Re-emergence of Pigeon Fever (Corynebacterium pseudotuberculosis) Infection in Texas Horses: Epidemiologic Investigation of Laboratory-Diagnosed Cases

In recent years, Texas has seen a dramatic increase in the number of clinical cases of Corynebacterium pseudotuberculosis (pigeon fever) infection in horses. Equine pigeon fever cases at Texas Veterinary Medical Diagnostic Laboratories (TVMDL) were analyzed with the objectives of investigating the spatiotemporal distribution and seasonal and annual trends of pigeon fever infection in horses in Texas between 2005 and 2011 and identifying high-risk areas and create a risk map for pigeon fever in horses in Texas. The study population consisted of horses culture-positive for C. pseudotuberculosis between January 1, 2005, and December 31, 2011 at TVMDL. The Poisson model of scan statistics was fitted to identify disease clusters. Empirical Bayesian smoothing was performed with the crude incidence estimates, followed by the geostatistical method of kriging to delineate high-risk areas. Cases increased 10-fold between 2005 and 2011. The annual cumulative incidence ranged from 9.3 to 99.5 per 100,000 horses at risk. Two seasonal peaks in the number of cases were observed in June and in December. Scan statistics identified a primary cluster in central Texas in 2011 (P < .0001 and relative risk of 9.2). Isopleth risk mapping also delineated a high-risk area in central Texas. High-risk areas were also detected in the panhandle and northern Texas. The epidemiological investigation supported anecdotal reports that pigeon fever is re-emerging in the Texas horse population. This study provides a baseline for future investigations of pigeon fever in the Texas horse population and serves as a reference for the disease distribution for veterinarians and horse owners.     

A Multiplex Polymerase Chain Reaction Assay for Direct Detection and Differentiation of β-Hemolytic Streptococci in Clinical Samples from Horses

Streptococcus equi subspecies equi, S equi subspecies zooepidemicus, and S dysgalactiae subspecies equisimilis are β-hemolytic Streptococci, often isolated from horses with respiratory or genital diseases. The aim of this study was (i) defining and validating a multiplex polymerase chain reaction (PCR) protocol for identifying these Streptococci in bacterial cultures and for detecting them directly in equine clinical specimens, and (ii) defining and validating a cheap DNA extraction protocol for clinical specimens. When respiratory and genital samples from symptomatic and asymptomatic horses were tested by bacterial culture and by multiplex PCR, all the 150 samples culture-positive for S equi, S zooepidemicus, or S equisimilis were also positive by PCR. Of 150 culture-negative samples, 143 were negative by PCR. Seven samples were positive by PCR but negative by bacteriology. The multiplex PCR protocol described in this study is proven suitable for a sensitive, specific, and rapid detection and identification of S equi, S zooepidemicus, and S equisimilis in cultured bacterial colonies, as well as in clinical specimens from symptomatic or asymptomatic horses. The inclusion of internal control primers in the PCR protocol excludes false-negative results. A cheap DNA extraction method has been also validated for swabs, tracheal aspirates, bronchoalveolar lavage, and guttural pouches lavage samples.     

Prevalence of Coxiella burnetii and Brucella spp. in tissues from subsistence harvested northern fur seals (Callorhinus ursinus) of St. Paul Island,Alaska

Background

The northern fur seal (Callorhinus ursinus) is an important cultural and nutritional resource for the Aleut community on St. Paul Island Alaska. In recent years, an increasing number of zoonotic pathogens have been identified in the population, but the public health significance of these findings is unknown. To determine the prevalence of Coxiella burnetii and Brucella spp. in northern fur seal tissues, eight tissue types from 50 subsistence-harvested fur seals were tested for bacterial DNA by real-time polymerase chain reaction.

Findings

Of the 400 samples tested, only a single splenic sample was positive for Brucella spp. and the cycle threshold (ct) value was extremely high suggesting a low concentration of DNA within the tissue. C. burnetii DNA was not detected.

Conclusions

Findings suggest that the risk of humans contracting brucellosis or Q fever from the consumption of harvested northern fur seals is low.     

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.     

Prevalence and characterization of Staphylococcus aureus and Staphylococcus pseudintermedius isolated from companion animals and environment in the veterinary teaching hospital in Zambia,Africa

The Republic of Zambia consists of only one veterinary teaching school at the University of Zambia (UNZA) where students and veterinarians are exposed to many bacterial pathogens including Staphylococcus aureus (SA) and Staphylococcus pseudintermedius (SP). The aim of this study was the characterization and antimicrobial susceptibility profile of eleven SA and 48 SP isolates from the veterinary hospitals’ in- and outpatients and the environment. No isolate was resistant to cefoxitin by disk diffusion test and the corresponding resistance gene mecA was not found. In contrast, the resistance rates of SA to penicillin (63.6%) and trimethoprim-sulfamethoxazole (36.4%) and SP to penicillin (52.1%) and tetracycline (25.0%) were the highest. A variety of sequence types (STs) without a predominant type including numerous novel types were determined, especially for SP (39.6%). The spa typing provided a clonal assignment for all SAs (100%) and 24 SPs (50%) with three and two novel types, respectively. This study has provided an overview of SA and SP in the veterinary teaching hospital at UNZA. However, for a better understanding of these species regarding pathogenesis and transmission, further studies on the prevalence and characterization of SA and SP from veterinary staff, pet owners, and farm animals in Zambia is needed.     

Phenotypic and genotypic characterization of Staphylococcus aureus isolated from raw camel milk samples

In the present study 320 milk samples collected from 160 apparently healthy camels of three different locations in Sudan were investigated for the presence of Staphylococcus aureus resulting in the isolation of this bacterial pathogen from 28 milk samples from 24 camels. Twenty-five S. aureus were identified phenotypically and by PCR mediated amplification of species-specific genes or gene segments. Investigation of the S. aureus for toxinogenic potential revealed that three S. aureus strains were positive for the enterotoxin encoding gene sec and the genes seg, sei, sem, sen and seo, representing the egc gene cluster. In addition all 25 S. aureus were positive for the superantigen-like encoding gene ssl7 (set1). Partial sequencing of gene sec of the three S. aureus strains yielded an almost complete sequence identity to the sequence of the sec variant sec2. However, all three sec2 genes of the present study showed a deletion of one base causing a frame shift and a corresponding earlier stop codon.According to the present results, the raw camel milk collected from three locations in Sudan seems to be, at least at this stage, of minor importance as vector causing staphylococcal food poisoning.     

Congenital infection with Anaplasma phagocytophilum in a calf in northern Germany

Anaplasma phagocytophilum is a Gram-negative, obligate intracellular tick-transmitted bacterium that replicates in neutrophils. It causes tick-borne fever (TBF) in sheep and cattle, but also elicits febrile disease in humans as well as in other domestic animals such as dogs, horses, and cats. Although increasingly recognized in Europe, the first laboratory-confirmed case of TBF in cattle from Germany has been published only recently. We here present the unusual case of an intrauterine transmission of A. phagocytophilum in a calf from northern Germany. To our knowledge, this is the first report of such an event occurring under field conditions in cattle.     

Enteric bacterial pathogen detection in southern sea otters (Enhydra lutris nereis) is associated with coastal urbanization and freshwater runoff

Although protected for nearly a century, California’s sea otters have been slow to recover, in part due to exposure to fecally-associated protozoal pathogens like Toxoplasma gondii and Sarcocystis neurona. However, potential impacts from exposure to fecal bacteria have not been systematically explored. Using selective media, we examined feces from live and dead sea otters from California for specific enteric bacterial pathogens (Campylobacter, Salmonella, Clostridium perfringens, C. difficile and Escherichia coli O157:H7), and pathogens endemic to the marine environment (Vibrio cholerae, V. parahaemolyticus and Plesiomonas shigelloides). We evaluated statistical associations between detection of these pathogens in otter feces and demographic or environmental risk factors for otter exposure, and found that dead otters were more likely to test positive for C. perfringens, Campylobacter and V. parahaemolyticus than were live otters. Otters from more urbanized coastlines and areas with high freshwater runoff (near outflows of rivers or streams) were more likely to test positive for one or more of these bacterial pathogens. Other risk factors for bacterial detection in otters included male gender and fecal samples collected during the rainy season when surface runoff is maximal. Similar risk factors were reported in prior studies of pathogen exposure for California otters and their invertebrate prey, suggesting that land-sea transfer and/or facilitation of pathogen survival in degraded coastal marine habitat may be impacting sea otter recovery. Because otters and humans share many of the same foods, our findings may also have implications for human health.     

The relationship between spotted fever group Rickettsiae and Ixodid ticks

Spotted fever group Rickettsiae are predominantly transmitted by ticks. Rickettsiae have developed many strategies to adapt to different environmental conditions, including those within their arthropod vectors and vertebrate hosts. The tick-Rickettsiae relationship has been a point of interest for many researchers, with most studies concentrating on the role of ticks as vectors. Unfortunately, less attention has been directed towards the relationship of Rickettsiae with tick cells, tissues, and organs. This review summarizes our current understanding of the mechanisms involved in the relationship between ticks and Rickettsiae and provides an update on the recent methodological improvements that have allowed for comprehensive studies at the molecular level.     

Fecal carriage of multi-drug resistant and extended spectrum β-lactamases producing E. coli in household pigeons,Bangladesh

Antibiotic resistance and ESBL constitute a risk to human and animal health. Birds residing close to humans could mirror the spectrum of human associated antibiotic resistance. Household pigeons were screened in Bangladesh to shed light on human associated, as well as, environmental antibiotic resistance. Escherichia coli from pigeons (n = 150) were tested against 11 antibiotics. 89% E. coli isolates were resistant to one or more critically important human antibiotics like ampicillin, cefadroxil, mecillinam, ciprofloxacin, gentamicin and tigecycline. No carbapenamase-producers were detected and the lower ESBL prevalence (5%) in pigeons. ESBL-producing E. coli isolates had bla_CTX-M-15 genes. Pigeons shared some bacterial clones and had bird associated sequence types like E. coli ST1408. Fecal carriage of bacteria resistance of critically important human antibiotics, together with examples of shared genotypes among pigeons, indicate the human-birds and bird to bird transmissions are important in the epidemiology of antibiotic resistance.     

Occurrence of Giardia and Cryptosporidium in pigs on Prince Edward Island, Canada

In a cross-sectional study of 633 pigs from 21 herds on Prince Edward Island, Canada (PEI), the prevalence of infection with Cryptosporidium and Giardia, and the genotypes and species of isolates were determined in order to establish the zoonotic potential of pigs in this region. As determined by direct immunofluorescence microscopy (DFA), 18 herds (86%) and 163 animals (26%; 95% CI: 22-29%) tested positive for Cryptosporidium, while just 3 herds (14%) and 6 animals (1%; 95% CI: 0.4-2%) tested positive for Giardia. Cryptosporidium spp. isolates were detected in 39% (95% CI: 34-44%) of weanlings (1-3 months of age) and 9% (95% CI: 6-13) of sows (>8 months of age). Molecular characterization using the 18S rDNA and HSP70 gene fragments revealed the presence of Cryptosporidium sp. pig genotype II, C. suis, C. parvum, and Cryptosporidium sp. mouse genotype. Among the 113 isolates of Cryptosporidium spp. successfully genotyped, pig genotype II (61%) predominated, with C. suis (36%) being the next most prominant isolate. C. parvum (2%; two isolates) and Cryptosporidium sp. mouse genotype (0.9%; one isolate) were only occasionally isolated. The only two Cryptosporidium-positive genotyped isolates from sows included one each of C. suis and Cryptosporidium sp. pig genotype II.All but one of the six Giardia positive isolates were detected in weanling pigs. None of the Giardia-positive isolates was amenable to PCR. This study demonstrates that Cryptosporidium spp. are highly prevalent in pigs on PEI, Canada, are found mostly in weanlings (1-3 months of age). Furthermore, the pigs are primarily infected by the host-specific genotypes and species, Cryptosporidium sp. pig genotype II and C. suis, whereas the zoonotic C. parvum is rare. Giardia duodenalis is only occasionally found in pigs. These findings suggest that domestic pigs on PEI, Canada, likely do not pose a significant health risk to humans from these parasites.     

Development of a multiplex PCR assay to detect Edwardsiella tarda,Streptococcus parauberis,and Streptococcus iniae in olive flounder (Paralichthys olivaceus)

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.     

Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host–pathogen interaction.     

Rickettsia lusitaniae sp. nov. isolated from the soft tick Ornithodoros erraticus (Acarina: Argasidae)

In this study a novel Rickettsia from the spotted fever group, isolated from Ornithodoros erraticus soft ticks collected from pigpens in the south of Portugal, is described. After initial screening revealed Rickettsia-positive ticks, isolation attempts were then performed. Successful isolates were achieved by shell-vial technique using Vero E6 cells at 28 °C. Molecular characterization of the isolate was performed based on analysis of five rickettsial genes gltA, ompA, ompB, sca1 and htr with their subsequent concatenation along with other rickettsial species resulting in a clustering of the new isolate with Rickettsia felis and Rickettsia hoogstraalii. The degree of nucleotide sequence similarity with other rickettsiae fulfills the criteria for classification of our isolate as a novel species. The name Rickettsia lusitaniae sp. nov. (= CEVDI PoTiRo) is proposed for this new species found in O. erraticus.     

Prevalence and risk factors for brucellosis in domestic yak Bos grunniens and their herders in a transhumant pastoralist system of Dolpo,Nepal

Disease caused by Brucella spp. represents the most common bacterial zoonotic infection worldwide. The distribution and public health impact of these infections in Nepal's mountain regions are poorly characterized. This cross sectional study assesses the burden of brucellosis on transhumant pastoralists and their yak in and around Shey Phoksundo National Park, Nepal. Objectives were to: (1) estimate individual animal prevalence of Brucella-seropositive yak, (2) identify herd- and individual-level risk factors associated with Brucella seropositivity in individual yak, and (3) identify herd-level risk factors associated with reported human brucellosis-like symptoms in a household. A case of household symptoms was defined as the reported occurrence within the previous year of at least one of three acute symptoms (chills, fever, night chills) and one of two chronic symptoms (joint pain, swollen joint(s)) in one or both of two individuals interviewed in a household. Two-hundred-ninety-seven yak from 61 herds were sampled, and 61 household questionnaires were completed. Estimated true prevalence was 0.22 (95% CI: 0.17; 0.28). Poisson regression with generalized estimating equations was used to account for repeated measures within a cluster (herd). Yak in herds reporting abortion occurrence within the previous year were 2.3 times more likely to be seropositive than those in herds not reporting abortion (95% CI: 1.2; 4.2, p = 0.01). For every 10 animal increase in herd number, individual animal seropositivity risk increased by 30% (95% CI: 10%; 50%, p = 0.001). Male yak were 0.7 times as likely to be seropositive as female yak (95% CI: 0.5; 0.9, p = 0.01). Three to five year old yak were 2 times more likely to be seropositive than yak <3 years old (95% CI: 1.3; 3.2, p = 0.003), and yak >5 years old were 4.9 times more likely to be seropositive than yak <3 years old (95% CI: 2.9; 8.1, p < 0.001). Risk of reported brucellosis-like symptoms at the household level was 2 (95% CI: 1.1; 3.5, p = 0.02) times greater for households with herds with >1 reactor, and was 3.6 (95% CI: 1.4; 9.2, p = 0.008) times greater for households reporting the practice of raw milk consumption. These results indicate that yak seropositivity for Brucella spp. is widespread in the region, and is associated with reported human disease. This epidemiologic understanding is essential to the identification of public health opportunities at the interface of Himalayan livestock populations and the transhumant pastoralists that depend on them.     

Efficiency of entomopathogenic fungi in the control of eggs and larvae of the horn fly Haematobia irritans (Diptera: Muscidae)

The present study assessed the pathogenic effect of isolates E9, IBCB425 and IBCB159 of the Metarhizium anisopliae fungus, JAB06, JAB07 and AM09 of Beauveria bassiana, IBCB133 and CB75 of Isaria fumosorosea (=Paecilomyces fumosoroseus) and CG189 and CG195 of Isaria farinosa (=Paecilomyces farinosus) against eggs and larvae of the horn fly Haematobia irritans. Eggs were inoculated with suspensions containing 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ of the fungal isolates and observed after 48 h to determine viability. In the larvae study, eggs were allowed to hatch into fresh bovine feces that had been treated with 10⁸, 10⁷ or 10⁶ conidia mg feces⁻¹. In both studies, 5 days after initial procedures, all formed pupae were transferred to an incubator at 27 ± 0.5 °C until the emergence of the adult flies was complete. The M. anisopliae isolates did not cause the death of H. irritans eggs, but they did promote the death of larvae that hatched from treated eggs, and therefore increased the total mortality. Isolate E9 promoted 100% mortality of treated larvae at a concentration of 10⁸ conidia ml⁻¹. For the B. bassiana isolates, no activity was observed against insect eggs or larvae. Both I. fumosorosea isolates promoted significant mortality (p < 0.05) of eggs at every concentration of conidia. Isolate CG195 of I. farinosa increased the mortality of larvae and pupae that hatched from treated eggs and promoted significant total mortality (p < 0.05) of the insect at every concentration of conidia.     

Equine Proliferative Enteropathy Caused by Lawsonia intracellularis in a Foal in Brazil

An 8-month-old foal in Brazil presented with fever, colic, diarrhea, hypoproteinemia (especially hypoalbuminemia), leukocytosis, and hyperfibrinogenemia and became lethargic and anorexic with a poor body score. A diagnosis of Lawsonia intracellularis proliferative enteropathy was confirmed by fecal polymerase chain reaction and serologic testing, and the foal was successfully treated with oxytetracycline followed by azithromycin.     

Effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 on ruminal pH and the lactic acid concentration of simulated rumen acidosis in vitro

The aim of this study was to examine the effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 (M. elsdenii H6F32) on ruminal pH and the lactic acid concentrations in simulated rumen acidosis conditions in vitro. A mixed culture of ruminal bacteria, buffer, and primarily degradable substrates was inoculated with equal numbers of M. elsdenii H6 or M. elsdenii H6F32. The pH and lactic acid concentrations in the mixed culture were determined at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 h of incubation. Acid-tolerant M. elsdenii H6F32 reduced the accumulation of lactic acid and increased the pH value. These results indicate that acid-tolerant M. elsdenii H6F32 could be a potential candidate for preventing rumen acidosis.     

DNA microarray-based detection of Coxiella burnetii,the causative agent of Q fever

Background

An easy-to-handle microarray assay based on the cost-effective ArrayTube™ platform has been designed for the rapid and unequivocal identification of Coxiella burnetii, the causative agent of Q fever. The gene targets include the chromosomally coded markers icd, omp/com1, and IS1111 as well as the plasmid coded markers cbbE and cbhE.

Results

A representative panel comprising 50 German C. burnetii isolates and 10 clinical samples was examined to validate the test. All tested isolates harboured plasmid QpH1 and were correctly identified, corresponding to 100% sensitivity. The assay’s limit of detection was 100 genome equivalents (GE) for icd, omp/com1, cbbE and cbhE and 10 GE for IS1111. Assay specificity was 100% as determined by analysing a panel of 37 non-Coxiella strains.

Conclusions

The present array is a rational assembly of established and evaluated targets for the rapid and unequivocal detection of C. burnetii. This array could be applied to the screening of vaginal swabs from small ruminants; screening of environmental samples e.g. on farms or screening of human samples.     

Molecular epidemiology of Mycobacterium avium subspecies paratuberculosis: IS900 PCR identification and IS1311 polymorphism analysis from ruminants in the Punjab region of India

Johne's disease is chronic granulomatous infectious enteritis of animals caused by Mycobacterium avium subspecies paratuberculosis. A total of 153 animals from 19 dairy farms, 2 gaushalas (unproductive-animal rehabilitation centers), 2 goat and 2 sheep farms from different districts of the Punjab region were selected on the basis of clinical signs of disease. All samples from cattle (n = 86), buffalo (n = 34), goat (n = 25) and sheep (n = 26) were subjected to Ziehl-Neelsen staining and DNA extraction by a freeze and thaw method. Ziehl-Neelsen staining detected 71% samples positive for acid-fast bacilli whereas IS900 PCR detected 55% positive for Map DNA. IS1311 PCR-REA analysis of IS900 positive samples revealed ‘Bison’ type as the most prevalent (82%) genotype of Map, infecting all domestic ruminants. ‘Cattle’ type was present in a minority of cases (15%) from cattle, buffaloes and goats. This is the first report of ‘Cattle’ type Map from buffalo and goat species in India.