E. del Cacho

Prevalence of Cryptosporidium and Giardia infections in cattle in Aragón (northeastern Spain)

Abstract Faecal samples from 554 bovines randomly selected at 30 farms in Aragón were examined to investigate the prevalence of Cryptosporidium and Giardia infections. C. parvum oocysts were identified by using the Ziehl-Neelsen modified technique in 109 (19.7%) bovines ranging from 3 days old to adults. Positive animals were found in 19 (63.3%) farms. As much as 44.4% of calves aged 3–4 days were infected, but infection rates peaked at 6–15 days of age (76.7%). Nevertheless, prevalence was also high in weaning calves aged 1.5–4 months (14%), fattening calves and heifers 4–24 months old (7.7%) and adults (17.8%). Diarrhoea was recorded in 78.6% of suckling and 29.4% of weanling calves infected by C. parvum, but it was only found to be statistically associated with infection in suckling calves (P < 0.01). All calves shedding moderate or many oocysts had diarrhoea, whereas asymptomatic infection was always correlated with few oocysts in faeces. Cryptosporidial infections were always asymptomatic in bovines older than 4 months. Giardia cysts were identified in 65 bovines (11.7%) from 16 (53.3%) of the farms surveyed. Infection rates were significantly higher in suckling (14.1%) and weanling calves (38%) than in bovines older than 4 months (2.2%) (P < 0.001). Diarrhoea was recorded in 45.5% of suckling and 10.9% of weanling calves infected by Giardia, but it was not found to be statistically associated with infection. In fact, infection rates were higher in non-diarrhoeic than in diarrhoeic calves.

Prevalence and analysis of potential risk factors for Cryptosporidium parvum infection in lambs in Zaragoza (northeastern Spain).

An epidemiologic study was carried out to investigate the prevalence of and to identify factors associated with the risk of Cryptosporidium infection in sheep in Zaragoza (northeastern Spain). Faecal samples from 583 lambs aged from 1 day to 3 months and 205 ewes older than 1 year were collected at 89 farms in the two regions of the province of Zaragoza with the highest sheep population (Zaragoza and Ejea de los Caballeros). In every sheep farm, data of the factors potentially associated with the likelihood of C. parvum infection were analysed: geographical location, season, size of herd, number of lambs in the herd at sampling time, lambing period, cleaning of lambing area and presence of diarrhoeic lambs in the farm. C. parvum oocysts were identified by using the Ziehl-Neelsen technique in 344 lambs (59%) from 75 farms (84.4%). Infected lambs ranged from less than 7 days to 90 days of age, although the percentage of animals shedding oocysts peaked at 8-14 days of age (76.2%). Statistical analysis showed that infection rates were significantly higher in lambs aged between 1 and 21 days (66.4%) than in those aged between 22 and 90 days (23%) (P<0.0001, chi(2)). Analysis of correlation between excretion of oocysts and diarrhoea revealed a relationship in all age groups and the probability of presenting diarrhoea was significantly higher for lambs shedding oocysts (86.3%) than for those which did not excrete the parasite (32.2%) (P<0.0001, chi(2)). Similarly, cryptosporidial infection rates were significantly higher in diarrhoeic (79.4%) than in non-diarrhoeic lambs (22.4%). Furthermore, infection intensity was correlated with the presence of clinical symptoms. Presence of diarrhoeic lambs in the farm was the only factor significantly associated with an increased risk of infection since the percentage of herds testing positive was significantly higher in farms with diarrhoeic lambs (91.3%) than in those without cases of neonatal diarrhoea (12.5%) (P<0.0001, chi(2)). Factors associated with a decreased risk of C. parvum infection in lambs included low numbers of lambs in the farm and cleaning of the lambing area. Additionally, lambs 8-14 days of age were less likely to be infected at the first lambing period and in spring/autumn. Cryptosporidial infection was also detected in 16 ewes (7.8%) which excreted few oocysts and without diarrhoea.

Cryptosporidium species and subtype analysis from dairy calves in Spain

Faecal specimens from 287 diarrhoeic calves younger than 21 days, collected over a 2-year period (2006-2007) from 82 dairy cattle farms in 14 provinces across the north of Spain, were examined for the presence of Cryptosporidium oocysts. Overall, 63 farms (76.8%) and 166 calves (57.8%) tested positive by microscopy. In order to elucidate the genetic diversity, selected positive specimens from 149 calves originating from 61 farms in the 14 provinces were examined by genotyping and subtyping techniques. Cryptosporidium parvum was the only species identified by PCR-RFLP of SSU rDNA from all 149 isolates and sequencing of a subset of 50 isolates, except for 2 specimens that were identified as C. bovis. Sequence analyses of the glycoprotein (GP60) gene revealed that most C. parvum isolates (98%) belonged to the subtype family IIa and 2 isolates were identified as the novel subtype IIdA23G1. Subtype IIaA15G2R1 was the most common and widely distributed (80.3% of the 61 farms), followed by subtype IIaA16G3R1 (14.7%), whereas the remaining IIa subtypes (IIaA16G2R1, IIaA17G2R1, IIaA18G3R1, IIaA19G3R1) were restricted to 1-3 farms. All these C. parvum IIa subtypes have previously been described in human patients, indicating that most isolates from diarrhoeic calves in northern Spain have zoonotic potential.

PREVALENCE OF CRYPTOSPORIDIUM INFECTIONS IN PIGS IN ARAGON (NORTHEASTERN SPAIN)

Faecal samples from 620 pigs randomly selected from 27 farms throughout Aragón were examined to determine the prevalence of Cryptosporidium infections. Detection of oocysts was performed using the ethyl-acetate stool concentration method and the modified Ziehl-Neelsen technique. Cryptosporidium parvum oocysts were identified in 136 (21.9%) pigs from 21 (77.8%) farms. Infected animals ranged from 1 to 6 months old and oocysts were not detected in suckling piglets or adults. Infection rates were significantly higher in weaned, 1-2 month old piglets (59.2%) than in fattening, 2-6 month old pigs (34.3%) (P < 0.001). Cryptosporidial infections were asymptomatic in most of the pigs (90.4%) and usually of low intensity, since 92.6% of the infected pigs excreted few oocysts (0-1 oocysts per field at x 200 magnifications). Although 24.1% of weaned and 5.6% of fattening pigs infected by C. parvum had diarrhoea, it was not found to be statistically associated with infection. In fact, infection rates were higher in non-diarrhoeic than in diarrhoeic pigs, in both weaned (64.7% and 46.7%, respectively) and fattening pigs (34.3% and 33.3%).

Field trial on the therapeutic efficacy of paromomycin on natural Cryptosporidium parvum infections in lambs.

The objective of this study was to evaluate the therapeutic efficacy of paromomycin against cryptosporidiosis in naturally infected lambs under field conditions. The 36 cross-bred neonatal lambs, 3-10 days old, were used. On the first day that lambs showed diarrhea (Day 1) they were randomly divided into three groups. The infected control group (14 lambs) remained unmedicated whereas the two other groups were orally medicated with paromomycin solution (Humatin((R)), Parke Davis, France): 12 lambs (Group A) at 100mg/kg per day for three consecutive days (Days 1-3) and 10 lambs (Group B) at 200mg/kg per day for two days (Days 1 and 2). Drug efficacy was assessed by evaluating the presence of diarrhea, oocyst shedding and weight gains from Days 1 to 23. The results show the efficacy of paromomycin in reducing both cryptosporidial oocyst output and severity of clinical signs. On Day 4, all unmedicated lambs remained infected and excreted large numbers of cryptosporidial oocysts (mean score: 2.5) whereas oocyst output had stopped in most medicated lambs (>60%) and low numbers of oocysts were excreted in the remaining lambs (mean score: 0.45 in Group A and 1 in Group B). Mean oocyst excretion was significantly reduced in medicated lambs from Days 2 to 5 (P<0.05). Treatment also reduced, but not completely prevented, clinical symptoms although diarrhea stopped in most medicated lambs just after drug withdrawal. The mean weight gains of Group A lambs were higher than that of unmedicated lambs throughout the study and statistically significant differences were found from Days 1 to 11 (1.99+/-0.81 versus 1.47+/-0.53) (P<0.05). By contrast, the growth rate of Group B lambs from Days 11 to 23 was impaired when compared with the two other groups (P<0.05) although no significant differences were found at the end of the study (Days 1-23).

Prevalence of intestinal parasites, including Cryptosporidium parvum, in dogs in Zaragoza city, Spain

Faecal samples from 81 dogs aged between 2 months and 13 years were collected in the small animal clinic (37 domestic dogs) and the animal shelter (44 stray dogs) located in the Faculty of Veterinary Sciences in Zaragoza city (northeast Spain) and screened for the presence of Cryptosporidium oocysts. Faeces were concentrated by the formalin-ethyl acetate method and smears of the sediment were stained by using the modified Ziehl-Neelsen technique. Cryptosporidium parvum oocysts were detected in six dogs (7.4%) aged from 2 months to 6 years. Infection was detected in both domestic (three) and stray (three) dogs and all of them excreted few oocysts (0-1 oocyst per 20 x field). No statistically significant differences in prevalence occurred between dogs younger than 6 months (11.8%) and the older dogs (6.2%). Prevalences were not significantly different between domestic (8.1%) and stray dogs (6.8%). Diarrhoea was recorded in three of the positive dogs (50%), although additional enteric parasites such as oocysts of Isospora spp. were also detected in their faeces. Nevertheless, prevalence was significantly higher in diarrhoeic (30%) versus non-diarrhoeic (4.2%) dogs (P < 0.05). Cryptosporidium was one of the parasites most frequently detected in the dogs surveyed.

Comparison of an acid-fast stain and a monoclonal antibody-based immunofluorescence reagent for the detection of Cryptosporidium oocysts in faecal specimens from cattle and pigs

A commercially available direct immunofluorescence (IF) assay with monoclonal antibodies (Monofluo Kit Cryptosporidium, Diagnostics Pasteur, France) was compared with the modified Ziehl-Neelsen (MZN) acid-fast technique for the detection of Cryptosporidium oocysts in faecal samples from cattle and pigs. Stool specimens individually collected from 108 bovines and 90 pigs were examined in a blind test. The results of the two procedures corresponded (both positive or negative) in 102 (94.4%) cattle samples and 80 (88.9%) pig faecal samples. However, the remaining six (5.5%) cattle specimens and 10 (11.1%) pig stool samples, all of them harboring few oocysts (0-1 oocysts per 20 x field), were negative by MZN and positive by IF. False-negative results of the acid-fast stain occurred in suckling (17.2% of discrepant results) and weaned calves (2.9%) as well as weaned piglets (43.7%) and fattening pigs (10%). Stool specimens from the remaining age groups were negative by both techniques. The MacNemars chi-square test showed that differences between both methods were statistically significant (P < 0.05). Compared with immunofluorescence procedure, the sensitivity of MZN technique in samples from cattle and pigs was 79.3% and 67.7% and the negative predictive value was 92.9% and 85.5% respectively. The specificity and positive predictive values of the acid-fast stain were 100% in both animal species. It is concluded that the monoclonal antibody-based immunofluorescence reagent evaluated is more efficient that the MZN technique, especially for detecting a low number of Cryptosporidium oocysts, in faecal specimens from both cattle and pigs.

Pathogenesis of Eimeria praecox in chickens: virulence of field strains compared with laboratory strains of E. praecox and Eimeria acervulina

The pathogenesis in chickens of the apicomplexan Eimeria praecox was compared with that of Eimeria acervulina, using intestinal lesions, mucosal integrity, body weight gain (BWG) and the feed conversion ratio (FCR) as criteria. Characteristics of each species were described by combinations of polymerase chain reaction assays and classic parasitological signs. There were considerable overlaps in lengths, breadths, shape indices and volumes of the oocysts of each species. Both species caused statistically significant reductions in BWG at the lowest inocula tested (500,000 sporulated oocysts per bird of E. praecox and 250,000 of E. acervulina). E. praecox was observed for the first time to cause actual body weight loss and marked increases in FCR, as did E. acervulina. E. acervulina caused gross, white pathognomonic lesions, but E. praecox caused micro-lesions, visible in fresh tissue only with a dissecting microscope. Occasionally, lesions of the Houghton strain of E. acervulina were observed to be rounded, rather than typically “ladder-like”. Both species caused villous erosion and atrophy. No mortality occurred in birds receiving up to 1 million sporulated oocysts of either species. Using BWG and FCR as criteria, the virulence of recent field strains of E. praecox from Wales (Tynygongl) and the USA (Raleigh) was compared with English laboratory strains of E. praecox (Houghton) and E. acervulina (Houghton). E. praecox (Tynygongl) was markedly more virulent than E. acervulina (Houghton), which was more virulent than E. praecox (Raleigh) and E. praecox (Houghton).

Immunohistochemical study of the cyst of Besnoitia besnoiti.

The present study has been undertaken in order to provide information on the molecular structure of the cysts of Besnoitia besnoiti. To that end, immunohistochemical techniques have been used to investigate the expression of several enzymes and proteins implicated in the cellular membrane permeability of bradyzoites. Paraffin and frozen sections, which were obtained from subcutaneous tissue samples taken from naturally infected cattle (coming from northeast Spain), were treated with a panel of antibodies. These were specific for Na(+), K(+)-ATPase, alkaline phosphatase, calmodulin, S100 protein, heat shock proteins, hsp60, and hsp70. Positive-cysts for the said antibodies were found in 23.3% of the cows studied. Bradyzoites showed a positive immunoreaction in every positive cyst with respect to all these antibodies. In addition to the low percentage of positive animals, it is worth noting that positive and unstained cysts were observed in the same tissue section. These results suggest that bradyzoites may pass through both active and dormant metabolic phases.

Comparison of oocyst shedding and the serum immune response to Cryptosporidium parvum in cattle and pigs

Abstract A comparison was made between oocyst shedding and the presence of specific serum IgG antibodies to Cryptosporidium parvum in 108 bovines and 90 pigs. Oocysts were detected by a commercial immunofluorescence assay in feces from 26.8% of bovines and 34.4% of pigs, whereas positive titers as determined by an indirect fluorescent antibody method were found in sera from 12.9% and 48.9% of the respective animals. Infection was significantly most frequent in suckling calves (82.7%) and weaned piglets (87.5%). By contrast, the numbers of seropositives were highest in weaned calves (17.1%) and fattening pigs (76.6%). The results of coprological and serological analysis corresponded in 65.7% of bovines and 56.7% of pigs. When used to diagnose the shedding of cryptosporidial oocysts, the detection of specific IgG antibodies had a sensitivity ranging from 10.3% (cattle) to 58.1% (pigs) and a specificity of 86.1% (cattle) and 55.9% (pigs).