Validation of a serum ELISA test for cyathostomin infection in equines.

Cyathostomins are ubiquitous equine nematodes. Infection can result in larval cyathostominosis due to mass larval emergence. Although faecal egg count (FEC) tests provide estimates of egg shedding, these correlate poorly with burden and provide no information on mucosal/luminal larvae. Previous studies describe a serum IgG(T)-based ELISA (CT3) that exhibits utility for detection of mucosal/luminal cyathostomins. Here, this ELISA is optimised/validated for commercial application using sera from horses for which burden data were available. Optimisation included addition of total IgG-based calibrators to provide standard curves for quantification of antigen-specific IgG(T) used to generate a CT3-specific 'serum score' for each horse. Validation dataset results were then used to assess the optimised test's performance and select serum score cut-off values for diagnosis of burdens above 1000, 5000 and 10,000 cyathostomins. The test demonstrated excellent performance (Receiver Operating Characteristic Area Under the Curve values >0.9) in diagnosing infection, with >90% sensitivity and >70% specificity at the selected serum score cut-off values. CT3-specific serum IgG(T) profiles in equines in different settings were assessed to provide information for commercial test use. These studies demonstrated maternal transfer of CT3-specific IgG(T) in colostrum to newborns, levels of which declined before increasing as foals consumed contaminated pasture. Studies in geographically distinct populations demonstrated that the proportion of horses that reported as test positive at a 14.37 CT3 serum score (1000-cyathostomin threshold) was associated with parasite transmission risk. Based on the results, inclusion criteria for commercial use were developed. Logistic regression models were developed to predict probabilities that burdens of individuals are above defined thresholds based on the reported serum score. The models performed at a similar level to the serum score cut-off approach. In conclusion, the CT3 test provides an option for veterinarians to obtain evidence of low cyathostomin burdens that do not require anthelmintic treatment and to support diagnosis of infection.

Equine tapeworm (Anoplocephala spp.) infection: evaluation of saliva- and serum-based antibody detection methods and risk factor analysis in Slovak horse populations.

A lack of accurate information on the prevalence and distribution of Anoplocephala spp. infections on horse farms has led to insufficient attention to tapeworm control and increasing horse anoplocephaloses in Europe. Our study aimed to examine the occurrence of Anoplocephala spp. infection using coprological, serum- and saliva-based antibody detection methods and to analyze the risk factors associated with tapeworm infection in domestic horses in Slovakia. Fecal, serum, and saliva samples were collected from 427 horses from 31 farms in Slovakia. Additionally, a questionnaire study was conducted to collect information on tapeworm distribution on horse farms and analyze risk factors associated with infection. Fecal samples were examined by the mini-FLOTAC and the double centrifugation/combined sedimentation-flotation techniques. Serum and saliva samples were analyzed by ELISA to determine antibody levels against Anoplocephala spp. The effects of variables associated with an individual horse were tested for the positive result of the saliva ELISA test on Anoplocephala spp. Cestode eggs were detected in 1.99% of fecal samples (farm prevalence 12.90%), with no differences between the two coprological methods. Serum-based tapeworm ELISA results revealed that 39.39% of horses tested positive (farm prevalence 83.87%); while saliva-based tapeworm ELISA results revealed 56.95% positive horses (farm prevalence 96.77%). Binary logistic regression analysis revealed four meaningful predictors that significantly impacted the likelihood of detecting tapeworm infection in horses: horse age, pasture size, anthelmintic treatment scheme, and access to pasture. The influences of other variables associated with an individual horse were not significantly associated with detecting tapeworm infection.

The Use of Innovative Diagnostics to Inform Sustainable Control of Equine Helminth Infections.

Helminths are commonly found in grazing equids, with cyathostomin nematodes and the cestode Anoplocephala perfoliata being the most prevalent. Most horses harbour low burdens of these parasites and do not develop signs of infection; however, in a small number of animals, high burdens can accumulate and cause disease. Cyathostomins are associated with a syndrome known as larval cyathostominosis. This occurs when large numbers of larvae emerge from the large intestinal wall. This disease has a case fatality rate of up to 50%. A. perfoliata infection has been associated with various types of colic, with burdens of >20 worms associated with pathogenicity. Anthelmintic resistance is a serious problem in cyathostomins and is emerging in A. perfoliata. Control methods that reduce reliance on anthelmintics now need to be applied, especially as no new dewormer compounds are on the horizon. Sustainable control methods must employ diagnostics to identify horses that require treatment. Coprological tests (faecal egg counts, FECs) have been used for several decades to inform treatment decisions to reduce helminth egg shedding. These tests cannot be used to assess host burdens as FECs do not correlate with cyathostomin or A. perfoliata burdens. In the last decade, new tests have become available that measure parasite-specific antibodies, the levels of which have been shown to correlate with parasite burden. These tests measure antigen-specific IgG(T) and are available in serum (cyathostomin, A. perfoliata) or saliva (A. perfoliata) formats. Tests for other helminths have been developed as research tools and need to be translated to support equine clinicians in practice. A key element of sustainable control strategies is that diagnostics must be used in combination with management approaches to reduce environmental transmission of helminths; this will help limit the proportion of horses harbouring parasite burdens that need to be targeted by treatment. This manuscript provides a review of the development, performance and general utility of various diagnostic methods for informing equine helminth management decisions.

Investigations on the occurrence of tapeworm infections in German horse populations with comparison of different antibody detection methods based on saliva and serum samples.

BACKGROUND: Effective and sustainable worm control in horses would benefit from detailed information about the current regional occurrence of tapeworms. Different diagnostic methods are currently available to detect Anoplocephala spp. infections in horses. However, the format as well as the sensitivity and specificity of the methods vary considerably. METHODS: A coprological, serological and questionnaire study was conducted to investigate the prevalence and risk factors of tapeworm infections on 48 horse farms in the region of Berlin and Brandenburg, Germany. In total, faecal samples of 484 horses were analysed using the double centrifugation/combined sedimentation-flotation and mini-FLOTAC. Serum (n = 481) and saliva (n = 365) samples were analysed by ELISAs to determine antibody levels against Anoplocephala spp. 12/13 kDa excretory/secretory (E/S) antigens. RESULTS: Cestode eggs were detected in 0.6% of faecal samples (farm prevalence 6.3%) without differences between the two methods. In contrast, antibodies against Anoplocephala spp. were detected in 16.2% (farm prevalence 52.1%) and in 29.5% (farm prevalence 75.7%) of the serum and saliva samples, respectively. Both ELISA based methods for detection of tapeworms reported a greater number of infected animals requiring treatment than were positively identified by coproscopy. Logistic regression analysis identified permanent pasture access, large pastures and regular pasture changes and high strongyle egg counts as risk factors for positive serum antibody responses to Anoplocephala spp. while last treatment with praziquantel was protective. Other protective factors were the presence of foals and high numbers of horses on the farm. Daily removal of faeces from the pasture and horse age did not have a significant effect. CONCLUSIONS: The findings of the present serological investigation indicate that tapeworm prevalence in Berlin/Brandenburg horse farms is much higher than would be anticipated by using conventional/coproscopic analyses. Moreover, the majority of tapeworm-positive horses had not received a cestocidal drug at their last treatment. Considering the already known low sensitivity of the coproscopic detection, the equine veterinary diagnostics can be enhanced by the use of antibody detection methods such as the saliva-based ELISA.

Validation of a novel saliva-based ELISA test for diagnosing tapeworm burden in horses.

BACKGROUND: Tapeworm infections pose a significant threat to equine health as they are associated with clinical cases of colic. Diagnosis of tapeworm burden using fecal egg counts (FECs) is unreliable, and, although a commercial serologic ELISA for anti-tapeworm antibodies is available, it requires a veterinarian to collect the blood sample. A reliable diagnostic test using an owner-accessible sample such as saliva could provide a cost-effective alternative for tapeworm testing in horses, and allow targeted deworming strategies. OBJECTIVES: The purpose of the study was to statistically validate a saliva tapeworm ELISA test and compare to a tapeworm-specific IgG(T) serologic ELISA. METHODS: Serum samples (139) and matched saliva samples (104) were collected from horses at a UK abattoir. The ileocecal junction and cecum were visually examined for tapeworms and any present were counted. Samples were analyzed using a serologic ELISA and the saliva tapeworm test. The test results were compared to tapeworm numbers and the various data sets were statistically analyzed. RESULTS: Saliva scores had strong positive correlations with both infection intensity (0.74) and serologic results (Spearman's rank coefficients; 0.74 and 0.86, respectively). The saliva tapeworm test was capable of identifying the presence of one or more tapeworms with 83% sensitivity and 85% specificity. Importantly, no high-burden (more than 20 tapeworms) horses were misdiagnosed. CONCLUSIONS: The saliva tapeworm test has statistical accuracy for detecting tapeworm burdens in horses with 83% sensitivity and 85% specificity, similar to those of the serologic ELISA (85% and 78%, respectively).

Solution structure of the Mycobacterium tuberculosis EsxG·EsxH complex: functional implications and comparisons with other M. tuberculosis Esx family complexes.

Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn(2+) binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn(2+)-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners.

Molecular features governing the stability and specificity of functional complex formation by Mycobacterium tuberculosis CFP-10/ESAT-6 family proteins.

The Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins play essential but poorly defined roles in tuberculosis pathogenesis. In this article we report the results of detailed spectroscopic studies of several members of the CFP-10/ESAT-6 family. This work shows that the CFP-10/ESAT-6 related proteins, Rv0287 and Rv0288, form a tight 1:1 complex, which is predominantly helical in structure and is predicted to closely resemble the complex formed by CFP-10 and ESAT-6. In addition, the Rv0287.Rv0288 complex was found to be significantly more stable to both chemical and temperature induced denaturation than CFP-10.ESAT-6. This approach demonstrated that neither Rv0287.Rv0288 nor the CFP-10.ESAT-6 complexes are destabilized at low pH (4.5), indicating that even in low pH environments, such as the mature phagosome, both Rv0287.Rv0288 and CFP-10.ESAT-6 undoubtedly function as complexes rather than individual proteins. Analysis of the structure of the CFP-10.ESAT-6 complex and optimized amino acid sequence alignments of M. tuberculosis CFP-10/ESAT-6 family proteins revealed that residues involved in the intramolecular contacts between helices are conserved across the CFP-10/ESAT-6 family, but not those involved in primarily intermolecular contacts. This analysis identified the molecular basis for the specificity and stability of complex formation between CFP-10/ESAT-6 family proteins, and indicates that the formation of functional complexes with key roles in pathogenesis will be limited to genome partners, or very closely related family members, such as Rv0287/Rv0288 and Rv3019c/Rv3020c.

Structure and function of the complex formed by the tuberculosis virulence factors CFP-10 and ESAT-6.

The secreted Mycobacterium tuberculosis complex proteins CFP-10 and ESAT-6 have recently been shown to play an essential role in tuberculosis pathogenesis. We have determined the solution structure of the tight, 1:1 complex formed by CFP-10 and ESAT-6, and employed fluorescence microscopy to demonstrate specific binding of the complex to the surface of macrophage and monocyte cells. A striking feature of the complex is the long flexible arm formed by the C-terminus of CFP-10, which was found to be essential for binding to the surface of cells. The surface features of the CFP-10.ESAT-6 complex, together with observed binding to specific host cells, strongly suggest a key signalling role for the complex, in which binding to cell surface receptors leads to modulation of host cell behaviour to the advantage of the pathogen.

Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility.

We have previously shown that the secreted M. tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 complex, which may represent their functional form. In the work reported here a combination of yeast two-hybrid and biochemical analysis has been used to characterise complex formation between two other pairs of CFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to determine whether complexes can be formed between non-genome paired members of the family. The results clearly demonstrate that Rv0287/Rv0288 and Rv3019c/3020c form tight complexes, as initially observed for CFP-10/ESAT-6. The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins are also able to form non-genome paired complexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding to the more distantly related CFP-10/ESAT-6 proteins.