclature is unlikely. Indeed, astute journal reviewers and article readers would surely have corrected the blunder at the manuscript stage if not in the subsequently published papers. Unfortunately, no such correction of the strain’s original name in the scientific literature seemed forthcoming.

Others contend that Williams was merely an underling in Park’s laboratory and that being such a scientific novice, Williams did not yet deserve the high-level recognition or even acclaim so early on in a fledging career. In the famous Hershey-Chase experiment, Martha Chase was a junior-level laboratory assistant, and she was included in the naming of the groundbreaking work. Chase did much of the actual experiments herself hands-on. The term “Hershey-Chase experiment” has withstood the test of time, as it is routinely mentioned as such in the textbooks of today. However, Chase did not receive much further acclaim, as she did not share the Nobel accolade with Hershey in 1969. The argument that equal credit to a student in the principal investigator’s laboratory “is just not done” does not hold water. There have been cases mentioned elsewhere in this book in which both student and laboratory mentor enjoy equal acclaims, such as with the Nobel Prize bestowments to both Joesph Taylor (mentor) and Russell Hulse (student).

Even in modern times, some scientific publications dealing with the famous Corynebacterium strain eight are denoted simply as “PW8.” It seems perhaps that both names could be potentially lost to science history because of a general unfamiliarity with history itself. Considering today’s workforce climate and much effort being placed in the academic discipline of scientific history, however, we think that an overall ignorance of history is, while not impossible, also somewhat unlikely.

Some historians assert that the exclusion of Williams in the strain was due to overt sexism that was most certainly and widely in play at the time, given the limited numbers of female scientists who were their heads of research laboratories. Given the climate or culture of the day and the relatively fewer numbers of women scientists who were laboratory directors, administrators, or policymakers, it seems safe to argue that sexism was a significant factor. Elements of sexism in the workplace are still present in scientific and other circles, even in the burgeoning years of the 21st century. Williams did not receive much notice about her contributions until later in life. Nevertheless, in a later interview, Williams related that she was proud to have had her name associated with such a significant aspect of scientific history.

5) In 1896, she was researching scarlet fever in Paris, France. What exactly is scarlet fever—and how does it affect the human body?

Scarlet fever is a bacterial malady caused by rare Streptococcus pyogenes strains called group C or G streptococcal microbes. Scarlet fever can occur as a complication of strep throat, which is caused by the same bacterium. The microorganisms secrete a battery of virulence factors, producing a distinctive skin rash and fever in scarlet fever patients. One of these toxins is similar in nature to the toxin produced by Corynebacterium diphtheriae. The Streptococcus pyogenes version of the toxin is called Spe, for streptococcal pyrogenic exotoxins, of which four types are known: SpeA, SpeB, SpeC, and SpeF. Clinically speaking, the rash is referred to as diffuse erythematous and is accompanied by a bright scarlet color, which starts in the body trunk and spreads to the arms and legs. Typically, the rash lasts about five to seven days and disappears. The outer layers of the affected skin area are then shed. Even today, it is unclear whether the rash is caused by Spe-toxin action or the immune response to any of the four toxin antigens.

Another hallmark of the Streptococcus pyogenes microbe is its ability to destroy red blood cells, producing yellow halos on blood agar plates. See Figure 3. In particular, two virulence factors are involved in blood lysis: streptolysin S (SLS) and streptolysin O (SLO). The SLS protein completely lyses any red blood cells it encounters, and bacteria harboring the SLS protein are responsible for the blood agar phenotype called β-hemolysis, i.e., complete blood destruction. The SLO-harboring Streptococcus bacteria are antigenic, eliciting antibody responses, a valuable property in their species group identification.

File:Streptococcus pyogenes (Lancefield Group A) on Columbia Horse Blood Agar - Detail.jpg

Figure 3. Streptococcus pyogenes on blood agar.

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After her success with diphtheria bacillus strain eight and its proclivity for toxin production, Williams moved to the Institute of Pasteur, where she had hoped to repeat her successes with scarlet fever. She had hope to learn about microbial toxins produced during scarlet fever. Instead, while on the sabbatical in Paris, Williams encountered a somewhat different work environment than previously known. The new settings were characterized by a culture rooted in secrecy in which knowledge of time-sensitive data was considered strictly off-limits until published, and the use of cadavers for research was prohibited. Thus, progress for Williams on scarlet fever research at Paris was stopped in its tracks. Nevertheless, upon returning to New York, she continued work on scarlet fever, where she had access to living patients and cadavers.

Back in Williams’ laboratory in New York, physician Bertha van H. Anthony undertook new studies in which the techniques used for rabies would also be used for scarlet fever. Namely, they obtained small pieces of tissue with scarlet fever rashes on them from 17 live clinical patients with the ailment and 33 cadavers. Williams also took healthy skin pieces from nine non-diseased individuals as negative controls for comparative purposes.

First, Anthony and Williams fixed the various tissue samples with the fixative agent called Zenker’s fluid and incubated the tissues between five to 10 hours. Next, they transferred the fixed tissues between iodine-alcohol and pure alcohol solutions alone, then cleared them in cedar oil and embedded them in a wax-like material called paraffin. Lastly, they used various tissue-specific stains to visualize the host cell structures and any microbial elements harbored in them. They found that the scarlet fever infected tissues showed tiny reticular bodies, probably indicative of bacteria. They felt, however, that better data could be had if they also studied the tissues of the nose and mouth and maybe even samples from the lymphatic system.

In addition, the investigators isolated the Streptococcus pyogenes bacteria from samples of blood, blister fluids, pus, and the throats of scarlet fever patients. Then, they examined the ability of the bacteria found in these regions to undergo hemolysis, that is, lytic destruction of red blood cells—a blood-bursting property. They soon found a great variety of hemolytic activities. However, in all cases of the Streptococcus pyogenes isolates, they exhibited complete blood hemolysis, β-hemolysis, a vital property of these pathogenic bacteria.

Though Williams had been stifled in her endeavor to study scarlet fever at the Pasteur Institute, she was not so stifled at New York. Another positive outcome occurred. Williams became interested in rabies and was, thus, able to enter the field of rabies vaccine research. Thus, Williams would also continue rabies work, as well as scarlet fever, upon her return home to New York.

6) Moving on to rabies—what did Williams have to offer in this realm? Any luck here?

Before the start of the 20th century, Williams would discover an efficient method for diagnosing rabies early on during the infection, and she would create a newer rabies vaccine for disease prevention. She is now more widely recognized for co-discovering Negri bodies, which are key diagnostic features in the Rabies virus species detection during the onset of its disease symptoms. The virion is a member of the Rhabdoviridae family of viruses. The genus is Lyssavirus which is Greek for madness. The Rabies virus structure has a bullet-shaped feature, with glycoprotein spikes sticking out through its envelope from its internal matrix protein, which covers its helical nucleocapsid.

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File:Figure 21 01 04ab.jpg

See Figure 4.

Figure 4. Rabies virus (portion selected from original image).

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In the day of Williams and her contemporaries, rabies was a terrifying disease. Indeed, rabies is dreadful if one acquires it in prevailing times, as it is still almost always fatal. The ailment was also known as hydrophobia because the patients frequently had an irrational fear of water. Another term often mentioned during these events was “mad dog” because these behaviorally affected animals whose bites on humans transmitted the disease and initiated the pathogenesis of virus infection. It did not help much that rabies patients often were recorded to have gone “mad” after being bitten by a rabid animal. The rabies aliment is known as a zoonotic disease because of the transmission of the virus by animals. On most occasions, the painful disease was alleviated only by death. It was clear that a means of prevention was necessary. Williams set out to find a better rabies vaccine than was available at that time.

In 1898, Williams had used the rabies culture from Paris to grow at her laboratory in New York. The cultured viruses were used to inoculate the brains of young laboratory rabbits. Once the rabies microbe was grown to total capacity and the animals had observable rabies, the brains of the animals were removed and preserved. The brain material containing viruses was treated with carbolic acid and incubated at 37 °C for a full day to inactivate the infectivity character of the Rabies virus. The inactivated virus was neither infectious nor pathogenic, but it was immunogenic. The ability of an inactivated virus to elicit an immune response in an individual is a telltale characteristic of immunogenicity. After culturing and inactivating the Rabies virus, it was tested for bacterial contamination. If free of other microbes, the inactivated virus was tested for its ability to protect test animals from infection with the Rabies virus. The new vaccine turned out to effectively provide a good measure of immunity against the Rabies virus. A vaccine for rabies was thus produced on a large-scale basis. The vaccine would be used to improve efforts towards the prevention of rabies.

However, a difficulty persisted where patients could still acquire rabies and succumb to it while waiting an unusually long time for the laboratory tests to confirm a diagnosis. There was, therefore, a need for a quicker diagnosis of rabies than was available at the time. Thus, Williams set out to develop a faster means for diagnostic testing.

Williams started with the so-called smear method for rabies diagnosis. The method involved laboratory animal inoculations with Rabies virus cultures brought from the Institute of Pasteur, followed by staining and examining brain tissue slices under the microscope.

First, the method required extracting brain tissue from freshly dead laboratory animals, such as dogs, cats, and horses. It was clear that the test animals had to be freshly dead. Otherwise, decomposed brain tissues could show inaccurate diagnoses. Later work would focus on salivary tissue taken from live human patients or brain tissue from cadavers. In the meantime, Williams removed the central nervous systems from animals, and she cut tissues from the cerebellum, the cerebral cortex, and the hippocampus, which was referred to at the time, the early 1900s, as Ammon’s horn.

The tissues from the brain regions were sliced into ultra-thin sections and each placed on a microscope slide. The brain slices were pressed firmly by hand to the slide using a small glass coverslip and left on the benchtop to air-dry. Next, the pressed brain slices were fixed to the slide using a solution of picric acid and methanol that was neutralized with sodium carbonate. Lastly, the fixed slices of the sectioned brain tissues on the slides were stained with two chemicals: fuchsin and methylene blue.

The fuchsin stain typically adheres to glycoproteins and cell wall parts of specific bacteria and macrophages, which can be found in the brain to turn these structures red. The methylene blue usually stains cell walls of bacteria like Corynebacterium, turning them blue. In Williams’ work, the fuchsin and methylene blue-stained an infected brain structure called Negri bodies, and they were used in the differential diagnosis of rabies in brain slices. See Figure 5. In February of 1908, Williams would publish an influential review article called “The Diagnosis of Rabies” in the American Journal of Public Hygiene. Her method for detecting rabies early on was better than the previous lab tests and widely used for the next 30 years in clinical settings.

Figure 5. Negri bodies in the brain.

Rabies

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7) Why is rabies so challenging to diagnose, or have things changed recently?

The scientific work of Williams regarding rabies would lead to a more rapid means of detection for earlier diagnosis than was possible before. However, even today, the microscopic evidence that the Rabies virus infects a person, the telltale symptoms, and the serological detection of anti-Rabies virus antibodies do not occur until it is too late to intervene medically.

The various laboratory tests invoked include direct immunofluorescence of anti-rabies antibodies using reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assays (ELISA), and visualization of Negri bodies in affected neurons. With the RT-PCR, immunofluorescence, and ELISA testing, the Rabies virus-specific antibodies in saliva, cornea, blood serum, cerebrospinal fluid, or brain tissue do not appear until late in the course of the disease.

Consequently, medical doctors rely on observing neurological symptoms in an individual whom a suspected rabid animal has bitten as the basis for rabies diagnosis. Therefore, the laboratory tests for the confirmation of rabies disease diagnoses occur too late for treatment. Furthermore, the logistics involved are immensely formidable in determining whether the suspected animal is rabid because a post-mortem evaluation is required. Frequently, rabies disease in humans is diagnosed only after death, during their autopsies!

The laboratory tests used to diagnose rabies infection are accurate but not fast enough to influence treatment. Only one successful case of rabies disease cessation has been reported in all medical history using the antiviral drug treatment with ribavirin. Clinical rabies is usually fatal unless treated with the vaccine early on during the disease course—this treatment is called post-rabies immunization. Otherwise, the patient suffers progressive, fatal meningoencephalitis.

If a supposed rabid animal bites a person, prophylactic measures are invoked straightaway until the rabid animal is tested directly for rabies, as well. Meanwhile, these protective measures include wound cleaning with soap, water, and an antimicrobial agent like povidone-iodine antiseptic (Betadine), plus immunizations with the rabies vaccine—four vaccine injections over two weeks.

In the present era, the rabies vaccine is composed of a human diploid cell vaccine. The vaccine is derived from culturing the virion repeatedly in human cultured cells. The virion is inactivated for its pathogenicity but is nevertheless highly immunogenic, meaning that an effective immune response can be provoked. The vaccination is a form of active immunization, where the patient mounts the immune response after receiving the antigen injection.

The patient also gets an infusion of human-derived anti-rabies immunoglobulin proteins on the wounded site and an injection of globulin near the wound. The latter two methods are passive immunization, where other individuals have performed the immune response.

At present, it is estimated that about 55,000 cases of rabies occur on an annual basis worldwide. The disease is rare in humans but is found in animals at about 6,000 cases per year in the U.S., providing an unwelcome challenge to veterinarians.

8) Negri bodies are the term used for rabies—and an Italian scientist working at the same time as Williams—beat her into a journal (I guess Negri got positive reviews from all three reviewers!). Is this all still relevant today?

The Negri bodies still have relevance in current times, especially when considering the laboratory diagnosis of rabies. The character of the Negri bodies is in the form of so-called inclusion bodies that can reside on the insides of host cells. See Figure 5. These intracellular cytoplasmic inclusions have aggregates of rabies viral nucleocapsid molecules inside of the infected neuronal cells. These intracellular structures, the Negri bodies, are a unique diagnostic feature of rabies disease. About 70% to 90% of brain tissue from infected patients has indicative Negri bodies in them. Therefore, even today, the Negri inclusion bodies inside infected tissue are identified by microscopy and are diagnostically indicative of much-feared rabies.

In general, when a virus infects a host cell, a degree of damage or abnormal changes in the structures and function of the host cell can occur. This viral effect on a cell of the host is called cytopathogenesis. Cell damage inflected by a virus characterizes the cytopathogenic or cytopathic effect. Usually, such cell damage can be seen with a microscope. In this cytopathic effect relationship between the Rabies virus and brain host cells, the Negri bodies are specific to the rabies disease. Thus, the presence of Negri bodies in a neuronal specimen is precisely indicative of rabies disease.

Inside the Negri bodies, an RNA-dependent RNA polymerase that is already packaged in the infecting virion makes the mRNA molecules of the Rabies virus. These new mRNA molecules are then used to make Rabies virus proteins by translation. Thus, besides their role in laboratory diagnosis of rabies, the Negri inclusion bodies serve significant roles in the viral life cycle: viral transcription and translation.

While it is widely known amongst the immunologists and virologists that Williams had observed the Negri bodies first, and hence, discovered them, she was nevertheless not the first to publish the work in 1903. Instead, Adelchi Negri, a microbiologist, and pathologist from Italy, published first and was given credit. Hence, with their rabies viral nucleocapsids, the abnormal nerve cells were named after Negri instead of Williams. Had Williams published first, the intracellular pathological presence of the Rabies virus in host cells could very well have entered the textbooks and history books as the “Williams bodies.” At the very least, one could argue that Drs. Negri and Williams are co-discoverers of these pathological structures. Hence, one could justifiably propose that these abnormal cellular structures be designated “Williams-Negri bodies.”

In any case, however, the diagnostic test that Williams would develop in 1905 was demonstrated to be more efficient, with definitive results provided in minutes rather than days, as in the case of the standard method used before. Thus, the laboratory method of Williams would now be the industry standard in clinical medical laboratories worldwide for the next two generations of rabies patients.

9) Forced into retirement in 1934 by N.Y. Mayor Fiorello La Guardia, no telling what additional contributions she could have made to the field. How is she recognized today?

According to a news article from the New York Times, the city acted to oust Williams and force her into retirement at 71 because she had exceeded the city’s mandatory age for retirement, which was 70 years. Her ousting occurred despite an uprising by peers, colleagues, students, and various healthcare providers. They had started a petition campaign to stop the forced retirement, but it was to no avail.

Today Anna Wessels Williams is noted for her significant contributions to the microbiological and immunological sciences. Thanks to Williams, much of the developed world has enjoyed a treatment for diphtheria (the Park-Williams strain no. 8!), a rapid diagnosis for rabies, a means for identifying blood-destroying scarlet fever-causing bacteria, and the co-discovery of the famous Negri bodies.

We anticipate that history will be kinder to Williams because of her permanent contributions to popular and scientific writings. One popular book published in 1905 by Williams and co-authored with Park, titled “Who’s Who among the Microbes,” enjoyed wide acclaim from reviewers as highly recommended for readers without specialization but who desired to understand the fundamental laws of good health and the mechanisms that underlie that health.

For the specialists, Williams and Park in 1929 published a classic book called Pathogenic Micro-Organisms Including Bacteria and Protozoa: A Practical Manual for Students, Physicians and Health Officers.” The definitive classic was a reference book for medical students, teachers, practicing medical doctors, and policymakers. The textbook enjoyed 11 editions and was affectionately known as the “Parks and Williams” book by its devoted readers. The book seemed to be the one lasting entity in which her name was not removed. We foresee that these written contributions to popular and medical science by Williams will last for millennia.

10) What have I neglected to ask about this unsung hero of science? 

It was reported by John Barry, author of “Influenza: The Epic Story of the Deadliest Plague in History,” that Anna Williams enjoyed accompanying stunt fliers in World War I vintage airplanes and reveled in the airplanes’ quick turns and out-of-control drops. Williams was known to provide both creativity and a degree of wildness to the laboratory, a stark contrast to her stoic lab colleagues.

In 1914, Williams published an influential review of the disease called trachoma, a neglected tropical disease of the eye caused by biting flies and sexual transmission of the bacterium Chlamydia trachomatis. A severe case of the trachoma ailment can lead to blindness in its patients. The microbe is also the causative agent of neonatal conjunctivitis.

Williams devised a highly accurate predictive laboratory test for trachoma. The test would be used to screen school children, thus, protecting them from the ailment and potential blindness. Williams examined specimens from clinical cases of inflammatory conjunctivitis using the microscope and microbial culturing.

She obtained the clinical specimen by a curettage procedure involving a scraping of specific eye tissue sections using a curette device. The curetted eye tissue was used for bacterial culturing. This method involved rubbing a cotton swab over the collected eye material and inoculating veal broth and horse or rabbit blood agar. The inoculants were then incubated for microbial growth, and the cultured microbes were used for counting their cell numbers.

Williams also grew the cultured bacteria in specialized media to enrich them for blood-eating bacilli and the so-called gonococcus. The curetted eye tissues were also used for film preparation on microscope slides, fixed, and stained with various chromogenic chemicals. Together, these tests were used to screen school children from largely poor sections of New York, publishing the results in The Journal of Infectious Diseases in 1914, just before the onset of the Great War.

With the arrival of the First World War, Williams focused on influenza, which was responsible for an estimated 30 to 50 million deaths during the 1918-1919 influenza pandemic. Williams was one of the very few women microbiologists devoted to identifying the disease’s causative agent.

Of all of the scientists devoted to this endeavor, Anna Wessel Williams, Oswald Avery, and William Park were the three most significant figures. In September of 1918, amid the worst flu pandemic ever in the world’s history, Park and Williams were summoned to Camp Upton on Long Island, where the disease had just arrived. She took autopsy specimens from the mucosal membranes, sputum, and tissue samples from influenza victims. Unfortunately, Williams and Parks managed to find only the so-called Pfeiffer’s bacillus, known later as Bacillus influenzae and today as Haemophilus influenzae, a bacterium later found not to cause the flu but rather severe infections of the ear and urinary tract. Williams and Park prepared a vaccine using Bacillus influenzae as the antigen. However, before the vaccine could be widely deployed, the pandemic subsided. It would not have worked anyhow.

Park and Williams tested their hypothesis that Bacillus influenzae was the causative microbe of influenza systematically. Their team cultured 20 isolates of the microbe from clinical cases. They injected each of the pure isolates into laboratory test animals of rabbits and waited for an immune response to be mounted. Next, Park and Williams collected blood sera from the rabbits, and they conducted an agglutination test, finding a positive result indicating that the rabbits made antibodies against the Bacillus influenzae isolates.

However, when Park and Williams tested their new anti-sera against different isolates of Pfeiffer’s bacillus, their results were primarily negative. Park and Williams discovered that they were dealing with the Pneumococcus bacteria, known today as Streptococcus, to their chagrin. In early 1919, Park and Williams no longer believed that any of these bacteria caused the flu. Instead, they published their belief that these various bacteria were merely contaminants. Williams would write in her diary that the evidence pointed to a filterable virus that caused influenza.

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