Equine Blood Samples Research Articles

Validation of a real-time polymerase chain reaction for the detection and quantification of the nucleic acid of Histoplasma from equine clinical samples.

Histoplasmosis is a neglected yet major cause of morbidity and mortality in both equids and people in resource-scarce settings. One of the major hindrances to the control of histoplasmosis is a lack of readily available diagnostic tests. Tests are needed to support clinical decision-making and to be applied in population-based research to further understand this disease in situ. This paper reports, for the first time, the validation and application of a qPCR to detect Histoplasma directly from equine clinical samples, bypassing the need to culture this notoriously difficult organism. We report and comment on the performance of the qPCR in comparison with our previously developed nested PCR.

PCR-Based Equine Gene Doping Test for the Australian Horseracing Industry.

The term 'gene doping' is used to describe the use of any unauthorized gene therapy techniques. We developed a test for five likely candidate genes for equine gene doping: EPO, FST, GH1, IGF1, and ILRN1. The test is based on real-time polymerase chain reaction (PCR) and includes separate screening and confirmation assays that detect different unique targets in each transgene. For doping material, we used nonviral (plasmid) and viral (recombinant adeno-associated virus) vectors carrying complementary DNA for the targeted genes; the vectors were accurately quantified by digital PCR. To reduce non-specific amplification from genomic DNA observed in some assays, a restriction digest step was introduced in the PCR protocol prior to cycling to cut the amplifiable targets within the endogenous genes. We made the screening stage of the test simpler and faster by multiplexing PCR assays for four transgenes (EPO, FST, IGF1, and ILRN1), while the GH1 assay is performed in simplex. Both stages of the test reliably detect at least 20 copies of each transgene in a background of genomic DNA equivalent to what is extracted from two milliliters of equine blood. The test protocol was documented and tested with equine blood samples provided by an official doping control authority. The developed tests will form the basis for screening official horseracing samples in Australia.

Accuracy and validation of a point-of-care blood glucose monitoring system for use in horses.

Abnormal blood glucose (BG) levels often seen in critically ill horses are significantly associated with adverse patient outcomes and increased mortality. Rapid and accurate BG monitoring is now considered an essential component of evidence-based equine practice and can provide critical information quickly for treatment. Although several point-of-care (POC) BG monitoring hand-held devices are commercially available for veterinary use, none contains a unique algorithm validated for use in horses. The AlphaTrak 3 (AT3) BG monitoring system is a first-of-its-kind device with an equine-specific algorithm that allows stall-side clinical decision making, and frequent monitoring at minimal cost. As such, AT3 is potentially a preferred alternative to more costly and time-consuming standard diagnostic reference laboratory methods. The objective of this study was to determine the accuracy of the AT3 device in measuring BG levels in equine whole blood samples in comparison to results obtained by the Beckman Coulter AU480 reference analyzer per ISO15197:2013 specifications. Accuracy of the AT3 equine algorithm were initially verified by testing equine blood samples with artificially adjusted blood glucose levels followed by its validation in a field study. Testing with artificially adjusted equine samples (n = 129) showed that 98.9% of glucose measurements ranging from 29 to 479 mg/dL fell within ISO accuracy threshold of ±15 mg/dL or ±15% of the average reference value. In addition, 100% of the AT3 measurements fell in consensus error grid (CEG) zone A, which indicates that test outcomes have a minimal likelihood of adverse clinical impact. In a follow-up field study involving 96 horses, 98.4% of AT3 measurements met the ISO accuracy threshold and 99.2% of AT3 measurements fell in CEG zone A. These results demonstrate that the AT3 glucometer has a high degree of accuracy in horses and is a dependable, convenient, and cost-effective device for accurately monitoring equine BG levels in farm or clinical settings.

Blood from Horses and Cows In Vitro Exposed to Quaternium-15 and Thiacloprid: Haematology and Erythrocyte Osmotic Fragility Alterations

Blood cells are constantly exposed to several pollutants, including xenobiotics, and they can be considered a useful target for pollution exposition of the animal. The aim of this study was to investigate the effects of two xenobiotics (Quaternium-15, a preservative used in personal care products, and Thiacloprid, a neonicotinoid pesticide) on the haematological profile and erythrocyte osmotic fragility (EOF) of equine and bovine blood samples. Ten blood samples from horses and cows were exposed for 24 h to Quaternium-15 at a concentration of 0.1 and 1 mg/L and to Thiacloprid at a concentration of 30 and 60 µg/mL. A decrease in the values of the red blood cells, white blood cells, haematocrit, haemoglobin, mean corpuscular volume, and platelets, and an increase of EOF were found in blood samples exposed to xenobiotics compared to the control. According to the results gathered in the current study, the two xenobiotic compounds herein tested negatively affect the haematological indices causing haemolysis both in cattle and horse blood. This study, despite being preliminary, emphasizes the concept that blood cells are an excellent target for evaluating the effects of xenobiotics.

Analytical Performance Evaluation of the New GEM® Premier™ 5000 in Comparison to the Epoc® Blood Gas Analyzer in Horses.

Different blood gas analyzers are used in equine practice. Every machine needs to be validated, as they have not been designed for use in horses. The aim of this study was to compare the newly marketed GEM5000 machine to the formerly validated epoc machine for blood gas analysis in horses. In this prospective, non-blinded, comparative laboratory analyzer study, 43 equine blood samples were analyzed on both analyzers and values were compared between the two machines via Lin's concordance analysis, Passing-Bablok regression analysis and Bland-Altman plots. Duplicate measurements were conducted on the GEM5000 machine to evaluate precision. The GEM5000 failed to achieve the required precision for tHb, Hct and iCa2+, but achieved acceptable precision for all other parameters. Concordance correlation analysis revealed poor correlation for Na+, Cl-, iCa2+, K+, Hct and tHb, while there was an at least moderate agreement for all other parameters. Passing-Bablok regression revealed significant constant bias for pCO2, pO2, Cl-, and iCa2+ and significant proportional bias for pCO2, iCa2+ and SO2. Bland-Altman analysis revealed significant systematic bias for Na+, Cl-, iCa2+, K+, Hct, tHb and SO2. This study shows that while precision of the GEM5000 is good, values should not be used interchangeably with data obtained from other blood gas analyzers.

The effects of cannabidiol on immune function and health parameters in senior horses

Cannabidiol (CBD) has potential to reduce pain and inflammation in humans leading to the interest of use in equine. The purpose of this study was to determine the effects of CBD on immune function by measuring inflammatory cytokines and antibody responses to vaccination, as well as other health parameters in senior horses. Horses were orally-dosed with CBD (2 mg/kg: 13 horses) or control (soy oil: 14 horses) daily for 90 days, from July 2021 to November 2021. Peripheral blood samples were collected on days 0, 30, 60, and 90 before administering treatments. On day 90 all horses were kept on treatment and vaccinated with an equine influenza vaccine and blood samples were collected post-vaccination on days 14 and 21. For all time points, plasma samples were analyzed for determination of CBD and metabolites, 7-OH CBD and 7-COOH CBD, using tandem mass spectrometry. For time points 0, 30, 60 and 90, blood samples were analyzed for CBC and chemistry. Additionally, peripheral blood mononuclear cells (PBMC) were isolated, stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, stained intracellularly for interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) then analyzed via flow cytometry. Real-time PCR (RT-PCR) analyzed both stimulated PBMCs and whole blood for cytokine gene expression. Inflammatory proteins C-reactive protein, interleukin 1 receptor agonist, and prostaglandin E2 were measured with equine-specific enzyme linked immunosorbent assays. Thyrotropin-releasing hormone stimulation test and oral sugar test were performed on all horses before and after the study to analyze metabolic function. Hemagglutination inhibition (HI) titers were measured for immune responses pre- and post-vaccination. All data were analyzed using either a paired t-test or a two-way repeated measures analysis of variance (significance P < 0.05). Plasma concentrations of CBD and metabolites were determined with 7-COOH CBD, the most significant metabolite, in CBD treated horses compared to control treated horses. A significant decrease was determined for whole blood inflammatory cytokine expression of IFN-γ at day 60, and for IL6 at day 60 and 90 for CBD-treated horses when compared to control horses. CBD did not significantly affect any other immune factors, HI titers, or health parameters. This study demonstrated that treatment with CBD reduced some inflammatory cytokine production with no negative side effects as measured by CBC or chemistry profiles. This study reveals the initial understanding of CBD in the horse, however more in-depth research is needed to fully understand its efficacy on the health of the horse.

Emergent and Neglected Equine Filariosis in Egypt: Species Diversity and Host Immune Response.

Equine filariosis (EF) is a neglected vector-borne disease caused by nematode species belonging to the Onchocercidae and Setariidae families. Aside from their zoonotic potential, some species are responsible for serious health problems in equids worldwide, leading to significant economic difficulties. Here, we molecularly investigated equine blood samples (320 horses and 109 donkeys from Egypt) and four adult worms isolated from the peritoneal cavity of 5 out of the 94 slaughtered donkeys. In addition, quantitative enzyme-linked immunoassays (ELISAs) targeting circulating cytokines were used to identify whether the immunological profile of the infected animals is a Th1 (i.e., INF-gamma as indicator) or Th2 (i.e., IL-5 and IL-10 as indicators) response type. Overall, 13.8% and 0.3% of the donkeys and horses, respectively, were scored as positive for filaroid DNA. The 18S phylogeny revealed the occurrence of three different filaroid species, identified here as Mansonella (Tetrapetalonema) sp., Setaria digitata and Dirofilaria repens. Th1 (INF-gamma and IL-5) and Th2 (IL-10) immune response types were identified in equines infected with S. digitata and Mansonella (T.) sp., respectively. These results provide new data on the species diversity of EF in Egypt and extend knowledge of the downregulation of the protective immune response by the potentially zoonotic Mansonella (T) sp. There is an urgent need to implement control measures to preserve equine health and limit the propagation of these vector-borne filaroids in Egypt.

Insights into equine piroplasmosis in Venezuelan sport horses: Molecular diagnosis, clinical, and cardiovascular findings.

Equine piroplasmosis (EP) is a tick-borne infectious disease highly prevalent in tropical and subtropical regions, such as Venezuela. EP affects wild and domestic equids leading to several clinical presentations, from asymptomatic to severely affected animals. In this study, thirty-three (33) sport horses under regular training activities and from endemic regions of north-central Venezuela were submitted to an observational survey, case-control, to describe the presence of clinical signs and natural EP infections. A conventional PCR assay targeting the SSU rRNA gene revealed EP etiologic agents in 13 out of 33 sampled horses (~ 39.4% infections). Nine (9) of these EP-positive samples were confirmed as infected with Babesia caballi (6/9=66.7%) or Theileria equi (3/9=33.3%) by DNA sequencing and BLASTN analyses. A phylogeny of SSU rRNA gene sequences revealed that these new B. caballi and T. equi sequences clustered within the worldwide distributed phylogenetic genotype A, respectively. No acute EP cases were observed in this study; however, six (6) PCR-positive animals displayed mild clinical signs compatible with EP, including a mild leukocytosis (P<0.05). The heart rate variability frequency domain analysis in four (4) of these EP-positive infected animals revealed a significant (P<0.05) higher low-frequency/high-frequency ratio suggesting a sympathovagal imbalance in these chronically infected animals. Other clinical and cardiovascular parameters were similar between the different groups. Sport horses are routinely submitted to intense training programs and sport-related activities that could lead to loss of the host-parasite equilibrium that characterizes enzootic regions, increasing the likelihood of infection reactivation and the risk of transmission. Heart rate variability analysis contributes to evaluate the sympathovagal balance and detecting homeostasis disturbances in sport horses. Molecular diagnostic tests for EP based on the detection of parasite DNA in equine blood samples should be included in the health programs of sport horses in endemic areas.

Risk and protective factors of Leishmaniasis in the rural area of the western border region of Rio Grande do Sul, Brazil

BackgroundThe Leishmaniases are on the top of the global list of tropical neglected diseases. The number of infected dogs in South America is estimated in millions and correlated to disease cases in humans, especially in Brazil. Equines may get infected too and can play a role in the epidemiological chain. Thus, the aim of the present study was to evaluate risk and protective factors of leishmaniasis in rural areas of the western border region of Rio Grande do Sul state, Brazil by Leishmania spp. protozoa molecular detection and serological evaluation (ELISA) in equine and canine blood samples. This work included nine farms around the city of Uruguaiana. Epidemiologic information regarding farm characteristics and biologic material collection of canine (22) and equine (91), totalizing 113 samples was collected. The polymerase chain reaction (PCR) technique was used to detect Leishmania spp. in biological samples. Variables related to the farm were collected and evaluated through descriptive analysis followed by chi-square test and a logistic regression analysis.ResultsNineteen positive samples (19/113 – 16,81%) were detected, being 18 equines and 1 canine, in six of the nine farms included in the study. No animal showed clinical signs of the disease. According to the variables analyzed, when compared each characteristic separately, the presence of abundant vegetation and poor hygiene demonstrated to be risk factors to Leishmania infection in rural areas. The logistic regression showed that excellent general hygiene, proximity to the weir and trimmed grass were protective factors (p=0.038, p=0.001 and p=0.014, respectively). Having excellent hygiene represents a 70% lower chance of getting infected, keeping the grass cut protects the animal by more than 90% and the proximity of the weir represents a protective factor of 96%.ConclusionsThe presence of Leishmania infection in the western border region of Rio Grande do Sul was 16,81% and it was influenced by farm characteristics. The role of the excellent general hygiene as a protective factor is extremely relevant in the leishmaniases prevention.

CO-oximetry measurements and antioxidant effects of ascorbic acid and methylene blue in equine methemoglobinemic blood.

To determine the effects of time after sampling on CO-oximetry measurements of equine blood samples and the effects of adding ascorbic acid (AscAc) and methylene blue (MetBlue) to samples with methemoglobinemia. Experimental study. University teaching hospital. Thirty healthy adult horses assigned to 5 groups. Repeated CO-oximetry determinations were performed on venous (n=6) and arterial blood samples (n=7) stored at 0°C for 48hours. Methemoglobinemia was induced in vitro in 17 additional blood samples. Six were used as untreated controls, 6 had AscAc added, and 5 had MetBlue added. Total hemoglobin, oxyhemoglobin, carboxyhemoglobin, methemoglobin (MetHb), and oxygen saturation of hemoglobin (SO2 ) were measured. Oxyhemoglobin and SO2 increased from 69.8%±10.2% and 90%±3% to 82.8%±7.9% and 99%±3%, respectively, after 8hours in venous blood (mean±SD, P<0.001). There was an effect of treatment (P=0.032) and of time (interaction P=0.003) on MetHb% in methemoglobinemic samples. The difference in absolute MetHb% from time 0 was as follows: 7.0% (interquartile range [IQR] = 21.2), -0.2% (IQR=3.5), and -4.4% (IQR=5.2) at 48hours in control, AscAc, and MetBlue groups, respectively (P<0.05). There was no effect of time on MetHb% in the AscAc group (23% [IQR = 52.6] at time 0 to 23.2% [IQR = 56.9] after 48h). Storage of blood in ice water to determine O2 Hb and SO2 using a CO-oximeter should not exceed 4hours. Measurement of MetHb% could be delayed by up to 48hours if AscAc is added to the sample. MetBlue significantly decreased MetHb% over time. The limitations of this study include the fact that the antioxidant effects of AscAc and MetBlue were evaluated in vitro and not in vivo. Further studies are needed to evaluate different storage temperatures and syringe types.

Quantification of 17 Endogenous and Exogenous Steroidal Hormones in Equine and Bovine Blood for Doping Control with UHPLC-MS/MS.

A simple and fast analytical method able to simultaneously identify and quantify 17 endogenous and exogenous steroidal hormones was developed in bovine and equine blood using UHPLC-MS/MS. A total amount of 500 µL of sample was deproteinized with 500 µL of a mixture of methanol and zinc sulfate and evaporated. The mixture was reconstituted with 50 µL of a solution of 25% methanol and injected in the UHPLC-MS/MS triple quadrupole. The correlation coefficients of the calibration curves of the analyzed compounds were in the range of 0.9932–0.9999, and the limits of detection and quantification were in the range of 0.023–1.833 and 0.069–5.5 ppb, respectively. The developed method showed a high sensitivity and qualitative aspects allowing the detection and quantification of all steroids in equine and bovine blood. Moreover, the detection limit of testosterone (50 ppt) is half of the threshold admitted in plasma (100 ppt). Once validated, the method was used to quantify 17 steroid hormones in both bovine and equine blood samples. The primary endogenous compounds detected were corticosterone (range 0.28–0.60 ppb) and cortisol (range 0.44–10.00 ppb), followed by androstenedione, testosterone and 11-deoxycortisol.

Assay variability and storage stability of the myeloperoxidase index of the ADVIA 2120i hematology analyzer in canine and equine whole blood samples.

The myeloperoxidase index (MPXI), on ADVIA hematology analyzers, reflects the mean neutrophil myeloperoxidase staining. It is used as a marker of inflammation in animals and people, but assay variability and storage stability are unknown. We aimed to determine MPXI precision and stability with refrigerated storage of canine and equine EDTA-anticoagulated blood and compared MPXI results between two analyzers. Inter-assay coefficients of variations (CVs) were determined from three human-based controls assayed before and after a 20- or 21-day calibration. Blood from 14-16 dogs and 26 horses was assayed 4-10 times within 1day for intra-assay CV measurements. Median control and single run results from 18 canine and 35 equine samples were compared between analyzers. Blood from 10-12 dogs and 10-11 horses was analyzed after collection, and 24, 48, and 72hours of refrigerated storage. Inter-assay CVs of controls were 10.7%-15.9% and 6.4%-9.6% before and 4.3%-7.7% and 2.8%-17.5% after calibration, for ADVIA 1 and 2, respectively. Calibration altered peroxidase gain settings and improved precision. Intra-assay CVs were 0.6%-64% and 3%-350% for canine and equine samples, respectively. Median MPXI results differed significantly between the analyzers, likely from calibration-associated changes in gains. MPXI decreased with storage, and with variable changes between animals and analyzers. Platelet clumps and lipid contributed to the variability in replicate MPXI measurements. MPXI has a higher variability in equine samples than in canine samples. Equivalent results might not be obtained between analyzers. Results change unpredictably with repeated analyses over 72hours. MPXI measurements might only be useful in controlled research settings.

Effects of storage time and temperature on thromboelastographic analysis in dogs and horses.

The accessibility of thromboelastography (TEG) to general practitioners is limited by short sample storage times (30minutes) and storage temperatures (20-23°C). We aimed to evaluate the stability of canine and equine citrated blood samples when stored for extended periods of time, both at room temperature (RT) (20-23°C) and refrigerator temperature (FT) (2-7.5°C). Citrated whole blood samples from healthy dogs and horses (n=10 for each) were stored for 30minutes (baseline) at RT before TEG analysis. Baseline values for TEG variables R, K, α, MA, LY30, and LY60 were compared with those from samples stored for 2, 8, and 22.5h, at RT and FT. Results were compared using an ANOVA (P<.05). Total allowable analytical error (TEa ) based on biological variation data was used to evaluate stability. In dogs, statistically significant differences included shorter R, longer K, decreased MA, and increased LY60 at various time points and storage temperatures from 2h onward. Only samples stored for 2h at FT showed acceptable stability compared with TEa . In horses, statistically significant differences included shorter R and K, and decreased α, LY30, and LY60 at various time points and storage temperatures from 2h onward. Samples were not stable at any time compared with TEa , regardless of the temperature. In this study, canine samples could be stored for up to 2h at FT without affecting TEG results; equine samples should be stored for 30minutes at RT.

Investigating the presence of equine piroplasmosis in Ireland

BackgroundEquine piroplasmosis (EP) is a notifiable disease in Ireland and a significant concern to domestic and international equine industries. Information regarding EP presence in Ireland is currently limited. This retrospective...

Phylogenetic analysis and geographical distribution of Theileria equi and Babesia caballi sequences from horses residing in Spain

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.

Effects of time and temperature on blood gas and electrolytes in equine venous blood

This study aimed to evaluate the viability time of horse venous blood samples kept at laboratory temperature (LT) and in water with ice (WI), to perform blood gas analysis. Eleven blood samples were collected in duplicates from 10 healthy horses. The samples were transported to the laboratory and subjected to one of the 24 h storage method. Each pair of syringes was distinctly kept at LT or submerged in WI. Blood gas tests were performed at times T0h, T1h, T2h, T3h, T4h, T5h, T6h, T8h, T10h, T12h and T24h after collection. Analyses of electrolytes were also performed from the same samples. A difference in blood pH was found between the treatments (P &lt; 0.05). From T4h, pH decreased in samples kept at LT, but in WI, pH did not change. For partial pressure of carbon dioxide (pCO2), a difference between treatments (P &lt; 0.05) was noted starting at T8h. In samples kept at LT, pCO2 increased; no changes occurred in samples stored in WI. There was a decrease in the base concentration beginning at T5h in samples kept at LT (P &lt; 0.05), but no variation in samples kept in WI. These changes can be attributed to the erythrocyte metabolism, still active in vitro, which generates lactic acid from anaerobic glycolysis. The potassium concentration increased in samples kept in WI from T4h, with a gradual increase until T24h. Conservation of equine venous blood samples in WI is efficient in reducing cellular metabolism, thereby increasing the viability of samples for examination and interpretation of results.

Analysis of Theileria equi diversity in The Gambia using a novel genotyping method.

Theileria equi, one of the primary pathogens causing equine piroplasmosis, has previously been sub-classified into a number of clades on the basis of 18S SSU rRNA gene sequence diversity. This partitioning of the parasite population has potential implications for host immunity, treatment and vaccine development. To detect and identify different clade genotypes among and within individual equine blood samples, a novel PCR-based technique was designed and optimized. Theileria equi has only recently been described in The Gambia, and the developed genotyping technique was used to analyse blood samples taken from 42 piroplasmosis-positive horses and donkeys within the country. Three different T.equi genotypes were detected within the population, including the same genotype as the recently described Theileria haneyi, with 61.9% of individuals found to be infected with more than one genotype. Overall, there was a trend that males were more likely to have a multiple genotype infection. Thus, the novel genotyping technique has been shown to be effective in analysis of field populations and offers researchers a rapid method of identifying multiple T.equi genotypes both within individuals and equine populations in epidemiological studies.

First Report Regarding the Simultaneous Molecular Detection of Anaplasma marginale and Theileria annulata in Equine Blood Samples Collected from Southern Punjab in Pakistan.

The present study was designed to check the molecular detection of Anaplasma marginale and Theileria annulata in blood samples of horses and donkeys collected from Dera Ghazi Khan District in Punjab and to document their phylogenetic origin and their association with studied epidemiological factors (sex and age) and complete blood count parameters, if any. A total of 195 blood samples were collected from apparently healthy horses (N = 141) and donkeys (N = 54). A. marginale DNA was detected by PCR in 4.9% (7/141) horse and in 9.2% (5/54) of donkey blood samples. Prevalence of T. annulata was 5.6% (8/141) and 11.1% (6/54) in horse and donkey samples, respectively. While 1.4% (N = 2) horses and 3.7% (N = 2) donkeys were found co-infected with both parasites. Representative amplicon for both parasites was confirmed by DNA sequenced and partial DNA sequence of the major surface protein-1b encoding gene of A. marginale and cytochrome b gene from T. annulata were submitted to the GenBank database under the accession number MK792344-MK792348. Epidemiological data analysis revealed that female horses were more prone to A. marginale (P = 0.02) while female donkeys were more susceptible to A. marginale (P < 0.001) and T. annulata (P < 0.001) infection. It was observed that horse and donkey infected either with Anaplasma marginale or Theileria annulata had significantly disturbed red and white blood cell counts and their associated parameters. This is a first ever study regarding molecular detection of A. marginale and T. annulata in equine blood samples from Pakistan. We recommend that this multiplex PCR protocol should be used for the detection of Anaplasma marginale and Theileria annulata in livestock for their proper diagnosis and treatment.

Genetic Diversity of Theileria equi from Horses in Different Regions of Brazil Based on the 18S rRNA Gene

Equine piroplasmosis stands out among the diseases that affect Equidae in Brazil and the world. It is caused by the protozoa Theileria equi and Babesia caballi. The objective of the present study was to carry out the molecular characterization of T. equi using equine blood samples collected in the 5 geographic regions of Brazil. Samples from all over the country were tested for the presence of T. equi by real-time PCR. The 18S rRNA sequences (∼1,600 bp) obtained from 23 samples taken from naturally infected horses were characterized by sequencing and analyzed to identify the genotypes and the possible sites of genetic variability. Thirteen different T. equi 18S rRNA sequences were identified, and 2 different genotypes were demonstrated to be in circulation in Brazil. Alignment entropy analysis demonstrated the existence of three hypervariable regions (V2, V4, and V8) within the 18S rRNA sequence of T. equi. The V2 region is located between nucleotides 63 and 75, V4 is located between nucleotides 524 and 586, and V8 is located between nucleotides 1,208 and 1,226. The hypervariable region V4 demonstrated the greatest variation within the 18S rRNA sequence of T. equi. Phylogenetic analysis based on the 18S rRNA sequences revealed the formation of 3 distinct clades (A, B, and C). The Brazilian samples belonged to 2 clades (A and C). The present study describes the characterization and heterogeneity of the circulating T. equi 18S rRNA sequences in Brazil. The results confirm that the country is an endemic area for the disease, and they indicate that at least 2 distinct T. equi genotypes are naturally infecting equines in Brazil.

An immuno polymerase chain reaction screen for the detection of CJC-1295 and other growth-hormone-releasing hormone analogs in equine plasma.

CJC-1295 is a 30 amino acid peptide-based drug that stimulates the release of growth hormone (GH) from the pituitary gland. It is unique among performance-enhancing peptides due to the presence of a reactive maleimidopropionic acid group that covalently links the peptide to free thiols on the surface of plasma proteins. Once conjugated, CJC-1295 remains active in the bloodstream for significantly longer than non-conjugated peptide-based drugs that are rapidly excreted. Conjugation of CJC-1295 to plasma proteins prevents its detection by top-down mass-spectrometry-based peptide screening protocols as it effectively becomes a macromolecular protein with an undefined molecular weight. Using a pair of monoclonal antibodies raised against the CJC-1295 peptide, we present an immuno-polymerase chain reaction (I-PCR) assay that is capable of detecting the CJC-1295-protein conjugate at concentrations down to 0.8pg/mL. Detection of endogenous equine GHRH necessitated a screening threshold for CJC-1295 in equine plasma of 50pg/mL. The effectiveness of the assay for controlling the illicit use of CJC-1295 was confirmed in equine blood samples after administration in thoroughbred race horses.