Retrospective study of canine endoparasites diagnosed by fecal flotation methods analyzed across veterinary parasitology diagnostic laboratories, United States, 2018.

BACKGROUND: Companion animal endoparasites play a substantial role in both veterinary medicine and public health. Updated epidemiological studies are necessary to identify trends in occurrence and distribution of these parasites, and their associated risk factors. This study aimed to assess the occurrence of canine endoparasites  retrospectively, using fecal flotation  test data available through participating academic veterinary parasitology diagnostic laboratories across the United States of America (USA). METHODS: Canine fecal flotation records from ten veterinary diagnostic laboratories located in nine states in the USA acquired from January 1, 2018, to December 31, 2018, were included. RESULTS: A total of 4692 fecal flotation test results were obtained, with a majority comprised of client-owned dogs (3262; 69.52%), followed by research dogs (375; 8.00%), and shelter dogs (122; 2.60%). Samples from 976 (20.80%) dogs were positive for at least one parasite, and co-infections of two or more parasites were found in 3.82% (179/4692) of the samples. The five most commonly detected parasites were: Giardia sp., (8.33%; 391/4692), Ancylostomatidae (5.63%; 264/4692), Cystoisospora spp. (4.35%; 204/4692), Toxocara canis (2.49%;117/4692), and Trichuris vulpis (2.43%; 114/4692). Various other internal parasites, including gastrointestinal and respiratory nematodes, cestodes, trematodes, and protozoans were detected in less than 1% of samples. CONCLUSIONS: These data illustrate the importance of parasite prevention, routine fecal screening, and treatment of pet dogs. Additionally, pet owners should be educated about general parasite prevalence, prevention, and anthelmintic treatment regimens to reduce the risks of environmental contamination and zoonotic transmission.

Evaluating the relationship between fecal egg count, FAMACHA score, and weight in dewormed and non-dewormed Katahdin rams during a parasite challenge.

The objective of this study was to evaluate and to estimate the relationship between fecal egg counts (FECs) and FAMACHA score and the body weight of growing Katahdin rams during a parasite challenge. One of the largest factors negatively influencing reproduction and economics in the sheep industry is gastrointestinal nematode (GIN) parasites. Due to anthelmintic resistance of these parasites, animals are selected for parasite resistance using FEC and FAMACHA scores. Data were used from the Virginia Tech Southwest Agricultural Research and Extension Center Ram Test in Glade Spring, VA, from the year 2012 to 2018 in which animals were tested in 14-d intervals for 70 d. Mixed models for repeated weight measurements were made from backward stepwise selection to evaluate the relationships between weight and GIN FEC. A total of 576 animals within 23 contemporary groups derived from test year and pasture group were analyzed. Ram, contemporary group, and consignor were considered random effects, and fixed effects were birth type, test day, age, age squared, starting weight, FEC, and FAMACHA score. Pairwise contrasts were used in the statistical analysis of parameters and their interactions. Weight and age were found to have a quadratic relationship. Increased FEC was associated with weight loss at a rate of 0.00030 kg/FEC (P < 0.0001). Animals dewormed at any point during the trial weighed less than those that were not and increased with test day to a maximum difference of 4.66 kg (P < 0.001). FAMACHA score was found to be significant (P < 0.05), but a direct relationship with weight was not conclusive. Overall, rams with severe enough parasite load to require deworming had lesser weights, which could impact the profitability of sheep production and reinforced the need to select animals that had greater innate parasite resistance.

Effect of birdsfoot trefoil cultivars on exsheathment of Haemonchus contortus in fistulated sheep.

Proanthocyanidin (PAC, condensed tannin) containing forages have well-documented anti-parasitic effects against gastrointestinal nematodes (GIN) of small ruminants. Although extensive research has been conducted on the inhibition of exsheathment of the L3 stage of Haemonchus contortus by in vitro exposure to the extracts of PAC containing plants, only one study has previously attempted to replicate this process in vivo and it was found that consumption of fresh sainfoin slowed the exsheathment rate. No similar studies have explored the effect of feeding condensed tannin forages in the form of hay on in vivo exsheathment of GIN. Another PAC containing forage, birdsfoot trefoil (Lotus corniculatus, BFT), has a large area of adaptation globally and feeding BFT has been shown to reduce fecal egg counts and total worm burdens. However, its effect on the in vivo exsheathment of H. contortus in the rumen is unknown. Recent work from this laboratory showed that BFT populations differ in the ability of aqueous extracts of freeze-dried plants to reduce exsheathment of H. contortus in vitro, and that the reduced exsheathment caused by BFT populations did not directly correlate with PAC content. Therefore, the objective of this study was twofold: 1) to evaluate the ability of birdsfoot trefoil hay to impair ruminal exsheathment of H. contortus in vivo and 2) to measure the difference in exsheathment between three commercially available cultivars of birdsfoot trefoil representing a broad range of in vitro efficacy against H. contortus. Four rumen fistulated ewes were fed three cultivars of birdsfoot trefoil (cv. Bruce, Empire, and Pardee) hay or a control hay (alfalfa/grass hay) in a Latin 4 × 4 design. The effect of consumption of birdsfoot trefoil on the exsheathment of H. contortus larvae in vivo was evaluated. For each exsheathment test, two capsules with 2000 ensheathed third-stage larvae per capsule were placed in the rumen of each ewe for eight hours. Larval containment capsules were made by capping each end of a short piece of Tygon® tubing (ID 9.5 mm, OD 14.3 mm) with an 8 µm NuncTM Cell Culture Insert. Larval exsheathment and motility were examined pre and post rumen exposure. Three exsheathment tests were run per diet cycle. Consumption of BFT hay did not significantly alter larval exsheathment. These results highlight the importance of further in vivo testing on the role of condensed tannins and other plant secondary compounds on larval exsheathment in the rumen.

Gut microbial signatures associated with moxidectin treatment efficacy of Haemonchus contortus in infected goats.

Parasitic infections are associated with profound changes in the structure and function of the gut microbiome in various host-parasite systems. Here we examined the microbial composition and function in the abomasum, proximal colon and feces of Haemonchus contortus-infected goats after a partial anthelmintic drug clearance. A single-dose treatment of H. contortus-infected goats with Cydectin (moxidectin) resulted in an 83.9 % and 61.8 % reduction in fecal egg counts (EPG) and worm burden, respectively (P < 0.01), and restored abomasal pH to a normal baseline level. The treatment significantly increased the abundance of Proteobacteria, particularly that of Campylobacter, in the proximal colon. It also significantly affected several basic pathways, including bacterial secretion, butyrate metabolism, and LPS biosynthesis, and seemingly reduced the cellulolytic capacity in the colon. Several network modules displayed a strong correlation with EPG and worm burden. The Mantel test indicated a strong correlation between treatment related network topologies of the operational taxonomic units (OTU) belonging to Actinobacteria and Rikenellaceae and EPG and worm burden levels, respectively. Furthermore, microbial signatures that may better predict anthelmintic efficacy were identified. A signature or balance represented by the log ratio of the abundance of Verrucomicrobiaceae and Camplyobacteraceae had a strong correlation with EPG (r = 0.80). These novel insights into the interactions between H. contortus and gut microbiome in the caprine host and the consequence of a partial anthelmintic clearance on animal health and well-being may facilitate the design of more effective next-generation anthelmintics.

Biology, Epidemiology, and Control of Gastrointestinal Nematodes of Small Ruminants.

Strongylid gastrointestinal nematodes are an important cause of disease and economic loss in small ruminants. These parasites are important in most of the United States, with the bloodsucking parasite Haemonchus contortus being the predominant species of concern. Sheep and goats are infected while grazing, and the biology of infective larvae on pastures is important in the design of parasite management programs. Widespread resistance to anthelmintics requires strategies designed to preserve remaining drug activity; these include combination treatments with multiple classes of anthelmintics and targeted treatments.

Comparison of diagnostic techniques for detection of Giardia duodenalis in dogs and cats.

BACKGROUND: An evaluation of currently available in-clinic diagnostic tests for Giardia duodenalis infection of dogs and cats has not been performed. In addition, there is discordance among published diagnostic comparisons. The absence of a true gold standard for detecting Giardia duodenalis also complicates diagnostic evaluations. OBJECTIVES: To evaluate diagnostic tests commercially available in the United States for detecting Giardia duodenalis in dogs and cats, in comparison to a widely used reference test, the direct immunofluorescent assay (IFA), and also to compare the results of 2 methods of analysis: comparison of diagnostic tests to a reference test (IFA) and Bayesian analysis. ANIMALS: Fecal samples from a convenience sample of 388 cats and dogs located in Colorado, Oklahoma, and Virginia. METHODS: Fecal samples were tested for Giardia duodenalis by zinc sulfate centrifugal fecal flotation and 4 different commercial diagnostic immunoassays. Results were analyzed via Bayesian analysis and by comparison to the IFA as the reference test. RESULTS: Sensitivity and specificity by comparison to IFA was ≥82% and ≥90%, respectively, for all diagnostic tests in dogs and cats. When analyzed via Bayesian analysis, sensitivity and specificity were ≥83% and ≥95%, respectively. When ZnSO4 centrifugal fecal flotation results were combined with immunoassay results, there was no longer a significant difference between the sensitivities of the commercial in-clinic immunoassays. CONCLUSION AND CLINICAL RELEVANCE: The Bayesian analysis validates using IFA as the reference test. Differences in commercial in-clinic immunoassay sensitivities can be mitigated when the results are combined with ZnSO4 centrifugal fecal flotation results.

Giardia duodenalis assemblages in cats from Virginia, USA.

Giardia duodenalis is considered a species complex that is divided into 8 genetically distinct but morphologically identical assemblages (A-H). Assemblages C-H are generally host adapted, while A and B infect both people and animals and are considered potentially zoonotic. Furthermore, within assemblage A there are four subtypes (AI, AII, AIII, and AIV) of varying zoonotic potential; human isolates belong to AI and AII, while animal isolates belong to AI, AIII and AIV. Assemblages A, B, C, D, and F have all been reported from cats. The objective of this study was to determine the assemblage(s) of G. duodenalis present in cats from Virginia using multilocus genotyping and to assess if there were any differences among the assemblage(s) found in the populations of cats surveyed (free-roaming, shelter, owned) or their geographic location within Virginia. Samples that were positive for G. duodenalis cysts by microscopy using centrifugal flotation with ZnSO4 solution and/or direct immunofluorescence assay were genotyped using PCR and sequencing targeting fragments of the SSU rRNA, gdh, bg, and tpi genes. In total, 54 cyst-positive samples were analyzed by PCR and sequencing: 43 produced amplicons, and 37 samples had interpretable sequence data at one or more loci. Assemblage F was detected in 21/37 samples, AI was detected in 12/37 samples, and in 4/37 samples both assemblages F and AI were detected. The potentially zoonotic assemblage AI was detected in cats from two widely separated animal shelters and from one free-roaming cat. These genotyping data demonstrate that potentially zoonotic G. duodenalis assemblages are present in cats in Virginia.

In vitro screening of 51 birdsfoot trefoil (Lotus corniculatus L.; Fabaceae) strains for anti-parasitic effects against Haemonchus contortus.

Secondary plant compounds have shown bioactivity against multi-drug resistant Haemonchus contortus in small ruminants. This study screened 51 strains of birdsfoot trefoil (BFT, Lotus corniculatus) crude aqueous extracts (BFT-AqE) for anti-parasitic activity in vitro against egg hatching, and of those 51 strains, 13 were selected for further testing of motility of first (L1) and third stage (L3) larvae, and exsheathment of L3. Proanthocyanidin content ranged between 1.4 and 63.8 mg PAC g-1 powder across the 51 BFT strains. When tested against egg hatching, 21 of the 51 aqueous extracts had an EC50 of 1-2 mg powder mL-1, 70% of the strains were >90% efficacious at 6 mg powder mL-1 and 11 of the strains were 100% efficacious at 3 mg powder mL-1 BFT-AqE. Across the 13 strains tested against L3, efficacy ranged from 0 to 75% exsheathment inhibition, and 17 to 92% L3 motility inhibition at a concentration of 25 mg powder mL-1 BFT-AqE. There was no correlation between the PAC content of BFT powders and the anti-parasitic activity of aqueous extracts, therefore other secondary compounds may have contributed to the observed anti-parasitic effects. Further testing of BFT using bioactivity-driven fractionation and screening of BFT populations for the identified anti-parasitic compounds is needed.

Anthelmintic efficacy of cranberry vine extracts on ovine Haemonchus contortus.

The discovery that plant secondary compounds, including proanthocyanidins (PAC), suppress gastrointestinal nematode (GIN) infection has provided promise for alternative methods of GIN control in small ruminants. This investigation is the first to examine the anthelmintic potential of cranberry vine (CV) against the GIN Haemonchus contortus. The purpose of this study was to explore the anti-parasitic activity of CV in the form of a specific organic proanthocyanidin extract (CV-PAC) and an aqueous extract (CV-AqE) containing PAC and other compounds. In vitro egg hatching, first (L1) and third (L3) stage larval and adult worm motility and L3 exsheathment were evaluated after a 24-h incubation with CV products. In addition, CV treated worms were observed via scanning electron microscopy, and a preliminary investigation of the efficacy of CV powder against an experimental infection of H. contortus was conducted. The in vivo effect on an experimental infection was determined by administering 21.1 g CV powder to lambs (n = 9 per group) for three consecutive days, and collecting fecal egg count data for four weeks post-treatment. The effect of CV-PAC on egg hatching, L3 motility and exsheathment was limited. However, a substantial effect was observed on motility of post-hatch L1 (EC50 0.3 mg PAC/mL) and adults (EC50 0.2 mg PAC/mL). The CV-AqE showed more effect on egg hatching (EC50 5.3 mg/mL containing 0.6 mg PAC/mL) as well as impacting motility of L1 (EC50 1.5 mg/mL with 0.2 mg PAC/mL) and adults (EC50 3.4 mg/mL with 0.4 mg PAC/mL), but like CV-PAC, did not substantially effect L3 motility or exsheathment. Scanning electron microscopy revealed an accumulation of aggregate on the cuticle around the buccal area of adult worms incubated in CV-AqE and CV-PAC. In the preliminary in vivo study, there was a significant effect of treatment over time (p = .04), although differences in individual weeks were not significant. In summary, both extracts inhibited motility of L1 and adult worms. The higher efficacy of CV-AqE than CV-PAC at levels that contained the same concentrations of PAC tested alone, suggest that other secondary compounds in the CV-AqE contributed to the observed effects on the parasites. This first study of the in vitro and in vivo effects of CV suggest that this readily available plant product may have utility in integrated control of H. contortus and support the need for additional testing to provide further information.

Prevalence of zoonotic parasites in feral cats of Central Virginia, USA.

Felis catus, the domestic cat, is the definitive host for parasites that may result in adverse health outcomes in humans. Prevalence data of zoonotic parasites in feral cats, which are free-roaming domestic cats that are born and live in the wild, are limited. The objective of this study was to assess seroprevalence of Toxoplasma gondii antibodies and copro-prevalence of potentially zoonotic parasites in feral cats and to evaluate risk factors for seropositivity and faecal excretion of parasites. In this cross-sectional survey, 275 feral cats at Trap-Neuter-Release clinics in Central Virginia were tested for parasites via faecal flotation, direct immunofluorescence assay (faeces) and modified agglutination testing (serum). Toxoplasma gondii seroprevalence was 22.35% (95% CI: 17.47-27.86). Faecal prevalence of T. gondii-like oocysts was 1.04% (95% CI: 0.13-3.71), Toxocara cati 58.85% (95% CI: 51.54-65.89), Ancylostoma spp. 18.75% (95% CI: 13.49-25.00), Giardia duodenalis 5.73% (95% CI: 2.89-10.02) and Cryptosporidium spp. 3.33% (95% CI: 1.37-7.24). Female cats were more likely than males to excrete faecal Ancylostoma spp. eggs (OR 2.88; 95% CI 1.34-6.17). Adults were more likely than immature cats to be seropositive (OR 2.10; 95% CI: 1.11-3.97) and to excrete faecal Ancylostoma spp. eggs (OR 2.57; 95% CI: 1.10-5.99). However, immature cats were more likely than adults to excrete T. cati eggs (OR 6.79; 95% CI: 3.31-13.90) and to excrete one or more potentially zoonotic species (OR 4.67; 95% CI: 2.28-9.55) in faeces. Results of this study have implications for human and animal health and highlight the importance of collaboration between public health, medical and veterinary communities in preventive efforts.

Immunologic detection of Giardia duodenalis in a specific pathogen-free captive olive baboon ( Papio cynocephalus anubis) colony.

Several commercial Giardia immunoassays were evaluated in baboons for sensitivity and specificity as well as ease of use in a large specific pathogen-free (SPF) colony. An additional objective was to identify the assemblage(s) of Giardia duodenalis present in this baboon colony. A direct immunofluorescent antibody test (IFAT) was used as the reference test. Tests evaluated were a patient-side rapid test for dogs and cats, a human rapid test, and a well-plate ELISA designed for use with humans. Test sensitivities and specificities were compared using the McNemar paired t-test and were further evaluated for agreement using an unweighted Cohen kappa statistic. When compared to the IFAT reference, both human tests were more sensitive than the veterinary test. Based on PCR and sequencing of the G. duodenalis small-subunit ribosomal RNA and glutamate dehydrogenase loci, assemblage AI was present in this baboon colony. We found that 10 of the 110 (9%) baboons in this SPF colony were infected with a zoonotic strain of G. duodenalis.

Flotation of Toxocara canis Eggs in Commercial Bleach and Effects of Bleach Treatment Times on Larval Development in These Eggs.

Toxocara canis is a common intestinal nematode of young dogs. Puppies contaminate the environment with large numbers of eggs that can embryonate and become infective in less than a month. Embryonated eggs are infectious for humans and other paratenic hosts. Most T. canis infections in humans are asymptomatic; however, migration of T. canis larvae in the eye and in the central nervous system can result in vision loss, blindness, and even death. The eggs of T. canis are highly resistant to harsh environmental conditions and routinely used chemical disinfectants. The objective of this study was to evaluate the effects of full-strength commercial bleach (5.25% sodium hypochlorite solution) treatment on development of T. canis eggs and to report our serendipitous finding that T. canis eggs in dog feces can float in passive fecal flotation tests using bleach. We also demonstrated that T. canis eggs could be identified using the McMaster's fecal eggs counting test using 100% bleach. Toxocara canis eggs collected from the feces of naturally infected 4-8 wk old puppies were treated with full-strength bleach (5.25% sodium hypochlorite solution) for 15 min, 30 min, 60 min, and 120 min; washed free of bleach smell by centrifugation; and resuspended in 0.1 N sulfuric acid solution to undergo larval development at room temperature for 18 days after exposure to bleach. Motile larvae were observed in T. canis eggs in all groups treated for 15-120 min and eggs continuously exposed to bleach for 18 days. Our results indicate that bleach may not be an appropriate disinfectant for dog kennels, cages, or laboratory utensils and work surfaces. Toxocara canis eggs are resistant to bleach treatment and continue to pose a risk for canine and human infections. Further study is needed to find the most appropriate methods for disinfection and removal of eggs to reduce the risk of transmission of this parasite.

Development and evaluation of a protocol for control of Giardia duodenalis in a colony of group-housed dogs at a veterinary medical college.

OBJECTIVE To develop and evaluate a protocol for control of Giardia duodenalis in naturally infected group-housed dogs at a veterinary medical college. DESIGN Prospective evaluation study. ANIMALS 34 dogs. PROCEDURES All dogs were tested for evidence of G duodenalis infection. Dogs were treated with fenbendazole on study days 1 through 10. On day 5, dogs were bathed and moved into clean, disinfected kennels in a different room to allow for disinfection and drying of their assigned kennels at 26.7°C (80°F) for 24 hours on day 6. After treatment, dogs were returned to their original housing; fecal samples were collected weekly from days 8 through 41 and then every 3 weeks until day 209. Samples were fixed in formalin and examined by direct immunofluorescence assay. Additionally, 1 pretreatment sample underwent PCR assay and DNA sequencing to determine the assemblage (genotype) of the organism. Normal handling routines for the dogs and their use in teaching activities were not changed. RESULTS Initially, all dogs in the colony shed G duodenalis cysts. During and immediately after treatment (days 8 and 13), no cysts were detected in any dogs. On day 20, 1 cyst was observed in the fecal sample from 1 dog; results for all subsequent fecal analyses were negative. The G duodenalis cysts collected from the pretreatment sample had an assemblage C genotype. CONCLUSIONS AND CLINICAL RELEVANCE The integrated protocol was successful in controlling G duodenalis infection in this dog colony, despite exposure of dogs to a variety of environments and frequent handling by multiple individuals. Sequence analysis identified an assemblage typically found in dogs but not in people, indicating that zoonotic transmission would be unlikely.

Risk factors and sources of foodborne hepatitis E virus infection in the United States.

Hepatitis E virus (HEV) is an important human pathogen with pigs and other species serving as natural animal reservoirs. Ample evidence documents sporadic cases of hepatitis E acquired via consumption of undercooked meat. Chronic hepatitis E cases in immunosuppressed individuals are mostly caused by zoonotic HEV of swine origin. We report here the identification of genotype 3 HEV from non-liver commercial pork from local grocery stores in southwest Virginia, and association of HEV seropositivity to the consumption of undercooked meat in healthy young adults at a university in the United States. These results raise concerns about foodborne HEV transmission in the United States. J. Med. Virol. 88:1641-1645, 2016. © 2016 Wiley Periodicals, Inc.

St. Croix sheep produce a rapid and greater cellular immune response contributing to reduced establishment of Haemonchus contortus.

The objective of this study was to determine breed differences in immune response shortly following Haemonchus contortus infection. Peripheral and local cellular and humoral immune responses were evaluated in 24 St. Croix hair lambs and 24 Dorset×(Finn-Rambouillet) wool lambs at 0, 3, 5 and 7 days after infection with 10,000 L3 H. contortus larvae. Blood samples taken immediately before harvest revealed no differences in circulating effector cell populations, yet there were significant differences in levels of circulating neutrophils. Across all time points, hair lambs had a higher average circulating neutrophil concentration (3018 cells/µl) than wool lambs (1818 cells/µl; P<0.05). Infected hair lambs also had greater serum total-IgA compared to wool lambs (1.8 vs 0.9 mg/ml; P=0.006). Breeds did not differ in eosinophil or globule leukocyte (GL) counts in abomasal tissue, but infiltration of these cell populations increased with time. Globule leukocyte counts peaked at day 3 after infection whereas eosinophil numbers continued to increase to day 7 after infection. When averaged across all time points, abomasal neutrophil counts were higher in hair lambs (831 cells/mm(2)), than wool lambs (561 cells/mm(2); P<0.0001). Total abomasal lymph node (ALN) weight increased exponentially from 2.60 g at day 0 to 6.57 g by day 7 in hair lambs whereas ALN weight only marginally increased in wool lambs and was significantly lower than hair lambs by day 7 (P=0.0003). This result suggests a greater expansion of lymphocytes in the ALN promoting early development of antigen-specific acquired immune responses in hair lambs. Greater IgA production and infiltration of immune cells to the abomasal mucosa at an earlier stage of infection may limit establishment of adult parasites and thereby shorten the duration and severity of infection.

Confirmation of Borrelia burgdorferi sensu stricto and Anaplasma phagocytophilum in Ixodes scapularis, Southwestern Virginia.

To determine the prevalence of Borrelia burgdorferi and Anaplasma phagocytophilum in a newly established population of Ixodes scapularis in the mountainous region of southwestern Virginia, questing adult ticks were collected and the identity and infection status of each tick was confirmed by PCR and sequencing. A total of 364 adult ticks were tested from three field sites. B. burgdorferi sensu stricto was identified in a total of 32/101 (32%) ticks from site A, 49/154 (32%) ticks from site B, and 36/101 (36%) ticks from site C, for a total prevalence rate of 33% (117/356). In addition, A. phagocytophilum was detected in 3/364 (0.8%) ticks, one from site A and two from site B. The prevalence of both pathogens in ticks at these sites is similar to that reported from established endemic areas. These data document the presence of I. scapularis and the agent of Lyme disease in a newly established area of the Appalachian region, providing further evidence of range expansion of both the tick and public and veterinary health risk it creates.

A study of the level and dynamics of Eimeria populations in naturally infected, grazing beef cattle at various stages of production in the Mid-Atlantic USA.

There is little information available on the species dynamics of eimerian parasites in grazing cattle in the central Appalachian region of the United States. Therefore, the objective of this study was to describe the level of infection and species dynamics of Eimeria spp. in grazing beef cattle of various age groups over the course of a year in the central Appalachian region. Rectal fecal samples were collected from male and female calves (n=72) monthly from May through October 2005, heifers only (n=36) monthly from November 2005 to April 2006, and cows (n=72) in May, July, and September, 2005. Eimeria spp. oocysts were seen in 399 of 414 (96%) fecal samples collected from the calves from May through October. Fecal oocysts counts (FOC) in the calves were lower (P<0.05) in May than all other months and no significant differences were detected from June through September. Eimeria spp. oocysts were detected in 198 of 213 (92%) of fecal samples collected from the 36 replacement heifers monthly from November to April and monthly mean FOC did not differ during this time period. The prevalence of oocyst shedding increased to 100% in calves in September and remained near 100% in the replacement heifers during the sampling period. Eimeria spp. oocysts were also detected in 150 of 200 (75%) samples collected in May, July, and September from the cows and mean FOC did not differ significantly over the sampling period. Eimeria spp. composition was dominated by Eimeria bovis in fecal samples collected from calves, replacement heifers and cows. Mixed Eimeria spp. infections were, however, common in all groups and 13 Eimeria spp. oocysts were identified throughout the sampling period.

Experimentally induced clinical Cystoisospora canis coccidiosis in dogs with prior natural patent Cystoisospora ohioensis-like or C. canis infections.

Diarrhea caused by intestinal coccidia (Cystoisospora species) is a common problem in pet dogs and in dogs in animal shelters. Cystoisospora canis has the largest oocysts of the 4 named species of coccidia infecting dogs. The present study examined an isolate of C. canis obtained from a dog from São Paulo, SP, Brazil. Oocysts sporulated within 2 days at room temperature, and 20 sporulated oocysts were measured at 37.6 by 28.6 µm (range 35-42 by 26-31 µm). Most sporulated oocysts contained 2 sporocysts, each with 4 sporozoites, although a few (<1%) were Caryospora-like and contained 1 sporocyst with 8 sporozoites. Two experiments using a total of 11 female 6-wk-old beagles were conducted to determine the pathogenicity of oral infection with 5 × 10(4) sporulated oocysts of this isolate of C. canis. Five of the 11 dogs had natural infections with Cystoisospora ohioensis-like (n = 4) or C. canis (n = 1) species prior to the predicted patent period of 9-10 days. Ten of the dogs developed diarrhea with occasional blood, and 3 dogs were affected to the extent that clinical treatment for coccidiosis using sulfadimethoxine was recommended. Dog CRU had a natural C. canis infection and did not develop clinical disease after oral infection with C. canis oocysts. This dog had a prepatent period of 9 days and a patent period of 3 days, corresponding to experimental infection with the new isolate of C. canis. It excreted fewer C. canis oocysts than did the other dogs. The 4 dogs with natural C. ohioensis-like infection all developed clinical disease, and 1 required treatment. The prepatent period was 9-10 days, and the patent period was 10-11 days in these dogs. All 6 dogs not naturally infected with Cystoisospora developed clinical disease, and 2 required treatment. The prepatent period was 9-10 days, and the patent period was 8-12 days. The present study confirms that C. canis is a primary pathogen for young dogs. It demonstrates that prior infection with C. canis but not C. ohioensis-like coccidia confers some resistance to clinical disases and a decrease in oocyst production in dogs challenged with C. canis.

Hair-type sheep generate an accelerated and longer-lived humoral immune response to Haemonchus contortus infection.

Antibody levels produced in response to gastro-intestinal nematode (GIN) parasite infection are typically higher in GIN-resistant breeds than susceptible breeds. Consequently, GIN-resistant ewes should generate greater parasite-specific antibody in colostrum and milk, potentially providing greater passive immunity to young lambs. To test this hypothesis, we monitored immunoglobulin levels in wool and hair-type sheep infected with Haemonchus contortus for 35 days following the end of a 45-day autumn breeding season and subsequently for 6 weeks around the time of parturition. Ten, first-parity ewes of each type were infected with 12,000 H. contortus L3 larvae following the end of breeding. In response to infection, hair ewes generated greater serum IgA (P<0.05), although the pattern of IgA production was similar between the types. Following experimental infection, wool ewes were incapable of clearing the parasite infection well in advance of parturition. Prior to parturition, hair ewes had lower FEC and greater circulating H. contortus-specific IgA. However, no difference was seen in total or antigen-specific IgA production in the colostrum and milk of either breed. These data further demonstrate that hair-type sheep can rapidly reduce fecal egg output and generate greater humoral immunity as evidenced by higher levels of circulating antigen-specific antibody, but there is no evidence to suggest GIN-resistant sheep preferentially mobilize antigen-specific IgA to colostrum or milk. Thus, no clear difference exists between types of sheep in ability to deliver parasite-specific IgA to their offspring.

Anthelmintic effect of plant extracts containing condensed and hydrolyzable tannins on Caenorhabditis elegans, and their antioxidant capacity.

Although tannin-rich forages are known to increase protein uptake and to reduce gastrointestinal nematode infections in grazing ruminants, most published research involves forages with condensed tannins (CT), while published literature lacks information on the anthelmintic capacity, nutritional benefits, and antioxidant capacity of alternative forages containing hydrolyzable tannins (HT). We evaluated the anthelmintic activity and the antioxidant capacity of plant extracts containing either mostly CT, mostly HT, or both CT and HT. Extracts were prepared with 70% acetone, lyophilized, redissolved to doses ranging from 1.0mg/mL to 25mg/mL, and tested against adult Caenorhabditis elegans as a test model. The extract concentrations that killed 50% (LC(50)) or 90% (LC(90)) of the nematodes in 24h were determined and compared to the veterinary anthelmintic levamisole (8 mg/mL). Extracts were quantified for CT by the acid butanol assay, for HT (based on gallic acid and ellagic acid) by high-performance liquid chromatography (HPLC) and total phenolics, and for their antioxidant activity by the oxygen radical absorbance capacity (ORAC) assay. Extracts with mostly CT were Lespedeza cuneata, Salix X sepulcralis, and Robinia pseudoacacia. Extracts rich in HT were Acer rubrum, Rosa multiflora, and Quercus alba, while Rhus typhina had both HT and CT. The extracts with the lowest LC(50) and LC(90) concentrations, respectively, in the C. elegans assay were Q. alba (0.75 and 1.06 mg/mL), R. typhina collected in 2007 (0.65 and 2.74 mg/mL), A. rubrum (1.03 and 5.54 mg/mL), and R. multiflora (2.14 and 8.70 mg/mL). At the doses of 20 and 25mg/mL, HT-rich, or both CT- and HT-rich, extracts were significantly more lethal to adult C. elegans than extracts containing only CT. All extracts were high in antioxidant capacity, with ORAC values ranging from 1800 µmoles to 4651 µmoles of trolox equivalents/g, but ORAC did not correlate with anthelmintic activity. The total phenolics test had a positive and highly significant (r=0.826, p ≤ 0.01) correlation with total hydrolyzable tannins. Plants used in this research are naturalized to the Appalachian edaphoclimatic conditions, but occur in temperate climate areas worldwide. They represent a rich, renewable, and unexplored source of tannins and antioxidants for grazing ruminants, whereas conventional CT-rich forages, such as L. cuneata, may be hard to establish and adapt to areas with temperate climate. Due to their high in vitro anthelmintic activity, antioxidant capacity, and their adaptability to non-arable lands, Q. alba, R. typhina, A. rubrum, and R. multiflora have a high potential to improve the health of grazing animals and must have their anthelmintic effects confirmed in vivo in both sheep and goats.