HISTOMONAS MELEAGRIDIS INFECTIONS IN TURKEYS IN THE USA: A CENTURY OF PROGRESS, RESURGENCE, AND TRIBUTE TO ITS EARLY INVESTIGATORS, THEOBALD SMITH, ERNST TYZZER, AND EVERETT LUND

Histomoniasis has been recognized for over a century as an economically important disease of turkeys; it is highly transmissible, commonly proves fatal, and has threatened the turkey industry in the past (Cushman, 1893a, 1893b; Smith, 1895). Management practices, including chemical prophylaxis, essentially controlled this disease in turkeys until the United States Food and Drug Administration (FDA) withdrew approval of treating turkeys with nitarsone or carbarsone in 2013 owing to concerns about residual arsenic in meat and viscera and to avoid the consequent soil contamination with arsenic, which ended up in poultry litter (Fisher et al., 2015). This has led to a resurgence of turkey histomoniasis in recent years, and the turkey industry is once again threatened (Chadwick and Beckstead, 2020; Jones et al., 2020; Beer et al., 2022). There are several excellent papers on this subject, including reviews of old literature (Reid, 1967; Lund, 1969; Levine, 1973; McDougald, 2005). Here we review past and current research performed at the United States Department of Agriculture (USDA) laboratories and pay tribute to early investigators.

Fredrick Rice, a veterinarian in the state of Rhode Island, published the first description of turkey histomoniasis in a short report in 1892. Rice wrote that an obscure and previously unknown disease initially presented as a spot on the heads of turkeys, spread rapidly through the flock, and killed turkeys in just a few days. In the following year, Cushman (1893a), an expert in raising poultry, detailed the effects of a mysterious disease affecting turkeys as well as hens. Cushman noted in August that affected fowl were weak, had no appetite, and died once the color of the comb changed from bright scarlet to dark purple. He illustrated the high mortality rate of the disease, noting that 78 out of 150 turkeys on a farm in Rhode Island died in a single day in September, and he saw that most of the dead turkeys had abnormal livers. Cushman (1893a) had no clue as to the etiology of the disease but made the astute observation that turkeys were affected where grounds had been used previously to raise chickens, and in another short report, Cushman (1893b) first used the term “Blackhead.” Cushman, who worked at the Rhode Island Agricultural Experiment Station, sent samples of preserved tissues from infected turkeys to Theobald Smith, who worked at the Bureau of Animal Industries (BAI), USDA, Washington, DC. After receiving these samples, Smith visited the Rhode Island laboratory for 3 wk and worked with Cushman to collect additional samples from affected turkeys. After examining the samples, Smith (1895) described both gross and microscopic lesions of the disease in the liver and ceca. Smith later collaborated with Cooper Curtice, a veterinarian, who continued to study Blackhead epidemiology in Rhode Island (Curtice, 1907a, 1907b, 1907c) and eventually joined Smith at BAI, USDA.

While working at BAI, USDA, Theobald Smith (1895) first recognized that the parasite in 17 of 18 affected turkeys was a protozoan; he named it Amoeba meleagridis. He determined that A. meleagridis primarily infected young turkeys, some as young as 3 wk old, and recommended disinfection of affected turkey houses with low concentrations of mercuric chloride and a mixture of sulfuric acid and carbolic acid. Meanwhile, his coworker Veranus Moore further described lesions in turkeys that had been orally inoculated with infected tissues and feces (Moore, 1896). This research was published in a bulletin by the BAI, USDA, after being approved by the Secretary of Agriculture, a requirement for publication at the time. After leaving USDA, Smith continued his research on histomoniasis for a few more years at the Rockefeller Institute for Medical Research (then located in Princeton, New Jersey) and then at Harvard University (Smith, 1915, 1917; Smith and Graybill, 1920a). He found that poults hatched from eggs of infected turkeys were free of A. meleagridis, and in collaboration with Dr. Graybill he postulated that histomoniasis, in turkeys and chickens, was associated with the presence of a worm, Heterakis sp.; however, the exact nature of this association remained undetermined (Graybill and Smith, 1920; Smith and Graybill, 1920b).

Theobald Smith deserves the reputation he earned as one of the most accomplished American microbiologists. He made the seminal discovery of the transmission of a protozoan (Babesia bigemina) via an arthropod vector (a tick of the genus Boophilus, Smith and Kilbourne, 1893). This discovery predated and inspired work that confirmed Plasmodium transmission by mosquitoes. Smith was born in New York State (Table I), and after graduating from college, he wanted to teach algebra, but he could not find a job (Zinsser, 1936). He changed his goals and decided to study medicine after coming face to face with death in a near-fatal boating accident. He was quite interested in research and had authored 2 publications before completing his MD degree. At the age of 25, Smith’s mentor at Cornell University, Professor S. H. Gage, encouraged him to accept a job as an Animal Inspector in the newly created BAI, in Washington, DC. This laboratory was headed by Dr. Daniel Salman, a veterinarian from Cornell University, who was seeking someone to study Texas Cattle Fever. Dr. Salman was a famous scientist as well; the bacterial genus Salmonella is named after him as is Salmon poisoning of dogs. This disease is caused by the rickettsial organism Neorickettsia helminthoeca, which is carried within the fluke Nanophyetus salmincola, whose larval stages (metacercariae) can be found in raw fish tissues. Smith was later joined at BAI by 2 veterinarians, Frederick Kilbourne and Cooper Curtice, also from Cornell University.

Smith made many discoveries during his 20-yr tenure at BAI, and his contributions to histomoniasis should be judged in the light of the fact that only meager research facilities existed at BAI when he joined the facility at the age of 25; there was only 1 light microscope, which had been imported from Germany by Dr. Salman, and there was no provision for housing animals.

Among Smith’s many discoveries 2 stand out: his contributions toward the discovery and description of Blackhead disease in turkeys as stated earlier, and his work on transmission and control of Texas Cattle Fever, an economically important disease caused by Babesia, which still requires surveillance at the Mexico–United States border. Smith meticulously documented his research on Babesia transmission in a 301-page monograph published in the USDA BAI Bulletin in 1893 (this is his longest published manuscript we are aware of). However, his demeanor did not suit him well for mission-oriented research at USDA (Dolman, 1984), and an author dispute and a limited circulation of his Babesia publication despite the production of 10,000 copies probably cost him a Nobel Prize (Dolman, 1984). But in our opinion, if any scientist at USDA was worthy of a Nobel Prize, it was Theobald Smith. Salient features of Smith’s life are summarized in Table I.

Ernest Tyzzer and Theobald Smith became acquainted while Smith was a professor at Harvard University and Tyzzer was earning his medical degree there (Table I). While studying the protozoan Amoeba meleagridis described by Smith, Tyzzer discovered that it exists in both a flagellated and amoebic form. Only the previously described amoebic phase described by Smith was present in tissues, but Tyzzer identified a second flagellated phase that could be found free in the cecal contents. Therefore, Tyzzer (1920c) created a new genus, Histomonas, to accommodate the parasite described by Smith; thus, the agent of Blackhead is properly designated as Histomonas meleagridis (Smith, 1895) Tyzzer, 1920. Tyzzer made seminal discoveries including work on transmission, prevention, and vaccination of histomoniasis, which are summarized in Table II. Tyzzer is credited with saving the turkey industry by advocating control measures that are still relevant.

Ernest Tyzzer had much in common with Theobald Smith: they were both brilliant MDs who did not brag about their contributions, they preferred to publish as sole authors, and their personalities dissuaded erstwhile graduate student mentees (Weller, 1978). Tyzzer was modest and kind, and spoke slowly, softly, and simply (Weller, 1978). After graduating from Brown University in 1897, he postponed entry to medical school for 1 yr and studied the central nervous system of the flounder for a master’s degree earned in part at the Woods Hole Oceanographic Institution. He entered Harvard Medical School in 1898 and trapped fur animals to supplement expenses for his education. After selling the furs, he brought the carcasses to the laboratory of Professor W. T. Councilman, where they were examined for parasites; little did Tyzzer know that he would thereafter research parasites for most of his life.

Both as a medical student and after obtaining his MD degree, Tyzzer worked on smallpox and vaccinia viruses and cancer. He learned histological techniques, and even designed cabinets for vertical storage of stained slides, a system still used today. He was a skilled microbiologist; he discovered and named the 2 Cryptosporidium species, Cryptosporidium muris and Cryptosporidium parvum, in the mice that he was using for cancer research and could differentiate the oocysts of these 2 species, which differ by less than 2 µm. The DBA (originally called dba and named for color coating genes) inbred strain of mouse, still used in research, was derived from 3 mice supplied to Tyzzer that he maintained during World War I.

Tyzzer succeeded Theobald Smith in 1916 as head of the Department of Comparative Pathology at Harvard University, where he conducted his monumental work on poultry parasites. He discovered that multiple species of coccidia (Eimeria) parasitize chickens and named 3 new species of Eimeria in chickens; he meticulously described their life cycles in a classic monograph (Tyzzer, 1929). He used a magnifying glass to help draw life cycle stages and used the histologic techniques he had learned as a medical student.

Tyzzer was elected as President of the American Society of Parasitologists in 1954. He remains one of the most famous microbiologists/parasitologists in the United States. Above all, although he was not a veterinarian, his work on the transmission and prevention of poultry parasitic disease saved the poultry industry.

Histomoniasis research at USDA was put on hold for about 4 decades beginning with Theobald Smith’s departure in 1915 from BAI until it was rejuvenated in the 1950s under the leadership of Everett E. Lund (Table III). During this interval, the BAI laboratory in Washington, DC, was dissolved, parasite research was moved to Beltsville, Maryland, and the laboratory was named the Beltsville Parasitological Laboratory (BPL), Animal Disease and Parasite Research Division, Agricultural Research Service (ARS). Details of the parasitology research conducted at USDA during this interval were described by Andrews (1987). That tradition continues to this day at the Animal Parasitic Diseases Laboratory (APDL), Beltsville Agricultural Research Center, Beltsville, Maryland. Poultry coccidiosis and other foodborne parasites are also studied in 2 other laboratories in Beltsville (namely, the Animal Biosciences and Biotechnology Laboratory and the Environmental Microbiology and Food Safety Laboratory).

Lund worked on the biology and control of histomoniasis for decades and devoted much effort toward improving turkey health (Williams, 2013). Lund had a strong personal motivation to improve the turkey industry; his family could not afford turkey when he was growing up, and he had never even tasted turkey as a child (Lund, 1977). He credited William Billings (Doc Billings, as he was popularly known) from Minnesota for spreading the message of Smith and Tyzzer to turkey growers regarding the prevention of histomoniasis (Lund, 1977). Although Billings produced no scientific publications, his efforts were widely recognized by turkey growers (Wallace et al., 1962). Billings is credited with saving the turkey industry in Minnesota. He traveled from farm to farm and had a very good rapport, notably with women (who more often raised turkeys than did men during the Depression; Wallace et al., 1962). The lesson here for us to learn is that dissemination of research results to customers is as important as making a discovery.

Lund’s main contributions (summarized in Table III) were the development and standardization of experimental Hi. meleagridis infections in the Beltsville Small White Turkey (BSWT; this breed was developed at the Beltsville Agricultural Research Center [USDA Circular CA 44-61, August 1965]), discovery of transmission through He. gallinarum eggs and earthworms, and examination of the role of different avian hosts in the transmission of histomoniasis in turkeys. Lund published most of his papers as the sole author, but near his retirement, he collaborated with Dr. P. Augustine, the husband and wife team of Anne and Barry Chute, and Gary Wilkins (Table III). Research on histomoniasis was terminated at USDA after the mandatory retirement of Lund at the age of 70 in 1977. He died at the age of 90.

Histomoniasis in turkeys has reemerged as a major problem because drugs effective against the parasite are no longer permitted by FD A for use in poultry. At the request of turkey growers, the United States Congress recently established a new USDA-ARS research project “Characterization, prevention, and mitigation of histomoniasis in turkeys.” This project is a collaborative effort between researchers at the Animal Parasitic Diseases Laboratory in Beltsville and the Department of Poultry Science, Division of Agriculture, University of Arkansas.

Here we summarize the biology and control of histomoniasis. Only limited references are cited because, as stated earlier, old literature has been reviewed by others.

Histomonas meleagridis is an extracellular, pleomorphic, biphasic protozoan, with an ameboid stage in tissues and a flagellate form in the cecal contents of birds. Its size is highly variable, stated to be 4–30 µm long (Levine, 1973). The ameboid stage found in tissue lacks flagella, and it divides by binary fission. The cytoplasm has an indistinct outer ectoplasm and an inner, denser endoplasm. The endoplasm contains a vesicular nucleus, a basal granule, and food vacuoles (Fig. 1). In addition to an eccentric nucleus, it contains an axostyle, parabasal bodies, Golgi apparatus, endoplasmic reticulum, phagocytic vacuoles, and centrioles, but neither a mitochondrion nor a flagellum (Lee et al., 1969). The flagellate form found in cecal contents or cultured in vitro has a single flagellum (Mielewezik et al., 2008; Munsch et al., 2009). A resistant cystic stage has been reported (Munsch et al., 2009; Zaragatzki et al., 2010) but has not been conclusively demonstrated. To date, no environmentally resistant stage has been documented in feces, and the trophozoite stage in feces survives outside the host only for a few hours; trophozoites are killed by turkey bile, making their ingestion innocuous to turkeys (Lund and Chute, 1970c).

Histomonas meleagridis is considered a symbiont/commensal of the cecal worm, He. gallinarum (designated He. gallinarum, hereafter; previously called He. gallinae). Heterakis gallinarum is a nematode that parasitizes the ceca of chickens, turkeys, and several other Galliformes species (Levine, 1973). It is up to 15 mm long and lives in the cecal contents (Cupo and Beckstead, 2019a). It has a 1-host direct life cycle. Unembryonated He. gallinarum eggs are passed in feces; within these eggs, the larva develops, molting once (retaining the cuticle); the second-stage larva is released in the duodenal lumen of birds upon ingestion of the embryonated egg. The larva traverses the small intestine lumen and reaches the cecal lumen, where it enters the epithelium where it remains for 5–10 days, molting twice before becoming sexually mature in the cecal lumen. The He. gallinarum eggs are environmentally resistant and can survive for months in the environment (Farr, 1961). They can be embryonated in solutions containing 1.5% nitric acid and 1% formalin. Heterakis gallinarum eggs can hatch and persist in coelomic cavities of several species of earthworms (Lund et al., 1966b). Histomonas meleagridis can invade the tissues of both male and female worms and can pass to the embryo of He. gallinarum before the shell forms around their egg (Lee, 1969, 1971; Lee et al., 1969). Histomonas meleagridis has been observed in He. gallinarum eggs (Lee, 1969). Viable Hi. meleagridis has been isolated in cell cultures from He. gallinarum larvae hatched in vitro (Ruff et al., 1970). In this study, the embryonated He. gallinarum eggs had been treated with 3% sodium hypochlorite before hatching; sodium hypochlorite removes the outer 2 layers of the egg and kills bacteria and protozoal trophozoites. This information is relevant for the transmission of Hi. meleagridis. Although up to 9 Hi. meleagridis trophozoites have been demonstrated in a He. gallinarum egg (Kendall, 1959), only a few (1 in 200 to 1 in 1,000) eggs harbor Hi. meleagridis (Lund and Burtner, 1957). Embryonated He. gallinarum eggs hatch second-stage larvae in the gut lumen of chickens. Histomonads are released from He. gallinarum larvae during molting (McDougald, 2005). Turkeys are a poor host for He. gallinarum, and most of the life cycle of Hi. meleagridis work involving worms was done in chickens.

Histomonas meleagridis that escape from these newly hatched larvae are spherical and contain a single flagellum, which probably assists the parasite to migrate to the cecum. In the cecum, Hi. meleagridis invades the epithelial layer without invading cells; instead, it forms pockets between the cecal villi, causing disruption and inflammation of the cecal tissue. Histomonads have also been found in the Bursa of Fabricius, are likely the result from a cloacal infection. Intrarectal inoculation of Hi. meleagridis is an efficient means of experimental transmission in turkeys (Lund, 1955). The parasite loses its flagellum and assumes an amoeboid shape with pseudopodia projections. At this point, Hi. meleagridis migrates to, and invades, the parenchymal cells of the liver through the intestinal-hepatic portal vein.

Chickens are a more suitable host for He. gallinarum than turkeys; thus, it was realized a century ago that to prevent transmission of histomoniasis, turkeys should never be raised with chickens. Prolonged outbreaks of histomoniasis have affected commercial turkey farms in the absence of chickens, and these outbreaks are difficult to explain based on current knowledge of the life cycle of the worm and the protozoan. One hypothesis is that the parasite is initially introduced to the premises as infected He. gallinarum eggs, possibly on boots or equipment previously used in an infected area. Horizontal transmission could then occur with close contact among turkeys. Horizontal transmission has been demonstrated between experimentally infected and naïve birds, housed on paper or in contact with litter (Hu and McDougald, 2003; McDougald and Fuller, 2005). A possible mechanism for this transmission is the phenomenon of reverse peristalsis, or cloacal drinking, which is thought to have evolved in avians as a means of conserving water (Sorvari et al., 1977). Experimental infection entails applying viable histomonads to the cloaca, allowing the parasite to invade the cecum and Bursa of Fabricius through such reverse peristalsis.

Since the removal of nitarsone from the market, histomoniasis cases in the United States have increased substantially per surveys conducted by Clark and Kimminau (2017) and Clark and Froebel (2020). In 2015, there were 55 reported cases (Clark and Kimminau, 2017), whereas 127 and 96 cases were reported in 2018 and 2020, respectively (Clark and Froebel, 2020). In a study focused on Hi. meleagridis–infected turkey flocks in California from 2000 to 2014, mortality ranged from 12 to 65% (Hauck et al., 2018). However, mortality in affected turkey flocks can approach 100%, especially without any means of chemoprophylaxis or therapeutics. The incubation period (onset of clinical signs) of Hi. meleagridis from ingestion is around 2–3 wk. Initially, turkeys appear depressed with no appetite, seem weak and drowsy, and have sulfur-colored diarrhea. Birds can die within a few days after the initial onset of clinical signs, and histomoniasis-associated mortalities, which can approach 100%, are most often seen in turkeys older than 3 wk of age. Cyanosis of the head (blackhead) is not a characteristic sign, and this term should be discarded—it was used before etiology was known. Lesions are typically confined to the ceca and liver although other organs may rarely be affected, and one or both ceca may be infected (Fig. 2). In the ceca, initially, there is inflammation due to the invasion and multiplication of the parasite in the mucosa; eventually the entire cecal wall may be affected producing transmural typhlitis (Fig. 3). As the disease progresses, drying of the foul-smelling yellowish exudate may produce a cheesy plug.

Lesions in the liver are pathognomonic and have been known since the disease was first recognized (Smith, 1895; Moore, 1896). Yellowish-green depressed areas can occur throughout the liver and extend deeper into the hepatic parenchyma (Fig. 4). They are discrete but can become confluent, and the parasite can indiscriminately destroy hepatic parenchyma. Lesions can disappear in turkeys that survive the acute disease; however, Hi. meleagridis can persist in liver parenchyma long after recovery from clinical signs.

Necropsy, histological examination of tissues, and PCR can aid diagnosis. As stated earlier, hepatic lesions are characteristic. In histological sections, amoeboid trophozoites occur in masses (Fig. 5). Histomonas meleagridis is extracellular; it secretes proteolytic enzymes to degrade host tissue. Histomonas meleagridis trophozoites stain poorly with hematoxylin and eosin but stain brilliantly with periodic acid Schiff (PAS) reaction (Fig. 3). Immunohistochemical staining with polyclonal anti–Hi. meleagridis antibodies can confirm the diagnosis (Fig. 5C).

PCR techniques based on the amplification of Hi. meleagridis ribosomal DNA have been used to detect Hi. meleagridis in cell culture media, litter, and infected tissue (Hafez et al., 2005; Hauck et al., 2006, 2010a, 2010b; Popp et al., 2011; Hauck and Hafez, 2012; Cupo and Beckstead, 2019b; Beckmann et al., 2021). Genotyping methods have also been used to identify strains of Hi. meleagridis and using PCR sequencing have been found helpful in characterizing outbreaks (Hauck et al., 2010b; Popp et al., 2011). PCR has also been used with success to detect Hi. meleagridis–containing He. gallinarum eggs on flies and darkling beetles inhabiting poultry houses, providing some evidence for these invertebrates serving as paratenic hosts for the parasite (Farr, 1961; Cupo and Beckstead, 2019b).

Histomonas meleagridis can be cultivated in vitro in a cell culture medium with killed bacteria (reviewed in Beer et al., 2022). Bacteria appear to be necessary for the growth of Histomonas in culture. However, this relationship is not fully understood.

The control measures that Tyzzer advocated a century ago are still relevant (Table II). The foremost advice is that chickens should never be raised on or near turkey farms, because they are reservoirs of Hi. meleagridis and the worm He. gallinarum. The eggs of He. gallinarum are highly resistant to environmental stressors and chemical treatments. A risk assessment study concluded that having a broiler breeding proximity within 1.5 km of a turkey farm is a risk factor for outbreaks of histomoniasis in turkeys (Jones et al., 2020). Persons who go from 1 flock to another can carry He. gallinarum eggs on their boots, and common disinfectants do not kill worm eggs. Waterers should be separated from feeders. Histomonas meleagridis can survive within worm eggs and earthworms for long periods; no known methods effectively decontaminate infected premises and grounds used for raising turkeys. Whether wild Galliformes can introduce histomoniasis is an open question.

Until the prohibition of antiprotozoal drugs by the FDA, many drugs were used prophylactically by medicating turkey feed. Arsenicals have been predominantly used to prevent histomoniasis since the 1950s. Drugs in the nitroimidazole class such as dimetridazole were also approved and available for use to control histomoniasis in the 1960s (McDougald, 1997). The European Council and the United States banned the use of nitroimidazoles in food-producing animals in 1995 and 1997, respectively, due to potential carcinogenicity (McDougald, 2005). As a result, for approximately 2 decades, an arsenical-based drug, nitarsone (Histostat), was the only compound approved and available to effectively prevent histomoniasis in commercial poultry flocks (Caillat et al., 2002). Continuous inclusion of the arsenic-based drug in the diet was common, followed by a 5-day withdrawal period before slaughter (McDougald, 2005). However, in 2015, nitarsone was voluntarily withdrawn from the market, and the FDA revoked its approval status because of issues related to inorganic arsenic deposition in tissues when consumed by poultry. This led to a resurgence of devastating outbreaks in commercial turkey operations since there were no other means of control available. Liebhart et al. (2017) published an extensive list of anti-histomonial drugs, but newer compounds are needed. Herbal products show some promise, and research toward finding a prophylactic medicine is in progress (Beer et al., 2022; Fodor et al., 2023).

Currently, there is no vaccine against histomoniasis, but research is in progress (Clark and Kimminau, 2017; Liebhart et al., 2017; Beer et al., 2022). It has long been known that turkeys inoculated with live attenuated culture-derived trophozoites can develop protective immunity against rectal challenge but not against the challenge with infected He. gallinarum eggs (Lund, 1959). Prolonged in vitro cultivation of Hi. meleagridis can alter immunogenicity and pathogenicity leading to attenuation of virulence. Application of Hi. meleagridis vaccine candidates (high passage) in a commercial setting is currently not feasible since intracloacal administration of the vaccine would be expensive, time-consuming, and stressful. Efficacy when administered orally to poults at hatch has had variable success (Beer et al., 2022). When considering administration at the flock level, large-scale in vitro propagation of histomonads would not be cost-effective.

The research team at USDA and the University of Arkansas is addressing some of the major issues facing the turkey industry. Horizontal transmission of histomoniasis among turkeys remains unexplained in the absence of Heterakis egg transmission. To determine if an environmentally resistant cyst stage of Hi. meleagridis exists is one of our objectives. We are also testing phytochemical and pharmacological compounds for in vitro and in vivo effects against Hi. meleagridis because there are no effective drugs for histomoniasis approved by the FDA. Efforts are underway to develop a recombinant vaccine for immunizing poults against Hi. meleagridis infection and develop a drug-sensitive strain of He. gallinarum carrying attenuated Hi. meleagridis as a vaccine against histomoniasis.

We thank Gary Wilkins for providing personal information concerning Dr. Everett Lund and Drs. Larissa Araujo, Aditya Gupta, Oliver Kwok, Chistina Yager, and Benjamin Rosenthal for their help in the preparation of this paper. We also thank Dr. Andrew Jansen for electron micrographs.