Interplay between Eimeria acervulina and Cryptosporidium parvum during In Vitro Infection of a Chicken Macrophage Cell Line (HD11)

Background: Eimeria acervulina is a frequent intestinal pathogen of chickens, causing economic impact on the poultry industry. Cryptosporidium parvum is a neglected parasite in chickens. However, because of its zoonotic potential, poultry cryptosporidiosis may pose a risk to public health. Little is known about the parasite–host interactions during coinfection with both parasites. In this study, we investigated the possible interactions during in vitro coinfection of E. acervulina and C. parvum in a chicken macrophage cell line (HD11). Methods: HD11 cells were inoculated with E. acervulina and C. parvum sporozoites and incubated 2, 6, 12, 24, and 48 h post infection (hpi). Mono-infections for each parasite were also investigated. Real-time PCR was used to quantify parasite replication. Additionally, macrophage mRNA expression levels of IFN-γ, TNF-α, iNOS, and IL-10 were measured. Results: For both parasites, multiplication was, in most groups, lower in the coinfection group (COIG) compared with mono-infections. However, at 6 hpi, the number of C. parvum copies was higher in co-infections. Intracellular replication started to decrease from 12 hpi onward, and it was almost undetectable by 48 hpi in all groups. Infections resulted in low expression of all cytokines, except at 48 hpi. Conclusions: Infection of avian macrophages with both E. acervulina and C. parvum seemed to hinder intracellular replication for both parasites in comparison to mono-infection. A clear reduction in intracellular parasites from 12 hpi onward details the important role potentially played by macrophages in host control of these parasites.


Introduction
Chicken coccidiosis is an intestinal disease caused by the ubiquitous Eimeria genus [1] with significant economic importance for the poultry industry [2]. The disease can range from mild infection to acute enteritis with high mortality. Eimeria sp. infection can predispose to infection by other opportunistic pathogens, such as Clostridium spp., Campylobacter spp., or Salmonella spp. which can exacerbate the clinical condition of the host [3][4][5][6]. Seven Eimeria species are usually associated with chicken coccidiosis [1,7], among which Eimeria (E.) acervulina is one of the most common [7]. E. acervulina infects the upper part of the small intestine (duodenum), and the disease is mostly moderated. However, single E. acervulina infections are not the norm [6,7] and mixed infections with other Eimeria species or bacteria can be fatal [6]. Cryptosporidium (C.) parvum is a zoonotic apicomplexan parasite and a major cause of mild to severe gastrointestinal disease in humans and domestic animals [8][9][10]. It can infect the small intestine of a broad range of species [10][11][12][13], including birds. C. parvum infection in domestic fowl is not very common and occurs mostly as a subclinical condition [12][13][14]. In chicken, Cryptosporidium species predominantly invade the airways (respiratory cryptosporidiosis) and the bursa of Fabricius or cecum (intestinal cryptosporidiosis) [15][16][17]. Although seldom reported, classical small-intestine infections related to C. parvum as seen in mammals can occur in chickens [17,18], and infected birds can shed oocysts in the feces, which could pose a risk for human infection [12,13,19].
One of the first lines of defense against invading pathogens is the innate immune system. Along with heterophils and dendritic cells, macrophages play an imperative role in the inactivation and elimination of avian pathogens [20][21][22]. They achieve this through chemotaxis and phagocytosis, amongst other functions [22]. Additionally, they are uniquely situated at the interface between the innate and adaptative immune responses [22]. The latter is due in part to their antigen-presenting abilities [22]. During eimeriosis, the macrophages not only actively inactivate and destroy Eimeria spp. but also serve to transport sporozoites to the main regions of infection [23][24][25]. C. parvum utilizes mammal macrophages in a similar fashion [26]. Yet, the role of the avian macrophage has not been thoroughly explored in Cryptosporidium spp. infections in birds [27].
C. parvum and E. acervulina are both intracellular parasites that can infect the same site of the small intestine. Moreover, possible coinfections may impact not only parasite invasion and replication, but also, more importantly, the host immune response. The objective of this study was to evaluate possible interplay between both parasites in terms of intracellular replication and immune response activation during coinfection of E. acervulina and C. parvum.

Parasite Maintenance, Excystation, and Purification of Sporozoites
E. acervulina oocysts were kindly provided by MSD Animal Health Innovation GmbH (Schwabeheim, Germany). The oocysts were maintained through biannual passages in healthy 11 day old chicks, and sporulated oocysts were purified according to Eckert et al. [28] Purity of the isolate was assessed before and after each passage by multiple PCR assays for seven Eimeria spp. According to Andreopoulou et al. [7]. Finally, sporozoite excystation and purification were carried out following Rentería-Solís et al.'s [29] methodology.
C. parvum oocysts were routinely passaged in neonatal calves; oocyst purification was conducted according to Najdrowski et al. [30]. The C. parvum oocysts were from an inhouse isolate belonging to the sub-genotypes IIaA14G1R1 and IIaA15G2R1 [31]. C. parvum sporozoite excystation was performed following the protocol of Berberich [32]. Briefly, C. parvum oocysts were pelleted at 10,000 rpm (9500× g) for 4 min at room temperature (RT) and then placed on ice. They were subsequently pretreated with 1 mL of 5.25% sodium hypochlorite (NaOCl) in cold PBS (1×) and incubated for 5 min on ice. The oocysts were then centrifuged at 10,000 rpm for 4 min at RT. After centrifugation, the supernatant was discarded, and the pellet was washed with PBS (1×) 2-3 times. Afterwards, the oocysts were resuspended in excystation medium consisting of 0.8% sodium taurocholate (Sigma, Steinheim, Germany) dissolved in RPMI 1640 culture medium. Oocysts were then preincubated at 15 • C for 1 h, and thereafter incubated at 37 • C with 5% CO 2 for 1:30 h. The excystation rate and sporozoite counts were determined using a Neubauer counting chamber (Mediparts, Oberhausen, Germany) for both E. tenella and C. parvum.

In Vitro Infection Assay
Confluent HD-11 monolayers (80% to 90% confluency) were infected with C. parvum (2 ×10 5 sporozoites/well, MOI = 1) and E. acervulina (2 × 10 5 sporozoites/well, MOI = 1), and then incubated at 41 • C in 5% CO 2 for 2, 6, 12, 24, and 48 h. Infections were visualized under a phase-contrast microscope after each timepoint and every 24 h thereafter. Three groups of infection were set for each timepoint: single infection of C. parvum (CPIG), single infection of E. acervulina (EAIG), and coinfection with both C. parvum and E. acervulina (COIG). Additionally, every timepoint included a negative control group consisting of uninfected HD-11 monolayers. All reactions were performed in triplicate. For the 48 h groups, monolayers were washed once with sterile PBS, and fresh medium was added to the wells 24 h post infection (hpi). After each incubation period, cells were trypsinized and centrifuged, and the pellet was used for DNA or RNA purification.

Quantification of E. acervulina and C. parvum by Real-Time Quantitative PCR (qPCR)
DNA was purified from the pelleted cells using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For quantification of E. acervulina, amplification of the SCAR marker gene was conducted as described by Taha et al. [34] using the primers published by Blake et al. [35] (Table 1). Furthermore, replication of C. parvum was estimated using a qPCR assay targeting the HSP70 gene [36]. All reactions were performed on a Bio-Rad CFX Connect Real-Time PCR Detection System (Bio-Rad, Feldkirchen, Germany). Standard curves were generated by serial dilutions of plasmid DNA as previously published [36,37].

Reverse-Transcriptase and Cytokine mRNA Quantification Using Real-Time PCR
Purification of RNA was performed using the RNeasy kit (Qiagen, Hilden, Germany) following the kit's instructions. Purified total RNA was DNase-treated (DNase I, RNase-free, Thermo Scientific, Dreieich, Germany), and complementary DNA (cDNA) was synthesized using a RevertAid first-strand cDNA synthesis kit (Thermo Scientific, Dreieich, Germany) as described by the manufacturer. Relative quantification of the following chicken cytokines was performed using SYBR green-based real-time PCR assays as previously published [38,39]: interferon gamma (IFN-γ), inducible nitric oxide synthase (iNOS), interleukin 10 (IL-10), and tumor necrosis factor alpha (TNF-α) (see Table 1). Relative quantification of mRNA was performed using the 2 −∆∆Ct method and expressed as n-fold differences. Normalization of Ct values was conducted with primers for the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Statistical Analysis
Kolmogorov-Smirnov and Shapiro-Wilk normality tests were used to determine the normal distribution of data. A two-way ANOVA test was used for comparison between different timepoints and groups of infection. Differences were considered significant when the p-value was ≤0.05. All statistical analyses were performed using the GraphPad Prism 9.2 software (GraphPad, San Diego, CA, USA).

Quantification of E. acervulina and C. parvum
Monolayer integrity was visually assessed by microscopy at all timepoints. Overall, E. acervulina intracellular multiplication in the single groups (EAIG) was significantly higher (p < 0.0001) than in the coinfection groups (COIG) at all times (Figure 1a). Across the timepoints, E. acervulina copies in EAIG started notably higher than the COIG during the first 2 and 6 hpi. However, they significantly reduced their numbers from 12 hpi onward (24 and 48 hpi).

Cytokine Analysis
The mRNA expression levels of four cytokines (Table 1) were measured in both single (EAIG and CPIG) and coinfection groups (COIG) at 2, 6, 12, 24, and 48 hpi by RT-qPCR ( Figure 2). No statistically significant differences were found between single infection groups and coinfection groups for all cytokines (p > 0.05). However, there was a significant change across timepoints in the cases of TNF-ɑ and IL-10 (p = 0.0334 and 0.0247, respectively). C. parvum multiplication between single and coinfection groups (CPIG and COIG, respectively) was not significant (p = 0.02381). Nevertheless, multiplication rates were higher in the CPIG than the COIG at all points except at 6 hpi ( Figure 1b). Differences between time points, however, were significant for all groups (p < 0.0001) (Figure 1b). Parasite copies gradually decreased with time in the CPIG groups. In the COIG groups, however, the number of C. parvum copies notably increased at 6 hpi. Furthermore, from 12 hpi onward, C. parvum numbers in COIG were significantly reduced in comparison with CPIG (p-value < 0.0001).

Cytokine Analysis
The mRNA expression levels of four cytokines (Table 1) were measured in both single (EAIG and CPIG) and coinfection groups (COIG) at 2, 6, 12, 24, and 48 hpi by RT-qPCR ( Figure 2). No statistically significant differences were found between single infection groups and coinfection groups for all cytokines (p > 0.05). However, there was a significant change across timepoints in the cases of TNF-α and IL-10 (p = 0.0334 and 0.0247, respectively).

Cytokine Analysis
The mRNA expression levels of four cytokines (Table 1) were measured in both si gle (EAIG and CPIG) and coinfection groups (COIG) at 2, 6, 12, 24, and 48 hpi by RT-qPC ( Figure 2). No statistically significant differences were found between single infecti groups and coinfection groups for all cytokines (p > 0.05). However, there was a significa change across timepoints in the cases of TNF-ɑ and IL-10 (p = 0.0334 and 0.0247, respe tively).

Discussion
We investigated the differences in parasite intracellular multiplication and host-c cytokine expression between single and coinfections of E. acervulina and C. parvum. D infections of Eimeria spp. and Cryptosporidium spp. have been previously reported [1 however, this is, to our knowledge, the first in vitro coinfection study in avian mac phages.
Zhang et al. [40,41] provided quantitative data during single and coinfection of

Discussion
We investigated the differences in parasite intracellular multiplication and host-cell cytokine expression between single and coinfections of E. acervulina and C. parvum. Dual infections of Eimeria spp. and Cryptosporidium spp. have been previously reported [18]; however, this is, to our knowledge, the first in vitro coinfection study in avian macrophages.
Zhang et al. [40,41] provided quantitative data during single and coinfection of E. tenella and Toxoplasma gondii in peripheral blood macrophages. E. tenella numbers were constantly reduced at all timepoints examined (2, 6, 12, and 24 hpi) in coinfected cultures [41]. Interestingly, the same authors measured the replication of E. tenella in poultry macrophages at 24, 48, and 72 hpi. In that study, E. tenella levels increased across timepoints, and detection of intracellular meronts was possible [40]. In our study, we did not investigate timepoints beyond 48 hpi. However, the number of parasites in our experiments was significantly reduced from 12 hpi onward. Michael [42] described the structural morphology of chicken macrophages 6 days after infection with E. acervulina. The author only found sporozoites within the macrophages [42]. It appears that E. acervulina simply fails to further develop into merozoites within macrophages, unlike E. tenella.
We detected a decrease in parasite DNA at 12 hpi. This could have been due to phagocytosis or removal of extracellular parasites after cell rupture during washing steps. During visual examination of monolayer integrity, we did not find noticeable damage to the cell structure. Other possible explanations for this discrepancy could be the different immunogenic potentials across Eimeria species [43], or the difference in MOI between studies. We used a lower MOI than Zhang et al., which may have impacted the macrophage response [40,41].
Zhang et al. [41] discovered that the phagocytic activity of the infected macrophages was hindered at 6 hpi for E. tenella infection but not for T. gondii. This could explain the peak in parasite replication we observed at 6 hpi in almost all cohorts of our study; the increment in intracellular E. acervulina at 6 hpi could have been the product of active cell invasion instead of phagocytosis. Moreover, the rapid decrease in parasite numbers after 6 hpi could indicate initiation of effective phagocytosis. However, macrophage phagocytosis was not investigated in our study.
C. parvum has the capacity to proliferate in mammal macrophages during in vitro infections [26,44]. Martinez et al. [26] were able to observe intracellular type I and II meronts of C. parvum in mouse macrophages at 24, 48, and 72 hpi. To the best of our current knowledge, no studies have previously been published that focused on intracellular mono-or coinfection dynamics of Cryptosporidium spp. in avian macrophages. In our study, coinfection with C. parvum and E. acervulina seemed to reduce the number of intracellular parasites for both species, except for C. parvum at 6 hpi in which coinfection showed a larger number of parasites than in the mono-infection. This was not the case for previous reports using E. tenella as a model, in which E. tenella showed a numeric advantage during coinfections of macrophages with T. gondii in comparison with single infections [40,41].
In addition to phagocytosis or their role as vehicles to infection sites [24][25][26]41], macrophages are also expert triggers of immune response, thanks in part to their ability to express cytokines [22]. For this study, we measured the expression levels of a cytokine involved in the inhibition of parasite replication in both E. acervulina and C. parvum: IFNγ [42][43][44]. In our study, IFN-γ was only expressed at 48 hpi with very mild expression in the E. acervulina single infection, whereas the highest values were recorded in the coinfection and the C. parvum mono-infection. According to Dallout et al. [43], IFN-γ was not expressed in E. acervulina-infected macrophages during the first 48 hpi. In the present study, 48 hpi was also the timepoint with the lowest parasite replication, which could be explained by the possible role of IFN-γ in hindering E. acervulina and C. parvum intracellular multiplication. Additionally, IL-10 expression levels peaked at 48 hpi in all groups and were their lowest at 2 and 6 hpi. Given the regulatory activity of IL-10 against IFN-γ and previous data from E. acervulina in vivo infections [45], the low levels of IFN-γ mRNA in most groups could be explained by the continuous expression of IL-10 across infections and timepoints. iNOS was continuously expressed across all infection settings and timepoints. Interestingly, iNOS was continuously expressed with fold change values >1.5 for all groups and timepoints. Similar results were obtained in most groups for TNF-α, a known promoter of iNOS [46], which could explain the continuous presence of the latter, except at 2 hpi, when TNF-α expression was downregulated. These discrepancies could be further elucidated using proteomic methods. Furthermore, macrophage in vitro replication ability or cell viability during infection were not studied in this project but could be of importance for future investigations. This could help to elucidate possible hampering of macrophage function. Lastly, while in vitro models are an important tool for infection research, replication of our results in an in vivo model can be of significance, as applied in previous examples with other coinfections such as Toxoplasma gondii and Eimeria tenella [47].

Conclusions
Both E. acervulina and C. parvum gradually reduced their intracellular presence under the conditions of this study, which suggests an active role of macrophages in parasite control. Coinfection with both parasites seemed to affect their multiplication. We also replicated a previously reported inhibition in phagocytosis at 6 hpi for other apicomplexan parasites. Competition of different parasites for intracellular resources might play a role in parasite replication and should be further studied. Since coinfection with various species of the genus Eimeria and with other avian Cryptosporidium spp. may occur under natural conditions, coinfection studies deserve more attention.