Molecular and serological detection of tick-borne pathogens in donkeys (Equus asinus) in Italy.

Donkeys, owing to the frequent outdoor activity, are exposed to a high risk of infection with tick-borne pathogens. This work aimed to detect exposure to Theileria equi, Babesia caballi, Anaplasma phagocytophilum and Borrelia burgdorferi s.l. of donkeys reared in Central Italy. For this purpose 122 adult donkeys were selected within 11 herds and submitted to blood collection. IgG antibodies to T. equi, B. caballi, A. phagocytophilum and B. burgdorferi s.l. were detected by IFAT. Conventional PCRs targeting the genes MSP2 and the flagellin were used for the detection of A. phagocytophilum and B. burgdorferi s.l. respectively and a Real Time PCR Sybr Green was used to detect Babesia/Theileria spp…. The species identity was determined by amplicons sequencing. Forty eight (39.3%) and 58 (47.5%) animals tested positive for T. equi and B. caballi antibodies, respectively; nine animals (7.4%) were found positive for antibodies against A. phagocytophilum whereas negative results were obtained for B. burgdorferi s.l. Twenty-six (21.3%) animals showed antibodies for both T. equi and B. caballi. Twenty-three (18.8%) donkeys were positive to Babesia/Theileria spp. PCR assay. Out of 21 sequenced amplicons, 20 were identified as T. equi, belonging to three main groups designated A, B and D and one as B. caballi group A. Neither A. phagocytophilum nor B. burgdorferi PCR results were positive. The study showed a high exposure of donkeys to tick-borne pathogens and provides information on the genetic identity of the T. equi strains circulating in Central Italy.

Evaluation of the performance of a rapid enzyme-linked immunosorbent assay in the detection of Anaplasma phagocytophilum antibodies in horses.

The aim of this study was to evaluate the performance of a commercially available rapid enzyme-linked immonosorbent assay, the Snap® 4Dx test, in the detection of Anaplasma phagocytophilum antibodies in horses. Two hundred apparently healthy horses (asymptomatic) and 244 animals showing clinical symptoms (symptomatic), were tested for A. phagocytophilum immunoglobulin G (IgG) antibodies using both the Snap® 4Dx kit and an indirect fluorescence antibody test (IFAT), with the latter serving as a comparative test. Horses belonging to the symptomatic group were also tested for evidence of active infection with A. phagocytophilum by analysis of IFAT IgM titers and PCR assay amplifying a specific fragment of the 16S rRNA gene. The overall agreement between the results obtained using the two tests, as well as the relative performance exhibited by the Snap® 4Dx test in the two groups, was assessed. Forty of the 45 animals (89%) testing positive for IgG antibodies using IFAT were correctly identified using Snap® 4Dx testing. The agreement between the results of the two tests was very high (k>0.9), with almost identical performances in both symptomatic and asymptomatic animals. Conversely, within the symptomatic group, only 44% (no. 11/25) of Snap® 4Dx positives appeared to be associated with a state of active infection, whereas the remaining 56% (no. 14/25) were related both to not infected animals (no. 1) and to horses whose status of infection needed further evaluations to be confirmed (no. 13/25). This study suggests that the Snap® 4Dx test could represent a valid screening method for use during epidemiological surveys of equine populations. Nevertheless, in-clinic application of the test does not appear to be merited.

Efficacy of toltrazuril 5 % suspension (Baycox®, Bayer) and diclazuril (Vecoxan®, Janssen-Cilag) in the control of Eimeria spp. in lambs.

Sheep coccidiosis is a pathology caused by protozoan parasites of the genus Eimeria spp. with clinical and economic effect especially in young animals (Ambrosi 1995; Pellérdy 1974). A field study was conducted to evaluate the efficacy of a metaphylactic treatment with toltrazuril 5 % suspension (Baycox®, Bayer) in comparison with diclazuril (Vecoxan®, Janssen-Cilag) and untreated controls against naturally acquired Eimeria infections in housed lambs. A total of 170 animals, aged 24 to 34 days and randomly divided in three homogeneous groups, were included in the study. The assessment of treatment efficacy was based on total faecal oocyst excretion (opg) and count reduction (FOCR) in the two groups of animals treated with toltrazuril (TOLT) and diclazuril (DICL) compared with untreated control group (C). The animals treated with toltrazuril showed a considerably lower mean opg to that of group C (5.78 opg versus 144.62 opg) (p < 0.05) and a FOCR of 97.7 %. The higher efficacy (99.23 %) was observed at 15 days post treatment; however, the average efficacy of the drug remained extremely high (> 90 %) for all the study. The lambs treated with diclazuril showed an intense, persistent oocyst excretion with average levels of 97.54 opg, (p < 0.05). This study demonstrates the good efficacy of toltrazuril administered orally to lambs in the prepatent period in subclinical natural Eimeria infections in housed lambs.

Influence of a metaphylactic treatment with Baycox(®) Bovis on the reproductive performances of Fresian heifers: a preliminary study.

A blind, randomised, controlled, multicentric field trial was conducted to assess the influence of a metaphylactic treatment with an oral solution of toltrazuril on some reproductive parameters of Italian Fresian heifers during the first 18-20 months of life. For this goal 40 calves were selected from two dairy farms and randomly divided into two homogeneous groups: MTol, treated with toltrazuril and NegC, left untreated. The calves were clinically and coprologically examined over the entire study period. The body condition scores, the body weights and the age at the first service were recorded and compared between the two groups, in addition to some other reproductive parameters including number of pregnancies, average service per pregnancy, conception rate, conception rate at first service and post first service conception rate. The analysis of the results showed that the metaphylactic treatment with toltrazuril influenced positively the average age of the first service (MTol 461.4 days versus NegC 485.45 days), the overall conception rate (MTol 95 % versus NegC 85 %), the success at first (MTol 60 % versus NegC 45 %) and second (MTol 75 % versus NegC 45 %) services and, consequently, the mean number of services to be carried out for each animal (MTol 1.4 ± 0.6 versus NegC 1.6 ± 0.79). Furthermore, the results confirmed that toltrazuril treatment, applied in accordance with the epidemiological aspects of each farm, is highly efficacious in persistent reduction of oocyst excretions with particular reference to Eimeria zuernii, Eimeria bovis, considered to be mainly responsible for clinical coccidiosis.

Prevalence of tick borne pathogens in horses from Italy.

In order to investigate the prevalence of tick-borne diseases, equine piroplasmosis, equine granulocytic anaplasmosis and Lyme borreliosis in Central Italy, blood samples from 300 horses were analyzed for the presence of antibodies against Babesia caballi, Theileria equi, Anaplasma phagocytophilum and Borrelia burgdorferi using the IFAT. The blood samples were also subjected to PCR assays in order to detect pathogen DNA. A total of 78 (26.0%) and 123 (41.0%) horses were found to be seropositive for B. caballi and T. equi, respectively, while 41 (13. 4%) and 21 (7.0%) horses were, respectively, seropositive for A. phagocytophilum and B. burgdorferi. Seropositivity for more than one agent was detected in 76 horses using IFAT. The most common association observed was between T. equi and B. caballi (14.7%). In addition, 54 horses (18.0%) were found to be positive for one or more tick-borne pathogens (TBPs) using PCR testing. Among these, 28 (9.3%) harbored single infections, while 26 (8.7%) were found to be co-infected with two or more pathogens. The correlation (K value) between IFAT and PCR results was 0.32 for T. equi, 0.34 for B. caballi, 0.62 for B. burgdorferi and 0.48 for A. phagocytophilum, reflecting an unprecedented degree of multiple exposures to TBPs in horses.

Evaluation of an immunofluorescence antibody assay for the detection of antibodies against Toxoplasma gondii in meat juice samples from finishing pigs.

Serum and meat juice analyses for the detection of Toxoplasma gondii antibodies by an immunofluorescence antibody assay were compared in 100 seropositive and 100 seronegative slaughtered heavy swine. Meat juice was obtained from diaphragm and gracilis muscles of the serologically tested animals. Seventy-two diaphragmatic meat juice samples (36%, 95% interval confidence [IC] 29.4%-43.1%) and 63 gracilis meat juice samples (31.5%, 95% IC 25.1%-38.4%) tested positive for T. gondii antibodies. The average concordance between serum and meat juice derived from both muscles was "substantial" (K=0.6-0.8). The K-value was 1 when considering serum samples showing a titer >1/16, whereas it decreased to 0.62 and to 0.49 when considering serum samples with a 1/16 titer in meat juice from diaphragm and gracilis muscle, respectively.

Long-term effect of toltrazuril on growth performances of dairy heifers and beef calves exposed to natural Eimeria zuernii and Eimeria bovis infections.

The long-term effects of a toltrazuril treatment against natural Eimeria bovis and/or Eimeria zuernii infections were investigated in comparison with diclazuril and untreated controls on two dairy (Italian Friesian breed) and two beef (Chianina breed) farms. At each trial site, 30 calves were allocated into three groups of 10 calves each: T (treated with toltrazuril), D (treated with diclazuril) and C (left untreated). For 40 weeks post-treatment, the calves were weighed and examined clinically and parasitologically. The oocyst counts as well as the number of scour days were significantly lower in the T group than in the D and C groups. Final bodyweights and body condition scores of the T group exceeded those of groups C and D. The results confirmed that toltrazuril was highly efficacious, safe and provided productive benefits in dairy and beef calves.

Pathological changes caused by Anoplocephala perfoliata in the mucosa/submucosa and in the enteric nervous system of equine ileocecal junction.

In this study, pathological changes caused by Anoplocephala perfoliata in the ileocecal junction were investigated in 31 regularly slaughtered mixed-breed horses of both sexes. Our results showed a significant relationship between parasite burden and grading of histopathological lesions in the mucosa and submucosa. Hypertrophy of the circular muscle layer was found in infected horses. Moreover, enteric nervous system evaluation showed a significant injury of intestinal nervous elements in the horses with moderate to high parasitism expressed as an increase of degenerative-regressive changes in neuronal cells and a decrease in the number of myenteric ganglia and neuronal cells. These findings can help to clarify the pathogenesis of intestinal motility disorders associated with A. perfoliata infection in horses.

Are macrocyclic lactones useful drugs for the treatment of Parascaris equorum infections in foals?

A study was carried out to assess the field efficacy of moxidectin (MOX) against Parascaris equorum in foals. A total of 70 foals from 2 Italian thoroughbred (Farms 1 and 2) stud farms and one trotter stud farm (Farm 3), aged 3-5 months and with faecal egg counts (FEC) f>or=300 eggs per gram (EPG) were included in the study. On each farm, foals were divided into 2 groups (of 10, 8 and 10 foals, respectively) and treated with either moxidectin (MOX, 400 mcg/kg b.w.) or ivermectin (IVM, 200 mcg/kg b.w.) on day 0 and examined for faecal count reduction (FECR) on day 14. Five, 4 and 5 foals, respectively, were kept as untreated controls. An individually based estimation of efficacy was assessed by a bootstrap simulation to evaluate the percent reduction of FECRs. Two thousand bootstrap resamples were constructed from individual FECRs and the parasite population was considered susceptible for FECs>or=90% and 95% lower confidence limit (CL)>90%, suspected resistant for FECRs>or=90% and 95% LCL<90% and resistant when FECR<90% and 95% LCL<90%. On day 14, foals with FECRs categorized as resistant or suspected resistant were treated with pyrantel pamoate (PYR, 13.2mg/kg b.w.) and examined for FECR on days 28 and 35. On day 14, MOX and IVM treatments were ineffective in all foals from both thoroughbred farms, while MOX ad IVM were effective (FECRs>90%) in 7 and 5 trotter foals, respectively, and remained >90% until day 35. PYR treatment given on day 14 to remaining 8 foals (3 MOX-treated and 5 IVM-treated) was fully efficacious at faecal examinations carried out on days 28 and 35.

Field effectiveness of pyrantel and failure of Parascaris equorum egg count reduction following ivermectin treatment in Italian horse farms.

A study was carried out to assess the field efficacy of ivermectin (IVM) and pyrantel pamoate (PYR) against Parascaris equorum. Seventy-three foals (3-18 months old) from 5 stud farms, not treated with anthelmintics in the previous 10 weeks and with individual faecal egg counts (FEC) >200, were included in the study. For each stud farm, 5-7 foals were included in the IVM-treatment group (IVM 0.2%, 200 mcg/kg body weight) or in the PYR-treatment group (PYR 38%, 13.2mg/kg body weight) and 3 were untreated as controls. For each foal, FECs were carried out before treatment (Day 0) and on Days 7 and 21. An individually based estimation of efficacy was assessed by a bootstrap simulation applied to different previously suggested formulae to evaluate the percent reduction of faecal egg counts (FECR). Two thousand bootstrap resamples were constructed from individual FECRs and the parasite population was considered susceptible for FECs >or=90 and 95% confidence interval (C.I.) >or=95%, suspected resistant for FECRs comprised between 80 and 90% and 95% C.I. <95% and resistant when FECR