An evaluation of the occurrence of pneumocystis infection through examination of pharyngeal and oral swabs using the nested PCR method

A method of collecting pharyngeal and oral swabs from humans and laboratory rats, to be examined later for Pneumocystis infection with simple and nested PCR, was developed. The swabs were obtained from 15 HIV-infected patients, including 5 suffering from PCP, and from 10 immunocompetent children aged 2 to 6 years. Furthermore, the swabs were taken from 30 healthy laboratory rats and 23 animals subjected to immunosuppressive treatment. DNA of Pneumocystis was detected in all the examined rats with nPCR method, but only in 47% of healthy animals when simple PCR was used. Nested PCR examination of swabs collected from human subjects revealed the infection with Pneumocystis in all HIV-infected patients with PCP, and in 8 out of 10 symptomless carriers of Pneumocystis; moreover, the examination was positive in 2 out of 10 immunocompetent children. It was concluded, that noninvasive method of collecting pharyngeal and oral swabs in conjunction with very sensitive method of amplification DNA by nPCR is suitable for measuring the prevalence of Pneumocystis infection in the populations of humans and laboratory animals. The developed method offers a possibility of safe diagnosis of pneumocystosis in patients whose clinical status precludes collection of BAL through bronchoscopy.     

Determination of phylogenetic relationships among methicillin-resistant Staphylococcus aureus recovered from infected humans and Companion Animals

Companion animals carry different microorganism of severely public health hazard for human; the kindness relation and contact between humans and companion animals may the route in the transmission of most zoonotic bacteria, including Methicillin-Resistant Staphylococcus aureus (MRSA). Therefore, the current study investigate the companion animals mainly dogs and cat as a reservoir for MRSA and the genetic similarity between the recovered strains of MRSA from such companion animals and their owners. One hundred swabs were collected under aseptic condition from companion animals and seventy swabs were collected from nasal and soft tissue of the infected owners in contact. All samples were examined with standard microbiological techniques, antimicrobial sensitivity, molecular typing and genetic finger printing using RAPD-PCR to determine the genetic finger printing of the recovered strains from humans and companion animals. The prevalence of the MRSA was higher in dog’s swabs than human swabs. Dog swabs showed a rate of (44.4%), cat’s revealed (27.3%), while the owner swabs could detect (42.8%). The antibiotics profiles were 69.2% and all MRSA strains were positive for mecA gene (100%), while only 25 strains (38.5%) were positive for Panton Valentine Leukocidin (PVL gene). Phylogenetic tree revealed 4 clusters with complete genetic relatedness and higher identity between the strains recovered from humans and companion animals. Our results revealed that there is great similarity between the recovered strains, indicating that pets play an important role in colonization and transmitting MRSA to humans, and vice versa.     

Viruses isolated from captive and free-ranging wild ruminants in Alberta.

Nasal secretions, leukocytes and preputial or vaginal swabs from a group of 15 captive wild ruminants, comprising six pronghorn antelope (Antilocapra americana), seven fallow deer (Dama dama) and two mule deer (Odocoileus hemionus), and from 50 free-ranging pronghorns in southern Alberta, were examined for viral agents. Captive animals were given injections of dexamethasone daily for 6 days in attempts to reactivate latent infections. Specimens were collected at 2-3 day intervals from days 0 to 18. Free-ranging pronghorns were sampled only once, at the time of capture. Fifteen viral isolates were obtained from the animals: six isolates of parainfluenza 3 (PI3) from nasal swabs from one fallow deer and one mule deer; five isolates of herpesvirus from leukocytes, vaginal, preputial and nasal swabs from three fallow deer; and four isolates of PI3 from nasal secretions of the 50 free-ranging pronghorns.     

Herpesvirus infection in woodland caribou in Alberta, Canada

Sera and genital swabs collected from 121 adult woodland caribou (Rangifer tarandus caribou) in five subpopulations in northern Alberta, Canada, between December 1997 and October 1999, were examined for evidence of infection with herpesviruses or pestiviruses. No virus was isolated from sera or swabs, and no antibodies against bovine viral diarrhea virus were detected. However, 63 (52%) of the 121 animals had neutralizing antibody titers against bovine herpesvirus 1. There was sufficient serum from 37 of the 121 caribou to allow parallel testing for antibodies against a new alphaherpesvirus isolated from an elk (Cervus elaphus nelsoni), and 20 animals had antibodies against this virus. Paired sera collected 11 mo apart from 14 caribou showed seroconversion in seven animals, indicating that an active herpesvirus infection was present. Virus neutralization data suggest that these caribou are infected with a distinct alphaherpesvirus.     

Volatile components of lemur scent secretions vary throughout the year

Olfactory signals can communicate a wide variety of information and are very important in many mammalian species. However, little is known about the olfactory communication of primates. This study used gas chromatography to examine the volatile components of the anogenital gland secretions of wild Milne-Edward's sifaka, Propithecus edwardsi (Primates, Indriidae) (n = 17), captured in Ranomafana National Park, Madagascar. We compared scent swabs of animals and examined differences between social group, age class, sex, and season. The only identified differences in the volatiles were between swabs from the different seasons; all other categories were statistically indistinguishable.     

Rapid diagnosis and management of parainfluenza I virus infection in common marmosets (Callithrix jacchus)

During 1983 a severe episode of respiratory infection occurred in a marmoset colony at these laboratories. Of 91 marmosets, 69 showed clinical signs of disease, one died and nine were so ill that euthanasia was necessary. Eight were examined post mortem and all showed consolidation of the lungs. Laboratory studies were carried out in an attempt to establish the cause of the outbreak and an interstitial pneumonia was found in seven animals which were examined histologically. Direct electron microscopy of nasal swabs and lung samples revealed the presence of a high titre of a paramyxovirus, and subsequent immunofluorescence studies established that the particular paramyxovirus involved was parainfluenza virus type I. Subsequent studies showed that surviving affected animals had seroconverted to parainfluenza I virus while animals that had not been implicated in the outbreak had not.     

Etiological investigation of multiple respiratory infections in cats

In order to evaluate the relevance of multiple infections in domestic cats with Upper Respiratory Tract Disease (URTD) one hundred animals with clinical signs were investigated for detection of Feline Herpesvirus type-1 (FHV-1), Chlamydophila felis, Feline Calicivirus (FCV) and Bordetella bronchiseptica from mucosal swabs. Forty-seven cats were positive for FCV, 42 cats for FHV-1, 26 for B. bronchiseptica and 8 for C. felis. Dual or multiple infections were found in 33 of examined animals. Our results document that FCV and FHV-1 are the major recognized cause of URTD, although infections associated with other pathogens such as B. bronchiseptica or C. felis are also common in cats.     

Cryptococcus gattii in wildlife of Vancouver Island, British Columbia, Canada

Although Cryptococcus gattii has emerged as an important pathogen of humans and domestic animals on Vancouver Island, Canada since 1999; its distribution in regional wildlife species is largely unknown. Opportunistic sampling methods were employed to obtain nasal swabs for fungal culture from wild mammal species residing within the coastal Douglas fir biogeoclimatic zone on the southeast coast of the island. Samples were collected from 91 animals representing 14 species. Cryptococcus gattii was isolated from the nasal swabs of two eastern gray squirrels (Sciurus carolinensis) trapped in Duncan, British Columbia. The relative proportion of nasal colonization in wild mammal species is consistent with findings in domestic animals, suggesting that animals may be good indicators of environmental organisms.     

Viral infections of the Kenya baboon (Papio cynocephalus) in its natural habitat

Stools, rectal swabs, throat swabs, and tissues were collected from 508 baboons (Papio cynocephalus) in 17 areas of Kenya and Tanzania during 5 field trips between 1961 and 1968. A total of 11 isolations were made: nine agents were recovered from 508 fecal samples and 2 from 468 throat swabs. These isolates were identified as adenovirus serotypes AA153, SA7, SV15 and SV23. SV23 was found only in throat swabs and never from fecal material while AA153, SA7 and SV15 were recovered only from fecal material. One rectal specimen produced 2 serotypes, SV15 and SA7. No isolates were recovered from necropsy samples from 19 animals.     

Natural Infection of Pregnant Cows with Schmallenberg Virus – A Follow-Up Study

Schmallenberg virus (SBV), an orthobunyavirus discovered in European livestock in late 2011 for the first time, causes premature or stillbirth and severe fetal malformation when cows and ewes are infected during pregnancy. Therefore, cattle of two holdings in the initially most affected area in Germany were closely monitored to describe the consequence for fetuses and newborn calves. Seventy-one calves whose mothers were naturally infected during the first five months of pregnancy were clinically, virologically, and serologically examined. One calve showed typical malformation, another one, born without visible abnormalities, was dead. Two cows aborted during the studied period; spleen and brain samples or meconium swabs were tested by real-time PCR, in none of the fetuses SBV-specific RNA was detectable and the tested fetal sera were negative in a commercially available antibody ELISA. In contrast, in nine clinically healthy calves high SBV-antibody titers were measurable before colostrum intake, and in meconium swabs of six of these animals viral RNA was present as well. The mothers of all nine seropositive calves were presumably infected between days 47 and 162 of gestation, which is within the critical timeframe for fetal infection suggested for SBV and related viruses.     

Prevalence of Salmonella in Australian reptiles

From January 2007 until June 2008, 504 reptiles of four families and 57 species were examined for Salmonella by using cloacal or intestinal swabs. Salmonella was identified in 139 (28%) of the 504 animals tested. Of the 504 reptiles examined, 210 were captive and 294 were wild. Ninety-eight (47%) of the captive reptiles were shedding Salmonella at the time of sampling. In contrast, only 41 (14%) of the wild reptiles were shedding Salmonella. The higher prevalence of Salmonella in captive reptiles was statistically significant (P<0.0001). No Salmonella was found in 60 wild, freshwater chelonians or 48 wild southern water skinks (Eulamprus heatwolei). Our results suggest that some species of wild reptiles in Australia are not natural carriers of Salmonella and that diet and captivity may influence Salmonella excretion in other species.     

Oral Disease in Animals: The Australian Perspective. Isolation and Characterisation of Black-Pigmented Bacteria from the Oral Cavity of Marsupials

A wide range of animals suffer from periodontal disease. However, there is very little reported on disease and oral micro-biota of Australian animals. Therefore, the oral cavity of 90 marsupials was examined for oral health status. Plaque samples were collected from the subgingival margins using curettes or swabs. Plaque samples were plated onto non-selective trypticase soy agar plates, selective trypticase soy agar, non-selective and selective Wilkens Chalgrens Agar. Plates were incubated in an anaerobic atmosphere and examined after 7–14 days for the presence of black–brown-pigmented colonies. A combination of morphological and biochemical tests were used (colonial morphology, pigmentation, aerobic growth, Gram reaction, fluorescence under long-wave UV light (360 nm), production of catalase, enzymatic activity with fluorogenic substrates and haemagglutination of sheep red cells) to identify these organisms. Black-pigmented bacteria were cultivated from the plaque of 32 animals including six eastern grey kangaroos, a musky rat kangaroo, a whiptail and a red-necked wallaby, 18 koalas, a bandicoot and five brushtail possums. No black-pigmented colonies were cultivated from squirrel or sugar gliders or quokkas or from marsupial mice. The majority of isolates were identified as Porphyromonas gingivalis -like species with the higher prevalence of isolation from the oral cavity of macropods (the kangaroos and wallabies). Oral diseases, such as gingivitis can be found in native Australian animals with older koalas having an increase in disease indicators and black-pigmented bacteria. Non-selective Wilkens Chalgren Agar was the medium of choice for the isolation of black-pigmented bacteria.     

Skin swabbing as a new efficient DNA sampling technique in amphibians, and 14 new microsatellite markers in the alpine newt (Ichthyosaura alpestris)

This study introduces a novel DNA sampling method in amphibians using skin swabs. We assessed the relevancy of skin swabs relevancy for genetic studies by amplifying a set of 17 microsatellite markers in the alpine newt Ichthyosaura alpestris, including 14 new polymorphic loci, and a set of 11 microsatellite markers in Hyla arborea, from DNA collected with buccal swabs (the standard swab method), dorsal skin swabs and ventral skin swabs. We tested for quality and quantity of collected DNA with each method by comparing electrophoresis migration patterns. The consistency between genotypes obtained from skin swabs and buccal swabs was assessed. Dorsal swabs performed better than ventral swabs in both species, possibly due to differences in skin structure. Skin swabbing proved to be a useful alternative to buccal swabbing for small or vulnerable animals: by drastically limiting handling, this method may improve the trade-off between the scientific value of collected data, individual welfare and species conservation. In addition, the 14 new polymorphic microsatellites for the alpine newt will increase the power of genetic studies in this species. In four populations from France (n=19-25), the number of alleles per locus varied from 2 to 16 and expected heterozygosities ranged from 0.04 to 0.91. Presence of null alleles was detected in two markers and two pairs displayed gametic disequilibrium. No locus appeared to be sex-linked.     

Microbiological Quality of Protein Feed Supplements Produced by Rendering Plants

Samples of protein feed supplements produced by rendering plants were examined for salmonellae, total aerobic bacterial counts, coliform counts, and enterococci. Isolations of salmonellae were more frequent from products with high counts; however, 6% of the samples with total counts of less than 1,000 per gram and 14% of the samples with coliform counts of less than 1 per gram contained salmonellae. Serotypes of Escherichia coli which have been associated with disease in domestic animals and poultry were also isolated from products. Although the distribution of serotypes of salmonellae isolated from environmental swabs and flies was similar to that isolated from products, the isolation of several serotypes from flies which had not been isolated in plants suggested that flies may be a potential source of contamination.     

Prevalence and distribution of Arcobacter species in various sources in Turkey and molecular analysis of isolated strains by ERIC-PCR

AIMS: To determine the prevalence of Arcobacter in various food, animal and water sources in Turkey and to subtype the isolated strains using enterobacterial repetitive intergenic consensus (ERIC)-PCR. METHODS AND RESULTS: A total of 806 samples consisting of chicken (100) and turkey meat (100); minced beef (27); rectal swabs from cattle (173), sheep (68) and dogs (62); cloacal swabs of broilers (100) and layers (100); gall bladders of cattle (50) and drinking water samples (26) were examined. A previously described membrane filtration method was used for the isolation. Isolates were identified at species level using multiplex-PCR and discriminated by ERIC-PCR for subtyping. Ninety-eight (12.1%) of the samples examined were found positive for arcobacters. Arcobacter spp. were isolated from 68%, 4%, 6.9%, 8% and 37% of chicken and turkey meats, rectal swabs and gall bladders of cattle and minced beef, respectively. No arcobacters were obtained from the rectal swabs of sheep and dogs, cloacal swabs of broilers and layers, and water samples examined. In total, 99 Arcobacter isolates were obtained. Of these isolates, 92 were identified as Arcobacter butzleri, five were Arcobacter skirrowii and two were Arcobacter cryaerophilus. Thirteen distinct DNA profiles among A. butzleri isolates were obtained by the ERIC-PCR. Of these profiles, eight were from chicken carcass, three from cattle rectal swab and two from minced beef meat isolates. Some of the isolates originated from different sources gave the same DNA profiles. All isolates of A. skirrowii and A. cryaerophilus gave different DNA profiles. CONCLUSIONS: Poultry carcasses, minced beef meat, rectal swabs and gall bladders of cattle were found to be positive for Arcobacter spp. A. butzleri was the predominant species isolated. In addition, large heterogeneity among the Arcobacter isolates was determined. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of the poultry carcasses and minced beef meat, rectal and gall bladder samples of cattle with arcobacters poses a risk for both human and animal infections. Detection of several different Arcobacter strains may suggest multiple sources for contamination and infection.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats.

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent. faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium-like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum, shared characteristics with Ent. columbae. The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol. Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

Identification and composition of the tonsillar and anal enterococcal and streptococcal flora of dogs and cats

Enterococcus faecalis was the most frequently isolated enterococcal species from anal swabs and tonsils of dogs and cats, although in the anal samples from dogs Ent. hirae was found almost as often as Ent. faecalis. Most Ent.faecium strains from dog tonsils differed from those associated with humans and other animals in that they fermented sorbitol. Typical Ent. avium as well as atypical Ent. avium -like strains were seen in dogs, while the related species Ent. raffinosus was associated with cat tonsils. Enterococcus cecorum also occurred mainly in cats. Certain atypical strains, presumptively identified as Ent. cecorum , shared characteristics with Ent. columbae.
The most frequent streptococcal species in tonsils of cats and dogs were Streptococcus suis and Strep. canis. Streptococcus canis and Strep. bovis predominated in anal swabs. The canine Strep. suis differed from the common porcine strains in fermenting mannitol.
Forty-seven of the 288 isolates examined could not be identified or related to known species. The characteristics of two groups of these bacteria, provisionally called 'Ton 31 group' and 'O7 group' are described.     

Safety studies of the oral rabies vaccine SAD B19 in striped skunk (Mephitis mephitis)

Safety of the modified live rabies virus vaccine, SAD B19, was studied in striped skunks (Mephitis mephitis). Seven skunks received 10(7.9) foci formatting units by direct oral administration. In four cages, a vaccinated animal was placed with a control animal, the other three vaccinated skunks were housed individually. Saliva and nasal swabs were collected 1, 2, 4, 24, 48, and 72 hr post-vaccination. From all vaccinated and control animals (n = 11) blood samples were collected 0, 28, 56, 84, and 296 days post-vaccination. Three of seven vaccinated skunks seroconverted. None of the control animals had detectable levels of rabies virus neutralizing antibodies. Also no vaccine virus was isolated from the nasal and saliva swabs collected from any animal. Thus, SAD B19 was innocuous for skunks in our study after direct oral administration at field concentration.     

Detecting nonhemolytic Pasteurella haemolytica infections in healthy Rocky Mountain bighorn sheep (Ovis canadensis canadensis): influences of sample site and handling

Effects of sampling procedures on ability to culture Pasteurella spp. from Rocky Mountain bighorn sheep (Ovis canadensis canadensis) were examined experimentally. Sample site influenced (P less than 0.0001) recovery of P. haemolytica in adult bighorn sheep. We isolated nonhemolytic P. haemolytica from 18 of 19 tonsillar swabs and 18 of 19 tonsillar biopsies from adult sheep, yet only four of 19 nasal swabs yielded isolates. Sample handling also affected (P less than 0.0001) recovery of P. haemolytica. Nonhemolytic P. haemolytica was cultured from 14 of 19 tonsillar swabs plated directly onto blood agar, but from only two of 19 swabs stored for 24 hr in modified Stuart's medium. We detected nonhemolytic P. haemolytica at least once in bronchial aspirates from four and in nasal swabs from three of six bighorn lambs. Based on direct cultures of tonsillar swabs and/or biopsies, all 26 bighorn sheep (seven lambs, 19 adults) sampled were infected with nonhemolytic P. haemolytica; only two lambs developed pneumonia during the study period. Thirty-four of 37 nonhemolytic P. haemolytica isolates tested were biotype T; three were biotype A. Serotypes 3; 4; 3, 4 and 3, 4, 10 were identified in a subsample of 17 isolates. Our data suggest tonsillar swabs or biopsies plated directly onto blood agar and incubated immediately offer the greatest probability of recovering nonhemolytic P. haemolytica from health bighorn sheep.     

Prevalence of vancomycin-resistant enterococci in hospitalized patients and those living in the community in the Czech Republic

Between July 1, 2002 and December 31, 2003, rectal swabs from both hospitalized patients and community subjects in the Czech Republic were taken to ascertain the prevalence of vancomycin-resistant enterococci (VRE). The swabs were used for isolating and identifying enterococci and their susceptibility to antibiotics. Vancomycin resistance phenotypes were verified by PCR detection of vanA, vanB, vanC1 and vanC2 genes. A molecular biology analysis was performed in Enterococcus faecium VanA strains. During the observed period, 2691 rectal swabs from the hospitalized patients and 6529 rectal swabs from the subjects in community setting were examined. In total, 31 VRE of hospital origin and 13 community-population strains were isolated. The prevalence of VRE in the gastrointestinal tract was 1.9% in the hospitalized patients and 0.4% in the community subjects. The prevailing strains were Enterococcus faecium VanA (61.3%) in the VRE of hospital origin and Enterococcus gallinarum VanC (46.2%) in the community VRE. Mutual comparison between the hospital and community Enterococcus faecium VanA strains showed no similarity.