Motility based assays using cultured fourth stage larvae fail to provide consistent discrimination between known avermectin-resistant and -susceptible isolates of Cooperia spp.

The fecal egg count reduction test (FECRT) is the only method commonly used for diagnosing anthelmintic resistance in gastrointestinal nematodes of cattle, but this method has several drawbacks that have limited its widescale implementation. Consequently, there exists a need to develop better methods for diagnosing resistance. Assays based on larval motility are used commonly for screening potential drug candidates, and for detecting drug resistance, but previous work in our lab demonstrated that the L3 stage failed to discriminate between avermectin-resistant and susceptible isolates of Cooperia spp. We hypothesized that the L4 may be a better stage for this purpose because it is a parasitic and actively feeding life stage without a double cuticle. L3 larvae of Cooperia spp. were exsheathed and cultured to L4 by maintaining them in media at 37 °C and 20 % CO2, with media changes and observation every 48 h for nine days. Three avermectin-resistant and two avermectin-susceptible GIN isolates (diagnosed by FECRT) containing >88 % Cooperia spp., were used. Three biological replicates were performed for each parasite isolate using both eprinomectin and ivermectin. Eleven drug concentrations from 0.01um to 40um and negative controls were evaluated. Motility readings were taken using the Worminator system before addition of the drug and at 24- and 48 -hs post drug exposure. Resistance ratios for ivermectin and eprinomectin ranged from 0.35 to 2.75 and 0.54-1.03, respectively. Though significant differences (p < 0.05) in percent inhibition were found at some drug concentrations in some assays, there were no consistent significant differences in the dose-response between susceptible and resistant isolates. Inhibition was greater in about half of the assays for the susceptible isolates, and in half the assays for the resistant isolates. The lack of consistency in these data indicate that motility of L4 is not a reliable diagnostic phenotype for measuring resistance to avermectin drugs in Cooperia spp.

Sparganosis due to Spirometra sp. (cestoda; Diphyllobothriidae) in captive meerkats (Suricata suricatta).

We report three cases of sparganosis due to plerocercoids of the tapeworm Spirometra sp. in captive meerkats (Suricata suricatta) from a zoo exhibit in the southeastern United States. Two meerkats were euthanized, one due to an uncontrollable seizure and the other due to trauma, and at necropsy cysts containing cestode larvae were observed. A third meerkat had a subcutaneous nodule surgically removed, which contained similar larvae. The third animal died years later, and had numerous cestode larvae in the pleural and peritoneal cavities. The larvae were morphologically identified as plerocercoids of diphyllobothriidean cestodes. On necropsy, multiple nodules, ranging in size from 2.5 to 3.0 cm, were observed in the subcutaneous tissue and muscles. Multifocally, separating skeletal muscle fibers were longitudinal and transversal sections of cestode larva. Histologically, parasitic cysts contained large numbers of neutrophils and macrophages, admixed with proteinaceous material. Molecular and phylogenetic analyses confirmed that specimens from one of the meerkats belonged to the genus Spirometra and was closely related to Spirometra plerocercoids isolated from a snake from the United States and wild felids from South America. Meerkats likely became infected by ingesting infected second intermediate hosts, such as amphibians and reptiles that may have entered the exhibit. Management practices that minimize access of meerkats and other susceptible hosts to intermediate hosts should be implemented.

Parasite survey on a captive wolf population using classical techniques and ELISA coproantigen detection, USA.

As laws change around the United States, wildlife that were once kept as companion animals are now often confiscated by local authorities. They are then euthanized unless a home is found for them at a sanctuary. Wolf sanctuaries are, therefore, becoming increasingly important for their conservation and management. However, little data is available on best practices for the health management of captive wolves, including data on parasitic diseases. Our objective was to assess the prevalence of parasites of captive wolves combining classical coprological techniques and immunoassays based on the detection of coproantigen of selected canid parasites. Fecal samples of 39 animals were collected upon observation of individual animals defecating. All samples were processed using the Fecal Dx® tests, a suite of coproantigen ELISAs for detection of ascarid, hookworm, whipworm, and Giardia (IDEXX Laboratories Inc.). Out of the 39 samples, 38 were processed using the double-centrifugation sugar flotation (DCSF) and 34 using a modification of the Baermann technique. Twenty-eight samples (71.8%) were positive for hookworm, and none positive for the other parasites tested using coproantigen ELISA. Ancylostoma sp. (26, 68.4%), Eucoleus boehmi (13, 34.2%), and Trichuris sp. (2; 5.3%), and Sarcocystis sp. (13, 34.2%) were detected using DCSF. No metastrongyloid lungworm larvae were found. The Cohen's kappa index (0.97) showed excellent agreement between the hookworm coproantigen ELISA and the DCSF using feces preserved in ethanol for a short period of time. This study provides a baseline on the parasites of captive wolves, and shows that recent innovative diagnostics in veterinary parasitology, developed and optimized for dogs, may be used for assessing the health of wolves.

Ectopic infection by Dioctophyme renale in a dog from Georgia, USA, and a review of cases of ectopic dioctophymosis in companion animals in the Americas.

We report a case of ectopic dioctophymosis in an outdoor, eight-year-old spayed female, Coonhound-mix dog from Murrayville, Hall County, Georgia, USA. The dog presented to the clinic with an apparent puncture wound on her right, most caudal mammary gland, draining a serosanguinous discharge along with significant edema and thickening of the surrounding tissues. After initial physical examination the dog was placed into a cage awaiting diagnostic procedures. A couple of hours later, a bright red, live nematode was found in the bottom of the cage and submitted to the Parasitology Diagnostic Laboratory, Department of Infectious Diseases of the University of Georgia College of Veterinary Medicine. The specimen was morphologically identified as a female Dioctophyme renale, measuring 30 cm in length. The wound was cleaned with chlorhexidine solution. The patient was started on cefpodoxime 100 mg orally, once daily for 10 days. The dog had recent history of a mammary tumor on the left chain. After a week, an ultrasound examination confirmed integrity of the kidneys. Herein, we also provide a review on cases of ectopic dioctophymosis in companion animals in the Americas. Such cases are not uncommon, and nematodes may be found in various organs and tissues including the abdominal and thoracic cavities, scrotum, uterus, and mammary glands.

Comparison of fecal egg counting methods in four livestock species.

Gastrointestinal nematode parasites are important pathogens of all domesticated livestock species. Fecal egg counts (FEC) are routinely used for evaluating anthelmintic efficacy and for making targeted anthelmintic treatment decisions. Numerous FEC techniques exist and vary in precision and accuracy. These performance characteristics are especially important when performing fecal egg count reduction tests (FECRT). The objective of this study was to compare the accuracy and precision of three commonly used FEC methods and determine if differences existed among livestock species. In this study, we evaluated the modified-Wisconsin, 3-chamber (high-sensitivity) McMaster, and Mini-FLOTAC methods in cattle, sheep, horses, and llamas in three phases. In the first phase, we performed an egg-spiking study to assess the egg recovery rate and accuracy of the different FEC methods. In the second phase, we examined clinical samples from four different livestock species and completed multiple replicate FEC using each method. In the last phase, we assessed the cheesecloth straining step as a potential source of egg loss. In the egg-spiking study, the Mini-FLOTAC recovered 70.9% of the eggs, which was significantly higher than either the McMaster (P = 0.002) or Wisconsin (P = 0.002). In the clinical samples from ruminants, Mini-FLOTAC consistently yielded the highest EPG, revealing a significantly higher level of egg recovery (P < 0.0001). For horses and llamas, both McMaster and Mini-FLOTAC yielded significantly higher EPG than Wisconsin (P < 0.0001, P < 0.0001, P < 0.001, and P = 0.024). Mini-FLOTAC was the most accurate method and was the most precise test for both species of ruminants. The Wisconsin method was the most precise for horses and McMaster was more precise for llama samples. We compared the Wisconsin and Mini-FLOTAC methods using a modified technique where both methods were performed using either the Mini-FLOTAC sieve or cheesecloth. The differences in the estimated mean EPG on log scale between the Wisconsin and mini-FLOTAC methods when cheesecloth was used (P < 0.0001) and when cheesecloth was excluded (P < 0.0001) were significant, providing strong evidence that the straining step is an important source of error. The high accuracy and precision demonstrated in this study for the Mini-FLOTAC, suggest that this method can be recommended for routine use in all host species. The benefits of Mini-FLOTAC will be especially relevant when high accuracy is important, such as when performing FECRT.

Dracunculus infections in domestic dogs and cats in North America; an under-recognized parasite?

We reviewed 62 new cases and 18 published reports of Dracunculus infections in domestic dogs and cats to describe the epidemiology of this parasite in dogs and cats in North America. We collected host and parasite data when available, including age, sex, and breed of dog, nematode location in the host, and any clinical signs at presentation and/or description of the apparent lesion. For dogs, infections were noted in six of the AKC breed groups, but none was reported from the toy group or the miscellaneous breed class. Age of infected dogs ranged from 7 months to 19 years (median 4 years; average 5.3 years), and infection rates were similar in male and female dogs. Most nematodes were associated with the distal extremities, but worms were also found in the chest/thorax, abdomen, head, and flank. Although most infected dogs had a single worm, three dogs had two or more worms that were collected from multiple lesions. Three new cat cases, with similar lesions, presentations and seasonality, were detected in Alabama, North Carolina and Texas. Cases were reported from a wide geographic range throughout eastern North America, during every month of the year, but 72% of infections were diagnosed in the late winter to early spring (December to May). All collected worms were larvigerous females which cannot be identified to species based on morphologic characters. Thus, we attempted to amplify and sequence a portion of the cytochrome c oxidase subunit I (COI) gene for specific identification. Although 13 worms from 12 cases were available, sequences were obtained for only eight worms from seven cases. These eight worms were D. insignis, a common parasite of raccoons (Procyon lotor) and other primarily carnivorous mammals. Female worms are the most likely to be detected in dogs and cats because male worms do not emerge, parasites should be preserved in ethanol for molecular identification. Although this study used convenience sampling of available data, we found that the parasite is widespread throughout the eastern US and Canada and that Dracunculus infections in dogs are more common than is revealed in published literature. However, more research is needed to understand the epidemiology, including transmission route(s), prevalence, and distribution of this parasite.

Utilization of composite fecal samples for detection of anthelmintic resistance in gastrointestinal nematodes of cattle.

Recent reports indicate that anthelmintic resistance in gastrointestinal nematodes of cattle is becoming increasingly prevalent worldwide. Presently, the fecal egg count reduction test (FECRT) is the only means available for detection of resistance to anthelmintics in cattle herds at the farm level. However, the FECRT is labor and cost intensive, and consequently is only rarely performed on cattle farms unless for research purposes. If costs could be reduced, cattle producers might be more likely to pursue drug resistance testing on their farms. One approach to reducing the cost of the FECRT, is the use of composite fecal samples for performing fecal egg counts (FEC), rather than conducting FEC on fecal samples from 15 to 20 individual animals. In this study FECRT were performed on 14 groups of cattle using both individual and composite FEC methods To measure how well the results of composite sampling reproduce those of individual sampling, Lin's Concordance Correlation Coefficient was utilized to describe both the linear relationship between methods and the slope and y-intercept of the line relating the data sets. There was little difference between the approaches with 98% agreement in mean FEC found between methods Mean FEC based on individual counts ranged between 0 and 670.6 eggs per gram of feces, indicating that the results of this study are applicable to a wide range of FEC levels. Standard error of the mean FEC and range of FEC are reported for each group prior to and following treatment to describe the variability of the data set. There was greater than 95% agreement in drug efficacy between individual and composite sampling methods, demonstrating composite sampling is appropriate to evaluate drug efficacy. Notably, for all groups tested the efficacy calculated by composite sampling was within the 95% confidence interval for efficacy calculated using individual sampling. The use of composite samples was shown to reduce the number of FEC required by 79%. These data demonstrate that pooling fecal samples from a group of cattle and then performing repeated FEC on that composite sample yields results very similar to performing individual FEC on those same animals, while substantially reducing the cost of performing a FECRT as compared to individual fecal samples. Furthermore, we have developed suggested methods for using composite samples in a FECRT, provided a cost comparison for this methodology, and described potential issues associated with the use of composite samples that must be considered.

Prevalence of antibodies to spotted fever group Rickettsia spp. and Ehrlichia spp. in coyotes (Canis latrans) in Oklahoma and Texas, USA.

Coyotes (Canis latrans) are commonly infested with ticks, including Amblyomma americanum, the predominant vector of Ehrlichia chaffeensis and Ehrlichia ewingii; Dermacentor variabilis, an important vector of Rickettsia rickettsii; and Amblyomma maculatum, a major vector of Rickettsia parkeri, a spotted fever group (SFG) Rickettsia. To determine the degree to which coyotes are infected with or exposed to tick-borne bacterial disease agents, serum samples collected from coyotes in Oklahoma and Texas were tested for antibodies reactive to R. rickettsii, Ehrlichia canis, E. chaffeensis, E. ewingii, Borrelia burgdorferi, and Anaplasma phagocytophilum by indirect fluorescent antibody (IFA) testing or enzyme-linked immunosorbent assay (ELISA). Of the coyotes tested, 60% (46/77) and 64% (47/74) had antibodies reactive to R. rickettsii and E. chaffeensis, respectively, on IFA. Additionally, 5% (4/77) had antibodies reactive to E. canis, but not B. burgdorferi or A. phagocytophilum, on SNAP(®) 4Dx(®) ELISA; subsequent serologic analysis by plate ELISA using species-specific peptides revealed antibodies to E. ewingii, E. canis, and E. chaffeensis in 46% (23/50), 18% (9/50), and 4% (2/50) of serum samples, respectively. Taken together, these data indicate that coyotes in this region are commonly exposed to SFG Rickettsia and E. ewingii and that further consideration of coyotes as a component of the maintenance cycle for these pathogens may be warranted.

Detection of Dirofilaria immitis and Ehrlichia species in coyotes (Canis latrans), from rural Oklahoma and Texas.

There is a lack of knowledge regarding the prevalence of Dirofilaria immitis and Ehrlichia spp. in coyotes in Oklahoma and Texas. Documenting the prevalence of these vector-borne disease agents in coyotes from Oklahoma and Texas underscores the importance of wild canids as reservoir hosts that infect companion animals and humans. To learn more about the sylvatic cycle of D. immitis and Ehrlichia spp. in coyotes from Oklahoma and Texas, we tested for infection with and exposure to, respectively, these disease agents. Coyote carcasses were collected opportunistically from animal control experts and hunters in seven counties in Oklahoma and Texas from January to March, 2010. Serum samples from 77 coyotes were tested with a commercial ELISA test. Five (6.5%) coyotes had D. immitis antigens, and four (5.2%) had antibodies to Ehrlichia spp. The overall prevalence of D. immitis was low relative to studies from the eastern United States. Little is known about the prevalence of Ehrlichia spp. throughout the United States, but coyotes from rural Oklahoma in the current study had a higher exposure rate than those reported from California, and a lower rate than data from an earlier study from Oklahoma.

Detection of Trichinella murrelli in coyotes (Canis latrans) from Oklahoma and North Texas.

We determined the prevalence and mean intensity of Trichinella sp. infection in coyotes from six counties in Oklahoma and one in northern Texas. Tongues from 77 coyotes were examined using histology and artificial tissue digestion. Histological examination showed a prevalence of 3.9% (3 of 77) whereas the prevalence was 6.5% (5 of 77) based on artificial digestion of 5.0 g of muscle from coyote tongues. One sample was positive for Trichinella sp. on histology but negative by artificial digestion. Combining data from both diagnostic techniques showed that six of 77 (7.8%) coyotes were infected with Trichinella spp. The mean intensity of Trichinella sp. larvae ranged from 0.2 to 66.2 with an average of 16.0 larvae per gram (LPG) of tongue. Genotyping results demonstrated that the coyotes were infected with Trichinella murrelli. This is the first report of T. murrelli infection in coyotes in Oklahoma. T. murrelli had previously been isolated from coyotes in Texas.