Extracellular traps development in canine neutrophils induced by infective stage Toxocara canis larvae.

Neutrophils, a crucial element of the host defense system, develop extracellular traps against helminth parasites. Neutrophils accumulate around the larvae of Toxocara canis (T. canis) in the tissues of the organism. This study aimed to determine the reaction in canine neutrophils after incubation with infective stage T. canis larvae (L3) in vitro. Most L3 were still active and moved between the extracellular traps (NETs) after 60-min incubation. NETs were not disintegrated by L3 movement. The L3 was only immobilized by NETs, entrapped larvae were still motile between the traps at the 24 h incubation. NETs were observed not only to accumulate around the mouth, excretory pole or anus but also the entire body of live L3. The extracellular DNA amount released from the canine neutrophils after being induced with phorbol 12-myristate 13-acetate was not affected by T. canis excretory/secretory products obtained from 250 L3. To the Authors'knowledge, the extracellular trap structures was firstly observed in canine neutrophils against T. canis L3 in vitro. NETs decorated with myeloperoxidase, neutrophil elastase and histone (H3) were observed under fluorescence microscope. There were not significant differences in the amount of extracellular DNA (P > 0.05), but the morphological structure of NETs was different in the live and head-inactivated T. canis larvae.

Determination of extracellular traps structures from sheep polymorphonuclear leukocytes to Echinococcus granulosus protoscoleces.

It was aimed to detect extracellular traps structures from sheep polymorphonuclear leukocytes (PMN) after being confronted with Echinococcus granulosus protoscoleces in vitro. Also, the effect of cyst fluid was examined on the development of extracellular traps. At the end of the incubation for 1 h, the extracellular traps augmented with neutrophil elastase, histone (H3) and myeloperoxidase were visualized in the protoscoleces-PMN co-culture microscopically. Some protoscoleces lysed and the chitinous hooks released were surrounded by the extracellular traps. The other protoscoleces were still intact and the extracellular trap structures were observed around them. The relationship between the extracellular DNA contents and the protoscoleces concentration was not found statistically significant (P > 0.05). The extracellular DNA amount in the co-cultures diluted in RPMI-1640 increased with the incubation time (P < 0.05). However, the time-dependent relationship was not found in the co-cultures diluted in the cyst fluid (P > 0.05). The difference in the extracellular DNA amount was detected as statistically significant (P < 0.05) between the two co-culture groups (diluted in RPMI-1640 or the cyst fluid), except for 30 min incubation. To the Author's knowledge, NETosis reaction was firstly observed in sheep PMN after being confronted with protoscoleces in vitro. The cyst fluid had some negative effects on the development of extracellular traps from sheep PMNs at the 1-h incubation time. It should be investigated which molecules are responsible for NETosis inhibition in hydatid cyst fluid. Future studies may clarify whether neutrophils fight with protoscoleces by using their different mechanisms.

Vaccines Currently Available in the Field of Veterinary Parasitology

Some parasite vaccines, developed for use in the veterinary field, are available in the market. Such vaccines usually contain live or attenuated parasites. Aside from these, a few parasite vaccines have also been prepared using recombinant technology. The objective of this review is to provide information about the antiparasitic vaccines available for use in the veterinary field globally.

Morphological and ecological features of some nematodes of the genus Rhabdochona in marinka obtained from Fergana Valley, Uzbekistan.

The aim is to study the morphological and ecological features of some nematode species of the genus Rhabdochona parasites of marinka fish in the Fergana Valley, Uzbekistan. Rhabdochona gnedini, Rhabdochona denudata and Rhabdochona hellichi turkestanica were detected as 5.3%, 7.9% and 3.7%, respectively. According to the Authors' knowledge, Rh. hellichi turkestanica was recorded in Uzbekistan and Central Asian republics for the first time. Rhabdochona spp. were only detected in the intestine of marinkas lived in the mountain rivers Rezaksay, Chodaksay and small tributaries of the Syrdarya river in western Fergana. The prevalence of Rhabdochona spp. in the marinka can be associated with a wide range of nutrition in the mountain rivers and small tributaries of the Syrdarya river, where it could eat large numbers of mayfly and caddis larvae that act as intermediate hosts of rhabdochona.

FELINE GIARDIASIS IN TURKEY: PREVALENCE AND GENETIC AND HAPLOTYPE DIVERSITY OF GIARDIA DUODENALIS BASED ON THE ß-GIARDIN GENE SEQUENCE IN SYMPTOMATIC CATS.

Giardia duodenalis is a common zoonotic protozoan parasite with a broad host distribution. The main objectives of the present study were to determine the prevalence of giardiasis and to reveal the genetic and haplotype diversity of G. duodenalis in symptomatic cats in Turkey. Fecal samples were collected from cats (n = 102) with diarrhea that were admitted to different pet clinics in the Central Anatolia region of Turkey. All samples were analyzed by microscopic examination (ME), rapid immunochromatographic test (ICT), and PCR targeting the ß-giardin (bg) loci of the parasite. Phylogenetic, haplotype, and network analyses of G. duodenalis based on the bg gene were carried out. Overall, G. duodenalis was detected in 70/102 (68.6%) of the cats with diarrhea by ME (38/102, 37.3%), ICT (51/102, 50%), and PCR (30/102, 29.4%). According to sequence analyses of the bg gene region, all isolates were identified as G. duodenalis assemblage B. Haplotype analyses revealed 2 known and 8 novel haplotypes for G. duodenalis assemblage B. This study provides first prevalence and genetic and haplotype diversity data on G. duodenalis assemblage B from cats in Turkey.

Development and validation of SYBR Green- and probe-based reverse-transcription real-time PCR assays for detection of the S and M segments of Schmallenberg virus.

Schmallenberg virus (SBV), discovered in Germany in 2011, causes congenital malformations in ruminants. Reverse-transcription real-time PCR (RT-rtPCR) assays based on various segments of SBV have been developed for molecular detection. We developed alternative RT-rtPCR assays for SBV detection to avoid earlier reported mutations and hypervariable regions of the S and M segments of the viral genome. For SYBR Green-based detection of the S segment, the R2 value and efficiency of the developed assay were 0.99 and 99%, respectively. For probe-based S segment detection, 2 assays were developed; the first had an R2 value of 0.99 and 102% efficiency, and the second had a R2 value of 0.98 and 86% efficiency. The probe-based M segment assay had an R2 value of 1.00 and 103% efficiency. Detection limits of the RT-rtPCR assays with new primer sets were 102 and 101 copies/µL for the S and M segments, respectively. Field samples from cattle and sheep were also used for primary validation of the developed assays. Our assays should be suitable for SBV detection in ruminants and for in vitro studies of various SBV strains.

The first record on Giardia muris from mice in Turkey.

Giardia muris has been reported from both laboratory and wild rodents in worldwide. During routine care, soft consistency was observed in feces of Swiss albino mice. Motile Giardia spp. trophozoites were observed microscopically on all fecal preparations after stained with Lugol's iodine solution. The trophozoites were broadly ovoid in a form with a pointed end. They possess two large nuclei and an adhesive disk whose length overlapped one-half of the trophozoite body length. The caudal flagella are unequal. ß giardin (bg) gene region of isolates was successfully amplified and sequenced. According to sequence analyses of the bg gene region, the mice isolate was identified as G. muris and deposited in GenBank (Accession Number: MK675656). The bg sequence of G. muris was shown 99.3-99.7% identity with the isolates of G. muris reported from different countries in GenBank. We have firstly demonstrated G. muris trophozoites in the fecal samples of naturally infected Swiss albino mice in Turkey.

Comparison of Picogreen and Sytox Orange Stains in Quantitative Analysis of Extracellular Trap Formed against Toxoplasma gondii in Polymorphonuclear Leukocytes from Sheep.

OBJECTIVE: The present study aimed to compare the effectiveness of two extracellular DNA dyes (Sytox Orange and PicoGreen) in quantitative measurement of the DNA that forms the backbone of the extracellular trap structures in vitro. Toward this aim, the co-culture of polymorphonuclear leucocytes (PMNs) and Toxoplasma gondii tachyzoites isolated from sheep was selected as model. METHODS: T. gondii tachyzoites and PMNs isolated from the sheep were incubated for varied durations (30, 60, 90 and 120 min); the extracellular DNA released following this incubation was stained using two different dyes. RESULTS: In the present study, no statistical significant difference was observed between the two extracellular DNA dyes with regard to the measurement of extracellular DNA released for all the incubation durations (p>0.05), except for the 30-min incubation (p=0.014). CONCLUSION: There was a statistically significant difference at 30 min incubation time. This difference may be attributed to the staining properties of the dye. Researchers studying in vitro netosis are recommended to use two different extracellular DNA dyes for the quantitative analysis of extracellular DNA formed during short-term incubation.

Neutrophil isolation from feline blood using discontinuous Percoll dilutions.

OBJECTIVE: Some studies have performed in vitro neutrophil isolation from feline blood. The major limiting factor for these studies is the small volume of blood that can be collected without development of potentially life-threatening complications. In the present study we attempted neutrophil isolation from feline venous blood samples using discontinuous Percoll gradients. MATERIAL AND METHODS: Blood was collected from the cephalic vein of clinically healthy adult cats. The blood samples were layered on Percoll dilutions (72 %, 63 %, 54 % and 45 %). After centrifugation, the feline polymorphonuclear leukocytes (PMN) accumulated as a band between 72-63 % Percoll dilutions. The total cell count was calculated using light microscopy counts. The percentage of the neutrophils was determined microscopically after staining with Diff-Quik stain. Neutrophil viability was evaluated with a 0.01 % Trypan blue assay. The activation was determined based on intact cell morphology in the isolated neutrophils. RESULTS: The mean PMN number was 22 x 105 per ml (minimum - maximum: 20-26 x 105/ml). Neutrophil homogeneity was > 95 % in the cell suspensions. The viability of isolated neutrophils was > 98 %. The technique did not result in neutrophil activation. CONCLUSION AND CLINICAL RELEVANCE: Discontinuous Percoll gradients (72 %, 63 %, 54 % and 45 %) can be used to isolate neutrophils from blood samples of cats. The technique was simple to perform and neutrophil activation was minimal.

Seroprevalence of Neospora caninum in Dairy Cattle Raised in Çiçekdagi District of Kirsehir Province.

OBJECTIVE: Neospora caninum is one of the most important causes of abortion in cattle worldwide and causes significant economic losses in the meat and dairy industries. This study aimed to determine the seroprevalence of N. caninum in dairy cattle raised in Çiçekdagi district of Kirsehir province. METHODS: One hundred sixteen serum samples collected from dairy cattle were analyzed for N. caninum antibodies by a commercial Enzyme-Linked ImmunoSorbent Assay (ELISA) kit (VMRD c-ELISA). RESULTS: The seropositivity rate was 18.1% in the cattle examined. The seroprevalence of N. caninum was 23.4% in dairy cattle with fertility problems, 33.3% in cattle with a history of abortion, and 7.8% in clinically healthy dairy cattle (p=0.006). Cattle breeds with highest seropositivity rates were Holstein, Simmental, and Brown Swiss (p=0.008). CONCLUSION: Control measures should be taken for both dogs as final host of the parasite cattle to prevent the spread of neosporosis in cattle in Çiçekdagi district.

Prevalence of Haemoproteus spp. in Tumbler Pigeons (Columba livia domestica) in Kirikkale Province, Turkey.

OBJECTIVE: Haemoproteus spp. are common blood parasites of pigeons. They have been reported in pigeons in many regions worldwide, including Turkey. Pigeon breeding is a popular hobby in Kirikkale province, and there is no information about the prevalence of Haemoproteus spp. The present study aimed to determine the prevalence of Haemoproteus spp. in tumbler pigeons in Kirikkale province (Kirikkale and Yahsihan district). METHODS: Blood samples were taken from the wing vein of pigeons (n: 173) through microcapillary (with/heparin) tubes between February and March 2016. Blood smears were stained with 5% Giemsa solution. Ectoparasites of the pigeons were collected in separate sealed boxes. Epidemiological data of the sampled pigeons (age and sex) were obtained from the breeders. RESULTS: In total, 23 (%13.2) of 173 pigeons were infected with Haemoproteus spp. Parasite was detected in 73.9% of pigeons over 1 year old and 26.1% of pigeon under 1 year age. Haemoproteus spp. was observed in 56.2% of females (13/23) and 43.4% of males (10/23), Sex-related differences were not observed (p = 0.821). Ectoparasites of the pigeons were identified as Columbicola spp. CONCLUSION: To the best of our knowledge, this is the first study in Kirikkale province that reported the prevalence of Haemoproteus spp. in pigeons.

Role of NETs in the difference in host susceptibility to Toxoplasma gondii between sheep and cattle.

The main aim of this study was to compare extracellular traps (NETs) formation by polymorphonuclear neutrophils (PMNs) of cattle and sheep when exposed to T. gondii tachyzoites in vitro. The effects of parasite concentrations and different incubation periods on NETs development in cattle and sheep PMNs were studied. The effect of NET structures on host cell invasion by tachyzoites was also studied. This is the first report of NETs development by sheep and cattle PMNs against T. gondii in vitro. T. gondii-induced extracellular DNA production from PMNs was dependent on tachyzoite concentrations and incubation time in both sheep and cattle. Many nuclear and cytoplasmic changes were observed in sheep and cattle PMNs after exposure to T. gondii tachyzoites. The typical appearance of NETs, with MPO, NE and histone (H3) attached to extracellular DNA, was observed. Tachyzoites were entrapped within this structure. Myeloperoxidase (MPO) activity was higher in the cattle PMN-tachyzoite co-cultures than sheep. NETs structures released from sheep PMNs caused mechanical immobilisation of T. gondii tachyzoites, however, NET structures released from cattle PMNs may be lethal to tachyzoites. Bovine MPO may have a lethal effect on T. gondii tachyzoites in vitro during a 3h incubation. Besides other mechanisms that effect on host susceptibility to T. gondii in sheep and cattle, extracellular traps formation as a part of immunological reactions may be play a role in host susceptibility to T. gondii.

Netosis: Alternative Defense Method Used by Neutrophils to Fight Pathogen.

Neutrophils form the first line of defense of the innate immune system in organisms. They use three fighting methods to combat pathogens: phagocytosis, degranulation, and netosis. In this review, we aim to provide information about netosis.

Prevalence of Intestinal Parasites in Cats and Their Importance in Terms of Public Health.

OBJECTIVE: This study aimed to detect the prevalence of intestinal parasites in cats by fecal examination. METHODS: A total of 100 fecal samples were collected from owned and stray cats in the Kirikkale province. Epidemiological data on the sampled cats (e.g., age, race, and sex) were recorded. The samples were macroscopically investigated for the presence of cestode proglottids. The fecal smears were stained with Giemsa and Carbol fuchsin stains. The samples were prepared by centrifugal flotation with a saturated sugar solution. The slides were examined using light microscopy. RESULTS: Parasites were detected in 47% of feces of cats examined. Protozoa (Isospora spp. and Cryptosporidium spp.) oocysts, gravid proglottid of cestode (Joyeuxiella spp.), and nematode (Toxocara spp. and hookworm) eggs were present in the fecal samples. In this study, 48.9% of cats were infected with one species, and 44.6% and 6.3% of cats were infected with two and three species, respectively. The more prevalent parasite species were Isospora spp. (65.9%) and Toxocara spp. (48.9%). The parasites were found to be more common in stray cats. CONCLUSION: Intestinal parasites were highly prevalent in the cats examined in this study. House cats can have significant parasitic infections, particularly because they are allowed outdoors. House cats should be regularly examined by a veterinarian.

Prevalence of intestinal parasites in hamsters and rabbits in some pet shops of Turkey.

OBJECTIVE: In this study, we aimed to determine the parasite species carried by hamsters and rabbits purchased from some commercial pet shops in Turkey. METHODS: For this purpose, the fecal samples of clinically healthy Syrian hamsters, dwarf hamsters, and crossbred rabbits were collected from 22 pet shops randomly selected in Ankara and Kirikkale provinces, located in Central Anatolia Region of Turkey. The fecal samples were examined with centrifuge flotation technique using saturated salt solution. RESULTS: Parasitic infection rate was 57.5% in dwarf hamsters, 54.9% in Syrian hamsters, and 56.3% in crossbreed rabbits. Trichurid eggs were the most prevalent parasite in the feces of Syrians hamsters (28.1%). The other parasites of Syrian hamsters were as follows: Eimeria spp. oocysts (15.4%) and the eggs of H. nana (11.2%), Syphacia spp. (11%). and Aspiculuris spp. (5.6 %). Only trichurid eggs were observed in the fecal samples of dwarf hamsters (51.5%). Oocysts of Eimeria spp. (52.7%) and the eggs of P. ambiguus (3.6%) were detected in the feces of rabbits. CONCLUSION: Within the scope of this study, the detection of H. nana eggs, a zoonotic parasite, in the feces of Syrian hamster was quite remarkable for public health.

[Seroprevalence of Toxoplasma gondii in sheep in Silopi district by using indirect fluorescent antibody test (IFAT)]. / Silopi'de koyunlarda Toxoplasma gondii'nin yayginliginin indirekt floresans antikor testi (IFAT) ile serolojik olarak belirlenmesi.

OBJECTIVE: In the present study, it was aimed to detect seropositivity of T. gondii in sheep raised in the Silopi district. METHODS: For this aim, blood samples were obtained from 100 female Hamdani sheep. The serum samples were examined using indirect fluorescent antibody (IFA) with respect of T. gondii specific antibody. RESULTS: Seropositivity was detected in 97 of sheep examined (97%). The seropositivity titers for the IFA test showed that 1:16 in 58 (59.7%), 1:64 in 22 (22.6%), 1:128 in 16 (16.4%) and 1:256 in 1 (%1) of sheep were found as seropositive. Seropositivity was observed as 96% in aborted sheep. Seropositivity was detected as 96% and 100% in 2-4 and 5-10 year old sheep, respectively. The relationship between age and seropositivity rate was not found significant (p > 0.05). CONCLUSION: The seropositivity of T. gondii was higher in sheep grown in Silopi.

In-vivo efficacy of toltrazuril on experimentally induced Toxoplasma gondii tissue cysts in lambs: a novel strategy for prevention of human exposure to meat-borne toxoplasmosis.

The aim of the present study was to investigate in vivo efficacy of toltrazuril on Toxoplasma gondii tissue cysts following induction of chronic toxoplasmosis in 4-week-old lambs (n=27) by inoculation of 1×10(5) T. gondii ME 49 strain oocysts (day 0). Beginning at the 15th day after inoculation, lambs in Group T20 and Group T40 were given toltrazuril orally 2 times, once every week (Baycox 5%, Bayer Animal Health) at a dose of 20 mg/kg and 40 mg/kg, respectively. Positive control (PC) lambs were not given any therapy, and 2 clinically healthy non-infected lambs were used as negative controls (Group NC). Two out of 9 lambs in PC group (oocyst inoculated but non-treated) were killed on toltrazuril treatment days (day 15 and 22) to evaluate the tissue cyst presence in their muscles. On day 90, the remaining 25 lambs were necropsied, and samples from the brain and 11 different muscle groups were collected. The tissues were examined for the presence of tissue cysts by histopathology, immunohistochemistry, nested-PCR and percoll gradient centrifugation. Anti-T. gondii antibodies were screened by IFAT throughout the experiment. The increased T. gondii seropositivity beginning from the 15th day of inoculation remained steady at Day 45 and Day 90 in Groups PC while it was significantly lower at Day 90 in toltrazuril receiving groups. In toltrazuril treated groups, histopathological findings included degenerative changes in the cyst wall, complete macrophage invasion to the cysts, and reduction or removal of the cysts in toto. Four out of 9 lambs (44.4%) in both toltrazuril treated group (Group T20 and T40) did not contain tissue cyst in any examined tissues while all positive control animals had T. gondii tissue cysts at least in one muscle group. The toltrazuril treatment efficacy on the cyst presence was determined as 44.4%. The number of the cysts in the musculature was significantly different between non-treated and toltrazuril treated lambs (X(2)=6.613; p=0.037). For the total number of cysts, the positive control lambs had higher number of cysts compared to both toltrazuril treated lambs (T20 and T40) (X(2)=5.629; p=0.018 and X(2)=5.629; p=0.018, respectively) while there were no differences between Group T20 and Group T40 (X(2)=0.000; p=1.000). According to PCR results, the brain and M. semitendinosus were positive in all 7 control lambs while 12 out of 18 lambs were positive in toltrazuril treated lambs. In conclusion, the results are promising as the toltrazuril treated lambs had markedly less parasite counts compared to those of untreated lambs. Further research should be conducted to reveal if toltrazuril treatment in sheep could be used as a strategy to minimize the cyst exposure of humans through consumption of raw or undercooked mutton.

Antiparasitic efficiency of Artemisia absinthium on Toxocara cati in naturally infected cats.

OBJECTIVE: The first aim of the present study was to determine the efficiency of A. absinthium extract on cats naturally infected with Toxocara cati. The second aim was to determine the efficiency of the extract on the embryonic development of T. cati eggs in vitro. METHODS: Artemisia absinthium extract was orally administrated to cats at the doses of 300 mg/kg and 600 mg/kg body weight in Group 1 and 2, respectively. It was given only once a day and the treatment continued 7 consecutive days. The faeces of the cats were examined both macroscopically and microscopically by flotation procedure with saturated salt solution pre-, during and post- treatment period. The faecal analysis was maintained during 8 days after completing the extract administration. The alteration of faecal egg numbers was performed by using the McMaster technique. RESULTS: The faecal egg numbers per gram were decreased gradually in cats in the trial groups. In the treatment period, the activities of ALT, AST, ALP, urea and creatinine were located within the physiological ranges in cats. In in vitro trials with A. absinthium extract, the embryonic development of T. cati eggs was identical in all groups (treatment and control). A. absinthium extract did not inhibit larval development in eggs in in vitro trials. CONCLUSION: This plant extract may be an alternative choice in the treatment of parasitic diseases in future.

[The effect of different temperatures on viability of Aelurostrongylus abstrusus first stage larvae in faeces of cats]. / Cesitli Sicaklik Derecelerinin Kedi Diskisindaki Aelurostrongylus Abstrusus 1. Dönem Larvasinin Canliligina Etkisi.

Aelurostrongylus abstrusus lives in the respiratory bronchioles and alveolar canals in the lungs of the cat and causes parasitic pneumonia. In present study, the effect of different temperatures on the viability of first stage larvae in feces of cats naturally infected with A. Abstrusus was investigated. Feces of cats were divided into two portions. One portion was left at room temperature (21-24 degrees C) and the other, left at +4 degrees C for three month-period. The fecal samples were examined by Baermann and McMaster techniques at 15 day-intervals. The number of living first stage larvae detected gradually decreased. The latest time of live larvae found in faeces left at room temperature was the 45th day and at +4 degrees C, the 60th day.

Seroprevalence of Bartonella vinsonii subsp. berkhoffii in urban and rural dogs in Turkey.

The seroprevalence of Bartonella vinsonii subsp. berkhoffii was investigated in stray urban dogs and shepherd and farm guard dogs from rural areas sampled from 10 provinces of Turkey. Sera from 855 dogs were examined for the presence of anti-B. vinsonii subsp. berkhoffii antibodies by indirect fluorescent antibody test. Overall, 56 (6.6%) of the 855 dogs examined, including 16 (3%) of the 522 stray dogs and 40 (12%) of the 333 rural dogs, were seropositive. This is the first report on prevalence of antibodies to B. vinsonii subsp. berkhoffii in dogs in Turkey.