Genotyping of bovine Prototheca mastitis isolates from Poland

Bovine mastitis due to unicellular, achlorophyllous algae of the genus Prototheca is a serious and complex ailment that accounts for high economic losses in the dairy industry. Bovine protothecal mastitis has been almost exclusively associated with only one species Prototheca zopfii. Recently, based on the 18S rDNA sequence analysis, P. zopfii has been divided into three genotypes (1-3), of which P. zopfii genotype 3 has been given the status of a new species Prototheca blaschkeae sp. nov. The aim of this study was to investigate the genotypic composition of the population of P. zopfii bovine mastitis isolates from Poland, according to that threefold classification. Of the 44 isolates tested, 43 (98%) were identified as P. zopfii genotype 2. Only one isolate was assigned to the newly established P. blaschkeae species. These findings clearly show the predominance of the P. zopfii genotype 2 in the etiology of bovine mammary protothecosis in Poland, but also underline the involvement of P. blaschkeae in the disease. The study is the first to provide molecular characterization of Polish P. zopfii mastitis isolates, collected from different regions of Poland.     

Diversity among isolates of Actinobacillus equuli and related organisms as revealed by ribotyping

Objective The objective of this work was to examine the diversity within Australian isolates of Actinobacillus equuli and related organisms by the genotypic method of ribotyping.
Design Ribotyping, performed using the enzyme Hae III, was used to examine the diversity in 12 field isolates of A equuli (five being capable of fermenting L-arabinose), one field isolate of Pasteurella caballi and two unclassifiable field isolates. Isolates were obtained from Australian horses, except for three isolates of A equuli (one L-arabinose positive and two L-arabinose negative) which were obtained from horses and a pig in Africa. In addition, the type strains for A equuli and P caballi and a reference strain for Bisgaard Taxon 9 were included in the study.
Results The ribotype patterns were analysed by computerised cluster analysis, yielding five clusters (A to E). All five of the L-arabinose positive A equuli were assigned to cluster A, with all the other seven A equuli isolates (all L-arabinose negative) and the type strain being assigned to cluster B. One of the two unclassified isolates formed cluster C along with the reference strain for Bisgaard Taxon 9. The remaining unclassified isolate formed cluster D. Cluster E consisted of the field isolate and reference strain of P caballi .
Conclusion The results of this study indicate that A equuli is a diverse species, with L-arabinose positive isolates of A equuli being quite distinct from typical L-arabinose negative isolates. Ribotyping appears to be a useful tool in confirming the identity of A equuli -like organisms from horses.     

Sources of Prototheca spp in a dairy herd environment

A source of Prototheca spp causing mastitis in a herd of 263 milking cows in North Carolina was investigated. Of 38 samples from the dairy environment, 18 (47%) contained Prototheca spp. Isolation sites included cattle drinking water; a feed trough; mud, dirt, and excreted feces from a dirt lounging area; water, sludge, mud, and vegetation from a creek in the lounging area; and the floor of a freestall barn. Samples were collected from 5 additional dairies, including one dairy with and 4 dairies without a history of protothecal mastitis. Prototheca spp were isolated from 48 (25%) of 190 samples from various sites on all 6 dairies. Isolates were P zopfii (45; 94%) and P wickerhamii (3; 6%). Isolation frequency ranged from 4 to 47% of samples/dairy. There was no apparent difference in the isolation frequency of Prototheca spp from samples from dairies with or without a history of protothecal mastitis. Sites characterized by wetness and the presence of organic matter most commonly yielded Prototheca spp. Because Prototheca spp appear to be common in the dairy environment, factors in addition to presence in the environment may be important in development of protothecal mastitis.     

Metastatic protothecal retinitis in a dog. Electron microscopic observations

Disseminated protothecosis, due to Prototheca wickerhamii, was present in a two-year-old female dog with a nine-month history of hemorrhagic colitis and diarrhea. Shortly thereafter, the dog developed "acute blindness" of the left eye. Euthanasia was done after medical therapy failed to control the disease. Histologically, the eye had multiple microabscesses and necrotic foci containing myriad protothecal organisms under the detached retina. Numerous organisms also were present in the mucosa and walls of the colon. The identification of P. wickerhamii was confirmed by the histologic appearance and immunofluorescent studies. The ultrastructural features of P. wickerhamii also were studied.     

Canine protothecosis: review of the literature and report of an additional case

A review of protothecosis in dogs revealed that this malady usually begins in the gastrointestinal tract and progresses to systemic involvement. Clinical signs generally include bloody diarrhea or blood-stained feces as well as blindness, ataxia, and polyuria. Histologically, myriads of protothecal organisms in different stages of development are found in the granulomatous lesions. Two main species have been culturally identified: Prototheca zopfii and P wickerhamii. In the absence of cultural studies, species identification can be accomplished readily by immunofluorescence. The present case involved P zopfii infection in a 5-year-old female Cocker Spaniel that had bloody diarrhea, with a history of bloody diarrhea 6 months earlier.     

Comparison and phylogenetic analysis of the heat shock protein 70 gene of Babesia parasites from dogs

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.     

Comparative sequence analysis of 16S rRNA gene of Pasteurella multocida serogroup B isolates from different animal species

The phylogenetic relationships of five isolates of Pasteurella multocida serotype B:2 belonging to buffalo, cattle, pig, sheep and goat were investigated by comparative sequence analysis of 16S rRNA gene. The 1468bp fragment of 16S rRNA gene sequence comparison showed that the isolates of cattle (PM75), pig (PM49) and sheep (PM82) shared 99.9% homology with the buffalo isolate (vaccine strain P52) whereas, the goat isolate (PM86) shared 99.8% homology with the vaccine strain. The 16S rRNA gene sequences of these isolates were also found monophyletic with type B reference strain NCTC 10323 of P. multocida subsp. multocida. The present study indicated the close relationships of haemorrhagic septicaemia causing P. multocida serotype B:2 isolates of buffalo and cattle with other uncommon hosts (pig, sheep and goat).     

Molecular heterogeneity in clinical isolates of Malassezia pachydermatis from dogs

Molecular investigation of 16 strains, conventionally identified to be Malassezia pachydermatis, isolated from dogs in Japan was carried out by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD band patterns of 13 clinical isolates were identical to that of standard strain of M. pachydermatis (CBS-1879). The other three clinical isolates were different from the standard strain of M. pachydermatis in RAPD patterns, and two of the three isolates were identical. About 620 bp genomic DNA fragments of the CHS2 gene were amplified from the same 16 clinical isolates of M. pachydermatis by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of the 16 clinical isolates revealed that the 13 strains were genetically very close to the standard strain of M. pachydermatis and the other two isolates were genetically close to the standard strain of M. furfur rather than M. pachydermatis. The remaining one isolate was phylogenetically distinct from all the seven Malassezia species reported so far.     

Restriction Enzyme Analysis of Indian Isolates of Egg Drop Syndrome 1976 Virus Recovered from Chicken,Duck and Quail

Egg drop syndrome 1976 (EDS-76) is caused by a haemagglutinating adenovirus belonging to group III of the genus Aviadenovirus in the family Adenoviridae. All isolates are serologically identical, but have been divided into three groups based on restriction endonuclease (RE) analysis. In this study the viral DNA of various Indian EDS-76 viral isolates (CEDS-A, CEDS-B, EDS-M, EDS-ML, EDS-1/AD/86, EDS-KC and QEDS) obtained from different avian species and different geographical regions were digested with restriction endonucleases viz., EcoRI, BamHI, HindIII and PstI. The results showed that one Indian isolate obtained from duck (DEDS-KC) was different from all other chicken and quail counterparts. All other isolates were identical to the reference viral strain BC-14, which belong to group I of EDS-76 viruses. The duck isolate EDS-KC could not be placed in any of the three groups reported earlier.     

Genetic diversity of avian infectious bronchitis coronavirus in recent years in China

Fifty-six isolates of avian infectious bronchitis virus (IBV) were obtained from different field outbreaks in China in 2010, and they were genotyped by comparison with 19 reference strains in the present study. The results showed that LX4-type isolates are still the predominant IBVs circulating in chicken flocks in China, and these isolates could be grouped further into two clusters. Viruses in each cluster had favored amino acid residues at different positions in the S1 subunit of the spike protein. In addition, a recombination event was observed to have occurred between LX4- and tl/CH/LDT3/03I-type strains, which contributed to the emergence of a new strain. The most important finding of the study is the isolation and identification of Taiwan II-type (TW II-type) strains of IBV in mainland China in recent years. The genome of TW II-type IBV strains isolated in mainland China has experienced mutations and deletions, as demonstrated by comparison of the entire genome sequence with those of IBV strains isolated in Taiwan. Pathogenicity testing and sequence analysis of the 3' terminal untranslated region revealed that TW II-type IBV strains isolated in mainland China have a close relationship with the embryo-passaged, attenuated TW2296/95.     

Generalized protothecosis in a fruit bat (Pteropus lylei)]

Prototheca organisms, histologically resembling Prototheca wickerhamii, were found in a fruit bat as a cause of a massive granulomatous lymphadenitis and splenitis and a widespread granulomatous meningoencephalitis. Focal lesions were also present in the myocardium, in skeletal muscles, and in the kidneys.     

Characterisation of a novel Mannheimia sp from Australian feedlot cattle

OBJECTIVE: To characterise eight isolates of a Gram-negative organism obtained from the upper respiratory tract of cattle showing evidence of mild upper respiratory tract disease. DESIGN: The isolates were compared with the five recognised species within the genus Mannheimia - M haemolytica, M glucosida, M granulomatis, M ruminalis and M varigena--using a range of phenotypic and genotypic methods. RESULTS: Phenotypic characterisation indicated that the isolates belonged to the trehalose-negative [Pasteurella] haemolytica complex. This complex has recently been reorganised into five species within the new genus Mannheimia. Ribotyping performed using HindIII and a computerised analysis system indicated that the eight Australian isolates formed a distinct cluster that was related to, but different from, the five recognised species of Mannheimia. The 16S rRNA sequence of one isolate (BNO311) was determined and a phylogenetic analysis performed. Isolate BNO311 was distinct from the five named Mannheimia spp but did join a larger cluster consisting of rRNA cluster IV (M varigena) and the unnamed rRNA cluster V of Mannheimia. DNA:DNA hybridisation between isolate BNO311 and M haemolytica NCTC 9380T, M granulomatis P411 and Actinobacillus ligniersii NCTC 4189T all suggested similarities of approximately 30%. CONCLUSIONS: These phenotypic and genotypic characterisation studies suggest that the eight Australian isolates represent a new species of Mannheimia. Until further characterisation studies are performed, we are unwilling to propose a name for this taxon, preferring to refer to this possible new species as Bisgaard taxon 39 of cluster V of Mannheimia.     

Prototheca zopfii genotypes isolated from cow barns and bovine mastitis in Japan

This study is the first investigation on Japanese isolates of Prototheca zopfii from bovine mastitis and the cow-barn surroundings by molecular characterization to clarify routes of infection for bovine protothecal mastitis. We performed isolation of Prototheca from cow-barn surroundings (drinking water, sewage and feces) and milk samples from cases of bovine mastitis. Genotypes of the 32 isolates of P. zopfii from cow-barn surroundings and 67 isolates from mastitis were analyzed by genotype-specific PCR assays and restriction fragment length polymorphism (RFLP) assays. All mastitis isolates were identified as P. zopfii genotype 2. Conversely, 29 isolates from cow-barn surroundings were identified as P. zopfii genotypes 1 and 3 isolates as genotype 2, respectively. Given these results, both genotypes of P. zopfii could exist in cow-barn surroundings, but no sites were identified as frequent sources of P. zopfii genotype 2. P. zopfii isolates should thus be further explored with regard to genotype to clarify the reservoir of etiological agents in bovine Prototheca mastitis.     

VP2 gene of a canine parvovirus isolate from stool of a puppy

VP2 gene of a canine parvovirus (CPV) isolate from the feces of a puppy which was diagnosed to be CPV infection was analysed. The result indicated that this clinical isolate was phylogenetically close to the isolate of wild-type CPV (strain CPV-T37) prevailing in Taiwan rather than isolates from Japan.     

Comparative phospholipid analogue distributions of Porphyromonas gingivalis isolated from cats in Australia and the USA

DNA-DNA homology measurements and phospholipid (PL) analogue profiling have shown heterogeneity of Porphyromonas gingivalis. The aim of this study was to determine whether there were differences between cat strains of P. gingivalis from Australia and USA with respect to PL analogue distribution. Lipids were extracted with chloroform-methanol and examined by fast atom bombardment-mass spectrometry (FAB-MS) in negative-ion mode, using published methods. For PL analogues, the major anions included those with mass-to-charge (m/z)=634, 648, 662, 705, 932, 946 and 960, respectively, corresponding to expected presence of PE (28:0), PE (29:0), PE (30:0), PG (32:1), and three unknown homologues of a glycero-phospholipid with a single nitrogen. Analyses were compared to calculate a matrix of Pearson coefficients of linear correlation from which a dendrogram was produced of strains clustered by single linkage. One cluster was comprised solely of Australian cat-to-cat bite isolates and a second cluster included exclusively USA cat- and dog-to-human bite isolates except for one Australian cat-to-cat bite isolate (VPB 5089). The US cluster included three outliers, one of which was the Australian cat isolate VPB 5089. The human type strain (ATCC 33277) was quite remote from all dog and cat strains. It was shown that P. gingivalis human and non-human animal isolates have distinct PL analogue profiles from each other. Furthermore, the cat strains from the USA and those from Australia showed quantitative differences in polar lipid profiles that correlated largely with country of isolation.     

Single nucleotide polymorphisms in the bovine Histophilus somni genome; a comparison of new and old isolates

Histophilus somni, a causative agent of the bovine respiratory disease complex, can also cause a variety of systemic disorders, including bronchopneumonia, myocarditis, pericarditis, arthritis, pleuritis, and infectious thrombotic meningoencephalitis. The purpose of this study was to determine if currently circulating strains differ from those of the 1980s by identifying genomic changes. Single nucleotide polymorphisms (SNPs) and insertion and deletion (INDEL) sites were examined by whole-genome sequencing in 12 samples, 6 old and 6 new. The 31 028 SNP/INDELs recorded were compared against the reference genome sequence of the pathogenic H. somni strain 2336. The distribution of about 75% of these SNPs within a specified gene differed between old and new isolates and did not follow any particular pattern. The other 25% clustered into 2 groups containing the same SNPs in various genes: group I included 5 old isolates and 1 new isolate; group II included 5 new isolates and 1 old isolate. For putative virulence genes there were more SNPs in group I compared with strain 2336, itself an older isolate, than in group II. Although only 25% of all the SNPs formed 2 clusters, the results suggest some genetic difference in various genes between old and new strains.     

Genetic and antigenic characterization of the matrix protein of two genetically distinct ovine lentiviruses

Small Ruminant Lentiviruses (SRLV) are a group of non-oncogenic retroviruses including Maedi-Visna virus (MVV) and Caprine Arthritis-Encephalitis virus (CAEV), which cause a chronic, multisystemic disease in sheep and goats, respectively. Phylogenetic analyses of SRLV are based in most cases on partial pol sequences. Several reports indicate that the species specificity of these viruses is not as strict as previously thought; MVV-like viruses have been found in goat populations and vice versa. Recently, the sequencing of some Italian ovine isolates has shown the presence of a new cluster more similar to classical caprine isolates (CAEV-like). Few data are available on the variability of structural proteins involved in the antibody response of infected animals. In this study, the gag gene of two genetically distinct ovine isolates, namely the MVV-like It-561 and the CAEV-like It-Pi1, was sequenced and the epitopes of matrix protein (MA) were mapped. Recombinant MAs and their subunits from both ovine aforementioned strains were tested against a panel of sheep and goat sera. Reactive epitopes were found in all three subunits of MA, although the central subunit displayed a more consistent reactivity. Epitope mapping of this subunit demonstrated that the amino acid sequence of at least one immunodominant epitope was quite different in the two strains. This antigenic variability may affect the sensitivity of a single strain-based immunoassay and suggests that both SRLV genotypes should be used in the development of future diagnostic tests, to avoid viral strain selection during the eradication programmes.     

Isolation and Identification of one Strain of H9 Subtype Avian Influenza Virus and Study on its Biological Characteristics

In 2013,one case of suspected H9 subtype avian influenza occurred in a chicken farm of Jilin povince.Clinical samples were collected from the diseased farm,inoculated into the allantoic cavity of 9-day-old SPF chicken embryo,and then one strain of virus was isolated.The results of HA test,HI test and molecular biology test all showed that the isolate belonged to H9 subtype avian influenza virus (AIV).The HA cleavage site of the isolate was RSSR↓GLF,which was consisted with the molecular characteristic of low pathogenic AIV.The HA peptide chain had 9 potential glycosylation sites which were same as other isolates of recent years.The isolate had 8 receptor binding sites,including 234 receptor binding site by glutamine (Q) mutating into threonine (T).The phylogenetic tree revealed the isolate belonged to Eurasian lineages and it had far genetic relationship with the earliest domestic isolate (A/Chicken/Beijing/1/94(H9N2)),but had close genetic relationship with the representative strain (A/Chicken/Guangxi/55/2005(H9N2)) of major epidemic branch since 2007.We prepared an inactivated oil-emulsion vaccine of the isolate,and then vaccinated SPF chicken.21 days after vaccination,the HI titer of chicken serum antibody reached up to 10log2.The result suggested the isolate had good immunogenicity.     

一株H9亚型禽流感病毒的分离鉴定及生物特性研究

2013年中国吉林某养鸡场发生疑似H9亚型禽流感疫情,采集该发病鸡场病料接种9日龄SPF鸡胚,分离得到一株病毒。经血凝(HA)试验、血凝抑制(HI)试验、测序分析,鉴定该毒株为H9亚型禽流感病毒(AIV)。对本试验分离株HA基因进行测序及序列分析,结果显示分离株HA基因的裂解位点为RSSR↓GLF,符合低致病性AIV的基因特征;HA肽链具有9个潜在糖基化位点,与近些年H9亚型AIV分离株的潜在糖基化位点相同;具有8个受体结合位点,其中234位受体结合位点由谷氨酰胺(Q)变异成苏氨酸(T);HA基因系统进化树结果显示本试验分离株属于欧亚进化分支,与中国最早分离株A/Chicken/Beijing/1/94(H9N2)亲缘关系较远,与2007年后中国H9亚型AIV主要流行分支的代表株A/Chicken/Guangxi/55/2005(H9N2)亲缘关系较近。将该毒株制成油乳剂灭活疫苗免疫SPF鸡,免疫后第21天免疫鸡血清抗体高达10log2,表明本试验分离株具有很好的免疫原性。