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The diagnostic methods for granting and maintenance of the official tuberculosis-free (OTF) status and for intra-Community movement of cattle are the tuberculin skin tests (single or comparative) and the interferon-γ (IFN-γ) release assay (IGRA). However, until now, IGRAs have been primarily applied in infected farms in parallel to the skin test to maximize the number of infected animals detected. Therefore, an evaluation of the performance of IGRAs in OTF herds to assess whether if their specificity is equal to or higher than that of the skin tests is needed. For this, a panel of 4365 plasma samples coming from 84 OTF herds in six European regions (five countries) was assembled and analysed using two IGRA kits, the ID Screen® Ruminant IFN-g (IDvet) and the Bovigam™ TB Kit (Bovigam). Results were evaluated using different cut-offs, and the impact of herd and animal-level factors on the probability of positivity was assessed using hierarchical Bayesian multivariable logistic regression models. The percentage of reactors ranged from 1.7 to 21.0% (IDvet: S/P ≥ 35%), and 2.1–26.3% (Bovigam: ODbovis–ODPBS ≥ 0.1 and ODbovis–ODavium ≥ 0.1) depending on the region, with Bovigam disclosing more reactors in all regions. The results suggest that specificity of IGRAs can be influenced by the production type, age and region of origin of the animals. Changes in the cut-offs could lead to specificity values above 98–99% in certain OTF populations, but no single cut-off yielding a sufficiently high specificity (equal or higher than that of skin tests) in all populations was identified. Therefore, an exploratory analysis of the baseline IFN-γ reactivity in OTF populations could help to assess the usefulness of this technique when applied for the purpose of maintaining OTF status. [ABSTRACT FROM AUTHOR]

Abstract A 2-month-old Andalusian colt in central Spain presented dyspnea, diarrhea, and depression. Despite the initial treatment for presumptive pneumonia caused by Rhodococcus equi, after 1 hour, the colt showed signs of shock and died. The necropsy revealed multiple abscesses in the lung, as well as enlarged mesenteric lymph nodes, and hemorrhagic peritonitis. Samples were taken from trachea, lung parenchyma, small intestine, and colon. All samples yielded a pure culture of Streptococcus equi spp. zooepidemicus, whereas R. equi was excluded by using a specific qPCR performed on all samples. Isolates from all samples were characterized by pulse-field gel electrophoresis and the representative strain was identified by MALDI-TOF and qPCR. Its genome was sequenced, assembled, and annotated; multilocus sequence typing data assigned the strain to sequence type 70 and genes related to adhesion, bacteriocins, invasion, antimicrobial resistance, toxins, and superantigens szeN and szeP were identified in the assembled genome. The strain was susceptible to all antimicrobials tested. Our findings support the inclusion of S. zooepidemicus on an endemic farm where R. equi has been ruled out via PCR testing. S. zooepidemicus can cause multiorgan disease in foals, similar to infection in humans. The presence of superantigens in this S. zooepidemicus strain may have contributed to the fatal outcome in this clinical case. Highlights • Streptococcus zooepidemicus should be in the differential diagnoses when R. equi is ruled out by PCR testing. • S. zooepidemicus can cause multiorgan disease in foals, similar to infection in humans. • The presence of superantigens in the strain may have contributed to fatal outcome. [ABSTRACT FROM AUTHOR]

Equine piroplasmosis (EP) is a tick-borne disease caused by Babesia caballi and Theileria equi , which causes severe economic losses to the equine industry, represented by the Spanish Purebred (SP) horse in Spain. The seroprevalence of EP is known in certain regions of Spain, but till date, there are no data in SP horses. The aim of this study was to determine the seroprevalence of EP in the SP breeding horse population in central Spain and to evaluate risk factors associated with the occurrence of the infection. A total of 536 horse serum samples were tested for antibodies against T. equi and B. caballi by competitive inhibition enzyme-linked immunosorbent assay. Data on possible risk factors were examined using two logistic regression models with and without a random effect at the stud farm level. The true seroprevalence results were 21.8% for T. equi and 4.7% for B. caballi . Increasing horse age and increasing numbers of breeding stallions on the stud farm were identified as factors associated with EP seropositivity. The implementation of vaccination programs in the horses and disinfection measures in the premises were associated with a lower T. equi and B. caballi seroprevalence, respectively. A moderate exposure to EP was found in SP horses in central Spain. Further investigations are necessary to ensure appropriate preventive measures for stud farms and to facilitate the development of an EP surveillance program in the SP breeding horse population. [ABSTRACT FROM AUTHOR]

The intraerythrocytic protozoans Theileria equi and Babesia caballi are the causative agents of equine piroplasmosis (EP), one of the most important equine tick-borne diseases due to its significant impact on global international horse trade. Although EP is known to be endemic in Spain, previous phylogenetic studies have only been conducted for limited geographical regions. Therefore, the objective of this study was to evaluate the genetic diversity and distribution of these parasite species nationwide. This was performed by amplification of the 18S small subunit (SSU) rRNA gene from 100 EP positive equine blood samples using a nested PCR protocol, and sequencing the obtained amplicons. Seventy-seven T. equi and six B. caballi isolates were successfully sequenced and phylogenetic analysis revealed that the T. equi isolates grouped into the previously described clades A (n = 21/77), D (n = 1/77) and E (n = 55/77), while B. caballi isolates were placed into clades A (n = 5/6) and B (n = 1/6). Isolates from T. equi clade D and B. caballi clade B have not previously been reported in Spain. A greater intra-clade diversity (97.3–98.3 % identity) was observed between T. equi clade E isolates compared to those within clade A (99.7–100 % identity). Additionally, a multivariable logistic regression model was used to analyse associations between the clade of T. equi infection and available epidemiological data. Horses residing in Spanish northern regions were statistically more likely to be infected with T. equi clade E (p = 0.01). We conclude that while extensive sequence variation of equine piroplasms exists in Spanish infected horses, a requirement for increased equine movement controls between Spain and EP-endemic countries should be considered. [ABSTRACT FROM AUTHOR]

Serological analysis of equine piroplasmosis (EP), caused by Theileria equi and Babesia caballi , is included in the export testing requirements for most of the countries worldwide, thus involving a high economic impact on equine industry of EP-endemic countries, such as Spain. A total of 3368 serum samples from healthy horses collected prior to export between 2015 and 2018 in Spain were tested for antibodies against T. equi and B. caballi by using a competitive inhibition enzyme-linked immunosorbent assay (cELISA). The overall seroprevalence results in Spain revealed that almost a quarter of the horses analysed (24.1 %; 95% CI 22.6–25.5) could not be exported to countries free from EP. The implementation of prevention measures such as the use of acaricides and daily checks for ticks in horses, as well as regular serological screening of horses in Spain would aid to increase the number of horses exported to other countries. [ABSTRACT FROM AUTHOR]