Competitive ELISA for the detection of antibodies to Rift Valley fever virus in goats and cattle

Rift Valley fever virus (RVFV) is one of the important emerging viral diseases of serious impact in public health and animal hygiene both in human and animal industries. In this study, we developed a monoclonal antibody-based competitive ELISA for the detection of antibodies to RVFV in goats and cattle. The recombinant N protein of RVFV was expressed in E. coli with a six-histidine tag, and the purified N protein was used for detecting antigen with a competitive monoclonal antibody against RVFV antibodies. The competitive ELISA (C-ELISA) could detect antibodies at 9-11 days after inoculation in goats and cattle with a sensitivity of 94.7% (virus neutralization titer >32) and specificity of 99.7%, respectively. In addition, the C-ELISA did not show any cross-reactivity with positive sera against arboviruses such as Akabane, Aino, Chuzan, Ibaraki and bovine ephemeral fever virus, which are prevalent viral agents in ruminant animals throughout Southeast Asia. The results of the present study indicate that the C-ELISA is a simple, rapid and convenient serodiagnostic method for RVFV in goats and cattle.     

Enzyme-linked immunosorbent assay for detection of antibodies to Rift Valley fever virus in ovine and bovine sera

An enzyme-linked immunosorbent assay (ELISA) was developed for the rapid detection of antibodies to Rift Valley fever virus (RVFV) in ovine and bovine sera. Conditions to reduce nonspecific reactions were optimized. The ELISA results correlated with those of a plaque-reduction neutralization test, revealing 100% specificity and 90.7% sensitivity. In sera from sheep and cattle inoculated against RVFV, the hemagglutination-inhibition test in combination with the ELISA provided a better indication of response to killed RVFV vaccine than did either test alone.     

Report of a serological study of Coxiella burnetii in domestic animals in Albania

The prevalence of Coxiella burnetii antibodies in domestic ruminants in Albania has been investigated. A total of 1656 serum samples taken from sheep, goats, and cattle housed on farms located in 20 different districts were tested by ELISA for the presence of specific antibodies to C. burnetii phase I and II antigens. Specific IgG antibodies were detected in 9.1% of the animals from both lowland and mountainous areas. In total, a slightly higher percentage of antibodies was detected in sheep and goats (9.8%) than in cattle (7.9%).     

Rinderpest Antibody Detected in Sheep and Goats Before an Outbreak of Rinderpest Reported in Cattle in Northern Tanzania

In January 1997, serum samples from 1346 adult sheep and goats were tested by a competitive ELISA to determine the prevalence of rinderpest in the northern zone of Tanzania. Seroconversion rates of 20%, 13%, 9%, 7% and 3% in sheep and goats were recorded in Ngorongoro, Monduli, Hai, Arumeru and Simanjiro districts, respectively. The low profile and insidious nature of the rinderpest virus involved caused very mild disease in cattle in some of these area. The mild signs associated with this outbreak of rinderpest resulted in difficulty in its diagnosis. In these circumstances, the presence of rinderpest antibody in sheep and goats served as a valuable and effective indicator of the rinderpest outbreak in cattle.     

Ehrlichia ruminantium seroprevalence in domestic ruminants in Ghana. II. Point prevalence survey

Serum samples collected on a single occasion from cattle, sheep and goats at sites in all 10 regions of Ghana were tested for antibodies to Ehrlichia (previously Cowdria) ruminantium, the causative agent of heartwater, by polyclonal competitive ELISA (PC-ELISA). The survey revealed the presence of heartwater-exposed ruminants throughout the country, with local seroprevalence up to 100%. Seronegative, and therefore presumably susceptible, animals were also present in all regions, in some areas in numbers high enough to indicate local endemic instability. Overall seroprevalences in cattle, sheep and goats were 61, 51 and 28% respectively, and were generally higher in the northern part of the country and lower in the forest zone. Amongst animals over 1 year old, two thirds of cattle and sheep, and around one third of goats throughout the country had been exposed to E. ruminantium. In the north, seroprevalence in sheep sampled with and without cattle was similar, whereas in the south seroconversion rates in sheep were significantly higher in areas where cattle were present.     

The detection of antibody against peste des petits ruminants virus in Sheep,Goats, Cattle and Buffaloes

Monoclonal antibody-based competitive ELISA (C-ELISA) has been used for the specific measurement of antibodies to peste des petits ruminants (PPR) viruses in sheep, goats, cattle and Buffalo. Serum samples from sheep (n = 232), goats (n = 428), cattle (n = 43), buffalo (n = 89) were tested. The animals had not been vaccinated against rinderpest or PPR. Findings suggested that the sero-positive cases were significantly higher in sheep (51.29%) than in goats (39.02%) (P = 0.002). The overall sero-prevalence of PPRV in small ruminants was 43.33%. The PPR antibodies seroprevalence was 67.42% in buffalo and 41.86% in cattle which was significantly higher in buffalo (P = 0.005). The overall sero-prevalence of PPRV in large ruminants was 59.09%. Cattle and buffalo sera showed a high prevalence of antibody against PPR virus which may explain the difficulty experienced in achieving high post-vaccination immunity levels against rinderpest. Because antibodies against PPR virus are both cross-neutralizing and cross-protective against rinderpest virus, further vaccination in the presence of antibodies against PPR virus may be a waste of national resources. It was also suggested that antibodies to PPR virus could prevent an immune response to the rinderpest vaccine. This paper presents serological evidence for the transmission of PPR virus from sheep and goats to cattle and buffalo and highlights the need to include PPR serology in the sero-monitoring programme to give a better indication of national herd immunity of sheep and goats against PPR.     

Cloning and expression of Rift Valley fever virus nucleocapsid (N) protein and evaluation of a N-protein based indirect ELISA for the detection of specific IgG and IgM antibodies in domestic ruminants

Serodiagnosis of Rift Valley fever (RVF) currently relies on the use of live or inactivated whole virus as antigens. The recombinant nucleocapsid (N) protein of RVF virus was tested for diagnostic applicability in an indirect enzyme-linked immunosorbent assay (I-ELISA), using sera from experimentally infected sheep (n=128), vaccinated sheep (n=240), and field-collected sera from sheep (n=251), goats (n=362) and cattle (n=100). The N-protein based I-ELISA performed at least as good as VN and HI tests. In goat the diagnostic sensitivity (D-Sn) and specificity (D-Sp) of the I-ELISA was 100% when using the anti-species IgG conjugate. Using protein G as a detection system, the D-Sn and D-Sp in goats were 99.4% and 99.5%, in sheep field sera both 100%, in cattle 100% and 98.3%, respectively. The I-ELISA based on recombinant N-protein has the potential to complement the traditional assays for serodiagnosis of RVF. Advantages of the N-protein are its safety, stability and cost-effectiveness in use and production.     

Diagnostic performance of PCR and ELISA on blood and milk samples and serological survey for small ruminant lentiviruses in central Spain

The diagnostic performance of an ELISA for the detection of antibodies to the small ruminant lentiviruses (SRLVs) maedi-visna virus and caprine arthritis-encephalitis virus in milk and corresponding blood samples was evaluated in 50 sheep. The agreement between ELISA results in blood and milk was 90 per cent, and the κ value was 0.79. In addition, a serological survey in the central zone of Spain was performed using milk samples from 413 animals (250 sheep and 163 goats) from 12 flocks/herds. All flocks/herds had some animals that were positive for SRLV. Among the animals, 60.0 per cent of the sheep and 8.0 per cent of the goats tested were seropositive. Each sample was also tested using a PCR technique, which increased the percentage of positive animals detected. Using a combination of ELISA and PCR gave a total of 72.2 per cent of sheep and 28.8 per cent of goats positive for SRLV.     

Serologic and virologic investigations into pestivirus infection in wild and domestic ruminants in the Pyrenees (NE Spain)

An outbreak of disease associated to a border disease virus was described in the Southern chamois (Rupicapra pyrenaica) in Spain in 2002. Sera and/or spleen samples from 57 mouflon, 15 red deer, 21 roe deer, 3 fallow deer, 55 sheep, 32 cattle, and 68 goats sharing the chamois habitat were studied. An antibody ELISA test yielded an inconclusive result in 2 mouflon and positive results in 5 goat sera. Comparative virus neutralization tests were performed on the 2 inconclusive mouflons, 3 of the 5 seropositive goats, 55 sheep and 32 cattle, using 6 pestivirus strains. Positive results were obtained in 1 mouflon, 2 goats, 69% of sheep and 78% of cattle. Virological investigations performed with an antigen ELISA test yielded negative results in 21 goats and 39 mouflons, the result in 1 mouflon being inconclusive. PCR performed on 12 goats and the inconclusive mouflon gave negative results. These results suggested that it is unlikely that chamois BDV is infecting wild and domestic ruminants.     

Comparison of the Q-fever complement fixation test and two commercial enzyme-linked immunosorbent assays for the detection of serum antibodies against Coxiella burnetii (Q-fever) in ruminants: Recommendations for use of serological tests on imported animals in New Zealand

Abstract

AIM: To make valid recommendations on the use of serological test methods for the detection of serum antibodies in ruminants against Coxiella burnetii (Q-fever), by comparing the performance of the complement fixation test (CFT) and two ELISA, and by identifying reasons for discrepancies between the test methods.

METHODS: A total of 73 serum samples from infected cattle, 69 from infected goats, and 100 samples from non-infected cattle and 57 samples from non-infected sheep, as well as 95 samples from infected cattle herds (mix of seropositive and seronegative samples), were tested using the CFT, the IDEXX ELISA (I-ELISA) and the Pourquier ELISA (P-ELISA). A mixed panel of 12 serum samples from sheep from inter-laboratory proficiency testing (proficiency panel) was also tested using the CFT and both ELISA, and further investigated using IgG- and IgM-specific ELISA.

RESULTS: Generally, the two commercial ELISA were more sensitive than the CFT for the detection of infected ruminants. Good agreement between ELISA for positive and negative results was found for samples from the infected herd, while results for the positive panels varied between the two ELISA. For the total of the positive serum panels, the I-ELISA detected 95% of samples as positive or suspicious, while the P-ELISA detected only 81%. In the P-ELISA, more samples were considered suspicious (18%) than in the I-ELISA (14%). All sera from noninfected sheep and cattle tested negative in the serological test methods employed, except for one positive sample from a sheep in the P-ELISA. Further investigation revealed that a CFT-positive but ELISA-negative result was due to high IgM and low IgG reactivity.

CONCLUSIONS: The two commercial ELISA were more sensitive than the CFT in all panels from infected ruminants. However, they could only detect IgG. The I-ELISA should be the serological test method of choice for cattle, sheep and goats for import testing of animals into New Zealand because it was more sensitive than the P-ELISA and was equally specific to the PELISA and the CFT. For other animal species, such as deer and camelids, the CFT should still be used since none of the ELISA has been evaluated for these species. This study has shown that the two commercial ELISA will detect the majority of infected ruminants but may miss animals that have not developed an IgG response.     

Re-emergence of foot-and-mouth disease in Botswana

The re-emergence of foot-and-mouth disease (FMD) in Botswana is reported. The disease outbreak occurred in the Matsiloje Extension Area of Francistown veterinary district situated in the northeastern part of the country in an Office International des épízooties (OIE) recognized FMD free zone without vaccination. The disease affected cattle only and did not spillover into sheep and goats resident in the same extension area, as demonstrated by lack of seroconversion to FMD when tested. The virus isolate associated with the outbreak was identified as FMD virus; Southern African Territories (SAT) type SAT-2. The disease outbreak is discussed in relation to FMD outbreaks that have occurred previously within and outside Botswana.     

Peste des petitis ruminants morbillivirus infection in lambs and young goats at Qassim region, Saudi Arabia

An outbreak of peste des petits ruminants (PPR) in lambs and young goats of Najdi breed of sheep and goats occurred during winter 2005 at Qassim region of central Saudi Arabia. The PPR infection was confirmed by demonstration of antibodies against the virus in the serum of clinically-ill young sheep and goats using competitive ELISA test. Clinical examination of infected animals showed fever, salivation, lacrimation, mucopurulent nasal discharge, difficult breathing and diarrhoea. The disease was particularly severe in the goats. Morbidity was about 20% and mortality was less than 3 percent. Autopsy showed necrotic and ulcerative lesions in the mouth, stomach and intestine. Mesenteric lymph nodes were swollen and congested. The lungs were patchy pneumonic mostly at the diaphragmatic and apical lobes. Liver and kidney lesions were seen in goats only and both organs were congested and necrotic. Histopathological examination revealed necrotic inflammation in the gastrointestinal tract. Intracytoplasmic viral inclusions were seen in the enterocytes of goats. Lung sections showed bronchopneumonia and syncytial and giant cells. The bronchial epithelium of goats had intracytoplasmic viral inclusions. Extensive coagulation necrosis, fatty degeneration and presence of intracytoplasmic viral inclusions were seen in hepatocytes and syncytial cells were evident in biliary epithelium of goats. Congestion, coagulation necrosis and syncytial cells were seen in the renal tubular epithelium of goats only. In a survey to determine the magnitude of the outbreak, PPR antibodies were evidenced in 363/996 (36.6%) sheep and 530/962 (55.1%) goats.     

Peste des petits ruminants infection in domestic ruminants in Sudan

The existence of peste des petits ruminants (PPR) in domestic ruminants and camels in Sudan during 2008–2012 was investigated. Lung tissues and serum samples were randomly collected from sheep, goats, cattle, and camels at different areas of Sudan. A total of 12,384 serum samples were collected from clinically healthy 7413 sheep, 1988 camels, 1501 cattle, 1459 goats, and 23 gazelles at different areas in the Sudan. They were examined for PPR antibodies using competitive ELISA (cELISA). The overall detected seroprevalence of PPR in tested sera was 49.4%; seroprevalence values within species were 67.1, 48.2, 25.8, 2.1, and 21.7% in sheep, goat, cattle, camels, and gazelles, respectively. The highest seroprevalence (68.1%) was observed in sera collected from Darfur states, then the central states (54.3%). A total of 1276 lung tissue samples (623 sheep, 324 cattle, 220 camels, and 109 goats) were collected. The majority of lung samples were collected from clinically healthy animals that showed lesions on PM in slaughterhouses (95%) and during PPR outbreaks; samples were tested for PPR antigen using immunocapture ELISA (IcELISA). PPR antigen was detected in 233 out of the 1276 tested samples (18.3%). Positive results were observed in samples collected from clinically healthy and diseased animals. The observed prevalence values in each species were 33.6, 21.1, 15.4, and 12.3% in camel, goat, sheep, and cattle, respectively. PPR antigen was detected in samples from different areas; however, the highest prevalence (63.9%) was found in samples collected from the eastern states, then Khartoum state (28%). Trials for virus isolation were done in different cell cultures. Out of 30 IcELISA-positive samples inoculated in primary bovine and ovine kidney cells, Vero cells, the PPR virus was successfully isolated from 15 (eight sheep, five camels, and two goats) samples in the three cell culture types. Using RT-PCR, PPRV nucleic acid was detected in all 25 IcELISA-positive tested samples.     

Clinical protection, sub-clinical infection and persistence following vaccination with extinction payloads of O1 Manisa Foot-and-Mouth Disease monovalent vaccine and challenge in goats and comparison with sheep

Small ruminants play an important role in the epidemiology of Foot-and-Mouth Disease (FMD). Small ruminants are vaccinated with one-half or one-third of cattle dose of oil-based or aqueous vaccines respectively. The extinction antigen payload in vaccine for protection in small ruminants is poorly studied. FMD seronegative Nellore sheep (n=30) and Osmanabadi goats (n=30) were vaccinated with different payloads of O(1) Manisa vaccine (0.45-5 μg). Vaccinated and sero-negative unvaccinated sheep (n=6) and goats (n=6) were challenged intradermally into the coronary band with O(1) Manisa virus. The sheep and goats were monitored for signs of FMD and samples were collected for measuring viraemia and virus associated with nasal swabs and probang samples. Clotted blood was collected for serology. Vaccines containing antigen payload up to 0.94 μg protected sheep and goats against challenge. Sheep and goats vaccinated with 0.45 μg antigen payload were poorly protected against challenge. An antigen payload of 0.94 μg was sufficient to offer complete protection and also absence of carrier status. Sheep and goats with no vaccination or with poor sero conversion to vaccination showed sub-clinical infection and became carriers. The results of the study suggest that vaccination offers protection from clinical disease even at a low payload of 0.94 μg and hence one-half of cattle dose of the oil-based vaccine formulations is sufficient to induce protective immune response in sheep and goats. Since no live virus could be isolated after 5 days post challenge from the nasal swab or probang samples even though viral RNA was detected, the risk of these animals transmitting disease was probably very low.     

Rift Valley fever in Madagascar in 1991. Sero-epidemiological studies in cattle]

Rift Valley fever (RVF) virus was detected for the first time in Madagascar in 1979, but without any impact on human and animal populations. However, in 1990 and 1991, several outbreaks with massive cattle abortions were described. Since that period, seroepidemiological surveys have been conducted on the East coast and in the highlands (outbreak areas), in the high cattle density regions, and in the national slaughter house in Antananarivo. A high RVF IgM antibody prevalence was detected in the outbreak areas during the epizootics. This IgM prevalence thereafter decreased and was followed by an IgG high prevalence in all tested regions. Hypotheses on the occurrence of the virus in Madagascar, outbreak origins and virus circulation on the island are discussed.     

Antibodies against certain bluetongue and epizootic haemorrhagic disease viral serotypes in Indonesian ruminants.

The orbiviruses contain several important viruses of livestock including bluetongue (BT) and epizootic haemorrhagic disease of deer (EHD) which share some group antigens. Preliminary screening of sera for antibodies to orbiviruses by the agar gel immunodiffusion (AGID) test has previously revealed widespread infections with the BT group in Indonesia. However serum neutralization (SN) tests give a more accurate estimate of exposure to each serotype in the BT and EHD groups, and in this study were applied to sera that had reacted previously in the AGID test. Five different serotypes of BT and one serotype of EHD virus were studied. Reactors to BT serotype 20 were the most prevalent, followed by EHD type 5 and BT types 21, 12, 1 and 17. Antibodies against BT serotype 20 were present in cattle, buffaloes, goats and sheep, but were most common in buffaloes. Buffaloes showed the highest exposure to the BT serotypes tested. Antibody to EHD type 5 occurred most frequently in cattle. Antibodies against all BT and EHD serotypes tested were found in buffaloes and cattle while goats had antibodies against BT types 20, 21 and EHD type 5 and sheep had antibodies only against BT type 20.     

Post-vaccination antibodies profile against Peste des petits ruminants (PPR) virus in sheep and goats of Punjab,Pakistan

A total of 70 sheep and 330 goats were selected randomly. All the animals were kept under same housing and management conditions. Serum samples were collected from all the animals and tested for the presence of antibodies against Peste des petits ruminants (PPR) virus using competitive ELISA (cELISA). All the animals were found negative showing percentage inhibition (PI) values <50. The animals were vaccinated against PPR with Nig/75/1 strain vaccine of PPR Serum samples were collected from randomly selected 12 sheep and 30 goats at 10, 30 and 45 days post-vaccination. The samples were subjected to cELISA to determine the presence of antibodies against PPRV. The samples with PI >50 were considered as sero-positive. The sheep found positive at 10, 30 and 45 days post-vaccination were 1(8.3%), 7(58.3%) and 12(100%) respectively. In case of goats 3(10.0%), 29(96.6%) and 27(90.0%) animals gave positive results at 10, 30 and 45 days post-vaccination respectively. Mean PI values in sheep at 10, 30 and 45 days post-vaccination were recorded as 37, 65 and 91 respectively, whereas in goats these values were 43, 78 and 86 respectively.     

Seroepidemiology of rinderpest in bovids in Sri Lanka using the enzyme linked immunosorbent assay (ELISA) technique

Approximately 0.2% (n=4397) of the bovids (cattle and buffalo) in Sri Lanka were sampled, from June 1992 using a multi-stage sampling procedure. Serum antibodies for the rinderpest virus were detected using the competitive enzyme-linked immunosorbent assay. The age, the agroclimatic zone, the management system practiced in the farms, and the vaccination history of the sampled bovids were studied as potential risk factors for being seropositive.

The prevalence of rinderpest antibodies in non-vaccinated bovids was 3.5% (n=4101). The prevalence was higher in the dry zone (9%; where the outbreak emerged in 1987), compared to bovids in the other zones (1%). Seropositive bovids over three years of age were approximately at fourfold higher chances of being seropositive compared to those that were ≤3 years old. The higher prevalence in older animals is probably due to exposure to the virus during the 1987 epidemic. Bovids from the dry zone (annual rainfall 20 to 35 inches) were at higher odds of being seropositive even after controlling for the possible effects of age, agroclimatic zone, management system and vaccination. The fact that 62% of bovids from the dry zone in this study were reared under extensive management system (free grazing) which allow unrestricted contact between animals, may be the reason for the above finding. A relatively poor response to vaccination observed in vaccinated bovids (seroprevalence=12%; n=296) could be attributed to difficulties in maintaining the vaccine at recommended temperatures in the field. This is the first island-wide study on seroprevalence of rinderpest in Sri Lanka.     

Seroprevalence of malignant catarrhal fever-related gammaherpesviruses in domestic ruminants in Turkey

The prevalence of malignant catarrhal fever (MCF) virus infection in cattle, sheep and goat populations and also the prevalence of recovered and chronic MCF cases in north-western Turkey are reported. A total of 600 animals, 200 individuals of each species, located in four provinces were sampled between December 2003 and July 2005. A monoclonal antibody-based competitive inhibition (ci) ELISA were used to detect infection status of the animals. Detected antibody prevalence was 97.5%, 96.0% and 15.0% in sheep, goats and cattle, respectively. These results showed that MCF related gammaherpesvirus infections are common in north-western Turkey. There was no significant difference between prevalences detected in sheep and goats, as well as various breeds of these species. There was also no significant difference among locations. Results of this study show that sheep and goats may equally be important in the epidemiology of MCF in Turkey. Seropositivity against MCF agents among cattle was 15.0%. The results indicate that MCF infections may be maintained in intensively managed cattle herds having no close contact with small ruminants.