The occurrence of Coxiella burnetii in sheep and ticks of the genus Dermacentor in Baden-Wuerttemberg

This study examined the occurrence of Coxiella burnetii (C. burnetii), the infectious agent of Q-fever, in sheep and sheep-ticks in Baden-Wuerttemberg, Germany, as a possible source of infection in Q-fever outbreaks. Using PCR, we examined a total of 1066 Dermacentor ticks from 23 herds and 49 samples of tick excrement from 18 herds for C. burnetii. We found the infectious agent in one non-engorged tick and in one sample of tick excrement from the same herd, in Efringen-Kirchen (district Loerrach). Sequencing the PCR-products confirmed the amplifications as specific for C. burnetii. Further serological tests of random samples of the four districts of Baden-Wuerttemberg showed a seroprevalence from 0 to 1.4% using complement fixation test (CFT), as well as a 0.9 to 10.2% seroprevalence, using ELISA test. Serum samples from a Q-fever-suspicious herd resulted, however, in 6% (CFT) and 53% (ELISA) positive reactions. A comparison between CFT and ELISA showed both a correlation of the two test methods that increased with higher CFT titration levels and positive reactions using ELISA for 9.4% of the serums that had tested negative using CFT. The results of the present study reveal that ticks and their excrements are important vectors of transmission of Q-fever in Baden-Wuerttemberg. Investigations on C. burnetii using PCR as well as serological surveys of sheep are important instruments for diagnosis and disease control of Q-fever.     

Evaluation of the recombinant Heat shock protein B (HspB) of Coxiella burnetii as a potential antigen for immunodiagnostic of Q fever in goats

Coxiella burnetii is an intracellular bacterium that causes a worldwide zoonosis, the Q fever. Currently, to diagnose the infection in ruminants, whole cell antigens-based ELISAs are used. In this study a heat shock protein, the HspB, was evaluated for its ability to be recognized by the goat immune system and its capacity to sign a stage of infection. The htpB gene of C. burnetii was cloned and sequenced. A high identity (>90%) was observed among the htpB genes of four ruminant strains tested. A recombinant protein was expressed in a prokaryotic expression system. The rHspB protein was used to determine the IgG reactivity by ELISA. Sera from experimentally and naturally infected goats were tested. The rHspB is recognized early during the infection course, at 18 days post-infection. Moreover, 80-90% of the animals tested were positive at 39-60dpi. In addition, animals presenting a reactivation of the infection displayed a higher reactivity, statistically significant (p<0.05), than that of the animals in latent infection. These findings suggest that the rHspB could be a good candidate for the development of an ELISA test making possible the detection of recent C. burnetii infection in goats as well as reactivation in those with latent infection.     

Diagnosis of Coxiella burnetii-related abortion in Italian domestic ruminants using single-tube nested PCR

Coxiella burnetii, an obligate intracellular parasite with a worldwide distribution, is the causative agent of acute and chronic Q fever in humans. Although infection is often unapparent in cattle, sheep and goats, there is increasing evidence that C. burnetii infection in these species is associated with abortion and stillbirth. This paper describes the introduction of a single-tube nested PCR protocol for the diagnosis of C. burnetii-related abortion in domestic ruminants in Italy. A total of 514 aborted foetuses from cattle (n = 138) and sheep and goat (n = 376), collected from 301 farms, were analyzed from January 2001 to March 2005. Ninety-seven of 514 (18.9%) animals tested PCR-positive, with 16/138 (11.6%) cattle and 81/376 (21.5%) sheep and goat. Eleven of 102 (10.8%) farms with reproductive disorders in cattle and 37/199 (18.6%) farms with reproductive disorders in sheep and goats were infected with C. burnetii. A greater incidence was observed in three of the seven investigated provinces (p < 0.01), with rates of infected farms of up to 23.8%. Data showed that almost all the C. burnetii-related abortions were recorded between October and April (p < 0.01). These findings suggest that Q fever in humans is largely underestimated in Italy, probably because its occurrence is obscured by flu-like symptoms in acute forms.     

Comparison of a maedi-visna virus CA-TM fusion protein ELISA with other assays for detecting sheep infected with North American ovine lentivirus strains.

A maedi-visna virus CA-TM fusion protein ELISA (MVV ELISA) was evaluated for the detection of antibody in sheep infected with North American ovine lentivirus (OvLV). The results of the MVV ELISA were compared with other assays for OvLV antibody and with viral infection in an intensively studied group of 38 sheep with a high prevalence of OvLV infection and disease. The sensitivity, specificity, and concordance of assays for OvLV antibody (MVV ELISA, indirect ELISA, Western blot, and AGID), virus (virus isolation, PCR, antigen ELISA), and OvLV-induced disease in each animal were compared with OvLV infection status as defined by a positive result in two or more of the assays. Five sheep met the criteria for absence of OvLV infection. The sensitivity of the MVV ELISA in detecting OvLV infected sheep was 64%, whereas the sensitivity of the other three tests for antibody ranged from 85 to 94%. All the antibody assays were 100% specific in this group of animals. Of the assays for virus, the PCR test had the highest sensitivity and the best concordance with OvLV infection, but it also had the lowest specificity of any of the virus or antibody assays. Among the antibody tests, the concordance of the MVV ELISA compared most favorably with the AGID test for detecting OvLV-infected sheep. Analysis of serum samples from 28 lambs experimentally-infected with one of three North American strains of OvLV suggested that there were no significant strain differences detectable by antibody assay. Twenty virus-inoculated lambs were positive by both the MVV ELISA and the AGID test, five lambs were MVV ELISA negative and AGID test positive, and three lambs were MVV ELISA positive and AGID test negative. No pre-inoculation samples were positive by either assay. In a longitudinal study involving seven lambs, antibodies to OvLV were detected by AGID 3-5 weeks post-inoculation, but were not detected by MVV ELISA until 5-10 weeks post-inoculation. Among 128 naturally and experimentally-infected sheep that were seropositive in the AGID test, the overall sensitivity of the MVV ELISA was higher in the naturally infected sheep (84%) than in the experimentally infected sheep (69%). The data indicated that the MVV ELISA represents a less sensitive, but specific alternative for the detection of OvLV antibodies.     

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%).     

Seroprevalence of Q fever (coxiellosis) in sheep from the Southern Marmara Region, Turkey

Little information is available in Turkey on Q fever, a zoonose caused by Coxiella burnetii and transmitted from domestic ruminants. This study aimed at investigating the seroprevalence in sheep flocks from three provinces (Bursa, Balikesir and Canakkale). Serosurvey was undertaken on 42 flocks, which were categorised by sizes. Sera were collected randomly from specific age groups within the young population. CHEKIT Q-fever ELISA kit was used to identify the infection in sheep. The results showed that 20% (n=151) of sheep were seropositive. A total of 34 flocks (81%) revealed at least one seropositive animal. Higher seroprevalence was observed in Balikesir region. Larger flocks resulted more infected than medium and small flocks. An association was found between seropositivity and age, when the primiparous ewes (1-year old) had higher antibodies rates than newborn sheep (aged less than 10 months) or biparous ewes (2 years old). These results showed that Q fever infection was common and circulating in the studied region, hence encourage efforts to propose measures that could reduce the spread and the zoonotic risk.     

Occurrence, distribution, and role in abortion of Coxiella burnetii in sheep and goats in Sardinia, Italy

Between 1999 and 2002, 9349 sera and 517 aborted samples (422 foetuses and 95 placenta) were analysed from 675 sheep and 82 goat farms distributed all over the island of Sardinia. After abortion notification, sera collected at random from adult animals were examined to detect antibodies specific to Coxiella burnetii by ELISA, whereas foetuses and placenta were analysed by PCR assay. Specific IgG antibodies were detected in 255 (38%) sheep farms and in 39 (47%) goat herds whereas 40 ovine (10%) and 3 (6%) caprine foetuses were C. burnetii PCR-positive. Although C. burnetii DNA was amplified from different types of tissues, placenta was the tissue with the highest detection rate. Seroprevalence analysis indicates that C. burnetii distribution in sheep and goats is very high, but PCR results demonstrate that C. burnetii has a relatively low role in abortion, especially in goats.     

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.     

Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis

OBJECTIVE: To determine the sensitivity and specificity of an absorbed ELISA and an AGID test for the detection of clinical and subclinical paratuberculosis in sheep. DESIGN: By testing a panel of sera from 1257 Australian Merino and crossbred sheep greater than 1 year of age, of which 1137 sheep were not infected with Mycobacterium avium subsp paratuberculosis and 120 sheep had paratuberculosis. PROCEDURE: Sera were collected from 457 sheep in Victoria and 800 sheep in Western Australia. Presence of M a paratuberculosis infection in Victorian sheep was determined by histological examination of intestinal tissues, whereas sheep from Western Australia were presumed to be free of Johne's disease. The ability of an absorbed ELISA to discriminate between infected and uninfected sheep was described by test sensitivity and specificity, the distribution of ELISA OD, and the area under a receiver operating characteristic curve. RESULTS: The absorbed ELISA had a specificity of 98.2 to 99.5% (CI) and a sensitivity of 35 to 54% (CI). In sheep from infected flocks in Victoria, the AGID test had a specificity of 99 to 100% (CI) and a sensitivity of 38 to 56% (CI). The sensitivity of serological tests was higher in sheep with a body condition representative of the lower quintile of their flock of origin. CONCLUSION: The AGID test and absorbed ELISA are useful tests for the detection of ovine paratuberculosis. Although the tests had a similar accuracy, they detected different subpopulations of infected sheep with only moderate overlap. The AGID test had a higher specificity than the absorbed ELISA.     

Paratuberculosis in sheep: an emerging disease in South Africa

During a serological survey for ovine paratuberculosis a total of 145934 ovine serum samples from 2019 farms throughout South Africa were tested by means of the AGID assay. Fifty-two infected farms were identified in the Western Cape and Eastern Cape provinces. Links between infected farms in the two provinces were established. Examination of the distribution of infected farms in the Western Cape indicated a positive correlation between acid soils and occurrence of infection. In an attempt to increase the sensitivity and facilitate screening of large numbers of sera two commercial ELISA systems were evaluated for their potential use in a future monitoring program. Sera from histologically positive sheep and known negative sheep flocks were used. The highest sensitivity (50. 9%) was found if both ELISA systems were run concurrently and the results of both systems combined.     

Evaluation of a commercially available enzyme-linked immunosorbent assay for detecting antibodies to Fasciola hepatica and Fasciola gigantica in cattle, sheep and buffaloes in Australia

A commercially available ELISA for detecting antibodies to liver fluke was evaluated for use in Australia. Milk and serum samples from cattle and sheep in which infection with Fasciola hepatica was confirmed by detection of eggs in faeces were used to estimate sensitivity. Similar samples collected from cattle and sheep outside the F. hepatica-endemic area were used to estimate specificity. The ELISA was also evaluated for detecting antibodies to F. hepatica in milk from sheep and antibodies to Fasciola gigantica in sera from cattle and buffaloes, but with small numbers of samples. In cattle, the sensitivity and specificity of the ELISA were 98.2% and 98.3% using serum and 97.7% and 99.3% using milk. In infected herds, 41.4% and 41.5% of animals were positive in the serum and milk ELISAs, respectively, whereas F. hepatica eggs were found in faecal samples from 26.5% of animals. In sheep, the sensitivity of the ELISA was 96.9% and the specificity was 99.4%. In infected flocks, 60.2% of animals were positive in the serum ELISA and F. hepatica eggs were found in faecal samples 52.2% of animals. There was perfect agreement in the ELISA between paired serum and milk samples collected from ewes. The assay detected antibodies in sera from cattle and buffaloes with natural and experimental F. gigantica infections. In the experimentally infected animals, antibodies were detected 2 weeks post-infection. We conclude that the ELISA will be a valuable tool for diagnosing F. hepatica infections in cattle and sheep. The assay may also be useful for diagnosing F. gigantica infections but further studies are required to establish sensitivity and specificity.     

Sensitivity and specificity of the agar-gel-immunodiffusion test, ELISA and the skin test for detection of paratuberculosis in United States Midwest sheep populations

Our objective was to estimate the sensitivity and specificity of the agar-gel-immunodiffusion test (AGID), the ELISA, and the skin test for the detection of Mycobacterium avium subspecies paratuberculosis (MAP) in sheep using Bayesian methods without a gold standard. Fourteen flocks (2 465 sheep) were used. Five flocks (450 sheep) were considered MAP non-infected and 9 flocks (2 015 sheep) had sheep infected with MAP. Sheep were skin tested and blood was collected for AGID and ELISA testing. Results were analyzed using a Bayesian 3-test in 1-population model fitted in WinBUGS. The model allowed for dependence (correlation) between the two serologic tests, but these two tests were assumed to be conditionally independent of the skin test. The estimated specificity was 99.5% (95% PI of 98.9-99.9%) for the AGID; 99.3% (98.4-99.8%) for the ELISA using an optical density measured cutoff of 0.20; 99.2% (98.1-99.8%) using a cutoff of 0.15; 97.5% (95.8-98.7%) using a cutoff of 0.10; and 98.7% (97.3-99.5%) for the skin test. The estimated sensitivities were 8.3% (6.2-10.7%) for the AGID; 8.0% (6.0-10.4%), 10.6% (8.3-13.1%), and 16.3% (13.5-19.4%) for the ELISA using the cutoffs 0.20, 0.15, and 0.10 respectively; and 73.3% (62.3-85.8%) for the skin test. The skin test was specific in non-infected populations and sensitive in infected populations, although in some cases a positive skin test might represent MAP exposure rather than infection. The AGID and ELISA were specific but lacked sensitivity. The AGID and ELISA consistently identified two different populations of infected sheep with only moderate overlap between positive test results.     

Enzyme-linked immunosorbent assay for detection of bluetongue virus antibodies

The immune response to bluetongue virus in sheep and cattle was studied by applying a newly developed indirect enzyme-linked immunosorbent assay (ELISA). Purified virus obtained by sucrose gradient centrifugation was used at a concentration of 0.01 optical density units (formula: see text) to coat individual wells (200 microliter) of a microtitration plate. Dilution of antigen was performed in 0.05 M carbonate buffer, pH 9.6, and adsorption lasted for at least 16 hours at 4 C. Coated plates retained their activity for 10 weeks when stored at 4 C. Sera recovered from experimentally infected sheep and cattle were tested together with known negative sera. A good correlation between results was obtained with the modified complement-fixation test and the ELISA; however, the ELISA proved to be more sensitive. The group specificity of the ELISA was proven by testing various type-specific sheep and cattle immune sera. The ELISA has potential for the detection of group-specific antibodies to bluetongue virus infection.     

Detection of Coxiella burnetii in the air of a sheep barn during shearing

Local epidemics of Q fever occur sporadically in Germany, mainly in rural residential communities. There is increasing evidence that these outbreaks, which are caused by Coxiella burnetii, are related particularly to the lambing season and shearing periods of nearby sheep holdings. It is assumed that this zoonotic agent is massively emitted from the placenta of infected ewes at birth and during shearing of wool contaminated with infected faeces of ticks. However, little is known about the airborne transmission and travel distance of this infectious agent, and only few attempts have been made to isolate it directly from the air. This paper describes for the first time the isolation and detection of C. burnetii in the air of an enclosed sheep barn during shearing of a herd which had tested positive for C. burnetii serologically and by PCR. Samples of inhalable dust samples were taken using I.O.M. samplers with glass fibre and polycarbonate filters at a flow rate of 2.5 l/min. The sampling time was nearly 4.5 h. Two sampling positions were set up on both sides of the shearing place at a distance of 3 m and 1.5 m above the ground. A third position with the same sampling equipment was not activated and served as a sampling and transport control. In the laboratory, the glass fibre filters were used to determine the dust concentration. The polycarbonate filters were treated in a specific breakdown procedure which inactivates PCR inhibitors, followed by amplification and sequencing of a specific DNA section of C. burnetii, which was found in the dust from both active sampling positions. The investigation clearly shows that the sampling and detection methods used in this small field study are suitable for the detection of C. burnetii in the air of sheep barns. The results confirm experimentally the high risk of airborne transmission of C. burnetii from sero-positive sheep herds during shearing. However, little is known about the effective travel distance of infective airborne C. burnetii particles. There is an urgent need for more detailed investigations on the emission and airborne dispersion of infectious C. burnetii particles in order to improve our understanding of the health risks caused by this zoonotic agent originating from sheep herds.     

Evaluation of an enzyme-linked immunosorbent assay for the serological diagnosis of Neospora caninum infection in sheep and determination of the apparent prevalence of infection in New Zealand

Neospora caninum has recently been shown to be a cause of abortions of sheep in New Zealand. A commercially available enzyme-linked immunosorbent assay (ELISA) was validated for use in sheep with sera from experimentally infected sheep. A cut-off threshold was established that demonstrated sero-conversion between 7 and 14 days post-infection. Higher inocula led to earlier sero-conversion. This ELISA was applied to 640 sera collected from rams across New Zealand and 0.625% (+/-0.61%) (4/640) were shown to be serologically positive. The four positive sera were also demonstrated to be positive by indirect fluorescent antibody test (IFAT). The ELISA evaluated here lends itself more readily to large-scale investigations than IFAT. The low background of N. caninum infection in the New Zealand sheep population suggests that N. caninum abortions could be more easily diagnosed by serological means than in populations with higher background sero-prevalence.     

Investigations concerning the prevalence of Coxiella burnetii and Chlamydia abortus in sheep in correlation with management systems and abortion rate in Lower Saxony in 2004

The intracellular bacteria Coxiella (C) burnetii and Chlamydia (Chl) abortus induce abortion in sheep and also affect humans. While Chl. abortus only infrequently infects humans, C burnetii is the aetiological agent of numerous Q fever outbreaks during the last decades. There is only limited knowledge about the prevalence of both pathogens in sheep, although sheep are involved in almost all Q fever outbreaks in Germany. The aim of our study was to investigate the prevalence of both pathogens in flocks located in Lower Saxony, Germany, in correlation to the management form and abortion rate. Serum samples of 1714 sheep from 95 flocks located in Lower Saxony were investigated by ELISA. 2.7% of these samples were positive, 1.3% showed inconclusive results in the C. burnetii-ELISA. Elevated intra-flock seroprevalences were only detected in three migrating flocks. Chlamydia-specific antibodies could be detected in 15.1% serum samples of mainly shepherded and migrating flocks. In one of these flocks with a high intra-flock seroprevalence for C burnetii (27%) and Chlamydia (44.9%), C burnetii was detected in 21.6% of the placenta samples of normal births and in 12.5% of the colostrum samples by PCR. Aborted fetuses and the corresponding placentas were negative in C burnetii-PCR, but in most of them and also in many other placenta samples Chl. abortus could be detected by PCR and DNA microarray. This survey shows a low overall prevalence of C. burnetii in sheep in Lower Saxony in the year 2004. However, three migrating flocks with a high intra-flock prevalence are localized in the southern parts of Lower Saxony. Spreading of C burnetii could occur, because of the large radius of grazing of all three flocks.     

Goats may experience reproductive failures and shed Coxiella burnetii at two successive parturitions after a Q fever infection

Q fever is a zoonosis caused by the obligate intracellular bacterium, Coxiella burnetii. Aborting domestic ruminants are the main source of human infection. In January 2003, an abortion episode occurred in a dairy caprine herd where 18/60 (30%) goats experienced reproductive problems: 4/60 (7%) aborted and 14/60 (23%) had stillbirths. Serological screening for abortion-related infectious diseases suggested Q fever. The diagnosis of C. burnetii infection was confirmed with PCR based on the occurrence of C. burnetii shedding into vaginal mucus, faeces and colostrums taken after kidding from the affected animals. The pregnancy following this episode resulted in one abortion and four stillbirths; three of those goats had already experienced reproductive failure during the previous kidding season. The seroprevalence of C. burnetii infection and the bacteria shedding were investigated using both ELISA and PCR assays, respectively, during the course of the initial and subsequent kidding seasons. Serological testing, performed on the whole herd 6 weeks after the abortion episode, showed 48/60 (80%) of ELISA positive goats. PCR assay performed on both vaginal swab and milk samples showed that the bacterium was shed for almost four months after the outbreak. C. burnetii DNA was also amplified from vaginal swab and milk samples taken from goats after the second kidding season. Furthermore, the bacteria were found into 14 vaginal swabs and 12 milk samples taken from infected females at both kidding seasons.     

Ovine brucellosis in alberta

Two parallel surveys of rams from Alberta sheep flocks were conducted to determine the presence of infection with Brucella ovis. In a retrospective study over a period of 24 months, using complement fixation test, 12 flocks out of 142 tested were considered infected. In another 17-month survey of slaughter rams by serology and culture methods 11 flocks out of 124 were found to be infected. The overall prevalence of ovine brucellosis was 8.6% of the flocks tested which represented 12.5% of the estimated sheep flocks in Alberta. Up to 67% of rams in infected flocks reacted to complement fixation test.

The complement fixation test was evaluated for its efficiency in the diagnosis of ovine brucellosis and compared with a limited number of an enzyme-linked immunosorbent assay (ELISA) results and clinical criteria. The complement fixation test as well as ELISA identified all culture positive rams. Both serological tests appeared satisfactory for the diagnosis of B. ovis epididymitis when the results could be interpreted in the light of flock history and clinical findings.

     

Evaluation of a modified Rose Bengal test and an indirect enzyme-linked immunosorbent assay for the diagnosis of Brucella melitensis infection in sheep

A modified Rose Bengal test (mRB) and an indirect ELISA (iELISA) with Protein G as the conjugate, were evaluated for the diagnosis of Brucella melitensis infection in unvaccinated sheep with a known bacteriological status, and their diagnostic efficacy was compared with that of the standard Rose Bengal (RB) and Complement Fixation (CF) tests used in the current eradication campaign in EU countries. All tests showed 100% specificity when testing the sera from 212 Brucella-free sheep. When testing the sera from 219 Brucella melitensis culture-positive sheep, both the mRB and iELISA tests were more sensitive (98.6% and 96.8%, respectively) than the RB and CF tests (95.0% and 92.7%, respectively). These results were similar when testing the sera from 181 animals belonging to infected flocks but found bacteriologically negative, suggesting that the mRB or iELISA tests could advantageously replace the current RB procedure used as the screening test.     

Experimental microcyst sarcocystis infection in lambs: serology and immunohistochemistry

Density gradient centrifugation using a performed self generated gradient of colloidal silica enabled the isolation of microscopic sheep sarcocystis cystozoites, free from heart muscle contamination. The efficiency of separation of cystozoites from residual heart muscle after digestion in pepsin and hydrochloric acid was 63 to 92 per cent. Antigens from cystozoites were used on enzyme-linked immunosorbent assays (ELISA) of plasma from six coccidia-free lambs infected once orally with 70,000 microcystic sheep sarcocystis sporocysts and for raising antisera in rabbits. Use of an anti-sheep IgM conjugate in the ELISA showed that anti-sarcocystis IgM production was transitory, appearing five to 10 days after infection, peaking in concentration at 42 days and following the peak of the acute phase of infection (32 and 33 days) in the lambs. In contrast, total anti-sarcocystis immunoglobulins, detected by ELISA, increased from five to 21 days after infection and continued to increase until the lambs were killed (the last at 81 days) and was more useful in diagnosing chronic infection. No cross reactions between microcystic sheep sarcocystis and Toxoplasma gondii or Eimeria species of sheep were observed. A peroxidase anti-peroxidase test, using rabbit anti-sarcocystis sera, detected second generation meronts and sarcocysts in fixed tissues from infected lambs making it useful for the diagnosis of acute or chronic disease post mortem.