Relationship between different enteric viral infections and the occurrence of diarrhea in broiler flocks in Jordan.

The aim of this study is to determine if enteric viruses are the cause of diarrhea in broiler flocks in Jordan. Intestinal content samples were collected from 101 broiler flocks from several regions of Jordan to detect the presence of astrovirus, coronavirus, reovirus, and rotavirus, by using reverse transcriptase polymerase chain reaction (RT-PCR). Forty-six of these flocks were clinically healthy with no enteric disease, and the other 55 flocks were clinically suffering from diarrhea. The samples were collected between 5 and 16 d of age. The results show that 79% of total 101 flocks tested were infected with one or more of the above enteric viruses. Coronavirus was the most common virus, detected in 56.4% of these flocks, with astrovirus in 29.7% of the flocks, and rotavirus (9.9%) and reovirus (5.6%) being the least common. None of these flocks were found to be infected with all four viruses, but one of the flocks was found to be infected with astrovirus, coronavirus, and rotavirus simultaneously. Individual infection was noted with astrovirus, coronavirus and rotavirus but not with reovirus, whereas all flocks infected with reovirus were also infected with coronavirus. There was no statistical evidence to link these viruses as the main cause of diarrhea in the flocks tested. This is the first study in Jordan to detect all of these viruses and to correlate their presence with diarrhea in chicken flocks.

West Nile virus infection in horses in Jordan: clinical cases, seroprevalence and risk factors.

The objectives of this study are to report clinical WNV infection in horses and to determine the seroprevalence of and risk factors for WNV infection in horses in Jordan. In late summer and early fall of 2012, two mares were presented for evaluation of neurological signs. The first mare had hind-limb ataxia. The second mare was slightly depressed and lethargic. She had ataxia in her four limbs and cranial nerves deficits. Both horses were found positive for WNV IgM antibodies using commercial IgM-capture ELISA test. Both horses were treated symptomatically and recovered uneventfully. The occurrence of clinical cases initiated the need for a seroprevalence and risk factors study. Two hundred and fifty-three normal horses were randomly enrolled in the study. Enrolled horses were grouped into five major regions according to the geographical proximity and climatic similarities. From each region, around 50 horses were sampled. The serum collected from each horse was screened by a competitive ELISA, and those that reacted positive using the previous ELISA test were further tested using commercial IgM-capture ELISA test. Sixty-three horses (24.9%) of the 253 surveyed were seropositive to WNV. Of the 63 horses, none had IgM antibodies for WNV. The region with the highest prevalence was Jordan Valley and Balqa. Horses used for polo (OR = 9.77; 95%CI = 1.32-25.44) and horses located in Jordan Valley and Balqa region (OR = 13.31; 95% CI = 2.33-32.54) were identified as risk factors for seropositivity to WNV in Jordan. These risk factors were attributed to the hot and humid weather, which enhance vector availability. West Nile virus appears to be endemic in Jordan. Future studies are warranted to evaluate the virus situation in the country during the next few years in an attempt to control it.

Equine babesiosis: seroprevalence, risk factors and comparison of different diagnostic methods in Jordan.

The purposes of this study were to estimate the seroprevalence and distribution of horse piroplasmosis, to evaluate risk factors associated with the occurrence of the disease and to compare the different diagnostic methods used for this disease. A total of 253 clinically normal horses were sampled, and a collection form was completed for each horse from five of six different climatic zones of Jordan. The sixth zone was not sampled because it did not include horse population. Competitive enzyme-linked immunosorbent assay (cELISA) revealed 37 horses (14.6%) positive for Theileria equi, and none of the horses was positive for Babesia caballi. Microscopic examination of thin blood smears and PCR test revealed no positive results for either parasite. Grazing was the only risk factor that was associated with being seropositive to the disease; horses that graze are 11.5 more likely to be seropositive (P<0.05, OR=11.5, CI: 3.292, 39.962). This is the first study to estimate the prevalence of horse babesiosis using serological test and to identify risk factors associated with the disease in Jordan. Competitive enzyme-linked immunosorbent assay (cELISA) test appears to be more reliable than microscopic examination and PCR in estimating the seroprevalence of the disease as well as identifying carrier horses to babesiosis.

Genotype and allelic frequencies of a newly identified mutation causing blindness in jordanian awassi sheep flocks.

A total of 423 blood samples were collected (during 2009 and 2010) from all the ram holdings at three major Jordanian governmental Awassi breeding stations (Al-Khanasry, Al-Mushairfa and Al-Fjaje) and two private flocks. All blood samples were screened for the presence of mutations at the CNGA3 gene (responsible for day blindness in Awassi sheep) using RFLP-PCR. The day blindness mutation was detected in all studied flocks. The overall allele and genotype frequencies of all studied flocks of the day blindness mutation were 0.088 and 17.49%, respectively. The genotype and allele frequencies were higher in station flocks than the farmer flocks (0.121, 24.15 and 0.012, 2.32, respectively). Al-Mushairfa and Al-Khanasry stations have the highest genotype and allele frequencies for the day blindness mutation that were 27.77, 30.00% and 0.14, 0.171, respectively. The investigated farmer flocks have low percentages (0.03, 5.88% at Al-Shoubak and 0.005 and 1.05%, at Al-Karak, respectively for genotype and allele frequencies) compared with the breeding stations. Ram culling strategy was applied throughout the genotyping period in order to gradually eradicate this newly identified day blindness mutation from Jordanian Breeding station, since they annually distribute a high percentage of improved rams to farmer's flocks.

Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta) as well as the pro-inflammatory cytokine IFN-gamma were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-gamma stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-beta prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN-gamma. Finally, the results suggest an important role of IL-10 and TGF-beta on the profile of NO production which may explain the low NO production in MAP clinically infected cows.

Prevalence and risk factors associated with bovine viral diarrhea virus infection in dairy herds in Jordan.

A cross-sectional study was carried out to determine the seroprevalence and to identify risk factors associated with bovine viral diarrhea virus (BVDV) infection in 62 non-vaccinated dairy herds (671 cows) in Jordan between January and June 2007. Information regarding herd management was recorded through a personal interview with farmers. Antibodies against BVDV were detected using an indirect ELISA test. Chi-square analysis and multivariable logistic regression model were used to identify risk factors for BVDV seropositivity. The true prevalence of antibodies against BVDV in individual cows and cattle herds was 31.6% and 80.7%, respectively. The seroprevalence of BVDV in medium and large size herds was significantly higher than that in smaller herds. There was no significant difference in BVD seroprevalence between different age groups. Random-effects logistic regression model revealed two major factors associated with seropositivity to BVDV; exchange of visits between adjacent farm workers and not isolating newly purchased animals before addition to the herd. The seroprevalence of BVDV in cows located in the northern Jordanian governorates was significantly higher than that in other studied governorates. Results of this study indicated that BVDV is highly prevalent in Jordan and BVDV infection could be controlled by livestock-trade control, and applying strict biosecurity measures in the dairy farms.

Metabolic profiles in goat does in late pregnancy with and without subclinical pregnancy toxemia.

BACKGROUND: Pregnant goat does can develop various metabolic diseases during late pregnancy that may have profound effects on their health and productivity, including subclinical pregnancy toxemia. OBJECTIVES: This study was performed to evaluate serum biochemical findings in goats in late pregnancy with and without subclinical pregnancy toxemia. METHODS: Serum samples were obtained from 153 clinically healthy goats in the last trimester of pregnancy from 14 herds in central and northern Jordan. Serum was analyzed for the following constituents: 3-hydroxybutyrate (3-HB), glucose, total protein, albumin, urea, creatinine, aspartate aminotransferase, alkaline phosphatase, cholesterol, triglycerides, cortisol, T3, T4, total calcium, magnesium, and phosphorus, using commercially available kits. Goats were classified as having subclinical pregnancy toxemia or as controls on the basis of 3-HB results. Biochemical results were compared between the 2 groups using nonparametric statistical tests. RESULTS: Fifty-three does had subclinical pregnancy toxemia (3-HB>0.86 mmol/L) and 100 were pregnant control does (3-HB< or =0.86 mmol/L). Does with subclinical pregnancy toxemia had significantly lower glucose concentrations (P<.001) and significantly higher urea (P=.042) and total protein (P=.048) concentrations compared with pregnant control does. 3-HB concentration was significantly correlated with glucose (r=0.290, P=.035), phosphorus (r=0.351, P=.01), and cortisol (r=0.394, P=.004) concentrations in goats with subclinical toxemia. CONCLUSIONS: Results of this study indicate that the biochemical profile of does with subclinical pregnancy toxemia had significantly lower glucose (P < .001) and total protein (P = .048)concentrations, whereas urea concentrations were significantly higher (P = .042) compared with pregnant control does [corrected].

External coaptation for the treatment of long bone fractures in cattle: a retrospective study of 35 cases (1996-2001).

The objective of this retrospective study was to report the history, signalment, frequency, types, complications, and long-term outcome of long bone fractures in cattle treated using external coaptation techniques. The sex, age, breed, history, physical examination findings, and radiographic findings were recorded. Of 35 cases with long bone fractures treated using external coaptation, 31 were discharged alive. The mean hospitalization time for these cattle was 2 days. Six to 12 months after treatment, 26 (84%) cases were considered satisfactorily healed. It was concluded that cattle with long bone fractures could be treated successfully using external coaptation with minimal effect on productivity.

Peritoneal fluid analysis in adult, nonpregnant Awassi sheep.

BACKGROUND: To the authors' knowledge, there is no information in the literature about normal peritoneal fluid values in ovine species. OBJECTIVES: The purpose of the study reported here was to establish reference intervals for peritoneal fluid from clinically normal Awassi sheep and to compare the values to those in blood. METHODS: Peritoneal fluid and blood samples were collected into tubes containing EDTA, from 40 clinically healthy, nonpregnant, female Awassi sheep, aged 2 to 7 years. Total nucleated cell count (TNCC) was determined using an electronic cell counter. Total protein, albumin, urea, creatinine, and glucose concentrations and aspartate transaminase activity were analyzed using commercially available kits. RESULTS: TNCC (mean +/- SD) of peritoneal fluid was 1.1 +/- 0.87 X 10(3)/microl, with neutrophils (3.9%), lymphocytes (33.5%), macrophages/monocytes (61.2%), and eosinophils (1.4%). Biochemical results in peritoneal fluid were: total protein, 1.7 +/- 0.74 g/dL; albumin, 1.0 +/- 0.04 g/dL; urea, 12.6 +/- 3.95 mg/dL; creatinine, 0.6 +/- 0.19 mg/dL; glucose, 54.8 +/- 6.11 mg/dL; and aspartate transaminase, 23.5 +/- 8.82 U/L. Eosinophil percentage and creatinine concentration did not differ significantly from blood values. CONCLUSION: Baseline values for cytologic and biochemical parameters in peritoneal fluid of Awassi sheep, with comparison to blood, have been generated. Such data may be applicable to other ovine species and can be used in the clinical investigation of ovine abdominal disorders.

Studies on the mechanism of diarrhoea induced by Escherichia coli heat-stable enterotoxin (STa) in newborn calves.

Enterotoxigenic Escherichia coli (ETEC) produces a heat-stable enterotoxin (STa) that binds to and activates a putative intestinal receptor, guanylate cyclase, causing an increase in the intracellular levels of cyclic guanosine monophosphate (cGMP). Using flow cytometry and 125I-STa binding assays, we studied the distribution of STa-receptors on enterocytes isolated from different segments of the newborn calf's intestinal tract. We also investigated the effect of STa on the intracellular levels of cGMP and ion transport to the intestinal lumen. More STa-receptors were found on enterocytes prepared from the ileum than on enterocytes obtained from the other segments of the intestinal tract. Guanylate cyclase activity was higher in the ileum of STa-challenged calves than in the ileum of control calves. No changes were observed in the guanylate cyclase activity of the other intestinal segments of the STa-challenged and control calves. Na+ levels, as measured by atomic absorption spectroscopy, were significantly increased in the luminal contents of the ileum of STa-challenged calves, whereas serum Cl- levels were significantly lower in the STa-challenged calves than in control calves. This study supports previous observations on the role of guanylate cyclase in the initiation of STa-induced secretory diarrhoea and suggests that Na+/Cl- coupling may be the major mechanism for the loss of ions in the diarrhoeal response that is mostly induced in the ileum of newborn calves.

Characterization of the interaction of Escherichia coli heat-stable enterotoxin (STa) with its putative receptor on the intestinal tract of newborn calves.

Enterotoxigenic Escherichia coli (ETEC) induces severe diarrhea in newborn calves through the elaboration of heat-stable enterotoxin (STa). We investigated the distribution and characteristics of the STa-specific receptors on enterocytes and brush border membrane vesicles (BBMVs) prepared from anterior jejunum, posterior jejunum, ileum and colon of newborn calves. We found that density of the receptors and their affinity to STa were higher on enterocytes and BBMVs that were derived from the ileum than enterocytes and BBMVs prepared from other segments of the calf intestine. This study suggests that, in newborn calves, the ileum is the major part of the intestinal tract that is affected in the course of secretory diarrhea caused by STa-producing ETEC strains.

Characterization of the interaction of Escherichia coli heat-stable enterotoxixn (STa) with its intestinal putative receptor in various age groups of mice, using flow cytometry and binding assays.

BACKGROUND AND PURPOSE: Enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) is a major cause of diarrhea in young animals. Age-dependent variation in the density and affinity of the mouse enterocyte receptors specific for STa was investigated. METHODS: Four age groups (2-day-, 1- and 2-week-, and 2-month-old) of Swiss Webster mice were studied (8 to 10 mice/group). Flow cytometry and radiolabeled STa (125I-STa) assays were used as reliable quantitative measures for characterization of STa-enterocyte receptor interaction. RESULTS AND CONCLUSIONS: Interaction of STa with its putative receptor was stronger for enterocytes of 2-day-old mice. Scatchard analysis of 125I-STa-receptor interaction suggested that STa-receptors exist at higher numbers on enterocytes from 2-day-old (7.2 nmol/mg) than older (0.30, 0.36, and 0.40 nmol/mg for 1-week-, 2-week-, and 2-month-old mice, respectively). Additionally, receptors from 2-day-old mice had greater affinity for STa (Kd = 75 nM) than did receptors from older mice (Kd = 125, 1,430, and 1,111 nM for 1-week-, 2-week-, and 2-month-old mice, respectively). Density of STa receptors on enterocytes and their affinity to STa may determine extent of binding and severity of the secretory response, and may explain the high susceptibility of newborn animals and human infants to STa-mediated diarrhea.

Use of flow cytometry to measure the interaction between Escherichia coli heat-stable enterotoxin and its intestinal receptor in mice.

Binding of Escherichia coli heat-stable enterotoxin (STa) to its putative receptor on the brush border membrane of enterocytes is a prerequisite for the induction of diarrhea in infected humans and animals. Humans and animals of different ages vary in their susceptibility to the effect STa, perhaps due to the difference in STa interaction with its intestinal receptor. Flow cytometry was compared to indirect immunofluorescence and 125I-STa binding assays to measure the STa-enterocytes receptor interaction in different age groups of Swiss Webster mice (2-, 7-, 14-day-old). Flow cytometry indicated stronger interaction between STa and its putative receptor on enterocytes from the 2-day-old mice than enterocytes from older mice. 125I-STa-binding assay suggested that the stronger fluorescence intensity on enterocytes from younger mice is due to higher STa receptor density and higher receptor affinity to STa. Flow cytometry is more sensitive quantitative assay to measure the interaction between STa and its intestinal receptor than indirect immunofluorescence microscopy.

Insulin modulates intestinal response of suckling mice to the Escherichia coli heat-stable enterotoxin.

Effect of insulin on the response of suckling mice to the enterotoxigenic Escherichia coli heat-stable enterotoxin (STa) was studied. Four groups (8-10 in each group) of two day old Swiss Webster suckling mice were used. Five, 10, 25, and 50 micrograms of insulin were given orally to half the mice in each group respectively. The rest of the mice in each group were given normal saline as intra-litter controls. After 7 days, the suckling mouse assay for STa was performed on three mice from each insulin-treated and control groups. Enterocyte suspensions were prepared from mice in all groups. Intestinal tissue samples were taken for electron microscopy. Interaction of STa with its putative receptor on the enterocytes was evaluated using indirect immunofluorescence and flow cytometry. The suckling mouse assay revealed a significant increase in the gut weight to body weight ratio in all mice in the insulin treated groups compared to control mice (p < 0.05). Flow cytometry and indirect immunofluorescence analyses suggested that insulin had an upregulatory effect on the STa receptor level. Similarly, insulin was found to increase intestinal brush border membrane differentiation as indicated by the increase in the inward movement of milk particles through the intestinal mucosa. Insulin seems to modify the structure-function of the brush border membrane including the response of suckling mice to STa. This study may provide further insights into the mechanism of STa/receptor interaction in diarrhea in newborn animals and human infants.

Age-dependent variation in the density and affinity of Escherichia coli heat-stable enterotoxin receptors in mice.

Enterotoxigenic strains of Escherichia coli that produce heat-stable enterotoxin (STa), are a major cause of diarrheal disease worldwide. Resistance to diarrheal disease in human infants and newborn animals has been attributed to a gradual turnover in the intestinal brush border membrane receptors to bacterial pili. In this study, we demonstrated age-dependent variation in the density and affinity of the mouse enterocyte receptors specific for STa. Flow cytometry and radiolabeled-STa (125I-STa) assays were used as more reliable quantitative measures for the characterization of STa-enterocyte receptor interaction. These assays indicated a stronger interaction of STa with its putative receptor on the enterocytes of the 2-day-old suckling mice than with enterocytes from 1-week, 2-week and 2-month-old mice. Scatchard plot analysis of 125I-STa-receptor interaction suggested that STa-receptors exist at a higher number on enterocytes from the 2-day-old mice than enterocytes of the older mice. Additionally, receptors from the 2-day-old mice had a greater affinity for STa ligand than receptors from the older mice. Density of STa receptors on enterocytes and their affinity to STa may determine the extent of binding and severity of secretory response. This may further explain the increased susceptibility of newborn animals and human infants to STa-mediated diarrheal disease.

Epidemiological studies of clinical and subclinical ovine mastitis in Awassi sheep in northern Jordan.

Forty-six Awassi sheep flocks selected by stratified random sampling were subjected to a cross-sectional study to determine the prevalence of intramammary infections, to assess the influence of flock size and parity on the prevalence of somatic cell count (SCC) and to identify major udder pathogens. Of the 3472 udder halves examined, 29.8% had over 10(6) SCC/ml and 0.03% had dry teats due to chronic mastitis. Flocks with 30-49 milking ewes (small flock size) were much younger (P < 0.001) than flocks with 50-99 ewes (medium) and flocks with > or = 100 ewes (large). Pairwise analysis of the InSCC of both halves of the udders revealed significant mean differences for small and large flock size (P < 0.05), and for medium and large flock size (P < 0.001). Mean InSCC was lower (P < 0.05) in samples obtained from the left half compared with samples of the right half of the udder. Multiparous ewes had higher (P < 0.001) mean InSCC than primiparous ewes. Also, ewes with twin lambs had higher (P < 0.001) mean InSCC in the right half of the udder compared with single-lamb ewes. Samples collected in January (winter) had lower (P < 0.05) mean InSCC compared with samples collected in June. The most common organisms isolated from subclinical mastitis cases were coagulase-negative Staphylococci (17.8%), E. coli (13.6%), Streptococcus agalactiae (6.8%) and Staphylococcus aureus (6.8%). Of the 46 flocks, 20 were monitored monthly for 9 consecutive months to determine the incidence of clinical mastitis diagnosed by shepherds or/and sheep farmers with major pathogens. The incidence of clinical mastitis (expressed as the number of clinical cases per 100 ewe-months) were 2.1 +/- 1.9 (SD), 1.9 +/- 1.1, and 1.2 +/- 2.1 for small, medium and large flocks size strata, respectively. The overall population estimate was 1.7 +/- 0.02 cases per 100 ewe-months. The most-common clinical isolates were S. aureus (22% of all clinical isolates) and E. coli (14.2%).