A Parasitologic and Molecular Survey of Hepatozoon canis Infection In Stray Dogs In Northeastern Iran.

Canine hepatozoonosis, caused by Hepatozoon canis, is a tick-borne disease in domestic and wild dogs that is transmitted by ingestion of Rhipicephalus sanguineus ticks. The aim of the study was to detect H. canis in stray dogs in Iran with the use of blood smear examination and molecular techniques. From October 2014 to September 2015, 150 EDTA blood samples were collected from stray dogs in northeastern Iran. Blood smears were microscopically examined for the presence of Hepatozoon gamonts; whole blood was evaluated by PCR with subsequent sequencing and phylogenetic analysis. Hepatozoon spp. gamonts were observed in the neutrophils of 5/150 (3.3%) blood smears, whereas Hepatozoon spp. 18S rDNA was detected in 12/150 (8.0%) blood samples from stray dogs. There was a good correlation between microscopy and PCR methods (κ = 0.756). The highest seasonal rate of infection was detected in the summer ( P < 0.05). The frequency of Hepatozoon spp. infection did not differ significantly by gender or age ( P > 0.05). The alignment analysis of the sequenced samples showed ≥99% similarity with other nucleotide sequences of Hepatozoon spp. in GenBank. The phylogenetic tree also revealed that the nucleotide sequences in this study were clustered in the H. canis clade and different from the Hepatozoon felis and Hepatozoon americanum clades. Accordingly, we conclude that H. canis infection is present among dogs in northeastern Iran.

Serological study of Toxoplasma gondii infection in Turkoman horses in the North Khorasan Province, Iran.

Toxoplasmosis is an important zoonotic disease with worldwide distribution. The infection is observed in an unusually wide range of warm-blooded animals, including most of the livestock and humans. Many studies have shown high prevalence of toxoplasmosis in man and animals in Iran. The present study was conducted to investigate the seroprevalence of toxoplasmosis in Turkoman horses in the North Khorasan Province. During 2011-2012, 100 blood samples from horses were collected and tested for antibodies against toxoplasmosis using indirect fluorescent antibody test. The seroprevalence of toxoplasmosis was detected in 14 % (14) horses. The antibodies titres were detected in the range of 1:20-1:160 dilution. The lowest and highest frequencies of toxoplasmosis were observed in the age groups of <1 year and 1-10 years, respectively (P < 0.05). No significant difference was observed between toxoplasmosis frequencies and gender and usage of horses. With regard to the high frequency of toxoplasmosis in the sampled horses, attention must be paid to the animal health for the control and prophylaxis of the disease.

Molecular genotyping of Echinococcus granulosus from dromedaries (Camelus dromedarius) in eastern Iran.

With the aim of genotyping Echinococcus granulosus cysts found in Iranian dromedaries (Camelus dromedarius), 50 cysts of E. granulosus were collected from five geographical regions in Iran. Cysts were characterized using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer 1 (ITS1) gene and sequencing fragments of the genes coding for mitochondrial cytochrome c oxidase subunit 1 (cox1). Morphological criteria using rostellar hook dimensions were also undertaken. The present results have shown that 27 out of 50 E. granulosus cysts (54%) were determined as the G1 strain, and the other (46%) were determined as the G6 strain. The molecular analysis of the ITS1 region of ribosomal DNA corresponded with the morphological findings. Because of its recognized infectivity in humans, the G1 genotype is a direct threat to human health and its presence in Iranian dromedaries is of urgent public health importance.

A serological study of Leishmania infantum in dogs of Khorasan Razavi province, Iran.

Leishmania infantum is the principal agent of visceral leishmaniosis (kala-azar) in all areas of Iran. The disease is endemic in Fars, Ardabil, East Azerbaijan and Bushehr provinces while it is sporadic in other parts of the country. Dog as a member of canidae family is the main animal reservoir host of kala-azar. The present study was carried out to determine the sero-prevalence of canine leishmaniosis in Mashhad, the capital city of Khorasan Razavi Province, in the north east of Iran. Two-hundred and nine dogs in two different classes including stray and sheepdogs were selected for this cross-sectional study. The blood samples were collected and tested by indirect immunofluorescent antibody test for detection of anti-Leishmania infantum antibodies. The results showed that 16/209 (7.6 %) of blood samples were positive. The sero-prevalence rates in stray and sheepdogs were 11/94 (11.7 %) and 5/115 (4.3 %), respectively (p < 0.05). Based on the results obtained, it is concluded that visceral leishmaniosis is distributed among dogs of this area, also sero-prevalence of canine leishmaniosis in stray dogs higher than sheepdogs.

Hyalomma anatolicum anatolicum and Hyalomma dromedarii (Acari: Ixodidae) imbibe bovine blood in vitro by utilizing an artificial feeding system.

Hard ticks have great importance because of blood feeding and transmitting dangerous human and animal diseases. Each year, they cause a lot of economical damage to the livestock industry. Control of ticks and tick-borne diseases is a major priority in most parts of the world and many studies have been done in this field. We know that studying haemoparasites and assessing the effect of different compounds on ticks requires a lot of money, support and sometimes it is time consuming. Considering all of these problems, today, in some research laboratories throughout the world, artificial in vitro feeding of ticks has become common. Development and application of such methods provide a cheap and accessible background for investigating haemoparasitic diseases under controlled conditions. For the first time we report successful in vitro feeding of two important ixodid ticks of the genus Hyalomma.

Study on ovine abortion associated with Toxoplasma gondii in affected herds of Khorasan Razavi Province, Iran based on PCR detection of fetal brains and maternal serology.

Toxoplasma gondii, an obligatory intracellular protozoan parasite, is one of the causative agents of ovine abortion, as reported in many countries. Different techniques are being used to detect this pathogen in infected ovine fetuses. One of the most sensitive and specific diagnostic techniques is Nested-PCR amplification of the B1 target gene of the organism. In total, 200 brain samples of aborted ovine fetuses and maternal sera submitted from different parts of Khorasan Razavi province, Iran were investigated to track the role of Toxoplasma gondii in ovine abortion by a slightly modified Nested-PCR and IFAT assays, respectively. Among all samples, 27 (13·5%) were PCR-positive and 31 (15·5%) were IFAT-positive and the Toxoplasma-induced abortion prevalence calculated was 8·8% to 18·2% with 95% confidence interval. Results show that high levels of congenital transmission may occur in 27/31(87%) of pregnancies with an excellent logical agreement (ĸ=0·9) between 2 different tests. According to the results of this study, the Nested-PCR employed in this investigation could be recommended as an applied routine test for the routine examination and confirmation of Toxoplasma gondii-induced ovine abortion.

A serological study and subsequent isolation of Toxoplasma gondii from aborted ovine fetuses in Mashhad area, Iran.

Toxoplasmosis is an important cause of abortion and stillbirth in sheep on a worldwide basis. In the present study, the role of Toxoplasma gondii in inducing abortion in sheep of the Mashhad area of Iran was addressed using serological and parasitological methods. In total, 325 aborted ovine fetuses were collected between 2006 and 2008 during lambing season. Thoracic and abdominal fluids of aborted fetus were serologically investigated with a T. gondii -IFAT. Antibody titers equal to or greater than 1:20 were detected in 17 (5.2%) of ovine fetuses. Processed brain samples of seropositive ovine fetuses were intraperitoneally inoculated into mice for isolation of T. gondii . The process yielded a single T. gondii isolate that remained avirulant for mice after several passages. The results document that T. gondii is among the important causes of ovine abortion in Iran.

A survey of Neospora caninum-associated bovine abortion in large dairy farms of Mashhad, Iran.

Neospora caninum is an apicomplexan protozoon causing abortion in cattle worldwide. The present study was designed to assess the importance of this parasite for causing abortion in dairy farms in the Mashhad area of Iran. Of the aborted bovine fetuses, 151 were collected from dairy farms between 2006 and 2008. First, brain samples were examined by polymerase chain reaction (PCR) for the presence of N. caninum DNA, diagnosis was complemented with immunohistochemistry (IHC) and fetal serology (ELISA). Twenty-two (14.5%) of bovine fetuses were considered to be infected with N. caninum with at least one diagnostic technique being positive. PCR yielded 18 (11.9%) positive out of 151 brain samples. Only 52 brain samples were suitable for IHC examination, and N. caninum organism was detected in six (11.5%) of these 52 brain samples. Fetal fluids (n = 151) were assessed with a N. caninum-ELISA, resulting in 15 (9.9%) seropositive fetal fluids samples. In the present study, a good agreement was observed between PCR and ELISA, and a fair agreement between PCR and IHC. The results indicated that abortion due to N. caninum infection is prevalent among large-size dairy farms in the Mashhad area of Iran, and that different complementary diagnostic techniques should be used to increase the chance to detect N. caninum.

First report of Neospora caninum-associated bovine abortion in Mashhad area, Iran.

Neospora caninum is an apicomplexan protozoan which causes abortion in cattle worldwide. The present study was designed to assess the importance of bovine neosporosis for causing abortion in Iranian cattle. Infection was primarily diagnosed by polymerase chain reaction (PCR), complemented with histopathology and immunohistochemistry (IHC). One hundred brains of aborted bovine fetuses were collected from Iranian dairy herd between 2003 and 2005 in Mashhad area. N. caninum was detected by PCR in the brains of 13 aborted fetuses. Lesions consistent with N. caninum infection were observed in 12 of fetal brains. Immunohistochemical examination of brain detected N. caninum organisms in three aborted fetuses, and a thick-walled (2 mum) cyst with 50 mum diameter was identified in one of the IHC-positive brain. The results indicated that neosporosis is an important cause of abortion in dairy cattle of Iran.

An epidemiological study on Anaplasma infection in cattle, sheep, and goats in Mashhad Suburb, Khorasan Province, Iran.

The prevalence of Anaplasma infection was studied in cattle, sheep, and goats in the Mashhad area from 1999 to 2002. A total of 160 cattle from 32 farms and 391 sheep and 385 goats from 77 flocks were clinically examined for the presence of Anaplasma spp. in blood smears. The study revealed that 19.37% of cattle were infected with Anaplasma marginale and 80.3% of sheep and 38.92% of goats were infected with Anaplasma ovis. Prevalence of Anaplasma infection between male and female and between different age groups of cattle, sheep, and goats were statistically nonsignificant. Seasonally, the prevalence of Anaplasma infection in sheep and goats reached its highest level in summer, while a decrease was observed in autumn, and reached the lowest level in winter. The seasonal prevalence of Anaplasma infection in cattle was not significantly different. Symptomatic cases were not observed in any of the cattle, sheep, and goats. The ranges of anaplasmatemia in infected cattle, sheep, and goats were 0.005-0.5%, 0.01-3%, and 0.01-3%, respectively.

Prevalence of Theileria spp. infection in sheep in South Khorasan province, Iran.

The prevalence of Theileria spp. infection was studied in sheep in the South Khorasan province in Iran from 2003 to 2004. A total of 840 sheep from 34 flocks were clinically examined and investigated for the presence of Theileria spp. in the appropriate blood smears and any tick species on the body of the animals. In this study, 11.9% of sheep were infected with Theileria spp., with a parasitemia of 0.02-0.1%. Differences in the infection rates were statistically significant among different areas of the South Khorasan province. The highest prevalence was found in the Ferdows area (31.4%) and the lowest rate in the Nehbandan area (0.7%). The prevalence of Theileria spp. infection in males and females and between different age groups of sheep were not statistically significant. Seasonally, the prevalence of Theileria spp. infection in sheep reached its highest level in June (26.3%), whereas it decreased in July and August. It was found that 50.5% of the animals harbored Rhipicephalus sanguineus, 48.5% harboured Hyalomma anatolicum and 0.89% harboured Hyalomma dromedari.

Seroepidemiology of Neospora caninum infection in dairy cattle herds in Mashhad area, Iran.

A seroepidemiology study of Neospora caninum was conducted in Mashhad area, Khorasan province, Iran on 337 cattle from 30 dairy using ELISA. All herds had seropositive cattle and antibodies were formed in 46% of the cattle. Abortion was significantly associated with seropositivity of cattle. The highest risk of abortion was observed in the 1-2-year-old cows. Our results indicate that Neospora infection is widespread in Iran.

Trials to induce protective immunity in mice and sheep by application of protoscolex and hydatid fluid antigen or whole body antigen of Echinococcus granulosus.

In the current study, soluble proteins prepared from 200 mature Echinococcus granulosus and protoscolices of sheep hydatid cysts were applied to immunize sheep and mice respectively. The samples were mechanically homogenized in a blender, sonicated and the final yield was maintained at -20 degrees C until analysis. Hydatid fluid was isolated from liver or lung of sheep under sterile conditions. In the first experiment, 15 mice were randomly allocated to three groups of five mice each. Each mouse in groups 1 and 2 was immunized with 100 microg of hydatid fluid and protoscolex proteins in 100 microl of phosphate-buffered saline (PBS) and emulsified with an equal volume of Freund's complete adjuvant (FCA) respectively. The mice of group 3 were immunized with adjuvant in PBS. The mice were boosted 4 weeks after the first vaccination with the same preparation except that FCA was replaced by Freund's incomplete adjuvant (FIA). In the second experiment, eight male or female lambs 4-6 months of age, were allocated to two groups of four lambs each. Each lamb in the test group was vaccinated subcutaneously in the neck with a 2-ml dose of vaccine (1 mg of whole body protein of E. granulosus dissolved in 1 ml of PBS plus 1 ml of FCA). Control lambs were vaccinated with adjuvant in PBS. Lambs were boosted the same way as in the first experiment. Three weeks after the second vaccination, each mouse and lamb received a challenge infection with 2000 protoscolices intraperitoneally and each lamb additionally received 10 gravid E. granulosus. All mice and sheep were killed after 7 months and examined for hydatid cysts. In these studies, protective immunity was induced in mice with protoscolex protein and with hydatid fluid, and in sheep with whole-body homogenate of E. granulosus and the levels of protection afforded were found to be 72.1, 82.6 and 90.9% respectively.

Toltrazuril treatment to control diaplacental Neospora caninum transmission in experimentally infected pregnant mice.

We addressed the question whether diaplacental transmission of Neospora caninum can be controlled by metaphylactic chemotherapy using toltrazuril or enrofloxacin. Female C57/BL6 mice, infected on day 10 of pregnancy, were medicated for 6 consecutive days p.i. with 52.5 mg toltrazuril or - as an out-group control medication--16.7 mg enrofloxacin per kg body weight per day. Other control groups received either infection but no medication or vice versa. Toltrazuril treatment significantly reduced pre- and perinatal losses (10 deliveries of healthy newborns, versus 1 abortion and 4 failures) when compared to control-enrofloxacin (2 deliveries, versus 1 abortion, 7 failures and 2 pre-parturient deaths of dams) and non-treated animals (3 deliveries, versus 6 abortions, 8 failures and 4 pre-parturient deaths). Simultaneously, PCR-based parasite detection in the brain of mothers, histopathological findings as well as clinical fatality were significantly less frequent in toltrazuril-treated dams. The overall toltrazuril treatment efficacy was determined as 87 %, that of enrofloxacin-treatment as 17 %. The progenies of toltrazuril-treated dams also exhibited a very low rate of PCR-positivity in their brain (3 out of 39), whereas untreated dams delivered litters with mostly PCR-positive brains (12 out of 14) and a relatively high death rate post-partum (5 out of 19 newborns died). Mice subjected to a second mating delivered newborns all negative by N. caninum-PCR, indicating that diaplacental tachyzoite passage does not occur in a later, repeated pregnancy. Overall, our experiments showed that toltrazuril-treatment of an acute N. caninum-infection--induced during pregnancy--results in a clear reduction of fetal losses and a marked reduction of diaplacental passage of the parasite to the fetal brain, whereas enrofloxacin, as an out-group control substance, failed to show the same effect.

A study about tick vectors of bovine theileriosis in an endemic region of Iran.

The aim of this study was to determine the population of ticks in infected cattle and to identify the tick vectors of bovine theileriosis in an endemic area of Iran from 1998 to 1999. A total of 120 suspected cattle suffering from theileriosis were clinically examined and investigated for the presence of Theileria annulata in blood smears and the presence of any tick species on the body of cattle. In this study, 680 ticks were collected from 107 cattle infected with T. annulata. The prevalence of ticks infesting cattle was 92.35% Hyalomma anatolicum excavatum, 5.14% H. marginatum marginatum, 1.17% H. asiaticum asiaticum and 1.32% Rhipicephalus sanguineus. The examination of 510 tick salivary glands revealed that 51% of H. a. excavatum and 1.3% of H. a. asiaticum were infected with sporozoites of T. annulata.

Identification of tick vectors of ovine theileriosis in an endemic region of Iran.

This study was made to determine the population of ticks in infected sheep and attempting to identify the tick vectors of ovine theileriosis in an endemic area of Iran from 2000 to 2001. A total of 188 suspected cases of ovine theileriosis from 28 flocks were clinically examined and investigated for the presence of Theileria lestoquardi in appropriate blood smears and any tick species on body of sheep. In this study, 36.17% of sheep were infected to T. lestoquardi with a parasitemia of 0.01-15%. There was no significant differences between the rate of parasitemia in sheep and the frequency of infected ticks. We found that 61.1% of the animals harboured Hyalomma a. anatolicum, 33.42% Rhipicephalus sanguineus and 0.05% Hyalomma m. marginatum. The examination of 345 tick salivary glands showed that (15%) of salivary glands of H.a. anatolicum and (4%) of R. sanguineus contained Feulgen positive bodies. Seasonally, the prevalence of Theileria infection and H.a. anatolicum infestation in sheep reached the highest level in July (62.5%) and June (23.6%), while a decrease was observed in September (24.5%) and August (17.39%), respectively. The prevalence of Theileria infection in all age groups and between male and female sheep was statistically non-significant.

An epidemiological study on ovine babesiosis in the Mashhad suburb area, province of Khorasan, Iran.

The prevalence of Babesia spp. infection was studied in sheep of the Mashhad area in Iran from 1998 to 2000. A total of 677 sheep originating from 115 flocks were clinically examined and investigated for the presence of Babesia spp. in appropriate blood smears and any tick species on the body of the animals. The study revealed that the infection rate for Babesia ovis and Babesia motasi were 167 (24.6%) and 4 (0.5%), respectively. Double (mixed) infections occurred in 21 (3%) sheep. Differences in infection rates were statistically non-significant between male and female sheep and between different age groups. Seasonally, the prevalence of Babesia spp. infection started to increase in April and reached highest values in August (56%), while a decrease was observed in September, reaching the lowest levels In February and March. The study demonstrated that 1.7% of sheep infected with B. ovis and 50% of sheep infected with B. motasi exhibited clinical signs. Sheep infected with B. motasi showed the highest levels of parasitemia. We found that 550 (73%) of the animals harbored Rhipicephalus sanguineus; 166 (21%) Hyalomma marginatum; 19 (2.5%) Dermacentor daghestanicus; 14 (1.8%) Hyalomma anatolicum; 6 (0.66%) Hyalomma asiaticum; and one (0.13%) Haemaphysalis punctata. The examination of 727 tick haemolymph samples and 52 tick egg smears showed that one sample (0.2%) of haemolymph of R. sanguineus, two (1.2%) haemolymphs of H. marginatum and two (2%) eggs of R. sanguineus harbored kinetes morphologically matching the criteria described for B. ovis.