Evaluation of effects of Tempol on testicular ischemia/reperfusion injury.

AIM: Tempol, a synthetic antioxidant compound, has received significant attention for its potential therapeutic applications in recent years, especially against ischemia/reperfusion (I/R) injury. The aim of the present research was to assess the protective effects of Tempol on testicular I/R injury caused by testicular torsion and detorsion (T/D) in rats. METHODS: The subjects were divided into five groups: sham, testicular T/D, testicular T/D with Tempol treatment at 50 and 100 mg/kg, and healthy rats treated with Tempol at 100 mg/kg. Testicular torsion was induced by rotating the left testicles for 2 h, followed by detorsion for 24 h. Testicular tissues were evaluated for gene expression, oxidative stress markers, and histopathology, epididymal sperms were stained and analyzed, and blood serum samples were collected to measure the testosterone hormone. RESULTS: The results showed that testicular I/R caused a significant decrease in sperm velocity parameters, viability, and count, as well as an increase in abnormal sperms (p < 0.05). However, treatment with Tempol significantly improved these parameters (p < 0.05). Histopathological analysis revealed severe damage to the testicular tissues, but treatment with Tempol improved the structural integrity of the seminiferous tubules. Testicular I/R also resulted in increased oxidative stress index and decreased testosterone levels significantly (p < 0.05), but Tempol administration mitigated these effects significantly (p < 0.05). Furthermore, the expression of Bax and Bcl2, genes associated with apoptosis, were significantly altered by testicular I/R (p < 0.05), but Tempol prevented these changes significantly (p < 0.05). CONCLUSION: These findings provide strong evidence that Tempol can effectively prevent testicular I/R injury.

Molecular and hematological investigation of Trypanosoma evansi infection in Iranian one-humped camels (Camelus dromedarius).

Trypanosoma species cause animal trypanosomiasis that infects many animals. Trypanosoma evansi is an organism that infects camels. There are many economic problems associated with this disease, including lower milk and meat yields and abortions. The purpose of the current survey was molecular study of the presence of Trypanosoma in dromedary camel blood in the south of Iran, and its effects on the hematologic, and some acute-phase protein changes. Blood samples were aseptically collected from the jugular vein of dromedary camels (n = 100; aged from 1 to 6 years) originating from Fars Province in EDTA-coated vacutainers. Genomic DNA from 100 µL of the whole blood was extracted and amplified using a PCR assay based on ITS1, 5.8S, and ITS2 ribosomal regions. Also, the PCR products obtained were sequenced. Moreover, the changes in hematological parameters and serum acute-phase proteins (serum amyloid A, alpha-1 acid glycoprotein, and haptoglobin) were measured. Among 100 tested blood, nine samples (9%, 95% CI: 4.2-16.4%) were found positive by the PCR assay. The phylogenetic tree and blast analysis showed four different genotypes closely related to the strains (accession numbers: JN896754 and JN896755) previously reported from dromedary camels in Yazd Province, center Iran. Based on hematological analysis, normocytic and normochromic anemia and lymphocytosis were detected in the PCR-positive cases compared with the negative group. Furthermore, alpha-1 acid glycoprotein was significantly increased in the positive cases. There was a substantial and positive relation between the number of lymphocytes, and the levels of alpha-1 acid glycoprotein and serum amyloid A in the blood (p = 0.045, r = 0.223 and p = 0.036, r = 0.234, respectively). A noticeable frequency of T. evansi infection was reported in dromedary camels in south Iran. This is the first report on the genetic diversity of T. evansi in this region. There was a significant association among Trypanosoma infection, lymphocytosis, and alpha-1 acid glycoprotein. Trypanosoma-positive camels had a significant decrease in hematocrit (HCT), hemoglobin (Hb), and red blood cell (RBC) values compared to the non-infected group. Further experimental studies are needed to elucidate the hematological and acute-phase protein alteration during a different phase of Trypanosoma spp. infection.

Class 1 integron causes vulnerability to formaldehyde in Escherichia coli.

In this study, the relationships of integron 1 element, formaldehyde dehydrogenase, and orfF genes with the level of formaldehyde resistance of isolated E. coli were investigated. E. coli bacteria were isolated from apparently healthy and colibacillosis-affected broilers of Fars Province, Iran. Formaldehyde resistance level and the presence of genetic markers were measured using MIC, and PCR tests, respectively. The prevalence of integron 1 element, orfF, and formaldehyde dehydrogenase genes in E. coli isolates were 61%, 8%, and 94%, respectively. In addition, according to our cut off definition, 15% and 85% of isolates were resistant and sensitive to formaldehyde, respectively. None of the genes had a statistically significant relationship with the formaldehyde resistance; however, the isolates containing integron 1 were significantly more sensitive to formaldehyde in the MIC test than those without integron 1. Integron 1 gene cassette could carry some bacterial surface proteins and porins with different roles in bacterial cells. Formaldehyde could also interfere with the protein functions by alkylating and cross-linking, and this compound would affect bacterial cell surface proteins in advance. Through an increase in the cell surface proteins, the presence of integron 1 gene cassette might make E. coli more sensitive to formaldehyde. As integron 1 was always involved in increasing bacterial resistance to antibiotics and disinfectants such as QACs, this is the first report of bacterial induction of sensitivity to a disinfectant through integron 1. Finally, integron 1 does not always add an advantage to E. coli bacteria, and it could be assumed as a cause of vulnerability to formaldehyde.

Effect of catecholamine stress hormones (dopamine and norepinephrine) on growth, swimming motility, biofilm formation and virulence factors of Yersinia ruckeri in vitro and an in vivo evaluation in rainbow trout.

In this study, we evaluated the impact of the catecholamines on growth, swimming motility, biofilm formation and some virulence factors activities of pathogenic Yersinia ruckeri. Norepinephrine and dopamine (at 100 µM) significantly increased the growth of Y. ruckeri in culture media containing serum. An increase in swimming motility of the pathogen was found following the exposure to the hormones; however, no effect was seen on caseinase, phospholipase and haemolysin productions. Further, antagonists for the catecholamine receptors were observed to block some of the influences of the catecholamines. Indeed, the effects of catecholamines were inhibited by chlorpromazine (the dopaminergic receptor antagonist) for dopamine, labetalol (α-and ß-adrenergic receptor antagonist) and phenoxybenzamine (the α-adrenergic receptor antagonist) for norepinephrine, but propranolol (the ß-adrenergic receptor antagonist) showed no effect. Pretreatment of Y. ruckeri with the catecholamines resulted in a significant enhancement of its virulence towards rainbow trout and the antagonists could neutralize the effect of the stress hormones in vivo. In summary, our results show that the catecholamines increase the virulence of Y. ruckeri which is pathogenic to trout through increasing the motility, biofilm formation and growth.

Effect of quorum quenching bacteria on growth, virulence factors and biofilm formation of Yersinia ruckeri in vitro and an in vivo evaluation of their probiotic effect in rainbow trout.

Five N-acyl homoserine lactone-degrading bacteria (quorum quenching (QQ) strains) were selected to evaluate their impacts on growth, virulence factors and biofilm formation in Yersinia ruckeri in vitro. No difference was observed among the growth pattern of Y. ruckeri in monoculture and coculture with the QQ strains. To investigate the regulation of virulence factors by quorum sensing in Y. ruckeri, cultures were supplemented with 3oxo-C8-HSL. The results indicated that swimming motility and biofilm formation are positively regulated by QS (p < 0.05), whereas caseinase, phospholipase and haemolysin productions are not influenced by 3oxo-C8-HSL (p > 0.05). The QQs were able to decrease swimming motility and biofilm formation in Y. ruckeri. QQ bacteria were supplemented to trout feed at 108  CFU/g (for 40 days). Their probiotic effect was verified by Y. ruckeri challenge either by immersion or injection in trout. All strains could significantly increase fish survival with Bacillus thuringiensis and Citrobacter gillenii showing the highest and lowest relative percentage survival (RPS) values (respectively, 85% and 38%). Besides, there was no difference between the RPS values by either immersion or injection challenge expect for B. thuringiensis. The putative involvement of the QQ capacity in the protection against Yersinia is discussed.

Evaluation of PCR and high-resolution melt curve analysis for differentiation of Salmonella isolates.

Consumption of poultry products contaminated with Salmonella is one of the major causes of foodborne diseases worldwide and therefore detection and differentiation of Salmonella spp. in poultry is important. In this study, oligonucleotide primers were designed from hemD gene and a PCR followed by high-resolution melt (HRM) curve analysis was developed for rapid differentiation of Salmonella isolates. Amplicons of 228 bp were generated from 16 different Salmonella reference strains and from 65 clinical field isolates mainly from poultry farms. HRM curve analysis of the amplicons differentiated Salmonella isolates and analysis of the nucleotide sequence of the amplicons from selected isolates revealed that each melting curve profile was related to a unique DNA sequence. The relationship between reference strains and tested specimens was also evaluated using a mathematical model without visual interpretation of HRM curves. In addition, the potential of the PCR-HRM curve analysis was evaluated for genotyping of additional Salmonella isolates from different avian species. The findings indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of Salmonella isolates to determine the serovar/serotype.

Morphological and molecular characterisation of Haemogregarina sp. (Apicomplexa: Adeleina: Haemogregarinidae) from the blood of the Caspian freshwater turtle Mauremys caspica (Gmelin) (Geoemydidae) in Iran.

To date, a number of species of Haemogregarina have been described from different turtle hosts, mainly based on the morphology of the developmental stages detected in the host erythrocytes. The diversity and overlapping morphological features in the old and recent descriptions has led to considerable complications in the taxonomy of Haemogregarina spp. In this study, different stages of maturity and developing gamonts of a putative new species of Haemogregarina were detected in erythrocytes of the Caspian turtle Mauremys caspica (Gmelin) (Geoemydidae) originating from a southern province in Iran. Although some of the morphological characteristics were consistent with Haemogregarina stepanowi Danilewsky, 1885, some new observations were made, particularly in the gamont stage. The phylogenetic analysis based on 18S rDNA sequences revealed that the present isolate appears as basal to a large clade of Haemogregarina spp. with sequences available in the GenBank database. In accordance with the phylogenetic results, the present Iranian isolate showed a higher degree of interspecific divergence (up to 3.3%) compared to the data for the taxa available in the GenBank database. Thus, molecular data indicate that this isolate may represent a new species. However, further genetic analyses are needed as a complementary tool to the morphological characterisation in order to elucidate the phylogenetic relationships of Haemogregarina spp.

Application of polymerase chain reaction on cerebrospinal fluid for diagnosis of cerebral coenurosis in small ruminants.

Sheep and goats serve as intermediate hosts for the canine tapeworm Taenia multiceps. The cysts produced by the intermediate stage of parasite are usually found in the cerebral hemispheres of small ruminants, and the resulting disease is commonly known as coenurosis. Coenurosis is clinically manifested in the form of various nervous symptoms, depending on the exact location of the cyst. The variety of neurological symptoms contributes to the complexity of clinical diagnosis and reinforces the need for a more specific and acceptable diagnostic approach. We demonstrated here, for the first time, that the T. multiceps DNA is present in the cerebrospinal fluid (CSF) of the infected sheep and goats. In addition, the molecular genetic marker of the mitochondrial DNA was applied phylogenetically to show that our isolates together with other T. multiceps strains comprised a monophyletic group that is a sister to Taenia krabbei. Pairwise comparison between the cox1 sequences of our study and other T. multiceps genotypes existing in the GenBank showed similarity ranging from 98 to 100%. Accordingly, the polymerase chain reaction (PCR) can be used for amplification of DNA of the parasite originated from the CSF and provides a valuable method for accurate identification of coenurosis cases.

Molecular and morphological comparison of two different types of Habronema muscae (Nematoda: Habronematidae) in horse.

Habronema muscae is a spirurid nematode that undergoes developmental stages in the stomach of equids, causing chronic catarrhal gastritis. Despite preceding investigations have developed polymerase chain reaction (PCR)-based assays for molecular diagnosis, we aimed to assess the applicability of cytochrome c oxidase subunit 1 (cox1) sequences to identify the H. muscae infection and to assess the level of intraspecific variations in this parasite obtained from affected horses in Southern Iran. According to the morphological characterizations, two different isolates of H. muscae were identified. Although the majority of the recovered specimens had normal characterizations of H. muscae, a number of parasites showed an abnormal feature as large, asymmetrical, and thick cuticular extensions was observed at their anterior end (head region) in gross and histologic examinations. Unexpectedly, molecular assay disclosed that both morphologically distinct samples were completely identical to each other based on cox1 sequence. Multiple alignment of the cox1 amino acid sequences showed that all polymorphism sites were silent. Also, phylogenetic analysis provided strong support that H. muscae form a sister group to Spirocerca lupi and Thelazia callipaeda.

The first molecular isolation of Halicephalobus gingivalis from horses in Iran.

BACKGROUND: Parasitic infections pose significant threats to humans' and animals' well-being worldwide. Among these parasites, Halicephalobus spp., a genus of nematodes, has gained attention due to its ability to cause severe infections in various animal species, including horses. OBJECTIVE: This study aimed to determine the prevalence of Halicephalobus spp., specifically focusing on Halicephalobus gingivalis in horses. MATERIALS AND METHODS: In July 2022, a cross-sectional study was conducted in northern Iran to determine the prevalence of Halicephalobus spp. Using standard coprological techniques, 141 fecal samples from randomly selected horses were analyzed for GI helminth eggs. The Halicephalobus spp. eggs present in faeces were identified by molecular methods. Polymerase Chain Reaction (PCR) was used to amplify the partial 5' variable region (~ 390 base pairs) of 18 S DNA using SSUA_F and SSU22_R primers. Furthermore, the PCR products obtained were sequenced, and phylogenetic analysis was performed using available sequences from GenBank. RESULTS: Microscopic examination of 141 fresh faecal samples revealed 5 fecal samples were infected with small ellipsoidal nematode eggs ranging between 40 and 50 × 50-60 µm. This study's PCR amplicons showed ~ 390 bp bands on 2.0% agarose gel. A partial sequence of 18 S DNA (363 bp) was obtained herein (GenBank accession no. OQ843456). CONCLUSION: Overall, using molecular tools represents a significant step forward in diagnosing and managing the Halicephalobus gingivalis infections in horses.

Clinicopathological findings of a natural outbreak of Theileria annulata in cattle: an emerging disease in southern Iran.

Theileriosis is an economically important hemoprotozoal disease with high morbidity and mortality in cattle. The present study reported the pathological features of a natural outbreak of tropical bovine theileriosis due to Theileria annulata in Fars Province, southern Iran. T. annulata was confirmed by the presence of T. annulata piroplasms in the blood smears and also by polymerase chain reaction test. On necropsy, pale mucous membranes and petechial and ecchymotic hemorrhages in the mucosal and serosal surfaces together with lymphadenopathy were observed. The liver was friable, yellowish, and larger than normal. Hemorrhages and punched-out ulcers were observed in the abomasal mucous membrane. Severe petechial hemorrhages were seen in the skin particularly in the hairless areas. Pulmonary edema and emphysema with petechial and ecchymotic hemorrhagic foci in the lungs were evident. The main histological changes were proliferation of lymphocytes in the lymph nodes and proliferation of macrophages, lymphocytes, and plasma cells in the spleen, Peyer's patches, portal tracts of the liver, and interstitial tissue of the kidneys. The mucous membrane of the abomasum showed numerous multifocal areas of necrosis and ulceration, and the submucosal area and lamina propria adjacent to these lesions showed hyperemia and hemorrhages, with mononuclear cell infiltration. The skin showed multifocal necrotic changes, petechial and ecchymotic hemorrhages, and chronic dermatitis. The schizonts of Theileria were evident in the cytoplasm of the lymphocytes and macrophages of the lymph nodes, spleen, and skin. Molecular examination revealed that these animals were infected with T. annulata. The present study describes the clinicopathological findings of bovine tropical theileriosis in an unpredictable weather condition.

Genetic characterization and phylogenetic analysis of Trypanosoma evansi in Iranian dromedary camels.

Whole blood samples were collected from 117 male clinically healthy Camelus dromedarius aged between 6 months to 18 years from several farms in Yazd Province of Iran. Trypanosoma evansi-affected camels were detected by Giemsa-stained blood smears, and the positive blood samples (4 out of 117) were submitted to PCR examination and phylogenetic analysis. Basic Local Alignment Search Tool data of the obtained complete internal transcribed spacer (ITS) sequences revealed that they corresponded to those of T. evansi, Thailand cattle isolate (AY912276) with the homology of 99 %. Both phylogenetic trees generated by ITS1 and complete ITS were unable to clearly show inter- and intraspecific genetic diversity of Trypanosoma spp. isolates. The phylogenetic tree inferred from the ITS2 nucleotide sequences (569 bp) clearly showed the genetic diversity of the parasites. Phylogenetic and molecular analyses of this region showed that two distinct genotypes of T. evansi in Iranian dromedary camels are present. In contrast to the ITS1 and ITS2 regions, multiple alignment of the nucleotide sequence of the 5.8S rRNA showed a high degree of sequence conservation during evolution in various Trypanosoma spp.

Detection of Hepatozoon spp. in dogs in Shiraz, southern Iran and its effects on the hematological alterations.

Canine hepatozoonosis is a tick-transmitted apicomplexan infection caused by two species of Hepatozoon, H. canis, and H. americanum. The present research aimed at detection of Hepatozoon spp. in dogs and its effects on hematological alterations. Blood samples were taken from 108 dogs to assess Hepatozoon spp. Phylogenetic analysis was performed based on the 18S rDNA marker by PCR assay and Giemsa-stained blood smear examination. Of the 108 blood samples of dogs tested in the present study, eight (7.40%, 95% CI: 3.25-14.07%) were positive by the Hepatozoon-specific PCR assay. However, in the microscopic examination, only one sample (0.93%) was positive. All of the sequenced samples were H. canis. The Hepatozoon sequences obtained from PCR amplicons in the canine-positive cases exhibited 100% similarity to each other and 98.47-100% similarity to other relevant sequences in GenBank. These findings represent the first molecular evidence of H. canis in dog populations in South Iran. Furthermore, according to the hematological analysis, significantly higher average numbers of neutrophils and lymphocytes were found in the infected group compared to the non-infected dogs. In this study, no statistically significant connection (P<0.05) was observed between H. canis infection and the examined risk factors.

Polymorphism in neutrophil cytosolic factor 4 (NCF4) of dairy cows had mastitis in previous lactations, and the relationship with the respiratory burst.

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase), as a key factor in innate immunity, consists of several components, one of them is p40phox which is encoded by neutrophil cytosolic factor 4 (NCF4). Respiratory burst and reactive oxygen species (ROS) production are antimicrobial mechanisms associated with NADPH oxidase. This study evaluated the effects of g.18174 A > G and g.18270C > T single-nucleotide polymorphisms (SNP) in NCF4 on bovine mastitis and the respiratory burst capacity of neutrophils. SNPs of 160 dairy cattle were determined using a novel PCR-RFLP protocol by employing restriction enzymes, MboI and FokI. Also, the flow cytometry measured respiratory burst in 82 blood samples. Our results indicated that only g.18174 A > G SNP reduced the respiratory burst capacity. However, both SNPs were not significantly correlated with clinical mastitis. We concluded that g.18174 A > G decreases the function of NADPH oxidase. However, both SNPs were not significantly correlated with clinical mastitis.

Characterization of Fasciola hepatica genotypes from cattle and sheep in Iran using cytochrome C oxidase gene (CO1).

The present study compared the genetic variation among 19 different isolates of Fasciola hepatica from cattle and sheep in different areas of Iran using sequence data for mitochondrial DNA gene, the subunit 1 of cytochrome C oxidase gene (CO1). Four different CO1 genotypes were detected among F. hepatica isolates that showed five variable nucleotide positions (accession nos.; GQ398051, GQ398052, GQ398053, GQ398054). Nucleotide sequence variation among 19 isolates for CO1 analyzed in this study ranged from 0% to 0.98% in Iran. Among the five polymorphism sites identified in this study, only one (T to G at position 51 in 5'end of GQ175362) resulted in putative amino acid alteration of phenylalanine (TTT) to leucine (TTG) in CO1. A phylogenetic analysis of the sequence data revealed that host associations and geographic location are likely not useful markers for Fasciola genotype classification. In addition, morphological analysis showed that the ratios of body length and body width of some (n = 5) of the 19 examined F. hepatica isolates were intermediate between F. hepatica and Fasciola gigantica, representing the substantial polymorphism of the F. hepatica species and the difficulty in the accurate recognition based on morphological features. In conclusion, Iranian F. hepatica exhibited the presence of considerable genetic diversity at CO1.

First report of ocular histoplasmosis in a horse from Iran: molecular, clinical and pathological findings.

Histoplasma capsulatum is a dimorphic fungus that is traditionally classified in three varieties: Hc var. capsulatum, Hc var. duboisii, and Hc var. farciminosum (HCF). Cytology, hematology, pathology, polymerase chain reaction (PCR), sequencing, and phylogenetic analyses were applied on samples collected from the blood and the eye of a horse with pustular lesions and ocular discharge. Physical examination and cytopathological tests showed H. capsulatum infection. Additionally, the results of two PCR tests confirmed H. capsulatum infection. The phylogenetic tree of the internal transcribed spacer sequence of Iranian H. capsulatum showed homology with the HCF variety. For the first time, H. capsulatum infection in the eye of a horse from Iran was detected and phylogenetically analyzed. This study revealed that H. capsulatum could establish infection in Iranian animals in addition to people, and indicated the role of soil enriched with bird dropping in the preparation of a favorable environment for H. capsulatum propagation. Further investigations are required to clarify the natural history and risk factors associated with histoplasmosis in Iran.

Theileria equi in the horses of Iran: Molecular detection, genetic diversity, and hematological findings.

In all equids worldwide, Theileria equi and Babesia caballi are believed to be two important erythrocytic protozoa that cause equine piroplasmosis. In addition, it was recently discovered that Theileria haneyi is another potential equine piroplasmosis (EP) agent. Ixodid ticks are the major vectors of these parasites. Equine piroplasmosis is of international importance and affects enormously the equine industry. In this study, for the first time, molecular prevalence and genetic diversity of piroplasma parasites (T. equi and B. caballi) in horses from Fars province (south of Iran) were determined. Also, hematological alterations of naturally infected horses were analyzed. PCR positive horses showed anemia, thrombocytopenia, leukocytosis with a left shift of neutrophilia, and monocytosis. PCR results revealed that, from 133 blood samples of horses, 40 samples were positive (30.07%). The occurrence of T. equi in this area (30.07%) was more than the national average prevalence of T. equi (24.11%), but B. caballi prevalence in study area (0%) was less than the average of previous studies in Iran (5.47%). Our findings revealed that the T. equi was widespread in Fars province of Iran. PCR products of 18S rDNA and EMA-1 genes of T. equi strains were sequenced successfully. All 18S rDNA sequences collected in this experiment revealed 100% similarity together. According to the phylogenetic tree constructed using the 18S rDNA gene, Iranian T. equi is clustered with strains from Cuba (KY111762, KY111761) and USA (CP001669, JX177672). So, this could be concluded that T. equi studied in this research, and those strains are initiated from a common T. equi ancestor at an unknown time ago. Also, the phylogenetic tree based on EMA-1 gene demonstrated a genetically diverse population of Iranian T. equi strains (10 different genotypes). As EMA-1 is one of the most immunogenic antigens in this parasite, such variability could be a concern about the efficacy of T. equi vaccines. Finally, more studies on equine piroplasmosis in the provinces of the southern region of Iran are recommended to create a better vision of disease in this region.

Intrauterine infusion of blood serum of dromedary camel improves the uterine health and fertility in high producing dairy cows with subclinical endometritis.

The blood serum of dromedary camels contains a unique type of antibodies with a high potency to neutralize toxins and to identify and inactivate some bacterial pathogens. The present study was designed to examine changes in the endometrial histology of cows with no subclinical endometritis (SE) (experiment 1) and changes in the uterine cytology and endometrial mRNA expression of COX2, IL-1ß, IL-8, and iNOS following intrauterine administration of DCBS in cows with SE as compared to different common treatments (experiment 2). In addition, the effects of the intrauterine administration of DCBS were examined on the pregnancy rate in dairy cows with SE (experiment 3). DCBS did not induce any histological reactions in the bovine endometrium. The mean ( ± SE) percentage of PMNs after intrauterine infusion of Pen-Strep, DCBS and double DCBS in cows with SE differed as compared to cows treated with PGF2α and no treated cows with SE (1.47 ± 0.87; 1.43 ± 1.08 and 1.31 ± 0.23 vs 3.00 ± 0.43 and 3.5 ± 0.75, P < 0.05, respectively) in experiment 2. The mRNA expression of COX2, IL-1ß, and iNOS was reduced (P < 0.05) after treatment with Pen-Strep, DCBS and double DCBS as compared with no treated-cows with SE. The pregnancy rate after the first AI was tended to be higher (49.2 vs 39.0%), while the overall pregnancy rate was greater (P < 0.05) in cows with SE when treated with DCBS as compared to the Pen-Strep group (76.9 vs 61.0%) in experiment 3. In conclusion, serum of dromedary camel, as a non-antibiotic preparation, can improve the uterine health and fertility when used for the treatment of bovine SE.

Genetic characterization of the causative agent of besnoitiosis in goats in Iran on the basis of internal transcribed spacer rDNA and its comparison with Besnoitia species of other hosts.

This study was conducted to identify genetic characteristics of Besnoitia spp. isolated from goat in Iran. Molecular analysis of two portions of nuclear ribosomal DNA (ITS1 and ITS2) was used for the genetic characterization of the Besnoitia species. Comparison of the sequencing data of the Iranian Besnoitia samples obtained in the present study (GenBank accession number HM008988) with those previously reported for other Besnoitia spp. in the GenBank database revealed a particularly close relationship between the present goat Besnoitia samples and the Besnoitia samples from the cattle, caribou, and equids (Besnoitia besnoiti, Besnoitia tarandi, and Besnoitia bennetti). This is the first use of a genetic approach to interrogate the identity of the species of Besnoitia infecting Iranian goats. Also, the results of the present study showed the occurrence of a similar sequence polymorphism for ITS1 and ITS2 in all Iranian isolates, which exhibit 100% identity in these ribosomal sequences, to those of B. besnoiti previously reported from Israel. Although the ITS1 sequence of Iranian goat isolates is identical to European cow isolates, the ITS2 sequences derived from present Besnoitia genotype differed in one nucleotide position compared with other European B. besnoiti. Further studies should be employed based on this molecular data to identify the natural definitive host in order to complete the life cycle of this distinct genotype of parasite.

Characterization of Sarcocystis fusiformis based on sequencing and PCR-RFLP in water buffalo (Bubalus bubalis) in Iran.

Four Sarcocystis species, i.e., Sarcocystis fusiformis and Sarcocystis buffalonis with cats as definitive hosts, Sarcocystis levinei with dogs as definitive host, and Sarcocystis dubeyi with unknown definitive host, have previously been described from water buffalo (Bubalus bubalis). The aim of the present study was genetic characterization of the causative agent(s) of water buffalo sarcocystosis in Khuzestan Province, western Iran. RFLP-PCR and partial sequence analysis of 18S rDNA gene were used for the genetic characterization of the specimens directly obtained from water buffalo. In RFLP-PCR, four restriction enzymes (Dra1, Ssp1, Fok1 and Bsl1) were used for species discrimination of Sarcocystis spp. in this host. Comparison of the molecular sequencing results and RFLP-PCR pattern of the samples obtained in the present study with those previously reported for different Sarcocystis spp. revealed that all positive Sarcocystis samples represented S. fusiformis. To our knowledge, this is the first demonstration of the existence of S. fusiformis in the Iranian water buffalo population by a genetic approach. In addition, comparison between the alignments between the Iranian 18S rDNA sequences (HQ703791), made in this study, and those previously reported for S. fusiformis in different geographical location (accession nos. AF176927, AF176926, and U03071) showed the occurrence of local genetic polymorphisms and heterogeneity in this ribosomal locus. Despite the occurrence of some genetic variations in the hypervariable regions of the 18S rDNA in S. fusiformis, Dra I restriction site was conserved among all sequences available. According to the present study, it seems that cats have a more significant epidemiological role than dogs in transmission of sarcocystosis agent to water buffalo in Iran.