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.

In vitro infection of Madin-Darby bovine kidney (MDBK) cells with Eimeria acervulina sporozoites: quantitative analysis of parasite cellular invasion and replication using real-time polymerase chain reaction (PCR).

Poultry coccidiosis causes considerable economical losses to the livestock industry. Eimeria parasites are responsible for this disease. On a global scale, E. acervulina and E. tenella are amongst the most common Eimeria spp. infecting broilers. E. tenella is commonly used as infection model in in vivo and in vitro studies. On the other hand, E. acervulina has barely been studied under in vitro conditions. A well established and widely used in vitro model for E. tenella infection is the Madin-Darby bovine kidney cell line (MDBK); however, little is known regarding suitability of MDBK cells as host cells for E. acervulina. We infected MDBK monolayers with two different doses, 5 × 104 and 2 × 105, of E. acervulina sporozoites and evaluated cultures at 24 and 96 h post infection (hpi). For comparison, we ran an identical infection assay using E. tenella sporozoites. To assess parasite reproduction, the number of DNA copies of E. acervulina SCAR marker and E. tenella ITS-1 gene was quantified using real-time quantitative PCR. We found that the number of E. acervulina copies increased significantly at 24 hpi in comparison to E. tenella (p < 0.05). After 96 hpi, E. acervulina gene copies were considerably reduced while E. tenella continued to multiply (p < 0.05). Our results show that MDBK monolayers could be used for in vitro research aimed to study E. acervulina sporozoite cell invasion. Nevertheless, modifications of in vitro cultivation appear necessary to allow qualitative and quantitative studies over longer periods of parasite reproduction.

A SYBR green I real-time polymerase chain reaction (PCR) assay for detection and quantification of Trichomonas gallinae.

Trichomonas gallinae are parasitic flagellates of importance in wild and domestic birds. The parasite is worldwide distributed, and Columbine birds are its main host. Current research focuses mostly on epidemiological and phylogenetic studies. However, there is still a lack of knowledge regarding parasite-host interaction or therapy development. Real-time PCR is a useful tool for diagnostic and quantification of gene copies in a determined sample. By amplification of a 113-bp region of the 18S small subunit ribosomal RNA gene, a SYBR green-based real-time PCR assay was developed. A standard curve was prepared for quantification analysis. Assay efficiency, linearity, and dissociation analysis were successfully performed. Specificity, sensibility, and reproducibility analysis were tested. This assay could be a useful tool not only for diagnostic purposes but also for future in vivo and in vitro T. gallinae studies.

A modified method for purification of Eimeria tenella sporozoites.

Coccidiosis is an economically important gastrointestinal disease in domestic fowl. Eimeria species are the causative agents of avian coccidiosis. Current challenges in management and prevention of eimeriosis enhance the need for research in this field. Sporozoite purification is a necessary step for Eimeria spp. in vitro infection models. Current alternatives such as DE-52 anion exchange chromatography and Percoll gradient require time and resources. We present a modified protocol consisting on vacuum filtration of sporozoites using a disposable 5-µL filter. Yield percentages were similar to those reported for Percoll gradient purification. By reducing time and efforts during sporozoite purification, it could be possible to increase resources in other areas of Eimeria studies.

Molecular characterization of bovine Cryptosporidium isolated from diarrheic calves in the Sudan.

Cryptosporidiosis is a common protozoan infection causing morbidity and mortality in young cattle and may be zoonotically transmitted to humans. So far, there is no data available on the presence of Cryptosporidium spp. in the Sudan. The aim of this study was to isolate, identify, and genotype Cryptosporidium oocysts sampled from diarrheic calves housed at different farms in three different municipalities in Khartoum State (Khartoum, Khartoum North, Omdurman). A total of 149 fecal samples were evaluated microscopically for the presence of Cryptosporidium oocysts using the modified Ziehl-Neelsen staining method and 87 (58.3%) samples tested positive. Positive and negative samples were further analyzed by nested PCR targeting the SSU rRNA region. Positive samples were subjected to restriction enzyme analysis of PCR amplicons (PCR-RFLP). Nested PCR identified Cryptosporidium DNA in 53 samples (35.5%); restriction digestion of the PCR products revealed the presence of C. parvum (73.5%), C. ryanae (13.2%), C. andersoni (7.5%), and C. bovis (1.8%). Species distribution was clearly related to age with C. parvum being the predominant species in dysenteric pre-weaned calves. Sequencing of three genes (SSU rRNA, COWP, and GP60) for three C. parvum isolates originating from the three different municipalities showed that all belong to C. parvum subtype family IId. Based on data obtained by GP60, sequencing the two C. parvum isolates from Khartoum and Omdurman represent subtype IIdA18G1, whereas oocysts isolated in Khartoum North belong to subtype IIdA19G1. The observed genotypes are zoonotic and thus C. parvum in calves is potentially a health risk to humans in Khartoum State, Sudan. To the best of our knowledge, this is the first reported attempt to characterize Cryptosporidium isolated from cattle in the Sudan.