Prevalence and age-related variation of Cryptosporidium species and genotypes in dairy calves

Fifteen dairy farms in seven states on the east coast of the US were each visited on two consecutive years to determinate the prevalence of Cryptosporidium species in pre-weaned (5 days to 2 months) and post-weaned calves (3-11 months), respectively. After each of 971 fecal specimens collected directly from each calf was sieved and subjected to density gradient centrifugation to remove debris and concentrate oocysts, specimens were examined by immunofluorescence microscopy, and polymerase chain reaction (PCR). For all PCR-positive specimens the 18S rRNA gene of Cryptosporidium was sequenced. Cryptosporidium was identified from all farms. Types of housing appeared to have no influence with regard to prevalence of infection. Of 971 calves, 345 were infected with Cryptosporidium (35.5%), but more pre-weaned calves (253 of 503; 50.3%) than post-weaned calves (92 of 468; 19.7%) were found to be infected. A total of 278 PCR-positive specimens characterized by gene sequencing revealed Cryptosporidium parvum, Cryptosporidium andersoni, and two unnamed Cryptosporidium genotypes Bovine B (AY120911) and deer-like genotype (AY120910). The prevalence of these Cryptosporidium species and genotypes appeared to be age related between pre- and post-weaned calves. C. parvum, the only zoonotic species/genotype, constituted 85% of the Cryptosporidium infections in pre-weaned calves but only 1% of the Cryptosporidium infections in post-weaned calves. These findings clearly demonstrate that earlier reports on the presence and prevalence of C. parvum in post-weaned cattle that were based solely on oocyst morphology must be reassessed using molecular methods to validate species and genotype. This finding also indicates that persons handling or otherwise exposed to calves under 2 months of age are at greater risk of zoonotic infection from Cryptosporidium than the risk of infection from exposure to older calves.     

Prevalence of Giardia duodenalis genotypes in pre-weaned dairy calves

To determine the prevalence of Giardia genotypes in pre-weaned dairy calves, fecal samples were collected from a minimum of 18, 1-7-week-old dairy calves per farm on two farms each in the states of Vermont, New York, Pennsylvania, Maryland, Virginia, North Carolina, and Florida. Samples cleaned of fecal debris and concentrated using CsCl density gradient centrifugation were stained and examined by immunofluorescence microscopy and also subjected to PCR and gene sequence analysis. Prevalence by PCR ranged from 9% on a farm in Pennsylvania to 93% on a farm in Vermont, with an average prevalence for 407 calves on 14 farms of 40%. Gene sequence analysis of the TPI, beta-giardin and 16S rRNA genes revealed 85% of the positive samples to be Assemblage E, while 15% were Assemblage A, although the percentages of these genotypes varied greatly from farm to farm. Some farms had no Assemblage A Giardia. Thus, while a majority of the calves were infected with a genotype that is not known to be infectious for humans, calves on 7 of 14 farms did harbor Assemblage A Giardia. Calves should be considered as a potential source of human infectious cysts in the environment.     

Quantification of Mannheimia haemolytica leukotoxin by indirect ELISA

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of extracellular leukotoxin (LKT) produced in chemostat culture of Mannheimia haemolytica in a serum-free culture medium. Leukotoxin purified with preparative SDS-PAGE was used for the production of chicken polyclonal antibodies (PAb) that served as the primary detecting antibody. Excising the LKT protein from an analytical SDS-PAGE gel proved an efficient technique for the purification of the toxin. Consequently, the 102 kDa LKT polypeptide purified in this manner served as reference toxin and the resulting calibration curve was modelled using a four parameter logistic fit to relate absorbance to LKT protein concentration. The lower detection limit corresponded to an LKT concentration of 14.5 ng ml(-1). The presence of SDS, serum albumin and the coating pH had a distinct effect on the absorbance values of the indirect ELISA.     

Age-dependent seroprevalence of antibodies against a Helicobacter pylori-like organism and Helicobacter pylori in commercially reared swine

OBJECTIVE: To determine the prevalence of antibodies against a swine-origin Helicobacter pylori-like organism (HPLO) and H pylori in conventionally reared swine. ANIMALS: 640 conventionally reared swine of various ages from 16 high-health farms in Canada, 20 sows from Ohio, and 35 gnotobiotic swine. PROCEDURES: Blood was collected from the cranial vena cava. Sera were collected and tested via ELISA for antibodies against antigen prepared from a swine-origin HPLO and human H pylori strain 26695. RESULTS: Antibodies reactive with a swine HPLO, H pylori, or both were detected in 483 of 640 swine from all 16 farms in western Canada. Seroprevalence varied with age and was low (5.6%) in suckling ( 4 weeks old to adulthood. CONCLUSIONS AND CLINICAL RELEVANCE: Findings suggested that colonization by a swine-origin HPLO, H pylori, or both and resultant seroconversion, like that of H pylori infection in humans, were common in commercial swine operations. Furthermore, data indicated that gastric infection was acquired at an early age. The relationships to gastric colonization by HPLOs and clinical manifestations of disease such as gastritis and gastroesophageal ulceration remain to be determined.     

Serological and clinical surveillance studies to validate reported foot-and-mouth disease free status in Tsirang district of Bhutan

Serological and clinical studies were conducted between March 2009 and August 2010 to validate the foot-and-mouth disease free status of Tsirang district of Bhutan as determined by the country's passive surveillance system. Randomised (first survey) and targeted (third survey) samplings, with subsequent follow-up samplings (second and fourth), were conducted on FMD-susceptible animals to detect the disease at a design prevalence of 25% and 20% at the individual animal-level and village-level, respectively. Sera from cattle, goats, pigs, and sheep were tested for the presence of non-structural protein (NSP) antibodies using two commercial (PrioCHECK(?) FMDV NS and CHEKIT(?)-FMD-3ABC-bo-ov) and one in-house NSP kit (c-ELISA, AAHL, Australia). The overall seropositivity (all species) at the animal-level was 3% (95% CI: 1.7, 4.8) and 3.5% (95% CI: 2.1, 5.4), for the randomised and targeted surveys, respectively. Except for one goat from the first survey, none of the small ruminants and pigs had NSP antibodies. The seropositives from the first and targeted surveys were distributed among 13 and 16 of 20 villages sampled, respectively. All repeat testing from the initial seropositive animals and their herd mates, for both the first and third surveys, were negative in the NSP tests 6-8 months later. Using the hypergeometric exact probability formula for two-stage analyses, the results enabled rejection of the null hypothesis and supported conclusion that the population was free from disease at the minimum expected prevalence of 20% at the 95.53% and 99.46% confidence levels, for the randomised and targeted surveys, respectively. Clinical surveillance also showed absence of disease or clinical signs suggestive of FMD. The few seropositives were likely to be false positives due to factors such as imperfect specificities of the tests and possible NSP-residues in the vaccines. The study has paved the way for initiation of zoning approaches for the progressive control of FMD in Bhutan.     

Porcine circovirus-2 and concurrent infections in the field

Porcine circovirus-2 (PCV-2) is the necessary cause of post-weaning multisystemic wasting syndrome (PMWS) in swine; however, a variety of co-factors, including other infectious agents, are thought to be necessary in the full expression of disease. Porcine parvovirus (PPV) was found in the inoculum used in the first experiments to reproduce PMWS in gnotobiotic swine. Retrospective and prospective studies in the field and laboratory have demonstrated PCV-2 can act synergistically with PPV to enhance the severity of PMWS. PCV-2 has been shown to play a role in the porcine infectious disease complex (PRDC). Other co-infecting agents with PCV-2 in the lung include, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV) and Mycoplasma hyopneumoniae. Exposure of pregnant sows to PPV, PRRSV, or encephalomyocarditis virus may interact with PCV-2 infected foetuses. The severity of hepatic lesions in PCV-2 infected pigs may be enhanced by co-infection with agents such as swine hepatitis E virus and Aujezsky's disease virus. Additional studies are required to determine the mechanistic basis for the interaction of PCV-2 with other agents in the pathogenesis of the various clinical syndromes that have been associated with PCV-2 infection.     

A comparative histochemical study of plant polyphenols in southern African grasses

Recent anatomical studies have shown that tannin‐like substances (TLS) occur in the epidermal cells of a number of southern African tropical grasses, and the presence of condensed tannins in grasses has been confirmed by chemical analyses. A number of species from four of the five subfamilies of the Poaceae were compared for their responses to a range of histochemical tests which differ in their specificity for phenolic compounds. These included: ferrous sulphate, acidified vanillin, diazotized sulphanilic acid, Fast Blue‐BB, dimethoxybenzaldehyde and nitrous acid, Safranin and Fast Green. In addition, the radial diffusion test for protein precipitation was used. Comparative histochemical tests indicated that most taxa known to contain TLS showed comparable responses to the tests used here, with variations in intensity and hue of the coloured products formed. These qualitative differences suggest the presence of a number of different compounds including oligomeric procyanidins, oligomeric prodelphinidins, monomeric and/or dimeric flavan‐3‐ols and flavan‐3,4‐diols. The presence flavan‐4‐ols has been confirmed in the andropogonoid grasses by previous workers. Histochemical tests are adequate to identify the presence of condensed tannins and their precursors in plant tissue. However, they do not provide a means to identify those compounds which precipitate protein and function as digestibility‐reducing compounds in plant‐herbivore interactions.     

The molecular identification and genetic diversity of economically important wireworm species (Coleoptera: Elateridae) in Canada

Click beetle larvae (wireworms) are significant crop pests in Europe and North America. In Canada, there are ~30 economically important species which are morphologically difficult to identify, but for which sequence data are lacking. Accurate knowledge of damage-causing species and the population genetics and phylogeography of elaterids will provide insight into their sustainable management. Here, we use interspecific variation in mitochondrial 16S rRNA as a robust method of identification, consider the intra- and interspecific genetic variation of some important Canadian wireworm pests and assess the genetic structure and isolation by distance for a re-emerging major pest species, Hypnoidus bicolor Eschscholtz. Wireworms were sampled from Canada and the USA, identified as morphospecies, and sequenced at the 16S rRNA region (294–442 bp). Within some species unusually high intraspecific genetic distances between samples suggested the possibility of cryptic wireworm species or misidentifications, though this was <1 % for most species. Phylogenetic analyses gave some indication of the likely identity of these ambiguous samples. There was a significant correlation between genetic and geographic distance and significant genetic structuring within and between H. bicolor populations, which appeared to be composed of two species comprising several haplotypes. These data provide a starting point for determining the distribution of damage-causing species throughout Canada. The inclusion of data from other nuclear and mitochondrial loci, and use of sequence data from known adult samples, would further aid identification and relationships of wireworm species.