Assessment of Neospora caninum infection in bulls using serological and molecular techniques.

Neospora caninum is a significant cause of abortion and economic losses in cattle worldwide. The main aim of the present work was to detect the prevalence of N. caninum infection in bulls in Hamedan (Iran) using serology and molecular techniques. All blood samples (n = 792) were screened for detecting the antibodies to N. caninum using enzyme-linked immunosorbent assay (ELISA). Then seropositive animals were rechecked using the immunofluorescent antibody test (IFAT). Also, blood, epididymis, and spinal cord samples were collected from animals for molecular analysis using nested PCR. In serology, using ELISA, 3.91% of animals were seropositive for N. caninum. Additionally, true prevalence based on the sensitivity and specificity of the ELISA was calculated 1.25% (95% CI: 0.48-2.02%). Neospora-infection in animals, calculated as the number of bulls seropositive and/or one sample positive to nested PCR, was 3.40%; and 19 bulls tested positive by both serology and molecular diagnostic methods. The overlaps between ELISA and molecular results were observed in 74.19% of whole blood samples, 80.64% of the epididymis, and 87.09% of the spinal cord. Using ELISA, the seroprevalence of N. caninum was detected 1.8% in ≤2 and 5.45% in >2 years old group of animals (p = 0.009, PR = 3.1). In addition, the seropositivity in Holstein and native breed animals was calculated 6.57% and 2.93%, respectively (p = 0.019, PR = 2.3). Seven sequences with 94.9-99.3% similarity were detected in multiple alignments of positive PCR products. Our work was the first comprehensive evaluation of Neospora-infection/neosporosis in Iranian bulls. We detected a low prevalence of infection in animals compared to previous reports. The ELISA is a sensitive serological technique for detecting the highest number of positive bulls in the present investigation and, the nested PCR is a reliable technique to identify Neospora-DNA.

Control measures to reduce Neospora caninum abortions in dairy farms: a pilot study in Iran.

Neospora caninum plays an important role in abortion and economic losses in dairy industry worldwide. The main target of this work was to detect the infection rate of N. caninum in various hosts in dairy farms for identifying the risk factors and applying appropriate control programs to reduce the number of abortions. The study was conducted in dairy farms with high incidence of abortion in Hamedan province, West of Iran. After the primary assessment, we conducted a controlling program for reducing the Neospora­infection rate and associated abortions. Before implementing the control program, the seropositivity was 24.8% in cows (N=476 distributed in 10 farms) and 8.6% (N=185) in dogs. Abortion occurred in 3.57% of pregnant cows. 94.1% of aborted cows were positive for Neospora­infection. Based on molecular technique, the infection rate was detected in 7.3% of cats (N=41), in 25% of pigeons (N=19)and in 11.8% of rodents (N=51). After the implementation of neosporosis control programs in the farms, the seropositivity of N. caninum decreased to 8.2% in cows and 2.9% in dogs. After the one­year follow­up, no cases of abortions were reported in the farms. This was the first parallel evaluation of Neospora­infection and controlling programs in Iranian dairy farms. Regular control of rodents, retesting of seronegative animals and farm biosecurity measures are recommended for reducing the abortion incidence. The access of dogs to the herd and to aborted materials should be restricted.

Brucellosis in livestock: First study on seroepidemiology, risk factors, and preventive strategies to manage the disease in Famenin, Iran.

Background and Aim: Brucellosis is an infectious disease in humans and livestock. The disease is endemic in many regions of Iran, for example, Hamedan Province. Knowledge of infection rate and associated risk factors is essential to control and prevent the disease. The study aimed to estimate the prevalence of brucellosis and associated risk factors in cattle, sheep, and goats in Famenin, Hamedan Province, West of Iran. Materials and Methods: Blood samples of 1758 animals (1470 sheep, 190 goats, and 98 cattle) were obtained in different rural regions of Famenin. The samples were evaluated to detect of Brucella-antibodies using rose Bengal plate test (RBPT), Wright standard tube agglutination test (SAT), and 2-Mercapto-Ethanol (2-ME) techniques. The risk factors associated with brucellosis such as age, gender, history of vaccination against brucellosis, and abortion history in animals were evaluated. In the sampling process, the critical gaps related to the distribution of brucellosis in the herds and regions are identified for designing the strategies to prevent and control the disease. Results: About 6.88% and 89.31% of animals had a history of abortion and vaccination against brucellosis, respectively. Most of the animals were female (92.49%) and in the range of 2-3 age old (39.8%). The antibodies to the Brucella-infection in animals were 2.73% with RBPT and 1.30% with SAT and 2-ME. The prevalence of brucellosis was detected 1.3% among individual animals and 11% among herds. This rate was 1.43% for sheep and 1.05% for goats, with no significant statistical difference. No seropositive case was detected in cattle samples using RBPT, STAT, and 2-ME. The highest rate of brucellosis (6.25%) was detected in Emamzadeh-Pirnahan region (22.2% goats and 5.6% sheep). In sheep, most cases of the disease were in 3-4 age-old group (1.92%), animals without a history of abortion (1.58%), and without a history of vaccination against brucellosis (2.80%). Furthermore, 5.94% of males and 1.11% of females were detected positive for brucellosis (p < 0.001). The chance of brucellosis in rams was 5.6 folds higher than in others (odds ratio = 5.64). Brucellosis in goats was detected 2.94% and 1.89% in the age groups <1 and 2-3 year-old. Furthermore, 1.22% of females and 1.34% of animals without a history of abortion were positive. Brucellosis was found in 0.61% of vaccinated and 3.85% of non-vaccinated goats. Except for gender in sheep, no significant statistical correlation (p > 0.05) was observed between prevalence of brucellosis and risk factors. In farmers, low level of information about the transmission and also control and preventive methods of the disease was dominant. Consumption of traditional and unpasteurized dairy products is also very common in the studied regions. Conclusion: This is a comprehensive evaluation of animal brucellosis parallel to humans' cohort study in the Famenin region for the first time. Although the rate of brucellosis in animals is low in the region, explaining the risk factors to farmers, mass vaccination, regular screening of animals, and culling the positive animals are very important for controlling and reducing the disease in the region.

Molecular detection and phylogenetic analysis of Neospora caninum in various hosts from Iran.

Different N. caninum strains with distinct biological characteristics and pathogenicity have been introduced from various areas of the world. This study aimed to detect Neospora-infection in different hosts from Iran using the nested-Polymerase Chain Reaction (PCR) technique and to perform the phylogenetic analysis of N. caninum isolates using the Nc-5 gene. Different species of parasite hosts (cattle, dogs, rodents, cats, and birds) from the dairy farms with history of abortion with intensive breeding systems in Hamedan Province, Iran were included for sampling. The stillbirth fetuses of cattle, sheep, and goats were also collected from the slaughterhouse. The nested-PCR technique was used to identify the Neospora-DNA in the aborted fetal materials. The PCR products of the positive samples were sequenced and compared with other isolates in GenBank. Antibodies to N. caninum was found in 19.1% of dogs. Also, 11.4% of dogs were positive for Neospora-like oocysts using microscopy method. The molecular prevalence of N. caninum was 10.6% in dogs, 13.6% in cattle, 35.4% in the aborted fetuses of cows, 31.9% in rodents, 7.6% in pigeons, 1.9% in sparrows, 5.1% in cats, and 7% in the semen sample of bulls. A sampled coyote was positive while the stillbirth fetuses of sheep and goats were negative. The multiple sequence alignments indicated that 34 sequences of N. caninum bore a 94.9-99.3% similarity to each other. Hence, these sequences were classified into two major clusters (clusters A and B) and seven sub-groups (isolates 1-7). The sequence of coyote had a high similarity to the sequences derived from dogs and cows. Also, root diagram showed the isolates of current study and previous reported from Iran have a similar origin. This study provided a phylogenetic analysis of Neospora-infection in different hosts, and for the first time, explored Neospora-infection in coyotes from Iran. Different isolates were detected in the studied population in the region. Comparison of the sequences showed the farm dogs play an important role in the life cycle and distribution of the parasite; so the relationship of dogs with the herd and their access to aborted materials should be restricted. Also, regular control of rodents' population and culling of seropositive cows with a history of abortion is essential for cutting the chain of transmission.

Herd-level contamination of Neospora caninum, Toxoplasma gondii and Brucella in milk of Iranian dairy farms.

The bulk milk examination is a reliable screening tool for monitoring the quality of milk in the farms. The infection to Neospora caninum, Toxoplasma gondii and Brucella sp. Was evaluated in bulk milk samples of dairy farms in Hamedan province, West part of Iran. All the dairy farms (n = 149) were examined for N. caninum, T. gondii and Brucella infections using milk ring test (MRT), microbiology, serology (Enzyme-linked Immunosorbent Assay), and molecular techniques. Based on molecular methods, Brucella-infection was negative in all farms; while, 55 %, 5.4 % and 2.7 % of samples were positive for N. caninum, T. gondii and mix infection, respectively. The highest Neospora-infection was detected in the farms with history of abortion in fall and winter. There was significant association between Neospora-infection and the presence of dogs and rodents in the farms, herd size, and age of the animals. Also, a significant association was seen between Toxoplasma-infection and the presence of cats and rodents in the farms, as well as age of the animals. Average total bacterial count (TBC) was calculated 1.14 × 106±1.1 × 106. The highest TBC was in the farms from Central locations of studied area (5.7 × 106±2.24 × 106), farms with more than 120 animals (7.9 × 106±2.8 × 106), and farms with ≥50-months age (1.74 × 106±6.3 × 105) in spring and summer (6.9 × 106±3.7 × 106). The number of somatic cells was estimated between 1 × 104 and 2 × 106 (Average = 4.2 × 105±3.39 × 105). The current study was a comprehensive evaluation of Neospora, Toxoplasma and Brucella infections in milk samples of Iranian dairy farms for the first time. Neospora-infection is responsible for economic losses in the region. Health education and milk pasteurization are so helpful for inhibiting the milk borne diseases. To reduce the risk factors, predict and design the appropriate schemes like redundant of heterogeneous animals are recommended.

Neospora caninum infection in Iran (2004-2020): A review.

Knowledge on neosporosis and associated risk factors in different species of animals are so important for designing the control programs and reduce the economic losses globally. This literature review targeted for evaluating the infection rate of Neospora caninum in animals in Iran. Until April 2020, all of published documents in the main English and Persian-language databases were searched. A total number of 110 documents (English = 85 and Persian = 25) were extracted. Most of reports were sero-epidemiological studies using ELISA in Iranian cattle population. The range of Neospora infection was 3.8-76.2% in cattle, 0-54.6% in dogs, 0.9-9.9% in sheep, 6.2% in goats, 19.2-55.9% in buffaloes, 20-42.2% in horses, 52% in donkeys, 3.2-27% in camels, 14% and 19% in cats, and 0-20.4% in rodents. This rate in birds was 17.3% in chicken, 9.8% and 30.4% in pigeons, 2.8% and 3.7% in sparrows, and 9.9% in hooded crows. This is a comprehensive literature review on Neospora infection in Iran at the first time. The infection of N. caninum is widespread in Iran especially in dogs and cattle population. This review can provide baseline information for future research. Study on other hosts especially on wild and exotic animals is recommended for exact estimate of neosporosis in Iran. Investigations into molecular diagnosis and genotyping of N. caninum strains are also needed; this will be helpful for developing vaccines and finding the connection among wild and domestic cycles of disease. Education on the risk factors associated with N. caninum infection for is suggested farmers and rural public.

Risk factors associated to Toxoplasma gondii infection in dairy farms in Hamedan suburb, Iran.

Toxoplasmosis is a zoonotic food-borne disease with global distribution. The principal aim of this research was to detect the seroprevalence of T. gondii infection in cattle, dogs and cats in dairy farms with high incidence of abortion, as well as to identify associated risk factors in Hamedan, Iran. A number of four large dairy farms with high history of abortion were selected and evaluated for risk factors associated to T. gondii infection. Blood samples were taken from 400 of pregnant cattle, 85 of dogs and 35 of cats (all of the dogs and cats population from interring and around of farms were sampled). The samples were examined for presence of antibodies to T. gondii infection using ELISA technique. Seroprevalence of T. gondii infection were detected 13% in cattle, 47.1% in dogs and 40% in cats. With regard to cattle seropositivity and associated risk factors, significant differences were found regarding abortion history (P = 0.026), race (P = 0.035), rodents' presence in farm (P = 0.006) and age groups (P = 0.011). Also, there were no significant differences between seroprevalence rate and stage of gestation (P = 0.998), type of breeding service (P = 0.561), stillbirth history (P = 0.302), dog contact with herd (P = 0.302) as well as poultry presence in the farm (P = 0.302). In both dogs and cats, the seroprevalence rate for females was higher than males (P < 0.05); also, no significant differences were seen in age groups (P > 0.05). This is the comprehensive evaluation of risk factors associated to toxoplasmosis in Iranian dairy farms at the first time. Our finding indicated that toxoplasmosis may partly be responsible for transmission to humans and also abortion and economic losses in dairy farms. Improve the sanitary strategies for reducing the risk factors in animals' husbandry and launching control programs are recommended.

Vertical transmission of Neospora caninum in Iranian dairy cattle.

Placentally, transmission of Neospora caninum plays significant role on retaining of infection and economic losses in dairy farms. The objective of present study was to assess placental transmission of N. caninum in Iranian dairy cattle. A total of 476 blood samples of pregnant dairy cattle were randomly collected for serology test using ELISA technique. Genomic DNA was extracted and subjected for conventional PCR and Nested-PCR to amplify 330bp and 100bp fragment length of NC5 gene, respectively. Positive animals were follow-up during gestation till abortion and/or full term delivery occurred. Molecularly, 25.3% (118/476; 95% CI: 21%-28.6%) of examined animals were seropositive for N. caninum infection in Holstein cattle (15.1%) with <2 years-old (75%) and history of abortion. The infection was detected. TT and abortion occurred in 13.6% and 3.57% of animals, respectively. Aborted cattle (94.1%, 16/17) were significantly infected to N. caninum and history of abortion was recorded in 42.4% of infected cattle. Placental transmission and abortion in naturally infected dairy cattle due to N. caninum was the first molecular evidence in all examined herds of cattle. It was also uncovered N. caninum had significant role on cattle abortion. Further studies for culling of seropositive cattle with history of abortion, associated risk factors related to neosporosis and lunch control program for reducing side effects of infection are essential in the region.

Neospora caninum infection in dairy farms with history of abortion in West of Iran.

Neospora caninum is a major cause of abortion and economic losses among dairy farms in Iran and other countries. The main goal of current investigation was to evaluate the presence of antibodies against N. caninum and associated risk factors in dairy herds with history of abortion in Hamedan province of West Iran. A total numbers of 476 and 185 blood samples of pregnant cattle and farm dogs from 10 dairy farms with history of abortion were randomly collected. Bulk milk sample was taken from each farm. All samples were subjected for detection of IgG antibody against N. caninum using ELISA technique. Of all examined animals, 24.8% of cattle and 8.65% of dogs were seropositive to N. caninum. The seroprevalence had significant differences with abortion, stillbirth, metritis, breed, close contact to dogs, wild carnivores, rodents, poultry, and pregnancy using artificial insemination method. There were no significant differences among seroprevalence and different age groups, number and stage of gestation, and herd population. In all investigated farms, bulk milk examination was positive. In examined dogs, there was significant difference between seroprevalence and sex. It was concluded that N. caninum infection may be responsible for abortion and economic losses in dairy farms of the region. This was also the first comprehensive report on associated risk factors to N. caninum infection in dairy farms in the region.

Prevalence of anti-Neospora caninum antibodies in Iranian goats.

UNLABELLED: Many species of animals play the role of intermediate host for Neospora caninum. Although the neosporosis has a global distribution, however there is no information on goats' infection in Iran. The main goal of present study was to determine the seroprevalence of N. caninum in goats of western Iran. A total of 450 sera were taken and analyzed for antibodies to N. caninum using ELISA technique. Of those, 28 (6.2%) examined goats were found to be seropositive (95%, CI=6.2±2.2%). In which male goats (11.1%) had the highest IgG against N. caninum with more than 2 years-old (54.4%). There was significant difference between prevalence and age. Positive association were in animals with history of abortion (18/30, 60%) and IgG against N. caninum. No correlations were found between male and female. This was the first report on N. caninum infection in Iranian goats and may be responsible for abortion and economic losses in goat husbandry in the region. KEY WORDS: goat, Neospora caninum, ELISA, Iran.

Prevalence of anti-Leishmania infantum antibodies in dogs from West of Iran.

The main aim of present study was to determine the sero-prevalence of canine visceral leishmaniasis (CVL) among stray and owner shepherd dogs in Hamedan province, West of Iran. A cross-sectional study was performed in 2013 using enzyme-linked immuno sorbent assay (ELISA). Blood samples (n = 380) were collected randomly from 170 stray and 210 owner shepherd dogs in different regions of Hamedan province. All of animals were asymptomatic. Antibodies to Leishmania infantum were found 3.95 % (0.95 CI: 2-5.9 %). The sero-prevalence rate of infection in stray dogs (6.47 %) was reported higher than owner shepherd dogs (1.9 %) (P = 0.023). There was significant differences among age groups (P = 0.003); unlike to different gender (P = 0.11). This is the first report of CVL in western Iran. Further comprehensive studies in infection rate and predominant Leishmania species in humans, sand flies and animals hosts (wild canines and rodents) is recommended in this region.

Study on gastrointestinal zoonotic parasites in pet dogs in Western Iran.

OBJECTIVE: Dogs are the definitive or reservoirs hosts of more than 60 zoonotic parasites. This study was conducted to investigate the prevalence of gastrointestinal (GI) parasites in pet dogs in Hamedan, Western Iran. METHODS: In cross-sectional study, 210 stool samples were collected randomly in pet dogs without clinical signs in Hamedan in April to December 2010. All samples were concentrated by formalin-ether technique. Smears of the feces were prepared and stained with Ziehl-Neelsen, trichrome, and iodine stains. RESULTS: During coproscopy, the overall proportion of GI parasitic infection was found in 6.7% (14/210) of samples. The detected parasites with their frequencies were Cryptosporidium spp. (3.8%), Toxocara canis (1.9%), and Giardia spp. (0.95%). A significant difference was not observed between infection rates in different age groups) p=0.617) or between genders (p=0.627). CONCLUSION: This is the first report of GI parasites in dogs from Western Iran. Although the rate of infection is low, the results showed that the pet dogs are reservoirs for zoonotic GI parasites and should be considered important to public health in this region. A combination of routine screening fecal samples for parasites, strategic anthelmintics regimens, and improved pet owner education is highly recommended for the control of GI parasites in pet dogs.

Prevalence of Cryptosporidium infection in sheep in Iran.

OBJECTIVE: Cryptosporidium is an important zoonotic parasite in humans and animals worldwide. This study was conducted to investigate the prevalence of Cryptosporidium infection in Iran. METHODS: Fecal samples (n=1.749) were collected randomly in asymptomatic sheep from different rural regions of Iran in 2011 to 2012. All samples were examined by using the cold modified Ziehl-Neelsen staining technique. RESULTS: Oocysts of Cryptosporidium was found in 11.3% (198/1749) of samples (9.8 < CI 95% < 12.8). There was a statistical differences among Cryptosporidium infection, age groups (p < 0.0001), and gender (p=0.02). CONCLUSION: This study is the first report of Cryptosporidium infection in sheep in different regions of Iran. Therefore, further comprehensive molecular studies in sheep to identify the source of contaminations (animals or humans) and designing control strategies is highly recommended.

Seroprevalence of Neospora caninum in slaughtered native cattle in Kurdistan province, Iran.

Neospora caninum is a worldwide distributed pathogen which causes abortion in cattle leading to economic loss in the cattle industry. The aim of this study was to determine the seroprevalence of N. caninum antibodies in the native cattle slaughtered in various areas of Kurdistan province (western Iran) from September 2010 to September 2011. Serum samples from 368 cattle slaughtered in seven slaughterhouses in this region were taken for detection of anti-N. caninum antibodies using commercial N. caninum ELISA kit. Antibodies to N. caninum were found in 29 samples (7.80%). The present study was the first report of Neospora infection in this region and indicated that native cattle of Kurdistan province were exposed to this parasite.

Prevalence of immunoglobulin G (IgG) antibody to Neospora caninum in dairy cattle of Hamedan province, west of Iran.

Bovine neosporosis caused by the apicomplexan protozoan parasite N. caninum, was initially recognized in 1989 and is now reported as a leading infectious cause of reproductive failure in dairy cattle in world wide. The aim of this study was to determine the seroprevalence of N. caninum infection in industrial dairy cattle of Hamedan province (west of Iran) by ELISA method. Blood samples were collected from 492 cattle in 41 farms. Antibodies to N. caninum were found in 63(12.80%) sera. A Significant difference was observed between seropositive cattle and dog presence in farm, dog contact with herd, abortion history and herd population. No significant differences were found between seropositive cattle and age as well as breed. This study is the first report of N. caninum infection in dairy cattle farms in Hamedan province. As per our knowledge, Neospora is an important factor in abortion of cattle in this region. Therefore, comprehensive studies for control strategies and improving management of dairy farms is necessary.

Prevalence of Coccidiosis in Broiler Chicken Farms in Western Iran.

The main goal of current study was to investigate the prevalence of coccidiosis in broiler farms in Hamedan province, western Iran. Chicks and fecal samples were collected in all of the 220 broiler farms in this region. All viscera were examined for gross pathological changes. The mucosa of small intestine and the caeca were examined for the presence and identification of parasitic forms using parasitology methods. The overall rate of coccidiosis was 31.8%; E. acervulina (75.7%), E. tenella (54.3%), E. necatrix (28.6%), and E. maxima (20%) were determined. Mixed infections were observed in all of the positive farms. There was a statistical significant difference (P < 0.05) among infection rate and age groups, dysentery, history of colibacilosis and clostridiosis in farm, and history of coccidiostats consumption, unlike to breed (P > 0.05). This is the first report of coccidiosis rate in broiler farms in this region. Further additional researches and design control strategies for improving management in farms are necessary.

Serological study of bovine herpes virus type 1 in dairy herds of Hamedan province, Iran.

A cross-sectional study with a random cluster sampling design was carried out to estimate the seroprevalence of bovine herpesvirus type 1 (BHV-1) in non-vaccinated dairy herds in Hamedan province, west of Iran. Simple random sampling was used for selection of cattle in each herd. Informative data about each herd and selected animals were recorded by the farm manager in a provided questionnaire. Blood samples were collected from 492 animals in 41 industrial herds. A commercial indirect ELISA test was used to determine the seropositivity against BHV-1. The individual and herd seroprevalence for BHV-1 were 58.74% and 82.93%, respectively. The intra-herd prevalences were ranged from 16.70% to 100%. Geographical characteristics of Hamedan province may explain the high sero-prevalence rates found in this study compared to those of others obtained from different parts of the country. The proportion of seropositive cows were increased with age (p <0.05). Animals from large and moderate sized herds had higher odds of seropositivity than those of small size herds. These findings could be related to the presence of a considerable number of BHV-1 carriers in this region. The high herd and animal prevalence found in the present study suggested necessity of implementing an intensive control program for reducing BHV-1 infection rates.