Mónica Luzón

Cryptosporidium and concurrent infections with other major enterophatogens in 1 to 30-day-old diarrheic dairy calves in central Spain

Abstract Faeces samples from 218, 1 to 30-day-old, diarrheic dairy calves in 65 dairy herds were screened for the presence of Cryptosporidium and concurrent infections with rotavirus, coronavirus, F5+ Escherichia coli and Salmonella spp. Calves were grouped according to their age as follows: 1–7, 8–14, 15–21 and 22–30 days. Cryptosporidium infection was detected in 43.8%, 71.9%, 63.2% and 6.9% of the calves in the respective age groups. Significant differences in the detection rate of Cryptosporidium were found between the age group 22–30 days and all other age groups, and between the age group 1–7 days and the age groups 8–14 days and 15–21 days. Cryptosporidium was the only enteropathogen detected in 60 of the 114 (52.6%) diarrheic calves. Concurrent infections with other enteropathogen(s) were detected in 64.3%, 46.3%, 39.5% and 0% of the Cryptosporidium-infected calves in the age groups 1–7, 8–14, 15–21 and 22–30 days, respectively. A significant age-associated decrease in the detection rate of mixed infections (p <0.05) was found. The detection rates of the other enteropathogens considered in calves with Cryptosporidium infection were 87% for rotavirus, 11.1% for coronavirus, 27.8% for F5+ E. coli and 1.8% for Salmonella.

Reliability of coprological diagnosis of Anoplocephala perfoliata infection

Three coprological methods were tested to establish the reliability of in vivo diagnosis of Anoplocephala perfoliata. A total of 107 faecal samples were analyzed, and the presence of tapeworms were confirmed postmortem in 24 animals with burdens that ranged from 1 to 248 worms; most of them (71%) with less than 100 parasites. Best results were obtained with a combination of two sedimentation/flotation methods, detecting only half the parasitized animals (54% sensitivity). No relationship could be established between tapeworm burden and egg detection, but results indicate that coprological methods have a lower likelihood of diagnosing cestode infection when horses have less than 100 tapeworms.

HYPODERMOSIS OF RED DEER IN SPAIN

From February 1998 to January 1999, 106 red deer in the Autonomic Organism National Park “Quintos de Mora” (Toledo, central Spain) were evaluated to determine the prevalence and dynamics of infection with Hypoderma sp. by detection of subcutaneous larvae. Between six and 13 deer shot in selective hunting were examined monthly. Hypoderma sp. larvae were detected throughout the period except in June, July and August of 1998. Excluding the period during which no subcutaneous larvae were detected, the number of animals sampled was 80 (52 males and 28 females), belonging to three age classes: 12 calves (<1-yr-old), 19 yearlings (1-yr-old), and 49 adults (2- to 10-yr-old). All the third instar (L3) collected were identified as H. actaeon. Total prevalence during the period of larval detection was 61%. Prevalence in yearling and adult deer shot during the official hunting season was 89%. Monthly prevalence increased from September to January and decreased from February to May. In September and October, a small percentage of larvae were classified as first instar (L1). The rest of larvae collected between September and December were second instar (L2). Third instar (L3) predominated in January and February and was the only stage collected from March to May. Intensity ranged from 1 to 145 larvae. Intensities were >100 larvae in 6% of animals. Possible relationships of intensity or prevalence of infection with sex or age of the animals were evaluated. Significant differences in prevalence were observed among different host age classes. Prevalence was higher in yearlings (84%) than in adults (63%) and lowest in calves (17%).

New multiplex PCR method for the simultaneous diagnosis of the three known species of equine tapeworm.

Although several techniques exist for the detection of equine tapeworms in serum and feces, the differential diagnosis of tapeworm infection is usually based on postmortem findings and the morphological identification of eggs in feces. In this study, a multiplex polymerase chain reaction (PCR)-based method for the simultaneuos detection of Anoplocephala magna, Anoplocephala perfoliata and Anoplocephaloides mamillana has been developed and validated. The method simultaneously amplifies hypervariable SSUrRNA gene regions in the three tapeworm species in a single reaction using three pairs of primers, which exclusively amplify the internal transcribed spacer 2 (ITS-2) in each target gene. The method was tested on three types of sample: (a) 1/10, 1/100, 1/500, 1/1000, 1/2000 and 1/5000 dilutions of 70 ng of genomic DNA of the three tapeworm species, (b) DNA extracted from negative aliquots of sediments of negative control fecal samples spiked with 500, 200, 100, 50 and 10 eggs (only for A. magna and A. perfoliata; no A. mamillana eggs available) and (c) DNA extracted from 80, 50, 40, 30, 10 and 1 egg per 2 μl of PCR reaction mix (only for A. magna and A. perfoliata; no A. mamillana eggs available). No amplification was observed against the DNA of Gasterophilus intestinalis, Parascaris equorum and Strongylus vulgaris. The multiplex PCR method emerged as specific for the three tapeworms and was able to identify as few as 50 eggs per fecal sample and as little as 0.7 ng of control genomic DNA obtained from the three species. The method proposed is able to differentiate infections caused by the two most frequent species A. magna or A. perfoliata when the eggs are present in feces and is also able to detect mixed infections by the three cestode species.

Differential diagnosis of equine cestodosis based on E/S and somatic Anoplocephala perfoliata and Anoplocephala magna antigens

The tapeworm responsible for equine colic, Anoplocephala perfoliata, is considered the most common intestinal tapeworm of horses worldwide. However, there is evidence that Anoplocephala magna has a similar prevalence in North America and Spain, and possibly in other countries, highlighting the need for diagnostic methods capable of distinguishing between these two species. Currently, immunodiagnosis of A. perfoliata is based on the identification of the 12/13 kDa excretory/secretory (E/S) A. perfoliata immunoreactive antigen, which while apparently specific, has never been tested in sera from A. magna-positive horses. Accordingly, we evaluated the specificity of 12/13 kDa E/S A. perfoliata antigen for the first time by testing this crude antigen against A. magna-positive sera in Western blot. In addition, we characterized a somatic (Som) crude antigen of A. perfoliata and for the first time, the E/S and Som crude antigens of A. magna, evaluating their potential utility for the differential serodiagnosis of equine anoplocephalosis in sera from horses of known parasitic status. SDS-PAGE revealed major low MW bands at: 14 and 12 kDa for E/S and Som-A. magna; 14 and 11 kDa for E/S A. perfoliata; and 11 and 10 kDa for Som-A. perfoliata. Protein regions at 12-14 kDa (E/S A. perfoliata), 10-15 kDa (Som-A. perfoliata) and 10-12 kDa (Som-A. magna) were recognized by Anoplocephala-positive sera at the genus but not the species level. These findings demonstrate cross-reactivity of these unpurified antigenic components, precluding their use in differential diagnosis between A. perfoliata and A. magna. Although these results do not directly indicate cross reactivity at the purified 12/13 kDa component of the E/S A. perfoliata antigen, it is possible that current immunodiagnostic methods based on this component might not accurately differentiate between these two tapeworm species, suggesting erroneous diagnosis of A. perfoliata in areas where A. magna is present.

Relationship between Iberian ibex (Capra pyrenaica) sperm quality and level of parasitism

This paper examines the relationship between parasite infection rates and reproductive function in wild Iberian ibexes. The animals examined were 43 adult males shot during the rutting season. Gastrointestinal and pulmonary nematodes, intestinal cestodes and intestinal coccidia were determined by coprological analysis. Protozoa in the muscles were detected by biopsy. Epididymal spermatozoa were collected from recovered testes. Sperm motility, the integrity of the plasma membrane, sperm viability, sperm morphology and acrosome integrity were all evaluated. Bronchopulmonary nematode larvae were detected with a prevalence of 100% (mean intensity 216.8 ± 65.8; index of dispersion 476.1, indicating an aggregated pattern). A negative correlation (R = -0.39; P < 0.05) was found between the shedding of larval lungworms and the percentage of sperm morphological abnormalities. Although directional relationships could not be identified, the present findings suggest that reproductive effort imposes a cost in terms of depressed parasite resistance.

Coprologically diagnosing Anoplocephala perfoliata in the presence of A. magna

Current copro-diagnostic tests for Anoplocephala perfoliata show high variation in their sensitivity and given the morphological similarity of Anoplocephala spp. eggs, this could be related to the presence of Anoplocephala magna alone or co-existing with A. perfoliata. In the present study, coprology was significantly more sensitive (p<0.01) at detecting A. magna than A. perfoliata. This difference was independent of the parasite burden and was greater when testing was limited to horses with mature or gravid tapeworms. A. magna infection was strongly linked to young horses (≤ 2 years). The eggs of A. magna are smaller. Using 15 and 70 μm cut-offs for oncosphere diameter and the major shell bisector length, respectively, the eggs of A. perfoliata were identified with 100% sensitivity, 97% specificity and 98% sensitivity, 84% specificity. The use of these two morphometric variables would therefore be useful for the copro-identification of A. perfoliata in countries where both species coexist.

Sarcocystosis in Cervus elaphus: Comparison of diagnostic methods

Red deer (Cervus elaphus) from a National Wildlife Reserve near Toledo in central Spain were surveyed for Sarcocystis infection. A total of 61 deer were examined. Tissue compression and histology were used to examine samples from diaphragm and heart from each animal included in the study, and results from the two techniques and the two tissues were compared to determine the tissue and technique that provide the most accurate measure of prevalence and intensity. Prevalence and intensity were then compared between calves, yearlings and adults. Sarcocystis was detected in 59 (97%) of the 61 deer. Comparison between tissues showed that (a) prevalence based on histology was similar for heart and diaphragm, (b) prevalence based on compression was significantly higher for heart than for diaphragm and (c) intensity was significantly higher for heart than for diaphragm, regardless of the technique used. Comparison between techniques showed that (a) both techniques rendered similar prevalences and intensities of Sarcocystis infection with heart samples and (b) both techniques were not comparable with diaphragm samples (compression rendered lower prevalence but higher intensity than histology). Together these data suggest that heart is the preferable tissue for estimating prevalence and intensity, regardless of the technique used. A preliminary species identification of isolated cysts from three animals showed two morph types, corresponding to Sarcocystis cervicanis (syn. S. cf. grueneri; S. wapiti) in the heart and diaphragm of three animals and S. hjorti, only in the diaphragm of two animals. Given the different location of those morph types, both heart and diaphragm should be sampled and preferably assessed using histology to most reliably detect infection. Based on histology of heart, prevalence and intensity of Sarcocystis were significantly lower in calves than in yearlings or adults.