Molecular prevalence, associated risk factors and phylogenetic evaluation of Theileria lestoquardi in the blood samples of small ruminants.

Raising small ruminants is the main source of income for farmers in Pakistan especially in rural areas of Dera Ghazi Khan in Punjab. Despite having large sheep population, the prevalence of intra-erythrocytic protozoa, Theileria (T.) lestoquardi, has never been reported from this area. This study was conducted to fill this knowledge gap and 333 blood samples of apparently healthy small ruminants (168 sheep and 165 goats) along with their epidemiological data were collected from Dera Ghazi Khan district during August till November 2022. The polymerase chain reaction (PCR) analysis amplified a 785 base pair amplicon specific for the Merozoite surface antigen (ms 1-2) gene of T. lestoquardi in 2 out of the 168 (3.3%) sheep blood samples, while no goat blood sample out of 165 was found to be infected with T. lestoquardi. DNA sequencing confirmed the presence of Theileria lestoquardi in both samples and phylogenetic analysis revealed that these amplicon resembled the partial ms 1-2 gene sequences detected in small ruminants from Pakistan, India Iran and Egypt. All the studied epidemiological factors (age, sex, breed, size of herd, dogs with herd, composition of herd, size of herd and Tick burden on sheep) were not found associated with the prevalence of T. lestoquardi. In conclusion, this study reports a low prevalence of T. lestoquardi infection in the Dera Ghazi Khan District of Punjab, Pakistan. The data generated from this work will help pave the way for the prophylactic detection and control of ovine and caprine theileriosis in the region.

First Broad-Range Serological Survey of Crimean-Congo Hemorrhagic Fever among Hungarian Livestock.

(1) Background: Crimean-Congo hemorrhagic fever (CCHF) is an emerging tick-borne disease endemic in Africa, Asia, the Middle East, and the Balkan and Mediterranean regions of Europe. Although no human CCHF cases have been reported, based on vector presence, serological evidence among small vertebrates, and the general human population, Hungary lies within high evidence consensus for potential CCHF introduction and future human infection. Thus, the aim of our pilot serosurvey was to assess CCHF seropositivity among cattle and sheep as indicator animals for virus circulation in the country. (2) Methods: In total, 1905 serum samples taken from free-range cattle and sheep in 2017 were tested for the presence of anti-CCHF virus IgG antibodies using commercial ELISA and commercial and in-house immunofluorescent assays. (3) Results: We found a total of eleven reactive samples (0.58%) from five administrative districts of Hungary comprising 8 cattle and 3 sheep. The most affected regions were the south-central and northwestern parts of the country. (4) Conclusions: Based on these results, more extended surveillance is advised, especially in the affected areas, and there should be greater awareness among clinicians and other high-risk populations of the emerging threat of CCHF in Hungary and Central Europe.

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.

Genomic Tools for the Identification of Loci Associated with Facial Eczema in New Zealand Sheep.

Facial eczema (FE) is a significant metabolic disease that affects New Zealand ruminants. Ingestion of the mycotoxin sporidesmin leads to liver and bile duct damage, which can result in photosensitisation, reduced productivity and death. Strategies used to manage the incidence and severity of the disease include breeding. In sheep, there is considerable genetic variation in the response to FE. A commercial testing program is available for ram breeders who aim to increase tolerance, determined by the concentration of the serum enzyme, gamma-glutamyltransferase 21 days after a measured sporidesmin challenge (GGT21). Genome-wide association studies were carried out to determine regions of the genome associated with GGT21. Two regions on chromosomes 15 and 24 are reported, which explain 5% and 1% of the phenotypic variance in the response to FE, respectively. The region on chromosome 15 contains the ß-globin locus. Of the significant SNPs in the region, one is a missense variant within the haemoglobin subunit ß (HBB) gene. Mass spectrometry of haemoglobin from animals with differing genotypes at this locus indicated that genotypes are associated with different forms of adult ß-globin. Haemoglobin haplotypes have previously been associated with variation in several health-related traits in sheep and warrant further investigation regarding their role in tolerance to FE in sheep. We show a strategic approach to the identification of regions of importance for commercial breeding programs with a combination of discovery, statistical and biological validation. This study highlights the power of using increased density genotyping for the identification of influential genomic regions, combined with subsequent inclusion on lower density genotyping platforms.

Serological investigation and genotyping of Mycobacterium avium subsp. paratuberculosis in sheep and goats in Inner Mongolia, China.

Paratuberculosis a contagious and chronic disease in domestic and wild ruminants, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Typical clinical signs include intractable diarrhea, progressive emaciation, proliferative enteropathy, and mesenteric lymphadenitis. Paratuberculosis is endemic to many parts of the world and responsible for considerable economic losses. In this study, different types of paratuberculosis and MAP in sheep and goats were investigated in Inner Mongolia, a northern province in China contiguous with two countries and eight other provinces. A total of 4434 serum samples were collected from six cities in the western, central, and eastern regions of Inner Mongolia and analyzed using the ELISA test. In addition, tissue samples were collected from seven animals that were suspected to be infected with MAP. Finally, these tissues samples were analyzed by histopathological examination followed by polymerase chain reaction (PCR), IS1311 PCR-restriction enzyme analysis (PCR-REA), and a sequence analysis of five genes. Among all 4434 ruminant serum samples collected from the six cities in the western, central, and eastern regions of Inner Mongolia, 7.60% (337/4434) measured positive for the MAP antibody. The proportions of positive MAP antibody results for serum samples collected in the western, central, and eastern regions were 5.10% (105/2058), 6.63% (85/1282), and 13.44% (147/1094), respectively. For the seven suspected infected animals selected from the herd with the highest rate of positivity, the gross pathology and histopathology of the necropsied animals were found to be consistent with the pathological features of paratuberculosis. The PCR analysis further confirmed the diagnosis of paratuberculosis. The rest of the results demonstrated that herds of sheep and goats in Inner Mongolia were infected with both MAP type II and type III. To the best of our knowledge, this is the first study of the two subtypes of MAP strains in sheep and goats in Inner Mongolia.

Mean corpuscular haemoglobin concentration as haematological marker to detect changes in red blood cells in sheep infected with Haemonchus contortus.

Haemonchus contortus is a nematode parasite that causes anaemia and affects the health of sheep. The mean corpuscular haemoglobin concentration (MCHC) is an excellent indicator to detect anaemia that could help to characterize resistant or susceptible lambs to gastrointestinal nematodes. The aim of this study was to evaluate the predictive value of MCHC in detecting changes in red blood cells and their relation to anaemia in lambs re-infected with H. contortus. An analysis of information was performed using 24 Pelibuey lambs previously infected in grazing, dewormed and experimentally re-infected with H. contortus. At the first haematological sampling (admission) the lambs were classified based on MCHC quartiles (Q). Subsequently, the lambs were housed for 56 days. Blood samples were taken every seven days to determine the haematological parameters using an impedance haematological instrument. Confidence limits were constructed with the records of the lambs that recovered their haematological parameters. Each quartile was analysed as a treatment in a repeated measures design over time. To know the optimal combination of sensitivity and specificity of MCHC to detect anaemia a curve of receiver operating characteristic (ROC) curve and the cut-off values were evaluated. In quartile 4 (Q4), lambs showed the highest faecal egg count (FEC, 764 eggs/g of faeces), mean corpuscular haemoglobin (17.0 pg) and MCHC (54.6 g/dL). This group also presented the lowest RBC values (5.8 × 106/mL), haematocrit (HCT, 18.3%), total plasma protein (5.7 g/dL), and HGB (9.7 g/dL). The optimal point of MCHC with ROC curve was 42.4 (sensitivity 88.2% and specificity 86.5%); the area under the curve was 0.91 (CI 95%, 0.86-0.96). These results are related to the haematological effects caused by H. contortus in susceptible lambs. In conclusion, the highest FEC and lower HCT in Q4 are important elements of the haematological damage caused by H. contortus and could identify susceptible lambs.

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.

Hematologic Conditions of Small Ruminants.

Anemia is a clinically important syndrome in small ruminants. Anemia can be divided into regenerative and nonregenerative forms. Differentials for regenerative anemia include hemorrhage owing to gastrointestinal or external parasitism or hemostatic disorders, and hemolysis owing to infectious, osmotic, toxic, and nutritional causes. Differentials for nonregenerative anemia include inflammatory and chronic diseases, renal failure, pancytopenia, copper deficiency, and heavy metal toxicosis. Iron deficiency anemia can be caused by chronic gastrointestinal and external hemorrhage or nutritional deficiency and may be mildly regenerative or nonregenerative. Appropriate diagnostic tests are described along with treatments, including blood transfusion, parasite control, and prevention.

Differences in the antibody response to adult Fasciola hepatica excretory/secretory products in experimentally and naturally infected cattle and sheep.

Fasciola hepatica (the liver fluke) is a common, global parasite of livestock. It can be highly pathogenic and has health and welfare implications for infected individuals. Typically, in ruminants, infections are sub-clinical, but if undiagnosed, they can lead to significant production losses. Accurate diagnosis is crucial to identify infection. Antibody detection ELISAs are commonly used to diagnose infection due to their high sensitivity and specificity and are typically based on native fluke excretory/secretory (ES) products or cathepsin L1 (CL1), the immunodominant antigen within ES products. These tests have been developed based on the antibody response of experimentally infected animals; however, this response has not been well characterised in naturally infected animals. We compared the antibody recognition of a recombinant CL1 (rCL1) antigen and native adult fluke ES products. Whilst samples from experimentally infected animals showed strong recognition of rCL1, serum antibodies from naturally infected animals did not. These results were confirmed by peptide array. Immunoblotting sera against ES products showed that experimentally infected animals had a strong, specific response to CL1/CL2 proteins whilst antibodies from naturally infected animals recognised multiple proteins and had a variable response to CL1/CL2. Mass spectrometry of proteins separated by 2D SDS PAGE, identified several antigens recognised by serum antibodies from a naturally infected cow, including cathepsins L1, L2 and L5, glutathione S-transferase and a dihydrolipoyl dehydrogenase. Overall, these results show that the antibody response in naturally infected animals to adult fluke ES products is qualitatively different to experimentally infected animals. This suggests that a diagnostic test based on CL1 alone may not be appropriate for diagnosis of natural F. hepatica infections in sheep and cattle.

Characterization of immunological, biochemical and inflammatory response of clinical and subclinical endometritis in ewes in the subtropics.

Pluriparus Ossimi (n = 50) ewes were used to investigate the immune profile of the affected ewes to accurately diagnose clinical and subclinical endometritis and associations with biochemical variables. Ewes were slaughtered and animals were classified into control (no fertility problems), subclinical endometritis (SCE) and clinical endometritis (CE) groups based on pre-slaughter determinations of conception failure. Serum was collected from ewes to estimate concentrations of pro-inflammatory cytokines including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) as well as nitric oxide (NO) concentration. The results from immunological evaluations indicated there were greater (P < 0.001) serum concentrations of IL-6, IL-8, TNF-α and NO in ewes classified with SCE and CE as compared to ewes of the control group. Furthermore, values for concentrations of TNF-α were positively correlated with IL-6 and IL-8 concentrations in ewes of the SCE and CE groups. In ewes classified with CE and SCE there were greater (P < 0.01) concentrations of blood glucose, ALT, AST, urea and creatinine than in ewes of the control group. It is concluded that serum pro-inflammatory cytokines IL-6, IL-8, and TNF-α are diagnostic markers for CE and SCE in ewes and serve as a criterion for different inflammatory complications in ewes classified as having CE or SCE.

Validation and field evaluation of a competitive inhibition ELISA based on the recombinant protein tSAG1 to detect anti-Neospora caninum antibodies in sheep and goats.

Neospora caninum is a protozoan parasite that causes abortion and reproductive failure in small ruminants. We validated and evaluated under field conditions a competitive inhibition ELISA based on the truncated SAG1 protein (tSAG1) from N. caninum for the detection of anti-N. caninum antibodies in sheep and goat flocks. The assay was validated using 80 positive and 142 negative serum samples from sheep and goats analyzed by IFAT and immunoblot (IB). ciELISAtSAG1 was then used to evaluate the prevalence of anti-N. caninum antibodies in 1449 goats from 143 flocks and 385 sheep from 40 flocks and compared to IFAT. The prevalence of anti-Toxoplasma gondii antibodies was evaluated by IFAT. The ciELISAtSAG1 cut-off was ≥ 36 percent inhibition, with a diagnostic sensitivity of 100.0 % (95 % CI = 95.4-100.0 %) and a diagnostic specificity of 98.6 % (95 % CI = 95.0-99.8 %) relative to the agreement between IFAT and IB. The field evaluation revealed a concordance between ciELISAtSAG1 and IFAT of 97.4 %, with an agreement (κ) of 0.90 for sheep sera, and a concordance of 96.5 % with κ = 0.85 for goat sera. The overall prevalence of anti-N. caninum antibodies in sheep was 14.3 % by IFAT and 15.8 % by ciELISAtSAG1. In goats, prevalence was 12.9 % by IFAT and 14.6 % by ciELISAtSAG1. The overall prevalence of anti-T. gondii antibodies was 28.8 % in goats and 43.8 % in sheep. The ciELISAtSAG1 could be useful for large-scale detection of anti-N. caninum antibodies in sheep and goats, and for seroepidemiological investigations due to its appropriate sensitivity and specificity, and the simplicity of production.

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.

Novel recombinant Mce-truncated protein based ELISA for the diagnosis of Mycobacterium avium subsp. paratuberculosis infection in domestic livestock.

Johne's disease (JD) is an infectious wasting condition of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) in domestic livestock of every country that has been investigated. Controlling JD is problematic due to the lack of sensitive, specific, efficient, and cost-effective diagnostic tests. A major challenge in the development of diagnostics like ELISA is the selection of an ideal antigen/(s) that is pathogen-specific and allows sensitive recognition. Therefore, the purpose of this study was to identify and use Mce-truncated protein-based ELISA assay for the diagnosis of MAP infection with high sensitivity and specificity. In silico epitope prediction by epitope mapping throughout the whole length of MAP2191 protein revealed that C-terminal portion of this protein presented potential T- and B-cell epitopes. Therefore, a novel Mce-truncated protein encoded by the selected region of MAP2191 gene was expressed, purified with Ni-NTA gel matrix and confirmed by SDS PAGE and western blot. A profiling ELISA assay was developed to evaluate sera from MAP infected and non-infected ruminant species for antibodies against Mce-truncated protein to infer the immunogenicity of this protein in the host. Using this Mce protein-based ELISA, 251 goats, 53 sheep, 117 buffaloes, and 33 cattle serum samples were screened and 49.4, 51.0, 69.2, and 54.6% animals, respectively, were found positive. Comparing with i-ELISA, the new Mce-based ELISA kit showed a relatively higher specificity but suffered from slightly reduced sensitivity. Mce-based ELISA excluded apparently false positive results of i-ELISA. Mce protein was found to be antigenic and Mce-ELISA test could be employed as a diagnostic test for JD in domestic livestock in view of the a relatively higher specificity and accuracy. The antigenic potential of Mce antigen can also be exploited for the development of a new vaccine for the control of MAP infection.

First Report on Molecular Characterization of Anaplasmosis in Small Ruminants in Pakistan.

Anaplasmosis is caused by a Gram-negative obligate intracellular bacterium of the genus Anaplasma with the pathogen having a zoonotic impact. The study aimed to estimate the prevalence of anaplasmosis in Pakistan, to unravel the association of potential risk factors, and to investigate the effect on hematological parameters in affected small ruminants. A total of 150 (n = 75 sheep; n = 75 goats) blood samples were initially screened microscopically and then subjected to PCR targeting the amplification of the 16S rRNA gene fragment of Anaplasma. The PCR-based positive samples were then processed for sequencing. Statistical analysis regarding risk factors was performed using R software. The study revealed an overall 29.33% (44/150) prevalence of anaplasmosis in small ruminants. Sheep had higher (P > 0.05) prevalence (32%) as compared to goats (25.30%). The final statistical model resulting from backward elimination showed only tick infestation as a significant predictor of infection status. The phylogenetic analysis of 16S rRNA gene of Anaplasma spp. revealed 9 study isolates clustered together and showed a close resemblance (99%) with Anaplasma ovis isolate (DQ837600) from Hungary. One of the isolates showed (99%) similarity with the isolate of Anaplasma marginale (MH155594) from Iraq. Furthermore, the hematological parameters pack cell volume, red blood cells, hemoglobin, white blood cells, granulocytes, monocytes, lymphocytes, and platelet count were decreased in Anaplasma-positive animals. This is the first study at the molecular level to characterize Anaplasma spp. in small ruminants of Pakistan, and it will be useful in developing control strategies for anaplasmosis.

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.

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.

The first discovery of severe fever with thrombocytopenia syndrome virus in Taiwan.

Severe fever with thrombocytopenia syndrome (SFTS), an emerging tick-borne zoonosis, has been rapidly spread in many Asian counties since 2010, which raises the great concern in East Asia. Nevertheless, the infection status of SFTS in Taiwan remains unclear. To investigate the existence of SFTSV in Taiwan, a total of 151 serum samples collected from 31 sheep, 63 bovine and 57 dogs were enrolled this study. Furthermore, 360 adult female Rhipicephalus microplus were also included. One-step RT-nested PCR and IgG ELISA were conducted to test SFTSV specific RNA and antibodies, respectively. The result provided the first evidence of the existence of SFTSV RNA and antibodies in ruminants and ticks in Taiwan.

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.

Anti-Sarcocystis Antibodies in Lambs Deprived of Colostrum.

INTRODUCTION: The objective of this study was to evaluate the presence of anti-Sarcocystis spp. specific IgG antibodies in serum samples from precolostral lambs to determine the occurrence of transplacental transmission of Sarcocystis spp. in sheep. METHODS: Blood samples were collected from 80 ewes and their respective lambs, immediately after lambing and before colostrum ingestion, respectively. The presence of anti-Sarcocystis spp. IgG was evaluated in serum samples using the indirect fluorescent antibody test (IFAT). Positive samples of the lambs were submitted to titration and IFAT to detect anti-T. gondii and anti-N. caninum specific IgG. RESULTS: Anti-Sarcocystis spp. IgG was detected in 62.5% of the ewes (50/80) and in 4% of the lambs of the seropositive ewes (2/50). None of the lambs from seronegative ewes were positive. The final titers of the positive lambs were 80. No cross reaction was detected among the positive samples to anti-Sarcocystis spp., anti-N. caninum, and anti-T. gondii IgG. The detection of anti-Sarcocystis spp. antibodies in serum samples of lambs deprived of colostrum suggests transplacental transmission of infection. Thus, the vertical transmission may be an alternative route of infection of Sarcocystis spp. also in sheep. Further studies are warranted to confirm transplacental transmission in sheep and to explain the importance of this infection pathway.