Leucocytozoon Caulleryi Research Articles

Molecular epidemiological studies of Leucocytozoon caulleryi in commercial layer flocks in Southern peninsular India reveal the presence of new subclusters.

The Leucocytozoon parasites are one of the important protozoa species affecting domestic poultry and wild birds. Though there are frequent reports about the incidence of Leucocytozoon in wild birds, the information regarding the occurrence of commercial poultry is underestimated. From October 2015 to August 2022, ailing and dead birds from 39 commercial layer flocks were submitted to the Poultry Disease Diagnosis and Surveillance Laboratory, Namakkal, with a history of brief illness, followed by mortality with oozing of blood from oro-nasal orifice. The presence of Leucocytozoon caulleryi was confirmed in all the flocks by laboratory examination and PCR. All the Leucocytozoon-positive cases were reported after heavy rainfall and the affected farms were inhabited with Culicoides flies. The infected birds were dull and depressed, exhibited labored breathing, and blood-tinted ropy mucous from the oro-nasal cavity. Necropsy examination revealed pale-anaemic comb and wattles, massive haemorrhagic clot over the surface of the kidney, peritoneum, and trachea, and scattering of greyish-white/reddish military megaloschizonts throughout the serosa of viscera and skeletal muscles. Histopathological examination revealed many spherical cysts or megaloschizonts in the intestine, pancreas, gizzard, spleen, liver, kidney, heart, and breast muscles. The cysts were deeply seated within the above organ's parenchyma or embedded in the serosa. There was extensive connective tissue proliferation with mononuclear cell infiltration around the cyst. Leucocytozoon-specific DNA was detected in all the flocks by PCR. Phylogenetic analysis of the cytochrome b gene of L. caulleryi reported from various parts of the world revealed that L. caulleryi is grouped into two major clusters (Cluster I & II). Further, Indian L. caulleryi sequences fall under sub-cluster I and II along with L. caulleryi reported from Taiwan, Egypt, China, Myanmar, Japan, South Korea, and Thailand.

Molecular and pathological characterization of natural co-infection of poultry farms with the recently emerged Leucocytozoon caulleryi and chicken anemia virus in Egypt.

In the summers of 2018 and 2019, a disease outbreak stroke 25 broiler chicken farms and 3 broiler breeder farms in different Governorates in Egypt. The disease caused a mortality rate ranging from 3.2 to 9%. Postmortem examination showed petechial hemorrhage in the breast and thigh muscles, thymus gland, and peritoneal cavity and extensive hemorrhages in the kidneys. A total of 140 liver, kidney, lung, skeletal muscles, thymus, and spleen samples were collected. Twenty-eight pooled samples were created and examined by PCR and histopathological examination to identify the causative pathogens. All collected samples were PCR-negative to Newcastle disease virus (NDV), avian influenza viruses (H5, H9, and H7), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and fowl adenovirus (FadV). Leucocytozoon caulleryi (L. caulleryi) genetic material was identified by PCR in 17 out of the 28 collected samples (61%). Five chicken farms (18%) showed positive PCR results for both L. caulleryi and chicken anemia virus (CAV). Histopathological examination revealed unilocular megaloschizonts in thymus, skeletal muscle, and lung as well as massive hemorrhages in parenchymatous organs. Nucleotide sequences of the identified pathogens were compared with other reference sequences available in the GenBank. The identified L. caulleryi has a close relationship with those previously detected in Asia, indicating potential transmission route of the parasite. The CAV has a close genetic relation with CAVs previously identified in Egypt. Furthermore, a real-time PCR for rapid, specific, and quasiquantitative detection of L. caulleryi was developed with a detection limit of 100 genome copies per reaction.

Leucocytozoon caulleryi

This datasheet on Leucocytozoon caulleryi covers Identity, Overview, Distribution, Hosts/Species Affected, Vectors & Intermediate Hosts, Further Information.

Effects of Artemisia annua on experimentally induced leucocytozoonosis in chickens

The biting midge Culicoides arakawae is the vector for the parasite Leucocytozoon caulleryi. Birds infected with L. caulleryi develop leucocytozoonosis. Given the food safety concern regarding drug residue in eggs, discovering a natural alternative to antibiotics is a worthy of exploration. Thus, we investigated the effects of the antimalarial herb Artemisia annua on experimentally induced leucocytozoonosis in chickens. We reared C. arakawae in the laboratory. Eggs were cultured, developing into larvae, pupae, and imagoes. Female midges sucked the blood of sick chickens and then were ground into a solution injected into healthy chickens. The control group was given empty capsules daily, whereas the 2 experimental groups were given 40 mg/kg sulfadimethoxine or 0.5 g of A. annua powder. Leucocytozoon gametocytes were detected in chicken blood through Giemsa staining. PCR detected the cytochrome b gene of L. caulleryi in the infected chickens. No significant among-group differences in body weight gain were observed before d 14 postinoculation (P > 0.05). Body weight gain in the control group was significantly lower from day 14 to 28 postinoculation (P < 0.05). After day 14, rectal temperature in the experimental groups decreased significantly compared with that in the control group. Lower rates of pale comb and green feces were observed in the animals receiving treatment from day 0. The experimental groups had a higher recovery rate and recovered earlier than did the control group. By day 31, all the animals had recovered. PCR detected L. caulleryi in the infected chickens with high sensitivity and accuracy. The animals receiving A. annua exhibited increased weight gain and reduced parasite concentrations in the blood. This in turn reduced mortality and the occurrence of pale comb and green feces. The findings are informative for research on leucocytozoonosis.

Leucocytozoon caulleryi in Broiler Chicken Flocks: Clinical, Hematologic, Histopathologic, and Molecular Detection.

Despite the vast Egyptian poultry production, scanty information is available concerning the infection of haemprotozoan parasites as pathogens in commercial broilers. In the present study, we provided the first detection of leucocytozoonosis in five broiler chicken flocks in El-Beheira Egyptian governorate. Despite the low mortality rates in the affected flocks (0.3%-1% as a 5-day mortality), severe postmortem (hemorrhagic spots and scars) and histopathologic lesions appeared in different organs including skeletal muscles, liver, kidney, pancreas, abdominal cavity, and bursa of Fabricius. Evaluation of blood smears revealed gametocytes in erythrocytes and leukocytes. Conventional reverse transcriptase-PCR and partial sequence analysis of mitochondrial cytochrome oxidase b gene detected Leucocytozoon caulleryi. GenBank accession numbers of the five Egyptian L. caulleryi isolates were obtained. The five L. caulleryi were 99.9% identical to each other and 99.14% similar to the L. caulleryi mitochondrial DNA gene of Asian strains from India, Japan, Malaysia, South Korea, Taiwan, and Thailand.

Development and application of a novel multiplex PCR assay for the differentiation of four haemosporidian parasites in the chicken Gallus gallus domesticus

Haemosporidian infections in domestic chickens (Gallus gallus domesticus) are not only widely prevalent but also cause economic loss. Diagnosis is usually made by microscopic examination; however, the method has several drawbacks such as requiring an experienced microscopist, being unreliable when parasitemia is low and being unable to accurately differentiate between co-infections from multiple parasite species. Therefore, the current extent of haemosporidian infections might be underestimated and neglected. We have developed a novel multiplex PCR assay to simultaneously detect and differentiate between four haemosporidian parasites: Leucocytozoon caulleryi, Leucocytozoon sabrazesi, Plasmodium juxtanucleare and Plasmodium gallinaceum. Primers in the present study specifically amplified the corresponding targets with no cross-species amplification detected. The multiplex PCR exhibited a significantly greater detection rate when compared with microscopic examination (p = 0.0001). The results demonstrate that the detection rate of multiplex PCR for L. sabrazesi, P. juxtanucleare, and P. gallinaceum are all greater than that of microscopic examination with p = 0.002, 0.0001 and 0.004, respectively. Co-infections were also detected more effectively by multiplex PCR. We applied the current method to field samples originating from Nan, Prachinburi, and Chachoengsao Provinces. The current study revealed that positive rates of haemosporidian parasites in chickens in the three study sites ranging from 39.5%–93.8%. The present assay offers a timesaving option for molecular diagnosis instead of using singleplex PCRs for detecting the parasites individually. Within a single reaction, this assay would be a useful tool for the detection of avian haemosporidian parasites either single or under co-infection conditions and for large-scale epidemiology studies.

Detection and molecular identification of Leucocytozoon and Plasmodium species from village chickens in different areas of Myanmar

Village chicken production, a traditional, small-scale, and extensive backyard poultry industry, has been profitable for local farmers in Myanmar. However, there is scanty information available concerning the infection of these chickens with avian pathogens, including haemoprotozoan parasites. In the present study, we provide the first report of microscopic detection and molecular identification of Leucocytozoon and Plasmodium parasites from seven different areas of Myanmar. Leucocytozoon gametocytes were detected in 17.6% (81/461) of the blood smears from village chickens. The nested polymerase chain reaction (PCR) for targeting Leucocytozoon mitochondrial cytochrome b (cyt b) genes had a 17.6% positive rate. Although the positive rate of nested PCR targeting Plasmodium/Haemoproteus cyt b was 34.3%, the PCR protocol was observed to possibly amplify DNA of a certain species of Leucocytozoon. There were no obvious clinical signs in the infected birds. Statistical analysis of the microscopic detection and PCR detection rates using the age and sex of birds as internal factors revealed that the statistical significances differed according to the study area. The sequencing of 32 PCR products obtained from each study area revealed infection by Leucocytozoon caulleryi in three birds, Leucocytozoon sabrazesi in two birds, Leucocytozoon schoutedeni in two birds, Leucocytozoon sp. in eighteen birds, and Plasmodium juxtanucleare in seven birds; however, Haemoproteus infection was not detected. While L. sabrazesi was detected in chickens from the central region of Myanmar, the other haemosporidians were detected in those from different areas. In the haplotype analysis, we detected 17 haemosporidian cyt b haplotypes, including two for L. caulleryi, one for L. sabrazesi, two for L. schoutedeni, nine for Leucocytozoon sp., and three for P. juxtanucleare. Phylogenetic analysis of the cyt b haplotypes revealed a considerably close genetic relationship among chicken haemosporidians detected in Myanmar, Thailand, and Malaysia. These results indicate that well-recognized widespread species of chicken Leucocytozoon and Plasmodium are distributed nationwide in Myanmar, providing new insights into the ecosystem and control strategies of haemosporidian parasites in domesticated chickens in Myanmar.

Variasi Morfologi dan Deteksi Leucocytozoon caulleryi dengan Metode PCR pada Ayam Ras di Wilayah Endemis Indonesia

The purpose of this study was to analyze the variation in morphology and detection Leucocytozoon caulleryi by PCR in mtDNA genes cty b that attacking bred chickens in endemic areas of Indonesia. This study was divided into three stages, data collection of Leucocytozoonosis cases in endemic areas of Indonesia; identification of parasites Leucocytozoon caulleryi morphologically through microscopic examination; and the identification of Leucocytozoon caulleryi cyt b genes that attack bred chickens in endemic areas by PCR. The results showed that there are variations in morphology of Leucocytozoon caulleryi that attacking chicken in endemic areas of Indonesia. Gamete morphometry of L. caulleryi had an average length and width 18.233 ± 4.672 dan 12.934 ± 3.349 µm. Nested PCR clearly showed positive reaction of Leucocytozoon infections by amplicons in 600 bp and 03 bp length.

The phylogenetics of Leucocytozoon caulleryi infecting broiler chickens in endemic areas in Indonesia.

Aim:The objective of this research was to determine the species and strains of Leucocytozoon caulleryi and study the phylogenetics of L. caulleryi of broiler chickens in endemic areas in Indonesia.Materials and Methods:Blood samples were collected from broiler chickens originated from endemic area in Indonesia, i.e., Pasuruan, Lamongan, Blitar, Lumajang, Boyolali, Purwokerto, and Banjarmasin in 2017. Collected blood was used for microscopic examination, sequencing using BLAST method to identify the nucleotide structure of cytochrome b (cyt b) gene that determines the species, and the phylogenetics analysis of L. caulleryi that infected broiler chickens in endemic areas in Indonesia, using Mega 5 software.Results:The results showed that Plasmodium sp. and L. caulleryi were infected broiler chickens in endemic areas in Indonesia. L. caulleryi in one area had very close phylogenetic relations with those in other areas. The genetic distance between L. caulleryi taxa from various endemic areas is very close (<5%).Conclusion:There is a very close phylogenetics among strains of L. caulleryi that infected broiler chickens in various endemic areas in Indonesia.

Pathology and molecular characterization of recent Leucocytozoon caulleryi cases in layer flocks.

Leucocytozoonosis was found in three layer farms in chickens with suspected fatty liver or fatty liver hemorrhagic syndrome in Korea between 2009 and 2011. These layer chicken flocks showed both mortality and decreased egg production for one or two weeks when they were between 59 and 82 weeks old. At the necropsy, the most prominent gross lesions were found in the liver, which was enlarged, had a fragile texture, exhibited yellowish discolorations, and had various hemorrhagic lesions. Tissue reactions associated with megaloschizonts specific for Leucocytozoon caulleryi were prominent upon microscopic examination of the liver without significant lipidosis. In addition, the ovaries and uterus were the most affected organs for Leucocytozoon caulleryi multiplication, which led to decreased egg productions. Molecular studies with formalin-fixed, paraffin-embedded tissues were performed in search of a partial region of the cytochrome b gene for hemosporidian parasites. Based on these results, the causal agent was determined to be closely related to Leucocytozoon caulleryi reported in Japan and Malaysia. In this study, we describe recently re-occurring leucocytozoonosis in layer chickens, which required histopathology for disease diagnosis. To prevent outbreaks and maintain chicken health and egg production, layer chickens need to be monitored for symptoms of leucocytozoonosis.

Diagnosis ofLeucocytozoon caulleryiInfection in Commercial Broiler Breeders in South Korea

This report confirms a recent outbreak of a Leucocytozoon caulleryi infection in a commercial broiler breeder flock in South Korea. Seven, 18-day-old broiler breeders (Gallus gallus) were necropsied following a history of depression, sudden death, and subcutaneous hemorrhages. On necropsy, subcutaneous hemorrhages were identified in the wings and legs, pectoral and thigh muscles, thymus, epicardium, pancreas, and kidneys. On histopathology, there were numerous schizonts and merozoits of a Leucocytozoon sp. noted in the heart, spleen, liver, kidneys, thymus, and bursa of Fabricius. Molecular analysis of the mitochondrial cytochrome oxidase b confirmed that the causative agent was Leucocytozoon caulleryi. Although L. caulleryi was diagnosed previously in South Korea, there had been no reports of L. caulleryi over the past several decades.

Loop-mediated isothermal amplification assay for detection of Histomonas meleagridis infection in chickens targeting the 18S rRNA sequences

Histomonas meleagridis is the causative agent of histomonosis, a disease of gallinaceous fowl characterized by necrotic typhlitis, hepatitis, and high mortality. To develop a rapid and sensitive method for specific detection of H. meleagridis, an assay based on loop-mediated isothermal amplification (LAMP) targeting the 18S rRNA gene was established. The detection limit of the LAMP assay was 10 copies for standard plasmids containing an 18S rRNA gene fragment, which was superior to that of a classical PCR method. Specificity tests revealed that there was no cross-reaction with other protozoa such as Trichomonas gallinae, Blastocytis sp, Tetratrichomonas gallinarum, Plasmodium gallinaceum, Toxoplasma gondii, Eimeria tenella, Leucocytozoon caulleryi, and Leucocytozoon sabrazesi. The assay was evaluated for its diagnostic utility using field liver and caeca samples collected from suspected cases, the detection rate was 100 and 97.92%, respectively. These results indicate that the LAMP assay may be a useful tool for rapid detection and identification of H. meleagridis in poultry.

The apicoplast genome of Leucocytozoon caulleryi, a pathogenic apicomplexan parasite of the chicken

Leucocytozoon caulleryi, a haemosporidian parasite of the chicken (Gallus gallus domesticus), can be highly pathogenic and often fatal. Although this parasite is extremely relevant to veterinary science, knowledge of its genomic features is limited. To gain information applicable to developing novel control methods for the parasite, we analyzed the apicoplast genome of L. caulleryi. This extranuclear organellar DNA of 85.1 % A + T and a unit of 34,779 bp was found to encode almost the same set of genes as the plastid genome of Plasmodium falciparum, including 16 tRNA and 30 protein coding genes, and except for one open reading frame, ORF91 absent in L. caulleryi. As in P. falciparum, the L. caulleryi apicoplast DNA contains two sets of a unique inverted repeat (IR), each one 5,253 bp and encoding genes specifying one large and one small rRNA subunit and nine tRNAs but no protein, and separated by a unique 13 bp sequence. Studies of several haemosporidian apicoplast DNA sequences have identified a corresponding IR region; however, none of these studies has looked at the complete sequence, even for well-studied species such as P. falciparum. Phylogenetic studies using a concatenated amino acid sequence based on the open reading frames confirmed the close relationship between L. caulleryi and Plasmodium spp. In this study, we determined the nucleotide sequence of the entire L. caulleryi apicoplast genome, including the region connecting the two IR units. This is the first report of the complete nucleotide sequence of a haemosporidian apicoplast DNA with a canonical IR.

Booster responses by oral vaccination with transgenic plants against chicken leucocytozoonosis

We developed a transgenic potato (TrP/R7) expressing the recombinant R7 (rR7) antigen for use as an oral vaccine to protect against a chicken protozoan disease, chicken leucocytozoonosis. The TrP/R7 potato was produced by Agrobacterium tumefaciens-mediated transformation and regeneration, and the R7 gene insertion into potato chromosomes was confirmed by genomic polymerase chain reaction and Southern hybridization. rR7 antigen expression in TrP/R7 potato was also confirmed by sandwich enzyme-linked immunosorbent assay and western blotting using an antibody against the second-generation schizont of Leucocytozoon caulleryi. A transgenic potato clone with the highest rR7 antigen expression (3 µg rR7 antigen per gram of fresh-weight potato leaves) was selected, cultivated, and used in oral administration experiments to examine its ability to boost immunity. Chickens were immunized with chicken leucocytozoonosis vaccine “Hokken” by injection, and chickens that developed moderate levels of antibody titres were fed with TrP/R7 leaves. Chickens fed with TrP/R7 leaves showed increased antibody responses. In contrast, chickens fed with non-transgenic potato leaves showed a continuous decrease in antibody titres. Furthermore, chickens fed with TrP/R7 potato leaves showed strong resistance against experimental challenge with L. caulleryi infection. This study demonstrates the use of a plant-based oral vaccine to boost immunity against a protozoan disease.

Highly conserved gene arrangement of the mitochondrial genomes of 23 Plasmodium species

Mitochondrial (mt) genomes from diverse phylogenetic groups vary considerably in size, structure and organization. The genus Plasmodium, the causative agent of malaria, has the smallest mt genome in the form of a tandemly repeated, linear element of 6 kb. The Plasmodium mt genome encodes only three protein genes ( cox1, cox3 and cob) and large- and small-subunit ribosomal RNA (rRNA) genes, which are highly fragmented with 19 identified rRNA pieces. The complete mt genome sequences of 21 Plasmodium species have been published but a thorough investigation of the arrangement of rRNA gene fragments has been undertaken for only Plasmodium falciparum, the human malaria parasite. In this study, we determined the arrangement of mt rRNA gene fragments in 23 Plasmodium species, including two newly determined mt genome sequences from P. gallinaceum and P. vinckei vinckei, as well as Leucocytozoon caulleryi, an outgroup of Plasmodium. Comparative analysis reveals complete conservation of the arrangement of rRNA gene fragments in the mt genomes of all the 23 Plasmodium species and L. caulleryi. Surveys for a new rRNA gene fragment using hidden Markov models enriched with recent mt genome sequences led us to suggest the mtR-26 sequence as a novel candidate LSU rRNA fragment in the mt genomes of the 24 species. Additionally, we found 22–25 bp-inverted repeat sequences, which may be involved in the generation of lineage-specific mt genome arrangements after divergence from a common ancestor of the genera Eimeria and Plasmodium/ Leucocytozoon.

Two extra chromosomal genomes of Leucocytozoon caulleryi; complete nucleotide sequences of the mitochondrial genome and existence of the apicoplast genome

We analyzed extra chromosomal genomes of avian blood protozoa, Leucocytozoon caulleryi. One of the genomes, the mitochondrial genome was completely sequenced resulting 5,959 bp in length. This genome contained the identical gene organization and contents to that of other avian blood protozoa previously analyzed: three functional genes for cytochrome c oxidase subunit I, III, and cytochrome b with following sets of discontinuous and scrambled 15 ribosomal subunit RNA genes. In addition, the mitochondrial genome was estimated to have the tandem repeated structure as well as previously found in avian Plasmodium species. Furthermore, we found partial gene sequences of apicoplast DNA, another extra chromosomal genome, from L. caulleryi. These sequences were estimated as partial caseinolytic protease C and elongation factor Tu A genes. This report is the first description of two extra chromosomal genomes of L. caulleryi.

A rapid assay for detecting antibody against leucocytozoonosis in chickens with a latex agglutination test using recombinant R7 antigen

A method for detecting antibody against leucocytozoonosis in chickens by latex agglutination (LA) was developed using latex beads coated with recombinant R7 (rR7), an outer membrane antigen of second-generation schizonts of Leucocytozoon caulleryi. Compared with the agar gel precipitation test, which is widely used in Japan, LA could detect antibody induced by L. caulleryi infection with greater sensitivity. No agglutination was detected with sera from specific pathogen free or layer chickens that had not been vaccinated with oil-adjuvanted rR7 antigen, or infected with L. caulleryi, nor with sera from chickens inoculated with other pathogens, establishing the specificity of the assay. The LA test was also able to be used to quantify serum antibody induced by vaccination, which is not possible with the agar gel precipitation test. This study has shown that LA using the rR7 antigen is a simple, quick, and useful method for detecting antibody against L. caulleryi.

Increase of Antibody Titer against Leucocytozoon caulleryi by Oral Administration of Recombinant R7 Antigen

In this study, we examined oral administration with recombinant R7 (rR7) antigen expressed in Escherichia coli using chicken leucocytozoonosis subunit vaccine (LV)-vaccinated and unvaccinated chickens. Only LV-vaccinated chickens showed re-induction of anti-second-generation schizont (2GS) antibody. Also, LV-vaccinated chickens whose anti-2GS antibody titer was middle-level showed increases of the antibody titer compared to vaccinated-control chickens (P>0.01, >0.05) after oral administration with rR7 antigen.

Cellular immune responses in chickens induced by recombinant R7 Leucocytozoon caulleryi vaccine.

We have recently developed recombinant subunit vaccine consisting of second-generation schizont (2GS) membrane protein (rR7) of Leucocytozoon caulleryi. Chickens immunized with rR7 antigen acquired clear resistance to challenge by Leucocytozoon sporozoites. We examined the induction of cellular immune responses in vaccinated chickens. Spleen adherent cells from vaccinated chickens showed significantly higher phagocytic activity against 2GS-coated latex particles than did cells from adjuvant-inoculated or untreated control birds. Anti-R7 chicken IgG significantly increased the phagocytic rate of adherent cells from these 3 groups. These results show that specific cellular immune responses are induced by recombinant R7 subunit vaccine consisting of L. caulleryi 2GS protein, which suppresses the growth of parasites in the host in association with antiparasite antibodies to 2GS antigen.

The correlation of protective effects and antibody production in immunized chickens with recombinant R7 vaccine against Leucocytozoon caulleryi.

Protective effects of recombinant R7 (rR7) vaccine against Leucocytozoon caulleryi in chickens were studied. After injection of oil-adjuvanted rR7 vaccine into chickens, antibody titers against second-generation schizonts (2GS) antigen of L. caulleryi (anti-2GS antibody) rapidly rose in all the immunized chickens, reached to a peak value 2 weeks after injection, and the titers persisted through 4 or 5 months after injection. Chickens having high levels of the anti-2GS antibody titers (> or = 102,400 ) at pre-challenge completely protected against sporozoites challenge of L. caulleryi. After the challenge inoculation, relatively high parasitemia of L. caulleryi was observed in all the inadequately immunized chickens having low levels of the antibody titers (< or = 3,200) at pre-challenge, although some of them seemed to be clinically normal. Correlation of protective effects in the immunized chickens was observed between both prevention of appearance of clinical signs and parasitemia after parasites challenge and anti-2GS antibody titers of the chickens at pre-challenge. The present study shows that chicken leucocytozoonosis can be prevented by vaccination, and humoral immunity may play an important role in the control of chicken leucocytozoonosis.