Molecular prevalence, phylogenetic characterization, and epidemiological risk factors of pulmonary nematodes in domestic cats in Türkiye.

Aelurostrongylus abstrusus, Oslerus rostratus, and Troglostrongylus brevior are nematodes found in the respiratory system of domestic cats and cause a variety of symptoms. All three parasites use the same hosts and niches, and the morphological measurements of their L1s excreted in faeces overlap with each other. In this study, 300 cats brought to Ondokuz Mayis University Veterinary Teaching Animal Hospital were screened for lungworms by morphological measurements and molecular analyses. The prevalence of the lungworms was found as 1.33% (4/300) for A. abstrusus, 0.66% (2/300) for O. rostratus, and 0.33% (1/300) for T. brevior. Molecular identification of A. abstrusus, T. brevior, and O. rostratus in domestic cats was carried out for the first time in Türkiye within the present study. This study also reveals the risk factors of lungworm positivity in domestic cats in Türkiye.

A novel one-step multiplex PCR protocol to detect avian haemosporidian parasites in the subgenus Haemoproteus (Kruse, 1890) used to quantify parasite prevalence in domestic pigeons (Columba livia) in Turkey.

Infections of avian haemosporidian parasites are regularly identified by molecular methods including multiplex PCR, which allows researchers to distinguish mixed infections of parasites from multiple genera. Here we extend the utility of a previously designed multiplex PCR by designing a primer set specific to parasites of the subgenus Haemoproteus (genus: Haemoproteus). The updated one-step multiplex PCR protocol we describe here allows for the detection of the genera Plasmodium and Leucocytozoon and the two subgenera (Haemoproteus and Parahaemoproteus) of the genus Haemoproteus. A sensitivity analysis showed that the multiplex PCR could amplify DNA of parasites in the subgenus Haemoproteus at very low levels of infection. We used this multiplex PCR to identify haemosporidian infections in 250 adult domestic pigeons (Columba livia) in Turkey. All samples were also screened by microscopy and a widely used nested PCR to compare with the results of multiplex PCR, to detect low levels of parasitemia, and to identify possible abortive infections. In total, 71 pigeons (28.4%) were found to be infected by all three methods. The multiplex PCR protocol successfully detected and discriminated both subgenera Haemoproteus and Parahaemoproteus infections. We compared our results with previous host species records to assess the host specificity of the parasite lineages we found. Our findings provide novel data on the prevalence of avian haemosporidians in domestic pigeons and demonstrate the utility of the new one-step multiplex PCR protocol for the determination of mixed avian haemosporidian infections. We expect that this protocol will contribute to a better understanding of the distribution, epizootiology, and ecology of avian haemosporidians.

Occurrence and molecular characterization of Enterocytozoon bieneusi in water buffaloes (Bubalus bubalis) in Turkey.

Microsporidia are obligate intracellular fungus-like parasites that infect humans and animals worldwide. However, there is limited epidemiological data on the occurrence and molecular diversity of microsporidia in buffaloes worldwide. In the present study, fecal samples of 300 water buffaloes (Bubalus bubalis) in Kayseri, Sivas, and Samsun provinces of Turkey were investigated using two nested PCR assays targeting the rRNA of E. bieneusi and Encephalitozoon spp. All the fecal samples from water buffalo were found to be negative for Encephalitozoon spp. PCR positive isolates of E. bieneusi were bidirectionally sequenced for genotyping and phylogenetic analyses. Enterocytozoon bieneusi was the only microsporidian species identified in 8 water buffaloes with an overall molecular prevalence of 2.7%. Two known genotypes, YNDCEB-90 (n = 5) and J (n = 3) were identified by ITS sequence analysis. The YNDCEB-90 and J genotypes fall into zoonotic Group 1 and 2 of E. bieneusi in the phylogenetic tree, respectively. These findings suggested that water buffalo in Turkey are harbouring zoonotic genotypes of E. bieneusi and may have a significant risk for zoonotic transmission to humans. This is the first report of detecting E. bieneusi genotypes J and YNDCEB-90 in water buffaloes. Further insight into the epidemiology of E. bieneusi in water buffaloes in different geographical areas in Turkey will be highly important to have determined the public health significance of this pathogen.

First report and genotyping of Dientamoeba fragilis in pet budgerigars (Melopsittacus undulatus), with zoonotic importance.

The protozoan Dientamoeba fragilis is one of the most common parasites in the digestive system of humans worldwide. The host range and transmission routes of D. fragilis, including the role of animals, are still ambiguous with few reports from non-human primates, sheep, rodents, pigs, a cat and a dog. In this study, we used microscopic and TaqMan qPCR analyses to investigate D. fragilisin 150 faecal samples from pet budgerigars (Melopsittacus undulatus) in the Central Anatolia Region of Turkey. Dientamoeba fragilis DNA was detected in 32 samples, resulting in a mean prevalence of 21.3%. In microscopic examination, trophozoites/cysts of D. fragilis were detected in 13 of 32 qPCR-positive samples. SSU rRNA sequence analyses of the qPCR-positive isolates identified genotype 1 of D. fragilis as predominant in budgerigars. Phylogenetic analyses of the SSU rRNA gene region clustered D. fragilis genotypes, as well as other trichomonads, in separate monophyletic clusters with bootstrap values ≥79.0. Our study provides the first evidence for the natural host status of pet budgerigars for D. fragilisand contributes to the knowledge of the epidemiology of this parasite. The high prevalence of genotype 1 of D. fragilis suggests that pet budgerigars are suitable reservoirs for zoonotic transmission. Our findings contribute to an increased awareness and knowledge of D. fragilis infections in the context of a one-health approach.

Investigation of Zoonotic Cryptosporidium and Giardia intestinalis Species and Genotypes in Cats (Felis catus)

Objective: Giardia intestinalis and Cryptosporidium spp. are important zoonotic protozoan parasites that infect humans and various animals. We investigated the occurrence of G. intestinalis and Cryptosporidium spp. infection in cats. To provide data on the zoonotic transmission dynamics of these parasites, genotypes of the detected isolates were investigated through DNA sequence characterization. Methods: A total of 100 fecal samples were collected from cats between June and October 2020 in Kayseri and Samsun provinces. Fecal samples were examined by nested polymerase chain reaction (PCR), targeting the ß-giardin gene of G. intestinalis and small subunit (SSU) rRNA gene of Cryptosporidium spp. All PCR products were sequenced for genotyping. Results: Of the samples examined, Giardia intestinalis was determined in 8 samples (8.0%), whereas none of the samples were found positive for Cryptosporidium spp. Sequence analyses of the ß-giardin PCR products indicated that all G. intestinalis isolates were classed into the zoonotic assemblage B. Conclusion: This study adds to the current data on the molecular epidemiology of cryptosporidiosis and giardiasis in cats. The findings also highlight the potential risk of cats for public health concerning the zoonotic transmission dynamics of G. intestinalis.

Determination of Serum Oxidative Stress, Antioxidant Capacity and Protein Profiles in Dogs Naturally Infected with Ehrlichia canis.

PURPOSE: Canine ehrlichiosis is an important tick-borne disease of dogs worldwide. In the present study, we aimed to determine the serum total antioxidant capacity (TAC), malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase, (SOD), glutathione peroxidase (GSH-Px), adenosine deaminase (ADA) activity and serum protein profiles in dogs affected with naturally acquired ehrlichiosis. METHODS: The animal materials had been consisted of ten dogs naturally infected with Ehrlichia canis, and ten controls negative for Ehrlichia canis. TAC, MDA, NO, SOD, GSH-Px, ADA activity and TP, ALB, GLOB levels were measured in sera of the animals. The serum protein concentrations were measured by autoanalyzer. The electrophoretic profiles of serum total protein were determined by native polyacrylamide gel electrophoresis (Native-PAGE). RESULTS: In dogs with ehrlichiosis, decreased TAC (P < 0.05) and GSH-Px (P > 0.05) levels were determined. However, NO (P > 0.05), SOD (P < 0.05), ADA (P > 0.05), MDA (P > 0.05), TP (P < 0.05) and GLO (P < 0.05) levels were found as increased in the Ehrlichia positive dogs. ALB levels were decreased without a statistical significance (P > 0.05). ALB, α1 and ß2 globulin strip densities were found as decreased in native-PAGE, while ß1 and γ globulin strip densities were significantly increased in the E. canis positive group when compared to the control. CONCLUSION: It was determined that the oxidative stress decreased high antioxidant activity in dogs naturally infected with E. canis, and consequently, pro-oxidant and antioxidant defense and serum protein profiles were affected. It was thought that antioxidant supplementation could be beneficial to the treatment of the disease.

Molecular identification and subtype distribution of Blastocystis sp. in farm and pet animals in Turkey.

A total of 1340 fresh fecal samples from farm and pet animals in Central Anatolia and the Middle Black Sea Region of Turkey were investigated using a PCR assay targeting the SSU rRNA of Blastocystis sp. An overall Blastocystis sp. prevalence of 19.4% (183/940) was found in farm animals, including cattle, sheep, water buffaloes, and chickens. Fecal samples of dogs, cats, and horses were negative. The highest prevalence of Blastocystis sp. was found in sheep (38.2%) among the farm animals. The SSU rRNA sequence analysis revealed two animal-specific subtypes, including ST10 in cattle and sheep and ST14 in water buffaloes. The zoonotic subtype ST7 was identified in chickens. Our results indicated a high prevalence of animal-specific subtypes in livestock and zoonotic subtype ST7 in chickens, highlighting the potential risk of chickens for zoonotic transmission of Blastocystis in the research area. This study is the first large-scale evaluation of Blastocystis in animal hosts in Turkey, and contributes to the molecular epidemiology and genetics of Blastocystis. Our results should be considered by authorities as an indication of the zoonotic importance of Blastocystis sp. and the need for surveillance in public health intervention programs.

First report and molecular prevalence of potential zoonotic Enterocytozoon bieneusi in Turkish tumbler pigeons (Columba livia domestica).

A total of 250 droppings of tumbler pigeons (Columba livia domestica, Columbidae) were collected individually from different breeders in Turkey, to investigate the presence and genotyping of microsporidian species by nested PCR and to reveal their zoonotic potential. In the present study, Enterocytozoon bieneusi was the only microsporidian species identified in 35 pigeons with an overall molecular prevalence of 14.0%. Only one known genotype zoonotic Peru6 was identified in all positive samples according to the sequence analyses of the internal transcribed spacer region of ribosomal DNA of E. bieneusi. This study represents the first report of E. bieneusi in pigeons in Turkey. Our study also confirms the competence of breeding pigeons as hosts for the zoonotic Peru6 genotype, corroborating its potential role as a source of human infection and environmental contamination. LAY SUMMARY: Microsporidia are spore-producing fungi defined as emerging opportunistic pathogens of humans. The occurrence of microsporidia in animals could be risky for human public health. Home kept breeding pigeons pose a high risk for transmission of the microsporidians to humans.

Occurrence and molecular identification of zoonotic microsporidia in pet budgerigars (Melopsittacus undulatus) in Turkey.

Encephalitozoon spp. and Enterocytozoon bieneusi are well-known microsporidian pathogens, recently classified as fungi, infecting humans and reptiles, mammals, and birds. Budgerigars (Melopsittacus undulates) are the most preferred captive pet birds in the households. Prevalence and molecular data on microsporidian species in budgerigars are scarce worldwide. The aim of the present study was to investigate the occurrence and genotypes of Encephalitozoon spp. and E. bieneusi in budgerigars, and to reveal their zoonotic potential. A total of 143 fecal samples were collected from owned healthy budgerigars in Turkey. Encephalitozoon spp. and E. bieneusi were examined by nested PCR targeting the ribosomal internal transcribed spacer (ITS) region and sequenced for identifying Encephalitozoon spp. and E. bieneusi. The overall prevalence of E. hellem and E. bieneusi was 14.7% (21/143) and 3.5% (5/143), respectively. Two genotypes of E. hellem were identified, including one known 1A (n = 18) and a novel TURK1B (n = 3). In addition, we determined two E. bieneusi genotypes, including one known N (n = 2) and a novel TURKM1 (n = 3). E. hellem 1A and novel TURK1B clustered as a sister taxon, and genotype N and novel TURKM1 genotypes fall into group 2 of E. bieneusi in the phylogenetic tree. Novel genotypes of E. hellem and E. bieneusi were described for the first time in the avian host. Moreover, E. bieneusi genotype N was first detected in avian hosts in the present study. This study contributes to the current knowledge on the molecular epidemiology and transmission dynamics of E. hellem and E. bieneusi. LAY SUMMARY: Spore producing microsporidia are ubiquitous, obligate, and intracellular fungus defined as emerging opportunistic pathogens of humans, livestock, companion animals, wild mammals, birds, and water worldwide. The occurrence of microsporidia in animals could be risky for human public health.

Molecular Detection of Zoonotic Microsporidia in Domestic Cats in Turkey: A Preliminary Study.

OBJECTIVE: This preliminary study was conducted to reveal that the molecular identity of Encephalitozoon spp. and Enterocytozoon bieneusi in indoor domestic cats' fecal samples from Turkey was screened using the PCR. MATERIALS AND METHODS: Nested PCR was performed using MSP and EBITS primers. All of the amplification products were sequenced to identify the microsporidia species. RESULTS: Four (5.5%) and three (4.1%) genomic DNA isolates of the fecal samples from 72 indoor domestic cats showed amplification of the ITS regions of E. bieneusi and Encephalitozoon spp., respectively. Two different genotypes, D and IV, of E. bieneusi were determined in two cats each based on the ITS sequence analyses. Moreover, Encephalitozoon spp. sequence analyses revealed three isolates belonging to E. intestinalis. CONCLUSIONS: This preliminary study has provided the first molecular data on the zoonotic genotypes of E. bieneusi and E. intestinalis in cats in Turkey. Furthermore, E. bieneusi genotype IV (accession number MG727664) was submitted to GenBank for the first time in the Western Palearctic Region as hosted by a domestic cat. Additionally, E. intestinalis (accession number MG570080) was also submitted to GenBank as a valid ITS sequence for the first time as hosted by a domestic cat worldwide.

Serum concentration and skin tissue expression of insulin-like growth factor 2 in canine generalized demodicosis.

BACKGROUND: There is increasing evidence that insulin-like growth factor-2 (IGF-2) levels are altered in skin injury; there are no data evaluating the serum concentration and skin tissue expression of IGF-2 in canine generalized demodicosis. HYPOTHESIS/OBJECTIVES: To assess serum concentrations of IGF-2 collected from dogs with generalized demodicosis compared to healthy dogs and to determine the location of IGF-2 in the skin of affected dogs. METHODS: Blood and skin samples were collected from 12 dogs of differing breeds and gender at 1-2 years of age that had a confirmed diagnosis of generalized demodicosis. Age-matched control skin and blood samples were collected from 11 normal dogs of different breeds and gender. Serum IGF-2 concentrations were measured by enzyme-linked immunosorbent assay. Skin tissue expression of IGF-2 was analysed by immunohistochemical methods. RESULTS: Serum concentration and skin tissue expression of IGF-2 were increased in dogs with generalized demodicosis compared with control dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: These findings indicate that keratinocytes, histiocytes and fibrocytes in the dermis are positive for IGF-2; they may be a source of the elevated serum IGF-2 levels in dogs with generalized demodicosis.

Investigation of the 8-hydroxy-2'-deoxyguanosine, total antioxidant and nitric oxide levels of serum in dogs infected with Babesia vogeli.

Oxidative stress was defined as corruption of balance between oxidant-antioxidant states in favor of oxidants. In this study, it was aimed to determine oxidative stress in naturally infected dogs with Babesia vogeli. The 8-hydroxy-2'-deoxyguanosine (8-OHdG), nitric oxide (NO) and total antioxidant capacity (TAC) levels in the sera were analyzed by ELISA. 8-OHdG levels increased in B. vogeli infected dogs compared to control group (P<0.05). Also, NO levels increased while TOC levels decreased in B. vogeli infected dogs but these variations were not found as statistically important (P>0.05).

A case of auricular, anal and umbilical myiasis caused by the larvae of Phormia regina (Meigen) (Diptera: Calliphoridae) in neonatal kittens.

The occurrence of feline myiasis is rare. Massive infestations of dipteran larvae can lead to death if not treated at an early stage. Auricular, anal and umbilical myiasis was detected in three neonatal kittens. The dipteran larvae were collected, fixed in 70% alcohol and clarified with 10% KOH for a few days. Later, larvae were dissected under the stereomicroscope, mounted on slides and then identified as the third instar of the black blowfly, Phormia regina (Meigen) (Diptera: Calliphoridae), according to their stigmatic and cephaloskeleton structures. Original measurements and figures are presented. Treatment included mechanical removal of larvae and cleansing of the area by applying polyvinylpyrrolidone-iodine complex. The presence of P. regina in cats has been reported here for the first time in Turkey.

Vitamin E and immunity.

Vitamin E is the most important chain-breaking, lipid-soluble antioxidant present in body tissues of all cells and is considered the first line of defense against lipid peroxidation and it is important for normal function of the immune cells. However, vitamin E deficiency is rare in well-nourished healthy subjects and is not a problem, even among people living on relatively poor diets, both T- and B-cell functions are impaired by vitamin E deficiency. While immune cells are particularly enriched in vitamin E because of their high polyunsaturated fatty acid content, this point puts them at especially high risk for oxidative damage. Besides its immunomodulatory effects, vitamin E also plays an important role in carcinogenesis with its antioxidant properties against cancer, and ischemic heart disease with limiting the progression of atherosclerosis. Supplementation of vitamin E significantly enhances both cell mediated and humoral immune functions in humans, especially in the elderly and animals.

Investigation of immunmodulatory effects of levamisole and vitamin E on Immunity and some blood parameters in newborn Jersey calves.

The immunmodulatory effects of dl-alpha tocopherol (vitamin E) and levamisole on the immune system and some blood parameters of newborn Jersey calves were investigated with the present study. Treatment groups 1, 2 and 3 were injected 13,3 ml isotonic saline solution (0,9% NaCl), 3 mg/kg levamisole HCl and 2000 IU vitamin E weekly, starting at birth until the age of two weeks. Average serum IgM levels of the control, levamisole and vitamin E calves were 111,7 +/- 9,3 mg/100 ml, 251,9 +/- 27,6 mg/100 ml, 202,2 +/- 43,3 mg/100 ml respectively on day 22. Average serum IgG levels of the levamisole and vitamin E groups elevated, compared to the control group on days 1, 8, 15 and 22. However, there were stastistically differences in treatment and control groups for serum total cholesterol, low density lipoprotein (LDL), high density lipoprotein (HDL), triglyceride and cortisol values and whole blood counts. All differences were in the reference ranges. Levamisole and vitamin E could be used as an alternative way for their beneficial effects such as improving the humoral immune responses of calves and their safety and practical use against the neonatal period infections in the field.

Trombiculidae larvae (Neotrombicula autumnalis) infestation in a Little Bittern (Ixobrychus minutus) in Turkey.

With this case report, we describe Trombiculidae larvae (Neotrombicula autumnalis) infestation in a Little Bittern (Ixobrychus minutus) which was brought to our clinics by the Directorship of Environmental and Forestry authorities of Samsun, Turkey in April, 2007. The male Little Bittern (I. minutus), with a black back and crown, and black wings with a large white patch on each wing, had thick Trombiculidae larvae infestation both on its legs. There were neither dermatological nor general conditional changes observed, except its superficial larval attachments to the skin. To our knowledge, this is the first report of Trombiculidae larvae (N. autumnalis) infestation in a Little Bittern (I. minutus) in Turkey.