Buffy Coat Smears Research Articles

Prevalence, risk factors, and hematologic changes in dogs from Baja California with presence of Ehrlichia spp., and coinfection with Anaplasma spp.

Ehrlichia and Anaplasma are obligate intracellular, gram-negative bacteria with tropism for hematopoietic cells, especially leukocytes and platelets. There are several Ehrlichia species that infect dogs. Ehrlichia canis is transmitted by Rhipicephalus sanguineus and replicates within monocytes and macrophages, leading to canine monocytic ehrlichiosis, a disease of worldwide distribution. The clinical signs are varied and non-specific. Anaplasma has two species that infect dogs, Anaplasma phagocytophilum and Anaplasma platys, the second also transmitted by Rhipicephalus sanguineus. This study aimed to evaluate the epidemiology and hematologic changes associated with the presence of Ehrlichia spp. and Anaplasma spp. coinfection in dogs from Baja California. Complete hematological analysis, examination of buffy coat smears, and ELISA tests were performed on blood samples from three veterinary diagnostic laboratories from Mexicali and Tijuana cities in Baja California, Mexico. A total of 5,469 dog samples were analyzed. The overall prevalence of Ehrlichia spp., was 4.79%, with a distribution of 6.3% in Mexicali (OR: 2.39 CI: 1.69-3.17) and 2.5% in Tijuana. The peak of infection was found in September. Contact with other dogs and tick infestation were the risk factors associated with infection (P < 0.05). There was 96% co-infection only in Tijuana and 0% in Mexicali. Anemia, thrombocytopenia, and hyperproteinemia are associated with Ehrlichia spp., and Ehrlichia/Anaplasma infection. In view of the foregoing, we have to maintain epidemiologic vigilance, as well as look further into the ticks present in the state and the possibility of transmission of unusual pathogens.

Role of buffy coat in detection of microfilaria in absence of eosinophilia: a case report

Filariasis is a very important public health problem endemic in tropical and subtropical countries. It is a debilitating disease with significant socioeconomic burden. In 2016, lymphatic filariasis was successfully eliminated from the Maldives, which was certified by WHO. We report a case of filariasis detected by a rapid antigen test while undergoing a screening program conducted by Health Protection Agency, Maldives in December 2023. The patient was asymptomatic and showed no peripheral blood eosinophilia. Conventional blood smears were negative. Nevertheless, buffy coat smear served as a promising test in detection of parasite, thus confirming the re-emergence of the disease in the country. Therefore, our study highlight the significance of buffy coat smears in detection of hemoparasites. We also focus on the importance of surveillance system in order to sustain the disease elimination goal that was already achieved.

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. Keywords: Buffy coat smear, Chickens, Cytochrome c oxidase I, Microfilaria, Nested-polymerase chain reaction.

Clinico-diagnostic and Therapeutic Management of Ehrlichiosis in Dog

Background: The research work was undertaken to investigate the efficacy of various diagnostic tests by using conventional microscopic examination, rapid Ab detection test and molecular technique, haemato-biochemical changes, Abdominal organs changes using ultrasonography, and therapeutic efficacy of different treatment protocols on Canine Monocytic Ehrlichiosis (CME) infected dogs from in and around Navsari, Gujarat, India. Methods: 69 dogs suspected of ehrlichiosis based on clinical signs and hematological values and then subjected to blood and buffy coat smear examination, rapid Ab detection test and PCR based assay targeting 16S rRNA gene. The pre and post-treatment haematology and biochemical analysis of treatment groups along with all the positive dogs were performed. Further, all the treatment group dogs were subjected to ultrasound for evaluating organ abnormalities. Modified Clinical Sign Grading (MCSG) score for CME-infected dogs was carried out to evaluate therapeutic responses based on minimizing the clinical score. Descriptive statistics along with Student t-test and One-way analysis of variance (ANOVA) followed by DMRT was performed to compare differences between means wherever applicable. Result: CME incidence among the 69 suspected cases was 49.28%. Out of 69 suspected dogs, Ehrlichial inclusions or morulae in the monocytes could be identified in only 4.35% of blood smears and 10.14% of buffy coat smears examined by Giemsa and or Field-stained and 15.94% of dogs were found positive for Ehrlichia genus by PCR. 72.5 per cent of dogs were found positive for antibodies to E. canis, out of 40 dogs. The sensitivity of blood smear examination, buffy coat smear examination, and thrombocytopenia was 10.34%, 24.14% and 89.66%, respectively. The specificity of all the diagnostic tests was 100%. The anemia along with leucopenia, were the most significant hematological alteration recorded while ALP, creatinine, and BUN were elevated and albumin was significantly decreased in CME-infected dogs. Significant improvement of haemato-biochemical parameters towards normalcy in both treatment groups. However, a higher percentage of reduction was observed in the clinical score of imidocarb treated CME affected dogs than in the oxy-doxycycline therapy after 14 days.

Diagnosis and treatment of Ehrlichiosis in a Pug dog

In addition to damaging the skin and causing inflammatory reactions and anemia, dog tick infestations are vectors for blood parasites such as Ehrlichia spp., known as Ehrlichiosis. This article reports the case of a five-year-old female Pug dog whose body was covered with ticks. The clinical examination found tick infestation on the face, ears, back, abdomen, legs, and interdigital areas. Supporting examinations included a complete blood count (CBC), buffy coat smear, and a test kit. CBC examination showed that the dog had leukocytosis, neutrophilia, hypochromic microcytic anemia, and thrombocytopenia. A buffy coat smear examination showed the presence of Ehrlichia morulae in the cytoplasm of the dog's monocytes, and the test kit results were positive for Ehrlichia antibodies; therefore, the dog was diagnosed with Ehrlichiosis. Treatment is done by administering Doxycycline, Intervetta Dr. Choice Ferro-B, and Fu Fang Ejiao Jiang orally for 21 days. After treatment, hematological parameters in leukocytes, neutrophils, erythrocytes, and platelets returned to normal ranges.

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.

Investigation of comorbidities in dogs with leishmaniosis due to Leishmania infantum

In endemic areas, dogs with leishmaniosis due to Leishmania infantum frequently have comorbidities, including mostly neoplastic, infectious, and parasitic diseases. The aim of this study was to compare the prevalence of comorbidities among dogs that are not infected by L. infantum, dogs that are infected but do not present leishmaniosis, and dogs with leishmaniosis, and to examine if certain comorbidities are independent risk factors for the infection by L. infantum and/or for the development of canine leishmaniosis (CanL). A total of 111 dogs, older than 1-year and non-vaccinated against CanL, were allocated into three groups: group A (n=18) included dogs that were not infected by L. infantum, group B (n=52) included dogs that were infected by L. infantum but did not present CanL, and group C (n=41) included dogs with CanL. Signalment and historical data were obtained using a structured questionnaire. Laboratory examinations included complete blood count, serum biochemistry, urinalysis, fecal parasitology, modified Knott's test, microscopic examination of capillary blood, buffy coat, lymph node, bone marrow and conjunctival smears, qualitative serology for Dirofilaria immitis, Anaplasma phagocytophilum/A. platys, Borrelia burgdorferi and E. canis, IFAT for L. infantum, ELISA for Babesia spp. and Neospora caninum, and real-time PCR for L. infantum in bone marrow, skin biopsies and conjunctival swabs. A variety of comorbidities were found in all three groups. No independent risk factors for infection by L. infantum were found. On the contrary, among dogs infected by L. infantum, being a mongrel [odds ratio (OR): 11.2], not receiving prevention for dirofilariosis (OR: 26.5) and being seropositive to N. caninum (OR: 17.1) or to Babesia spp. (OR: 37.6), were independent risk factors for presenting CanL. Although no comorbidities influence the probability of canine infection by L. infantum, certain comorbidities may be precipitating factors for the transition from the subclinical infection by L. infantum to the overt CanL.

Seasonal prevalence of bovine trypanosomosis and trypanosome species distribution in Jimma Horo district, Oromia regional state, Western Ethiopia

A study was conducted to determine the prevalence of bovine trypanosomosis in rainy (June 2019) and dry (February 2020) seasons in Jimma Horoo district, Kellem Wollega Zone, Oromia Regional State, Ethiopia. A total of 720 blood samples were examined using buffy coat and thin blood smear techniques. The packed cell volume (PCV) of each animal was determined. The overall bovine trypanosomosis prevalence was 4.3% (χ2 = 1.25, P = 0.26). The prevalence was 5% (95% CI = 4.1–8.3) and 3.3% (95% CI = 2.7–6.3) in the dry and rainy reasons, respectively (P > 0.05). Trypanosoma congolense, T. vivax and T. b. brucei were detected in (60%), (33.3%) and (6.7%) of infected cattle, respectively. The highest trypanosome prevalence was observed in Burka Gudina (7.6%), and the lowest in Melka Nega village (2.1%). There were significant variations between trypanosome prevalence in relation to body condition scores (χ2 = 23.16; P = 0.0.00) of examined cattle. No significant difference (P > 0.05) was observed between seasons, age, and sex categories of cattle. The PCV values of trypanosome infected (22.94%) was significantly lower than non-infected cattle (26.47%) (χ2 = 19.60; (P < 0.05). The prevalence of bovine trypanosomosis in Jimma Horo district was low and it can be controlled by treatment of infected cattle using sanative pairs of trypanocidal drugs with additional application of deltamethrin pour-on in the dry season. Further, in view of poor sensitivity of buffy coat technique used the use of molecular techniques should be encouraged.

Prevalence and Associated Risk Factors of African Animal Trypanosomiasis in Cattle in Lambwe, Kenya.

Background African animal trypanosomiasis (AAT) affects livestock productivity in sub-Saharan Africa. This study aimed to determine cattle AAT's prevalence and associated risk factors in Lambwe Valley, Kenya. Methods In a cross-sectional survey, livestock owners were recruited from four villages of Lambwe in Homa Bay, Kenya. Blood samples were collected from the jugular veins of cattle, and buffy coat smears were examined under a microscope. Parasites were further detected using polymerase chain reaction (PCR). Using a semistructured questionnaire, livestock owners were interviewed on their knowledge of AAT and control practices. Chi-square and multilevel models were used for the analysis. Results The overall prevalence was 15.63% (71/454). Trypanosoma vivax 10.31% and T. congolense Savannah 6.01% were the common species and subspecies. A total of 61 livestock keepers were involved in the study. Of these, 91.80% (56/61) knew AAT, and 90.16% (55/61) could describe the symptoms well and knew tsetse fly bite as transmission mode. Self-treatment (54.09%; 33/61) was common, with up to 50.00% of the farmers using drugs frequently. Isometamidium (72.13%; 44/61) and diminazene (54.09%; 33/61) were drugs frequently used. Although 16.39% (10/61) of the farmers claimed to use chemoprophylactic treatment, 6/10 did not use the right drugs. Animals (92.1%; 58/63) with clinical signs had positive infections. Villages closer to the national park recorded a higher prevalence. Infections were higher in cattle owned by those self-treating (27.23%; 58/213), those using drug treatment without vector control (27.62%; 50/181), those using single-drug therapy, and those practicing communal grazing (20.00%; 59/295). Clinical signs strongly associate with positive infections under multilevel modeling. Conclusion Cattle trypanosomiasis is prevalent in the Lambwe region of Kenya. This is influenced by inappropriate control practices, communal grazing, and the proximity of farms to the national park. In addition, clinical signs of the disease have a strong association with infections.

Evaluation of an Indirect Immunofluorescence Assay for the Detection of Anaplasma phagocytophilum Antigen in Ovine Buffy Coat Smears.

Diagnosis of anaplasmosis is challenging considering the great variation in clinical signs and the limitations of the available diagnostic assays, while the detection of carrier animals that play a significant role in disease epidemiology as reservoirs is of great significance. In this study, we evaluated the diagnostic accuracy of a newly developed indirect immunofluorescent assay (Ag-IFAT) for the detection of A. phagocytophilum antigens in buffy coat specimens, alone and in combination with cytology, using PCR as a reference. Blood samples were collected from 138 sheep of the Chios breed from six farms in Greece. A buffy coat was extruded from the centrifuged blood. Buffy coat smears were used for cytological examination and the Ag-IFAT assay. The Ag-IFAT assay presented excellent specificity (100%) and high sensitivity (85.4%) for the detection of A. phagocytophilum antigens in buffy coats, and it has an almost perfect agreement with PCR and cytology (κ value = 0.88 and 0.85, respectively). A. phagocytophilum antigens are likely to be detected using Ag-IFAT in a PCR-positive animal, as indicated by the good performance of the assay. Overall, this assay presents high diagnostic accuracy, and it could be used for the detection of animals during the early stage of infection.

Molecular characterization of Trypanosoma evansi, T. vivax and T. congolense in camels (Camelus dromedarius) of KSA

BackgroundTrypanosoma evansi is the leading infectious Trypanosoma spp. in camels (Camelus dromedarius) present in the Kingdom of Saudi Arabia (KSA) that could lead to extensive economic losses. The present study was aimed to assess the prevalence rate of T. evansi in Taif governorate, Makkah province, KSA using parasitological and molecular evaluations, and analyze their genetic relationship targeting internal transcribed spacer 1 (ITS1) and variable surface glycoprotein (VSG) genes. For evaluation, we have used 102 blood samples of camels obtained from three different regions in Taif.ResultsResults show a considerable prevalence rate of trypanosomosis 2/102 (2.0%) according to Giemsa-stained buffy coat smear, and 16/102 (15.7%) according to touchdown PCR. T. evansi (n = 10/102, 9.8%) was the main infectious species found in camels then T. vivax (n = 3/102, 2.9%). Mixed infections were detected in three camels with T. evansi, T. vivax, and T. congolense (n = 3/102, 2.9%). Regarding gender, the results indicate that female camels (11/66, 16.7%) show higher prevalence of Trypanosoma than males (5/36, 13.9%). Sequencing and phylogenetic analyses of ITS1 and VSG showed their relationships with T. evansi in other hosts from different countries.ConclusionsIn our peer knowledge, it is the first time to report a research-based prevalence of trypanosomosis in the camels of Taif governorate, Makkah province, KSA.

Evaluation of techniques for diagnosis of Trypanosoma vivax infections in naturally infected cattle in the Zona da Mata Mineira.

This study aimed to evaluate diagnostic techniques for trypanosomiasis, caused by Trypanosoma vivax, in naturally infected cattle in Minas Gerais, Zona da Mata. The deaths of six lactating cows with similar clinical conditions-characterized by hyporexia, hypogalactia, and recumbency-had been reported from one property. Initially, two animals were examined and diagnosed with trypanosomiasis through identification of the protozoan in a blood smear. After the initial diagnosis, all lactating cows (n=37) on the property were examined, and blood samples were collected for tests including whole blood smear, buffy coat smear, Woo's technique, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR). Woo's test, buffy coat smears, and whole blood smears indicated that 4/37 (10.81%) animals were positive for trypanosomiasis, whereas ELISA and PCR indicated that 33/37 (89.19%) and 27/37 (72.97%) animals, respectively, were positive. The agreement obtained between parasitological techniques was classified as high, while between ELISA and PCR, no agreement. In conclusion, parasitological techniques have a low capacity to identify infected animals in the chronic stage of T. vivax infection. Therefore, techniques such as PCR and/or ELISA should be used to minimize the occurrence of false negatives.

Prevalence of Bovine Trypanosomosis and Associated Risk Factors in Jima Geneti District of the Horo Guduru Wollega Zone in Ethiopia

Introduction: Parasitic diseases are significant causes of morbidity and mortality in humans and animals throughout the world. Methods: A cross-sectional study was performed in Ethiopia’s Jima Geneti area of the Horo Guduru Wollega Zone from December 2017 to April 2018. The prime goal of the study was to determine the prevalence of trypanosomosis in cattle and assess potential risk factors. Buffy coat and thin blood smears were employed to identify the trypanosome species. The research district and kebeles were chosen using a purposive selection strategy, while cattle were selected by a simple random sample method. Results: In total, 25 of the 384 investigated cattle were infected with trypanosomes, leading to an overall prevalence of 6.5%. Biqiltu Qidame town had a high prevalence of 10.4%, while Adileqa Tuluchali had a low prevalence of 3.5%. The study area had the highest prevalence of Trypanosoma congolense (60%), followed by Trpanosoma vivax (28%) and Trpanosoma brucei (12%) The prevalence of trypanosomosis in cattle was statistically significant (P&lt;0.05) with age, body condition, and packed cell volume (PCV) in the study area. Conclusion: Bovine trypanosomosis was prevalent in the researched area that had a severe impact on livestock production. To increase the livestock’s health and production in the study area, strategic disease prevention and control programs are necessary.

Microscopic, Serological and Molecular Screening of Theileria annulata in Camels (Camelus dromedarius) of Saudi Arabia

The present study was aimed to evaluate the prevalence rate of Theileria spp., especially T. annulata, in camels of Taif Governorate, Saudi Arabia. A total of 102 blood samples were collected from camels. Parasitological examination of buffy coat smear, ELISA-IgG, and PCR (targeting 16S rRNA and cytochrome b gene sequences) assays were done for T. annulata infection detection. The results report a high prevalence rate of T. annulata in the examined dromedary’s population using ELISA-IgG (32.4%), followed by targeted PCR (25.5%). Then lower detection was reported by buffy coat Giemsa stained (10.8%). Statistical analysis using one-way ANOVA followed by Tukey’s at P < 0.001 showed signifcant variation in values within different diagnostic tools and age categories. Both females and males were at risk of parasitic infections, but females have a higher incidence (17.6%, 15.7%, 6.8%). Young age (X≤3 years) camels were the most infected category (50.0%, 44.4%, 22.2%) according to age from elder ones. These results could be a baseline for easing a later large-scale epidemiological screening on tropical theileriosis in the camel population in Saudi Arabia.

Retrospective analysis of canine monocytic ehrlichiosis in Thailand with emphasis on hematological and ultrasonographic changes.

Background and Aim:Canine monocytic ehrlichiosis (CME) is a tropical endemic tick-borne disease that causes fatality or chronic infection involving many organs in dogs. This study aimed to examine the prevalence, risk factors, and hematological and ultrasonographic changes in the liver, gallbladder, kidneys, and spleen following CME infection.Materials and Methods:This retrospective study used 30,269 samples collected from dogs at the hematology section of the pathology unit of a university veterinary hospital and 35 samples collected from dogs at the diagnostic imaging unit. CME was determined using the buffy coat smear method. Data were analyzed using descriptive statistics and odds ratios.Results:The data revealed that the average yearly prevalence of CME was 1.32%. Risk factors contributing to CME infection were a tick on the body during physical examination, lack of ectoparasite control, and outdoor living. All 148 dogs with CME infection had low platelet counts. The percentages of CME-infected dogs with elevated serum alanine aminotransferase, alkaline phosphatase, and both enzymes above the normal range were 33.6%, 65.9%, and 29.8%, respectively. The rates for elevated serum levels of blood urea nitrogen, creatinine, and both compounds were 33.1%, 19.1%, and 17.3%, respectively. The most common ultrasonographic changes were liver abnormalities (hyperechogenicity or hypoechogenicity, hepatomegaly, and hypoechoic nodules), hyperechogenicity of the kidneys, and an enlarged spleen. These ultrasonographic changes were consistent with the hematology results, which showed a greater elevation of serum liver enzyme levels than renal enzymes.Conclusion:Ultrasonographic changes during CME infection and after treatment with doxycycline can help to monitor and identify persistent pathological changes in the target organs resulting from immune response to CME.

Molecular detection of Trypanosoma species and haematological alterations in four trypanosome-infected Nigerian horses

Trypanosomes cause anaemia and are responsible for widespread morbidity and mortality particularly in imported breeds of animals found in sub-tropical and tropical parts of the world. Light microscopy and polymerase chain reaction (PCR) were used to detect trypanosomes in naturally infected Nigerian crossbred horses at Obollo-Afor abattoir, Udenu Local Government Area, Enugu State Nigeria. Blood was collected via the jugular outflow from a total of 200 horses of varying ages and either sex. Conventional procedures were followed during the PCR assay, parasite identification in wet mount, Leishman-stained thin blood and buffy coat smears on glass slides. Light microscopy revealed Trypanosoma species with an elongated, streamlined and tapered body, highly suggestive of T. brucei brucei or its subspecies T. evansi or T. equiperdum. PCR assay produced the expected fragment size of 700 bp specific for ITS-1 region of the 18SrRNA gene of Trypanosoma species in 4 (2%) of 200 blood samples against the routine blood and buffy coat smear examination, which revealed trypanosomes in 3 (1.5%) out of 200 blood samples. Sex and age were not significantly (p&gt;0.05) associated with the trypanosome infection. One of the Trypanosoma infected anaemic horses had microcytic normochromic anaemia, high erythrocyte sedimentation rate and normal leukocyte count, while one of the Trypanosoma species infected non-anaemic horses had erythrocytic parameters and ESR values that are within the reference range, with leukocytosis. It was concluded that the prevalence of equine trypanosomosis was very low, and it’s characterized by mild to moderate anaemia in clinical cases.

Molecular Identification of Trypanosoma theileri and Biology of Trypanosomes

Trypanosoma theileri (T. theileri ) is a non-pathogenic, cosmopolitan, and commensal protozoa of cattle. The main objective of the current study was to investigate the biology and feasibility of T. theileri as a model candidate for the discovery of a novel drug. In the present study, the isolates of T. theileri obtained from the Institute of Tropical Medicine (ITM) in SDM 79 were cultivated at 26oC. Eight experiments with different inoculum and different times were grown. The growth curve was plotted to check the growth trends. The doubling time in the logarithmic phase was determined to be 17.43 hours. In addition, an experimental infection was done on a 3-month-old Holstein Friesian calf to isolate the blood-streaming shape; however, it was not successful after the blood buffy coat smear and PBMC culture in RPMI 1640 and HMI 9. Furthermore, the viability was determined by quantitative colorimetric Resazurin assay in 96-well fluorescence Microplates containing 0.4 to 2.4 mM of Resazurin. On the other hand, the response to Pentamidine (1-100 ng/mL) showed a strong negative correlation between the fluorescence signal and the highest Pentamidine concentration. IC50 was 9.25 ng/mL. Genomic DNA was extracted using the phenol-chloroform method. The gradient PCR amplification using T. theileri specific PCR (Tth625-PCR) primers was detected at 465 base pair (bp). In addition, the full-length 18S rDNA sequence was detected at 730 bp. In the silico analysis using common anti-trypanosome drug targets, no significant similarity could be found on either the DNA or the protein level. Nevertheless, homologous sequences have been identified among the drug targets for Ornithine decarboxylase. Therefore, the analysis might show the possibility of using T. theileri as a model for the search of new drugs once they have entire genome sequences. Analysis of the whole genome and transcriptome indicated a phylogenetic relationship between T. theileri and other pathogenic trypanosomes which can be the basis for novel drug development.

Morphological and Numerical Variations in WBC Distribution Pattern during Differential Count Estimation from Buffy Coat Smears

Morphological and Numerical Variations in WBC Distribution Pattern during Differential Count Estimation from Buffy Coat Smears

Epidemiological investigation of bovine trypanosomosis in Bedele district, Buno Bedele zone, Oromia regional state, Ethiopia

A cross-sectional study was conducted in December 2018 in four purposively selected villages of Bedele district, Oromia Regional State, Southwest Ethiopia. The study aimed to determine the prevalence of bovine trypanosomosis and associated risk factors of trypanosome infections in cattle. A total of 384 blood samples were collected from systematically selected cattle and examined using buffy coat and thin blood smear examination methods. The overall prevalence of bovine trypanosomosis was 8.3%. Trypanosoma congolense (68.8%) and T. vivax (31.2%) were the prevailing trypanosome species identified in the area. A statistically significant difference (P < 0.05) was observed in the prevalence of bovine trypanosomosis between body condition scores of cattle. The prevalence of bovine trypanosomosis was slightly higher in female (9%) as opposed to male (7.3%) cattle, but the difference was not statistically significant (P > 0.05). Similarly, no statistically significant difference (P > 0.05) was observed between age categories of cattle. The mean PCV value of trypanosome infected cattle (21.4 ± 3.6) was significantly (P < 0.05) lower than that of non-infected (25.6 ± 4.6). The current study indicated that, despite vector control measures implemented for several years, bovine trypanosomosis persists and continues to be a core problem to cattle health and production in the Bedele district. Therefore, more attention should be given to control the disease and its vectors based on temporal and spatial distribution. Also, the use of molecular techniques should be encouraged in view of their greater sensitivity when compared to the buffy coat method.

Serum protein profiles and C-reactive protein in natural canine filariasis.

Background and Aim:Canine filariasis is caused by several species of filarial worms. The pathophysiological response to infection is mainly due to the filaria lifecycle. Laboratory detection methods to assess the pathological alterations characteristic of filariasis are needed urgently. Serum protein profiles and C-reactive protein (CRP) levels are used widely to diagnose several animal diseases. This study aimed to determine the serum protein profiles and CRP levels in dogs infected with Dirofilaria immitis or Brugia pahangi or both parasites.Materials and Methods:Blood samples were collected from 980 dogs presenting at animal hospitals and veterinary clinics in Bangkok and its vicinity. The presence of microfilaria in samples was determined using a buffy coat smear and staining with Wright–Giemsa. The sheathed and unsheathed microfilaria species were identified by acid phosphatase staining. Forty positive samples were tested. The serum protein profiles were identified by agarose gel electrophoresis. The CRP concentration was measured using a fluorescent immunoassay.Results:Albumin levels and albumin-to-globulin ratios were significantly lower, and total protein, β2 globulin, and γ globulin levels were significantly elevated in dogs infected with D. immitis and B. pahangi compared with reference values in normal dogs. The average CRP concentrations in dogs infected with D. immitis or B. pahangi were 69.9 and 12.9 mg/L, respectively.Conclusion:The total protein and γ globulin levels increased in canine filariasis compared with the normal reference range. The CRP concentration in dogs infected with D. immitis was extremely high, whereas that in dog infected with B. pahangi was normal.