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Marinopyrrole Derivatives As Putative Antiparasitic Molecules Promoting Initiation Of Programmed Cell Death In Toxoplasma Gondii, Matthew C. Martens

Theses & Dissertations

Toxoplasma gondii is a globally-distributed obligate intracellular parasite and the causative agent of toxoplasmosis in humans, a biphasic disease often culminating in lifelong chronic infection. Though often mild, toxoplasmosis can lead to severe clinical presentation in immunocompromised patients and developing fetuses. Current therapeutic options are severely limited, with a combinatorial pyrimethamine/sulfadiazine regimen acting as the current standard of care for toxoplasmosis; however, both of these compounds can elicit serious adverse reactions in patients, and, additionally, are only effective against the acute stage of infection. To address these limitations, screenings of several analogs derived from the marine natural product marinopyrrole A …

Atlas Of Ticks (Acari: Argasidae, Ixodidae) In Germany, Franz Rubel, Katharina Brugger, Lidia Chitimia-Dobler, Hans Dautel, Elisabeth Meyer-Kayser, Olaf Kahl

Harold W. Manter Laboratory of Parasitology: Library Materials

An updated and increased compilation of georeferenced tick locations in Germany is presented here. This data collection extends the dataset published some years ago by another 1448 new tick locations, 900 locations of which were digitized from literature and 548 locations are published here for the first time. This means that a total of 3492 georeferenced tick locations is now available for Germany. The tick fauna of Germany includes two species of Argasidae in the genera Argas and Carios and 19 species of Ixodidae in the genera Dermacentor, Haemaphysalis, and Ixodes, altogether 21 tick species. In addition, three species …

Parasite Of The Month. Hepatocystis, Imran Ejotre, Deeann Reeder, Kai Matuschewski, Juliane Schaer

Faculty Journal Articles

No abstract provided.

Recent Advances In Understanding Tick And Rickettsiae Interactions, Shahid Karim, Deepak Kumar, Khemraj Budachetri

Faculty Publications

Ticks are haematophagous arthropods with unique molecular mechanisms for digesting host blood meal while acting as vectors for various pathogens of public health significance. The tick's pharmacologically active saliva plays a fundamental role in modulating the host's immune system for several days to weeks, depending on the tick species. The vector tick has also developed sophisticated molecular mechanisms to serve as a competent vector for pathogens, including the spotted fever group (SFG) rickettsiae. Evidence is still inadequate concerning tick–rickettsiae–host interactions and saliva-assisted transmission of the pathogen to the mammalian host. Rickettsia parkeri, of the SFG rickettsia, can cause a …

215— Schistosomiasis Infections In School Aged Children In Ghana, Katerina Pisciotta, Maia Mazzaferro

GREAT Day Posters

Schistosomiasis is a Neglected Tropical Disease caused by a parasitic blood fluke. There are currently over 240 million people affected globally. Humans become infected after coming into contact with water containing the infectious stage of the parasite. Our data consists of egg counts for Schistosoma haematobium and Schistosoma mansoni collected from school-aged children in Tomefa, Ghana, a marginalized informal community, between 2014 and 2019. Our research explores whether or not reinfections and continuous infections were occurring over the six-year period. Although the government conducts periodic mass drug administration, reinfections are likely given the social and environmental conditions in the community. …

A Guide To Helminth Parasites Reported From Shorebirds (Charadriidae) From The Americas, Albert G. Canaris, Sofia Capasso, Gay J. Canaris

MANTER: Journal of Parasite Biodiversity

The shorebird family Charadriidae in the Americas consists of 21 native and 7 vagrant species. Members of the family occupy a diversity of open habitats, ranging from Arctic tundra during nesting, coastal sands, and mudflats to inland prairies, savannas, and wetlands. Some native plovers migrate from nesting grounds in North America to wintering grounds in South America (Hayman et al., 1986; Paulson, 2005; Winkler et al., 2020). Our search of the literature revealed the following: 17 of 28 host species infected with helminth parasites, 153 helminth species, and 199 infections involving 13 geographic areas. The purpose of this guide is …

Effectiveness Of Pathogen-Specific Passive Antibodies To Mitigate Infectious Diseases In Apis Mellifera, Tanner Nordseth

Honors Thesis

Honeybees (Apis mellifera) are widely recognized as a vital part of the global ecosystem and the world's food supply due to their pivotal role in the pollination of both natural and agricultural flora. Colony Collapse Disorder (CCD) is an emerging phenomenon characterized by a colony's worker bees deserting the hive and leaving the queen behind. This usually results in colony failure. CCD is a multifactorial issue, with many environmental stressors and pathogens playing a role. Deformed Wing Virus (DWV) has been identified as a leading cause in this phenomenon. Paenibacillus larvae (P. larvae) is another lethal pathogen that is …

Exploring Anti-Schistosoma Analog Sa01, Sarah Alsuleiman

UNO Student Research and Creative Activity Fair

Schistosomiasis is a waterborne parasitic worm infection able to infect in the larval stage through direct skin penetration. This disease affects approximately 270 million people worldwide and is widespread in tropical as well as subtropical regions with higher distribution in areas lacking sufficient sanitation and safe drinking water. Although some possible alternatives are emerging, currently, the most effective drug treatment is praziquantel (PZQ). However, PZQ is only effective against the adult stage of the worm, allowing juvenile worms to progress in the infection. Furthermore, Schistosoma is developing resistance to this drug as reduced efficacy has been noted. As a result, …

Multi‐Country Investigation Of The Diversity And Associated Microorganisms Isolated From Tick Species From Domestic Animals, Wildlife And Vegetation In Selected African Countries, Emanuela Olivieri, Edward Kariuki, Anna Maria Floriano, Michele Castelli, Yohannes Mulatu Tafesse, Giulia Magoga, Bersissa Kumsa, Matteo Montagna, Davide Sassera

Harold W. Manter Laboratory of Parasitology: Library Materials

In many areas of Africa, recent studies highlighted the great impact of ticks on animal and human health throughout the continent. On the other hand, very limited information on the bacterial endosymbionts of the African ticks and their pattern of co-infections with other bacteria are found in literature, notwithstanding their pivotal role in tick survival and vector efficiency. Thus, we investigated the distribution of selected pathogenic and symbiotic bacteria in hard ticks collected from wild, domestic animals and from vegetation in various ecological zones in Africa and their co-occurrence in the same tick host. Overall, 339 hard ticks were morphologically …

Insights Into Leptopilina Spp. Immune-Suppressive Strategies Using Mixed-Omics And Molecular Approaches, Brian Wey

Dissertations, Theses, and Capstone Projects

Host-parasite interactions influence the biology of each over the course of evolution. Parasite success allows for the passage of potent virulence strategies from generation to generation. Host success passes stronger immunity and resistance strategies to the following generations as well. Only by studying both partners within their natural contexts can we begin to understand the relationship between the two and how immune mechanisms and virulence strategies interact as a molecular arms race.

In this work, we focus on a natural host-parasite pair, the Drosophila-Leptopilina model. Leptopilina species are parasites of several fruit fly species, including Drosophila melanogaster. This model …

Malaria In Southern Venezuela: The Hottest Hotspot In Latin America, Maria Eugenia Grillet, Jorge E. Moreno, Juan V. Hernández-Villena, Maria F. Vincenti-González, Oscar Noya, Adriana Tami, Alberto Paniz-Mondolfi, Martin Llewellyn, Rachel Lowe, Ananias A. Escalante, Jan E. Conn

Harold W. Manter Laboratory of Parasitology: Library Materials

Malaria elimination in Latin America is becoming an elusive goal. Malaria cases reached a historical ~1 million in 2017 and 2018, with Venezuela contributing 53% and 51% of those cases, respectively. Historically, malaria incidence in southern Venezuela has accounted for most of the country’s total number of cases. The efficient deployment of disease prevention measures and prediction of disease spread to new regions requires an in-depth understanding of spatial heterogeneity on malaria transmission dynamics. Herein, we characterized the spatial epidemiology of malaria in southern Venezuela from 2007 through 2017 and described the extent to which malaria distribution has changed country-wide …

Prevalence Of Cymothoidae (Isopoda) Infestation In Bigeye Scad (Selar Crumenophtalmus) From Batangas, Philippines, Tanya Faye S. Muji, Joaquin R. Sorreta, Janice A. Ragaza

Biology Faculty Publications

Cymothoid isopod research is relatively scarce in the Philippines, despite the local economic significance of bigeye scad as an inexpensive source of protein and other nutrients. Isopod parasitism has also been shown to have detrimental effects on their fish hosts. The current study aimed to define the host-parasite relationship between cymothoid isopod and bigeye scad by determining cymothoid isopod prevalence, intensity, and host-parasite length correlations in bigeye scad (Selar crumenophthalmus) hosts sourced from Batangas, Philippines. Fish samples were sampled from the Tagaytay City Market in Cavite, which sources fish directly from Batangas. Fish samples were immediately measured and …

Dirofilaria Immitis Prevalence In Canis Latrans In Kentucky, Melanie Ann Brandon

Murray State Theses and Dissertations

Dirofilaria immitis (canine heartworm) was found in forty-two (42) of the two hundred seventy-five (275) Canis latrans (coyote) necropsied in the state of Kentucky from November 27, 2019 through March 3, 2021. Thirty-five (35) of the positive cases were from western Kentucky region with the other seven spread across the state. With this group of coyotes, one hundred fifty-eight (158) were male and the other one hundred six-teen (116) were female. The estimated age ranged from a pup to senior dogs. A little over forty percent of the dogs were obtained through coyote/predator tournaments; the remaining were acquired from pest …

Occurrence Of Pulmonary Arteritis With Lungworm Infections In Gray Seals From New England, 2013-2020, Jillian R. Broadhurst, Inga Sidor Dvm, Ms, Dacvp

Honors Theses and Capstones

Gray seals (Halichoerus grypus) are a common pinniped species in New England coastal waters. Parasitic infection is an important cause of mortality for this species, and lungworm species are frequently implicated in cases of respiratory infection, especially in pups and weanling seals. The most common of these is Otostrongylus circumlitus, a large nematode found in the bronchi and bronchioles of affected seals. The full life cycle is unknown but its presence in lungs and sometimes pulmonary arteries suggests that migration via arterial circulation occurs. Signs and microscopic findings are widely variable; our cases included dyspnea, bloody mucoid …

Tick Ecdysteroid Hormone, Global Microbiota/Rickettsia Signaling In The Ovary Versus Carcass During Vitellogenesis In Part-Fed (Virgin) American Dog Ticks, Dermacentor Variabilis, Loganathan Ponnusamy, Haley Sutton, Robert D. Mitchell Iii, Daniel E. Sonenshine, Charles S. Apperson, Richard Michael Roe

Biological Sciences Faculty Publications

The transovarial transmission of tick-borne bacterial pathogens is an important mechanism for their maintenance in natural populations and transmission, causing disease in humans and animals. The mechanism for this transmission and the possible role of tick hormones facilitating this process have never been studied. Injections of physiological levels of the tick hormone, 20-hydroxyecdysone (20E), into part-fed (virgin) adult females of the American dog tick, Dermacentor variabilis, attached to the host caused a reduction in density of Rickettsia montanensis in the carcass and an increase in the ovaries compared to buffer-injected controls. This injection initiates yolk protein synthesis and uptake …

Trends And Opportunities In Tick-Borne Disease Geography, Catherine A. Lippi, Sadie J. Ryan, Alexis L. White, Holly D. Gaff, Colin J. Carlson

Biological Sciences Faculty Publications

Tick-borne diseases are a growing problem in many parts of the world, and their surveillance and control touch on challenging issues in medical entomology, agricultural health, veterinary medicine, and biosecurity. Spatial approaches can be used to synthesize the data generated by integrative One Health surveillance systems, and help stakeholders, managers, and medical geographers understand the current and future distribution of risk. Here, we performed a systematic review of over 8,000 studies and identified a total of 303 scientific publications that map tick-borne diseases using data on vectors, pathogens, and hosts (including wildlife, livestock, and human cases). We find that the …

Antibodies Against Egf-Like Domains In Ixodes Scapularis Bm86 Orthologs Impact Tick Feeding And Survival Of Borrelia Burgdorferi, Juraj Koči, Sandhya Bista, Payal Chirania, Xiuli Yang, Chrysoula Kitsou, Vipin Singh Rana, Ozlem Buyuktanir Yas, Daniel E. Sonenshine, Utpal Pal

Biological Sciences Faculty Publications

Ixodes scapularis ticks transmit multiple pathogens, including Borrelia burgdorferi sensu stricto, and encode many proteins harboring epidermal growth factor (EGF)-like domains. We show that I. scapularis produces multiple orthologs for Bm86, a widely studied tick gut protein considered as a target of an anti-tick vaccine, herein termed as Is86. We show that Is86 antigens feature at least three identifiable regions harboring EGF-like domains (termed as EGF-1, EGF-2, and EGF-3) and are differentially upregulated during B. burgdorferi infection. Although the RNA interference-mediated knockdown of Is86 genes did not show any influences on tick engorgement or B. burgdorferi sensu stricto persistence, the …

Biology And Molecular Biology Of Ixodes Scapularis, Daniel E. Sonenshine, Ladislav Šimo, Justin D. Radolf (Ed.), D. Scott Samuels (Ed.)

Biological Sciences Faculty Publications

This chapter describes the biology of the tick Ixodes scapularis in relation to its role as the vector of the Lyme disease agent, Borrelia burgdorferi. Following a review of the internal anatomy of the tick, we review basic molecular processes that contribute to an understanding of the dynamics of the tick's specialized parasitic processes, including attachment behavior, salivation; silencing of host anti-inflammatory responses to enable blood ingestion at the dermal feeding site; hemoglobin digestion and reproduction. The chapter is divided into three parts: 1) systematic and anatomical characteristics of ticks; 2) host finding, attachment, salivary disruption of host defenses, blood …

Double Anus In An Ixodes Scapularis Nymph, A Medically Important Tick Vector, Vikas Taank, Frank A. Lattanzio, Hameeda Sultana, Girish Neelakanta

Biological Sciences Faculty Publications

Background: Ixodes scapularis ticks are medically important arthropod vectors that transmit several pathogens to humans. The observations of morphological abnormalities, including nanism, missing leg, extra leg, and gynandromorphism, have been reported in these ticks. In this study, we report the presence of two anuses in a laboratory-reared I. scapularis nymph.

Results: Larval ticks were allowed to feed on mice and to molt to nymphs. Two anuses were observed in one of the freshly molted nymphs. Stereo and scanning electron microscopy confirmed the presence of two anuses in one nymph within a single anal groove.

Conclusions: This report confirms the rare …

Spatial And Temporal Variation In Aedes Albopictus Prevalence Across Arkansas, Alyssa N. Jones, Eric C. Lovely, Douglas G. Barron

Journal of the Arkansas Academy of Science

Aedes albopictus is a well-known vector species of mosquito that is responsible for the transmission of many arboviruses such as Zika, chikungunya, and dengue. The objective of this study was to quantify spatial and temporal variation of Ae. albopictus prevalence in Arkansas. We used egg abundance as a proxy for mosquito prevalence. Across 2 years, we worked with the Arkansas Department of Health to collect mosquito eggs using oviposition traps. Eggs were desiccated, counted, and later rehydrated in rearing chambers and raised through adulthood for species determination (>99% Ae. albopictus). We determined mean egg abundance by month, year, …

Development Of Environmental Metabarcoding Analysis For Use In Ecological Studies Of Aquatic Fungal And Oomycete Communities Using Nanopore Sequencing, Douglas Bennett

All Master's Theses

One of several factors contributing to amphibian decline are organisms called water molds (oomycetes), which parasitize and kill amphibian and fish eggs, larvae, and occasionally adults. Amphibian decline is one of the most difficult issues facing ecologists and conservationists to date; half of amphibian species are in severe decline and one third of amphibian species are facing extinction, threatening the stability of ecosystems globally. Multiple oomycete species are known to cause the disease saprolegniasis and contribute to amphibian decline; however, the full range of species involved is not presently known. This study evaluated the Oxford Nanopore minION DNA sequencer (R9.2 …

Global Distribution, Host Range And Prevalence Of Trypanosoma Vivax: A Systematic Review And Meta-Analysis, Eyerusalem Fetene, Samson Leta, Fikru Regassa, Philippe Büscher

Harold W. Manter Laboratory of Parasitology: Library Materials

Background

Trypanosomosis caused by Trypanosoma vivax is one of the diseases threatening the health and productivity of livestock in Africa and Latin America. Trypanosoma vivax is mainly transmitted by tsetse fies; however, the parasite has also acquired the ability to be transmitted mechanically by hematophagous dipterans. Understanding its distribution, host range and prevalence is a key step in local and global efforts to control the disease.

Methods

The study was conducted according to the methodological recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist. A systematic literature search was conducted on three search engines, namely PubMed, …

A Study On The Pathological Effects Of Trypanorhyncha Cestodes In Dusky Groupers Epinephelus Marginatus From The Canary Islands, Carolina De Sales-Ribeiro, Miguel A. Rivero, Antonio Fernández, Natalia García-Álvarez, Jorge Francisco González, Oscar Quesada-Canales, María José Caballero

Harold W. Manter Laboratory of Parasitology: Library Materials

Trypanorhyncha are cestodes commonly infecting marine fish. Numerous studies have detailed the biology of Trypanorhyncha species, but information on the pathological changes produced by these parasites is limited. Dusky groupers are keystone species necessary for the preservation of several marine ecosystems. Considering their vulnerable state of conservation and the efforts being made to culture them, identification of the effects caused by Trypanorhyncha is vital. Here, we aimed to determine the prevalence and pathological changes produced by Trypanorhyncha in dusky groupers from the Canary Islands. The prevalence of trypanorhynch plerocerci was 96%. Grossly, in the abdominal cavity, there were numerous larvae-filled …

Molecular Identification Of Parasites In An Intestinal Coprolite From A Mummified Religious Dignitary Of The Piraino Mother Church Crypt, Sicily, Amanda Rollins, Krystiana Krupa, Georgia Millward, Dario Piombino-Mascali, Karl Reinhard, Frederika Kaestle

Karl Reinhard Publications

Intestinal contents were sampled from a spontaneously enhanced mummy from the Sepulcher of the Priests of the Piraino Mother Church in the Province of Messina, Sicily. This adult male mummy, Piraino 1, is an unidentified religious dignitary dating from the late-18th to mid-19th centuries. Immunological and molecular diagnostics were used to test for common and clinically significant parasites. A morphological diagnosis of Trichuris trichiura (whipworm) was confirmed genetically. A previously undetected Enterobius vermicularis (pinworm) infection was also identified genetically. These data indicate that the Piraino 1 individual was simultaneously infected with multiple intestinal parasite species indicative of poor hygiene. This …

Focus Stacking Images Of Morphological Character States For Differentiating The Adults Of Ixodes Affinis And Ixodes Scapularis (Acari: Ixodidae) In Areas Of Sympatry, Robyn M. Nadolny, Marcée Toliver, Holly D. Gaff, John G. Snodgrass, Richard G. Robbins

Biological Sciences Faculty Publications

Adult females and males of Ixodes affinis and Ixodes scapularis are illustrated by focus stacking image photography, and morphological character states are described that reliably differentiate the two species. In conjunction with other environmental cues, such as the questing phenology of adults, these characteristics will enable the rapid identification of adults of either sex along the southern Coastal Plain of the United States, where these species are sympatric.

Tetraspanins As Potential Therapeutic Candidates For Targeting Flaviviruses, Waqas Ahmed, Girish Neelakanta, Hameeda Sultana

Biological Sciences Faculty Publications

Tetraspanin family of proteins participates in numerous fundamental signaling pathways involved in viral transmission, virus-specific immunity, and virus-mediated vesicular trafficking. Studies in the identification of novel therapeutic candidates and strategies to target West Nile virus, dengue and Zika viruses are highly warranted due to the failure in development of vaccines. Recent evidences have shown that the widely distributed tetraspanin proteins may provide a platform for the development of novel therapeutic approaches. In this review, we discuss the diversified and important functions of tetraspanins in exosome/extracellular vesicle biology, virus-host interactions, virus-mediated vesicular trafficking, modulation of immune mechanism(s), and their possible role(s) …

Tick Extracellular Vesicles Enable Arthropod Feeding And Promote Distinct Outcomes Of Bacterial Infection, Adela S. Oliva Chávez, Xiaowei Wang, Liron Marnin, Nathan K. Archer, Holly L. Hammond, Erin E. Mcclure Carroll, Dana K. Shaw, Brenden G. Tully, Amanda D. Buskirk, Shelby L. Ford, L. Rainer Butler, Preeti Shahi, Kateryna Morozova, Cristina C. Clement, Lauren Lawres, Anya J. O'Neal, Choukri Ben Mamoun, Kathleen L. Mason, Brandi E. Hobbs, Glen A. Scoles, Eileen M. Barry, Daniel E. Sonenshine, Utpal Pal, Jesus G. Valenzuela, Marcelo B. Sztein, Marcela F. Pasetti, Michael L. Levin, Michail Kotsyfakis, Steven M. Jay, Jason F. Huntley, Lloyd S. Miller, Laura Santambrogio, Joao H.F. Pedra

Biological Sciences Faculty Publications

Extracellular vesicles are thought to facilitate pathogen transmission from arthropods to humans and other animals. Here, we reveal that pathogen spreading from arthropods to the mammalian host is multifaceted. Extracellular vesicles from Ixodes scapularis enable tick feeding and promote infection of the mildly virulent rickettsial agent Anaplasma phagocytophilum through the SNARE proteins Vamp33 and Synaptobrevin 2 and dendritic epidermal T cells. However, extracellular vesicles from the tick Dermacentor andersoni mitigate microbial spreading caused by the lethal pathogen Francisella tularensis. Collectively, we establish that tick extracellular vesicles foster distinct outcomes of bacterial infection and assist in vector feeding by acting …

Spatio-Temporal Modelling Of Tick Life-Stage Count Data With Spatially Varying Coefficients, Thabo Lephoto, Henry Mwambi, Oliver Bodhlyera, Holly Gaff

Biological Sciences Faculty Publications

There is a vast amount of geo-referenced data in many fields of study including ecological studies. Geo-referencing is usually by point referencing; that is, latitudes and longitudes or by areal referencing, which includes districts, counties, states, provinces and other administrative units. The availability of large geo-referenced datasets for modelling has necessitated the development and application of spatial statistical methods. However, spatial varying coefficients models exploring the abundance of tick counts remain limited. In this study we used data that was collected and prepared by researchers in the Department of Biological Sciences from the Old Dominion University, Virginia, USA. We modelled …

Microbiomes Of Blood-Feeding Arthropods: Genes Coding For Essential Nutrients And Relation To Vector Fitness And Pathogenic Infections. A Review, Daniel E. Sonenshine, Philip E. Stewart

Biological Sciences Faculty Publications

Blood-feeding arthropods support a diverse array of symbiotic microbes, some of which facilitate host growth and development whereas others are detrimental to vector-borne pathogens. We found a common core constituency among the microbiota of 16 different arthropod blood-sucking disease vectors, including Bacillaceae, Rickettsiaceae, Anaplasmataceae, Sphingomonadaceae, Enterobacteriaceae, Pseudomonadaceae, Moraxellaceae and Staphylococcaceae. By comparing 21 genomes of common bacterial symbionts in blood-feeding vectors versus non-blooding insects, we found that certain enteric bacteria benefit their hosts by upregulating numerous genes coding for essential nutrients. Bacteria of blood-sucking vectors expressed significantly more genes (p < 0.001) coding for these essential nutrients than those of non-blooding insects. Moreover, compared to endosymbionts, the genomes of enteric bacteria also contained significantly more genes (p < 0.001) that code for the synthesis of essential amino acids and proteins that detoxify reactive oxygen species. In contrast, microbes in non-blood-feeding insects expressed few gene families coding for these nutrient categories. We also discuss specific midgut bacteria essential for the normal development of pathogens (e.g., Leishmania) versus …

Biodiversity Of The Coccidia (Apicomplexa: Conoidasida) In Vertebrates: What We Know, What We Do Not Know, And What Needs To Be Done, Donald W. Duszynski

Harold W. Manter Laboratory of Parasitology: Faculty Publications

Over the last two decades my colleagues and I have assembled the literature on a good percentage of most of the coccidians (Conoidasida) known, to date, to parasitise: Amphibia, four major lineages of Reptilia (Amphisbaenia, Chelonia, Crocodylia, Serpentes), and seven major orders in the Mammalia (Carnivora, Chiroptera, Lagomorpha, Insectivora, Marsupialia, Primates, Scandentia). These vertebrates, combined, comprise about 15,225 species; only about 899 (5.8%) of them have been surveyed for coccidia and 1,946 apicomplexan valid species names or other forms are recorded in the literature. Based on these compilations and other factors, I extrapolated that there yet may be an additional …