Infected Ponies Research Articles

Species interactions, stability, and resilience of the gut microbiota - Helminth assemblage in horses

The nature and strength of interactions entertained among helminths and their host gut microbiota remain largely unexplored. Using 40 naturally infected Welsh ponies, we tracked the gut microbiota-cyathostomin temporal dynamics and stability before and following anthelmintic treatment and the associated host blood transcriptomic response. High shedders harbored 14 species of cyathostomins, dominated by Cylicocyclus nassatus. They exhibited a highly diverse and temporal dynamic gut microbiota, with butyrate-producing Clostridia likely driving the ecosystem steadiness and host tolerance toward cyathostomins infection. However, anthelmintic administration sharply bent the microbial community. It disrupted the ecosystem stability and the time-dependent network of interactions, affecting longer term microbial resilience. These observations highlight how anthelmintic treatments alter the triangular relationship of parasite, host, and gut microbiota and open new perspectives for adding nutritional intervention to current parasite management strategies.

Use of Equine Herpesvirus 1 glycoprotein pseudotyped lentiviral particles for the development of serological tests and assessment of lyophilisation for transport and storage

Equine herpesviruses (EHVs) are enveloped DNA viruses predominantly infecting members of the Equidae family. EHVs primarily cause respiratory disease, however EHV-1 can produce cases of a neurological disease, abortion and neonatal death. Thus, these viruses represent a welfare issue for the equine industry and scientific focus for researchers. EHV-1 exhibits a complex array of 12 glycoproteins on its surface envelope, but it is unclear precisely which are important for virus cell entry and the role of each in host immune response. In order to investigate the contribution of these glycoproteins, pseudotype viruses (PVs) could provide a useful study tool. We have successfully generated functional EHV-1 pseudotyped lentiviruses bearing four glycoproteins, gB, gD, gH and gL (sequences derived from an aborted foetus during a large EHV1 outbreak strain in Normandy, France). PVs were employed in a pseudotype virus neutralisation test (PVNT) to measure levels of specific neutralising antibodies serum samples (n=52) taken longitudinally from experimentally infected ponies, compared with uninfected controls. PVs routinely require -80oC for long term storage and a dry ice cold-chain during transport which can impede dissemination and utilisation in other laboratories. Consequently, we further investigated whether freeze-drying (lyophilisation) of EHV-1 PV could address this issue. PVs were lyophilised and pellets either reconstituted immediately or stored under various temperature conditions, sampling at different timepoints. The recovery and functionality of these lyophilised PVs was compared with standard frozen aliquots in titration and neutralisation tests.

Development of an assay using real time cell analysis for the measurement of equid herpesvirus 1 specific neutralizing antibody in horses after experimental infection or field vaccination

Background: Equid herpesvirus-1 (EHV-1) is one of the most important horse pathogens, which induces respiratory disease, myeloencephalopathy, abortion or still-birth. The measurement of EHV-1 neutralising (EHV-1-VN) antibodies using a neutralisation assay (NA) gives an indication of the immune response of horses in the field. Objectives: To develop a real time neutralisation assay (RTNA) using the xCELLigence® technology for the measurement of EHV-1-VN antibodies in serums from EHV-1 experimentally infected or vaccinated horses. Study design: Assay development and field trial. Methods: The RTNA based on E. Derm cells was developed using serums (n=48) taken daily (from Days 0 to 18 post-infection) from ponies experimentally infected with a field EHV-1 strain. This method was further evaluated using field serums (n=63) taken during an EHV-1/-4 vaccination campaign. RTNA results were compared to those obtained with the conventional EHV-1 NA on RK13 cells. Two-fold serial dilutions of serums were incubated with a defined concentration of the KyD EHV-1 strain before infection of E. Derm cells. The normalised cell index was monitored pre- and post-infection using the xCELLigence® technology. Conventional NA was performed using the same experimental methodology on RK13 cells and cytopathic effects were measured 72 hours post-infection. Results: Serum titres which induced 50% of EHV-1 neutralisation (NT50) were determined using a dose-response approach. The increase of the EHV-1 NT50 was similar to the increase in titre measured with the conventional NA (R²=0.79). Seroconversion was detected between Days 0 and 18 post-infection for all infected ponies with both methods. The NT50 values measured for EHV-1/-4 vaccinated horse serums were also positively correlated with conventional NA titres (R²=0.83). Main limitations: EHV-1/-4 cross-reactivity will require further investigation. Each assay used different cells. Conclusion: While the conventional NA is time consuming, requires subjective measurements and provides semi-quantitative discrete ranked titres (e.g. 4, 8 etc.), the EHV-1-VN RTNA is an automated, sensitive and objective measurement that reports continuous titres.

Clinical and Hematological Profiles of Malaysian Ponies Experimentally Infected with a Field Strain of Trypanosoma evansi

Background:No outbreak has been reported onTrypanosoma evansiinfection in Malaysia ponies since 1983, and little is known about the interaction betweenT. evansiand ponies in the country. Therefore, an experimental study was designed to evaluate the pathogenicity of a local strain ofT. evansiin the local ponies.Method:For this purpose, four healthy local ponies were inoculated with 102live trypanosomes/kg body weight, whereas two ponies served as negative control. Blood samples and rectal temperature were collected on alternate days from both groups for 54 days. Physical examination comprised visible mucous membrane and any appearance of clinical signs were observed daily. The number of trypanosomes was estimated using the Neubauer haemocytometer method. Complete haemogram measurements were performed immediately using an automated blood cell counter and the data obtained was evaluated using the general linear model as linear regression. All infected ponies were salvaged treated with 7 mg/kg of diminazene diaceturate.Results:The four infected ponies developed parasitaemia on the 4thday post-infection (DPI), whereas the first high mean of parasites count was recorded on the 8thDPI. Parasitaemia was detected at a level that fluctuated throughout the infection period (30 days) in all infected ponies with a mean of 13.5x106 trypanosomes/ml blood on the 30thDPI. Successive peaks of pyrexia were accompanied by the peaks of parasitaemia and the highest temperature (39.4°C) was observed on the 20thDPI. Excessive weakness and a reduction of appetite fluctuated in the infected ponies during the infection and one animal died unexpectedly on the 23rdDPI. The mean values for RBC, PCV, Hb and thrombocyte count were significantly lower in the infected ponies than the control groups. Neutrophil and eosinophil were significantly declined after the onset of parasitaemia, whereas monocyte increased significantly in the infected group.Conclusion:The appearance of clinical signs and changes in haematological parameters suggests that Malaysian local ponies are susceptible toT. evansiinfection. Treatment of the infected ponies with the recommended dosage of diminazene diaceturate was successful in the surviving ponies.

African horse sickness virus (AHSV) with a deletion of 77 amino acids in NS3/NS3a protein is not virulent and a safe promising AHS Disabled Infectious Single Animal (DISA) vaccine platform

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. Currently, nine serotypes have been defined showing limited cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African Horse Sickness (AHS) in equids with a mortality up to 95% in naïve domestic horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are competent vectors of closely related bluetongue virus. AHS outbreaks cause huge economic losses in developing countries. In the developed world, outbreaks will result in losses in the equestrian industry and will have an enormous emotional impact on owners of pet horses. Live-attenuated vaccine viruses (LAVs) have been developed, however, safety of these LAVs are questionable due to residual virulence, reversion to virulence, and risk on virulent variants by reassortment between LAVs or with field AHSV. Research aims vaccines with improved profiles. Reverse genetics has recently being developed for AHSV and has opened endless possibilities including development of AHS vaccine candidates, such as Disabled Infectious Single Animal (DISA) vaccine. Here, virulent AHSV5 was recovered and its high virulence was confirmed by experimental infection of ponies. ‘Synthetically derived’ virulent AHSV5 with an in-frame deletion of 77 amino acids codons in genome segment 10 encoding NS3/NS3a protein resulted in similar in vitro characteristics as published NS3/NS3a knockout mutants of LAV strain AHSV4LP. In contrast to its highly virulent ancestor virus, this deletion AHSV5 mutant (DISA5) was completely safe for ponies. Two vaccinations with DISA5 as well as two vaccinations with DISA vaccine based on LAV strain AHSV4LP showed protection against lethal homologous AHSV. More research is needed to further improve efficacy, to explore the AHS DISA vaccine platform for all nine serotypes, and to study the vaccine profile in more detail.

Comparative evaluation of recombinant HSP70 (N & C-terminal) fragments in the detection of equine trypanosomosis

Trypanosomosis (Surra) is an economically important disease caused by Trypanosoma evansi which is an extracellular parasite present in the plasma, tissues and other body fluids of a wide range of hosts including domesticated animals. Currently, serological reports are based on detection of antibodies by ELISA using whole cell lysate (WCL) antigen, which has a limitation of persistence of anti-trypanosomal antibodies after successful treatment of the disease. Moreover, it has some ethical issues also like requirement of mice for in vivo maintenance of parasite for preparing the antigen. Therefore, in the present study, an attempt was made to evaluate the in vitro production of recombinant heat shock protein 70 (HSP70) for detection of antibodies in experimentally infected ponies. The amino acid sequence analysis of HSP70 revealed that N-terminal region of the protein was highly conserved while the C-terminal region was most divergent. The four different regions of HSP70 protein viz. HSP-1, HSP-2, HSP-3 and HSP-4 were cloned and expressed, among which HSP-1 (N-terminal region) & HSP-2 (C-terminal region) were truncated while HSP-3 & HSP-4 were complete C-terminal proteins. The recombinant fragments were probed with sequentially pooled experimental serum samples where antibodies were detected in these fragments from 10th day post infection till the termination of the experiment. Further, these recombinant fragments were also comparatively evaluated with WCL antigen in ELISA using experimental as well as field serum samples. It was observed that after successful treatment of infected ponies, there was a sharp fall in antibodies (within 90 days) when tested with recombinant HSP’s fragments, while antibodies persisted even after 469 days when tested against WCL antigen. The sensitivity and specificity of all HSP70 fragments were also estimated from field serum samples with reference to WCL antigen ELISA. The HSP-1 showed minimum sensitivity (41.03%) among all the recombinant fragments. Among the C-terminal fragments, maximum sensitivity was observed with the HSP-2 (61.54%) while minimum was observed with HSP-4 (48.72%). The specificity increases for recombinant fragments from N-terminal to C-terminal region of protein and maximum specificity was observed with HSP-4 fragment (91.3%).

Evaluation of melarsamine hydrochloride (Cymelarsan®) efficacy for the treatment of dourine nervous form on experimentally infected ponies

Evaluation of melarsamine hydrochloride (Cymelarsan®) efficacy for the treatment of dourine nervous form on experimentally infected ponies

Economic assessment of FEC-based targeted selective drenching in horses

In the face of an increased prevalence of drug-resistant cyathostomin populations, a targeted selective treatment (TST) strategy based on Faecal Egg Counts (FECs) has been proposed as an alternative management strategy. However, associated costs may be a barrier to the uptake of this strategy. Our study aims to provide an economic assessment of FEC-based TST. FECs were determined in a Welsh pony herd thrice a year from 2010 to 2014. This database was used to explore the impact of FEC price, sampling strategy (individual or pooled) and labour-associated costs. Drug price was set at the cheapest level, hence providing a conservative framework to determine the maximum viable FEC price in the context of a cost-driven horse industry. The maximum viable FEC price for a cost-efficient individual based strategy was determined by an in silico bootstrap approach consisting of randomly sampling 1000 virtual pony herds of various sizes (1 to 100 ponies) from the available database and estimating the associated costs (FEC price ranging from €1 to €10, anthelmintic costs and labour-associated costs). The costs and benefits of the pooling strategy that consists of basing the decision to treat on group FEC values were also investigated. This is thought to reduce FEC-based costs but may result in highly infected individuals being left undrenched, i.e. in false-negatives, as a result of FEC overdispersion. For various pool-sizes (1–20 ponies) and various cut-off thresholds (50–200eggs/g), we sampled 1000 pony herds in silico to estimate the associated costs and determine the number of positive ponies within a negative pool. Following these simulations, pool-based FECs of various sizes were performed on 40 ponies to compare predictions with real data. Within 4 years, anthelmintic costs were cut by 80%, albeit with free FECs. In silico estimations suggested that an individual FEC-based TST would not be cost-efficient in this context for an FEC price above €5. With a pooled FEC strategy, the proportion of false-negatives never exceeded 15% of the pool size. The combination of a 14-pony pool and a cut-off value of 150eggs/g minimized total costs while keeping the number of false negatives to a reasonable level. Real data obtained from infected ponies however, suggested that pool size should not exceed 10 individuals, since the inhomogeneous mixing of faeces of larger pools probably reduced the correlation between average pooled FECs and the mean of individual FECs. Our study provides an economic framework that could be valuable for emphasizing the use of FEC-based approaches in the field.

Cyathostominae distribution in experimentally infected ponies

Summary Nine ponies were experimentally infected with third stage cyathostome larvae. These animals were examined post-mortem for small strongyle infections from October 2011 to January 2012. Seventeen species of the following Cyathostominae genera were identified: Coronocyclus, Cyathostomum, Cylicocyclus, Cylicostephanus, Gyalocephalus and Petrovinema. Of the 17 species of Cyathostominae recovered, Cyathostomum catinatum, Cyathostomum alveatum and Cylicocyclus nassatus were the most prevalent (100 %). From the entire population of Cyathostominae, 65.6 % were located in the caecum and only 34.4 % in the colon. Only 1,736 (24,6 %) females and 5,329 (75,4 %) males were observed. There was a significant difference between the number of males and females in the genus Cyathostomum, Cylicocyclus and Gyalocephalus.

The Recent European Isolate (08P178) of Equine Arteritis Virus causes Inflammation but not Arteritis in Experimentally Infected Ponies

In the last two decades, outbreaks of equine viral arteritis (EVA) have been reported in Europe, but little is known about these European isolates of equine arteritis virus (EAV). EAV European strain (08P178, EU-1 clade) isolated from one of these recent outbreaks is able to cause clinical signs on experimental infection. The aim of the present study was to investigate the microscopical lesions induced by this isolate after experimental infection of ponies. Animals were killed at 3, 7, 14 and 28 days post infection (dpi). At 3 dpi, lesions were essentially restricted to the respiratory tract and intestines and were characterized by mild multifocal epithelial degeneration and associated mononuclear cell infiltration. Lesions were more severe at 7 dpi and by 14 dpi, respiratory lesions were even more severe and lymphoplasmacytic infiltrates extended to other organs. At 28 dpi, lesions were still present in the viscera. In all specimens the most prominent histological change was intraepithelial, subepithelial and perivascular lymphoplasmacytic infiltration, ranging from mild and multifocal to extensive and diffuse. No signs of arterial damage such as infarcts, haemorrhages or necrosis were found. In conclusion, infection of naïve animals with the European 08P178 strain of EAV is associated with inflammation, but not arteritis.

Treatment failure of ivermectin for Oxyuris equi in naturally infected ponies in Czech Republic

Abstract Nine ponies (20 months of age) with naturally acquired cyathostome and pinworm infections were treated orally with ivermectin at a dosage of 400 μg /kg. Ivermectin was highly effective on the cyathostome infection. However, adult Oxyuris equi were present in six horses. In recent years, anthelmintic treatment with macrocyclic lactones have not appeared to deliver the expected efficacy against equine pinworms (Oxyuris equi) in the USA. This is the first European study to demonstrate anthelmintic resistance in Oxyuris equi to macrocyclic lactones in naturally infected ponies (Czech Republic).

Identification of target cells of a European equine arteritis virus strain in experimentally infected ponies

Currently, little is known on the cellular pathogenesis of equine arteritis virus (EAV). The purpose of the present study was to identify the target cells in ponies experimentally inoculated with EAV 08P178 (EU, clade-1). EAV-target organs (respiratory tissues with associated lymphoid tissues and large intestines), collected at 3 and 7 days post inoculation (dpi) and with virus titers≥105.0 TCID50/g, were processed with double immunofluorescence staining for the simultaneous detection of EAV N-protein and one of the following cell markers: CD172a (myeloid cells), CD3 (T lymphocytes), IgM (B lymphocytes) and von Willebrand factor (endothelial cells). In the different analyzed organs, 31–58% and 47–63% of the EAV-positive cells were mononuclear leukocytes (mainly CD172a+ followed by CD3+) at 3 and 7 dpi, respectively. EAV-positive endothelial cells were not detected in 3.200 large blood vessels (≥3 endothelial cells/vessel cross section). However, in terminal capillaries (1–2 endothelial cells/vessel cross section) of the different organs, 15–51% of the endothelial cells were EAV-positive. In conclusion, the present study demonstrates that EAV 08P178 (i) has a main tropism for CD172a+ and CD3+ mononuclear leukocytes and (ii) infects a large number of endothelial cells in terminal capillaries. EAV 08P178 infection in capillaries is most probably the cause of an increased vascular permeability leading to leakage of fluid (edema-serous exudate) but not to severe vasculitis and hemorrhages.

Humoral immune response and spreading of Encephalitozoon cuniculi infection in experimentally infected ponies

A total of 9 (8 stallions and 1 mare) 1 year old ponies were used for the experimental infection caused by Encephalitozoon cuniculi genotype II (107 spores per animal). Subsequently, individual horses were slaughtered 7, 14, 21, 28, 35, 42, 49, 56, and 63 days post infection. Immediately after slaughter, tissues samples of stomach, duodenum, jejunum, ileum, caecum, colon, spleen, liver, kidney, bladder, heart, lungs, and brain were sampled. In addition, urine, feces and blood specimens were collected. Enzyme-linked immunosorbent assay was used for determination of humoral immune response and nested PCR targeting 16S rDNA, whole ITS, and 5.8S rDNA was used for detection of E. cuniculi in collected organs, blood, feces and urine. No clinical signs of microsporidiosis including diarrhea or colic, neurological signs and fever were observed in any horses during whole experiment. Acute microsporidiosis in ponies was characterized by the dissemination of microsporidia into almost all organs and significant increase of concentration of specific antibodies in blood was observed from 28 to 42 DPI. After this acute stage microsporidia disappeared from most organs with the exception of the kidney, which was positive up to 63 DPI when the experiment was terminated. No pathological changes were observed in any organs with exception of one mare's brain, where E. cuniculi-positive cavity measuring 5cm×3cm in diameter formed in the lobus piriformis.

Changes in Borrelia burgdorferi ELISA antibody over time in both antibiotic treated and untreated horses

Changes in ELISA serology are frequently used to determine antibiotic treatment success for Lyme disease in horses. This concept was based upon a previous report showing a marked decline in ELISA values in experimentally infected and antibiotic-treated ponies. Changes in Lyme serology following antibiotic treatment in naturally infected horses have not been reported. The objective of this study was to compare Borrelia ELISA antibody concentrations in naturally exposed horses both before and following antibiotic treatment for Lyme disease. A retrospective study was performed comparing oxytetracycline- or doxycyclinetreated (n = 68) and untreated (n = 183) horses from a single equine practice and their change in Borrelia ELISA values over a similar time period. Antibiotictreated horses had a decline in ELISA values in comparison to control horses (P ≤ 0.05) and untreated horses were twice as likely to have their ELISA values increase (OR = 0.5; 95% C.I. = 0.3-0.9) compared to treated horses. The magnitude of the decline in ELISA units following treatments was small compared to that previously reported in experimentally infected and treated ponies. Field-exposed horses with high Borrelia burgdorferi ELISA values who are treated with either oxytetracycline or doxycycline can be expected to have only a small decline in ELISA values following treatment. Persistently high ELISA titres following appropriate treatments for Lyme disease may not, without appropriate clinical signs, be a reason for more prolonged treatment.

Genomic and Immunologic analysis of the Chinese attenuated EIAV vaccine

the infection period. Since the virus undergoes rapid mutation following infection, the adaptive immune response must also evolve to meet this challenge. In order to fully understand the evolution of the T cell response during infection, we synthesized forty-four peptides, spanning the entire surface unit protein (gp90) of EIAV, and monitored the peptide-specific T cells responses both in vivo and in vitro over a six month period following challenge. One inapparent carrier (D64) and four recent EIAV infected ponies were included in this study. Peripheral blood mononuclear cells (PBMC) were isolated and stimulated separately with all 44 peptides. The EIAV gp90 epitopes-specific immune responses in vitro were determined by ELISPOT-IFNg assay. In parallel, all peptides were injected intradermally and punch biopsies were collected for real-time PCR tomonitor the cellular immune responses in vivo. As shown by both in vivo and in vitro assays, two of the four recently infected ponies recognized gp90 peptides three weeks post challenge. Similar to the CMI response to HIV infection, peptide-specific T cell recognition patterns changed over time. While some peptides were recognized throughout the sampling period, other peptides were only recognized at the later time points. Also, the response to some specific peptides disappeared after 6 months post infection. By contrast, peptide recognition by the inapparent carrier (D64) was more stable. The mechanisms responsible for this change remain unclear, but this dynamic shift in the immunodominant epitopes hierarchy in the newly infected horses may be the result of mutations in specific epitopes leading to an escape from T cell recognition. In the inapparent carrier, persistent recognition focusing on the more conserved peptides results in a more effective T cell response where virus replication is tightly controlled. These results indicate that T cell responses evolve during the early stage of EIAV infection. This interaction between virus mutation and T cell evolution needs to be considered when designing vaccines.

Cardiac Troponin I Concentrations in Ponies Challenged with Equine Influenza Virus

Myocarditis is thought to occur secondary to equine influenza virus (EIV) infections in horses, but there is a lack of published evidence. We proposed that EIV challenge infection in ponies would cause myocardial damage, detectable by increases in plasma cardiac troponin I (cTnI) concentrations. Twenty-nine influenza-naïve yearling ponies: 23 were part of an influenza vaccine study (11 unvaccinated and 12 vaccinated), and were challenged with 10⁸ EID₅₀ EIV A/eq/Kentucky/91 6 months after vaccination. Six age-matched healthy and unvaccinated ponies concurrently housed in a separate facility not exposed to influenza served as controls. Heparinized blood was collected before and over 28 days after infection and cTnI determined. Repeated measures analysis of variance, chi-square, or clustered regression analyses were used to identify relationships between each group and cTnI. All EIV-infected ponies developed clinical signs and viral shedding, with the unvaccinated group displaying severe signs. One vaccinated pony and 2 unvaccinated ponies had cTnI greater than the reference range at 1 time point. At all other times, cTnI was < 0.05 ng/mL. All control ponies had normal cTnI. There were no significant associations between cTnI and either clinical signs or experimental groups. When separated into abnormal versus normal cTnI, there were no significant differences among groups. This study demonstrated no evidence of severe myocardial necrosis secondary to EIV challenge with 10⁸ EID₅₀ EIV A/eq/Kentucky/91 in these sedentary ponies, but transient increases in cTnI suggest that mild myocardial damage may occur.

Evaluation of immune responses following infection of ponies with an EHV-1 ORF1/2 deletion mutant

Equine herpesvirus-1 (EHV-1) infection remains a significant problem despite the widespread use of vaccines. The inability to generate a protective immune response to EHV-1 vaccination or infection is thought to be due to immunomodulatory properties of the virus, and the ORF1 and ORF2 gene products have been hypothesized as potential candidates with immunoregulatory properties. A pony infection study was performed to define immune responses to EHV-1, and to determine if an EHV-1 ORF1/2 deletion mutant (ΔORF1/2) would have different disease and immunoregulatory effects compared to wild type EHV-1 (WT). Infection with either virus led to cytokine responses that coincided with the course of clinical disease, particularly the biphasic pyrexia, which correlates with respiratory disease and viremia, respectively. Similarly, both viruses caused suppression of proliferative T-cell responses on day 7 post infection (pi). The ΔORF1/ORF2 virus caused significantly shorter primary pyrexia and significantly reduced nasal shedding, and an attenuated decrease in PBMC IL-8 as well as increased Tbet responses compared to WT-infected ponies. In conclusion, our findings are (i) that infection of ponies with EHV-1 leads to modulation of immune responses, which are correlated with disease pathogenesis, and (ii) that the ORF1/2 genes are of importance for disease outcome and modulation of cytokine responses.

Validation of an In-Clinic Enzyme-Linked Immunosorbent Assay Kit for Diagnosis of Borrelia Burgdorferi Infection in Horses

Confirmation of Borrelia burgdorferi infection in horses has required enzyme-linked immunosorbent assay (ELISA) or Western blot tests performed by reference laboratories. An in-clinic C6 ELISA SNAP kit has been marketed for dogs. This canine kit was evaluated for horses using serum from experimentally infected ponies. Serum samples originated from 2 previous studies. In the first study, 7 ponies were exposed to B. burgdorferi-infected ticks; 4 ponies served as uninfected controls. Serum samples were obtained bimonthly for 9 months. In the second study, 16 ponies were exposed to B. burgdorferi-infected ticks. After confirmation of infection by skin culture, polymerase chain reaction (PCR), and serology, the ponies were allocated to 4 groups that received tetracycline, doxycycline, ceftiofur, or no treatment. Serum samples were obtained monthly, both before and after antibiotic treatments, for 11 months. For the current study, selected samples (n = 220) from both studies were tested with IDEXX SNAP Heartworm Ab/Borrelia burgdorferi Ab/Ehrlichia canis Ab Test Kits. Tested samples included samples taken before infection, from various times postinfection, and after antibiotic treatments. Results from confirmed positive or negative samples were used to determine sensitivity and specificity of the assay. Results indicate that the test kits have fair sensitivity (63%) and very high specificity (100%) for horses recently infected with B. burgdorferi. Validation of this test provides equine practitioners with an inexpensive, in-clinic method to confirm infection, although its moderate sensitivity may result in a moderate chance of a false negative test.

Passive transfer of Theileria equi antibodies to neonate foals of immune tolerant mares

Equine babesiosis, a tick transmitted haemoprotozoan disease caused by Theileria equi is globally distributed and responsible for heavy economic losses to the equine husbandry. Equids reared in endemic areas usually pick up infection at an early age and become immune tolerant throughout their life span. We studied the level of passively transferred antibodies in neonate foals born from pre-immuned mares. Latently T. equi infected pre-immuned pony and donkey mares (three each) were selected and T. equi antibody titres in neonates was monitored till 90 days post foaling (DPF) by applying Dot-ELISA on sequentially collected serum samples from foals and their dams. A very high antibody titre was observed in pre-immuned pony and donkey mares. The maximum antibody of 1:60 to 1:80 was observed in pony's and donkey's foal on 2–16 and 2–10 DPF, respectively and thereafter it declined to less than 1:20 on 63–77 and 56–63 DPF. Simultaneously parasite carrying status in neonate foals and their dam was also monitored by applying PCR on blood samples. We could demonstrate PCR amplification in dam's blood samples while no amplification was recorded in neonate's blood samples. This study indicated that new-born foals were born naïve and passively transferred immunity was transitory which wanes after 63–77 DPF.

Envelope variation as a primary determinant of lentiviral vaccine efficacy

Lentiviral envelope antigenic variation and associated immune evasion are believed to present major obstacles to effective vaccine development. Although this perception is widely assumed by the scientific community, there is, to date, no rigorous experimental data assessing the effect of increasing levels of lentiviral Env variation on vaccine efficacy. It is our working hypothesis that Env is, in fact, a primary determinant of vaccine effectiveness. We previously reported that a successful experimental attenuated equine infectious anemia virus vaccine, derived by mutation of the viral S2 accessory gene, provided 100% protection from disease after virulent virus challenge. Here, we sought to comprehensively test our hypothesis by challenging vaccinated animals with proviral strains of defined, increasing Env variation, using variant envelope SU genes that arose naturally during experimental infection of ponies with equine infectious anemia virus. The reference attenuated vaccine combined with these variant Env challenge strains facilitated evaluation of the protection conferred by ancestral immunogens, because the Env of the attenuated vaccine is a direct ancestor to the variant proviral strain Envs. The results demonstrated that ancestral Env proteins did not impart broad levels of protection against challenge. Furthermore, the results displayed a significant inverse linear correlation of Env divergence and protection from disease. This study demonstrates potential obstacles to the use of single isolate ancestral Env immunogens. Finally, these findings reveal that relatively minor Env variation can pose a substantial challenge to lentiviral vaccine immunity, even when attenuated vaccines are used that, to date, achieve the highest levels of vaccine protection.