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Molecular identification of a novel deltaproteobacterium as the etiologic agent of epizootic bovine abortion (foothill abortion).

Epizootic bovine abortion (EBA) is endemic in California's coastal range and the foothill regions of the Sierra Nevada, where it has been the primary diagnosed cause of abortion in beef cattle for >50 years. Investigation of these losses has defined a specific fetal syndrome characterized by late-term abortion or birth of weak or dead calves. Although the unusual clinical presentation and unique fetal pathology associated with EBA have been recognized since the 1950s, the identity of the etiologic agent is unknown. In this study, suppression-hybridization PCR was used to identify a fragment of the 16S rRNA gene of a previously undescribed bacterium in thymus tissue derived from affected fetuses. Phylogenetic analysis revealed that this pathogen was a deltaproteobacterium closely related to members of the order Myxococcales. A specific PCR was subsequently developed to detect the presence of this bacterium in DNA extracted from fetal thymuses. Using histopathology as the definitive diagnosis for EBA, this PCR demonstrated 100% specificity and 88% sensitivity. The bacterium was also detected in the argasid tick Ornithodoros coriaceus, which is the recognized vector of EBA. These data imply a close association between this novel agent and the etiology of EBA.

Abortion, Veterinary↗

Neosporosis in bovine dairy herds from the west of France: detection of Neospora caninum DNA in aborted fetuses, seroepidemiology of N. caninum in cattle and dogs.

Neospora caninum is considered one of the major causes of abortion in cattle in most parts of the world. In this study, the role of N. caninum was investigated in groups of aborted cattle and dairy herds from the west of France. Good correlation was found between parasite DNA detection in fetuses and serologic statuses of dams. In groups with documented abortion status and no antibodies to other pathogens, 17-45% of aborted animals were seropositive for N. caninum, and significant relationship between prevalence of Neospora antibodies and frequency of abortions was found. Neospora-associated abortions were observed all the year round, with a peak in summer. Higher ratios of seropositive abortions were found before the 6th month of gestation. In 12 herds studied in the field, serologic prevalence ranged 6-47%. No difference in age was found between seropositive and seronegative cows. Results indicate that N. caninum is an important and stable cause of abortion in cattle in France.

Abortion, Veterinary↗

Traceback systems used during recent epizootics in Asia.

Traceback systems in most countries of Asia are not well developed, as indicated by responses to a questionnaire by veterinary officials in thirteen countries. Marking of animals for traceback is practised only in a limited number of countries in specific areas or zones and for specific purposes only. In Malaysia, traceback has been undertaken by marking farm code tattoos on pigs. This enables the identification of the farm of origin of pigs found to be infected by Nipah virus in sero-surveillance programmes. The origin of the foot and mouth disease (FMD) virus that surfaced in the Republic of Korea in March 2000 was investigated through several epidemiological studies of suspected sources of contamination such as imported hay, yellow sand, milk collection trucks and feed delivery trucks. None of these studies gave results that indicated the origin of the FMD virus. The origin of the FMD virus that was recorded in Japan in March 2000 was also investigated in epidemiological studies; in this case, imported wheat straw was incriminated as the most likely source of infection. Comparative studies of the pathogenicities of FMD (type O) viruses isolated in Taipei China, the Republic of Korea and Japan, suggest that these viruses might have originated as vaccine strains used in a third country.

Abattoirs↗

Recombinant bovine interleukin-2 and dry cow therapy: efficacy to cure and prevent intramammary infections, safety, and effect on gestation.

Cows from eight commercial dairies were randomly assigned to intrammamary infusions of cephapirin or cephapirin plus interleukin-2 at the end of lactation. During the first phase of the trial, interleukin-2 was administered to 159 cows at a dose of 1 mg per gland by intracisternal infusion immediately after 300 mg of cephapirin were administered. One hundred sixty-one cows received infusions of a placebo (phosphate-buffered saline; PBS) immediately after cephapirin. In the second phase of the trial, 70 cows received 2 mg of interleukin-2 per gland, and 78 cows received the placebo. Cows were observed daily by the participating farmer for 72 h after infusion and also during routine feeding and care during the dry period and at calving. Potential side effects from udder infusions, particularly gross abnormalities of the udder and signs of systemic side effects were monitored. During the first phase, cure rates for intramammary infections caused by Staphylococcus aureus that were present at the end of lactation were 33.3% for quarters treated with cephapirin and 53.6% for quarters treated with cephapirin and interleukin-2. Cure rates did not differ between treatment groups for all other pathogens or during the second phase of the trial. The incidence of new intramammary infections during the dry period was not affected by intramammary infusion of interleukin-2. Eighteen of 229 (7.9%) cows treated with interleukin-2 aborted within 49 d of treatment compared with 4 of 239 (1.7%) cows treated with PBS. Eleven of the 18 (61.1%) abortions by cows treated with interleukin-2 occurred 3 to 7 d after infusion; none of the abortions by cows treated with PBS occurred until wk 7 after infusion.

Abortion, Veterinary↗

[The use of molecular fingerprinting techniques in environmental hygiene].

The analysis of epidemiological pathways for the transmission of pathogens via the environment is a fundamental task in the field of environmental hygiene. The task demands for a precise differentiation between isolates. This can be done by sophisticated techniques for differentiation which are based on phenotypic or genotypic features. The fingerprinting of DNA is a powerful tool in this context. A survey is given for different kinds of RFLP-analysis, plasmid profiling, and PCR fingerprinting as well regarding their principles, methodical variations and published applications. Notable limitations of the applications are also discussed.

Animal Diseases↗

Yersinia enterocolitica : a review of its role in food hygiene.

Since Yersinia enterocolitica, now classified as a member of the Enterobacteriaceae, was recognized as a distinct species in 1964 it has been isolated with increasing frequency from man and animals (including dogs and pigs) and from some human foods. Y. enterocolitica infections are now seen as a cause for some concern in both human and veterinary medicine. The organism is commonly found in specimens from swine slaughterhouses and has been isolated from samples of market meat, vacuum-packed beef, mussels, oysters, and ice-cream. It has also been found in nonchlorinated well water used for drinking purposes. Infections in man therefore probably have an alimentary origin. Only 23 human infections were recorded in 1966 but the number increased to over 4000 in 1974. However, reported incidence is affected by growing awareness about the role of the organism in human and animal disease and by intensive laboratory analyses. While knowledge about the geographical distribution of Y. enterocolitica is still fragmentary it is clear that infections are very frequent in some parts of the world and probably common but unrecognized in many countries. The most common symptoms of Y. enterocolitica infections in man are fever, abdominal pain, and diarrhoea. In the USA most isolations in human infections were made from blood and mesenteric lymph node samples. The pathogenic mechanism is not known. In one experiment involving a human volunteer subject a dose of 3.5 x 10(9) organisms was required to produce an infection. Only recently has some success been obtained in establishing experimental infections in mice, guinea-pigs, rats, and rabbits. Laboratory cultivation techniques for Y. enterocolitica are described together with a table of minimal tests for characterizing the organism and two biotyping schema. Little is known about methods for controlling this disease, but environmental hygiene and sanitation with regard to food and water should apply.

Animals↗

Genomic comparisons of Salmonella enterica serovar Dublin, Agona, and Typhimurium strains recently isolated from milk filters and bovine samples from Ireland, using a Salmonella microarray.

Salmonella-induced enterocolitis is the leading food-borne illness with a lethal outcome and causes millions of cases of gastroenteritis each year. We examined genomic variation among 12 environmental, veterinary, and clinical Salmonella enterica serovar Dublin, Agona, and Typhimurium strains isolated in Ireland between 2000 and 2003, as well as two clinical isolates from Canada and four archival isolates, which belonged to serovars Dublin and Agona. Using DNA-DNA hybridization to a microarray consisting of most of the predicted protein-encoding sequences of the S. enterica serovar Typhimurium LT2 genome, we identified a number of genomic regions that were absent in one or more serovars. The 34 genomic regions encoded proteins involved in sugar metabolism, transport, fimbrial and phage biogenesis, and transcriptional regulation, as well as inner and outer membrane-associated proteins. Two of the four prophages identified in strain LT2, prophages Gifsy-1 and Gifsy-2, were present in all six serovar Typhimurium strains examined. Prophage Fels-1 was absent from all 18 isolates examined, and Fels-2 was completely absent from the serovar Typhimurium isolates and the Salmonella Reference Collection B serovar Dublin strain Du2. All five Salmonella pathogenicity islands were present in all isolates. Plasmid pSLT was absent from all serovar Agona isolates, and only homologues of the spv genes were present in eight of the nine serovar Dublin strains. Only limited intraserovar diversity was found among the nine serovar Dublin, three serovar Agona, and six serovar Typhimurium isolates examined even though these isolates had extensive geographic, temporal, and source differences.

Animals↗

Evaluation of rotavirus infection and diarrhea in Iowa commercial pigs based on an epidemiologic study of a population represented by diagnostic laboratory cases.

Group A, B, and C rotaviruses were identified in 9% (96/1,048) of pig fecal specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory during 1987 and 1988. Six of the rotaviruses were group B, 5 were group C, and the remaining 89% were group A. Of the rotavirus cases with more than 1 serotype, 5 were multiple group A serotypes, 1 involved a group A and B serotype, and 1 included 2 group C serotypes. A retrospective epidemiologic evaluation of pig diarrhea in herds of origin was done using data obtained from the accession records of the rotavirus and 88 matched nonrotavirus pig diarrhea control cases. Herds from which rotavirus cases were derived experienced lower morbidity, mortality, and case fatality rates than matched control herds. The incidence of diarrhea decreased rapidly among all pigs from birth to 3 weeks of age. The peak incidence for piglet diarrhea occurred in February, and a moderate rise occurred in August-September. Definitive evidence for transmissible gastroenteritis virus was found in 12% of nonrotavirus cases but none of the rotavirus cases in which it was sought. Other pathogenic microorganisms were identified less frequently and inconsistently.

Animals↗

Validation of an internal method for the diagnosis of infections with Chlamydophila abortus and Coxiella burnetii by real-time multiplex PCR.

Chlamydophila abortus and Coxiella burnetii are one of the major pathogens implicated in abortion in cattle. Their characteristic of obligate intracellular bacteria, and of zoonotic agents, makes their culture difficult in diagnostic laboratories, and the traditional tools of diagnosis (detection of sera antibodies by ELISA, Stamp's coloration) encounter specificity, sensitivity and interpretability limits. Individual PCR have recently been developed. Nevertheless, their income/cost is a limiting factor for breeders. As the symptoms are not specific, the request for analysis often concerns the two valences. Consequently, the development and the validation of an internal multiplex PCR appears to be a suitable solution.

Abortion, Veterinary↗

Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia.

A highly pathogenic avian influenza virus, H5N1, caused disease outbreaks in poultry in China and seven other east Asian countries between late 2003 and early 2004; the same virus was fatal to humans in Thailand and Vietnam. Here we demonstrate a series of genetic reassortment events traceable to the precursor of the H5N1 viruses that caused the initial human outbreak in Hong Kong in 1997 (refs 2-4) and subsequent avian outbreaks in 2001 and 2002 (refs 5, 6). These events gave rise to a dominant H5N1 genotype (Z) in chickens and ducks that was responsible for the regional outbreak in 2003-04. Our findings indicate that domestic ducks in southern China had a central role in the generation and maintenance of this virus, and that wild birds may have contributed to the increasingly wide spread of the virus in Asia. Our results suggest that H5N1 viruses with pandemic potential have become endemic in the region and are not easily eradicable. These developments pose a threat to public and veterinary health in the region and potentially the world, and suggest that long-term control measures are required.

Animals↗

Differential detection of Bartonella species and strains in cat scratch disease diagnostics by polymerase chain reaction amplification of 16S ribosomal RNA gene.

Cat scratch disease (CSD) has been difficult to diagnose in animals because of the protracted clinical course of infection and the quiescent phases when the microbial culprit lies dormant. The causative agent in CSD appears to be multiple species and strains of Bartonella. Using polymerase chain reaction (PCR) techniques for amplification of highly variable regions of the 16S ribosomal RNA (rRNA) gene sequence, a very sensitive species- and strain-specific assay for CSD-causing Bartonella species was developed. PCR primers were designed to specifically amplify the 16S rRNA gene of Bartonella species but not of other microbial pathogens. This initial PCR was multiplexed with a universal primer set, based on conserved sequence regions in the 16S rRNA gene, that provides a 162-bp fragment in all species tested. Subsequently, 3 distinct nested PCR primer sets enabled the individual amplification and specific detection of Bartonella henselae type 1, B. henselae type II, and B. clarridgeae. Thus, this 2-step PCR procedure enabled the sensitive detection and identification of these species and the B. henselae genotype by exploiting minor sequences differences. Verification of these results were demonstrated with both sequencing and ligase chain reaction techniques. The diagnostic usefulness of this CSD test has been demonstrated by the analysis of specimens from control and infected cats. The diagnosis was confirmed and the specific B. henselae strain was correctly identified in peripheral blood specimens obtained from control and strain-specific CSD-infected cats. Such an accurate and sensitive diagnostic tool for the detection and identification of CSD causative agents should be a useful for the medical, veterinary, and scientific communities.

Animals↗

Antimicrobial-resistant Salmonella serovars isolated from imported foods.

A total of 187 Salmonella isolates representing 82 serotypes recovered from 4072 imported foods in the year 2000 by the U.S. Food and Drug Administration field laboratories were tested for their susceptibility to 17 antimicrobials of human and veterinary importance. Fifteen (8%) isolates were resistant to at least one antimicrobial, and five (2.7%) were resistant to three or more antimicrobials. Most of the isolates (n=9) exhibited resistance to tetracycline. Four isolates from catfish or tilapia from Taiwan or Thailand also demonstrated resistance to nalidixic acid. These nalidixic acid-resistant Salmonella isolates possessed a point mutation at the Ser83 or Asp87 position in DNA gryase, resulting in amino acid substitutions to phenylalanine, tyrosine, or asparagine. One Salmonella Derby isolated from frozen anchovies imported from Cambodia was resistant to six antimicrobials including ampicillin, amoxicillin/clavulanic acid, chloramphenicol, sulfamethoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Of seven isolates displaying resistance to sulfonamides, only one S. Derby and one Salmonella Agona contained class 1 integrons that were further shown to possess the aadA and pse-1 genes conferring resistance to streptomycin and ampicillin, respectively. This study indicates that antimicrobial-resistant Salmonella are present in imported foods, primarily of seafood origin, and stresses the need for continued surveillance of foodborne zoonotic bacterial pathogens from imported foods entering the United States.

Anti-Bacterial Agents↗

Stillbirths, mummies, abortions, and early embryonic death.

Stillbirths, mummies, abortions, and early embryonic death have a substantial impact on the profitability of a farm in both endemic and epidemic conditions. Fetal death is highly dependent on stage of gestation. Implantation occurs around day 14 postmating in sows, and fetal death of an entire litter at this time usually results in a regular return to service. If more than four embryos remain alive, the sow may go on to farrow normally. If fetal death occurs after implantation but before calcification (around 35 days gestation), the sow will either return to estrus at an irregular interval or will farrow a normal litter of reduced size. Although fetuses are normally resorbed prior to calcification, fetal death after that stage of development leads to mummification. Abortions are more directly related to maternal control of pregnancy than fetal failure. Stillbirths are those pigs that appear normal at birth but have lungs that do not float in water. Causes of fetal death can be divided into infectious and noninfectious categories. Infectious causes perhaps are overemphasized but are certainly important in epidemic situations. Some infectious causes of fetal death are primarily systemic maternal pathogens, whereas others may attack the fetus and/or placenta, directly such as PPV, PEV, PRV, SIRS virus, and Leptospira sp. Several other infectious agents have been associated with fetal death. Noninfectious causes of stillborns, mummies, abortions, and early embryonic death are most common in endemic situations. Most stillbirths are due to difficulty at or around parturition, primarily extended duration causing fetal anoxia. Environmental factors such as increased ambient temperature and seasonal infertility affect death rates, as do specific individual sow characteristics, nutritional factors, and toxicities. The causes of stillborns, mummies, abortions, and early embryonic death are often difficult to ascertain, but the potential rewards make investigation efforts worthwhile.

Abortion, Veterinary↗

Identification and characterization of merozoite antigens of a Theileria species highly pathogenic for small ruminants in China.

A new pathogenic Theileria species transmitted by Haemaphysalis qinghaiensis was identified in the Northwestern part of China and was shown to be highly pathogenic for small ruminants. The present article aimed at identifying merozoite antigens that might be suitable for developing diagnostic methods and designing a potential vaccine. Absence of other theilerial or babesial infections was confirmed by reverse line blot in all antigen samples used. Extensive Western blot analyses using serum from infected and noninfected animals led to the identification of four potential merozoite immunoreactive proteins at different molecular weights. Further protein characterization using peptide mass mapping by matrix-assisted laser desorption/ionization (MALDI) followed by database searching resulted in two significant hits that identified two proteins of parasite origin, one homologous to a possible MO25-family protein from Cryptosporidium parvum and the other with an HSP70 from Theileria annulata. Another protein was also identified as a parasite protein but without significant homology. Immunization of rabbits with selected proteins produced antisera that reacted specifically on Western blots with merozoite antigens of the corresponding sizes. This article represents the first identification and characterization of potential antigenic proteins of Theileria sp. (China) for veterinary purposes.

Animals↗

Low prevalence of antibodies against the zoonotic agents Brucella abortus, Leptospira spp., Streptococcus suis serotype II, hantavirus, and lymphocytic choriomeningitis virus among veterinarians and pig farmers in the southern part of The Netherlands.

Serum samples from 102 veterinarians and 191 pig farmers from the southern part of the Netherlands were investigated for antibodies against Brucella abortus, Leptospira spp, Streptococcus suis serotype II, Hantavirus (HV), and lymphocytic choriomeningitis virus (LCMV). All samples were collected in 1993 and stored until this study was performed. The prevalence of antibodies against B.abortus in veterinarians (4.5%) was significantly higher (P = 0.01) than in pig farmers (0%). None of the veterinarians (0%) and only one pig farmer (0.5%) had antibodies against Leptospira spp. Furthermore, significantly (P = 0.015) more veterinarians (6%) than pig farmers (1%) had antibody titres against muramidase-released protein (MRP),a protein of pathogenic S. suis serotype II strains. None of the veterinarians and a total of 3 (1.6%) pig farmers had antibody titres against HV. The prevalence of antibodies against LCMV tended to be higher in pig farmers (2.6%) than in veterinarians (0%) (P = 0.10). It can be concluded that the prevalence of antibodies against the investigated zoonotic agents in veterinarians and pig farmers is low.

Agriculture↗

Histological findings in corneal stromal abscesses of 11 horses: correlation with cultures and cytology.

Histopathology was compared to culture results and cytology from horses with corneal stromal abscess at the Auburn University and the Ohio State University Veterinary Teaching Hospitals. Significant bacteria were not isolated in culture or seen on histopathology in any of the horses. Although most bacteria infecting equine corneas can be isolated with blood and MacConkey's agars, failure to detect bacterial growth may not rule out infection because anaerobic or intracellular bacteria would not be isolated. The inability to visualise bacterial organisms on histological sections did not rule out their presence in the tissue, because there is often destruction of bacteria by neutrophils, macrophages and antibiotic therapy greatly reducing their numbers. Fungal keratitis was diagnosed by histopathology in 4 of 11 eyes (36%) and keratitis with no aetiological agent in 7 of 11 eyes (64%). Nine of 11 horses (82%) had a prominent neutrophilic stromal infiltrate and 2 (18%) had a predominantly pyogranulomatous reaction. Two of the 4 lesions that showed histological evidence of fungal infection were positive for identifiable fungi on culture and cytology. Fungal cultures of the other 2 cases with histological evidence of mycotic keratitis were negative or grew unidentifiable fungi which were considered pathogenic because, on histopathological sections, fungal hyphae were found deep in the corneal stroma surrounded by an inflammatory reaction. In 3 of 6 cases where fungi were recovered on culture, they were considered contaminants based on lack of evidence of organisms in histopathological sections. Histopathology and the use of special stains were important in the interpretation of culture and cytology results.

Abscess↗

Identification and characterization of an idiopathic pulmonary fibrosis-like condition in cats.

Interstitial lung diseases are a heterogeneous group of disorders with a variety of causes. In veterinary medicine, such lung diseases with a prominent fibrotic component of unknown etiology are often called idiopathic pulmonary fibrosis (IPF). In human medicine, this term is reserved for a distinct disease entity with specific histologic findings labeled as usual interstitial pneumonia (UIP). We identified 23 cats displaying histologic criteria of UIP The purpose of this retrospective study is to describe the presentation and response to therapy of these cats to better define this disease entity. All but 2 cats were middle aged to older (median 8.7 years), with no apparent sex or breed predisposition. Complaints included respiratory distress (n = 18) and cough (13). Duration of signs was less than 6 months in 17 cats. Physical-examination abnormalities included tachypnea, inspiratory or mixed inspiratory and expiratory effort, and adventitial lung sounds. No consistent hematologic or biochemical abnormalities, parasites, or positive serologic results for feline retroviruses, heartworms, or toxoplasmosis were present. Radiographic changes included dense patchy or diffuse interstitial, bronchiolar, and alveolar infiltrates. Analysis of bronchial lavage fluid revealed mild neutrophilic inflammation (n = 6) with no consistent pathogen growth. Clinical condition of 5 cats worsened after lavage. Coincident pulmonary neoplasia was identified in 6 cats. Response to therapy (corticosteroids, antibiotics, bronchodilators, and diuretics) was poor, and most cats died within days to months. Cats with histologic changes compatible with UIP had signs that mimicked many of the clinical findings of human IPF, and treatment response was similarly unrewarding.

Animals↗

Applications of competitor RNA in diagnostic reverse transcription-PCR.

Detection of RNA viruses by reverse transcription (RT)-PCR has proven to be a useful approach for the diagnosis of infections caused by many viral pathogens. However, adequate controls are required for each step of the RT-PCR protocol to ensure the accuracies of diagnostic test results. Heterologous competitor RNA can be used as a control for a number of different aspects of diagnostic RT-PCR. Competitor RNA can be applied to assessments of the efficiency of RNA recovery during extraction procedures, detection of endogenous RT-PCR inhibitors that could lead to false-negative results, and quantification of viral template in samples used for diagnosis; competitor RNA can also be used as a positive control for the RT-PCR. In the present study, heterologous competitor RNA was synthesized by a method that uses two long oligonucleotide primers containing primer binding sites for RT-PCR amplification of porcine reproductive and respiratory syndrome virus or West Nile virus. Amplification of the competitor RNA by RT-PCR resulted in a product that was easily distinguished from the amplification product of viral RNA by agarose gel electrophoresis. Assessment of a variety of RNA samples prepared from routine submissions to a veterinary diagnostic laboratory found that either partial or complete inhibition of the RT-PCR could be demonstrated for approximately 20% of the samples. When inhibition was detected, either dilution of the sample or RNA extraction by an alternative protocol proved successful in eliminating the source of inhibition.

Animals↗