Indirect immunofluorescence test using polyclonal antibodies for the detection of Taylorella equigenitalis

Contagious equine metritis is a horse disease that causes endometrial inflammation due to Taylorella equigenitalis. Since Taylorella asinigenitalis was characterized, genital swab culture has proved to be an insufficient method for distinguishing between the two Taylorella species. Here, we developed an indirect immunofluorescence (IIF) test using polyclonal antibodies. Specificity, sensitivity, and detection limit were assessed using isolated bacteria (55 T. equigenitalis strains, 46 T. asinigenitalis strains and 18 other bacterial species), experimental and genital swabs in comparison to bacterial culture and polymerase chain reaction (PCR) testing. Our results indicated that IIF using polyclonal antibodies allows T. equigenitalis to be discriminated from T. asinigenitalis. This test constitutes a rapid, sensitive and specific tool for confirming presumptive colonies of T. equigenitalis.     

Identification of Taylorella equigenitalis responsible for contagious equine metritis in equine genital swabs by direct polymerase chain reaction

A direct-PCR assay was developed for the rapid detection of Taylorella equigenitalis, a Gram-negative bacterium responsible for contagious equine metritis (CEM) in Equidae. The bacteria may be detected in equine genital swabs without need for a preliminary step of DNA extraction or bacterial isolation. Specificity was determined with 125 isolates of T. equigenitalis, 24 isolates of Taylorella asinigenitalis, five commensal bacteria of the genital tract and a facultative intracellular pathogen of foals found in large concentration in soil. Our PCR is specific and amplified a 413-bp 16S ribosomal DNA product only in all T. equigenitalis.     

Contagious Equine Metritis Within the United States: A Review of the 2008 Outbreak

Contagious equine metritis (CEM) is a reportable foreign animal disease in the United States caused by the organism Taylorella equigenitalis. Import and export regulations regarding transport of horses into the United States from countries which either have a high prevalence of CEM or those that trade freely with countries having a high prevalence of CEM have failed to prevent the 2008 outbreak, which has not yet been traced to the original horse. It is important to recognize the clinical signs of acute CEM infections (endometritis, infertility, and abortion) to prevent further outbreak of disease. With early recognition, proper testing, and quarantine measures, the outbreaks and economic losses to the United States can be kept to a minimum. However, there are certain characteristics of the disease that are difficult to diagnose and control, such as the ability of the organism to infect an animal without creating clinical signs and the difficulty in culturing it. A review of the transmission, clinical signs, diagnostic methods, treatment, and prevention of CEM as well as a brief summary of the 2008 outbreak of CEM within the United States has been discussed in this article.     

A two-step species-specific 16S rRNA PCR assay for the detection of Taylorella equigenitalis in horses

: A two-step PCR assay was developed for the molecular detection of Taylorella equigenitalis, a Gram-negative genital bacterial pathogen in horses. Two specific oligonucleotide primers (TE16SrRNABCHf [25mer] and TE16SrRNABCHr [29mer]) were designed from multiple alignments of the 16S rRNA gene loci of several closely related taxa, including T. asinigenitalis. Subsequent enhanced surveillance of 250 Thoroughbred animals failed to detect the presence of this organism directly from clinical swabs taken from the genital tract of mares and stallions. Such a molecular approach offers a sensitive and specific alternative to conventional culture techniques, and has the potential to lead to improved diagnosis and subsequent management of horses involved in breeding programmes.     

Genotyping of isolates ofTaylorella equigenitalis from thoroughbred brood mares in Japan

Profiles of the genomic DNA of 104 strains ofT. equigenitalis isolated from brood mares with contagious equine metritis in Hokkaido during the breeding seasons from 1980 to 1993, as well as those of five strains (SS28, EQ56, EQ59, EQ70 and HH139) previously isolated in Japan were examined after restriction digestion and crossed-field gel electrophoresis. These profiles were essentially identical to each other and the various isolates and strains appeared to have a common genotype, designated genotype J, with respect to two restriction enzymes,ApaI andNotI. These results suggest a common source for all these isolates obtained over the course of more than 10 years in Japan.Abbreviations CEM contagious equine metritis - CFGE crossed-field gel electrophoresis - PFGE pulsed-field gel electrophoresis     

Nucleotide Sequencing and Analysis of 16S rDNA and 16S-23S rDNA Internal Spacer Region (ISR) of Taylorella equigenitalis, as an Important Pathogen for Contagious Equine Metritis (CEM)

The primer set for 16S rDNA amplified an amplicon of about 1500 bp in length for three strains of Taylorella equigenitalis (NCTC11184^T, Kentucky188 and EQ59). Sequence differences of the 16S rDNA among the six sequences, including three reference sequences, occurred at only a few nucleotide positions and thus, an extremely high sequence similarity of the 16S rDNA was first demonstrated among the six sequences. In addition, the primer set for 16S-23S rDNA internal spacer region (ISR) amplified two amplicons about 1300 bp and 1200 bp in length for the three strains. The ISRs were estimated to be about 920 bp in length for large ISR-A and about 830 bp for small ISR-B. Sequence alignment of the ISR-A and ISR-B demonstrated about 10 base differences between NCTC11184^T and EQ59 and between Kentucky188 and EQ59. However, only minor sequence differences were demonstrated between the ISR-A and ISR-B from NCTC11184^T and Kentucky188, respectively. A typical order of the intercistronic tRNAs with the 29 nucleotide spacer of 5'-16S rDNA-tRNA^Ile-tRNA^Ala-23S rDNA-3' was demonstrated in the all ISRs. The ISRs may be useful for the discrimination amongst isolates of T. equigenitalis if sequencing is employed.     

Evaluation of the in vitro growth-inhibitory effect of epoxomicin on Babesia parasites

Epoxomicin potently and irreversibly inhibits the catalytic activity of proteasomal subunits. Treatment of proliferating cells with epoxomicin results in cell death through accumulation of ubiquinated proteins. Thus, epoxomicin has been proposed as a potential anti-cancer drug. In the present study, the inhibitory effects of epoxomicin on the in vitro growth of bovine and equine Babesia parasites were evaluated. The inhibitory effect of epoxomicin on the in vivo growth of Babesia microti was also assessed. The in vitro growth of five Babesia species that were tested was significantly inhibited (P < 0.05) by nanomolar concentrations of epoxomicin (IC₅₀ values = 21.4 ± 0.2, 4 ± 0.1, 39.5 ± 0.1, 9.7 ± 0.3, and 21.1 ± 0.1 nM for Babesia bovis, Babesia bigemina, Babesia ovata, Babesia caballi, and Babesia equi, respectively). Epoxomicin IC₅₀ values for Babesia parasites were low when compared with diminazene aceturate and tetracycline hydrochloride. Combinations of epoxomicin with diminazene aceturate synergistically potentiated its inhibitory effects in vitro on B. bovis, B. bigemina, and B. caballi. In B. microti-infected mice, epoxomicin caused significant (P < 0.05) inhibition of the growth of B. microti at the non-toxic doses of 0.05 and 0.5 mg/kg BW relative to control groups. Therefore, epoxomicin might be used for treatment of babesiosis.     

Comparison of in vivo and in vitro survival and fecundity rates of the poultry red mite, Dermanyssus gallinae

To assist in the testing of possible antigens in developing a vaccine against the poultry red mite (Dermanyssus gallinae De Geer), a rapid and reliable in vitro screening method is critical. This short paper describes how D. gallinae survival and fecundity rates in an in vivo feeding device compared to that of mites fed using an in vitro method. Results showed that survival of fed D. gallinae females and mites overall was greater in vitro, although there was no difference between male survival and fecundity between in vivo and in vitro designs. The in vitro feeding device described therefore has the potential to provide reliable results, comparable to those obtained by in vivo testing, to allow for the rapid screening of D. gallinae antigens.     

Effects of anesthetic protocols on electroejaculation variables of Iberian ibex (Capra pyrenaica)

The effects of three intramuscular anesthesia protocols - detomidine 190 μg/kg plus ketamine 2 mg/kg, detomidine 270 μg/kg plus ketamine 1.4 mg/kg, and tiletamine 3.4 mg/kg plus zolazepam 3.4 mg/kg - on penis protrusion and ejaculation variables were compared in nine captive Spanish ibex (Capra pyrenaica) subjected to electroejaculation. Body temperature, heart, and respiratory rates, as well as a number of plasma biochemical variables were also recorded prior to and during anesthesia. The detomidine plus ketamine protocols induced bradycardia and increased respiratory rate. However, the tiletamine/zolazepam protocol did not affect heart and respiratory rates. None of the three protocols caused a substantial change in rectal temperature, yet all protocols caused a significant increase in plasma glucose levels. Differences in anesthetic protocols did not affect sperm quality or quantity. However, choice of anesthetic protocol affected (P < 0.05) the degree of penis protrusion and the electrical pulse sequence required to achieve ejaculation. Results of this study support a recommendation of detomidine 270 μg/kg plus ketamine 1.4 mg/kg for anesthesia of Spanish ibex undergoing electroejaculation.     

Molecular detection of Theileria and Babesia infections in cattle

This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina.     

Prevalence and antibiotic resistance profiles of Enterococcus species in chicken at slaughter level; absence of vanA and vanB genes in E. faecalis and E. faecium

The prevalence of enterococci in neck skin samples of poultry from Ankara region in Turkey was investigated and their antibiotic resistance patterns were determined. In the study, 83 of 106 analyzed neck skin samples were positive for Enterococcus, with E. faecium as the most prevalent species (48%) followed by E. durans (23%) and E. faecalis (19%). Lower numbers were detected for E. gallinarum, E. hirae, E. mundtii and E. casseliflavus. Using the disc diffusion method, it was established that over 90% of E. faecium and E. faecalis isolates were high-level resistant against erythromycin and tetracycline. Four E. faecium isolates were additionally resistant to chloramphenicol, gentamicin and streptomycin, though they were susceptible to penicillin G. The most frequently observed multiple resistance in E. faecium (25%) was against erythromycin, tetracycline, chloramphenicol and streptomycin. Of the E. faecalis isolates, 44% were multiple resistant to erythromycin, tetracycline and streptomycin. Vancomycin resistance could not be demonstrated phenotypically and vanA or vanB genes were not detected by multiplex PCR in any of the isolates. Nevertheless, the observed resistance patterns are of concern for public health.     

Anthelmintic activity of Cocos nucifera L. on intestinal nematodes of mice

In this study, we evaluated the anthelmintic activity of the liquid extracted from the bark of the green coconut (LBGC), as well as butanol extract obtained from LBGC, on mouse intestinal nematodes. Thirty-six naturally infected mice were distributed into six groups receiving the following treatments: Group I: 1000 mg/kg of LBGC; Group II: 2000 mg/kg of LBGC; Group III: 500 mg/kg of butanol extract; Group IV: 1000 mg/kg of butanol extract; Group V: 0.56 mg/kg febendazole; and Group VI: 3% dimethylsulfoxide. The chemical composition of the LBGC and its butanol extract was determined by phytochemical tests. A dose of 1000 mg/kg of butanol extract had 90.70% efficacy in reducing the mouse worm burden (p < 0.05). Phytochemical tests revealed the presence of triterpens, saponnins and condensed tannins in the LBGC and butanol extracts. These results suggest that Cocos nucifera extracts may be useful in the control of intestinal nematodes.     

Immunological features of LPS from Ochrobactrum intermedium on sheep experimentally infected with Fasciola hepatica

The effects of the lipopolysaccharide (LPS) from Ochrobactrum intermedium in sheep with fasciolosis was reported previously, resulting in lower fecal egg counts and fluke burden. In the current study, we analyzed its immunological effects in two groups of sheep, treated (T) and controls (C). Fasciolosis induces a T helper (Th) type-2 response, characterized by IL-4 and IL-10 production; however, at the beginning of the infection, the IFN-γ production predominates (Th type-1 response). Although we did not find differences in IL-4 production or in the expression level of this gene in the hepatic lymph nodes, the expression level of IL-10 was higher (P < 0.05) in the T group at 4 wpi. The IFN-γ production was higher (P < 0.01) at 12 wpi as well as its level of expression at 4 wpi (P < 0.05) in the T group. We found a higher expression level of TGF-β at 4 wpi in the T group (P < 0.05), associated with the previous report of thicker fibrous tracks in a treated group. Immunoglobulin G1, related with a Th type-2 response, was higher (P < 0.01) in the T group at 4 and 12 wpi. In conclusion, the effects of LPS from O. intermedium could have resulted from a predominant Th type-2 immune response.     

Horizontal transmission dynamics of White spot syndrome virus by cohabitation trials in juvenile Penaeus monodon and P. vannamei

White spot syndrome virus (WSSV), a rod-shaped double-stranded DNA virus, is an infectious agent causing fatal disease in shrimp farming around the globe. Within shrimp populations WSSV is transmitted very fast, however, the modes and dynamics of transmission of this virus are not well understood. In the current study the dynamics of disease transmission of WSSV were investigated in small, closed populations of Penaeus monodon and Penaeus vannamei. Pair cohabitation experiments using PCR as a readout for virus infection were used to estimate transmission parameters for WSSV in these two species. The mortality rate of contact-infected shrimp in P. monodon was higher than the rate in P. vannamei. The transmission rate parameters for WSSV were not different between the two species. The relative contribution of direct and indirect transmission rates of WSSV differed between the two species. For P. vannamei the direct contact transmission rate of WSSV was significantly lower than the indirect environmental transmission rate, but for P. monodon, the opposite was found. The reproduction ratio R₀ for WSSV for these two species of shrimp was estimated to be above one: 2.07 (95%CI 1.53, 2.79) for P. monodon and 1.51 (95%CI 1.12, 2.03) for P. vannamei. The difference in R₀ between the two species is due to a lower host mortality and hence a longer infectious period of WSSV in P. monodon.     

The molecular epidemiology of Cryptosporidium and Giardia infections in coyotes from Alberta,Canada, and observations on some cohabiting parasites

Coyotes from southern Alberta and Saskatchewan, Canada, were examined for the presence of Giardia and Cryptosporidium and cohabiting helminths. Toxascaris was present in over 90% of the 70 animals examined, and Taenia sp. in 6.5–25% of the two groups of animals studied. Giardia (12.5–21.7%) and Cryptosporidium (0–17.4%) were also common and molecular characterisation revealed both zoonotic and host-adapted genotypes of Giardia, whereas the Cryptosporidium proved to be a variant of the canine species C. canis. The seasonal variation observed in the occurrence of Cryptosporidium may be related to stress-induced shedding of the parasite.     

Gastrointestinal parasites of cavy (Cavia aperea aperea) in southern Brazil

The aim of this study was to evaluate the gastrointestinal parasitism in Cavia aperea aperea (cavy), captured in the central region of Rio Grande do Sul State. Fecal samples from five free-living cavies were collected for research of parasites. Samples were analyzed by the centrifugal-flotation method with zinc sulfate and parasites were identified microscopically based on (oo)cyst and egg size and morphology. Cysts of Giardia sp. and (oo)cysts of Cryptosporidium sp. and Cystoisospora sp. were observed in one or more cavies. Eggs of Paraspidodera uncinata were observed in three of the five rodents. All infected animals showed mild infection by parasite. This is the first report of Giardia sp., Cryptosporidium sp. and Cystoisospora sp. in Cavia a. aperea.     

Screening of microfilaricidal effects of plant extracts against Dirofilaria immitis

Canine dirofilariasis is a common tropical parasitic disease of companion animals, caused by infestation of Dirofilaria immitis filarids within the pulmonary arteries and extending into the right heart. Increased reports of adverse reactions elicited by current microfilaricidal agents against D. immitis such as neurological disorders, circulatory collapse and potential resistance against these agents, warrant the search for new agents in forms of plant extracts. The use of plant extracts in therapeutic medicine is commonly met with scepticism by the veterinary community, thus the lack of focus on its medical potential. This study evaluated the presence of microfilaricidal activities of the aqueous extracts of Zingiber officinale, Andrographis paniculata and Tinospora crispa Miers on D. immitisin vitro at different concentrations; 10 mg/ml, 1 mg/ml, 100 μg/ml, 10 μg/ml and 1 μg/ml within 24 h, by evaluation of relative microfilarial motility as a measure of microfilaricidal activity. All extracts showed microfilaricidal activity with Z. officinale exhibiting the strongest activity overall, followed by A. paniculata and T. crispa Miers. It is speculated that the microfilaricidal mechanism exhibited by these extracts is via spastic paralysis based upon direct observation of the microfilarial motility.