Veterinary Research Forum

Abstract This study is the first to apply the behaviour, immunity, nutrition and environment (BIENE) protocol to assess dairy ewe welfare in intensive farms in Algeria. A total of 1,964 ewes from 50 farms in the Algiers region were evaluated between December 2022 and March 2023 using 13 animal-based indicators related to nutrition, housing, health, and behavior. Moderate tooth loss affected 54.67 ± 3.32% of ewes, and 20.38 ± 2.78% had a low body condition score (< 2). Housing problems included fleece moisture (11.28 ± 2.55%) and over 40.00% soiling of flanks and hindquarters. Health issues, such as mild anemia (23.55%), lameness (26.45%), nasal discharge (35.47 ± 4.43%), and labored breathing (8.79 ± 1.47%), were observed. In terms of behavior, most ewes appeared attentive and active, although 40.55% showed fear and 19.90% aggression. Flight time increased from 2.50 ± 0.07 sec to 3.80 ± 0.07 sec, and animals maintained greater distance from the corridor (2.79 ± 0.08 m) than from the observer (1.85 ± 0.06 m). Principal component analysis of qualitative behavior assessment identified three clusters, including well-being, discomfort, and vitality. Spearman correlation analysis showed that relaxed ewes had better body condition score (r = 0.29) and were less likely to be lame (r = – 0.32) or anemic (r = – 0.37). Vigorous ewes showed fewer signs of respiratory distress (r = – 0.32), while apathetic ones were more prone to poor conditioning (r = 0.31) and injuries (r = 0.30).The BIENE protocol proved effective in detecting major welfare issues in intensive Algerian sheep farming.

Abstract Pseudorabies (PR), an infectious disease caused by PR virus (PRV), has had a significant impact the development of the swine industry in China. Moreover, the public threat posed by PRV has raised widespread concern, with over 30 documented cases of PRV infection in humans in China. Various vaccines have been developed to prevent and control PR in China, primarily including inactivated vaccines and attenuated live vaccines. However, the extensive use of attenuated live vaccines and the presence of the field PRV strain in pig farm may increase the likelihood of recombination. In this study, we isolated a PRV variant strain (designated HuN-YY) from a piglet that had been immunized with the HB-98 vaccine strain in Hunan province and explored its genetic and biological features. The results showed that the gE, TK and gD genes of HuN-YY exhibited the highest sequence similarity with those of PRV variants, while the gC gene showed high homology with classical PRV strains. In addition, HuN-YY strain exhibited similar replication features compared to those of HB-98 and variant strains. Its pathogenesis in a mouse model was significantly lower than that of the variant strain. Collectively, these results provide clear evidence of genomic recombination of the HuN-YY strain, which will offer guidance for the future prevention and control of PR.

Abstract Hepatitis E virus (HEV) is a zoonotic pathogen that can cause acute viral hepatitis in both humans and animals. Some animals, such as swine, rabbits, cattle, and sheep are recognized as primary reservoirs of HEV. Recently, HEV has emerged as a significant public health concern in both industrialized and developing countries. Up to now, there is no information about zoonotic sources and prevalence of HEV in wild and domestic animals in Iran. This cross-sectional study aims to determine the prevalence of HEV. Multi-stage cluster sampling approach was used to collect 400 fecal swabs and 400 milk samples between September and February 2022. These samples were collected from rural cattle dairy farms in northern Iran. The viral genome was extracted, and semi-nested reverse transcription polymerase chain reaction was used to detect the HEV RNA genome. The overall estimated herd-level prevalence of HEV was found to be 6.25% (5/80, 95.00% CI: 2.06 - 13.99%). Among the 400 fecal and 400 milk samples obtained from 80 farms, the prevalence of HEV was 3.75% (3/80, 95.00% CI: 0.78 -10.57%), and 2.50% (2/80, 95.00% CI: 0.80 - 30.74%), respectively. Positive fecal samples were detected in Abbas-abad and Amol cities, while positive milk samples were found in Tonekabon City. Based on our findings, Hepatitis E is present in the cattle population of Mazandaran province, Iran. Further, more in-depth investigations are recommended due to the zoonotic nature of this disease. It is crucial to consider control and preventive measures in order to mitigate the spread of HEV.

Abstract Porcine respiratory disease complex is a clinically lethal condition and is the leading cause of mortality in weaned piglets as well as growing and fattening pigs. Pasteurella multocida (Pm) and Streptococcus suis (SS) are common respiratory pathogens in porcine respiratory disease complex. This study combined the recombinase polymerase amplification (RPA) technique with the lateral flow dipstick (LFD) technique. The dual Basic-RPA detection method for Pm and SS and the dual RPA-LFD rapid visualization detection method for Pm and SS were constructed, respectively. The detection limit of RPA-LFD was 10^-6 ng μL^-1, which was higher than the detection limit of RPA at 10^-5 ng μL^-1 and much higher than the detection limit of polymerase chain reaction at 10^-4 ng μL^-1. There was no cross-reactivity with other pathogens which indicated that the method had good specificity and high sensitivity. The detection rate of RPA-LFD was much higher than that of conventional Polymerase chain reaction in 60 clinical samples collected in 2023 with suspected Pm and SS. This method could avoid the complicated temperature cycling instruments and does not require professional laboratory skills, which makes it suitable for on-site detection.

Abstract Several bacteria, such as Escherichia coli, Pseudomonas, and Staphylococcus are considered as indicators of anti-microbial resistance (AMR) in a wide range of hosts and environments, because they may transfer AMR genes to important pathogenic bacteria. Hedgehog is one of the most important wild species living in urban areas. So, this study aimed to determine AMR against β-lactams and prevalence of colicin genes among various phylotypes of E. coli isolates from hedgehogs in the Kerman, Iran. Totally, 105 E. coli isolates were obtained from 21 hedgehogs (five isolates from each hedgehog). Resistances to the ampicillin (79.10%), cefotaxime (66.70%), and amoxicillin-clavulanate (62.00%) were the most prevalent, and resistance against ceftiofur (39.10%), ceftazidime (39.10%), and ceftriaxone (34.30%) had the lowest prevalence rates. In phenotypic tests, 2.90% of the isolates were extended spectrum β-lactamase producers. The prevalence of β-lactam resistance genes was 26.60% for bla_TEM, 3.80% for bla_CTX-M, 8.50% for bla_SHV, and 1.90% for bla_CMY. The frequency of colicin genes, including E1, V, E2-E9, and Ia.Ib was 5.71, 4.76, 10.47, and 11.42%, respectively. All E. coli isolates were negative for 5.10.K, Y.U, and A.N.S4 genes. Phylogenetically, B1 (49.50%), A (40.90%), and D (5.70%) were identified among the isolates, and 3.80% remained unknown. Wildlife could be considered as a bio-marker to determine the environmental dissemination of AMR. Also, hedgehog may be an important reservoir of antibiotic-resistant and non-pathogenic E. coli strains in urban environments. This study highlights the necessity of E. coli surveillance among domestic and wild animals.

Abstract Infecting to Melissococcus plutonius, the primary cause of the European foulbrood (EFB) disease, can be followed by infecting to the secondary bacteria, such as Enterococcus faecalis and Brevibacillus laterosporus. The aim of this research was to diagnose EFB disease by tracking the causes of the disease in apiaries all over Iran. From 260 apiaries, honey bee samples were randomly collected. After samples preparation, the genomic DNA was extracted and specific primers were selected for interested bacteria. Using the conventional polymerase chain reaction (PCR) method for E. faecalis and B. laterosporus and nested-PCR method for M. plutonius, the target fragments were amplified. Desired standard bacteria and distilled water were used as positive and negative controls, respectively. Results showed that out of 260 samples from apiaries, 74 and three samples were positive for E. faecalis and B. laterosporus bacteria, respectively. Also, the results of nested-PCR showed that 58 samples were positive, of which only 12 samples were positive in the evaluation of E. faecalis. Results demonstrated that the highest and the least levels of the infection for M. plutonius and E. faecalis were in the south and east of the country, respectively. Results indicated that sometimes due to the excessive growth of secondary bacteria, the main bacteria can be removed from the environment. Also, findings proposed that those provinces with higher number of populations, followed by higher amount of air pollution, had more infected samples than others.

Abstract Infectious canine cyclic thrombocytopenia or canine anaplasmosis is an infectious disease caused by Anaplasma platys. In Mexico, cases of human infection have been reported. The present cross-sectional study aimed to determine the frequency of A. platys infection in the municipality of Veracruz, Mexico, by nested polymerase chain reaction method. A total of 100 blood samples from dogs living in the municipality were collected and analyzed between March and June 2022. A descriptive analysis of blood samples for hemoparasites frequencies was performed with the free online software VassarStats. The evaluated variables were sex, street access, usage of ticks control methods, and living environment. The free online software WinEpi software was used to calculate odds ratio (OR) and confidence interval (CI: 95.00%). Out of 100 analyzed blood samples, 27 were confirmed positive for A. platys. The only risk factor found was the absence of tick prevention methods (OR = 9.81; 95.00% CI: 23.00 - 44.50). In conclusion, the frequency of A. platys was 27.00% and no risk factors were identified.

Abstract African swine fever (ASF) is considered as one of the most threatening diseases for the pig farming industry all over the world. Due to the lack of an effective vaccine, organized farms and backyard rearing must strictly enforce control measures in order to combat the disease. The present report describes the ASF epidemic in a piggery in Uttar Pradesh state, India. The pathological samples were collected from the affected pigs and processed for histopathological and molecular studies. Gross lesions comprised of cyanosis of ear pinna, multi-focal hemorrhagic spots on ventral abdomen and inner aspect of thigh, highly congested mesenteric lymph nodes with marbling, marked congestion, hemorrhages and splenomegaly, interstitial pneumonia, and multi-focal endocardial hemorrhages on papillary muscles and wall of ventricle in heart. Histopathological investigation revealed marked congestion and hemorrhages of mesenteric lymph node, liver and spleen. Depletion of lymphocytes from the splenic white pulp was visible in the splenic parenchyma. The virus was confirmed by polymerase chain reaction and phylogenetic analysis revealed a distinct clustering of the Uttar Pradesh virus isolates from Vietnam with other Ib group isolates, indicating a close genetic relationship between these samples. Additionally, the mutant Chinese virus isolate showed clear genetic differences with the Vietnamese Ib group, confirming its suitability as an out-group for comparison. The study represents the first report of ASF outbreak in North India, establishing the phylogenetic relationship between ASF virus circulating in the study area and other regions.

Abstract Foot-and-mouth disease (FMD) is considered as one of the most important contagious viral diseases affecting cloven-footed animals. For effective control of FMD, immunization along with herd immunity is essential in the field conditions. To assure and track the coverage and effectiveness of the vaccination program, the serological studies are very much required after the vaccination program. The present study was aimed to investigate the prevalence of antibodies against structural proteins of FMD virus (FMDV) serotypes of O, A and Asia-1 in seven districts of western Uttar Pradesh, India, and assure the efficacy of vaccination under National Animal Disease Control Program. A total of 308 sera samples were collected from apparent healthy vaccinated cattle and buffaloes from seven districts including Amroha, Baghpat, Bareilly, Bulandsahar, Gautam Budh Nagar, Meerut and Muzaffarnagar of western Uttar Pradesh, India. Determination of antibodies against structural proteins of FMDV was carried out using solid-phase blocking enzyme-linked immunosorbent assay. The protective level of the FMDV serotypes O, A and Asia-1 included in the inactivated trivalent vaccine was 66.55, 48.05 and 47.08% in bovines, respectively. To provide the higher level of protection against the circulating FMDV, the present study recommended the thorough investigation of the immunogenic interaction between the vaccine strains and the field strains. Further investigations should also be conducted with larger sample size and across diverse geographical regions to gain a more comprehensive understanding of herd immunity.

Abstract The interval between parturition and subsequent pregnancy is called the days open or calving to conception interval and is affected by several factors, especially dystocia. Dystocia is an ab-normal or difficult calving that may require assistance during labor. This study is a field trial in health education and the research team developed a comprehensive training program for farmers to educate them about the normal process of parturition in dairy cows and when and how to assist in parturition or dystocia. A series of classes was held for farmers and the study covered 486 multi-parous dairy cows, with 173 belonging to the group of trained farms (educated farmers) and 313 to the control group (non-educated farmers). Although dystocia was lower in the educated group, there were no significant differences in retained placenta between two groups. However, cows in the educated group had a better conception rate (lower service per conception) in sub-sequent parturitions. Hence, the median number of days open for cows from trained farmers was significantly lower than other farmers (85 days compared to 120 days, respectively). Based on Cox regression analysis, uterine prolapse, retained placenta, and dystocia could significantly impact subsequent pregnancies. Dystocia affects days open, and training on parturition and dystocia management can effectively reduce the numbers of days open in dairy cows.

Abstract ‎Nosemosis is one of the most prevalent bee diseases in the world causing significant ‎economic losses in the global bee-keeping industry. This cross-sectional study was conducted during April-September, 2016 to investigate the prevalence of nosemosis in different climatic regions of Iran. A total of 183 ‎apiaries were selected based on cluster sampling and the climate of apiaries under study was classified using Domarten method. In each apiary, five percent of the colonies were ‎ randomly sampled. A total of 183 adult bee samples were taken and examined by microscopic and polymerase chain reaction (PCR) methods for the presence of Nosema infections. According to the results, infection caused by Nosema ceranae was observed in all regions under study. The prevalence of N. ceranae was 46.40% (42.70–50.10). However, infection with Nosema apis was not observed in the samples in either pure form or as ‎associated infection. Based on the results of PCR, the prevalenceof N. ceranae was ‎53.80% (46.60– 61.00) ‎ in ‎humid, ‎71.00% (53.70–77.50) ‎ in semi-humid‎, ‎68.10% (61.40–74.80) ‎ in very humid, ‎29.40% (22.70–36.10) in arid, ‎34.30% (27.40–41.20) in semi-‎arid and ‎24.00% (17.90–30.00)‎ in Mediterranean climates. The prevalence of ‎infection in different climatic zones of the country was found to have significant ‎differences (p < 0.001). ‎ According to the findings, N. ceranae ‎ was the only Nosema species in honeybees with a broad geographical dispersion in Iran. It seems that climate can influence the prevalence of mentioned parasite. ‎

Abstract Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and phylogenetic analysis were used for molecular identification of lactic acid bacteria (LABs) isolated from Apis mellifera. Eighteen honeybee workers were collected from three different apiaries in West Azerbaijan. LABs from the gut of honeybees were isolated and cultured using routine biochemical procedures. Genomic DNA was extracted from LABs and a fragment of 1540 bp in size of 16S rRNA gene was amplified. PCR products were digested using HinfI endonuclease and digested products with different RFLP patterns were subjected to nucleotide sequencing and phylogenetic analysis. The results revealed that Lactobacillus and Bifidobacteria spp. are were the most abundant LABs in honeybee gut. Phylogenetic analysis showed that both Lactobacillus and Bifidobacterium were closely clustered with high similarity percentage with the same bacteria isolated from honeybees’ gut elsewhere. It was concluded that LABs isolated from honeybees had low sequence divergence in comparison with LABs isolated from other sources such as dairy products.

Abstract Dirofilaria immitis is an important filarial nematode in dogs. In this study, age and sex distribution of this zoonotic nematode among dogs were investigated in northwest of Iran in Meshkin-Shahr city. Molecular characteristics of the isolates, based on cytochrome oxidase subunit 1 (COX1) gene were compared to the isolates from other areas of the world.Blood samples were collected from 91 dogs which were selected by simple classified accidental sampling. Thin and thick blood smear examinations were used to find out infectivity with D. immitis. DNA extraction was performed from adult D. immitis recovered from heart of infected dogs. The COX1 gene was amplified and sequenced. Phylogenetic analysis was carried out using sequences obtained in this study along with relevant sequences deposited in the GenBank. Phylogenetic analysis and sequence variation was performed using MEGA software in comparison with those COX1 sequences deposited in GenBank. Out of 91 dogs, 19 (20.87%) were found positive for infection with D. immitis. There was no statistically significant difference between males and females of dogs in terms of D. immitis infection. However, the rate of infection in dogs more than 2 years old was significantly higher than those with lower age. Both sequences analyzed in this study showed 100% homology to each other. Intra-species variation of these isolates with those from other areas of the world amounted to 0 to 0.50%. Phylogenetic analysis of the COX1 gene suggested that it is conserved, and can be used for study on genetic diversity and classification of filarial nematodes.

Abstract Althoughpoultry meat is considered as the main source for human Campylobacter infections,there is limited information about non-poultry sources. The present study was aimed to investigate the prevalence and the antibiotic resistance of thermophilic Campylobacter spp. in fecal samples of the cattle and sheep in Shiraz, Iran. A total of 302fecal samples were obtained from clinically healthy, slaughtered cattle and sheep from Shiraz slaughterhouse. The animals were clinically healthy before being slaughtered. The samples were cultured according to the specific cultivation method under thermophilic conditions. The susceptibility of Campylobacter isolates were determined for 13 antimicrobial agents. All enriched samples and cultured isolates were targeted for polymerase chain reaction (PCR) detection of 16S rRNA and multiplex PCR for determining their species. Among 302 fecal samples, 65 (21.5%) and 205 (67.8%) samples were positive for the presence of Campylobacter species with the cultivation and PCR techniques, respectively. All 65 distinct isolates were susceptible to neomycin and colistin and the isolates showed high resistance to cephalotin (83.0%) and ciprofloxacin (67.7%). After the multiplex PCR, 78.5% of total positive samples showed the simultaneous presence of Campylobacter jejuni and Campylobacter coli. In conclusion, the results emphasized that non-poultry farms are important as a possible source of Campylobacter infections.

Abstract This study was conducted to investigate the prevalence of subclinical mastitis caused by Staphylococcus spp. in ewes in West-Azerbaijan province of Iran. Molecular characterization of isolated Staphylococcus spp. from diseased ewes were performed using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and DNA sequencing of glyceraldehyde-3-phosphate dehydrogenase (gap) gene. Also, antibiotic resistance of staphylococcal isolates against different antibiotics was investigated. A total number of 900 milk samples from 450 native ewes in their mid-lactation period were examined by the California mastitis test (CMT). The CMT positive samples were cultured and bacteria were isolated from 86 (9.50%) glands and 74 (16.40%) ewes. The prevalence of subclinical mastitis in the examined ewes was 16.40%. Microbiological analysis of milk samples revealed that 27 out of 74 sheep with subclinical mastitis were infected with Staphylococcus spp. Amplification of gap gene of 27 Staphylococcus isolates generated a single amplicon of 933 bp in size confirming that isolates were belonged to Staphylococcus genus. Digestion of PCR products by AluI endonuclease generated different RFLP patterns for each species. Nucleotide sequencing of gap gene followed by phylogenetic analysis showed that the most dominant Staphylococcusspecies were S. epidermidis, S. xylosus and S. chromogenes. Staphylococcal isolates showed the highest resistance to penicillin and ampicillin. In conclusion, Staphylococcus species, except for the southern parts of the province, play an important role in the development of subclinical mastitis in sheep in West-Azerbaijan province of Iran. Also, chloramphenicol, ciprofloxacin and neomycin are the most effective antibiotics for treatment of this disease.

Abstract 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.

Abstract Maternal testosterone has been indicated to affect sex ratio of offspring. The present study was conducted to elucidate the role of androgen receptor in this regard by blockade of androgen receptor using flutamide in female mice. Mice were randomly assigned to two experimental groups. Mice in the control (n = 20) and treatment (n = 20) groups received 8 IU equine chorionic gonadotropin (eCG) followed by human chorionic gonadotropin (hCG) injection (8 IU) 47 hr later. In addition, mice in the control and treatment groups received four injections of ethanol-saline vehicle and flutamide solution (2.50 mg), respectively, started from 1 hr before eCG injection until hCG injection at 12-hr intervals. Conception rate was not different between the treatment (18/20: 90.00%) and control (19/20: 95.00%) groups (p > 0.05). Litter size was higher in the treatment (8.22 ± 0.26) than control (7.21 ± 0.28) group (p < 0.05). Male sex ratio was lower in the flutamide-treated mice (67/148: 45.30%) as compared with the untreated ones (80/137: 58.40%; odds ratio = 1.69; p < 0.05). In conclusion, the results showed that androgen receptor blockade could skew sex ratio of offspring toward females implying that the effect of testosterone on sex ratio might be through binding to androgen receptor. In addition, the blockade of androgen receptor using flutamide appeared to enhance litter size.

Abstract The importance of cats in the Leptospira epidemiology is due to the possibility of transferring leptospirosis to wild and domesticated animals. The purpose of this survey was to determine the prevalence of Leptospira infection in shorthair cats in different location of Mashhad, Iran. Totally, 147 blood samples were taken from 42 (28.57%), 52 (35.37%) and 53 (36.05%) households, stray and cats which lived in industrial dairy cattle herds of Mashhad, Iran, respectively. Sera were tested with seven live Leptospira antigens using microscopic agglutination test (MAT). Sera with 50.00% agglutination at the dilution of ≥ 1/100 were considered as positive samples. Agglutination at dilutions of < 1/100 considered as suspected to Leptospira infection. Overall, 19 (12.92%) out of 147 cats showed reaction in MAT. The seroprevalence at a titer ≥ 1:100 and < 1:100 were 10 (6.80%) and 9 (6.12%), respectively. Serum samples showed positive reaction against Leptospira intterogans hardjo (no = 10; 52.63%), pomona (no = 5; 26.31%) and icterohaemorrhagiae (no = 4; 21.05%). Eight cats (42.10%) belong to dairy cattle herds had the most infection only by L. I. hardjo with 1:200 titer. There were no significant differences among the weight‚ age and sex of infected cats. However, there were significant differences between the infected cats in dairy cattle herds and the cats in the urban area (p < 0.05). It is concluded that cats can be infected by Leptospira spp. especially in commercial dairy cattle herds. Cats can be considered as a sanitation hazards in the area for this zoonotic disease.

Abstract Paratuberculosis (John’s disease) is infectious and chronically progressive granulomatous disease which affects domestic and wild ruminants. The causative agent is Mycobacterium avium paratuberculosis (MAP), a slow growing mycobactin dependent acid-fast bacillus. We investigated the detection and frequency of MAP in apparently healthy dromedary and Bactrian camels by insertion sequence 900 (IS900) polymerase chain reaction (PCR) and acid fast staining of fecal samples in Iran. Acid fast staining results showed that 6/50 (12.0%) samples of dromedary camels and 4/26 (15.3%) samples of Bactrian camels were suspected to MAP. Although the percentage of positivity for PCR assay of fecal dromedary camel was 8.0%, no bands corresponding to MAP detect in all samples of Bactrian camels. In conclusion, Although the incidence of MAP infection was low, further studies should be conducted to get more information on MAP infection in camel population, especially in areas where camels are close to other ruminants such as dairy cow, sheep and goat.

Abstract Maedi-Visna (MV) virus and caprine arthritis encephalitis (CAE) virus known as small ruminant lentiviruses (SRLVs) cause chronic diseases in susceptible animals. The main reservoirs of these viral agents are sheep and goat. In sheep, MV virus causes a disease as the same name of the virus. This is the first seroprevalence survey of SRLVs in sheep population of Khorasan-e-Razavi province in Iran. Two hundred and twenty sheep from 30 flocks in 12 regions of the province were selected by random cluster sampling method. Serum samples were analyzed for the presence of antibodies against MV/CAE viruses. The seroprevalence in sheep was 34.5% (95.0% CI: 28.3 to 40.7%). Totally, the seroprevalence was in the range of 6.7 to 72.2 %. In 26 flocks of sheep (89.6%; 95.0%CI: 74.4 to 98.8%), at least one seropositive case was detected. The relationship between seropositivity and age, sex, flock size and breeds of sheep were statistically analyzed. In logistic regression model, only age was correlated with SRLV seroprevalence (p < 0.05). This study showed relatively high seroprevalence against SRLVs in sheep population in this area of the country. Due to difficulty in clinical diagnosis, chronic course of the disease, the absence of effective vaccine and treatment and huge economic loss, more epidemiological studies with regards to prevention and control of the disease are necessary.