Preliminary study on buffy coat smear and molecular detection of microfilaria in domestic chickens (Gallus gallus domesticus) raised in Southern Thailand.

Background and Aim: Filarial nematode typically produces a larval stage (microfilariae) in the bloodstream of vertebrate hosts, where microfilariae reside in the blood or subcutaneous tissues. Filarial nematodes cause human diseases, such as river blindness and elephantiasis, which are widely studied. However, in avian species, they are overlooked because they are nonpathogenic. In Thailand, microfilaria can be found in wild birds and domestic chickens. Recently, an increase in the number of blood samples submitted to veterinary diagnostic laboratories may have increased the number of microfilariae. Therefore, knowledge about filarial species and reliable detection methods are important. Therefore, this study aimed to investigate the efficacy of buffy coat smear and polymerase chain reaction (PCR)-based methods for the detection of microfilaria in domestic chickens. In addition, parasites were identified using the sequence of the cytochrome c oxidase subunit 1 (COX1) gene. Materials and Methods: Giemsa-stained buffy coat smears from a previous study were reanalyzed. These available buffy coat smears were prepared from 55 domestic chickens raised as backyard free-ranging in Southern Thailand. Fifty-seven frozen genomic DNA extracted from chicken blood were used to detect the presence of the COX1 gene in Onchocercidae nematodes. The nested PCR protocol for amplification of the OnchoCOI_R2-OnchoCOI_R2 fragment of the COX1 gene was applied from a previous report. Sequences of COX1 were analyzed to identify Onchocercidae nematodes and if they were single or mixed infections. We constructed Bayesian phylogenetics to identify parasites and assessment of the relationship between filarial nematodes in avian species and other vertebrate hosts. Results: Buffy coat smears from 15 samples revealed microfilaria. Of these 15 samples, only eight were positive for COX1 nested-PCR amplification. The other two buffy coat-negative samples were also positive for nested-PCR. Sequencing of these 11 nested PCR-positive samples revealed that almost all of them were Onchocercidae nematodes. Bayesian phylogenetic analysis showed that chicken Onchocercidae spp. were grouped with other avian filarial nematodes. However, all chickens Onchocercidae spp. showed a double peak in the sequencing chromatogram, indicating mixed filarial infection (species or haplotypes). Therefore, no chicken Onchocercidae sequence was deposited on National Center for Biotechnology Information, GenBank. Conclusion: Giemsa-stained buffy coat smear was a reliable method for the detection of chicken microfilaria in routine veterinary diagnostic laboratories. Development of a new PCR-based method is necessary. This method may provide greater sensitivity and specificity of detection. In addition, the PCR method allowed us to access the genetic characteristics of nematodes, which helped us maximize our knowledge of nematodes. Further investigations, such as the pathogenicity of filarial nematodes in chickens and their potential vectors, are required.

Prevalence and Diversity of Blood Parasites (Plasmodium, Leucocytozoon and Trypanosoma) in Backyard Chickens (Gallus gallus domesticus) Raised in Southern Thailand.

Avian malaria and leucocytozoonosis can cause fatal diseases, whereas avian trypanosomiasis is reported to be harmless in chickens. Backyard chickens can be infected by several pathogens, including blood parasites, that may shed to industrial poultry production, with a consequently higher economic impact. This study aimed to investigate the presence of several blood parasites (Plasmodium, Leucocytozoon and Trypanosoma) in backyard chickens raised in Southern Thailand, using PCR-based detection and microscopic methods. From June 2021 to June 2022, 57 backyard chickens were sampled. Fresh thin blood smears were prepared from 11 individuals, and buffy coat smears were prepared from 55 of them. Both thin blood smears and buffy coat smears were used for microscopic analysis. Two nested PCR protocols that amplify a fragment of cytochrome b (cytb) and small subunit rRNA (SSU rRNA) genes were used to identify Haemosporida and Trypanosoma parasites, respectively. The number of positive samples was higher with the application of nested PCR than when buffy coat smears were used. Three new Plasmodium lineages (GALLUS47-49) and thirteen Leucocytozoon lineages (GALLUS50-62) were found. Trophozoites, meronts and gametocytes of Plasmodium gallinaceum (GALLUS01) were present in one thin blood smear. All thin blood smears revealed Leucocytozoon infections, but only three samples were a single infection. These three samples revealed the presence of fusiform host cell-parasite complexes, of which the morphological features resembled those of Leucocytozoon macleani (possible synonym is Leucocytozoon sabrazesi), while the cytb showed that this parasite is closely related to the lineage GALLUS06-07, described as Leucocytozoon schouteni. The Trypanosoma prevalence was 33.33%; it was present in only one of the thin blood smears, and it resembles Trypanosoma calmettei. This study showed the prevalence of a high diversity of Plasmodium (64.91%) and Leucocytozoon (89.47%) in Thai chickens. Both nested-PCR and buffy coat smear can be used as the diagnostic tool for the testing of Plasmodium, Leucocytozoon and Trypanosoma for parasitic control in backyard chickens and poultry farms. The information on the parasite species that can be found in chickens raised in Southern Thailand was also considered as the baseline information for further study.

Hematology, Ultrastructure and Morphology of Blood Cells in Rufous-Winged Buzzards (Butastur liventer) from Thailand.

In attempt to treat injured raptors and promote conservation awareness, the Kasetsart University Raptor Rehabilitation Unit (KURRU) was established in 2007. The complete blood counts (CBCs) are a manual tool used for the screening of raptor health. These tests require knowledge of blood cell morphology. This study aimed to describe the preliminary information of the hematology, ultrastructure, and morphology of blood cells in rufous-winged buzzards (RWB). There were 17 RWBs admitted into the KURRU. CBCs were manually performed by veterinary technicians. The morphology and morphometry of blood cells were observed from Wright-stained blood smears. Ultrastructure was observed from uranyl acetate and lead citrate-stained sections. The hematologic values were analyzed and described from individual RWBs that were clinically healthy, negative for blood parasites, and had PCV > 0.30 L/L. Consequently, CBCs of 12 out of 17 RWBs were included for descriptive hematologic values. Heterophils were the most prevalent white blood cells in RWBs. Of these 17 RWBs, 1 non-parasitized RWB showed hypochromic erythrocytes with PCV 0.18 L/L, which indicated that anemia in RWBs resulted from non-parasitic causes. The morphology of blood cells in RWBs was similar to those in other diurnal raptors, except that the lymphocytes showed pale or colorless cytoplasm. The electron micrographs highlighted that the basophil contained two types of granules: homogeneous electron-dense granules and reticulated electron-dense granules. The photomicrographs in this report are the scientific reference for identification of blood cells in RWBs. The CBCs from non-parasitized RWBs (clinically healthy) can be used as a cage mate reference in the KURRU. Additionally, we found evidence that evaluations of blood smears together with CBC examination were important in raptors.

Prevalence and genetic diversity of Haemoproteus and Plasmodium in raptors from Thailand: Data from rehabilitation center.

The diurnal raptors (Family: Accipitridae and Falconidae) are important as ecosystem bioindicators. Unfortunately, the global number of these birds has fallen, and they are close to extinction. This study reports the molecular prevalence and genetic diversity of Haemoproteus and Plasmodium in raptors admitted to the Kasetsart University Raptor Rehabilitation Unit over a period of 6 years. A total of 198 raptors, including 22 species from 30 provinces in Thailand, were admitted. The prevalence of parasites in raptors was low: Haemoproteus was 4.04% (95% CI: 1.29-6.78), and Plasmodium 2.53% (95% CI: 0.34-4.71). Eleven lineages of haemosporidian parasites were identified, and four lineages (ACCBAD02, NISALB01, NISALB02, and AEGMO03) are new globally. Interestingly, six lineages were isolated from birds belonging to the Accipitridae and Falconidae families (TYTAL4, TYTAL6, GLACUC08, MILANS06, OTUSCO02, and ORW1), indicating host shift of these parasites. Furthermore, the low prevalence of Haemoproteus and Plasmodium in raptors compared with that in previous reports suggests a relationship between the activity of avian hosts and vectors. This information is valuable for application in raptor rehabilitation and further research.

Molecular prevalence and phylogenetic relationship of Haemoproteus and Plasmodium parasites of owls in Thailand: Data from a rehabilitation centre.

Owls are nocturnal raptors that are prevalently infected with haemosporidian parasites wordwide. These birds were commonly submitted to the Kasetsart University Raptor Rehabilitation Unit, Kasetsart University, Thailand and were examined using PCR-based methods for the presence of haemosporidian infections of by the genera Plasmodium and Haemoproteus. Blood samples from 167 individual owls belonging to 12 species common in Thailand were collected between September 2012 and February 2018. The overall prevalence of haemosporidians was 34.1%, with Haemoproteus infections (25.1%) being more prevalent than Plasmodium infections (9.0%). The prevalence of both Haemoproteus and Plasmodium parasites was similar in all seasons of the year. Molecular characterization revealed 17 new haemosporidian parasite lineages (11 Haemoproteus and six Plasmodium), with genetic variation among partial cytochrome b sequences ranging from 0.0% to 3.6% in Haemoproteus lineages and 0.2%-8.8% in Plasmodium lineages. Phylogenetic analysis showed that all Haemoproteus lineages detected in owls appeared in one well-supported clade together with other parasites belonging to the Parahaemoproteus subgenus, indicating their close evolutionary relationship and common transmission modality by Culicoides biting midges. This study showes the existence of prominent non-described haemosporidian parasite diversity in Thai owls and provides baseline molecular information for further research on the genetic diversity of owl haemosporidian parasites. New DNA sequence information can be used for the diagnosis of owl infections, which have been often reported during rehabilitation planning.

Morphological and molecular characterization of avian trypanosomes in raptors from Thailand.

From September 2012 to May 2018, blood samples from 364 raptors (mostly adults) were collected and screened for trypanosomes and haemosporidians by microscopic examination and nested polymerase chain reactions (PCR). Trypanosoma spp. were identified in 15 birds from eight different species. Light microscopy revealed 14 cases of infection with Trypanosoma cf. corvi, including one each in black-shouldered kite (Elanus caeruleus, n = 49), Brahminy kite (Haliastur indus, n = 50), and spotted owlet (SO, Athene brama, n = 27); two mountain hawk-eagles (Spizaetus nipalensis, n = 3); and three each in Asian barred owlets (ABO, Glaucidium cuculoides, n = 27), barn owls (BO, Tyto alba, n = 65) and collared scops owls (CSO, Otus lettia, n = 41). In addition, one case of infection with T. avium was identified in an oriental scops owl (OSO, Otus sunia, n = 2). All infected raptors showed very low parasitemia levels. The PCR detected more three positives in one CSO, one Japanese sparrowhawk (Accipiter gularis), and one OSO. The sensitivity and specificity of the PCR method were 93.3% and 99.1%, respectively. The overall infection rate was very low (4.9%). The highest infection rate was recorded in cold-dry season (9.9%). Coinfection of Plasmodium with trypanosomes was found in all three ABOs. Coinfection with Haemoproteus spp. was found in one BO, three CSOs, and one SO. Coinfection with Haemoproteus spp. and Leucocytozoon danilewskyi was found in the OSO. Microfilarias were detected in one ABO and one CSO. The ultrastructure of trypomastigotes of T. cf. corvi in an ABO revealed fine structures. All small subunit ribosomal RNA (SSU rRNA) sequences belong to two clades: T. avium and T. corvi-culicavium complex/group. SSU rRNA gene amplification was not successful in one BO. The raptors with trypanosome infections showed normal hematological values and healthy appearance. Furthermore, this is the first report of T. avium in a nocturnal raptor from Thailand.

Quantitative and qualitative characteristics of blood cells in black-shouldered, Brahminy, and black kites.

BACKGROUND: Black-shouldered kites (BSK, Elanus caeruleus), Brahminy kites (BrK, Haliastur indus), and black kites (BK, Milvus migrans govinda) are medium-sized hawks found in Thailand, and little is known about the hematology of these three kite species. OBJECTIVE: This study reports basic hematologic values and describes the light microscopic, cytochemical, and ultrastructural characteristics of blood cells in these kites. METHODS: Blood samples were collected from 113 healthy kites (50 BSKs, 53 BrKs, and 10 BKs) from January 2012 to December 2017. Complete blood cell counts, cytochemical staining (Sudan black B, peroxidase [PO], periodic acid-Schiff, α-naphthyl acetate esterase, and ß-glucuronidase), and transmission electron microscopy were performed using standard methods. RESULTS: Hematology, morphometry, and cytochemical staining patterns of blood cells were tabulated. BSK erythrocytes were smaller than BrK and BK erythrocytes. Heterophils, the largest granulocytes, were the most prevalent leukocytes in all kites. Cytochemical reactions in blood cells from these three kite species were the same, except that heterophils from BrKs were the only cells positive for PO. The ultrastructure of heterophil and eosinophil granules from the BSKs were similar in their homogeneous electron densities but differed in shape. The eosinophil granules from BrKs and BKs revealed heterogeneous electron densities with central pallor in some granules. Basophils had different granular electron densities, and some granules were electron-lucent. CONCLUSION: The 23 baseline hematologic values and morphologic, cytochemical, and ultrastructural characteristics of all blood cell types in this study provide reference data for future kite healthcare.

Haemoproteus in barn and collared scops owls from Thailand.

The barn owl (BO) and the collared scops owl (CSO) are common nocturnal raptors throughout Thailand. Blood samples from 23 adult BOs and 14 CSOs were collected and processed for complete blood cell counts and parasite morphological examinations. Two Haemoproteus-positive samples were processed for ultrastructural observation. Polymerase chain reaction (PCR) analysis for a partial cytochrome b gene (cytb) from Haemoproteus was performed in all samples. Haemoproteus presence detected by light microscopy was lower than that detected by PCR (30.4% and 34.8%, respectively, in BO; and 50.0% and 78.6%, respectively, in CSO). Comparative hematology revealed that Haemoproteus-positive BOs had higher mean cell hemoglobin concentration, total leukocyte, absolute heterophil, basophil, and monocyte counts than Haemoproteus-negative BOs, but no significant differences between Haemoproteus-negative and -positive CSOs. Monocyte ultrastructure analysis revealed a role in the elimination of gametocytes. Morphologically, the Haemoproteus in 3 BOs and 6 CSOs were identified as H. noctuae, while that in 1 CSO was identified as H. syrnii. Phylogenetic analysis indicated the Haemoproteus spp. in 8 BOs and 7 CSOs were not closely related to H. noctuae or H. syrnii, and the cytb of 2 CSOs was that of H. syrnii. These results should be useful for study of Haemoproteus.