Comparative susceptibility of planktonic and 3-day-old Salmonella Typhimurium biofilms to disinfectants.

AIMS: To compare the susceptibility of a 3-day-old biofilm and planktonic Salmonella to disinfectants at different exposure times. We hypothesize that Salmonella biofilms are more resilient to disinfectants compared to planktonic Salmonella. METHODS AND RESULTS: The susceptibility of planktonic cells to disinfectants was tested by a modified version of the Council of Europe suspension test EN 1276. Salmonella biofilms were formed using the Calgary Biofilm Device. Results show that 3-day-old Salmonella biofilms are less susceptible to the disinfectants benzalkonium chloride, chlorhexidine gluconate, citric acid, quaternary ammonium compounds, sodium hypochlorite (SH) and ethanol, compared to planktonic Salmonella. Surprisingly, the results also demonstrate that low concentrations of SH were more effective against a 3-day-old biofilm compared to high concentrations of SH. CONCLUSIONS: While all the disinfectants evaluated were able to reduce biofilm-associated cells at concentrations and contact times sufficient to eliminate planktonic cells, there were still sufficient viable cells remaining in the biofilm to cause further contamination and potential infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Protocols for the use of chemical disinfectants need to include biofilm susceptibility testing. There is a requirement for an effective and standardized tool for determining the susceptibility of biofilms to disinfectants.

Relationship between Toxoplasma gondii seroprevalence and lamb marking in South Australian sheep flocks.

OBJECTIVE: Toxoplasmosis in sheep has negative impacts on reproductive performance. This study aimed to estimate the prevalence in Toxoplasma gondii infection in the South Australian sheep population, and assess any association between within-flock prevalence and reproductive efficiency (measured by lamb marking percentage), climatic region and rainfall. METHODS: A total of 875 individual mixed-age breeding ewes from 29 South Australian properties were blood sampled with an average of 30.2 ewes per property (min 28, max 32). Sera were tested for T. gondii-specific IgG antibody using a commercial Modified Agglutination Test kit. RESULTS: Overall, 209 of 875 (23.9%; 95% confidence interval [CI] 16.3% to 31.4%) of individual ewes tested seropositive for T. gondii-specific IgG antibodies, with a flock level seroprevalence of 28/29 (96.6%, 95% CI 96.6% to 100%). On individual farms, the seroprevalence ranged from 0% to 93.3%. Analysis showed that Kangaroo Island properties had significantly higher mean seroprevalence than any mainland climatic regions, and that the mainland regions did not significantly differ from each other. Linear regression revealed a significant association between seroprevalence and lamb marking percentage, with a slope of -5.4% lamb marking per +10% seroprevalence.

Vertical transmission of Toxoplasma gondii in Australian marsupials.

To date, little is known about the dynamics of vertical transmission of Toxoplasma gondii in Australian marsupials. Studies in mice demonstrate that vertical transmission of T. gondii is common and that chronically infected mice can transmit T. gondii to successive generations. In this study, PCR and immunohistochemistry were used to detect T. gondii in chronically infected marsupial dams and their offspring. T. gondii was detected in the unfurred pouch young of 2 out of 10 chronically infected western grey kangaroos (Macropus fuliginosus) and in the unfurred pouch young of a brush-tailed bettong (Bettongia penicillata). Results of the study suggest that vertical transmission of T. gondii can occur in chronically infected Australian marsupials.

Is canine heartworm (Dirofilaria immitis) endemic to South Australia?

BACKGROUND: Canine heartworm (Dirofilaria immitis) was reported as endemic to South Australia over 25 years ago, but the current status of canine heartworm infection in South Australia is unknown. METHODS: We surveyed South Australian veterinary practices to collect evidence about canine heartworm cases, prevention, screening and treatment practices. RESULTS: In total, 141 veterinary practices operating in South Australia (92.3% response rate) completed a 12-question telephone survey. Five confirmed cases of heartworm and one suspected false-positive case were reported from separate practices over the 2016-17 financial year. All confirmed cases had recently relocated to South Australia from interstate. Incidence of canine heartworm was estimated at 0.00% (95% confidence interval (CI): 0.00-0.0006) and prevalence at 0.002% (95%CI: 0.00-0.004) using conservative estimates of the South Australian dog population. Attitudes and protocols regarding heartworm differed significantly between metropolitan and rural veterinary practices. All metropolitan practices recommend systematic medicated prevention against heartworm, while some rural practices (20.7%) recommend prevention only when travelling interstate (P < 0.001). CONCLUSION: Although there is a minor source of heartworm from introduction of infected dogs into South Australia, there is no evidence that the infection is transmitted to other dogs within the state. Therefore, it is unlikely that heartworm is currently endemic to South Australia as reported in the past. This new evidence provides a strong case to refine current prevention practices to reflect the current negligible risk of heartworm infection within South Australia.

Seroprevalence of antibodies to Pestivirus infections in South Australian sheep flocks.

OBJECTIVE: Bovine viral diarrhoea virus (BVDV) and border disease virus (BDV) are of the genus Pestivirus. They are known to cause significant reproductive and production losses, with BVDV acknowledged as a major source of economic loss to the Australian cattle industry. Very little is currently known about the prevalence and effect of pestiviruses in the Australian sheep industry. The present study aimed to examine the seroprevalence and effect of both BVDV and BDV in South Australian sheep flocks. METHODS: In total, 875 breeding ewes on 29 properties were serologically tested by ELISA, AGID and VNT assays for the presence of Pestivirus-specific antibodies. RESULTS: Three (0.34%) individual animals returned serological results suggestive of previous BDV infection. All three positive animals were collected from one property, giving a property level seroprevalence of 3.45% and a within-flock seroprevalence of 10%. CONCLUSION: The results suggested that BDV infection is present, albeit at a very low incidence, in the South Australian sheep flock and BVDV infection appears to be absent. Consequently, pestiviruses are unlikely to impair production in South Australian sheep populations.

Evaluation of four serological techniques to determine the seroprevalence of Neospora caninum in foxes (Vulpes vulpes) and coyotes (Canis latrans) on Prince Edward Island, Canada.

The objectives of this study were (1) to evaluate the performance and agreement of serological assays (ELISA, IFAT, Neospora caninum agglutination test and immunoblot) using reference sera and field sera from foxes and coyotes and (2) to estimate the N. caninum seroprevalence in foxes and coyotes on Prince Edward Island, Canada. With fox and coyote reference sera the test performance of the ELISA, IFAT and IB was excellent (100% sensitivity and specificity). NAT showed a low sensitivity (50%). Serum was collected from 201 coyotes and 271 foxes. The seroprevalence observed in the different assays ranged from 0.5 to 14.0% in coyotes and 1.1 to 34.8% in foxes. The seroprevalence, when taking more than one test positive as cut-off value was 3.3 and 1.1% for coyotes and foxes, respectively. From the N. caninum-positive group, all coyotes were older than 3 years. Agreement among assays (measured as prevalence-adjusted bias-adjusted kappa) using the field sera ranged from 0.17 to 0.97. Best agreement was observed between ELISA and IFAT, poor agreement was observed between NAT and the other assays. Positive agreement was moderate to poor among all assays utilized in this study. Although the seroprevalence observed was low, N. caninum antibodies are present in foxes and coyotes on Prince Edward Island (PEI) and their role in the N. caninum epidemiology needs further study.

Comparison of serological methods for the diagnosis of Neospora caninum infection in cattle.

The aims of this study were to evaluate the performance and agreement of various commercial and in-house Neospora caninum antibody assays used in dairy cattle in North America, and to investigate reproducibility of two assays performed in different laboratories. From 1998 to 2005, three enzyme linked immunosorbent assays (ELISAs, a competitive ELISA-VMRD Inc., an indirect ELISA-Biovet Inc., and another indirect ELISA-Herdchek IDEXX Corp.), two indirect fluorescent antibody tests (IFATs, VMRD Inc., and in-house USDA) and one N. caninum agglutination test (NAT, in-house USDA) were utilized to test 397 randomly selected dairy cattle serum samples from 34 herds in eastern Canada for antibodies to N. caninum. The manufacturers' recommended cut-off values were used to evaluate test performance and agreement between tests. One IFAT (VMRD Inc.) performed well (sensitivity and specificity: 0.97 and 0.97, respectively) using reference sera (n = 452), therefore, results from this IFAT on the 397 samples could subsequently be used as the reference standard to calculate test characteristics for the other assays. Only 11% of the 397 sera were found to be N. caninum-positive with the IFAT. Prevalence-adjusted bias-adjusted kappa (PABAK) ranged from 0.06 to 0.99. Positive agreement was moderate to very good (P(pos) = 0.25-0.96). Negative agreement was very good for all assays (P(neg) > 0.94) except NAT (P(neg) = 0.66). Sensitivity was > or =0.89 for all assays except the NAT, which had a significantly lower sensitivity (0.66). Specificity was high (>0.94) for all assays except for one indirect ELISA (specificity = 0.52). This indirect ELISA did not perform satisfactorily when used in 1998, but an improved version of the ELISA performed as one of the best assays in 2004. Reproducibility of the competitive ELISA was excellent, but the reproducibility of the indirect ELISA that was improved was low (concordance correlation coefficient = 0.90 and 0.36, respectively). The performance characteristics observed for most assays in this study make them useful for screening antibodies to N. caninum in cattle.

Giardiasis in dairy calves: effects of fenbendazole treatment on intestinal structure and function.

Twelve Giardia duodenalis-infected Holstein dairy calves were allocated into a treatment (n=6) and placebo group (n=6) according to pre-study faecal cyst counts. Calves in the treatment group received an oral dose of 5 mg/kg fenbendazole once daily for 3 days, while placebo calves received a sterile saline solution. Calves were euthanised 7 days following the initiation of treatment and intestinal were collected and prepared for trophozoite quantitation, histology, electron microscopy, and disaccharidase assays. In all calves treated with fenbendazole, intestinal trophozoites were below detection limits, while in saline-treated calves, trophozoites were observed in all intestinal segments. Histologically, no significant difference was observed between treatment groups with respect to intestinal villus height or crypt depth. However, a significant decline in the number of intraepithelial lymphocytes (IEL) was observed in fenbendazole-treated calves when compared with placebo-treated calves in the duodenum (13.9+/-1.2 vs. 17.0+/-1.1 IEL/100 enterocytes) and jejunum (21.6+/-0.8 vs. 30.7+/-1.0 IEL/100 enterocytes). In addition, measurements from TEM micrographs demonstrated a significant increase in microvillus surface area in the jejunum of fenbendazole-treated calves compared with saline-treated calves (31.2+/-10.2 vs. 22.8+/-7.6 microm(2)). This increase in microvillus surface area was also associated with an increase in jejunal maltase activity in fenbendazole-treated calves compared with calves treated with saline. These results demonstrate that fenbendazole is an effective treatment for giardiasis in calves. fenbendazole treatment eliminated Giardia trophozoites from the small intestine of calves resulting in increased microvillus surface area and greater intestinal enzyme activity. This study also demonstrates that the pathogenesis of giardiasis in calves is similar to that observed in humans and laboratory animals, and provides further evidence that Giardia is a pathogen of cattle with potential economic importance.

Prevalence and genotypic characterisation of Giardia in dairy calves from Western Australia and Western Canada.

In this study, the prevalence of Giardia duodenalis infections was determined in Western Canadian and Western Australian dairy calves. Faecal samples were collected from Holstein calves located on a commercial dairy near Lethbridge, Alta., Canada (N=28) and from calves located on two commercial dairies located near Perth, WA, Australia (N=36). Faecal samples were examined for the presence of Giardia cysts using sucrose gradient centrifugation, followed by immunofluoresence microscopy. DNA was then extracted from Giardia isolates obtained from positive samples. A PCR based method was employed to amplify and sequence a 292bp region of the 16S-rRNA gene. Genetic sequences obtained from Giardia isolates were compared to each other and to previously sequenced isolates. Following a single faecal sample, 58% of Western Australian calves and 57% of Western Canadian calves were positive for Giardia. Geometric mean cyst counts/g of faeces were 839 for Western Australian calves and 3475 for Western Canadian calves, but these values did not differ significantly. Genetic sequences were obtained from 10 calves from Western Canada, while six sequences were obtained from Western Australian calves. Of the Western Canadian isolates, eight aligned with the proposed 'Hoofed livestock' genotype. Of the five isolates obtained from Western Australian calves, four sequences were identical to the 'Hoofed livestock' genotype. Two isolates from the Western Canadian calves and one isolate from the Western Australian calves had the identical genetic sequence to the Genotype (Assemblage) A sequence, a common human genotype. The results of this study demonstrate, for the first time, that Giardia infections occur in Western Australian calves. Also, calves from different geographical locations appear to be primarily infected with a Giardia genotype unique to hoofed livestock. However, calves can shed Giardia cysts potentially infective for humans. Thus, Giardia infections should be considered important to Australian dairy producers, and infections in calves may pose a risk to public health regardless of geographical location.

Effects of repeat fenbendazole treatment in dairy calves with giardiosis on cyst excretion, clinical signs and production.

In this 90-day study, 60 male Holstein dairy calves were experimentally infected with Giardia duodenalis. Calves were randomly blocked by weight into treatment (N=30) and placebo (N=30) groups. Beginning on study Day 0, calves in the treatment group were administered an oral dose of 5mg/kg of fenbendazole once daily for three consecutive days. Calves in the placebo group received a daily oral treatment of 5 ml of saline for 3 days. These treatments were repeated on Days 30 and 60 of the study. Fecal samples were collected from calves once per week and examined for the presence of Giardia cysts. Calves were monitored daily for clinical signs of intestinal disease and all episodes of diarrhea recorded. Calves were weighed once per week and total feed intake, on a dry matter basis, was calculated daily. Following each treatment, the number of calves shedding Giardia cysts in the fenbendazole group was reduced (p<0.001) compared to the saline group. Also, calves in the fenbendazole group had fewer cysts (p<0.05) detected in their feces following treatment compared with calves that received saline. Within 2 weeks post treatment, the number of infected animals and fecal Giardia cysts returned to placebo levels. This pattern of reinfection was consistent after every treatment period. Calves receiving fenbendazole had fewer total days with diarrhea (p<0.01) and the average number of days each calf had diarrhea was reduced (p<0.05), compared to the placebo group. There were no differences in mean body weight, average daily gain, or feed intake between the treatment or placebo groups. This study demonstrates that fenbendazole is an effective treatment for giardiosis, resulting in a clinical benefit and reducing the number of infective cysts shed by calves. However, this treatment regime had no impact on production parameters and reinfection occurred rapidly in these calves.

Vaccination of ewes for prevention of vertical transmission of Neospora caninum.

OBJECTIVE: To evaluate the immunologic response of a killed tachyzoite vaccine against Neospora caninum and its effectiveness in preventing vertical transmission of N caninum in sheep. ANIMALS: 40 Dorset ewes seronegative for N caninum. PROCEDURE: Group-A ewes (n = 20) were vaccinated on days 1 and 126 with a killed N caninum tachyzoite preparation in a commercially available adjuvant. Group-B ewes (n = 20) were sham vaccinated. Blood samples were collected from ewes every 2 weeks and a recombinant ELISA (rELISA) was used to determine serum antibody titers against N caninum. During pregnancy, ewes were challenged with live N caninum tachyzoites. Precolostral serum was collected from lambs and tested for antibodies against N caninum by use of an indirect fluorescence antibody test and the rELISA. Tissue specimens from stillborn lambs or lambs that died within 2 weeks of birth were collected and examined for N caninum antigen and DNA by use of immunohistochemistry and polymerase chain reaction assay, respectively. RESULTS: Serum antibody titers against N caninum were significantly higher in group-A ewes, compared with group B ewes, following vaccination. Serum antibodies against N caninum were detected in 100% (33/33) of group-B lambs and 75% (18/24) of group-A lambs. In tissue specimens, N caninum DNA was detected in 9 of 11 group-B lambs and 0 of 10 group-A lambs. Histologically, N caninum tachyzoites were observed in 4 group-A lambs and 3 group-B lambs. CONCLUSIONS AND CLINICAL RELEVANCE: The killed tachyzoite vaccine against N caninum stimulated a humoral immune response in sheep and provided partial protection against vertical transmission.

Efficacy of fenbendazole for treatment of giardiasis in calves.

OBJECTIVE: To determine efficacy of fenbendazole for treatment of giardiasis in calves. ANIMALS: Twenty male and 15 female Holstein calves (100 to 180 kg), naturally infected with Giardia sp. PROCEDURE: In vitro fenbendazole susceptibility and resistance development was determined for a ruminant Giardia isolate by use of an adherence assay. Calves were treated as follows: group 1, a single administration of 5 mg of fenbendazole/kg of body weight; group 2, a single administration of 10 mg of fenbendazole/kg; group 3, 5 mg of fenbendazole/kg, every 24 hours for 3 days; group 4, 10 mg of fenbendazole/kg, every 24 hours for 3 days; group 5, 20 mg of fenbendazole/kg, every 24 hours for 3 days; group 6, 0.833 mg of fenbendazole/kg, every 24 hours for 6 days; and group 7, saline solution. Fecal Giardia cysts were counted on days -3 through -1 and 1 through 7, 9, 11, 13, 21, and 28 by use of sucrose gradient concentration and staining with a fluorescent monoclonal antibody. RESULTS: The 50% adherence inhibition concentration was 0.024 +/- 0.002 microgram/ml, and resistance could not be detected after 5 weeks of continuous culture at sublethal concentration of fenbendazole (0.01 microgram/kg). Fenbendazole was 100% effective in eliminating cysts from the feces within 6 days for calves in treatment groups 2-6. Reinfection was observed in some calves within the 28-day study period. CONCLUSIONS: Fenbendazole is effective in the elimination of Giardia infections in calves, but repeat treatments may be required in reinfected animals. CLINICAL RELEVANCE: Fenbendazole is an effective and economical treatment for Giardia-associated diarrhea and growth rate reduction in calves.

Duration of naturally acquired giardiosis and cryptosporidiosis in dairy calves and their association with diarrhea.

OBJECTIVE: To determine duration of infection and association of infection with diarrhea for dairy calves with naturally acquired cryptosporidiosis and giardiosis. DESIGN: Cohort study. ANIMALS: 20 Holstein calves on a single dairy farm. PROCEDURE: Fecal samples were collected 3 times/wk for the first 45 days after birth, then weekly until calves were 120 days old and examined for Giardia duodenalis cysts and Cryptosporidium parvum oocysts. Calves were monitored for diarrhea during the first 45 days after birth; during each episode of diarrhea, fecal samples were examined for parasitic, bacterial, and viral pathogens. RESULTS: All 20 calves shed Giardia cysts and Cryptosporidium oocysts at some time during the study. Mean ages at which Giardia cysts and Cryptosporidium oocysts were first detected were 31.5 and 16.3 days, respectively. Mean number of Giardia cysts in feces remained high throughout the study, whereas Cryptosporidium occysts decreased to low or undetectable numbers 2 weeks after infection. Eighteen calves had a total of 38 episodes of diarrhea during the first 45 days after birth. Giardia duodenalis was the only pathogen identified during 6 (16%) episodes, C parvum was the only pathogen identified during 9 (24%) episodes, and G duodenalis and C parvum were identified together during 10 (26%) episodes. CONCLUSIONS: Prevalences of giardiosis and cryptosporidiosis were high in these calves, and both parasites were associated with development of diarrhea. Cryptosporidium parvum was an important pathogen when calves were < 1 month old, but G duodenalis was more important when calves were older. Calves cleared C parvum infections within 2 weeks; however, G duodenalis infections became chronic in these calves.

Giardia and Cryptosporidium in dairy calves in British Columbia.

A study was undertaken to determine the prevalence of Giardia infections in dairy calves and to compare Giardia and Cryptosporidium infections in calves of different ages. Fresh fecal samples were collected from 386 male and female Holstein calves (newborn to 24 wk) in 20 dairies located in the lower Fraser river valley area of British Columbia. Giardia intestinalis, Cryptosporidium parvum, and Cryptosporidium muris were enumerated in each sample after concentration by sucrose gradient centrifugation and immunofluorescent staining. Giardia was identified at all farm locations. The overall prevalence of Giardia in calves was 73% with a geometric mean cyst count of 1180 cysts per gram of feces (CI, 41 to 5014). Cryptosporidium parvum and C. muris were identified in 80% and 40% of the farms, respectively. The prevalence of C. parvum was 59%, and the geometric mean for oocysts was 457 oocysts per gram of feces (CI, 18 to 160). The prevalence of C. muris was only 2% and the mean oocyst counts were 54 oocysts per gram of feces. Giardiasis was not age dependent, and approximately 80% of the calves from 2 to 24 wk were infected. In contrast, C. parvum infections were predominant in calves 2 to 4 wk, while C. muris was demonstrated in calves older than 4 wk. Fourty-seven percent of calves with diarrhea had high numbers of Giardia cysts in their feces. Giardia infections are highly prevalent in dairy calves and should be considered in animals with diarrhea or failure to thrive.

World Association for the Advancement of Veterinary Parasitology (WAAVP): Guideline for the evaluation of drug efficacy against non-coccidial gastrointestinal protozoa in livestock and companion animals.

The current guideline was written to aid in the design, implementation and interpretation of studies for the assessment of drug efficacy against non-coccidial gastrointestinal protozoan parasites, with Giardia spp. as the leading example. The information provided in this guideline deals with aspects of how to conduct controlled studies using experimental infection models (dose determination and dose confirmation) and efficacy studies in commercial facilities (field effectiveness studies). Furthermore, the selection of suitable animals, housing, infection procedure, choice of diagnostic technique and data analysis are discussed. This guideline is intended to assist investigators in conducting specific studies, to provide specific information for registration authorities involved in the decision-making process, to assist in the approval and registration of new drugs and to facilitate the worldwide adoption of uniform procedures. The primary parameter for drug efficacy is the reduction in either parasite excretion or parasite counts and a minimum efficacy of 90% is required against non-coccidial gastrointestinal protozoa. A supporting efficacy parameter is a significant difference in the proportion of infected animals between treated and non-treated groups. Persistent efficacy is considered as an additional claim to therapeutic efficacy.

Seroprevalence of Toxoplasma gondii in wild kangaroos using an ELISA.

Infection with Toxoplasma gondii is a significant problem in Australian marsupials, and can lead to devastating disease and predispose animals to predation. T. gondii infection in kangaroos is also of public health significance due to the kangaroo meat trade. A moderate seroprevalence of T. gondii was observed in a study of western grey kangaroos located in the Perth metropolitan area in Western Australia. Of 219 kangaroos tested, 15.5% (95%CI: 10.7-20.3) were positive for T. gondii antibodies using an ELISA developed to detect T. gondii IgG in macropod marsupials. When compared with the commercially available MAT (modified agglutination test), the ELISA developed was in absolute agreement and yielded a kappa coefficient of 1.00. Of 18 kangaroos tested for the presence of T. gondii DNA by PCR, the 9 ELISA positive kangaroos tested PCR positive and the 9 ELISA negative kangaroos tested PCR negative indicating the ELISA protocol was both highly specific and sensitive and correlated 100% with the more labour intensive PCR assay.