Comparison of blood ΒHBA measurement devices for diagnosis of subclinical pregnancy toxaemia in sheep: A field study.

Pregnancy toxaemia is a serious disease that occurs during the last trimester of pregnancy in sheep. Yet, in most cases, the disease may have a subclinical course. This study was aimed at comparing blood ßHBA measurement devices for diagnosis of subclinical pregnancy toxaemia in late pregnant sheep. In the study, a total of 50 blood samples were collected from Romanov (n = 30) and cross-bred Hamdani (n = 20) sheep. Blood ßHBA levels were measured using TaiDoc TD-4235 and CentriVet ßHBA hand-held meter. Randox ßHBA (Ranbut) assay was used as a reference laboratory method to compare hand-held meters. ßHBA value of 0.8 mmol/L was set as the cut-off value for diagnosis of subclinical pregnancy toxaemia. Statistical analyses were carried out using Minitab 21 and Jamovi software. In the study, the correlation of Randox-TaiDoc TD-4235 and Randox-CentriVet was .822 (p < .001) and .728 (p < .001), respectively. Based on the Ranbut assay, nine ewes out of 50 were diagnosed with subclinical pregnancy toxaemia. Specificity (detection of healthy ewes) and sensitivity (detection of ewes with subclinical pregnancy toxaemia) for TaiDoc TD-4235 and CentriVet hand-held meters were 100%, 77.8%, and 100%, 66.7%, respectively. In the receiver operating characteristic (ROC) analysis, areas under the ROC curve (AUC) were 0.976 and 0.920 for TaiDoc and CentriVet, respectively. Bland-Altman analysis revealed a bias of 0.092 mmol/L for TaiDoc and a bias of 0.132 mmol/L for CentriVet. TaiDoc hand-held meter shows a better correlation with the Randox Ranbut assay and greater sensitivity compared to the CentriVet hand-held meter. In conclusion, both TaiDoc and CentriVet hand-held meters can be securely used in the diagnosis of subclinical pregnancy toxaemia in sheep. For these reasons, subclinical pregnancy toxaemia and these devices will be evaluated within the scope of herd management programme in the sheep industry. It should also be taken into account that these conditions will affect the future fertility of the mother and offspring.

Sero-epidemiology and associated risk factors of brucellosis among sheep and goat population in the south western Nepal: a comparative study.

BACKGROUND: Brucellosis is a zoonotic disease caused by Brucella spp. In Nepal, the presence of brucellosis in small ruminants, namely sheep and goats, has impacted farmers' livelihood and the food safety of consumers. A cross-sectional study was conducted in Rupandehi district of Nepal during January to March 2020 to investigate the seroepidemiology and associated risk factors of brucellosis in the sheep and goat population. Altogether, 19 sheep and 60 goat farms in the district were visited. Owners were interviewed to get information on animals, including their management and movement patterns. Three hundred fifty-seven samples (80 sheep and 277 goat samples) were collected proportionately based on farm sizes. Each serum sample was tested with Rose Bengal Test and ELISA to estimate the seropositivity of brucellosis. Logistic regression was carried out to calculate corresponding odds ratios of each variable associated with detection of brucellosis. RESULTS: At the farm level, 31.6% (6/19; 95% CI: 12, 54%) of sheep farms and 3.3% (2/60, 95% CI: 0.9, 11.4%) of goat farms were seropositive to brucellosis. Out of 80 sheep serum samples, 12 (15%; 95% CI: 8.79-24.41%) and out of 277 goat serum samples, three (1.1%; 95% CI: 0.37-3.14%) were seropositive to brucellosis. Age greater than 1.5 years (OR = 5.56, 95% CI: 1.39, 29.38; p = 0.02) and herd size of greater than 100 (OR = 4.74, 95% CI: 1.23, 20.32, p = 0.03) were identified as significant risk factors for seropositivity of brucellosis in the sheep population. While in the goat population, none of the variables was identified as a significant risk factor. CONCLUSION: The study provides evidence that the older sheep and the sheep from the large herds were at higher risk of brucellosis. A control program should be put in place immediately in the sheep population because they may transmit infections to other livestock as they were regularly moved for grazing and selling purposes. Also, strict biosecurity measures should be implemented among pastoralists to prevent brucellosis transmission in them. We suggest further one health-based study to reveal the transmission dynamics of brucellosis between animals and humans.

A highly sensitive semi-nested real-time PCR utilizing oligospermine-conjugated degenerate primers for the detection of diverse strains of small ruminant lentiviruses.

Small ruminant lentiviruses (SRLVs) are highly diverse retroviruses infecting sheep and goats. Although PCR-based testing is being utilized for diagnostics, its application is hampered by various factors. These include, among others, the exceptionally high genetic variability of SRLVs, as well as the low number of infected blood monocytes. For this reason, a highly sensitive and specific semi-nested real-time PCR for proviral DNA detection and quantification was developed. The method is innovative in that a) its design is based on selecting the preferred codon usage in the targeted conserved genomic regions and b) oligospermine-conjugated degenerate primers with increased Tm were utilized. Modifications permitted primer/template duplex formation in the cases of mismatches due to sporadic nucleotide polymorphisms in a number of variant SRLV strains and consequently, the detection of highly diverse SRLV strains. The potential loss of analytical sensitivity and specificity was counterbalanced by including a semi-nested step in combination with LNA probes. An in silico procedure for the evaluation of hybridization efficiency of the designed oligonucleotides to all known targeted variants was also implemented. The method presents a linear range of quantification over a 3-log10 range and a limit of detection of 3.9 proviral dsDNA copies per reaction. Its diagnostic performance was evaluated by testing field samples from seropositive and seronegative animals, followed by phylogenetic analysis of the strains detected. To further increase the diagnostic sensitivity, a DNA extraction protocol for blood leukocytes was developed and evaluated. A minimum of 500 ng input DNA is recommended for PCR-based detection of SRLV proviral DNA, given the low numbers of infected blood monocytes. The developed methodology may serve as a useful tool, which can be adjusted for the quantitative detection of viruses exhibiting high genetic variability.

Serological evidence of Coxiella burnetii infection in slaughtered sheep and goats at Kumasi Abattoir, Ghana.

Q fever, caused by Coxiella burnetii, is an important zoonosis worldwide. Q fever is documented in many parts of the world; however, information on the disease in Ghana is scanty. This study was therefore conducted to provide evidence of exposure of sheep and goats slaughtered at the Kumasi Abattoir to Coxiella burnetii. A total of 350 serum samples collected from 175 sheep and 175 goats were analyzed for the presence of C. burnetii antibodies using a commercial ELISA kit (ID Vet). Results of the study established a seroprevalence of 28.57% in goats, 16.57% in sheep and an overall seroprevalence of 22.29% in sheep and goats; 20.57% for male sheep, 23.86% for female sheep, 26.44% for male goats and 30.68% for female goats. Results showed that goats are more at risk to the infection than sheep however sex is not a risk factor. This study confirms the existence of Q fever in sheep and goats in Ghana hence, the disease should be considered as a public health risk to workers at the abattoir and other stakeholders in the sheep and goat production chain.

Measurement of serum zinc concentration in ruminants: a comparison of results from standard serum and specific trace element collection tubes.

Aims: To evaluate agreement between the concentration of Zn in serum from samples collected from cattle and sheep into standard collection tubes for serum and from samples collected into tubes developed for trace element measurement. Methods: Eighty-eight animals (78 cattle and 10 sheep) on eight farms had paired blood samples collected into standard serum and trace element vacutainers. The paired samples were submitted the same day to the laboratory to be tested for the concentration of Zn in serum using atomic absorption spectrophotometry. The agreement between the paired results was then assessed using limits of agreement analysis. On one farm an additional 10 pairs of samples was taken from the same animals; this second set of paired samples was refrigerated for 48 hours prior to laboratory submission to identify the impact of delaying submission on the apparent concentration of Zn in serum. Results: The limits of agreement analysis found no evidence of a systematic difference between Zn concentrations in serum collected into standard serum tubes and into trace element tubes as neither the intercept nor the slope on the mean-difference plot were significantly different from zero. The SD of the difference between results increased as the concentration of Zn increased, so at the lowest Zn concentration reported in this study (6.9 µmol/L) the limits of agreement were ±1.07 µmol/L, while at the highest (23.5 µmol/L) they were ±3.39 µmol/L. Refrigerating the sample (as whole blood) for 48 hours prior to submission increased the apparent concentration of Zn in serum in both standard serum tubes and trace element tubes by 1.3 µmol/L (95% CI = 0.75-1.85). Conclusions: There was no evidence that the concentration of Zn in serum from standard serum tubes were artificially elevated. In contrast, delaying sample submission by 48 hours did elevate Zn concentrations. Clinical relevance: While these data apply only to the batch of vacutainers used in this study, there is unlikely to be much between batch variation in the potential for contamination. Thus these results suggest that monitoring zinc status in ruminants, by measuring the concentration of Zn in serum from samples collected into standard serum tubes does not result in clinically relevant alterations in Zn concentration compared to using specific trace element tubes. However delaying submission to the laboratory may result in significantly elevated concentrations of Zn in serum so should be avoided.

Seroprevalence and associated risk factors of Coxiella burnetii (Q fever) in goats and sheep in northern Jordan.

This cross-sectional study aimed to study animal, farm, and within-farm seroprevalence of C. burnetii and to identify associated risk factors in goat and sheep farm in northern Jordan. Questionnaire was developed to collect information about risk factors and farms management practices. Blood samples from 730, ≥ 1-year-old females (goat n = 250; sheep n = 480) were randomly collected from 20 goat herds and 40 sheep flocks. IDEXX ELISA Kit was used to detect C. burnetii antibodies. The overall goat and sheep seroprevalence level was 32.5% (237/730) and was significantly higher in goats (43.3%, 108/250; 95% CI 37-49.6) than sheep (27%, 129/480; 95% CI 29.1-36.2) (χ2 test, p ≤ 0.001). Eighty percent (16/20) of goat herds and 60% (24/40) of sheep flocks had at least one seropositive animal (p ≥ 0.05). The average within goat herds and sheep flock seroprevalence were 36.4% (ranged: 0-91%) and 23.4% (ranged: 0-82%), respectively. Multivariate logistic regression model revealed that seroprevalence increased 1.79 times in goat herds compared with sheep flocks, 3.2 times more in farms containing ≥ 100 animals, and 1.7 times higher in farms with their animals that were ≥ 2 years of age than in farms with their animals that are < 2 years of age. In addition, seroprevalence significantly increased 1.52 times in farms loaning bucks or rams during breeding season and 1.63 times in farms containing cats on premises (p ≤ 0.05). Farm biosecurity measures are essential to prevent introduction and minimize transmission of C. burnetii infection to humans and animals.

Effects of Acacia mearnsii supplementation on nutrition, parasitological, blood parameters and methane emissions in Santa Inês sheep infected with Trichostrongylus colubriformis and Haemonchus contortus.

Gastrointestinal nematodes are a main problem for ruminant production, reducing animal performance and increasing environmental impact per unit of animal product generated. Tannin supplementation may lead to positive results regarding aspects such as parasitic infections and methane (CH4) emissions. Therefore, the objective of this experiment was to evaluate the effects of the condensed tannins (CT) extract made of powdered Acacia mearnsii bark (PAB) on nutrition, parasitic status and CH4 emissions in sheep artificially infected with Trichostrongylus colubriformis and Haemonchus contortus. Twenty 10-month old Santa Inês lambs (24.7 ± 3.14 kg of initial body weight) were used in a 50-day trial. Animals were divided in four treatment groups according to parasitic infection and PAB supplementation: two control groups without infections, one without PAB (C-) (n = 4) and one with PAB (C+) (n = 4); two infected groups, one without PAB (I-) (n = 6) and another receiving PAB (I+) (n = 6). Initially, animals were kept in individual pens where they were fed ad libitum chopped tifton 85 hay (Cynodon spp.) and 210 g/animal/day of concentrate. On the first day of experiment, animals of I- and I+ groups were artificially infected with infective larvae (L3) of T. colubriformis and H. contortus. Lambs were weighed fortnightly to calculate average daily body weight gain (ADG). Blood and faeces samples were also collected in the same moment of weighing for the evaluation of blood parameters and faecal egg count (FEC) respectively. After 40 days of experiment, measurements of CH4 emissions in small chamber system started and following that, apparent total tract digestibility (ATTD) assay was carried out in metabolic cages. In the end of experimental period (50 days), lambs were slaughtered and samples of abomasum and small intestine content were collected for worm count, identification, and eggs/female count. No significant (p > 0.05) treatment effects were verified for ADG, ATTD and worm count. Blood parameters were affected in both infected groups (p < 0.05) from the 28th experimental day onwards, when these animals started to show reduced red blood cells, haemoglobin and packed cell volume when compared to C- and C+. Decreased FEC was verified in I+ when compared to I- and also, H. contortus eggs/female worm for I+ was lower than for I- (p < 0.05). Both infected groups showed higher CH4 emissions than the control groups (p < 0.05). Results highlighted the anthelmintic potential of PAB and indicated methanogenic effect of parasitic nematode infections.

Effects of oral administration of copper capsules on helminth control in lactating dairy sheep: An effective alternative to replace conventional antiparasitics during lactation.

Two experiments were performed to determine whether oral administration of copper oxide capsules controlled helminthic infections in Lacaune sheep without acute collateral effects on animal health. In experiment 1, 48 multiparous lactating sheep (60.1 ±â€¯8.5 kg) were stratified according to initial number of eggs (Haemonchus contortus) per gram of feces (EPG) and were assigned randomly to 1 of two treatments (24 sheep/treatment): no oral administration (control) or oral administration of two copper capsules (treated; approximately 58 mg copper/kg body weight). Blood and fecal samples were collected on days 0, 15 and 45. Animals treated with copper capsules showed lower of EPG, eosinophils, acetylcholinesterase (AChE) in whole blood, and lower butyrylcholinesterase (BChE) activity in serum. Treated sheep had higher erythrocyte numbers, hemoglobin concentrations, hematocrit, and lymphocyte numbers. In experiment 2, 12 male lambs negative for helminths and coccidia were assigned randomly to one of two treatments (six lambs/treatment): control or treated (one copper capsule; approximately 58 mg copper/kg body weight); the experiment was designed to determine whether the results of experiment 1 were due to treatment or parasitism. Blood samples were collected on days 0, 5, 10 and 15 and fecal samples were collected on days 0, 7 and 15. Treated animals showed greater concentrations of lymphocytes; however, treatment had no effect on other hemogram variables, AChE and BChE activities, or levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, creatinine, urea, albumin, total protein, and reactive oxygen species. These data suggest that copper capsules in dairy sheep efficiently controlled H. contortus infections. Treatment was not harmful to lambs during the first 15 days, i.e. there were no signs of acute toxicity.

Benefits of the inclusion of açai oil in the diet of dairy sheep in heat stress on health and milk production and quality.

The objective of this study was to determine whether the addition of açai (Euterpe oleracea) oil in the diets of lactating sheep under heat stress exerted beneficial effects on health as well as milk production and quality. Eighteen multiparous Lacaune sheep (2 or 3 parities; 28-30 days of lactation; average milk production of 1.7 L/sheep/day) were stratified by parity and milk production and were assigned randomly to 1 of 2 treatments (9 sheep/treatment): diet supplemented with 2% of soybean oil (SOY) or 2% of açai oil (AÇAI) in the concentrate for 14 days. The amount of oil added in the diet was equivalent to 0.65% of the total diet (dry matter basis). Blood and milk samples were collected on days 1, 10 and 14. On day 14, the AÇAI group sheep had lower serum concentrations of leukocytes, neutrophils, and lymphocytes than did the SOY group sheep. On day 14, AÇAI group sheep had lower serum concentration of triglycerides and urea, milk concentration of fat and total solid and milk lipid peroxidation than did SOY group sheep. However, on day 14, AÇAI group sheep had higher serum concentrations of glucose and globulin, serum and milk antioxidant capacity against peroxyl radicals, milk production and productive efficiency than did SOY group sheep. The fatty acids profile in milk did not differ between groups. These data suggest that açai oil improved the antioxidant activity in serum and milk and improved milk production and quality in dairy sheep under heat stress.

Stress biomarkers and proteomics alteration to thermal stress in ruminants: A review.

Heat stress may adversely affect physiochemical and immune responses of livestock and alter biological functions. The comfort or thermoneutral zone for livestock, which has long been a subject of research, mainly depends on species, breed, and health. Heat stress is associated with impaired livestock productivity due to reductions in feed intake, growth rates and immunity and changes in blood constituents and biological pathways. In ruminants, elevated temperatures have deleterious consequences on protein synthesis. Exposure of ruminant animals to elevated temperatures may induce release of heat shock proteins (HSPs); HSPs usually enter the blood circulation during tissue damage and causes cell necrosis or death. Additionally, hyperthermia is associated with augmented production of cellular reactive oxygen species (ROS), which cause protein degradation and further decrease protein synthesis by preventing protein translation. Moreover, it has been suggested that high environmental temperatures lead to increased inflammatory signalling in tissues via activation of the nuclear factor kappa B (NF-κB) and tumor necrosis factor alpha (TNF-α) pathways as well as via alteration of skin colour gene (melanocortin 1 receptor (MC1R) and premelanosome protein (PMEL)) expression. Previous proteomics analyses have suggested that heat stress can reduce adenosine triphosphate (ATP) synthesis, alter gluconeogenesis precursor supply, and induce lipid accumulation in the liver with subsequent disturbance of liver structure. This review focuses on the scientific evidence regarding the impact of heat stress on immune and inflammatory responses, antioxidant status, stress biomarkers, skin colour gene (PMEL and MC1R) expression and proteomic profiles in ruminants.

Development and evaluation of a colloidal gold immunochromatographic assay based on recombinant protein CatL1D for serodiagnosis of sheep fasciolosis.

Fasciolosis is a zoonotic parasitic disease that seriously endangers the development of animal husbandry and human health. In order to develop a rapid serological diagnostic method for fasciolosis in ruminants, the CatL1D and CatB4 genes of Fasciola hepatica were amplified by reverse transcription polymerase chain reaction (PCR) and cloned, respectively, and then the CatL-B fusion gene (MeCatL-B) was constructed by gene splicing by overlap extension PCR technique. The recombinant rCatL1D, rCatB4 and rMeCatL-B proteins were then prepared by prokaryotic expression, respectively, and the recombinant protein with high specificity and sensitivity was screened via indirect enzyme-linked immunosorbent assay. Using the selected recombinant protein rCatL1D as a diagnostic antigen, we developed a colloidal gold immunochromatographic assay (CGIA) for detecting F. hepatica-specific antibodies, and 426 serum samples of slaughtered sheep were used to evaluate the sensitivity and specificity of F. hepatica CGIA assay. The results showed that the sensitivity and specificity of rCatL1D protein (100%, 96.67%) were higher than those of rCatB4 (94.29%, 80%) and rMeCatL-B (91.43%, 90%). Compared with the gold standard post-mortem inspection, the specificity and sensitivity of the CGIA method was 100% and 97%, respectively, and the consistency rate between these two methods was 99.3%. These results confirmed that the CGIA method based on rCatL1D protein could be a promising approach for rapid diagnosis of sheep fasciolosis because of its high sensitivity and specificity.

Changes in novel gastrointestinal and renal injury markers in the blood plasma of sheep following increasing intravenous doses of tolfenamic acid.

The administration of high doses of non-steroidal anti-inflammatory drugs (NSAID), such as tolfenamic acid (TA), has undesirable effects on different organs. Some novel biomarkers have been reported that can determine the gastrointestinal and renal injury caused by a high dose of NSAIDs or other toxic substances. This study was aimed at determining the changes in gastrointestinal (TFF2 and HYP), renal (NGAL and KIM-1) and cardiac (cTn-I, CK-MB) injury markers after the use of increasing intravenous doses of TA in sheep. TA was administered intravenously to groups of six sheep each, at the dose levels of 0 (Group 0, i.e., G0), 2 (G2), 4 (G4), 8 (G8) and 16 (G16) mg/kg. The concentrations of the studied biomarkers were measured at 3, 9, 18 and 36 h after administration of TA. The TFF2 and NGAL concentrations in G16 were found to be significantly higher (P < 0.05) than in the other groups except for G8 at different sampling times. HYP concentration in G16 was observed to be significantly (P < 0.05) lower than that in all other groups at 36 h. KIM-1 level in G16 was significantly (P < 0.05) higher than in all other groups at different sampling times. An increase in the renal markers, KIM-1 and NGAL, in G8 was observed before any change in plasma creatinine and urea. The cardiac marker cTn-I in G16 was significantly (P < 0.05) higher than in other groups at different sampling times. The results showed that the novel biomarkers (HYP, TFF2, NGAL, and KIM-1) can be used to determine gastric and renal injury in sheep.

Sensitivity and specificity of the FAMACHA© system in tropical hair sheep.

The aim of this research was to evaluate the sensitivity and specificity of the FAMACHA© (F©) system in Morada Nova ewes. The conjunctivae of 562 ewes were evaluated using the F© system. Packed cell volume (PCV) served as the gold standard for clinical F© evaluation. To calculate the sensitivity and specificity of the F© system, different criteria were adopted: animals classified as (I) F© 4 and 5 or (II) 3, 4, and 5 were considered to be anemic and animals classified as (I) F© 1, 2, and 3, or (II) 1 and 2 were considered to be non-anemic. Three standard values of PCV, namely, ≤ 19%, ≤ 18%, or ≤ 15%, were used to confirm anemia. The percentage of correct treatments was always high when the F© values 4 and 5 were used as criteria for positive tests. For all the PCV cut-off values, more animals were classified as false positives when evaluated using F© 3, 4, and 5 as criteria for a positive test and more true negative animals when evaluated using only F© 4 and 5 as criteria for a positive test. For both sets of criteria for the positive tests, few animals were classified as false negatives and true positives. Eliminating the classification of F© 3 as anemic decreased the sensitivity and increased the specificity for all the PCV cut-off values for the ewes. The F© system can be used as a reliable alternative to reduce selection pressure for anthelmintics in relation to routine non-selective blanket treatment for worm control in the Morada Nova ewes.

Serological survey for anti-Toxoplasma gondii antibodies in sheep of northeastern Algeria.

Although T. gondii is of considerable both public and veterinary importance worldwide, studies on its existence in sheep in Algeria, either through serology and or parasitology is scarce. To this end, a cross-sectional study was carried out in Tébessa, an Algerian eastern province, to, firstly, investigate the seroprevalence of T. gondii infection in sheep and, secondly, determine the potential risk factors that may be associated with seropositivity. A total of 376 serum samples from 39 flocks, collected between September 2015 and October 2017, were tested for anti-T.gondii antibodies via the enzyme-linked immunosorbent assay technique (ELISA). A T. gondii seroprevalence of 35.37% (95% CI 30.54-40.21%) was recorded, and 84.61% (95% CI 73.29-95.94%) of the flocks sampled had, at least, one seropositive animal. The multivariable logistic regression analysis showed that abortion during the latest pregnancy (OR = 1.56; 95% CI 1.02-2.44; p = 0.05), presence of goats in sheep flocks (OR = 1.76; 95% CI 1.04-2.98; p = 0.037), and the sampling period were the variables significantly associated with seropositivity. The present study reports, for the first time in this part of Algeria, the seroprevalence of T. gondii infection and bears out the highly dissemination capacity of the parasite. This is of a great importance for veterinarians in charge of veterinary public health, veterinary practitioners, and breeders in order to improve the control and prophylactic measures of toxoplasmosis. Nevertheless, further study should be conducted to explore the impact of the parasite on public and animal health.

Expression of Rift Valley fever virus N-protein in Nicotiana benthamiana for use as a diagnostic antigen.

BACKGROUND: Rift Valley fever virus (RVFV), the causative agent of Rift Valley fever, is an enveloped single-stranded negative-sense RNA virus in the genus Phlebovirus, family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing abortion storms in pregnant ruminants, high neonatal mortality in animals, and morbidity and occasional fatalities in humans. The disease is endemic in parts of Africa and the Arabian Peninsula, but is described as emerging due to the wide range of mosquitoes that could spread the disease into non-endemic regions. There are different tests for determining whether animals are infected with or have been exposed to RVFV. The most common serological test is antibody ELISA, which detects host immunoglobulins M or G produced specifically in response to infection with RVFV. The presence of antibodies to RVFV nucleocapsid protein (N-protein) is among the best indicators of RVFV exposure in animals. This work describes an investigation of the feasibility of producing a recombinant N-protein in Nicotiana benthamiana and using it in an ELISA. RESULTS: The human-codon optimised RVFV N-protein was successfully expressed in N. benthamiana via Agrobacterium-mediated infiltration of leaves. The recombinant protein was detected as monomers and dimers with maximum protein yields calculated to be 500-558 mg/kg of fresh plant leaves. The identity of the protein was confirmed by liquid chromatography-mass spectrometry (LC-MS) resulting in 87.35% coverage, with 264 unique peptides. Transmission electron microscopy revealed that the protein forms ring structures of ~ 10 nm in diameter. Preliminary data revealed that the protein could successfully differentiate between sera of RVFV-infected sheep and from sera of those not infected with the virus. CONCLUSIONS: To the best of our knowledge this is the first study demonstrating the successful production of RVFV N-protein as a diagnostic reagent by Agrobacterium-mediated transient heterologous expression in N. benthamiana. Preliminary testing of the antigen showed its ability to distinguish RVFV-positive animal sera from RVFV negative animal sera when used in an enzyme linked immunosorbent assay (ELISA). The cost-effective, scalable and simple production method has great potential for use in developing countries where rapid diagnosis of RVFV is necessary.

Expression and evaluation of recombinant P32 protein based ELISA for sero-diagnostic potential of capripox in sheep and goats.

The study is aimed to develop and evaluate a recombinant P32 protein based ELISA for sero-monitoring and sero-surveillance using known and random/suspected serum samples for capripox infections from sheep and goats. Truncated P32 gene of goatpox virus (with an ORF of 750 bp) was expressed in E. coli BL-21 CodonPlus (DE3)-RIPL cells using pET32a vector and characterized by SDS-PAGE analysis and confirmed by western blotting as 48 kDa polyhistidine-tagged fusion protein. The protein was purified under denaturing conditions using 8M urea and characterized by SDS-PAGE and immunoblotting. The purified protein was used for optimizing ELISA in a chequerboard titration method using anti-GTPV serum as known positive. The optimized conditions were found to be 300 ng of protein/well, 1:10 dilution of antibody, 1:10000 dilution of rabbit anti-goat/sheep conjugate with 3% skim milk powder and 2% gelatin in phosphate buffer saline containing tween-20 as blocking buffer. The expressed protein was specific only for goatpox virus and sheeppox virus but did not react with related viruses of sheep and goats namely orf virus, peste de petits ruminants virus, bluetongue virus and foot and mouth disease virus. The optimized ELISA was evaluated using pre-vaccinated, post-vaccinated and also post-challenge sera. The assay was found to have a diagnostic specificity of 100/98.7% and sensitivity of 97.1/98.1% when compared to whole virus antigen based ELISA/SNT by receiver operating characteristic (ROC) analysis. The optimized ELISA is able to determine the progression of antibody response against GTPV and SPPV following vaccination and challenge in sheep and goats. The rP32 protein based ELISA was evaluated using random field serum samples (n = 1008) suspected for sheeppox and goatpox and it has shown positivity rate as 24.4%. The rP32 protein based ELISA was found to be specific and sensitive for sero-evaluation of sheeppox virus and goatpox virus following vaccination and infection in sheep and goats.

Thermoregulatory response to outdoor heat stress of hair sheep females at different physiological state.

Forty Dorper × Pelibuey sheep females were used to evaluate the effects of physiological state on physiological variables and serum concentrations of metabolites, thyroid hormones, and electrolytes under outdoor heat stress conditions. Females were selected as follows (n = 10 per group): weaning ewe lambs (WEL; 3 months old), replacement nulliparous ewes (RNE; 8 months old), non-pregnant and non-lactating multiparous ewes (NME; 3-4 years old) and lactating multiparous ewes (LME; 3-4 years old). While physiological variables were measured both morning and afternoon, blood samples were collected before feeding in the morning to determine all blood components. Three contrasts were constructed: (1) WEL vs. older ewes, (2) RNE vs. multiparous ewes, and (3) NME vs. LME. Compared with older ewes, WEL had higher (P < 0.01) rectal temperature (RT) and hair coat temperatures through the day, and also higher (P < 0.01) respiratory rate (RR) only in the afternoon. Serum levels of glucose and cholesterol were lower (P ≤ 0.02) in WEL than in older ewes. Nulliparous ewes compared with multiparous had always similar RT but higher (P ≤ 0.05) hair coat temperatures in most of the body regions by the morning and higher (P < 0.01) RR, without difference for hair coat temperatures in the afternoon. Only serum glucose (P = 0.07) and urea nitrogen (P < 0.01) levels were affected by parturition number, being lower in multiparous ewes. Regarding the effect of lactation, while RR was unaffected, afternoon RT and hair coat temperatures in most of the body regions through the day were higher (P ≤ 0.03) in lactating ewes. In addition, LME had lower (P < 0.01) serum levels of glucose, cholesterol, and urea nitrogen, but higher (P = 0.02) triiodothyronine levels than NME. In conclusion, ewe lambs and lactating ewes were less tolerant to heat stress, while nulliparous and multiparous ewes showed similar thermoregulatory ability.

Seroprevalence and herd-level risk factors for seroprevalence of Leptospira spp. in sheep, beef cattle and deer in New Zealand.

AIMS To determine seroprevalence of Leptospira borgpetersenii serovar Hardjo and L. interrogans serovar Pomona in beef cattle, sheep and deer in New Zealand and the association between farm-level risk factors and seroprevalence. METHODS Between June 2009 and July 2010, 20 serum samples per flock or herd were collected from 162 sheep flocks, and 116 beef cattle and 99 deer herds from 238 farms, along with farm data by interview. Samples were tested for antibodies to serovars Hardjo and Pomona by microscopic agglutination testing, with a titre ≥48 being positive. Species-specific associations between herd-level seroprevalence (number of seropositive animals, for each serovar, divided by the number of animals tested) and herd-level risk factors were determined by multivariable logistic regression analysis. Vaccinated animals were excluded from seroprevalence estimates but included in multivariable analyses. RESULTS For sheep (n=3,339), animal-level seroprevalence was 43.6 (95% CI=41.9-45.3)% for serovar Hardjo and 14.1 (95% CI=12.9-15.3)% for serovar Pomona; for beef cattle(n=1,886), it was 45.6 (95% CI=43.3-47.9)% for Hardjo and 19.6 (95% CI=17.9-21.5)% for Pomona; and for deer (n=1,870), it was 26.3 (95% CI=24.3-28.4)% for Hardjo, 8.8 (95% CI=7.6-10.2)% for Pomona. In sheep flocks (n=161), flock-level prevalence for Hardjo varied from 77.9-91.3%, and for Pomona from 40.4-73.9%, when ≥1, ≥2 or ≥3 animals were seropositive. In beef herds (n=95), herd-level prevalence for Hardjo varied from 79.0-90.5%, and for Pomona from 42.1-68.4%. In deer herds (n=93), herd-level prevalence for Hardjo varied from 45.2-59.1%, and for Pomona from 22.6-48.4%. For sheep flocks, herd-level seroprevalence for Hardjo was associated with flock size (OR=1.56) and number of dogs (OR=0.75), and for Pomona, seroprevalence varied with region. For beef cattle, herd-level seroprevalence for Hardjo was associated with herd size (OR=1.4), presence of dams (OR=0.6) and vaccination (OR=2.9), and for Pomona, co-grazing with deer (OR=0.4), vaccination (OR=3.22), presence of dams (OR=0.2) and streams (OR=2.7). For deer herds, seroprevalence for Hardjo or Pomona was associated with herd size (OR=1.6 and 1.8) and varied with region, and for Pomona seroprevalence varied with season (summer vs. winter: OR=4.8). CONCLUSIONS Serovars Hardjo and Pomona were highly prevalent at herd and animal levels, with serovar Hardjo highest in all species. Larger herd size was the common risk factor for seroprevalence in all livestock species.

Coxiella burnetii Circulation in a Naturally Infected Flock of Sheep: Individual Follow-Up of Antibodies in Serum and Milk.

The control of Q fever, a zoonotic disease caused by the Coxiella burnetii bacterium, remains a scientific challenge. Domestic ruminants are considered the main reservoir, shedding C. burnetii essentially through parturition products during abortion or birth. Sheep are particularly frequently associated with human outbreaks, but there are insufficient field data to fully understand disease dynamics and to instigate efficient control measures. A longitudinal follow-up study of a naturally infected sheep flock was performed (i) to investigate relationships between seropositivity and bacterial shedding in the vaginal mucus, (ii) to describe the kinetics of antibodies, including responses to vaccination, (iii) to monitor maternal antibodies in ewe lambs, and (iv) to compare serological results for milk and serum samples. For 8 months, we collected blood samples every 3 weeks from 11 aborting and 26 nonaborting dairy ewes, 20 nonaborting suckler ewes, and 9 ewe lambs. Individual milk samples were also obtained from lactating females. All serum and milk samples were tested by enzyme-linked immunosorbent assay (ELISA), whereas vaginal swabs were tested by quantitative PCR. We found that some dairy females did not seroconvert despite shedding C. burnetii in their vaginal mucus. Overall, antibody levels in adult females were found to remain stable over time, with exceptions during the mating and lambing periods. Maternal antibodies decreased during the first month after birth. Interestingly, antibody levels in milk were correlated with those in serum. This study provides valuable field data that will help improve Q fever surveillance and within-flock management measures.IMPORTANCE Field data are necessary to improve the surveillance, diagnosis, and sanitary management of Q fever in livestock. Here, we provide extensive serological data obtained from serum and milk samples from infected and vaccinated ewes belonging to a naturally infected flock of sheep. We show that antibody levels are stable over time and seropositivity and vaginal shedding are not clearly correlated, whereas antibody levels in milk are strongly correlated with those in serum. Accordingly, we find that antibody levels in bulk tank milk are consistent with the variations observed in the serum of dairy females over time. We report the existence of maternal antibody transmission to ewe lambs and we show that the presence of maternal antibodies at birth does not prevent the development of a serological response to vaccination at the age of 4 months. Finally, we report that adult ewes generally seroconvert after vaccination, including during pregnancy.

Identification of new Corynebacterium pseudotuberculosis antigens by immunoscreening of gene expression library.

BACKGROUND: Caseous lymphadenitis (CLA) is a disease that affects sheep, goats and occasionally humans. The etiologic agent is the Corynebacterium pseudotuberculosis bacillus. The objective of this study was to build a gene expression library from C. pseudotuberculosis and use immunoscreening to identify genes that encode potential antigenic proteins for the development of DNA and subunit vaccines against CLA. RESULTS: A wild strain of C. pseudotuberculosis was used for extraction and partial digestion of genomic DNA. Sequences between 1000 and 5000 base pairs (bp) were excised from the gel, purified, and the digested DNA fragments were joined to bacteriophage vector ZAP Express, packaged into phage and transfected into Escherichia coli. For immunoscreening a positive sheep sera pool and a negative sera pool for CLA were used. Four clones were identified that strongly reacted to sera. The clones were confirmed by polymerase chain reaction (PCR) followed by sequencing for genomic comparison of C. pseudotuberculosis in GenBank. The genes identified were dak2, fagA, fagB, NlpC/P60 protein family and LPxTG putative protein family. CONCLUSION: Proteins of this type can be antigenic which could aid in the development of subunit or DNA vaccines against CLA as well as in the development of serological tests for diagnosis. Immunoscreening of the gene expression library was shown to be a sensitive and efficient technique to identify probable immunodominant genes.