Oral inoculation of turkeys with in vitro cultured Histomonas meleagridis in cecal contents results in histomonosis

These studies were approved by the Institutional Animal Care and Use Committee at North Carolina State University.

All three experiments used day-old male Nicholas poults from a commercial Aviagen hatchery (Lewisburg, West Virginia, USA). Experiment 1 was conducted in battery cages [89 cm x 56 cm x 56 cm] at the Talley Turkey Education Unit at North Carolina State University. Experiments 2 and 3were conducted in isolator cages [63 cm x 90 cm x 76 cm] located in Scott Hall in the Prestage Department of Poultry Science at North Carolina State University. Feed and water were provided ad libitum. A 24-hour light schedule was applied for the initial three-day period, followed by 16 hours of light with 8 hours of darkness for the remainder of each experiment. The environmental temperature was set at 33°C on day 1 and lowered by 3°C each week. All mortalities throughout the experiments were necropsied to determine the cause of death and recorded.

H. meleagridis cultures were collected previously from outbreaks in multiple locations in the Southeast US and Mid-West US, and cryopreserved. Three isolates from Michigan (ZMI), Georgia (BF), and Arkansas (ARK) were used. Isolates were removed from liquid nitrogen, thawed, and passed into 10 mL of modified Dwyer’s media consisting of 0.8 % (wt/vol) rice powder (Bob’s Red Mill, Milwaukie, OR, USA), 5 % horse serum (Cytiva HyClone, Waltham, MA, USA), in Medium 199 with Hank’s balanced salt solution (Sigma-Aldrich, St. Louis, MO, USA) (Hauck et al., 2010). Cultures were incubated at 42°C for 48 hours and passed into a new flask. This was repeated three times. Before each infection, H. meleagridis numbers were determined using a hemocytometer (Hausser Scientific, Horsham, PA, USA). Each isolate was counted and diluted to 50,000 histomonads/mL. All three reconstituted isolates were mixed into a single container of inoculum with approximately 1:1:1 ratio and stored in a portable incubator (Azzota Corp, Claymont, DE, USA) at 42°C during the inoculation. Cecal contents (Cargill Turkey) were warmed to 42°C and well-mixed to homogeneity. A 1:1 ratio of cecal content and 100, 000 histomonads/ml culture was mixed, and 1 mL of the mixture was administered to each poult using a syringe to deliver the inoculum into the esophagus. The beak of each poult was gently held to ensure inoculum was swallowed before the poults were placed back in their respective cages.

CIM and OICC treatments were repeated in isolator cages, a completely isolated environment, to avoid potential environmental vector effects and to confirm the findings from Experiment 1. Sixty one-day-old Nicholas Tom poults were placed randomly into isolators and assigned to 2 treatments (3 replicate cages/group x 10 birds/cage). Poults were inoculated cloacally with H. meleagridis in fresh cecal content (CI), or poults were inoculated orally with H. meleagridis in fresh cecal content (OI) (Table 1). The rest of the procedures were the same as in Experiment 1.

Ninety one-day-old Nicholas tom poults were placed randomly into isolator cages assigned to 3 treatments (3 replicate cages/group x 10 birds/cage). Treatments consisted of H. meleagridis (BUF) isolate in cecal content, Arkansas H. meleagridis (ARK) isolate in cecal content, or Michigan H. meleagridis (ZMI) isolate in cecal content (Table 1). The inoculation method for the OICC group in Experiment 1 was used for Experiment 3. Poults were inoculated at 8 AM and 5 PM from day 14 to 18. The Experiment was terminated on day 32.

All poults were examined daily for clinical signs of histomonosis. Mortalities were necropsied throughout the experiments and examined for signs of histomonosis. Ceca and livers were examined for lesions associated with histomonosis, scored, and recorded. Cecal and liver lesions were assigned a score of 0 to 4. For cecal lesions: 0 – no infection, 1 – slight thickening of the ceca but still functioning, 2 – thickened cecal wall, small cecal core formation, still partially functioning, 3 – Severely inflamed cecal wall, caseous core filling most of the ceca, 4 – severe inflammation of the cecal wall, necrotic, friable, with the entire cecal lumen filled with a caseous core, complete loss of function. For liver lesions: 0 – no infection, 1 – less than 5 small foci, 2 – multiple foci throughout the liver lobes, 3 – large and small foci, 4 – many large foci, necrotic lesions. Birds with a score of greater than 1 in the ceca or liver were considered positive for histomonosis.The infection rate was calculated based on birds showing positive signs of histomonosis in the ceca or the liver compared to the total number of birds inoculated with H. meleagridis.

Differences in mortalities, infection rates, and ceca and liver scores were analyzed in SAS 9.4 (Statistical Analysis System, Cary, NC, USA). Percentage mortality and infection rate data were transformed to arcsin values before statistical analysis. Data were analyzed by one-way analysis of variance (ANOVA). Duncan’s Multiple Range Test was used to separate significant treatment means. Statistical significance was accepted at P ≤ 0.05.

In Experiment 1, three methods of inoculation were compared: cloacal inoculation (CIM), oral inoculation with H. meleagridis in culture (OIM), and oral inoculation with H. meleagridis in cecal content (OICC). Cloacal inoculations led to the highest infection rates as well as mortality rates. Cloacal inoculations using H. meleagridis grown in culture have been the common route of infection for laboratory models since it was described by Hu et al. (2004), in which H. meleagridis culture was placed on the cloaca and taken up into the large intestine and deposited in the paired ceca. Previous researchers noted high infection rates when cecal lesions, liver lesions, and other H. meleagridis infected tissue inoculated into the large intestine of turkeys (Swales, 1950). The action of reverse peristalsis is a unique ability of birds (Duke, 1982; Hill, 1971). The bursal drinking ability to move materials from the cloacal lips of the anal portal allows birds to sample environmental antigens, when an aqueous contact is made. This is fortuitous for the researcher exploring a means of testing the bird’s ability to mount an immune response to an antigen (Sorvari and Sorvari, 1977). The consistent success with trans-cloacal inoculation of histomonads, producing high infection rates of 80–90 %, makes this procedure a very effective means for induction of histomonosis due to the innate cloaca to ceca reverse peristalsis reaction.

Oral inoculation with H. meleagridis in culture media produced low infection and mortality rates possibly due to the strong acidic environment in the proventriculus and especially in the ventriculus of turkeys, with the gizzard pH being approximately 2.19 (full-fed turkey; Farner, 1942) 3.5 (fasted Ross 308 chicken; Mabelebele et al., 2014). These pH levels fall below the minimum pH that unprotected H. meleagridis can tolerate. Due to H. meleagridis instability outside of a neutral pH (Hauck et al., 2010), it is not likely that H. meleagridis can survive the caustic pH of the upper intestinal tract; this may have affected cellular survival in the oral inoculation with media treatment group, where infection rates and mortality fell below 10 %. However, oral inoculation with cecal content containing identical numbers of histomonads led to higher infection rates. The difference in infection rates were attributed to the physical protection from cecal content and reduced exposure of H. meleagridis to the acidic environment in the upper GI tract. Similarly, Moore (1896) utilized the infected ceca and livers of turkeys that died of histomonosis and fed these organs 3 times over a 6-week period to presumed healthy turkeys in which four out of six birds became infected. Moore also mixed H. meleagridis infected excrement into feed, where 3 of 4 turkeys became infected. Lund (1956) attempted to infect turkeys orally with the use of saline and cecal content, with cecal content still producing around a 10 % infection rate. The higher infection rate in the current experiment might be due to the higher frequency and consistency of infection compared to the previous attempt. Likewise, in chickens, oral inoculations with H. meleagridis were successful after an 18-hour feed withdrawal or after an alkali mixture was fed to the birds in which 3 out of 7 birds developed histomonosis (Horton-Smith and Long, 1956). This successful oral inoculation may be due to the fact both feed withdrawal and alkali mixture will increase the alkalinity of the upper GI tract allowing for H. meleagridis survival. Liebhart and Hess (2009) were able to infect successfully 6 of 8 one-day-old and 5 of 10 two-week-old turkeys infected orally with a mono-eukaryotic culture of H. meleagridis after a 5-hour feed withdrawal. Liebhart and Hess hypothesized the success of their oral infections compared to others may be due to the differences in strain genetics and pH resistance. Though Hess reported greater infections in the one-day-old poults, who’s intestinal pH is above 4, pointing to the importance of intestinal environmental pH for H. meleagridis survival (Rynsburger, 2009). Beer et al. (2022) produced histomoniasis in one-day-old poults prior to consuming feed when orally inoculated with 2,000 and 20,000 H. meleagridis cells but poults orally inoculated at 21-days-old did not develop histomoniasis. However, oral infections of chickens at 14-days-old with 200,000 H. meleagridis cells triggered histomoniasis in 6 out of 10 birds when a 6-hour feed withdrawal was applied prior to infection while 50,000 and 100,000 cells failed to produce histomoniasis (Chen et al., 2024). On the contrary, Hu et al. (2004) attempted to infect turkeys orally with 100,000 H. meleagridis cells after a 6-hour feed withdrawal or without feed withdrawal. No infections were reported in either group of birds, which might be due to insufficient pH reduction by feed withdrawal, as 6 hours may not be a long enough length of time for clearance of feed content in the upper and middle GI tract. Feed consumption decreases the pH of the crop, ventriculus and gizzard, with large particle sizes of feed stimulating a more acidic environment (Nir et al., 1994). Prior to consumption of feed, the pH of the upper intestines has been measured between 4.4 to 5.0, potentially basic enough for the survival of H. meleagridis (Beer et al., 2022). Current and previous research suggests that protecting H. meleagridis from the low pH in the upper GI tract is the key to inducing successful infection. Though a cyst-like stage was not observed in this study, a cyst-like stage has been reported in vitro when cells were exposed to gradually reduced incubation temperatures, suggesting that histomonads excreted in feces may transform into a cyst to survive outside of the host (Zaragatzki et al., 2010). H. meleagridis in a cyst-like stage may be able to survive a lower pH, though no current in vivo work has been done with this stage (Landim De Barros et al., 2022; Zaragatzki et al., 2010).

It is also important to note that in Experiment 2, the oral infection group showed an even higher infection rate (90 %) compared to the treatment in Experiment 1 orally inoculated with cecal content (43 %). We hypothesize that the difference between the two experiments might be due to the viscosity and/or consistency of the cecal content between the two experiments. Cecal content used for Experiment 1 was previously collected and stored at −20°C for 4 months. In Experiment 2, fresh contents from turkey ceca harvested at a processing plant were used. One observation was the viscosity of the fresh versus defrosted cecal content, where fresh content was tackier and thicker than defrosted inoculum, which might have impaired the defrosted cecal content’s constituents to protect H. meleagridis. Both fresh and frozen cecal content had a similar pH of 6.3-6.7, within the normal range of cecal content (Asare et al., 2021). Similarities in pH between fresh and frozen content suggest that the buffering capacity between them was not altered. Although we did not analyze the fresh versus frozen cecal content’s bacterial constituents, the bacterial load might have been reduced in number/species, possibly contributing to the difference in infection rate (Bilic and Hess, 2020). Previous studies in gnotobiotic turkeys showed the presence of bacteria is necessary for the development of histomoniasis indicating a mutualistic relationship between H. meleagridis and bacteria (Springer et al., 1970). In vitro H. meleagridis growth has been shown to require bacteria and this relationship between bacteria and H. meleagridis may be similar in cecal content but additional investigations in this realm is needed (Hauck, 2017).

Because of the high infection rate of cecal content through the fecal/oral route, Experiment 3 aimed to examine the pathogenicity and virulence of three different isolates of H. meleagridis (BUF, ARK, and ZMI) when administered via the oral route. Variation in infection rates and cecal and liver lesions were observed among various isolates, revealing that BUF and ARK isolates produced higher infection rates compared to ZMI. Earlier research examined virulence factors in three isolates of H. meleagridis administered via the cloacal route (Wei et al., 2020). Two of the isolates (BUF and ZMI) were also used by Wei et al., 2020, who found that under varying conditions, the virulence factors of each isolate may vary, affecting the liver and ceca scores. Sulejmanovic et al. (2016) reported variation in lesion scores between isolates originating from different locations. The BUF and ZMI isolates produced increased disease severity compared to the ARK isolate when administered cloacally. In this investigation, we found that BUF was significantly more virulent than ZMI when administered orally. This suggests that there may be genetic differences among isolates in terms of survivability on exposure to a more acidic pH or stability after its entry into the GI tract. These differences in oral-inoculation infection rate based on characteristics of isolates from diverse geographical locations may be responsible for the incidence and severity of histomonosis outbreaks in various locations throughout the US. Recently, H. meleagridis has been separated into genetically distinct groups (Bilic et al., 2014). Further analysis is needed to understand the differences among isolates, genetic variations, virulence, and the mechanism of genetic variation of isolates passed through the full length of the avian gastrointestinal tract.

It is important to note that cecal content will change in viscosity, bacterial population, and quantity as histomonosis progresses. This may affect the shed rate of cecal histomonads and cecal content ability to protect H. meleagridis consumed by birds. Even with our observations on fecal/oral transmission of H. meleagridis, which confirm other research results, additional work is required to determine factors that support the natural fecal/oral method of transmission.