Development and evaluation of an indirect ELISA for detection of Teladorsagia circumcincta infection in sheep.

BACKGROUND: The gastrointestinal helminth, Teladorsagia circumcincta, is one of the major health risks and production-limiting diseases in small ruminant populations, particularly in temperate regions. With the increasing importance of disease management and recruited anthelmintic resistant types, accurate approaches are needed for the diagnosis of the infection in the host. Due to uncertain results using faecal examinations, the ELISA method was indicated for the detection of nematode antigenic materials. Despite some promising results, problems were described in terms of test specificity and cross-reactions. Therefore, this study aimed to evaluate the IgG response to worm somatic and excretory/secretory (ES) products using western blot analysis and an indirect ELISA for the detection of T. circumcincta infection in sheep. RESULTS: Based on the immuno-reactivity analysis, immunogenic fractions with molecular weights (MWs) of approximately 60, 75 and 100 kDa were detected in somatic content and two antigens of about 63 and 75 kDa in ES material. Accordingly, a specific product at 75 kDa had the strongest reaction and appeared as the most common antigenic protein. In ELISA, all the sera from the infected sheep revealed the OD rates above the calculated cut-off value with about two-fold greater average. Negative control samples were also specifically recognized with the mean OD rate of about 1/3 of the estimated cut-off value. The cross-reaction test, using rabbit anti-T. circumcincta IgG, did not show reactivity with the ES antigens of other prevalent nematodes including Haemonchus contortus, Protostrongylus rufescens and Marshallagia marshalli. In contrast, a strong positive reaction was observed with the somatic antigens of M. marshalli. CONCLUSIONS: The results of this study indicated that the indirect ELISA method using the ES content enables distinguishing the T. circumcincta infected sheep with high specificity. Those antigenic ES peptides with 63 and particularly 75 kDa MWs should be further investigated due to the potential for serological diagnostic methods and immunoprotective targets in the host.

Molecular evidence on the emergence of benzimidazole resistance SNPs in field isolates of Marshallagia marshalli (Nematoda: Trichostrongylidae) in sheep.

The infection with members of the Trichostrongylid nematodes has been frequently reported from sheep and goats. Because of the widespread use of Benzimidazoles (BZs), the resistance suspected to occur in some worms populations. In this study, we focused on the prevalent nematode, Marshallagia marshalli, from the abomasa of sheep. Samples were obtained from at least 10 infected farms and diagnosed with morphological and molecular methods. For resistance analysis, genomic DNA from pooled adult samples of all farms were analysed for the beta tubulin gene to detect any polymorphisms at codon positions of F167Y, E198A and F200Y. According to the results, seven farms (70%) revealed resistance (R) allele at F200Y with relatively high frequency. No other mutations were identified at the other two positions. Also, except for one homozygous (RR) occasion, the isolates with R allele had heterozygous (RS) genotype. This finding indicates that the worm populations are still affected by drugs of the BZ class. However, the genetic data also notes on developing resistance mechanisms in M. marshalli populations in sheep.

Detection and characterization of the Isospora lunaris infection from different finch hosts in southern Iran.

This study was conducted to investigate the Isosporoid protozoan infections in finch types. Fecal samples were collected from marketed domestic Java sparrows (Lonchura oryzivora), colored and white Zebra finch (Taeniopygia guttata), and European goldfinch (Carduelis carduelis) in southern Iran. The coccidial oocysts were recovered and investigated according to the morphological features and the ribosomal gene markers. Additionally, a challenge infection was conducted with 5 × 104 and 5 × 103 sporulated oocysts in four java sparrows to estimate the clinical manifestations. Based on the morphology, the oocysts of Isospora lunaris were identified in all sampled bird types; however, the molecular method revealed the isolates had considerable similarities with some of Isospora and systemic Isospora-like organisms named as Atoxoplasma. Phylogenetic data also constructed an Atoxoplasma/Isospora clade with high sequence identities. High dose of the challenge with the parasite led to severe depression and sudden death, but it did not coincide with remarkable lesions and parasitic invasion in visceral organs. Contrary to molecular results, this feature is consistent with the common Isospora infections in passerines and differs from those described for Atoxoplasma species. Because of the prevalence, possibility of transmission, and clinical consequences, preventive measures are necessary to avoid outbreaks of isosporoid infections among finch type birds.

Molecular characterization of ß-tubulin gene associated with benzimidazole resistance in larvae of field isolates of Parascaris (Nematoda: Ascarididae).

Since the past 2 decades, an increasing number of resistance to the Benzimidazoles (BZs) have been reported in nematode parasites of livestock. More recently, detection of single-nucleotide polymorphisms (SNPs) at codons of 167, 198 or 200 of the ß-tubulin gene has been attributed to the occurrence of resistance. In the present study, we investigated the presence of those SNPs in the ß-tubulin isotype-1 gene in different isolates of Parascaris in horse. Also, the mitochondrial (mt) and ribosomal genes were sequenced for species confirmation of the isolates. The analysis of sequences inferred from COII gene confirmed that those isolates were P. equorum. The distance between mt genes obtained here and several ascarid species in equids and other hosts suggests the need for the combination of more genetic data with morphologic and other diagnostic measures. The analysis on ß-tubulin isotype-1 gene revealed no resistance-related SNPs or substitutions at the expected codon positions and selection pressure with BZs has not occurred for Parascaris worms. Although the molecular data showed the susceptibility of Parascaris isolates against BZs, other mechanisms of resistance should be also investigated to confirm the validity of molecular results.

Phylogenetic analysis of goat warble fly (Przhevalskiana silenus) based on mitochondrial COI gene.

The larvae of the genus Przhevalskiana (Diptera: Oestridae) are the causative agents of subcutaneous myiasis in goats. Several species have been grouped under this genus based on the morphology of different larval stages, albeit with a lot of uncertainties. Thus, application of genetic tools seems to be helpful for taxonomy. During this study, the cytochrome oxidase I (COI) gene was targeted for the characterization of larval stages of goat warble fly. Fragments of 606 bp were amplified for all the specimens. Based on the COI gene analysis, all the recovered specimens were identified as larvae of Przhevalskiana silenus. Molecular data on the genus is relatively rare but present isolates revealed about 87-89% identity with previous isolates of P. silenus. According to the phylogenetic data, the present isolates branched (as a sister group) with a number of Hypoderma spp. including H. bovis, H. diana, H. lineatum and H. sinense. The present findings confirmed that the COI gene could be a suitable marker for genetic characterization and identification of larvae up to the species level.

Tick-borne zoonoses in the Order Rickettsiales and Legionellales in Iran: A systematic review.

BACKGROUND: Tick-borne zoonoses in the Order Rickettsiales and Legionellales cause infections that often manifest as undifferentiated fevers that are not easy to distinguish from other causes of acute febrile illnesses clinically. This is partly attributed to difficulty in laboratory confirmation since convalescent sera, specific diagnostic reagents, and the required expertise may not be readily available. As a result, a number of tick-borne zoonoses are underappreciated resulting in unnecessary morbidity, mortality and huge economic loses. In Iran, a significant proportion of human infectious diseases are tick-borne, with anecdotal evidence suggesting that tick-borne zoonoses are widespread but underreported in the country. Epidemiological review is therefore necessary to aid in the effective control and prevention of tick-borne zonooses in Iran. The aim of this review is to provide an in-depth and comprehensive overview of anaplasmosis, ehrlichiosis, spotted fever group rickettsioses and coxiellosis in Iran. METHODS: Using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, all relevant publications on tick-borne zoonoses in the Order Rickettsiales and Legionellales in Iran were searched using a number of search terms. The search was confined to authentic resources from repositories of popular data bases among them PubMed, Web of Science, Google Scholar, Science Direct, SpringerLink and SCOPUS. The search items included peer reviewed journals, books and book chapters published between 1996 and 2017. RESULTS: A total of 1 205 scientific publications and reports were sourced, of which 63 met the search criteria and were reviewed. Of the 63 articles reviewed, 36 (57.1%) reported on coxiellosis, 15 (23.8%) on anaplasmosis, 11 (17.5%) on ehrlichiosis and 1(1.6%) on spotted fever group rickettsiae in a large scale study involving four countries, among them Iran. The existence of tick-borne pathogens in the Order Rickettsiales and Legionellales was confirmed by molecular, serological and microscopic techniques conducted on samples obtained from sheep, cattle, goats, camels, poultry, animal products (milk and eggs), dogs, ticks and even human subjects in different parts of the country; pointing to a countrywide distribution. DISCUSSION: Based on the review, coxiellosis, anaplasmosis, ehrlichiosis, and SFG rickettsiae can be categorized as emerging tick-borne zoonotic diseases in Iran given the presence of their causiative agents (C. burnetii, A. phagocytophilum, A. marginale, A. bovis, A. ovis, A. central, E. canis, E. ewingii, E. chaffeensis and R. conorii) collectively reported in a variety of domestic animals, animal products, arthropods and human beings drawn from 22 provinces in Iran. CONCLUSION: Given the asymptomatic nature of some of these zoonoses, there is a high likelihood of silent transmission to humans in many parts of the country, which should be considered a public health concern. Presently, information on the transmission intensity of tick-borne zoonoses caused by pathogens in the Order Rickettsiales and Legionellales to humans and its public health impact in Iran is scanty.

Histopathological changes in the upper digestive tract of pigeons infected with Hadjelia truncata.

Thirty-five pigeons from ten different farms in Fars area, southern Iran were submitted for post mortem inspection. Based on the clinical observations and gross pathological examinations, all the birds showed severe weight loss, diarrhea and to some extent ventricular enlargement. Furthermore, all the cases demonstrated large numbers of nematodes attached to the mucosa and submucosa of the ventriculus. Parasitological examinations revealed that the recovered parasites were Hadjelia truncata. The histopathological changes showed necrosis of the mucosal cells with moderate infiltration of lymphocytes, macrophages, heterophils and eosinophils in the lamina properia and muscularis mucosa in the infected animals. Based on the parasitological and pathological findings it can be concluded that the nematode H. truncate could be assigned as a pathogenic agent in the upper tract of pigeons.

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.

Molecular characterization of bot flies, Oestrus spp., (Diptera, Oestridae), from domestic and wild Bovidae hosts.

The identification of Oestrus spp. larvae from Bovidae hosts is a difficult task due to the great morphological similarity between species. The lack of unambiguous identification criteria could have also serious epidemiological implications since domestic and wild hosts are sympatric in many natural areas. In order to accurately identify the Oestrus parasitizing hosts, we characterized two different genetic markers, 28S (rRNA) and COI, in larvae collected from domestic sheep and goats, European mouflon and Iberian ibex. Our sequence analyses demonstrate that all samples, except those from Iberian ibex, greatly resembles O. ovis and so we conclude that the species parasitizing this ibex is not O. ovis. Further studies will be needed to confirm whether it is in fact O. caucasicus, as previously suggested, or even a new species.

A study on the status of inflammatory systems in camels naturally infected with Toxoplasma gondii.

Toxoplasma gondii is a unique intracellular parasite with a worldwide distribution. This parasite infects a variety of cells in a wide range of animal species such as dromedary camels (Camelus dromedarius). In order to evaluate the pattern of possible changes in the blood level of some inflammatory mediators and antioxidant enzymes in camels infected with T. gondii, blood samples were taken from a total of 493 dromedary camels and serum concentrations of inflammatory mediators, acute phase proteins and antioxidant enzymes were measured. According to serological data, no seropositivity was found for anti-T. gondii IgM in serum samples; however, 49 camels (9.93 %) showed positive titrations for anti-Toxoplasma IgG. The analyses of data in seropositive animals showed significant increases (P < 0.05) in the serum level of IL-1ß and adenosine deaminase activity; however, IFN-γ and TNF-α demonstrated no significant changes in serum samples of the infected camels. In addition, while major acute phase proteins (haptoglobin (Hp) and serum amyloid A (SAA)) were markedly elevated in infected camels, the activity of antioxidant enzymes (SOD and GPX) was remarkably decreased in the blood samples of infected animals. Thus, during the chronic infection in camels, T. gondii can promote significant rises in concentrations of some cytokines (such as IL-1ß), acute phase proteins and adenosine deaminase.

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.

In vitro viability test for the eggs of Echinococcus granulosus: a rapid method.

In this study an attempt was made to develop an efficient, rapid, simple, and reproducible method for the in vitro viability test of Echinococcus granulosus eggs. The eggs were obtained from an experimentally infected dog and kept at 4°C until use. To prepare the dead or damaged eggs, the eggs were heated in hot water (69-72°C for 10 min), preserved in 70% ethyl alcohol (16 days) or exposed to direct sunlight (18 h). Sodium hypochlorite (0.5-0.7%) was used for the hatching process, and the hatched oncospheres were stained with 0.1% eosin for the viability test. With 0.5% sodium hypochlorite, the hatching rates for viable eggs and eggs killed or damaged by heat (69°C), 70% ethyl alcohol, and direct sunlight were 96%, 97.5%, 91.5%, and 94.6% respectively and there was no significant difference between the hatching rate for viable and dead or damaged eggs (p > 0.05). After staining with 0.1% eosin, the rates of the viable oncospheres hatched from viable eggs and the eggs killed or damaged by heat (69°C), 70% ethyl alcohol, and direct sunlight were 97.5% 3.6%, 7%, and 10.5%, respectively. The difference between the rates of viable oncospheres hatched from viable and dead or damaged eggs was extremely significant (P < 0.0001). With 0.7% sodium hypochlorite, the hatching rates for viable and dead eggs (killed by 72°C for 10 min) were 99.1% and 99.9%, respectively. In this condition, the rate of viable oncospheres was an average of 98.5% for viable eggs and 0.0% for dead ones. The results of this study showed that hatching of eggs by 0.7% sodium hypochlorite and staining of hatched oncospheres by 0.1% eosin are practical methods for the differentiation of viable and nonviable (dead) eggs of Echinococcus granulosus.

The correlations among serum tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and sialic acids with peripheral lymphocytes in bovine tropical theileriosis.

The infection with protozoan parasite Theileria annulata induces changes triggering the activation and/or proliferation of the host lymphocytes. In order to find out the possible correlations among peripheral circulatory lymphocytes, cytokine activities and the level of sialic acids, 50 dairy Holstein cattle, naturally infected with T. annulata, were divided into 4 subgroups according to their parasitemia rates (<1%, 1-3%, 3-5% and >5%). Also, ten non-infected cattle were sampled as control group. Blood samples were taken from jugular vein into acid citrate dextrose-containing tubes for measuring hematological parameters and B and T (CD(4) and CD(8)) cell populations and without anticoagulant for TNF-alpha, IFN-gamma and sialic acid concentrations. Remarkable decreases observed in red blood cells (RBCs), white blood cells (WBCs) and packed cell volume (PCV) in infected cattle compared to healthy ones (P < 0.05). Also, with increase in parasitemia rate, total lymphocytes and monocytes alleviated in the diseased groups. By contrast, total neutrohpils and the concentrations of TNF-alpha, IFN-gamma and total sialic acids were significantly elevated (P < 0.05) in infected animals. Accordingly, the circulatory populations of CD(4) and CD(8) T cells and B cells showed a substantial decrease, while a significant increase was observed in T (CD(4) and CD(8)) cells in cattle infected with <1% parasitemia rates. Decreased circulatory T cell population shows the ineffective responses of T cells to the stimulatory cytokines such as IFN-gamma or TNF-alpha. On the other hand, the elevation of cytokines (particularly IFN-gamma) and sialic acids have presumably an inhibitory role on circulatory B cell population in infected cattle. In addition, a high level of sialic acid concentration indicates the probable role of sialic acid to regulate the parasite-host cell adhesion during sporozoites invasion.