Preparation and application of fluorescent monoclonal antibodies recognizing goat CD4+CD25+ regulatory T cells.

Regulatory T cells (Tregs) are a subset of T cells participating in a variety of diseases including mycoplasmal pneumonia, contagious ecthyma, and so on. The role of Tregs in goat contagious ecthyma is not completely understood due to the lack of species-specific antibodies. Here, we developed a combination of CD4 and CD25 fluorescence monoclonal antibodies (mAb) to recognize goat Tregs and assessed its utility in flow cytometry, immunofluorescence staining. Using immunofluorescence staining, we found that the frequency of Treg cells was positively correlated with the viral load during orf virus infection. These antibodies could serve as important tools to monitor Tregs during orf virus infection in goats. KEY POINTS: • A combination of fluorescent mAbs (C11 and D12) was prepared for the detection of goat Tregs. • C11 and D12 are effective in flow cytometry, immunofluorescence staining, and C11 has excellent species specificity. • The frequency of Treg cells was positively correlated with the viral load during orf virus infection.

Diagnostic evaluation of nested PCR and microscopy for Cryptosporidiosis in goats: can COWP gene based qRT-PCR be useful in assessment of active Cryptosporidial infections?

The present research delved into the transmission patterns, diagnostic methods, molecular traits, and phylogenetic analysis of Cryptosporidium species. The research was undertaken to enhance comprehension of the epidemiology and the potential for zoonotic transmission. A total of 80 goat-kid samples were tested, 7 were confirmed positive by mZN microscopy and 12 by nested-PCR. By PCR, 18SSUrRNA, HSP70, and GP60 amplicons were tested for Cryptosporidium. The restriction enzymes viz., SspI, VspI and MboII were used to genotype 12 Cryptosporidium positive samples by which C. parvum and C. bovis mixed infections were detected. Quantitative reverse transcription real-time PCR was used to transcriptionally screen the COWP-subunit genes to assess the severity of the infection in goat-kids, which showed upregulation of COWP6 and COWP4, while COWP9 and COWP3 genes were downregulated. A silent mutation was found at the codon CCA→CCC, which is being reported for the first time in goat field isolates. Phylogenetic and sequencing analyses confirmed the presence of the anthropozoonotic IIe subtype.

The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants.

This study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used in the detection of infection in small ruminants. These recombinant proteins, obtained by genetic engineering and molecular biology methods, were tested for their reactivity with specific anti-Toxoplasma IgG antibodies contained in serum samples of small ruminants (192 samples of sheep serum and 95 samples of goat serum) using an enzyme-linked immunosorbent assay (ELISA). The reactivity of six recombinant trivalent chimeric proteins (SAG1-SAG2-GRA5, SAG1-SAG2-GRA9, SAG1-SAG2-MIC1, SAG1-SAG2-MIC3, SAG1-SAG2-P35, and SAG1-SAG2-ROP1) with IgG antibodies generated during T. gondii invasion was comparable to the sensitivity of TLA-based IgG ELISA (100%). The obtained results show a strong correlation with the results obtained for TLA. This suggests that these protein preparations may be a potential alternative to TLA used in commercial tests and could be used to develop a cheaper test for the detection of parasite infection in small ruminants.

Development of an indirect ELISA against Orf virus using two recombinant antigens, partial B2L and F1L.

Orf is a highly contagious viral disease affecting goats and sheep. It is caused by Orf virus (ORFV) and has caused severe economic losses to the global goat industry, including in China. In this study, an indirect ELISA method for recombinant proteins based on truncated dominant antigenic epitopes of B2L and F1L genes of ORFV was established. A series of conditions and its performance were comprehensively evaluated. The optimized ELISA reaction conditions were: the optimal coating amount of antigen was 0.25 µg/mL, 5% skim milk powder was closed for 1 h, the optimal dilution of serum was 1:200, the optimal incubation time of the rabbit anti-goat IgG was 1:8000, the optimal color development time of TMB was 15 mins, and the threshold value of negative-positive was 0.358. The method specifically detects anti-ORFV antibodies and does not cross-react with positive sera for other common goat pathogenic bacteria antiserum. ORFV-positive sera were still positive after 1:512 dilution, with intra-batch coefficient of variation (CV) between 7.1% and 9.5% and inter-batch CV between 5.0% and 7.6%; 51% (92/180) of immunized goat serum samples were tested positive and 14.44% (14/63) of non-immunized goat serum samples were positive. The results show that the indirect ELISA antibody assay established in this study has good specificity, sensitivity and reproducibility, and provides a technical tool for clinical ORFV serum antibody detection and epidemiological investigation.

Spinal lymphoma in a goat.

A 3-year-old Pygmy Wether was presented for chronic hindlimb paralysis. A neurological exam revealed nonambulatory paraplegia with absent deep pain nociception, lack of hindlimb withdrawal reflexes, and paraspinal pain on palpation with T3 to L3 neurolocalization. MRI of the lumbar spine revealed an extensive, dorsal to dorsolateral, severely compressive, heterogeneously contrast-enhancing extradural lesion of the lumbar spine with intervertebral foraminal extension into the surrounding paraspinal musculature. Vertebral bone marrow involvement was also noted in the L5 and L6 vertebrae. A diagnosis of lymphoma was obtained after cytological sampling. This is the first case report describing specific MRI findings (signal characteristics, enhancement pattern, and perilesional changes) in a goat with paraspinal lymphoma.

Development of in-house ELISA based on recombinant gag proteins of small ruminant lentiviruses isolated from goats in Thailand.

Small ruminant lentiviruses (SRLVs), are grouped in Retroviridae family, remain a significant loss in the small ruminant husbandry. As a result of unavailability of vaccine and effective treatment, the diagnosis plays a crucial role for the control of SRLV infection. However, the major challenge of diagnosis of SRLV infection is the genetic and antigenic variability of the viruses that can lead to a failure in serological detection. This study investigated the circulating strains of the viruses in goats in Thailand and an in-house ELISA was developed. The coding sequences for gag protein were optimized, synthesized, and expressed in Escherichia coli for increasing the sensitivity of ELISA test. A total of 365 serum samples were examined against the recombinant protein in an in-house ELISA. The results showed that the recombinant gag achieves 96.67% sensitivity and 93.18% specificity as compared with the commercially available ELISA test kit.

Detection of Coxiella burnetii in the mammary gland of a dairy goat.

The zoonotic bacterium Coxiella (C.) burnetii can be excreted by infected goats through birth products and milk. The detection of C. burnetii DNA in the mammary gland tissue of infected dairy goats and intermittent milk shedders has been reported, but confirmation of C. burnetii bacteria in the udder remained pending. The pathogen caused abortions in a 152-head dairy goat herd, resulting in the vaccination against C. burnetii of the entire herd with annual boosters. To monitor the C. burnetii shedding at herd level, monthly bulk tank milk (BTM) samples were analyzed using PCR (IS1111). Despite vaccination, C. burnetii DNA was detected in BTM samples within the first 16 months of the study. Therefore, individual milk samples were tested on four different occasions several months apart to identify potential intermittent milk shedders. Only one goat (#67455) tested positive three times. This goat was necropsied to investigate the presence of C. burnetii in the udder and other organs. PCR detected C. burnetii DNA solely in both mammary glands and the left teat cistern. Immunohistological examination identified C. burnetii antigen in mammary gland tissue, confirmed by the detection of C. burnetii bacteria in the mammary epithelial cells using fluorescence in situ hybridization. The removal of goat #67455 led to negative BTM samples until the end of the study. The findings demonstrate the occurrence of C. burnetii in the mammary gland of a naturally infected and vaccinated goat. The presence possibly contributed to intermittent milk shedding of goat #67455, and the mammary gland tissue may serve as a replicative niche for C. burnetii.

Malignant catarrhal fever in a goat: manifestation of virus-induced erythema multiforme.

Malignant catarrhal fever (MCF), caused by ovine herpesvirus 2 (OvHV2; Orthoherpesviridae, Macavirus ovinegamma2), has sheep as natural hosts. OvHV2 is an important macavirus globally that induces fatal disease in dead-end hosts. Goats, which can be infected subclinically with OvHV2, rarely develop MCF. A 28-wk-old female goat was presented with fever and multifocal crusty skin lesions. Histologic examination of a skin biopsy suggested erythema multiforme (EM), with pyoderma and dermal vasculitis. The doe was euthanized and subjected to postmortem and histologic examination. MCF was suspected and PCR assays for macaviruses were performed, followed by immunohistochemistry (IHC) for OvHV2 latency-associated nuclear antigen (oLANA), RNA in situ hybridization for Ov2.5 mRNA, and IHC to characterize infiltrating leukocytes. The main postmortem finding was severe multifocal ulcerative dermatitis with macrophage- and T cell-mediated arteritis. The latter was also detected in kidney, spleen, heart, and intestinal wall. The PCR assay detected high loads of OvHV2 in tissues. OvHV2 oLANA and Ov2.5 mRNA were expressed within the lesions in leukocytes, endothelial cells, fibroblasts, and/or keratinocytes. Our case confirms that MCF can initially manifest clinically as a skin disease in goats and as EM with confirmed viral etiology.

A novel case of cutaneous, nasal and systemic fusariosis in a goat.

The clinical findings associated with nasal, cutaneous and systemic fusariosis in a 3-year-old billy Boer goat are summarised. The clinical features, treatment, postmortem findings and laboratory diagnostics are reported and discussed in the context of existing knowledge on mycoses of small ruminants. The goat presented primarily for respiratory signs (inspiratory dyspnoea) with unilateral left-sided mucopurulent nasal discharge, and multifocal variably ulcerative and necrotic cutaneous nodules. Histopathology of nasal and cutaneous biopsies revealed necrotising pyogranulomatous inflammation with intralesional septate hyphal elements that correlated with culture of Fusarium oxysporum. The patient continued to deteriorate clinically during treatment with oxytetracycline and meloxicam, with the addition of sodium iodide and potassium iodide, and was humanely euthanased. Postmortem examination revealed multifocal nodular lesions throughout the kidneys, abdominal lymph nodes and lungs. These lesions were consistent with those identified antemortem from which F. oxysporum was cultured. Although treatment was unsuccessful, to the author's knowledge, no instance of rhinofacial or systemic caprine infection with Fusarium spp. has been documented in the veterinary literature, making this the first recognised instance of this form of infection in small ruminant species.

Changes in free amino acid profile in goats with pregnancy toxemia.

Pregnancy toxemia is a metabolic disorder that afflicts goats when the heightened energy requirements preceding parturition are not sufficiently satisfied. At present, the potential association between pregnancy toxemia and the free amino acid composition in hair goats remains uncharted territory. The purpose of this study is to investigate the free amino acid profile in goats during the pivotal three weeks preceding delivery, distinguishing among those with subclinical pregnancy toxemia (SPT), clinical pregnancy toxemia (CPT), and those in the control group (CG). Additionally, the study aims to investigate any potential relationship between the amino acid profile and beta hydroxy butyric acid (BHBA) levels. The researchers analyzed a total of 50 goats, comprising 20 goats with SPT, 20 with CPT, and 10 in the CG. The serum free amino acid profile was determined using a gas chromatography flame ionization detector (GC-FID) device. BHBA concentration in goats with CPT and SPT was significantly higher than KG (p < 0.001). Furthermore, in goats with CPT, the glucose concentration was significantly lower than in CG (p < 0.012). In goats with CPT and SPT, the concentration of valine, one of the gluconeogenic amino acids, was significantly higher than in control group (p < 0.001), while histidine concentration was significantly lower (p < 0.020) than in control group. Specifically in goats with CPT, the concentrations of alanine (p < 0.002), serine (p < 0.001), and threonine (p < 0.043) were significantly lower than in control group. Moreover, the concentration of phenylalanine, which is both a glycogenic and ketogenic amino acid, was significantly lower (p < 0.028) in goats with SPT compared to the control group. The Fisher ratio (p < 0.010) and Glycine/Alanine ratio (p < 0.001) were significantly higher in pregnancy toxemia goats with than in control group goats, indicating a poor nutritional and energy status of the goats during the prepartum period. In summation, the findings of this study underscore that amino acids exhibiting marked concentration variations hold considerable promise in the diagnosis, prognosis, and therapeutic management of pregnancy toxemia.

Nocardia farcinica abortion in a goat.

A female goat fetus was received by the Colorado State University-Veterinary Diagnostic Laboratory following an isolated abortion of twins by a reportedly healthy doe. Postmortem examination did not reveal any gross abnormalities. Histologic evaluation revealed pyogranulomatous and necrotizing bronchopneumonia with intracellular and extracellular gram-positive and non-acid-fast filamentous bacilli. Aerobic culture of the stomach contents and pooled lung and liver tissue yielded light growth of Nocardia sp., which was identified by MALDI-TOF MS and 16s rDNA sequencing as Nocardia farcinica.

Molecular detection and internal transcribed spacer-1 sequence diversity of Trypanosoma evansi in goats from Cavite, Philippines.

Goat production is an important source of livelihood and food. Goats may serve as reservoir of surra affecting livestock production. Here, forty-two free-roaming goats from Cavite, Philippines were screened using two primer sets, Trypanosoma brucei minisatellite chromosome for initial detection and the internal transcribed spacer 1 (ITS-1) to determine phylogeny. Initial PCR detection showed that 19/42 (45%) goats were positive, much higher than the rate previously reported in goats from Cebu (34%). The infectivity rate was higher in male (56%) than in female (42%) and the rate was higher in young ≤1 year old (100%) than in adult >1 year old (43%). Phylogenetic analysis of the ITS-1 sequences between T. evansi goat samples and other isolates indicate potential interspecies transmission.

Optimization of a foot-and-mouth disease virus Southern African Territories-specific solid-phase competitive ELISA for small ruminant serum samples.

We optimized and verified a single-spot solid-phase competitive ELISA (ss-SPCE) to detect antibodies against structural proteins of Southern African Territories (SAT) serotypes of foot-and-mouth disease virus (FMDV) in small ruminants. Sera from goats vaccinated and experimentally challenged with a SAT1 FMDV pool were tested in duplicate at 4 dilutions (1:10, 1:15, 1:22.5, 1:33.8) to optimize the assay. To assess the performance of the assay in naturally infected animals, we evaluated 316 goat and sheep field sera collected during active SAT2 outbreaks. Relative to results of the virus neutralization test, the optimal serum dilution and cutoff percentage inhibition (PI) were 1:15 and 50%, respectively. At these values, the Spearman rank correlation coefficient was 0.85 (p < 0.001), and the sensitivity and specificity (95% CI) were 80.3% (72.6, 87.2) and 91.1% (84.1, 95.9), respectively. Relative to the liquid-phase blocking ELISA and the nonstructural protein ELISA, the ss-SPCE exhibited divergent performance characteristics between the goat and sheep field sera. Repeatability was better for goats, but the correlation and agreement among all 3 assays were better for the sheep sera. The prevalence of SAT2 FMDV infection in the sampled sheep was 23.6%; sampled goats were seemingly FMDV-free. The ss-SPCE is an appropriate FMDV detection tool to investigate the role of small ruminants in the epidemiology of FMD in Africa.

Case report of congenital goitre in a goat kid: Clinical and pathological findings.

Congenital goitre is a deadly thyroid metabolic disorder characterised by low thyroid hormone levels, subsequent secretion of excess Thyroid-Stimulating Hormone (TSH) from the pituitary gland, and compensatory thyroid gland hyperplasia. This study aimed to summarise the clinical and pathological features of congenital goitre in a goat kid. In April 2019, a dead female goat kid with a history of dystocia was referred to Ferdowsi University of Mashhad Veterinary Teaching Hospital, Mashhad, Iran, to examine the carcass and find the reason for death. The necropsy were performed, along with histopathology examination, and clinical signs were recorded. Examination of the foetus revealed the presence of an enlarged thyroid gland, and the skin was thick with myxedema, pale, and without hair. After cutting the skin, the swelling showed a significantly enlarged thyroid gland with two asymmetrical lobes, with the right lobe 3.9×7.1 cm and the left 3.7×7.5 cm in size. In the histopathological examination, a severe proliferation of follicular cells was observed, which caused the thyroid gland to be microscopically dense. In conclusion, this study highlights the importance of recognising and addressing congenital goitre in goat kids. To prevent such tragic outcomes, it is crucial to focus on early detection and intervention. Furthermore, the agents of goitre need to find out and be clear.

A comparative study of serological tests used in the diagnosis of Toxoplasma gondii infection in small ruminants evidenced the importance of cross-reactions for harmonizing diagnostic performance.

Toxoplasma gondii is a major foodborne zoonotic pathogen that can be transmitted through the consumption of raw or undercooked meat of small ruminants, among others. Serology has been suggested as an epidemiological indicator and several tests are available nowadays. However, there is no comparative study with the most used ones. Therefore, the objective of this study was to develop and validate two in-house tests (Western blot -TgSALUVET WB- and ELISA -TgSALUVET ELISA 2.0-) and perform a comparative study including such tests and four commercial ELISA kits (IDScreen®, PrioCHECK®, Pigtype® and IDEXX). First, a specific pattern of recognition of immunodominant antigens by TgSALUVET WB was determined with serum panels of noninfected sheep and sheep infected with T. gondii or Neospora caninum. Next, TgSALUVET WB was used as a reference to preliminary validate TgSALUVET ELISA 2.0 using sera from sheep and goats naturally infected with T. gondii. Then, the abovementioned sheep serum panels were analyzed by all tests and subjected to TG-ROC analyses and agreement tests, and cross-reactivity with the anti-N. caninum IgGs was studied. All the techniques were accurate enough for the cutoff values initially suggested with all serum panels (Se and Sp ≥ 94%), except for PrioCHECK®, which showed 83% Sp. However, a cutoff readjustment improved their diagnostic performance. Additionally, cross-reactions between anti-N. caninum antibodies and T. gondii antigens were detected with all tests. Thus, a second cutoff readjustment was carried out and the use of both readjusted cutoff values is recommended to obtain comparable data and avoid false-positive results.

Biphasic pleural mesothelioma in a goat.

An 8-year-old Saanen goat doe was seen for inappetence, tachycardia, and intermittent bluish-grey discoloration of the oral mucous membranes. On physical examination, the goat was mildly tachypneic and tachycardic, with reduced sounds auscultated on the left side of the thorax. Euthanasia was elected. Necropsy revealed an infiltrative, multinodular mass within the left thoracic cavity and innumerable small, tan nodules disseminated across the pleura of the lungs, thoracic walls, and diaphragm. Upon histologic examination, the mass was composed of highly pleomorphic, fusiform to polygonal cells. Neoplastic cells exhibited positive immunoreactivity for both cytokeratin and vimentin, consistent with a diagnosis of biphasic pleural mesothelioma. Key clinical message: Mesothelioma has rarely been described in the goat but should be considered as a differential diagnosis for thoracic masses in small ruminants, along with thymoma; metastatic neoplasia; carcinomatosis; and granulomatous lesions caused by parasites, bacteria, and fungi.

Mésothéliome pleural biphasique chez une chèvre. Une chèvre Saanen âgée de 8 ans a été vue pour de l'inappétence, une tachycardie et une décoloration gris bleuâtre intermittente des muqueuses buccales. À l'examen physique, la chèvre était légèrement tachypnéique et tachycardique, avec des sons réduits auscultés du côté gauche du thorax. Il a été décidé d'euthanasier l'animal. L'autopsie a révélé une masse multinodulaire infiltrante dans la cavité thoracique gauche et d'innombrables petits nodules brun clair disséminés à travers la plèvre pulmonaire, les parois thoraciques et le diaphragme. À l'examen histologique, la masse était composée de cellules hautement pléomorphes, fusiformes à polygonales. Les cellules néoplasiques ont présenté une immunoréactivité positive pour la cytokératine et la vimentine, compatible avec un diagnostic de mésothéliome pleural biphasique.Message clinique clé:Le mésothéliome a rarement été décrit chez la chèvre mais doit être considéré comme un diagnostic différentiel des masses thoraciques chez les petits ruminants, avec le thymome, la néoplasie métastatique, la carcinomatose et les lésions granulomateuses causées par des parasites, des bactéries et des champignons.(Traduit par Dr Serge Messier).

Differential diagnosis of theileriosis through blood smear examination and polymerase chain reaction in small ruminants from Pakistan.

Background: Ovine and caprine theileriosis is a tick-borne hemoprotozoan disease, caused by Theileria spp., responsible for heavy economic losses in terms of high mortality and morbidity rates. Diagnosis of ovine theileriosis is primarily based on clinical symptoms, microscopic screening of stained blood smears, and lymph node biopsy smears, but the limitations of these detection methods against Theileria spp. infection limits their specificity. Aim: To overcome these limitations, the current study reports the differential diagnosis of theileriosis through a blood smear examination and polymerase chain reaction (PCR) in small ruminants from Pakistan. Methods: The study was conducted on 1,200 apparently healthy small ruminants (737 sheep and 463 goats). First, blood smears were screened for the presence of Theileria piroplasms in red blood cells. Second, PCR amplification based on 18S rRNA gene was performed by using primers specific to Theileria spp. Results: Out of the 1,200 samples of examined blood smears, 100 animals (8.33%) were found positive for Theileria species, which showed intra-erythrocytic bodies in the form of dot and comma shapes. Amplification of the isolated DNA from randomly collected blood samples of 737 sheep and 463 goats showed that an amplicon size of 1,098 bp was positive for Theileria spp. In total, 315 out of the 1,200 small ruminants examined in this study were found positive for Theileria spp. DNA through PCR amplification. Notably, out of the 885 blood samples negative by PCR amplification, only 15 blood samples were found positive by the blood smear test. Conversely, 230 blood samples that tested negative in the smear technique produced a specific band through PCR amplification. Overall, the sensitivity and specificity rates were 26.98% and 98.31% for the blood smear method and 73.01% and 100% for the PCR assay, respectively. Conclusion: Our finding suggests that PCR is the gold standard method compared to the conventional method of smear examination for the diagnosis of ovine and caprine theileriosis in Pakistan.

Investigation of the Clinical and Diagnostic Aspects of Peste des Petits Ruminants (PPR) in Sheep from the Southern Region of Iraq.

In the southern region of Iraq, Peste des petits ruminants (PPR) has been identified and diagnosed. The study was done on (300) local sheep breeds of varying ages and sexes exhibiting PPR symptoms, while (25), healthy sheep breeds served as the control group. Additionally, the diagnosis of PPRV was confirmed by PCR. Infected sheep exhibit a variety of clinical symptoms. However, DNA sequencing was used to detect genetic links and genetic variation, and the results revealed a closed genetic relationship with the NCBI BLAST PPRV India isolate (GU014574.1) at total genetic variation (0.02-0.01%). Results indicate a large rise in PCV and ESR in conjunction with leukocytopenia and lymphocytopenia, a significant difference in clotting factor indices, and a significant increase in ALT, AST, and CK. In addition, there was a substantial variation in acute phase response. Postmortem examinations revealed various erosive lesions on the upper and lower gums, severe hemorrhagic enteritis, particularly of the small intestine, and obvious congestion of the lungs. Histopathological changes revealed an obvious flattening of the intestinal mucosa as well as an enlargement of the villi. In addition to a granuloma in the sub-mucosa, chronic inflammatory cells, primarily lymphocytes, were seen invading the mucosa. It has been determined that the sickness was circulating in the southern region of Iraq and severely afflicted sheep, which might result in significant economic losses owing to the detrimental effects of the virus that causes the disease on the various bodily parts.

Dot-ELISA based on recombinant Hypodermin C of Przhevalskiana silenus for field diagnosis of goat warble fly infestation.

Goat warble fly infestation (GWFI) is an economically important myiasis caused by larvae of Przhevalskiana silenus (Diptera, Oestridae), prevalent in countries of the Mediterranean Basin and Indian subcontinent. GWFI is characterized by the presence of subcutaneous warbles at the lumbar and sacral region of dorsum in the infested animal. The early larval instars (L1 and L2) remain inaccessible to physical detection due to their small size and subcutaneous presence thus causing hindrance in the diagnosis. The objective of present study was to develop a field applicable early diagnostic intervention for GWFI monitoring and prophylactic management for effective control of the disease. Recombinant Hypodermin C (rHyC) antigen of P. silenus was expressed in Escherichia coli. The purified protein was used for optimizing dot-ELISA in a checkerboard titration using goat warble fly infested serum as known positive. The optimized assay was further tested for lower temperature (18°C) and incubation time (30 min). The optimized assay was assessed for inter-rater reliability and field samples. The optimized conditions require 188 ng of protein/dot, 1:800 dilution of serum sample, 1:4000 dilution of anti-goat IgG conjugate and 5% skim milk powder in phosphate buffer saline as blocking buffer. The assay was found to have a diagnostic sensitivity and specificity of 97.3% and 95.8%, respectively. The inter-rater reliability of dot ELISA with rHyC indirect ELISA was found to be almost perfect with a Cohen's kappa index of 0.973. Further testing at ambient temperature (18°C) and shorter incubation steps (30 min) supported suitability of the assay for field diagnosis of GWFI. The present study provides the first report of a sensitive and specific dot-ELISA for early diagnosis of GWFI which is rapid and cost effective. The test may provide an effective field applicable tool for sustainable control of GWFI.

Prokaryotic expression of the V protein of the peste des petits ruminants virus and development of an indirect ELISA.

In this study, we recombinantly expressed the V protein of the peste des petits ruminants virus (PPRV) and evaluated its diagnostic value for PPRV infection using an indirect ELISA (i-ELISA). The optimal concentration of the coated antigen of V protein was 15 ng/well at a serum dilution of 1:400, and the optimal positive threshold value was 0.233. A cross-reactivity assay showed that the V protein-based i-ELISA was specific to PPRV with consistent reproducibility and showed a specificity of 82.6% and a sensitivity of 100% with a virus neutralization test. Using the recombinant V protein as an antigen in ELISA is useful for seroepidemiological studies of PPRV infections.