مجله تحقیقات دامپزشکی
سال هفتادم شماره 3 (تابستان 1394)

Cryptosporidium parvum is a protozoan parasite which belongs to apicomplexa phylum. The parasite infects both wild and domesticated animals and human beings as well. The purpose of the present study was to detect oocyst shedding and diarrhea pattern in experimental cryptosporidiosis and their correlation with weight loss in neonatal calves. Twelve Holstein calves of both sexes were obtained at birth from dairy farm and randomly divided into two groups of 6 calves. Six calves were orally infected with 107 C. Parvum oocysts at the 12h post parturition. The control group was not infected. Clinical signs were examined and fecal samples were collected by the rectal examination twice a day. All calves were weighed from day 0 to day 30 with 3 days intervals to determine effects of cryptosporidiosis on weight gain. All infected calves were noticeably depressed and had a decreased appetite from 3 days post inoculation (DPI) while they received colostrum. Subsequently، watery diarrhea with clumps of mucus and yellow or pale changes of feces color were observed. The infected calves have had diarrhea for 5-8 days that remarkably had got dehydrated. The most severity of diarrhea was 4-6 DPI. Oocyst excretion started 4 DPI، peaked at 6 DPI (60. 48×106±9. 03oocysts/g feces) and continued until 11 DPI. Control calves had no diarrhea and other clinical signs during the whole period of the trial. The mean weight gain of control group was significantly higher than inoculated group during experiment (p<0. 001). The Weight of the infected calves was retarded until 9 days old and then risen subsequently. Present study showed the role of C. Parvum as the primary cause of diarrhea and weight loss among neonatal calves.

Theileriosis is a blood protozoan disease with high mortality in cattle in tropical and subtropical regions of the world. Several studies were conducted to identify Theileria species in infected cattles and vector ticks by microscopic examination. However, microscopic technique has lower sensitivity compared to molecular method. This study was carried out to identify Theileria species and its carriers in cattles of Yazd city, sing semi nested PCR. Between June to September of 2012, 100 EDTA blood samples and 249 ticks were collected from Holstein breed with no history of vaccination against the ileriosis in Yazd area. The collected samples were transported to the laboratory, then prepared the blood smears and stained with Giemsa method. Also, the collected ticks were separated into 50 tick pools, according to their species. Then their salivary glands were removed using stereomicroscope in 0.85% saline. DNA of blood and salivary glands was extracted using a commercial kit and analyzed by Semi-nested PCR. PCR-RFLP was also used to differentiate Theileria lestoquardi from Theileria annulata in positive samples of ticks. Ring forms of Theileria spp. were found in 4 (4%) of blood smears. All ticks were Hyalomma a.anatolicum. Results of PCR were indicated that 11 (11%) of blood samples and three pools of tick’s salivary glands were infected with Theileria. Theileria annulata were only detected in all positive samples by Semi-nested PCR. The results of PCR-RFLP using MspI enzyme showed that the tick’s salivary glands were negative to Theileria lestoquardi infection. Based on the results, it is concluded that Theileria annulata and Hyalomma a.anatolicum are important agent and vector tick of tropical theileriosis in dairy cattles of Yazd area.

Newcastle disease is one of the most serious viral diseases in the poultry worldwide.

Since the traditional strategies have been hardly effective in controlling the disease, the purpose of this study was to introduce new methods for early and rapid diagnosis of Newcastle. The present study helps to reduce further damage to the poultry industry.

RNA extraction was performed, using RNease mini kit, according to the manufacturer’s instructions. Extracted RNA with 68.23×109 copy numbers was prepared as serial dilutions of 100 μL for RT-PCR and RRT-PCR reactions. RRT-PCR and RT-PCR were performed, using commercial kit and RNease mini kit, respectively.

Results showed that amplification was done according to prepared dilution equal 10-34 for RRT-PCR reaction and a visible band observed on 1.5% Agarose gel up to 10-20 for RT-PCR reaction. Based on the results observed, RRT-PCR and RT-PCR reactions are able to detect 10-34 and 10-20 copy numbers of primary sample, respectively.

The sensitivity of RRT-PCR reaction is almost twice compared with RT-PCR reaction, also RRT-PCR reaction is able to diagnose Newcastle disease virus in infected samples with 10,000 copy numbers of the RNA virus less than RT-PCR.

Eimeria infection is an important part of protozoal infection in ruminants which causes economic lost in animal husbandry of Iran and worldwide. Therefore it is important to implement effective control programs on the prevalence and diversity of Eimeria species in livestocks. This study was aimed to determine prevalence and Eimeria species diversity in dairy cattle with different age groups under industrial management. The present study was carried out in industrial cattle husbandry of Kangavar suburban of Kermanshah province, Iran, from spring 2011 to 2012. A total of 307 fresh fecal samples were randomly collected from the rectum of examined animals. The fecal specimens were subjected for flotation technique and collect Eimeria oocysts. The intensity of infection was determined using McMater method. Eimeria species diversity and frequency was also determined by using oocyst sporulation technique in 2.5% potassium dichromate. The overall prevalence was 31.92% (98 out of 307). The highest prevalence was found in dairy cattle (37%) with 1-3 years old. There was no significant difference between prevalence and different age groups. The highest frequency was significantly in spring (41.66%). The intensity was variable in examined animals which ranged from 3.18×103 to 2.71×105. There was no significant difference between the prevalence and intensity in all age groups. Fecal consistency findings revealed that the highest infection was significantly in dairy cattle with normal fecal consistency in all age groups. According to Laboratory identification, a number of seven Eimeria species were detected in all infected animals. The most common specie was E. zuernii (28.25%) in 5% dairy cattle (1-3 years-old) in spring (30%) and summer (30%). There was significant association between frequency of Eimeria infection and each season during the course of the study. All infected cattle had mixed infections with two (42.85%) and three (3.15%) species of Eimeria. The results of this study indicated that cattle Eimeria infection was a problem in dairy cattle of industrial farms in the region. Attention should be paid within seasonal infection in young cattle to avoid probable clinical coccidiosis, particularly in farms with poor hygienic conditions and no prophylactic treatments.

CASE HISTORY: Equine coital exanthema caused by Equine herpesvirus 3 (EHV3) was reported in 1900. At the present time, it has become epidemic among horses. The virus could be transmitted via insemination, iatrogenic, and it basically infects the squamous epithelium of the epidermis. CLINICAL PRESENTATION: In the first week of January 2012 two mature male horses and a mare were imported from Netherland and kept in a stable of a horse riding club located around Tehran. In the first week of December, these animals manifested clinical signs of high fever with repeated intermittently for two times. During this period, one of the male horses developed exanthematous lesions on the penis and perinea tissues which depigmented later. Biopsy specimens obtained from the lesion processed for histopathological examination were stained with Harris H α E and nuclear fast Red methods.DIAGNOSTIC TESTING: Microscopically, ballooning degeneration of squumous epithelium, acantholysis, epithelial ulcers accompanied by multiple bacterial colonies and the herpes virus intranuclear inclusion bodies with margination of nuclear chromatin were evident. Obviously the horse had EHV3 infection and the stresses resulted from high fever led to the reappearance of genital exanthematous lesions. ASSESSMENTS: In spite of the contagious nature of the infection, the native horses kept in the same stable did not manifest any clinical sings of the infection and therefore the disease could be native. Since the EHV2 infection first affects upper respiratory infection with a high fever course and then induces viremia with another high fever period so it might be speculated that the primary disease of the Netherland horses caused by the EHV2 infection.

Despite the use of prophylactic chemotherapy and vaccination, coccidiosis is still one of the most devastating diseases in poultry industry. Understanding the immune mechanism helps researchers to prevent this parasitic infection more effectively.

The purpose of this study was to evaluate the antibody response in chickens, induced by a live attenuated vaccine (Livacox Q), before and after challenge, by means of ELISA.

One hundred and twenty one-day-old male Ross 308 broiler chicks were randomly divided into 4 groups of 30 birds. In 4th day of age, half the birds were orally vaccinated. The challenged groups received the infective dose at 14th day of age via oral administration. Besides recording weight gain, lesion score and oocyst count in 21st day old birds, humoral immunity was assessed by means of ELISA on serum samples taken from 7 and 21day-old birds.

Three days post vaccination, the average of optical density (OD) showed significant difference between vaccinated (0.553) and unvaccinated (0.686) birds (p≤0.05). In 21 day-old birds, the OD of unvaccinated-unchallenged (negative control) groups (0.331) differed significantly with vaccinated-unchallenged (0.663) birds. The average of lesion score in vaccinated-challenged birds (2.22) showed significant dissimilarity with unvaccinated-challenged groups. No difference and correlation were observed in comparing average of weight gain and oocyst count with serum optical density among treatment and control groups.

The results indicated that ELISA can be used for evaluating immunity uniformity of a flock after vaccination. Besides inducing antibody responses comparable to challenge with wild oocysts, vaccination with live attenuated coccidiosis vaccines may have inhibitory effects in intestinal lesion scores which are responsible for pathogenesis and economic loss during coccidial infections.

The immunity and health of newborn calf are related to immunoglobulins uptake from colostrum where treatment of colostrum may improve the uptake. The purpose of this study was to investigate the effect of controlled heat-treating of colostrum and antibiotic addition on immunoglobulins absorption, performance and health parameters of calf. The colostrum pool was prepared from multiparous dams and was frozen after the treatments. Colostrum was fed immediately after the birth three times a day for 2 days. Twelve newborn Holstein calves were divided into three treatment groups including 1) raw colostrum (control), 2) heat-treated colostrum, and 3) colostrum with oxytetracyclin in a completely randomized design. Blood samples were obtained in days 0, 7, 14, 21 and 28 via jugular vein. Health score and fecal point were determined daily. Results showed that heat-treating of colostrum increased immunoglobulins uptake and their apparent efficiency of absorption (p<0.01), where a 24% increase in blood immunoglobulins concentrations and a 15% in their apparent efficiency of absorption were observed by heating the colostrum compared with control group and a lower increase was observed compared with antibiotic group. No significant differences were observed in weight gain, weaning time and weight as well as the amount and time of beginning starter intake among groups. Feed conversion ratio did not affected by the treatments. There were no significant differences in blood cells counts among groups. Calves fecal point was not affected by the treatments but health score was increased by using heat-treated colostrum where scour incidence was reduced (p<0.05). Results of the study showed that controlled heat-treating of colostrum is an effective and practical method to improve immunoglobulins absorption and reduce scour incidence in calf.

Outbreak and spread of bacterial infections resulted in increasing use of antibiotics which subsequently leaded to bacterial resistance to conventional antibiotics.

To evaluate the effects of organic acids (Selko-pH®), Probiotic (PrimaLac®) and Echinacea purpurea (EP) alcoholic extract on the immune system of the broiler chickens, a completely randomized design experiment was conducted over 120 one-day-old male broiler chicks (Ross 308) with 4 treatment groups that each treatment consisted of 3 replications with 10 birds in each pen.

Groups were designed as follow: group one received organic acid constantly in drinking water (1:1000 v/v) until 14th day and then received 8 hours in a day consecutively (OA); group two received Probiotic (PrimaLac®) water soluble (by 14th day) and mix in the feed (until the end of the rearing period) (PM); group three received EP alcoholic extract in drinking water (1:1000 v/v) like organic acid group and group four were considered as control (received no feed additives).

All groups except the control increased the relative weight of the spleen and Bursa of Fabricius as lymphoid tissues; however, this increase was statistically significant only in the case of EP group (p≤0.001). Data of antibodies test against sheep red blood cells and heterophil to lymphocyte ratio also showed that all groups except the treatment of organic acid had significant differences in terms of specific and cellular immune responses (p<0.01). The data of total count of bacteria and also Lactobacilli bacteria in cecal’s content obviously showed that the number of Lactobacilli bacteria significantly increased in PM and OA treatments (p≤0.05).

Feed additives used in these experiments have the ability to improve the immune system.

The main part of ovary is consisted of follicles which certain drugs may cause change in them. The purpose of this study was to investigate the effects of Methylphenidate on ovarian follicle of mice, treated by Ritalin before puberty. 40 immature female mice at 3 weeks of age were divided into 4 groups, consisting of one control and 3 experimental groups. The experimental groups were gavaged by 2, 5 and 10 mg/kg methylphenidate respectively and the control group received only distilled water with the same method for 60 days. At the end of the experiment, the mice were weighed and then the serum levels of FSH and LH were assessed and structural changes of ovarian follicles and corpora lutea were studied. The mean difference of body weight in experimental groups compared with the control group which showed a significant reduction (p<0.05). In experimental groups compared with the control group, a significant reduction in pre enteral, enteral follicles, corpora lutea and a significant increase in atretic follicles were observed (p<0.05). Ritalin intake for a long period may increase the number of atretic follicles and decrease corpora lutea, so subsequently results in reduction of the growth of follicles and oocytes as well as inducing the atypical appearance of the cells in the luteinized cells.

K99 pilus antigen is one of the major adherence factors found on enterotoxigenic Escherichia coli (ETEC) of neonatal calves. It causes severe diarrhea in newborn calves via the production of heat-stable enterotoxin (STa).With increasing concern over the spread of antimicrobial resistance, the development of alternative to conventional antibiotics such as colicin is urgently needed. Colicin is an antimicrobial peptide produced by one strain of E. coli to suppress the growth of other strains of E.coli. The purpose of this study was to investigate the control of E.coli k99 and the efficacy of colicinogenic E.coli (CEC) in adult mice. The mice, used antibiotic were divided into four groups. The first group did not receive any inoculation. The second group was fed just with 0.5 ml colicin solution. The third group was fed just with 0.5 ml E.coli k99 suspension. The fourth group was first fed by 0.5 ml E.coli k99 suspension immediately after oral administration of CEC suspension. Fecal samples of mice in four groups were taken 1, 2, 3, 4, 5, 6 and 7 days after inoculation and colony forming units (CFUs) were monitored per gram feces. The results showed that CEC has inhibitory effect against E.coli k99. There were observed significant differences between the amounts of E.coli k99 recovered from the feces of mice in fourth group with the amount of E.coli k99 recovered from the feces of mice in third group. The data presented here support this claim that CEC plays a significant role against E.coli k99. Furthermore, the study suggested colicin warrants further evaluation as a potential alternative to conventional antibiotics for use to control of E.coli k99.

Causing site direct mutation can be one of the efficient methods to evaluate the characteristics and properties of various genes. Brucellosis is the most common zoonotic infectious disease that would cause great economic losses. Thus, recognition of pathogenic and immunogenic factors in the genus Brucella can lead to control this health problem.

Considering the importance of site direct mutation in identification of genome structure and numerous ways to achieve this goal, Overlap Extension PCR is introduced as an improved technique for the removal and replacement of the gene target.

For this study, with two-step PCR using specific primers, upstream and downstream fragments from target gene and antibiotic resistance cassette from plasmid pET28a (+), were reproduced and were connected to each other. The resulting fragment was cloned in specific position of pBluescriptIISK(-) plasmid by the restriction enzymes. Then, the construction was transferred into the genome of Brucella abortus by electroporation method.

Fusion PCR product was obtained without any change in the nucleotide sequence and then it was cloned into pBluescriptIISK (-) plasmid, finally the construction was replaced and the target gene was deleted.

The results of this study show that the Overlap Extension PCR is an optimized and modified technique to create mutations in the bacterial genome structure and can easily be used in the family Brucella.

Considering the difference between the rate of multiple ovulation and ovarian activities of different breeds of ewes in different regions, especially out of seasonal breeding condition, finding efficient and effective protocols to induce multiple ovulation out of seasonal breeding would be much appreciated. This study was conducted to determine fertility and multiple ovulation rates during summer and investigate the effect of supplementary feeding, and eCG injection prior to mating in the reproductive performance of Zel ewes. METHODES: At the beginning of summer, 135 ewes in Savadkooh region (north of Iran) were randomly selected and divided into six groups. The estrus cycles of ewes, except in control groups, were synchronized using a vaginal sponge for 14 days. In groups 1 (n=22) and 2 (n=22) ewes were fed with supplementary feeding for 21 and 8 days, respectively, prior to mating. In Group 3 (n=26) ewes were fed with the same ration as Group 2 and were injected (IM) with 400 IU eCG at the time of sponge withdrawal. Ewes in Group 4 (n=23) just grazed on the pasture and injected with eCG as Group 3. In Group 5 (n=20) the estrus of ewes was synchronized as other groups, but they were not received any other treatment. Ewes in Group 6 (n=22, Control) received no treatment, and as the same Group 1 and 2 were mated with fertile rams for eight weeks. Experimental ewes were mated with fertile rams (1:8) 48 hrs after sponge removal for 96 hrs. The findings showed that the fertility in group 3 was significantly more than group 2 and 5, but there was no significant difference between the others (p>0.05). The prolificacy and fecundity were significantly higher in groups treated with eCG than the others (p<0.01), however there was no significant difference between groups 3 (with flashing) and 4 (without flashing). It could be concluded that vaginal sponge and eCG treatment may improve the multiple lambing and reproductive performance of Zel ewes during summer, and supplementary feeding prior to mating, as described in this experiment, cannot make any significant improvement in multiple lambing.

Sheep are a form of investment and a quickly liquidatable resource, particularly in traditional and low income production systems. Tropical and long warm-season regions always affect sheep production negatively. In this experiment 15 female and 7 male sheep were chosen and their body temperature, heart rate, respiration rate measurements and blood sample for biochemical parameters analysis were taken during May 5 to September 5. Heart rate and respiration rate in male sheep were a little higher compared with female sheep but there was no significant difference between them respiration (56 vs. 55) and beat (120 vs. 118 per min). Rectal temperature wasn’t significant between two sexes (40.6-40.09 C˚). Also skin temperature wasn’t significantly different between two sexes (36.02- 36.08 C˚). The only difference was related to month effect (p<0.05). Sex and month hadn’t significant different effects on blood urea, creatinine, glucose and potassium concentration. Blood urea concentration of female sheep was not significantly higher than male’s (p>0.05). Blood Sodium concentration was significantly different between two sexes so that male sheep had the highest minimum average (p<0.05). There was no significant increase in blood Potassium concentration of female sheep compared to male sheep (p>0.05). These results indicated that sheep of this region had been well adapted to summer heat stress and they showed usual changes of blood metabolites in response to heat stress.

Heat stress declines the performance through physiologic changes of the poultry. This experiment was conducted to evaluate the effects of different levels of L-carnitine on performance, carcass characteristics and some blood parameters of broiler chickens fed with high fat diets under heat stress. Two hundred one-day-old broiler chicks (Ross 308) were used in a completely randomized design by four treatments and five replicates (pen) 10 birds in each replicate. The chickens of experimental treatments were fed with basal diet (control group) and the diets plus the different levels of 100, 200 and 300 mg/kg L-carnitine. The experimental diets were used during the finisher period (day 24 to 42 of age) and under heat stress (32±1ºC as cyclic from 9.00 AM to 5.00 PM). The results showed that the chicks fed with the highest L-carnitine had the higher weight gain during the finisher period as compared to control and the lower L-carnitine fed chicks (p<0.05). Furthermore, the feed conversion ratio of birds fed with the medium and high L-carnitine levels (100 and 300 mg) was lower than that of control birds (p<0.05). In orthogonal contrasts, L-carnitine supplementation caused the improvement in weight gain and feed conversion ratio during both the finisher and whole experimental periods (p<0.05). The consumption of highest L-carnitine decreased the abdominal fat as compared to control (p<0.05). In orthogonal contrasts, L-carnitine consumption caused the higher glucose and lower abdominal fat in comparison to control (p<0.05). CONLUSIONS: Totally, the results of recent experiment show that consumption of 300 mg/kg L-carnitine improves the performance of broiler chickens through the increase in dietary fatty acids and energy.

The use of feed withdrawal for molt induction can negatively affected the structure and microbial environment of intestine and lead to greater intestinal colonization by salmonella infection. The aim of the present experiment was to determine the effects of alfalfa as a high fiber ingredient for molt induction on intestinal morphometric characteristics and performance of commercial laying hens. In this experiment 108 Hy-line (W36) laying hens aged 74 weeks in a completely randomized design with 3 treatments and 6 replicates were used. Using treatments for 12 days molt period were included: 1- control group fed with layer ration (FF), 2- feed withdrawal group (FW), 3- group fed with 90% alfalfa and 10% layer ration (A90). Performance of birds was monitored for 12 weeks after the end of the molting period. Feed withdrawal hens had lowest villus height in all three regions of the intestine (p<0.05).The highest average of deudenal crypt depths was observed in FW hens. In all three regions of the intestine, the lowest amounts of villus index and villus surface was observed in FW hens (p<0.05). In all three regions of the intestine, the highest and lowest average of goblet cells was seen in the A90 and FW hens respectively. The highest mean of post molt egg mass was observed in FW hens (p<0.05). The best FCR was seen in the A90 group. The use of alfalfa-riched feed for molt induction of laying hens results in improvement of morphometric characteristic of intestine and post molt performance.