Seasonal and Age-Associated Pathogen Distribution in Newborn Calves with Diarrhea Admitted to ICU.

Calf mortality constitutes a substantial loss for agriculture economy-based countries and is also a significant herd problem in developed countries. However, the occurrence and frequency of responsible gastro-intestinal (GI) pathogens in severe newborn diarrhea is still not well known. We aimed to determine the seasonal and age-associated pathogen distribution of severe diarrhea in newborn calves admitted to the intensive care unit (ICU) of Erciyes University animal hospital over a year. Fecal samples were collected during the ICU admissions, and specimens were subjected to a diarrheal pathogen screening panel that included bovine coronavirus (BCoV), Cryptosporidium spp., ETEC K99+, and bovine rotavirus, using RT-PCR and conventional PCR methods. Further isolation experiments were performed with permissive cell cultures and bacterial enrichment methods to identify the clinical importance of infectious pathogen shedding in the ICU. Among the hospitalized calves aged less than 45 days old, the majority of calves originated from small farms (85.9%). The pathogen that most frequently occurred was Cryptosporidium spp. (61.5%) followed by rotavirus (56.4%). The frequency of animal admission to ICU and GI pathogen identification was higher during the winter season (44.9%) when compared to other seasons. Most calves included in the study were 1-6 days old (44.9%). Lastly, co-infection with rotavirus and Cryptosporidium spp. occurred more frequently than other dual or multi-infection events. This study was the first to define severe diarrhea-causing GI pathogens from ICU admitted newborn calves in Turkey.

Newly identified Cryptosporidium parvum virus-1 from newborn calf diarrhoea in Turkey.

Cryptosporidium is a common enteric parasite that primarily affects those immunocompromised susceptible individuals and newborns. Detailed investigations have revealed that Cryptosporidium (C.) oocysts contain dsRNA segments which are recently classified under the Partitiviridae family. The relationship between parasite and virus whether or not affect the clinical outcomes of newborn calf diarrhoea is not apparent. The aim of this study was the identification and characterization of Cryptosporidium parvum virus-1 (CSpV1) from newborn calves. We also aimed to understand that parasite-virus symbiont relationship role in the severity of disease cases. Parasitic screening was performed with the help of morphological examinations, immunoassay and molecular polymerase chain reaction (PCR) methods. To further identification of C. parvum oocysts, confocal laser, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) image analysis were used for the morphological investigations. Software-based in silico comparison and identity analyses were conducted from the CSpV1 genome for the genomic sequence characterizations. Cryptosporidium prevalence was 56.2% in newborn calf diarrhoeal cases. Virus dsRNA segments isolated from purified and clarified oocysts. Sequence results showed that we have successfully isolated CSpV1 from C. parvum oocysts. Virus RNA-dependent RNA polymerase (RdRp) was found to be highly variable and showed a species-specific relationship with their carriers. We also identified that CSpV1 frequency was around 8.8% from diarrhoea-showing newborn calves. Cryptosporidium was strongly associated with diarrhoea at early ages of newborns, but the parasite and CSpV1 relationship is not associated with the severity of newborn calf diarrhoea. The current study provides the first report and molecular characterization of CSpV1 in Turkey.

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.

Investigation of Anisakis larvae in different products of ready-to-eat fish meat and imported frozen fish in Turkey.

Globalization opens new market areas and affects food consumption habits, resulting in rapid and remarkable cultural change. Food habits such as consumption of raw fish meat have become popular, resulting in increased risk of emerging infectious diseases. Anisakis simplex sensu stricto (s.s) and A. pegreffii are the most common and important fish-borne zoonotic nematodes responsible for human anisakiasis, which occurs through the consumption of raw or undercooked fish as well as cooked fish due to their heat-stable allergens. Here, we investigated the prevalence, intensity, and abundance of Anisakis larvae in imported fish and ready-to-eat local fish products in Turkey. A total of 205 ready-to-eat fish products, 100 imported frozen Atlantic salmon (Salmo salar) fillets, and 100 imported frozen whole Atlantic mackerel (Scomber scombrus) were sampled from supermarkets, sushi restaurants, and fish markets. All samples were individually examined using a pepsin digestion technique. In total, 602 Anisakis type I larvae were recovered from 98/100 mackerel. No larvae were found in ready-to-eat products or frozen Atlantic salmon fillets. Overall, 8.8% of the larvae were found in the muscle tissue. The overall mean intensity and abundance of infection in mackerel were 6.14 and 6.02, respectively. The larvae were molecularly identified and their phylogenetic relationships with the relevant Anisakis sequences in GenBank were investigated. For this purpose, a subsample of randomly selected 100 Anisakis larvae were analyzed with PCR-RFLP of the ITS region. The larvae were identified as A. simplex (s.s.) (n = 87) and hybrids (n = 13). ITS and cox2 gene regions of all hybrids and randomly selected 50 A. simplex (s.s.) larvae were sequenced for species confirmation and phylogenetic analyses. No intraspecific nucleotide variation was found among the ITS sequences of either species. Seven and three haplotypes, respectively, were identified for A. simplex (s.s.) and hybrid species according to DNA polymorphism of the cox2 gene. Hybrids in our study clustered within the common A. simplex (s.s.) clade in the cox2 phylogenetic tree indicating the dominance of A. simplex (s.s) in the catching area of Atlantic mackerel. Consequently, our study indicates high occurrence of A. simplex (s.s.) larvae with an overall 98.0% prevalence in imported Atlantic mackerel, and highlights the importance of these fish as potential reservoirs for human allergic anisakiasis in Turkey and possibly in other countries.

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.

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.

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.