A Preliminary Parasitological Survey of Hepatozoon Spp. Infection in Dogs in Mashhad,Iran

Background

We attempted to determine the prevalence of Hepatozoon spp. infection in Mashhad, northeast of Iran, via blood smear parasitology.

Methods

The prevalence was investigated by examination of blood smear parasitology, using blood samples collected from 254 dogs (51 strays and 203 privately owned-dogs).

Results

Two stray dogs (2/51; 3.92%) and two privately-owned dogs (2/203; 0.98%) were infected with Hepatozoon spp. Therefore, as per blood smear parasitology, the prevalence of Hepatozoon spp. infection was 1.57% (4/254). Sixteen out of 254 dogs (6.29%) were infested with ticks; all of which were Rhipicephalus sanguineus. One of the dogs infected with Hepatozoon spp. exhibited ticks at the time of examination. Concurrent infection with Ehrlichia canis and Leishmania infantum was not detected in the four Hepatozoon spp. infected dogs.

Conclusion

This is the first epidemiological study on the prevalence of Hepatozoon spp. infection in dogs in Iran.     

Application of the Reverse Line Blot Assay for the Molecular Detection of Theileria and Babesia sp. in Sheep and Goat Blood Samples from Pakistan

Background

The present study was designed to detect the presence of tick-borne parasites (Theileria and Babesia spp.) in 196 blood samples collected from apparently healthy sheep and goats from two provinces, Punjab and Khyber Pukhtoon Khwa, in Pakistan.

Methods

Reverse line blot (RLB) assay was applied for the parasitic detection by the amplification of hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene. A membrane with covalently linked generic and species specific oligonucleotide probes was used for the hybridization of amplified PCR products.

Results

Parasites were detected in 16% of the ruminant blood samples under study. Two Theileria species, T. lestoquardi and T. ovis, were identified in samples. 25, of the total 32, infected animals were from Khyber Pukhtoon Khwa.

Conclusion

Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001). Risk factor analysis revealed that male (P = 0.03), animals infested by ticks (P = 0.03) and herd composed of sheep only (P = 0.001) were more infected by blood parasites.     

Prevalence and Molecular Diagnosis of Babesia ovis and Theileria ovis in Lohi Sheep at Livestock Experiment Station (LES), Bahadurnagar,Okara, Pakistan

Background

Babesia ovis and Theileria ovis are among the important and main etiological agents causing ovine babesiosis and ovine theileriosis, causing severe economic losses among sheep and goats. The aim of the present study was to determine the prevalence and molecular diagnosis of B. ovis and T. ovis in Lohi sheep at Livestock Experiment Station Bahadurnagar, Okara, Pakistan.

Methods

The prevalence of B. ovis and T. ovis was investigated in 200 Lohi sheep of mixed age and sex by PCR during 2011. The assay was employed using primers Bbo-F & Bbo-R, specific for a 549-bp fragment in B. ovis genomic DNA and primers TSsr 170F & TSsr 670R, specific for a 520-bp fragment in T. ovis genomic DNA. The animals were also screened for both haemoparasites through stained thin blood smears.

Results

Thirty two (16%), 48 (24%) and 26 (13%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites, respectively, through microscopy. Sixty eight (34%), 73 (37%) and 42 (21%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites, respectively, through PCR test.

Conclusion

The results indicate the high sensitivity of PCR for surveying babesiosis and theileriosis and there is noteworthy prevalence of these diseases in sheep at an experimental station where environmental conditions are relatively controlled as compared to field conditions.     

Molecular and Biomorphometrical Identification of Ovine Babesiosis in Iran

Background

Ovine babesiosis is the most important haemoparasitic tick-borne disease of small ruminants in Iran caused by Babesia ovis, B. motasi, and B. crassa. The aim of this study was to characterize the species of ovine Babesia species isolated from different geographical region of Iran.

Methods

One hundred fifty four blood samples collected from animals, which demonstrated the pale mucous membranes or hyperthermia. The specimens were transferred to the laboratory and the blood smears stained with Geimsa, the morphological and biometrical data of parasite in any infected erythrocyte have been considered. Extracted DNA from each blood samples were used in PCR and semi nested- PCR in order to confirm the presence of the species.

Results

Microscopical observation on 154 blood smears determined 38 (24.67%) and 40 (26%) samples were infected by Babesia and Theileria respectively. The mixed infections occurred in four (2.6%) samples. The results of the PCR assays showed nine (5.85%), 81 (53%) and 18 (11.7%) were distinguished as Babesia, Theileria and mixed infection, respectively. Semi nested- PCR did not confirm the presence of B. motasi.

Conclusion

The causative organism of many cases of haemoprotozoal diseases, which recorded in previous studies, could be B. ovis or Theileria lestoquardi. The result confirmed that B. ovis was only species which causes babesiosis in the study areas. It seems that the biometrical polymorphisms could exist in B. ovis in Iran. This polymorphism could be a main problem in differentiation between B. ovis and B. motasi and it could be dissolved by specific PCR analysis.     

Hematologic and Clinical Aspects of Experimental Ovine Anaplasmosis Caused by Anaplasma ovis in Iran

Background

Anaplasma ovis infections can cause clinical symptoms in acute phase and lead to huge economic losses in flocks. The aim of the present study was to investigate the hematological and parasitological changes in experimental anaplasmosis in sheep with Iranian strain of A. ovis.

Method

Five male sheep without any blood parasite infection were selected. One hundred ml heparinized blood was collected from splenectomised sheep that showed 6% A. ovis parasitemia. Inoculums of 20 ml blood were administered intravenously to each test animal. Hematological, parasitological and clinical changes of experimental anaplasmosis were studied in 0-38 days post infection.

Result

Parasitemia was detected 3 days post infection and reached its maximum level on the day 12 of experiment in test animals. Then the parasitemia was declined, but the organism could be found persistently until the last day of study. The red cell counts, packed cell volume and hemoglobin concentration were decreased and mean corpuscular volume was increased significantly during the infection period. Reticulocytosis and basophilic stippling were also detected. No significant changes were observed in total and differential leukocyte count and animal body temperature.

Conclusion

Experimental A. ovis infection in sheep resulted in marked normocytic normochromic anemia at the beginning of the infection which became macrocytic normochromic by the development of the disease. There were negative correlations between parasitemia and RBC, PCV and Hb values, therefore hematological assessment can be considered as a practical diagnostic tool in ovine anaplasmosis.     

Identification of Tropomyosin and Its Immunological Properties from Larvae of Cattle Tick,Boophilus annulatus

Background

Boophilus annulatus is an obligate blood feeder tick that can cause great losses in animals due to anemia and its ability to injure its host skin directly. The aim of this study was identification of cattle humoral immune response to some tick proteins during experimental infestation.

Methods

Immune sera against tick were collected from experimentally infested cattle with ticks. One and two-dimensional electrophoresis and Western blotting methods were used for the detection of immunogenic proteins in larval tick extract and eight of these proteins were identified by MALDITOF and MALDI-TOF-TOF mass spectrometry.

Results

In non-reducing one-dimensional SDS-PAGE, some bounds between 12 to more than 250-kDa appeared. In two-dimensional SDS-PAGE, numerous spot appeared and the identified immunogenic proteins by parallel immunoblotting weighted between 14 and 97 kDa. Amino acid sequences of protein spot with 37-kDa molecular weight had identity to tropomyosin based on Mascot search in NCBI.

Conclusion

Anti tropomyosin antibodies can be induced in experimentally infested hosts with ticks and it seems that tropomyosin can be useful for the development of anti tick vaccines.     

Trichinella Infection in Wildlife of Northeast of Iran

Background

The objective of this investigation was to detect the presence of Trichinella in some carnivores of Mashhad in northeast of Iran and to identify Trichinella species circulating in this area.

Methods

The present study was carried out using muscle tissue collected from 120 stray dogs, 26 wild boars, 25 rodents, two foxes and two hyenas captured in Mashhad City, province of Khorasan Razavi, Iran.

Results

Trichinella larvae were detected in three stray dogs by artificial digestion and compression. All larvae were identified as T. britovi using multiplex PCR.

Conclusion

This is the first report of identification of T. britovi in stray dog in Iran.     

Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA,ITS2)

Background

In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.

Methods

The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species.

Results

The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.

Conclusion

The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates.     

Molecular Study of Sheep Malignant Theileriosis at Barka Region in the Sultanate of Oman

Background

We used the PCR technique based on the abovementioned primer pair and sequencing to demonstrate the Theileria infection in the sheep samples collected from Sultanate of Oman.

Methods

According to the frame work of “integrated control of ticks and tick borne diseases in globalized world managed by EU-ICTTD-3 project, the samples from blood, liver, spleen, lymph node and lung were sent to the laboratory of Iranian Research Center for Ticks and Tick-borne Diseases (IRCTTD). Samples from blood smear and impression smears from liver, spleen, lymph node, and lung were analyzed by Geimsa staining. The DNA was extracted from the abovementioned samples and analyzed by PCR technique using specific primers derived from the nucleotide sequences of 18S rRNA gene of T. lestoquardi, which can amplify the common region in other Theileria and Babesia spp. Subsequently the amplified DNA was sequenced.

Results

The analysis of blood smears of the sheep was negative for piroplasmosis performed through the Giemsa staining. The impression smears prepared from liver, spleen, lymph node, and lung showed suspicious structures mimicking Theileria schizonts in some cells. The results showed an expected PCR product of 428 bp in length, which is specific for Theileria spp. The PCR products were subsequently sequenced. The corresponding nucleotide sequence is registered under accession number {"type":"entrez-nucleotide","attrs":{"text":"JF309152","term_id":"323695911","term_text":"JF309152"}}JF309152 in GenBank. The sequence alignment in GenBank showed that the PCR products had 99% homology to the known T. lestoquardi registered under accession number {"type":"entrez-nucleotide","attrs":{"text":"AF081135","term_id":"7576455","term_text":"AF081135"}}AF081135 in the GenBank.

Conclusion

Oman sheep are highly susceptible for Theileria infection and the infected sheep mostly die before the microschizonts or erythrocytic form of Theileria appears in the nucleated or erytrocytic cells respectively.     

In-Vitro Assessment of the Acaricidal Properties of Artemisia annua and Zataria multiflora Essential Oils to Control Cattle Ticks

Background

The aim of this study was to investigate the ‘acaricidal effect’ of Zataria multiflora and Artemisia annua essential oils on Rhipicephalus (Boophilus) annulatus.

Methods

This study was carried out in 2009 in the Laboratory of Parasitology of the Faculty of Veterinary Medicine of Shahrekord University, west central Iran. Six dilutions (5, 10, 20, 40, 60 and 80 µL/cm³) of both essential oils were used against engorged female R. (Boophilus) annulatus ticks using an in vitro immersion method. The mortality rates for each treatment were recorded 6, 15 and 24 hours post inoculation (hpi). Mortality rate was analyzed using Repeated Measures Analysis of Variance, and comparison of means was carried out using General Linear Models Procedure.

Results

The mortality rate caused by different dilutions of Z. multiflora essential oil ranged from 26.6% (using 10 µL/cm³) to 100% (using 40 µL/cm³) and for A. annua essential oil it was 33.2 to 100% (using 20 and 80 µL/cm³, respectively) by the end of the experiment (36 hpi). No mortality was recorded for the non-treated control group or for dilutions less than 5 and 10 µL/cm³ using Zataria and Artemisia essential oils, respectively. For Z. multiflora mortality peaked at 15 hpi for all concentrations other than 20 µL/cm³ and took 24 h to achieve its maximum effect while for A. annua the two highest concentrations needed 24 hpi to reach their full effect. In addition, essential oils applied at more than 20 and 60 µL/cm³ caused 100% egg-laying failure in engorged female ticks by Zataria and Artemisia, respectively while no failure was observed for the non-treated control group. The mortality rate in both botanical acaricides was dose-dependent.

Conclusion

Both these medicinal plants have high potential acaricidal effects on the engorged stage of R. (Boophilus) annulatus in vitro.     

Seroprevalence of Toxoplasma gondii in Sheep,Cattle and Horses in Urmia North-West of Iran

Background

Toxoplasma gondii is a zoonotic protozoan parasite found worldwide and responsible for major economic losses in most classes of livestock. This study was aimed to determine the prevalence of T. gondii infection in sheep, cattle and horses in Urmia, north-west of Iran, using MAT.

Methods

Blood samples of 276 livestock and 26 horses were collected from July 2009 till April 2010. The data were analyzed by the Chi-square, Fisher''s Exact and Cochran''s and Mantel-Haenszel Tests. The level of significance was set at P<0.05.

Results

Thirty-three (21.1%) sheep, 2 (1.6%) cattle and 3 (11.5%) horses were seropositive to T. gondii. Analysis showed that sheep were 15 times more likely to be seropositive comparing to cattle also 2 times more likely to be seropositive than horses.

Conclusion

This study showed seroprevalence of equine T. gondii infection with a considerable rate in sheep in Urmia, northwest of Iran. More comprehensive studies on livestock toxoplasmosis are required for further analysis of the parasite reservoir for human infection.     

Prevalence and Molecular Identification of Cryptosporidium Spp. in Pre-Weaned Dairy Calves in Mashhad Area,Khorasan Razavi Province,Iran

Background

Cryptosporidium parvum is a zoonotic pathogen transmissible from a variety of animals to humans and is a considerable public health concern. Dairy cattle have been identified in numerous reports as a major source of environmental contamination with this pathogen. The aim of study was to detect and isolate the Cryptosporidium spp. from fecal samples of naturally infected pre-wean calves in the Mashhad area

Methods

Overall, 300 fecal specimens from 1 to 30 days pre-weaned calves were collected from 10 farms in the Mashhad area the capital center of the Khorasan Razavi Province, Iran and microscopically examined for Cryptosporidium spp. All infected samples were also analyzed using nested –PCR. A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the small-subunit (SSU) rRNA gene was also used to detect and identify Cryptosporidium spp. in PCR- positive samples.

Results

Eighty five (28.3%) of the specimens were positive for Cryptosporidium spp. The prevalence of Cryptosporidium spp. in 8-14 days old and diarrheic calves were significantly higher than other groups. Restriction digestion of the PCR products by SspI, VspI restriction enzymes and sequence analysis revealed the presence of C. parvum bovine genotype in all isolates.

Conclusions

Our results suggest that pre-weaned calves are likely to be an important reservoir of zoonotic C. parvum.     

Seroprevalence of Neospora spp. in Horses in North East of Iran

Background

Neospora caninum, an obligate intracellular protozoan parasite, is recognized as a major cause of abortion in cattle, while limited information is presently available on the seroprevalence of Neospora antibodies in horses’ worldwide. The aim of the present study was to determine serologic prevalence of Neospora infection in horses in Iran.

Methods

Sera from 150 horses from Mashhad suburb in Razavi Khorasan Province, northeast Iran were examined for antibodies to Neospora spp. using Neospora modified direct agglutination test (N-MAT).

Results

Antibodies to this parasite were detected in 45 (30%) of the examined serum samples. Thirty four percent of the samples had titer of 1:40 while then reduced to 30% when 1:80 serum dilution was applied as significant cut off titer.

Conclusion

This study is the first investigation carried out on the Neospora in horses in Iran and indicates that horses in Iran are exposed to this parasite.     

The Effects of Babesiosis on Oxidative Stress and DNA Damage in Anatolian Black Goats Naturally Infected with Babesia ovis

Background

A reactive oxygen and nitrogen intermediate produced during an inflammatory response is the important part of host-defense strategies of organisms to kill the parasite. However, it is not well known whether these intermediates cause DNA damage and oxidative stress in goats infected with Babesia ovis. The purpose of this study was to clarify the effects of babesiosis on basal levels of DNA damage and oxidative status of goats naturally infected with B.ovis.

Methods

DNA damage and antioxidant parameters were determined in B. ovis infected goats. Ten infected Anatolian Black Goats with B. ovis diagnosed via clinical signs and microscopic findings and ten healthy were used in the study.

Results

The Babesia infection increased the levels of DNA damage, malondialdehyde (MDA), protein carbonyl content (PCO) and plasma concentration of nitric oxide metabolites (NOx), and decreased total antioxidant activities (AOA) and reduced glutathione (GSH). A significant positive correlation between DNA damage, MDA, PCO, and NOx concentrations was found in the infected goats. DNA damage showed a negative association with AOA and GSH concentrations in the infected goats.

Conclusion

The Babesia infection increases oxidative stress markers and DNA damage and decreases AOA in goats. These results suggest that the increases in the production of free radicals due to Babesia infection not only contribute to host-defense strategies of organisms to kill the parasite but also induce oxidative damage in other cells.     

Morphological and Morphometrical Description of Trichostrongylus Species Isolated from Domestic Ruminants in Khuzestan Province,Southwest Iran

Backgrounds

Genus Trichostrongylus (Nematoda: Trichostrongylidae) is one of the most important zoonotic nematodes with wide geographic distribution in the world. The purpose of the present study was to describe morphological and morphometrical characteristics of male Trichostrongylus species, currently prevalent in domestic ruminants of Khuzestan Province, southwest Iran.

Methods

Gastro-intestinal organs of 1600 sheep, goats, cattle, and buffalos, slaughtered in Khuzestan Province, southwest Iran, were examined for infectivity with Trichostrongylus species. For examination and measurements of helminthes, Azo-carmine staining was performed, followed by camera lucida drawings of morphological characters and measurements of morphometrical criteria with a calibrated microscope. Using valid nematodes systematic keys, almost all the parasites were identified at the level of species.

Results

Overall, 114 animals were found infected with at least one species of Trichostrongylus. Considering morphological characteristics of male Trichostrongylus, six species were identified including T. colubriformis, T. vitrinus, T. probolorus, T. capricola, T. longispicularis and Trichostrongylus sp.

Conclusion

Although, compared to the previous decades, currently Trichostrongylus is much less prevalent in the domestic ruminants of the study area, but still different species occur in these animals.     

Active and Passive Surveillance and Phylogenetic Analysis of Borrelia burgdorferi Elucidate the Process of Lyme Disease Risk Emergence in Canada

Background

Northward expansion of the tick Ixodes scapularis is driving Lyme disease (LD) emergence in Canada. Information on mechanisms involved is needed to enhance surveillance and identify where LD risk is emerging.

Objectives

We used passive and active surveillance and phylogeographic analysis of Borrelia burgdorferi to investigate LD risk emergence in Quebec.

Methods

In active surveillance, we collected ticks from the environment and from captured rodents. B. burgdorferi transmission was detected by serological analysis of rodents and by polymerase chain reaction assays of ticks. Spatiotemporal trends in passive surveillance data assisted interpretation of active surveillance. Multilocus sequence typing (MLST) of B. burgdorferi in ticks identified likely source locations of B. burgdorferi.

Results

In active surveillance, we found I. scapularis at 55% of sites, and we were more likely to find them at sites with a warmer climate. B. burgdorferi was identified at 13 I. scapularis–positive sites, but infection prevalence in ticks and animal hosts was low. Low infection prevalence in ticks submitted in passive surveillance after 2004—from the tick-positive regions identified in active surveillance—coincided with an exponential increase in tick submissions during this time. MLST analysis suggested recent introduction of B. burgdorferi from the northeastern United States.

Conclusions

These data are consistent with I. scapularis ticks dispersed from the United States by migratory birds, founding populations where the climate is warmest, and then establishment of B. burgdorferi from the United States several years after I. scapularis have established. These observations provide vital information for public health to minimize the impact of LD in Canada.     

Superoxide Dismutase (SOD) Enzyme Activity Assay in Fasciola spp. Parasites and Liver Tissue Extract

Background

The purpose of this comparative study was to detect superoxide dismutase (SOD) activities in Fasciola hepatica, F. gigantica parasites, infected and healthy liver tissues in order to determine of species effects and liver infection on SODs activity level.

Methods

Fasciola spp. parasites and sheep liver tissues (healthy and infected liver tissues), 10 samples for each, were collected, homogenized and investigated for protein measurement, protein detection and SOD enzyme activity assay. Protein concentration was measured by Bradford method and SODs band protein was detected on SDS-PAGE. SODs activity was determined by iodonitrotetrazolium chloride, INT, and xanthine substrates. Independent samples t-test was conducted for analysis of SODs activities difference.

Results

Protein concentration means were detected for F. hepatica 1.3 mg/ ml, F. gigantica 2.9 mg/ml, healthy liver tissue 5.5 mg/ml and infected liver tissue 1.6 mg/ml (with similar weight sample mass). Specific enzyme activities in the samples were obtained 0.58, 0.57, 0.51, 1.43 U/mg for F. hepatica, F. gigantica, healthy liver and infected liver respectively. Gel electrophoresis of Fasciola spp. and sheep liver tissue extracts revealed a band protein with MW of 60 kDa. The statistical analysis revealed significant difference between SOD activities of Fasciola species and also between SOD activity of liver tissues (P<.05).

Conclusion

Fasciola species and liver infection are effective causes on SOD enzyme activity level.     

Evaluation of Benzimidazole Resistance in Haemonchus contortus Using Comparative PCR-RFLP Methods

Background

In order to deworm the ruminants especially of sheep in Iran, consumption of benzimidazoles has more than 2 decades history and today farmers are using imidazothiazoles, macrocyclic lactones and mostly benzimidazole compounds (BZs) to treat infected farm animals. It has been demonstrated that the most common molecular mechanism leading to BZsresistance in Haemonchus contortus is a single mutation at amino acid 200 (phenylalanine to tyrosine) of the isotype 1 of beta tubulin gene. According to the report of such mutations in Iranian Teladorsagia circumcincta isolates with Restriction Site Created PCR-RFLP, we decided to evaluate the frequency of such mutations in H. contortus in three different geographical areas of Iran.

Methods

A total of 102 collected adult male H. contortus were evaluated with PCR-RFLP (using PSP1406I as restriction enzyme). By means of a second step to compare function of different methods and to increase sensitivity of detection mechanism, a third of samples were examined by another PCR-RFLP method (using TaaI as restriction enzyme) and finally beta tubulin gene of two samples was sequenced.

Results

All of samples were detected as BZss homozygote. Finally, beta tubulin gene sequencing of two samples showed no point mutation at codon 200.

Conclusion

It seems that BZresistance of H. contortus in Iran is not a serious problem as anticipated before.