Adhesion-Regulating Molecule from Haemonchus contortus: Potential Antigen for Diagnosis of Early Infection in Goats

Haemonchus contortus, a blood-sucking nematode of ruminants, causes large economic losses worldwide. Diagnosis of infection mainly depends on the evaluation of clinical signs and fecal examination. However, this has limitations for the diagnosis of early or light infections, where serological diagnosis seems to be more accurate and reliable. In this study, the recombinant H. contortus adhesion-regulating molecule protein (rHCADRM) was expressed and purified, and its diagnostic potential was evaluated. Serum samples from goats experimentally infected with H. contortus (n = 5) were collected at 0 (before infection, negative control), 7, 14, 21, 35, 49, 63, 85, and 103 days post-infection (DPI). The reactions between rHcADRM and goat serum were tested using Western blot (WB) analysis. The results show that rHcADRM can be recognized in the serum as early as 14 DPI, and the antibody against rHcADRM in infected goat could be maintained for over 89 days. No reaction was found between rHcADRM and antibodies against Trichinella spiralis, Fasciola hepatica, or Toxoplasma gondii. An indirect enzyme-linked immune sorbent assay (ELISA) was developed based on rHcADRM. The optimal coating antigen (279 ng of rHcADRM/well) and serum dilutions (1:50) were determined by checkerboard titration. A total of 64 serum samples, including 32 from H. contortus infection goats and 32 from helminth-free goats, were used to determine the positive (0.362) and negative (0.306) cut-off values for the ELISA. The results show this serological diagnosis method is highly sensitive (90.6%) and specific (93.75%). The coefficient of variation within run and between runs was less than 11%. To apply this indirect ELISA during field examination, 51 serum samples were randomly collected from goat farms and tested using this method. The result showed that 19.6% (10/51) of goats were infected with H. contortus, which was 100% consistent with the necropsy result, higher than that of fecal examination (15.7%, 8/51). These results indicate that rHcADRM could be a potential antigen for diagnosis of H. contortus infection in goats.

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Development and Potential Application of Ras Domain Containing Protein from Haemonchus contortus for Diagnosis of Goat Infection

Animals ◽

10.3390/ani10010138 ◽

2020 ◽

Vol 10 (1) ◽

pp. 138 ◽

Cited By ~ 2

Author(s):

Kalibixiati Aimulajiang ◽

Man Cao ◽

Shuyi Liao ◽

Muhammad Ali-ul-Husnain Naqvi ◽

Xiaowei Tian ◽

...

Keyword(s):

Haemonchus Contortus ◽

Indirect Elisa ◽

Fasciola Hepatica ◽

Trichinella Spiralis ◽

Clinical Signs ◽

Small Ruminants ◽

Gastrointestinal Nematode ◽

Enzyme Linked Immunosorbent Assay ◽

Fecal Examination ◽

Diagnostic Potential

Haemonchus contortus is an important gastrointestinal nematode of small ruminants that causes significant mortality in goats worldwide. Diagnosis of this infection mainly depends on the evaluation of clinical signs and fecal examination. However, limitations often occur in early or mild infections. For this purpose, serological diagnosis seems to be more accurate and reliable. Ras domain-containing protein (Ras) is one of H. contortus’s excretory and secretory products (ESPs) that can be isolated from different larval stages of the nematode. In this study, the recombinant H. contortus Ras domain-containing protein (rHcRas) was expressed and purified and its diagnostic potential was evaluated. Reactions between rHcRas and goat sera were tested using Western blotting (WB). The results showed that rHcRas could be recognized by sera as early as 14 days post infection (DPI), and antibodies against rHcRas in infected goats could be maintained for over 89 days. No reaction was found between rHcRas and antibodies against Trichinella spiralis, Fasciola hepatica, or Toxoplasma gondii. An indirect enzyme-linked immunosorbent assay (ELISA) was produced based on rHcRas. The optimal coating antigen (157 ng of rHcRas/well) and serum dilutions (1:50) were determined via checkerboard titration. Indirect ELISA based on rHcRas showed 87.5% sensitivity and 90.6% specificity. The cut-off values for this experiment were determined to be 0.324 (positive) and 0.273 (negative), respectively, and the variation coefficient (CV) was less than 15%. The results of the indirect ELISA in-field examination showed that 17.6% (9/51) of the goats were infected with H. contortus, higher than the fecal examination results (15.7%, 8/51). When compared the results of the indirect ELISA and necropsy testing, 98.0% (50/51) consistency was found. These results indicated that rHcRas was a potential antigen for the diagnosis of H. contortus infection in goats.

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Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat

10.21203/rs.2.12443/v4 ◽

2020 ◽

Author(s):

Muhammad Ali-ul-Husnain Naqvi ◽

KaLiBiXiaTi Aimulajiang ◽

Muhammad Ali Memon ◽

Muhammad Waqqas Hasan ◽

Sana Zahra Naqvi ◽

...

Keyword(s):

Haemonchus Contortus ◽

Cold Shock ◽

Fasciola Hepatica ◽

Trichinella Spiralis ◽

Early Stage ◽

Cross Reactivity ◽

Antibody Detection ◽

Economic Losses ◽

Diagnostic Potential ◽

Specificity And Sensitivity

Abstract Background : Haemonchus contortus ( H. contortus ) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. Results : Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21-103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis , Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28μg/well at 37℃ 1h and overnight at 4°C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD 450 ). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. Conclusion : These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

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Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat

10.21203/rs.2.12443/v2 ◽

2019 ◽

Author(s):

Muhammad Ali-ul-Husnain Naqvi ◽

KaLiBiXiaTi Aimulajiang ◽

Muhammad Ali Memon ◽

Muhammad Waqqas Hasan ◽

Sana Zahra Naqvi ◽

...

Keyword(s):

Post Infection ◽

Haemonchus Contortus ◽

Cold Shock ◽

Fasciola Hepatica ◽

Trichinella Spiralis ◽

Early Stage ◽

Cross Reactivity ◽

Economic Losses ◽

Diagnostic Potential ◽

Specificity And Sensitivity

Abstract Background: Haemonchus contortus (H. contortus) is the most abundant nematode causing haemonchosis with major economic losses to the small ruminant industry farming worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods are available to identify prepatent H. contortus infection. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H .contortus in goat. Results: Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. Fecal egg count technique was unable to detect H. contortus eggs in feces collected between 0 and 14 days post infection. Eggs were detected at 21, 28 and 35 days post infection. However, Specific anti rHc-CS antibodies were detectable in sera of all infected goats during early stage (2nd week of infection) and late stage (3rd to 14th week of infection) using immunoblotting assay. Furthermore, no cross reactivity was observed against most commonly found helminths (Trichinella spiralis, Fasciola hepatica, and Toxoplasma gondii) and uninfected goats. The format variables for rHc-CS indirect-ELISA were optimized. The optimum antigen coating concentration was found 0.28μg/well at 37℃ 1h and overnight at 4°C. Optimum dilution ratio of serum and rabbit anti-goat IgG was recorded 1:100 and 1:4000 respectively. The best blocking buffer was 5% bovine serum albumin (BSA) and the best time for blocking, serum incubation and TMB reaction was recorded as 60, 120 and 10 minutes respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded 100%. Conclusion: These results demonstrated that rHc-CS is a potential immunodiagnostic antigen to detect specific antibodies at early and late H. contortus infections in goat.

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Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat

10.21203/rs.2.12443/v1 ◽

2019 ◽

Author(s):

Muhammad Ali-ul-Husnain Naqvi ◽

KaLiBiXiaTi Aimulajiang ◽

Muhammad Ali Memon ◽

Muhammad Waqqas Hasan ◽

Sana Zahra Naqvi ◽

...

Keyword(s):

Haemonchus Contortus ◽

Cold Shock ◽

Fasciola Hepatica ◽

Trichinella Spiralis ◽

Early Stage ◽

Cross Reactivity ◽

Economic Losses ◽

Diagnostic Potential ◽

Specificity And Sensitivity ◽

Clear Band

Abstract Background: Haemonchus contortus (H. contortus) is the most abundant nematode causing haemonchosis with major economic losses to the small ruminant industry farming worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods are available to identify prepatent H. contortus infection. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H .contortus in goat. Results: Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. No eggs of H. contortus were detected in feces collected at 14 days post infection. However, Specific anti rHc-CS antibodies were detectable in sera of all infected goats during early stage (2nd week of infection) and late stage (3rd to 14th week of infection) using immunoblotting assay. Furthermore, no cross reactivity was observed against most commonly found pathogens (Trichinella spiralis, Fasciola hepatica, and Toxoplasma gondii) and uninfected goats. The format variables for rHc-CS indirect-ELISA were optimized. The optimum antigen coating concentration was found 0.28μg/well at 37℃ 1h and overnight at 4°C. Optimum dilution ratio of serum and rabbit anti-goat IgG was recorded 1:100 and 1:4000 respectively. The best blocking buffer was 5% bovine serum albumin (BSA) and the best time for blocking, serum incubation and TMB reaction was recorded as 60, 120 and 10 minutes respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded 100%. Conclusion: These results demonstrated that rHc-CS is a potential immunodiagnostic antigen to detect specific antibodies at early and late H. contortus infections in goat.

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Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat

10.21203/rs.2.12443/v3 ◽

2019 ◽

Author(s):

Muhammad Ali-ul-Husnain Naqvi ◽

KaLiBiXiaTi Aimulajiang ◽

Muhammad Ali Memon ◽

Muhammad Waqqas Hasan ◽

Sana Zahra Naqvi ◽

...

Keyword(s):

Haemonchus Contortus ◽

Cold Shock ◽

Fasciola Hepatica ◽

Trichinella Spiralis ◽

Early Stage ◽

Cross Reactivity ◽

Antibody Detection ◽

Economic Losses ◽

Diagnostic Potential ◽

Specificity And Sensitivity

Abstract Background: Haemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. Results: Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21-103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28μg/well at 37℃ 1h and overnight at 4°C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. Conclusion: These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

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Acute fasciolosis in cattle in southern Brazil

Pesquisa Veterinária Brasileira ◽

10.1590/s0100-736x2013000600003 ◽

2013 ◽

Vol 33 (6) ◽

pp. 705-709 ◽

Cited By ~ 6

Author(s):

Maria de Lourdes Adrien ◽

Ana Lucia Schild ◽

Clairton Marcolongo-Pereira ◽

Letícia Fiss ◽

Jerônimo L. Ruas ◽

...

Keyword(s):

Fasciola Hepatica ◽

Abdominal Cavity ◽

Clinical Signs ◽

Early Spring ◽

Eosinophil Infiltration ◽

Coagulation Necrosis ◽

Economic Losses ◽

Southern Brazil ◽

Bile Duct Proliferation ◽

Mild Anemia

This study describes the epidemiological and pathological aspects of an outbreak of acute fasciolosis in cattle in southern Brazil. Fifteen out of 70 three-year-old pregnant cows lost weight in the 30-40 days prior to calving. Clinical signs included diarrhea, weakness, mild anemia and jaundice. Dark yellow fluid in the abdominal cavity was observed at necropsy. Fibrin and clotted blood were adhered to the pericardium and lung, primarily in the diaphragmatic lobes. The liver was enlarged, and the capsular surface was irregular with clear areas and petechiae. At the cut surface, the liver was irregular, firm and edematous, and several hemorrhagic channels could be observed. Areas of fibrosis through the parenchyma and whitish thrombi occluding the great vessels were also observed. The livers of 10 cows that not died were condemned at slaughter for lesions of fasciolosis similar to those observed at necropsy. Microscopically, the liver showed areas of coagulation necrosis, extensive hemorrhages in the streaks or foci and disruption of the parenchyma with neutrophil and eosinophil infiltration. Fibrosis and bile duct proliferation were also observed. Immature Fasciola hepatica flukes were observed in the parenchyma surrounded by degenerated hepatocytes, neutrophils, eosinophils, and hemorrhages. The outbreak occurred on a farm located in an area endemic for fasciolosis, although the acute form of the disease is not common in cattle in this region. It is likely that the cows were infected by F. hepatica metacercariae released in the late fall or early spring in the rice stubble where the herd was grazing prior to calving. Although mortality due to fasciolosis in cattle is infrequent, outbreaks can occur and treatments that are effective in both the immature and adult forms of the parasite should be administered to prevent economic losses.

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A Field Study on the Prevalence of and Risk Factors for Endoparasites in Beef Suckler Cow Herds in Germany

Agriculture ◽

10.3390/agriculture8090132 ◽

2018 ◽

Vol 8 (9) ◽

pp. 132

Author(s):

Kathia Gillandt ◽

Jenny Stracke ◽

Tatiana Hohnholz ◽

Ralf Waßmuth ◽

Nicole Kemper

Keyword(s):

Fasciola Hepatica ◽

Eimeria Species ◽

Clinical Signs ◽

Parasite Burden ◽

Gastrointestinal Nematodes ◽

Economic Losses ◽

Pasture Management ◽

The Status ◽

Detailed Statistical Analysis ◽

Beef Suckler

Endoparasites are one of the major health issues in beef suckler cows and can cause economic losses. As studies on the parasitological status of beef suckler cow herds are rare, the aim of this study was to evaluate the status quo of the parasite burden in herds at four representative locations in Germany. Additionally, the farmers’ pasture management and deworming strategies were documented. Based on these data, the second aim of the study was to develop recommendations for improved deworming and pasture hygiene management. A total of 708 faecal samples were examined with parasitological routine methods. Results revealed Fasciola hepatica, gastrointestinal nematodes (GIN), Eimeria species (spp.), Moniezia spp. and Dictyocaulus viviparus as the most frequent findings. Clinical signs of parasitic diseases were not found during the farm visits. Statistical analyses showed a significant effect of the age status of the animal on the parasitological status in general. Due to the percentage of occurrence, detailed statistical analysis was performed for Eimeria, GIN and Fasciola hepatica, confirming the effect of age status. Assessing the parasitological status of beef suckler cows as routine procedure could help to establish an improved parasite-control management on a farm-individual basis.

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SEROLOGICAL DIAGNOSIS OF EQUINE INFECTIOUS ANEMIA VÍRUS INFECTION IN THE CENTRAL REGION OF THE RIO GRANDE DO SUL STATE

Ciência Rural ◽

10.1590/s0103-84781992000200012 ◽

1992 ◽

Vol 22 (2) ◽

pp. 191-196 ◽

Cited By ~ 2

Author(s):

Marlon Cezar Rebelatto ◽

Clóvis de Oliveira ◽

Rudi Weiblen ◽

Saul Fontoura da Silva ◽

Luiz Sérgio Segala de Oliveira

Keyword(s):

Equine Infectious Anemia Virus ◽

Serological Diagnosis ◽

Rio Grande Do Sul ◽

Serum Samples ◽

Agar Gel ◽

Blood Sucking ◽

Equine Infectious Anemia ◽

South Of Brazil ◽

Anemia Virus ◽

Santa Maria

This paper consists of a brief review on equine Infectious anemia added of the results of serological diagnosis of this infection performed at the Federal University of Santa Maria, RS, Brazil, between 1979 to 1990. A total of 7,035 serum samples were tested by the agar gel immunodiffusion test (Coggins test), to which 40 (0,57%) reacted positively. This percentage of positivity is lower than in other regions of Brazil, probably due to the lack of predisposing factors for equine infectious anemia virus spread, such as, a low density of blood-sucking insects in the South of Brazil and the seldom use of massive therapies and vaccinations in the region.

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Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in Goat

Animals ◽

10.3390/ani9080548 ◽

2019 ◽

Vol 9 (8) ◽

pp. 548 ◽

Cited By ~ 7

Author(s):

Muhammad Ali-ul-Husnain Naqvi ◽

Sana Zahra Naqvi ◽

Muhammad Ali Memon ◽

Kalibixiati Aimulajiang ◽

Muhammad Haseeb ◽

...

Keyword(s):

Western Blot ◽

Western Blotting ◽

Haemonchus Contortus ◽

Indirect Elisa ◽

Fasciola Hepatica ◽

False Negative ◽

Cross Reactivity ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Combined Use

Haemonchus contortus is recognized as one of the important health problems in small ruminants, leading to reduced production and economic loss for farmers worldwide. Prepatent diagnosis of H. contortus infection is crucial to improve control strategies as this helminth may remove up to one-fifth of total erythrocytes and may cause anemia, edema, diarrhea, and ultimately death in young animals. In this study, one of the excretory and secretory products, rHc-HCA59, was purified and used as antigen to detect specific antibodies in H. contortus infected goats during prepatent stage of infection using indirect enzyme linked immunosorbent assay (ELISA) as screening test. All goats (n = 38) were housed indoor, experimentally infected with 8000 infective larvae (L3) of H. contortus, and serum samples were collected prior to infection and at 14th day of infection. Immunoblotting was performed to confirm the results of indirect ELISA, evaluate the cross reactivity against rHc-HCA59 in sera of most common co-infecting parasites and rectify the false negative samples. Furthermore, three different batches of rHc-HCA59 were produced to evaluate the repeatability of ELISA. No eggs were detected in feces of all goats collected at 7th and 14th day of infection but, H. contortus eggs were detected at 21 days post infection in the feces. Indirect ELISA performed in this study showed 87% sensitivity and 100% specificity. The western blot analysis confirmed immunoreactivity in serum samples which scored positive in indirect ELISA and recognized the samples as negative which had OD450 lower than negative cut-off value in indirect ELISA. Furthermore, all false negative sera (n = 5) that had OD450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot. Moreover, no cross-reactivity was detected in ELISA and western blotting against rHc-HCA59 in positive sera of Toxoplasma gondii, Fasciola hepatica, and Trichinella spiralis. The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool for the detection of H. contortus infection during prepatent period in goats.

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Review on Epidemiology, Control and Public Health Importance of Bovine Fasciolosis

Austin Journal of Veterinary Science & Animal Husbandry ◽

10.26420/austinjvetscianimhusb.2021.1082 ◽

2021 ◽

Vol 8 (2) ◽

Author(s):

Feyisa TT ◽

Keyword(s):

Public Health ◽

Life Cycle ◽

Intermediate Host ◽

Bile Ducts ◽

Fasciola Hepatica ◽

Previous History ◽

Clinical Signs ◽

Farm Animals ◽

Economic Losses ◽

Public Health Importance

Fascioliasis is a trematode zoonotic snail-borne disease of public health and economic importance. Fasciolosis is a parasitic disease that affects most population of cattle and it exists in almost all parts of the world. It is caused by commonly known species of liver fluke that are Fasciola hepatica and Fasciola gigantica, which mainly affects domestic ruminants. Fasciolosis is more apparent in young cattle and is usually chronic in nature. Adult flukes in the bile ducts cause inflammation, biliary obstruction, distraction of liver tissue and anemia. Fasciolosis is the major burden for Animal production and by direct or indirect economic losses at different part of our country. Diagnosis of fasciolosis is based primarily on clinical signs and seasonal occurrence in endemic areas but previous history of fasciolosis on the farm or identification of snail habitats; postmortem examinations, hematological tests and examination of faeces for fluke eggs are useful. The life cycle of Fasciola spp. is a typical of digenetic treamatodes. Eggs laid by the adult parasite in the bile ducts of their hosts pass into the duodenum with the bile. The life cycle of Fasciola goes through the intermediate host and several developmental stages in the intermediate host. On a herd basis, clinical signs of fluke infection are usually vague (i.e., reduced productivity) and can be difficult to differentiate from the effects of less-thanoptimal management or other chronic disease conditions. Transmission of fasciolosis infection is depending on the presence of “lymnea snail”, host and final host. Among many parasitic problems of farm animals, fasciolosis is a major disease which imposes economic impact on livestock production particularly of cattle and sheep and it has public health importance as it have zoonotic value.

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