First Report of Isolation and Characterization of Acanthamoeba spp. from the Milk Used for Calf Feeding.

PURPOSE: Acanthamoeba spp. can be found in natural and artificial environments, which reflects their high adaptability to different conditions. Based on the available data, there is scarce information about the isolation of amoeba from milk. This study aimed to investigate the probable presence of Acanthamoeba in milk used for calf feeding. METHODS: 200 milk samples from 50 industrial and traditional farms were collected. The samples were filtered and cultured on the 1.5% Non-nutrient agar medium. The amoebic growth was examined with an inverted microscope daily. DNA was extracted from the positive plates, and a PCR reaction was undertaken using the primers amplifying the Acanthamoeba 18 S rRNA gene. Five samples were purified and sequenced using specific primers. Maximum likelihood reconstructions were performed using the phylogenetic program MEGA software. The osmo and thermotolerance of isolated trophozoites were examined as well. RESULTS: Out of 200 milk samples, Acanthamoeba was isolated from 27 (13.5%). The phylogenetic tree represents that all the isolates belonged to the genotype T4. Results of thermo and osmotolerance tests showed that isolates could develop at 37 and 43 ◦C. Besides, trophozoites survived at 0.5 M mannitol and 1 M. CONCLUSION: For the first time, Acanthamoeba spp. were isolated from milk used to feed dairy calves. Due to Acanthamoeba's neglected role in pathogen persistence and survival, hygiene instructions should be reconsidered.

Determination of the fate of Cholecalciferol injected by the basis of 25D3 plasma concentration.

Sun exposure in bovines is believed to be the most important route of 25D3 synthesis in suitable latitudes. In some situations, e.g. breeding systems, solar radiation cannot reach or penetrate into the skin and thus causes the 25D3 deficiency. Because of the critical effect of vitamin D on the immune and endocrine systems, the plasma must be enriched with 25D3 in a short period of time. In such a condition, injection of Cholecalciferol has been recommended. However, to our knowledge, the certain dose of Cholecalciferol injection for rapid 25D3 plasma enrichment has not been verified. On the other hand, it seems that the basis 25D3 concentration can influence or shift the 25D3 metabolism at the injection time. In the same line, the present study, designed to induce the different basis 25D3 concentration in treatment groups, aimed at investigating the effect of Cholecalciferol intramuscularly injection with the intermediate dose (11,000 IU/kg) on the calves' plasma 25D3 with different basis 25D3. Besides, an attempt was made to clarify the time that 25D3 reaches the sufficient concentration after injection in different treatment groups. To do this, twenty calves of 3 to 4 months old were chosen for the farm with semi-industrial elements. Furthermore, the effect of optional sun exposure/deprivation and Cholecalciferol injection on the 25D3 concentration variations was assayed. To do this, the calves were divided into four groups. Groups A and B were unconstrained to choose sun to expose or shadow in a semi-roofed place, but groups C and D were restricted to the completely dark barn. The interference of the digestive system in supplying vitamin D was minimized through dietary. All groups had a different basic concentration (25D3) on the day 21 of the experiment. At this time, groups A and C received the intermediate dose of (11,000 IU/kg) Cholecalciferol intramuscularly (IM). After Cholecalciferol injection, the effects of basis 25D3 concentration on the details of variation and fate of plasma concentration of 25D3 were investigated. The data collected from the two groups C and D showed that sun deprivation without any vitamin D supplementation, could rapidly and severely deplete the plasma from 25D3. Cholecalciferol injection could not immediately increase the 25D3 in the groups C and A. However, this injection enriches the 25D3 to sufficient value after two weeks if the basis 25D3 of plasma is insufficient, i.e. less than 30 ng/mL. Moreover, the injection of Cholecalciferol could not significantly increase the 25D3 concentration in the group A that had a sufficient basis 25D3 concentration. Therefore, it is concluded that the variation of 25D3 in plasma, after injection of Cholecalciferol, depends on its basic level at the time of injection.

The role of small ruminants in the epidemiology of leptospirosis.

Leptospirosis is a common global zoonotic disease of man and all farm animals. Although most leptospiral infections in sheep and goats are asymptomatic, they may play a role in the epidemiology of the disease by the spread of Leptospira through the urine. This study was carried out to evaluate the role of sheep and goats in the epidemiology of leptospirosis. Blood and urine samples were taken from 210 goats and 246 sheep. To detect antibodies, sera samples were tested with 8 live serovars of L. interrogans (Hardjo, Pomona, Grippotyphosa, Canicola, Ballum, Icterhemorrhagiae, Tarasovi, and Australis) by MAT. Then, urine samples were tested by Nested PCR targeting 16S rRNA gene for detection of pathogenic Leptospira. Results of MAT showed that 10.95% of goats and 8.53% of sheep had antibodies against at least one examined serovars. In both species, the highest reacting was L. i. Pomona with a rate of 68.18% and 56% in sheep and goats, respectively. Moreover, in PCR, 2 (0.95%) urine samples of goat and 12 (4.87%) urine samples of sheep were positive. All of the MAT positive studied animals were PCR negative and, statistical analysis showed that there was no relationship and agreement between the results of PCR and MAT in sheep (kappa = - 0.07, p > 0.05) and goats (kappa = - 0.02, p > 0.05). Finally, it is concluded that sheep and goats can excrete L. interrogans in the urine and thus transmit them to other animals and humans.

Comparison of Mycobacterium avium subsp. paratuberculosis infection in cattle, sheep and goats in the Khuzestan Province of Iran: Results of a preliminary survey.

BACKGROUND: Paratuberculosis or Johne's disease, the chronic infectious granulomatous enteritis of ruminants, is a worldwide infection, which is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The most common symptoms of this disease in cattle are loss of milk production, weight loss and diarrhoea, whereas in sheep and goats, the symptoms are emaciation, anorexia and severe disability. OBJECTIVES: The aim of this study was to compare the seroprevalence of MAP in cattle, sheep and goats in the southwest of Iran. METHODS: Blood samples were randomly collected from 530 cattle, 568 sheep and 368 goats in southwest of Iran. Sera were tested by a commercial ELISA kit (ID vet; ID Screen® Paratuberculosis Indirect) for detection of antibodies of MAP. RESULTS: Overall apparent and true seroprevalence rate of MAP was 6.00% (95% CI: 4.90%-7.30%) and 13.25% (95% CI: 11.55%- 14.95%). Apparent and true seroprevalence of MAP, respectively, was 4.34% (95% CI: 3.88%-6.46%) and 9.19% (95% CI: 6.98%-11.98%) in cattle, 6.87% (95% CI: 5.05%-9.27%) and 15.37% (95% CI: 12.60%-16.60%) in sheep and 7.07% (95% CI: 4.82%-10.18%) and 15.86% (95% CI: 12.41%-20.01%) in goats, respectively. As a result, there was no significant relationship between animal species and MAP infection. Moreover, multivariate logistic regression showed that the infection rate is not associated with age, gender and geographical location in cattle, sheep and goats (P > 0.05). CONCLUSION: This study confirms that the seroprevalence of MAP is relatively considerable in the cattle, sheep and goats in the southwest of Iran, although in cattle, it is less than goats and sheep. Therefore, preventive and control measures should be considered by animal health authorities and meat and dairy processing units.

Designing an enzyme-linked immunosorbent assay for detection of anti-penicillin antibodies levels in various species of animals in Khuzestan province, Iran.

The penicillin allergy is being increasingly recognized as a significant public health problem. Immunological responses to penicillin and other beta-lactam antibiotics are classified as immediate and non-immediate responses. This research aimed to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of the reactive antibody value against penicillin in various species of animals. The serum samples were collected from nine species (forty mature animals in each species) including horse, dog, goat, sheep, buffalo, cattle, donkey, chicken, and fish. The concentrations of total antibody and immunoglobulin M (IgM) against penicillin were detected using an in-house ELISA test. The total anti-penicillin antibodies concentration from high to low in animals was as chicken, horse, fish, donkey, dog, goat, sheep, buffalo, and cattle, respectively. In cattle and sheep, the level of anti-penicillin IgM (APM) was significantly higher than non-IgM antibodies (APNM). Moreover, levels of APNM were very low in chicken and fish serums; no difference was seen regarding these values in buffalo and goat. The other species had significantly lower APM than the APNM. The ani-penicillin antibody levels in the noted animals were successfully detected using the developed ELISA. Most of the species have anti-penicillin antibodies; however, they have reactive antibodies with differences in levels and isotypes.

The role of sheep in the epidemiology of Bovine alphaherpesvirus 1 (BoHV-1).

Bovine alphaherpesvirus 1 (BoHV-1) as a cusative agent for some diseases in cattle infects sheep and goat; and it is believed that these animals may be reservoir host for this virus. Thus, BoHV-1 infection in sheep and goat should be considerd when there is a program for control and eradication of this virus in cattle. Therefore, the aim of this study was to determine the seroprevalence of BoHV-1 in sheep, relationship between host and environmental factors with infection, and the role of sheep in the epidemiology of the BoHV-1. Blood samples were randomly collected from 310 healthy sheep in 6 cities of Khuzestan province (Southwest of Iran) including Ahvaz, Hendijan, Shushtar, Dezful, Masjed Soleyman and Behbahan. Sera were analyzed by virus neutralisation (VN) test for detection antibodies to BoHV-1. According to VN test, apparent and true seroprevalence seroprevalence of BoHV-1 infection was 28.4 % (95%CI: 23.4-33.4%) and 28.4 % (95%CI: 23.3-33.4%), respectively. Logistic regression revealed that the odds of infection between the age was 1.06 (95%CI: 0.9-1.25) (P > 0.05), implying that the odds of infection increased 6 % with rising one year of age. Besides, the relative frequency of infection in males was more than females', and the odds of infection in male sheep was identified to be 1.13 (95%CI: 0.47-2.71) (P > 0.05), compared to that in females. Moreover, in comparison to sheep with no history of abortion, the odds of infection in sheep with a history of abortion was 1.28 (95%CI: 0.57-2.87) (P > 0.05). The seroprevalence in Shushtar, Masjed Soleyman, Dezful, Ahvaz, Hendijan, and Behbahan were found to be 48.3, 46.7, 31.7, 20, 16.7, and 12 percent, respectively and 13.1 of fluctuation in infection can be justified by different geographical locations investigated in this study (P < 0.001). Considering the significant seroprevalence of BoHV-1, present study confirmed the role of sheep in the epidemiology of this virus and control of BoHV-1 in sheep should be considered by animal health authorities in areas where BoHV-1 is prevalent.

Isolation and identification of bubaline herpesvirus 1 (BuHV-1) from latently infected water buffalo (Bubalus bubalis) from Iran.

In order to isolate buffaloes herpesvirus 1 (BuHV-1) from latently infected water buffalo (Bubalus bubalis), 16 buffalo heifers were selected from a herd. At first, animals were bled and their sera were tested by virus neutralization (VN) test, using bovine herpesvirus 1 (BoHV-1). According to the results of VN test and dexamethasone injection (0.1 mg/kg BW) for 5 consecutive days, the examined buffaloes were divided into 4 groups. Vaginal and nasal swabs were daily collected from all buffaloes from day 0 to 10 days later. Based on the cytopathic effects in cell culture, a herpesvirus was isolated only from nasal swabs of three seropositive buffaloes which they had received dexamethasone. The nasal swabs of these three buffaloes were also positive in PCR, using primers specific for ruminant herpesviruses gD gene. The identity of the isolated viruses was determined according to partial amino acid sequences of gD, deduced from the nucleotide sequences of the PCR products. On the basis of sequence alignment, phylogenetic analysis, and genetic distances, the three buffalo virus isolates were more closely related to BuHV-1 and BoHV-5 than to BoHV-1.

Simple Indirect Enzyme-Linked Immunosorbent Assay to Detect Antibodies Against Bovine Viral Diarrhea Virus, Based on Prokaryotically Expressed Recombinant MBP-NS3 Protein.

BACKGROUND: Bovine viral diarrhea (BVD) is an economically important disease of cattle distributed worldwide. Diagnosis of BVD relies on laboratory-based detection of its viral causing agent or virus specific antibodies and the most common laboratory method for this purpose is Enzyme-Linked Immunosorbent Assay (ELISA). OBJECTIVES: The current study was aimed to develop a simple indirect ELISA to detect antibodies against Bovine Viral Diarrhea Virus (BVDV) in the sera of infected cattle. MATERIALS AND METHODS: A new simple indirect ELISA method was developed to detect BVDV infection by prokaryotically (Escherichia coli, BL21 strain) expressed recombinant whole nonstructural protein 3 (NS3) of BVDV (NADL strain). Four hundred bovine serum samples were evaluated by the newly developed NS3-ELISA and virus neutralization test (VNT) as the gold standard method to diagnose BVD. Among these samples, 289 sera had been previously tested by a commercial ELISA kit. RESULTS: Statistical analyses showed a very high correlation between the results of the developed NS3-ELISA and VNT (kappa coefficient = 0.935, P < 0.001), with the relative sensitivity and specificity of 94% and 98.8%, respectively. There was also a high correlation between the results of NS3-ELISA and the commercial ELISA kit (kappa coefficient = 0.802, P < 0.001) with the relative sensitivity and specificity of 90.72% and 91.15%, respectively. CONCLUSIONS: The newly developed simple indirect ELISA showed high sensitivity and specificity with respect to VNT. Developing such a simple, sensitive, and specific ELISA which is much less expensive than the available commercial ELISA kits can improve the detection of BVDV infections, help to eliminate the disease from herds, and decrease economic losses caused by this disease.