Molecular identification, risk factor assessment, and phylogenetic analysis of tick-borne pathogens in symptomatic and asymptomatic cattle from South-Eastern Iran.

Tick-borne pathogens (TBPs) represent a substantial threat to cattle globally, exerting adverse impacts on production, health, and economic viability. This study delves into the prevalence and implications of TTBPs in cattle sourced from resource-limited smallholder livestock farms situated in southeastern Iran, proximate to Afghanistan and Pakistan. Blood and tick specimens were systematically collected from a cohort of 230 cattle, comprising 150 asymptomatic and 80 symptomatic individuals. Genomic DNA isolated from blood samples underwent rigorous examination for the presence of key TBPs, including Anaplasma marginale, A. phagocytophilum, A. bovis, A. centrale, Babesia bigemina, and Theileria annulata, utilizing multiple genetic markers. Nucleotide sequence analysis facilitated the reconstruction of phylogenetic relationships. The study also evaluated various potential risk factors, such as clinical status, gender, age, breed, tick infestation, and management practices, to elucidate their associations with TTBPs. Among the cattle cohort, a staggering 87.8% (202/230) tested positive for at least one pathogen. Prevalence statistics encompassed A. marginale (72.2%), T. annulata (68.3%), A. phagocytophilum/A. platys-like complex (66.1%), A. centrale (16.7%), B. bigemina (10.0%), and A. bovis (6.1%). Remarkably, mixed infections involving two, three, and four pathogens were detected in 23%, 52.1%, and 2.2% of animals, respectively. Notably, all asymptomatic cattle were positive for at least one TBP. Tick infestation was observed in 62.2% (143/230) of cattle, predominantly caused by Hyalomma anatolicum (82.5%), Rhipicephalus (Boophilus) annulatus (13.1%), and R. sanguineus sensu lato (4.4%). Risk factors linked to TBPs encompassed tick infestation, older age, and crossbred animals. Clinical presentations among symptomatic cattle encompassed fever, anemia, weight loss, anorexia, jaundice, and enlarged superficial lymph nodes. This study underscores the pivotal role of asymptomatic carriers in the propagation of TTBPs within endemic regions. Furthermore, it emphasizes the potential for the implementation of molecular diagnostics to unmask subclinical infections, thereby affording the opportunity for targeted interventions aimed at ameliorating the burden of TTBPs in resource-constrained smallholder dairy farms.

Anaplasma ovis infection in sheep from Iran: molecular prevalence, associated risk factors, and spatial clustering.

The aim of this cross-sectional study was to determine the molecular prevalence and associated risk factors in sheep populations of Iran. To this end, between March 2017 and February 2018 jugular vein blood samples were collected from 1842 apparently healthy sheep from 327 herds in nine provinces in four ecological zones of Iran. A specific nested-PCR targeting the msp4 gene of A. ovis was employed. Fourteen variables were subjected to logistic regression analyses (univariate and multivariate) to specify the potential risk factors for infection. Statistically significant variables in univariate analyses (P ≤ 0.20) were assessed by multivariable logistic regression to control the confounding factors. Anaplasma ovis DNA was detected in 51.1% of herds (167/327) and 28.3% of animals (521/1842). Among geographical zones, herd and animal prevalence was highest in the Persian-Gulf zone (P < 0.001), and among provinces, Lorestan (in west) and Khuzestan (in south-west) had the highest prevalence (P < 0.001). Analysis of factors associated with A. ovis infection revealed that distance from other farms (OR = 2.52, P < 0.001), presence of other animal species in the farm (OR = 2.03, P = 0.046), season (OR = 1.40, P = 0.005), breed (OR = 3.762, P < 0.001), and age of sheep (OR = 1.20, P = 0.049) are potential risks in Iran. The spatial scan statistic in SaTScan recognized two high risks clusters for A. ovis infection in central (Semnan province) and the Persian-Gulf (Khuzestan province) zones amongst the study areas (P < 0.001). Sequence and phylogenetic analysis of the msp4 gene confirmed the detection of A. ovis. This research is the largest study focusing on ovine anaplasmosis in Iran and shows that infected sheep are present in all geographic zones, bioclimatic areas, and provinces.

Molecular survey on prevalence and risk factors of Anaplasma spp. infection in cattle and sheep in West of Iran.

Tick-borne pathogens (TBPs) in general and Anaplasma spp. in particular are known as economically important agents of diseases of domestic ruminants worldwide. Little information exists regarding the prevalence and the role of various factors affecting the occurrence of anaplasmosis in the west of Iran. The present study was carried out to determine the prevalence and risk factors associated with Anaplasma pathogens in Kurdistan province, west of Iran. During spring and summer 2016, blood samples were collected from a total of 401 livestock animals from different farms distributed in the Kurdistan province. Furthermore, data on general husbandry traits and management practices were recorded. PCR examination showed an overall prevalence of 37.3 and 10% in cattle and sheep, respectively. Cattle were infected with A. phagocytophilum (23.8%), A. bovis (15.4%), and A. marginale (13.9%) while sheep were infected with A. ovis (10%) and A. phagocytophilum (3.5%). The occurrence of anaplasmosis was considerably higher in cattle compared to sheep (P < 0.0001). Based on results of logistic regression models, risk of Anaplasma infection was significantly higher in crossbreeds (odds ratio (OR) = 1.93, 95% confidence interval (CI) = 1.025-3.65, P = 0.042) compared with local cattle breeds. Furthermore, being older than 1 year was associated with increased risk of Anaplasma infection in cattle (OR = 2.18, 95%, CI = 1.022-4.68, P = 0.044) and sheep (OR = 2.86, 95%, CI = 1.006-8.22, P = 0.049) compared to younger individuals. No difference was observed between the counties. Identifying potential risk factors and potential vectors and reservoirs contribute to the successful control and prevention of tick-borne diseases.

First report of Babesia occultans in two symptomatic cows in Iran.

This study reports the identification and first molecular characterization of Babesia occultans from naturally infected cows in Iran. Microscopic examination showed pyriform trophozoites, and ring-shaped merozoites (>2.5 µm) in Giemsa-stained blood smears obtained from two symptomatic cows in West-Azarbaijan province, Iran. PCR amplification of the partial 18S rRNA gene including the V4 hypervariable region were carried out on DNA extracted from blood samples. BLAST analyses of the partial 18S rRNA (approximately 400 bp) obtained from two cows revealed the presence of B. occultans and the detected sequences were identical to each other. Comparisons of the partial 18S rRNA sequence of the current isolate with other B. occultans sequences from Tunisia, South Africa, Turkey, Pakistan, and China confirmed the relation of the Iranian isolate to the species B. occultans. Sequence analysis of the obtained B. occultans showed 99.5-100% identity to the previously reported isolates. The sequences of B. occultans had 100% identity to a sequence obtained from ticks in Tunisia. This report is the beginning of a path to further research about B. occultans in vectors and reservoirs throughout Iran.

Epidemiological study on Anaplasma phagocytophilum in cattle: Molecular prevalence and risk factors assessment in different ecological zones in Iran.

Anaplasma phagocytophilum is a tick-borne pathogen affecting humans and domestic animals worldwide. This study aimed to determine the molecular epidemiology and its associated risk factors of A. phagocytophilum infection in cattle in four ecological zones of Iran. A multi-stage stratified random sampling method was utilized during 2017-2018. A total of 1851 blood samples from 320 cattle farms were collected and examined using specific nested polymerase chain reaction (nPCR) based on the 16S rRNA gene. The overall prevalence of A. phagocytophilum was 15.5% (286/1851) by using nPCR. All four zones were A. phagocytophilum positive, the presence of A. phagocytophilum DNA was detected in eight out of nine tested provinces. Univariable analysis of risk factors indicated that climate, altitude, longitude, latitude, season, farm-type, feeding method, hygiene of the farm, tick infestation, use of acaricides by the farmer, distance from other farms, contact with wild animals, race, sex, and milk yield were significant determinants (P < 0.05) for A. phagocytophilum infection. The multivariable analysis determined that longitude, latitude, season, feeding method, and hygiene of the farm remained as significant risk factors for A. phagocytophilum infection (P < 0.05). Specific (SaTScan) cluster analysis identified two high risks and four low risks statistically significant clusters for A. phagocytophilum infection amongst the study areas (P < 0.001). Phylogenetic analysis indicated that A. phagocytophilum 16S rRNA isolates were 96-99% identical to sequences deposited in the GenBank. To the best of our knowledge, this is the first comprehensive molecular study on the epidemiology and risk factors analysis of A. phagocytophilum infection in cattle in different climatic zones of Iran. Further investigations are necessary to be performed regarding the tick vectors, reservoir animals, and the zoonotic potential of the A. phagocytophilum in the endemic region of Iran.

Seroprevalence and risk factors assessment of the three main infectious agents associated with abortion in dairy cattle in Isfahan province, Iran.

This study aimed to determine the seroprevalence and identify the risk factors associated with Neospora caninum, Bovine herpesvirus type 1 (BHV-1), and Bovine viral diarrhea virus (BVDV) infection on industrial Holstein dairy cattle farms in Isfahan province, Central Iran. Blood samples were taken from 216 apparently healthy cattle from 16 randomly selected Holstein dairy farms in the North, South, East, and West of Isfahan in the summer of 2017. The antibodies to N. caninum, BHV-1, and BVDV were detected using a commercially available ELISA kit. The overall seroprevalence for N. caninum, BHV-1, and BVDV was 19%, 72.2%, and 52.8%, respectively. The significant major risk factors of BHV-1 in cattle were identified as farm direction, age groups, parity, and milk yield by the univariate analysis (p < 0.05). The significant major risk factors of BVDV in cattle were identified as age groups, parity, milk yield, and stage of pregnancy (p < 0.05). The only significant major risk factor of N. caninum was farm direction (p < 0.05). A significant association of concurrent infection with BVDV and BHV-1 has shown in the current study (p < 0.05). This study is the first to report the risk factors for N. caninum, BHV-1, and BVDV infection in the central part of Iran and allows us to conclude that these agents are widely distributed in this region.

First insight into Encephalitozoon cuniculi infection in laboratory and pet rabbits in Iran.

Encephalitozoon cuniculi infects a wide variety of domestic and wild mammalian species including humans. Although the infection status has been studied in laboratory and pet rabbits worldwide, there is shortage of information regarding the disease in Iran. In the present study, the occurrence of infection in brains of 117 asymptomatic rabbits from six breeding and experimental units with highest population of rabbit colonies in the country (n = 60) as well as pet rabbits of pet stores in two cities (n = 57) were examined by nested-PCR. Histological sections of brains and kidneys were also studied by light microscopy. PCR results revealed that 3.3% of laboratory rabbits (2/60) and 59.6% of pet rabbits (34/57) harboured E. cuniculi in their brains. Histopathology on the other hand showed spores of the parasite in kidney and brain of one and kidney of another pet rabbit. As encephalitozoonosis may interfere with results of experiments performed on laboratory rabbits, routine screenings for identification and culling of infected animals is recommended. Furthermore, infected companion rabbits can transmit E. cuniculi to people in close contact with them, therefore, improving public knowledge of this zoonotic infection is suggested.

A Rare Case of Non-cerebral Coenurus Cyst on the Heart of a Ewe

During the routine postmortem inspection from carcasses and offal of slaughtered ewes in an abattoir in Isfahan (Iran), an ovine heart was discovered with a firm nodule in the myocard on palpation. In closer examination, a liquid containing cyst (1x1 cm) was recognized on left part of the heart. The cyst had thick fibrotic capsule in outer surface and a thin inner layer containing few white clusters of scolices. White clusters contained scolices that adhered to the inner transparent layer, like white beads. Based on the gross and microscopical characteristics, the cyst was diagnosed as non-cerebral coenurus cyst. This is a rare report of heart coenurosis in a sheep.

Molecular study on infection rates of Anaplasma ovis and Anaplasma marginale in sheep and cattle in West-Azerbaijan province, Iran.

This study was carried out to determine the presence and frequency of Anaplasma ovis and Anaplasma marginale in sheep and dairy cattle in West-Azerbaijan province, Iran. A total number of 200 blood samples were randomly collected via the jugular vein from apparently healthy cattle (100) and sheep (100). The extracted DNA from blood cells was screened using genus-specific (Anaplasma spp.) nested-polymerase chain reaction (PCR) based on 16S rRNA gene primer sets. Species-specific PCR was set up using major surface protein 4 (MSP4) gene primer set. None of cattle blood samples were positive for Anaplasma spp. by the first nested PCR. Five samples among the 100 sheep blood samples were both positive in the first nested PCR and A. ovis -specific PCR, based on MSP4 gene. In total, 5.00% of animals were A. ovis positive. This study identified a low prevalence of A. ovis in the blood of apparently healthy sheep in West Azerbaijan province.

Comparison of molecular and microscopic technique for detection of Theileria spp. in carrier cattle.

In Iran, theileriosis is normally diagnosed with traditional Giemsa staining method. This is not applicable for identification of the carrier animals. The aim of this study was to compare conventional Giemsa staining method with the PCR technique in the detection of Theileria organisms. In this study, examinations were performed on 150 blood samples from cattle without clinical signs. Sensitivity and specificity of 50 microscopic fields were compared with Theileria specific PCR. The degree of agreement between PCR and microscopic test was determined by Kappa (κ) values with 95 % confidence intervals. PCR showed that 42 samples were Theileria spp. positive, while routine microscopy showed erythrocytes harboring Theileria like structures in 11 blood samples. Examination of 50 microscopic fields showed 57 % sensitivity and 99 % specificity compared to 100 % sensitivity and specificity for PCR. The κ coefficient between PCR and Microscopy (50 fields) techniques indicated no level of agreement. Our results showed that the microscopic examination remains the convenient technique for day-to-day diagnosis of clinical cases in the laboratory but for the detection of carrier animal containing low parasitemia. Therefore, molecular methods such as PCR can be used as a safe method for identifying cattle persistently infected with Theileria spp.

Echocardiographic assessment of cardiac structural and functional indices in broiler chickens treated with silver nanoparticles.

270 Ross broiler chickens of twenty days old were housed in 18-floor pens in a completely randomized design with six treatment groups and three replicate groups and fifteen chicks per each pen. The treatment groups (1-6) consisted of 0, 10, 20, 30, 50, and 70 ppm of nanocide in drinking water, respectively. At 26 days of age, 3 chickens were selected randomly for echocardiography using a 7.5 MHz linear probe, and the left ventricular internal diameter at the end of diastole (LVIDd), left ventricular internal diameter at the end of systole (LVIDs), left ventricular fractional shortening (LVFS), ejection fraction (EF), stroke volume (SV), interventricular septum thickness at the end of systole (IVSTs), and interventricular septum thickness at the end of diastole (IVSTd) were evaluated. LVIDd and LVIDs in group six were of higher rate than other groups and showed statistically significant differences with groups two, three, and four (P < 0.05). LVFS, percentage of EF, and IVSTd were minimum in group six and had significant difference with other groups (P < 0.05). The results of this study showed that prescription of high dosage of nanocide leads to cardiovascular problems with decrease in myocardial contractility and increase in the internal diameter of left ventricle.

Serological surveillance of bluetongue virus in cattle in central Iran.

The aim of this study was to evaluate the seroprevalence and distribution of antibodies to the bluetongue virus (BTV) among dairy Holstein cattle of central Iran. From September 2010 to August 2011, 892 blood samples from Holstein dairy cattle were collected from healthy animals. Blood samples were divided according to type of farm (industrial and non-industrial), season (warm and cold), location (North, South, East, and West), cattle production groups (calf, heifer, dairy and dry) and age groups (under 6 months, 6 months-2 years and over 2 years). The sera were screened using a commercially competitive enzyme-linked immunosorbent assay (c-ELISA) kit. Twenty-four sera (2.69 %) were found to be positive for BTV. Bluetongue virus seroprevalence was significantly higher (χ(2)=8.29, df=3, p < 0.05) in cattle in southern locations as compared to those in other locations. Older animals (>2 years) showed a relatively higher seroprevalence, but the difference was not statistically significant (p=0.06). No statistically significant difference in BTV seroprevalence was noted between farming systems, seasons and cattle production groups (p > 0.05). The results demonstrate that the seroprevalence of BTV is low in cattle from the Isfahan province, central Iran. Further studies are needed to determine the serotypes and vectors of BTV in the central region of Iran.

A molecular study on Theileria and Babesia in cattle from Isfahan province, Central Iran.

Bovine theileriosis and babesiosis are important hemoprotozoal diseases of cattle in tropical and subtropical regions that lead to economic losses in these animals. From March 2009 to July 2009, 176 blood samples of Holstein and crossbred cattle without any signs of disease were prepared from Isfahan province, Central Iran. The extracted DNA from blood cells were analyzed for members of the genera Theileria and Babesia by polymerase chain reaction (PCR) using a set of primers derived from the 18s rRNA gene. 42 out of 176 blood samples (23.9 %) were positive for Theileria spp. and none of them was positive for Babesia spp.. The present study showed that Theileria is detectable in cattle without any sign of infection but maintained a persistant sub-clinical state in the carrier cattle, which can serve as reservoirs of infection for ticks and cause natural transmission of the disease.

Seroprevalence of bovine leukemia virus (BLV) infection in dairy cattle in Isfahan Province, Iran.

Bovine leukemia virus (BLV), the causative agent of enzootic bovine leukosis (EBL) is an exogenous C-type oncovirus in the Retroviridae family. It causes significant economic losses associated with the costs of control and eradication programs due to carcass condemnation at slaughter and restrictions of export of cattle and semen to importing countries. The main objective of this research was to determine the seroprevalence of BLV infection in cattle herds in central region of Iran (Isfahan province) using a commercial enzyme-linked immunosorbent assay (ELISA) to detect serum antibodies against BLV. Samples of blood serum were collected from 403 female dairy cattle (Holstein-Friesian) from 21 livestock farms and 303 animals (81.9%) were BLV seropositive. A significant association was found between age as a potential risk factor and BVL seroprevalence with animals ≥ 4 years (86.6%) having a significantly (χ(2) = 35.6, p < 0.001) higher seroprevalence compared to those < 4 years (54.2%). We found no significant statistical association between seroprevalence and pregnancy, lactation status and farming systems as potential risk factors in this study (p > 0.1). It is concluded that BLV infection is a very common problem in the study area. Hence, control measures should be instituted to combat the disease and further studies are required to investigate the impact of this disease on dairy production in the country.

Seroepidemiological study of bovine respiratory viruses (BRSV, BoHV-1, PI-3V, BVDV, and BAV-3) in dairy cattle in central region of Iran (Esfahan province).

Respiratory diseases in calves are responsible for major economic losses in both beef and dairy production. Several viruses, such as bovine respiratory syncytial virus (BRSV), bovine herpes virus-1 (BoHV-1), bovine parainfluenza virus-3 (BPI-3V), bovine viral diarrhea virus (BVDV), and bovine adenoviruses (BAV), are detected in most clinical cases with respiratory signs. The aim of this study is to define seroprevalences of five major viral causes of bovine respiratory infections in cattle in central region of Iran (Esfahan province). The population targeted was 642 dairy cows (Holstein-Friesian) from 25 farms. Samples of blood serum from female cattle were examined. Sera were tested by commercial ELISA kits to detect antibody against BRSV, BoHV-1, BPI-3V, BVDV, and BAV-3. The results were analyzed by Chi-square test. In the present study, seroprevalences of BRSV, BoHV-1, PI3V, BVDV, and BAV-3 were 51.1%, 72%, 84.4%, 49.2%, and 55.6%, respectively. The present study shows that infections of bovine respiratory viruses are very common in cattle in Esfahan.

Copper, zinc, and iron concentrations in blood serum and diet of dairy cattle on semi-industrial farms in central Iran.

The aim of this study was to evaluate the blood serum and diet concentrations of copper, zinc, and iron in Holstein dairy cattle kept under semi-industrial farming in Isfahan province, central Iran. Moreover, the effects of season, pregnancy, and daily milk yield on serum Cu, Zn, and Fe concentrations were also evaluated. The study was carried out on 12 semi-industrial Holstein dairy cattle farms. A total of 120 blood serum samples (60 in each season) and 24 diet samples (12 in each season) were collected in the summer and winter. The Cu, Fe, and Zn contents were assessed in samples using atomic absorption spectrophotometer. In the summer, the mean concentrations of Cu and Fe in serum samples were lower and higher than the critical level, respectively (P < 0.05). In total diet samples, the mean concentrations of Zn were significantly higher than critical level (P < 0.05). In summer, winter, and total diet samples, the mean concentrations of Fe were significantly higher than critical level (P < 0.05). The serum Cu and Zn concentrations were significantly higher in the winter than those determined in the summer (P < 0.05). The serum Cu concentrations were significantly higher in nonpregnant than those in pregnant dairy cattle (P < 0.05). No significant difference was observed in serum Cu, Zn, and Fe concentrations of dairy cattle in different daily milk yield groups. It can be concluded that Holstein dairy cattle reared under semi-industrial dairy farming were deficient in serum Cu concentrations, especially in summer. Further, high level of Fe in blood serum might be due to feeding of cattle with diet containing excess quantity of Fe.

A new PCR-RFLP method for detection of Anaplasma marginale based on 16S rRNA.

Species of the genus Anaplasma (Rickettsiales: Anaplasmataceae) are obligate intracellular tick borne pathogens. Three species of Anaplasma that infect cattle and sheep (A. marginale, A. centrale and A. ovis) are well recognized. Of these erythrocytic Anaplasma, A. marginale can cause diseases in the livestock with high economical losses. Species-specific PCR based on 16S rRNA gene is commonly used for detection of Anaplasma species but can not differentiate A. marginale, A. centrale and A. ovis because of sequence similarity. In this study DNA extraction was performed on 50 blood samples with presence of Anaplasma spp. in marginal point of erythrocytes in their blood smears. The extracted DNA from blood cells was analyzed by PCR and PCR-RFLP using primers derived from 16S rRNA gene and restriction endonuclease Bst1107 I. The restriction endonuclease Bst1107I only recognizes the sequence (GTATAC) in corresponding PCR product of A. marginale and cut it. The nucleotide sequence of the A. marginale 16S rRNA gene was determined and compared with the sequences of A. marginale in GenBank. The 16S rRNA of A. marginale in Iran was completely similar to the related sequence deposited in GenBank at accession number of M60313. In the present study we propose a new PCR-restriction fragment length polymorphism analysis (RFLP) method based on 16S rRNA gene for specific detection of A. marginale.