Evaluation of three serological tests for brucellosis in naturally infected cattle using latent class analysis

Serological methods are traditionally used in diagnosis of brucellosis. However, the comparative performance of these tests and their accuracy under the local environment in Zambia has not been assessed. Thus, the objective of our study was to evaluate the diagnostic performance of three serological tests for brucellosis; Rose Bengal Test (RBT), competitive ELISA (c-ELISA) and Fluorescence Polarisation Assay (FPA) in naturally infected cattle in Zambia without an appropriate reference test to classify animals into truly infected and non-infected. Serological test results from a study to determine sero-prevalence were used to compare the performance of RBT, c-ELISA and FPA in diagnosing brucellosis in traditional cattle. Since none of the tests can be seen as a perfect reference test or gold standard, their performance in a population of naturally infected cattle was evaluated using latent class analysis which allows the sensitivity (Se) and specificity (Sp) to be estimated in the absence of a gold standard. The highest Se was achieved by the c-ELISA (97%; Credible Posterior Interval (CPI)=93-100%) and the highest Sp by the FPA (93%; CPI=85-99%), conversely these tests also had the lowest Sp and Se, respectively, with the RBT performing well in both the Se (93%; CPI=84-98%) and Sp (81%; CPI=61-97).     

Longitudinal study describing the clinical signs observed in horses naturally infected with equine influenza

We describe the clinical signs and disease course during an outbreak of equine influenza (EI) in na?ve horses in a police stables in Sydney, New South Wales, Australia.     

Immune response to Sarcocystis neurona infection in naturally infected horses with equine protozoal myeloencephalitis

Equine protozoal myeloencephalitis (EPM) is one of the most common neurologic diseases of horses in the United States. The primary etiologic agent is Sarcocystis neurona. Currently, there is limited knowledge regarding the protective or pathophysiologic immune response to S. neurona infection or the subsequent development of EPM. The objectives of this study were to determine whether S. neurona infected horses with clinical signs of EPM had altered or suppressed immune responses compared to neurologically normal horses and if blood sample storage would influence these findings. Twenty clinically normal horses and 22 horses with EPM, diagnosed by the presence of S. neurona specific antibodies in the serum and/or cerebrospinal (CSF) and clinical signs, were evaluated for differences in the immune cell subsets and function. Our results demonstrated that naturally infected horses had significantly (P<0.05) higher percentages of CD4 T-lymphocytes and neutrophils (PMN) in separated peripheral blood leukocytes than clinically normal horses. Leukocytes from naturally infected EPM horses had significantly lower proliferation responses, as measured by thymidine incorporation, to a non-antigen specific mitogen than did clinically normal horses (P<0.05). Currently, studies are in progress to determine the role of CD4 T cells in disease and protection against S. neurona in horses, as well as to determine the mechanism associated with suppressed in vitro proliferation responses. Finally, overnight storage of blood samples appears to alter T lymphocyte phenotypes and viability among leukocytes.     

A serological survey for equine influenza in New Zealand horses

Sheep and alpacas of similar age groups (6, 18 and 36+ months) were grazed for 16 weeks on pasture contaminated by lambs. Faecal egg counts, bulked larval cultures, lungworm larvae in faeces, dag scores, liveweight changes and nematode larvae on pasture were measured. Chabertia, Oesophagostomum, Cooperia, Ostertagia and Haemonchus and Trichostrongylus larvae were cultured from both the sheep and the alpacas. For the respective age groups, the alpacas had lower liveweight gains (10, 32 and 47 g/d vs 88, 84 and 120 g/d), peak faecal egg counts (384, 50 and 60 epg vs 1500, 500 and 140 epg) and faecal contamination of the perineum than the same ages of sheep. These results suggest alpacas became less affected with gastrointestinal nematodes than sheep.     

Sensitivity and specificity of various serological tests for the detection of Toxoplasma gondii infection in naturally infected sheep

Comparative serological examination of 300 serum samples from sheep slaughtered in the main abattoir in Cairo, Egypt revealed a higher prevalence of toxoplasmosis (43.7%) with the modified agglutination test (MAT), followed by the enzyme linked immune-sorbant assay (ELISA) (41.7%) and the indirect fluorescent antibody test (IFAT) (37%), while the lowest prevalence was detected with the dye test (DT) (34%). When the data from the first three serological tests were compared with that of the DT test, which was used as a reference test for toxoplasmosis, MAT had the highest sensitivity (96%), followed by ELISA (90.1%) and IFAT, which demonstrated the lowest sensitivity (80.4%). Conversely, IFAT had the highest specificity (91.4%), followed by MAT (88.9%) and ELISA (85.9%).     

African horse sickness in naturally infected,immunised horses

To determine whether subclinical cases, together with clinical cases, of African horse sickness (AHS) occur in immunised horses in field conditions, whole blood samples were collected and rectal temperatures recorded weekly from 50 Nooitgedacht ponies resident in open camps at the Faculty of Veterinary Science, University of Pretoria, Onderstepoort, during 2008–2010. The samples were tested for the presence of African horse sickness virus (AHSV) RNA by a recently developed real‐time RT‐PCR. It was shown that 16% of immunised horses in an AHS endemic area were infected with AHSV over a 2 year period, with half of these (8%) being subclinically infected. The potential impact of such cases on the epidemiology of AHS warrants further investigation.     

Characterization of trypanosome isolates from naturally infected horses on a farm in Kenya

Following an outbreak of trypanosomosis in horses on a farm in Kenya, 18 trypanosome isolates were collected from the infected animals over a period of one and a half years and cryopreserved for characterization. The characterization was done on the basis of morphology using Giemsa-stained blood and buffy coat smears, infectivity to mice, recombinant DNA hybridization, and chromosome separation by orthogonal field alternation gel electrophoresis (OFAGE). Morphologically, all the trypanosome isolates were identified as belonging to the subgenus Nannomonas, and a total of 16 out of the 18 isolates grew in mice. Using the recombinant DNA hybridization technique, the isolates were further classified as the 'savannah' type of Trypanosoma congolense. Furthermore, chromosome separation by OFAGE, carried out on six clones derived from different isolates, exhibited a profile characteristic of T. congolense, 'savannah' type. However, there were differences in the number and positions of the medium-sized and minichromosomes indicating a diversity of serodemes within the isolates. Hence the infecting trypanosomes in this disease outbreak were T. congolense, 'savannah' type, and comprised several serodemes or strains.     

Comparison of serological tests for detection of Mycoplasma gallisepticum antibodies in eggs and chicks hatched from experimentally infected hens

Specific pathogen free hens and males were experimentally infected with Mycoplasma gallisepticum. Eggs were then collected, and a part was incubated and set for hatching. Mycoplasma cultures were performed on infected adults and antibodies to MG were analysed by use of slide agglutination (SA) test and commercial ELISA tests on adults and chicks sera and on yolks from non incubated eggs. Both ELISA tests could detect antibodies in yolks from non incubated eggs laid three weeks after infection. SA and the three ELISA tests revealed positive sera in chicks hatched from eggs laid as soon as one week after infection.     

Oxidant-antioxidant imbalance in horses infected with equine infectious anaemia virus

This study assesses the impact of equine infectious anaemia virus (EIAV) infection on the oxidant/antioxidant equilibrium of horses. Blood samples from 96 Romanian horses aged 1-25 years, were divided into different groups according to their EIAV-infection status, age, and time post-seroconversion. The effect of infection on oxidative stress was estimated by measuring enzymatic antioxidants (superoxide dismutase [SOD], glutathione peroxidase [GPx] and catalase), non-enzymatic antioxidants (uric acid and carotenoids), and lipid peroxidation (malondialdehyde [MDA]). Infection modified the oxidant/antioxidant equilibrium in the horses, influencing GPx and uric acid levels (P<0.05). Time post-seroconversion also contributed to oxidative stress imbalance, exhibiting a significant influence on both SOD and MDA concentrations in the blood (P<0.05). Animal age did not have a significant influence on oxidative stress. Recently infected horses (<1 year following seroconversion), and horses >5 years old, represented the most vulnerable category in terms of oxidative stress, followed by recently infected animals <5 years old. The results of this study are novel in implicating EIAV infection in the development of oxidative stress in horses.     

A serological and clinical follow-up in horses with confirmed equine granulocytic ehrlichiosis

For diagnosis of equine granulocytic ehrlichiosis (EGE) serological testing of antibodies to Ehrlichia equi is frequently used. An elevated antibody level is often misinterpreted as confirmative of active infection and results in treatment with antibiotics. If only seropositivity is considered as the diagnostic criterium, many horses showing convalescence titres will be treated. This study was undertaken to obtain information about the kinetics of antibodies during the course of infection and, for this purpose, 45 horses with clinical signs of EGE and confirmed ehrlichiaemia were monitored serologically and clinically over time. For a correct handling of cases with suspected EGE, the following results should be helpful: (i) 44% of the horses in the acute ehrlichiaemic stage were found to be serologically positive to E. equi; (ii) all horses showed a rapid increase in antibody titre, reaching maximum value within a month after the ehrlichiaemic stage; (iii) when 8 months had passed, titres had decreased, but 18 of 24 examined horses were still serologically positive; (iv) after 12-15 months most of the horses (n = 10) were serologically negative; and (v) the period required for complete clinical recovery varied from one day up to 6 months after antibiotic treatment.     

Comparison of three serological tests and an interferon-gamma assay for the diagnosis of paratuberculosis in experimentally infected sheep

OBJECTIVE: To compare the diagnostic performance of a complement fixation test, an agar gel immunodiffusion test, an enzyme-linked immunosorbent assay, and a whole-blood interferon-gamma assay for paratuberculosis in 14 sheep experimentally infected with Mycobacterium avium subsp paratuberculosis. EXPERIMENTAL DESIGN: Longitudinal study. RESULTS: The IFN-gamma assay detected more experimentally infected sheep, and earlier, than any of the serological tests. None of the antibody assays was able to detect all sheep with histologically confirmed paratuberculosis. CONCLUSIONS: The superior performance of the IFN-gamma assay in detecting infected sheep in this small experimental population warrants its further evaluation in a larger population of sheep naturally exposed to M a paratuberculosis.     

Haematological and biochemical values in horses naturally infected with Strongylus vulgaris

The concentrations of serum proteins (beta 1, beta 2, gamma, alpha 1, alpha 2 globulins and albumin) and absolute numbers of eosinophils, neutrophils and lymphocytes were examined in 64 naturally infected horses and ponies in which the number of larvae of Strongylus vulgaris in the cranial mesenteric artery and the severity of the lesion of verminous arteritis could be determined. The horses were grouped according to the number of larvae found and the severity of the arteritis. The results demonstrated that, although some significant deviation from a random distribution occurred in certain of the values (chi 2 test), there was considerable individual variation in the values obtained for individual animals within groups and overlap of the range of values between groups. Also the number of larvae present in the artery did not necessarily accurately reflect the severity of the arterial lesion. Thus, the parameters examined could not be used reliably to estimate the intensity of infection with S vulgaris in an individual animal.     

Intradermal skin tests in equine dermatology: a study of 83 horses

Allergic diseases are often diagnosed clinically in the horse without performing diagnostic tests. The purpose of this work was to contribute to the validation of intradermal skin tests in the horse. Eighty-three horses, 14 showing skin or respiratory signs of supposed allergic origin, were subjected to an intradermal skin test using 6 different allergens, positive and negative controls. The tests were read for all animals after 20 min, and for 29 horses after 1 and 4 h. Additionally, 19 horses were tested a few months apart. The comparison after 20 min of the cutaneous reactions to allergens and to positive and negative controls allowed us to propose a threshold of positivity; skin reactions with a diameter above or equal to 15 mm, or with at least a 13 mm diameter and a skin thickness similar to the positive control. There was a marked difference between the healthy group and the allergic group for Culex pipiens and Dermatophagoides farinae, although positive reactions were not rare in the healthy group. Tabanus sp. gave positive reactions in both healthy and allergic animals. There was no significant variation in the reactions observed after 20 min and after 60 min. After 4 h, the progression of the reactions was highly variable and negative controls showed a certain number of positive reactions, which negated their reliability at the point in time, and made it difficult to interpret the other allergens. Finally, the repeatability of intradermal skin tests reactions after 20 min was poor, and probably related to the influence of seasonality for some of the allergens. Further studies are required, notably with others allergens, to interpret intradermal skin tests' responses in clinical practice.     

Comparison of culture versus quantitative real-time polymerase chain reaction for the detection of Taylorella equigenitalis in field samples from naturally infected horses in Canada and Germany

A quantitative real-time polymerase chain reaction method (qPCR) was developed and tested for the detection of Taylorella equigenitalis. It was shown to have an analytical sensitivity of 5 colony-forming units (CFU) of T. equigenitalis when applied to the testing of culture swabs that mimicked field samples, and a high analytical specificity in not reacting to 8 other commensal bacterial species associated with horses. As designed, it could also differentiate specifically between T. equigenitalis and T. asinigenitalis. The qPCR was compared to standard culture in a study that included 45 swab samples from 6 horses (1 stallion, 5 mares) naturally infected with T. equigenitalis in Canada, 39 swab samples from 5 naturally infected stallions in Germany, and 311 swab samples from 87 culture negative horses in Canada. When the comparison was conducted on an individual sample swab basis, the qPCR had a statistical sensitivity and specificity of 100% and 96.4%, respectively, and 100% and 99.1% when the comparison was conducted on a sample set basis. A comparison was also made on 203 sample swabs from the 5 German stallions taken over a span of 4 to 9 mo following antibiotic treatment. The qPCR was found to be highly sensitive and at least as good as culture in detecting the presence of T. equigenitalis in post-treatment samples. The work demonstrates that the qPCR assay described here can potentially be used to detect the presence of T. equigenitalis directly from submitted sample swabs taken from infected horses and also for determining T. equigenitalis freedom following treatment.     

The importance of vertical transmission of Neospora sp. in naturally infected horses

Neospora spp. is a intracellular protozoan phylogenetically closely related to Toxoplasma gondii and Sarcocystis neurona, and it can infect horses leading to the development of reproductive or neurological diseases. We determined the presence of antibodies to Neospora sp. in mares at their parturition time and determine the frequency of vertical transmission in healthy foals to verify the importance of transplacental transmission. The samples were analyzed by indirect immunofluorescence antibody test, showing that seroprevalence in mares is higher than in foals and seropositive mares are likely to transmit the neosporosis to their offspring. This shows that endogenous challenge occurs in horses, and it suggests that this protozoan can be disseminated by means of transplacental transmission in horse species.