A survey of Neospora caninum-associated bovine abortion in large dairy farms of Mashhad, Iran

Neospora caninum is an apicomplexan protozoon causing abortion in cattle worldwide. The present study was designed to assess the importance of this parasite for causing abortion in dairy farms in the Mashhad area of Iran. Of the aborted bovine fetuses, 151 were collected from dairy farms between 2006 and 2008. First, brain samples were examined by polymerase chain reaction (PCR) for the presence of N. caninum DNA, diagnosis was complemented with immunohistochemistry (IHC) and fetal serology (ELISA). Twenty-two (14.5%) of bovine fetuses were considered to be infected with N. caninum with at least one diagnostic technique being positive. PCR yielded 18 (11.9%) positive out of 151 brain samples. Only 52 brain samples were suitable for IHC examination, and N. caninum organism was detected in six (11.5%) of these 52 brain samples. Fetal fluids (n = 151) were assessed with a N. caninum-ELISA, resulting in 15 (9.9%) seropositive fetal fluids samples. In the present study, a good agreement was observed between PCR and ELISA, and a fair agreement between PCR and IHC. The results indicated that abortion due to N. caninum infection is prevalent among large-size dairy farms in the Mashhad area of Iran, and that different complementary diagnostic techniques should be used to increase the chance to detect N. caninum.     

First report of <Emphasis Type="Italic">Neospora caninum</Emphasis>-associated bovine abortion in Mashhad area,Iran

Neospora caninum is an apicomplexan protozoan which causes abortion in cattle worldwide. The present study was designed to assess the importance of bovine neosporosis for causing abortion in Iranian cattle. Infection was primarily diagnosed by polymerase chain reaction (PCR), complemented with histopathology and immunohistochemistry (IHC). One hundred brains of aborted bovine fetuses were collected from Iranian dairy herd between 2003 and 2005 in Mashhad area. N. caninum was detected by PCR in the brains of 13 aborted fetuses. Lesions consistent with N. caninum infection were observed in 12 of fetal brains. Immunohistochemical examination of brain detected N. caninum organisms in three aborted fetuses, and a thick-walled (2 μm) cyst with 50 μm diameter was identified in one of the IHC-positive brain. The results indicated that neosporosis is an important cause of abortion in dairy cattle of Iran.     

Extraction of genomic DNA from paraffin-embedded tissue sections of human fetuses fixed and stored in formalin for long periods

The advent of polymerase chain reaction (PCR) technology has increased the interest in fetal specimens housed in anatomy museums, as they may represent a unique source of genetic material for the study of uncommon or rare pathological conditions such as congenital malformations, neoplastic processes and parasitic as well as other infectious diseases. The aim of this study is to evaluate the quality of genomic DNA extracted from paraffin-embedded tissue sections of human fetuses that have been maintained in formalin for several years. Fetal tissues were embedded in paraffin, and tissue sections were submitted to ethanol/xylene dewaxing, followed by DNA extraction with ammonium acetate. DNA fragments were amplified from DNA extracted from formalin-fixed tissue sections, but not from Bouin-fixed tissues (average yield of 13.7mug/ml from 10 umbilical cord sections of 10mum; A(260):A(280)=1.55,). The addition of bovine serum albumim increased the yield of PCR amplification. Genomic DNA can be reliably amplified from paraffin-embedded human fetal tissues that had been fixed in formalin during 19 years and used for microdissection studies. This simple, cost-effective, and non-laborious method should facilitate the molecular analysis of a large number of specimens fixed for long periods of time.     

Validation of a Commercially Available Monoclonal Antibody-Based Competitive-Inhibition Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibodies to Neospora caninum in Cattle

A previously described monoclonal antibody (MAb)-based competitive-inhibition enzyme-linked immunosorbent assay (cELISA) was modified to optimize performance, and the assay was validated in various defined cattle populations for detection of serum antibody to Neospora caninum, a major cause of bovine abortion. Modifications to the cELISA included capturing native N. caninum antigen with a parasite-specific MAb (MAb 5B6-25) and directly conjugating the competitor MAb (MAb 4A4-2), with both MAbs binding different epitopes of a conserved, immunodominant 65-kDa tachyzoite surface antigen. The assay was validated using three serum sets, a "gold standard" set of 184 cow sera defined by fetal histopathology and N. caninum immunohistochemistry and by maternal N. caninum indirect fluorescence assay (IFA) at a 1:200 serum dilution, a relative standard set of 330 cow sera defined by IFA alone, and a set of 4,323 cow sera of unknown N. caninum status. A test cutoff of 30% inhibition was identified. The diagnostic sensitivity was 97.6%, and diagnostic specificity was 98.6% for the gold standard abortion-defined sera. The diagnostic sensitivity was 96.4%, and diagnostic specificity was 96.8% for the relative standard IFA-defined sera. Testing of the 4,323 bovine sera of unknown N. caninum status revealed a distinct bimodal distribution and steep sigmoid frequency curve with only 1.8% of samples within 5% of the test cutoff, indicating a sharp discrimination between test-positive and test-negative samples. In summary, the modified N. caninum cELISA provided a simple, rapid, and versatile method to accurately identify N. caninum infection status in cattle using a single cutoff value.     

Detection of Neospora from tissues of experimentally infected rhesus macaques by PCR and specific DNA probe hybridization.

Neospora is a newly recognized Toxoplasma-like cyst-forming coccidian parasite that causes abortion or congenital infections in naturally or experimentally infected animals. In this study, pregnant rhesus macaques were inoculated with culture-derived tachyzoites of a bovine Neospora isolate, and tissue samples from various major organs were collected from dams and fetuses for the detection of parasite DNA by using oligonucleotide primers COC-1 and COC-2 for PCR amplification of a conserved coccidial nuclear small-subunit rRNA gene sequence, and amplification products were confirmed by hybridization with a Neospora-specific DNA probe. PCR products were amplified from DNAs of different fetal monkey tissues, including brain, heart, lung, liver, spleen, skeletal muscle, skin, and placenta. In addition, Neospora DNA was amplified from the brain, heart, and lung tissues of infected rhesus macaque dams. The PCR and probe hybridization system may provide an effective method for the detection of Neospora infection in fetuses and dams from nonhuman primates and may be useful in determining the zoonotic potential of Neospora.     

Placental pathology associated with fetal death in cattle inoculated with Neospora caninum by two different routes in early pregnancy

Pregnant cattle were inoculated with N. caninum strain NC-1 tachyzoites intravenously (iv) (group 1, n = 8) or subcutaneously (sc) (group 2, n = 8) at 70 days' gestation. Control animals (group 3; n = 8) received uninfected Vero cells iv. Two animals from each group were killed at 14, 28, 42 and 56 days post-inoculation (dpi). Fetal mortality was 100% and 50%, respectively, in groups 1 and 2 from 28 dpi. In group 1 foci of degenerative fetal placental villi were observed at 14 dpi, with clusters of N. caninum tachyzoites in the affected mesenchyme. There was also inflammation of maternal septal tissues, with necrotic cell debris and serum exudate at the interstitium. At 28 dpi pregnancy had ended and the fetal cotyledons had become detached from the maternal caruncles. Immunohistochemically, particulate N. caninum antigen was detected in the cotyledons. At 42 and 56 dpi, fetal tissues had disappeared, the caruncles were greatly reduced in size, and the uterine epithelium had been largely restored. In group 2, lesions were either severe or absent ("all or nothing" response). In one animal carrying a dead fetus at 28 dpi, placentitis was much more severe than that seen in group 1 at 14 dpi. Lesions contained neutrophils, eosinophils and N. caninum antigen. In animals carrying dead fetuses at 42 and 56 dpi, fetal remains were found and the cotyledons contained N. caninum antigen. Antigen was also detected in fetal tissues. No significant pathological changes were detected in group 2 animals carrying live fetuses or any animal in group 3. Thus, N. caninum administered iv or sc in early pregnancy resulted in rapid fetal death, with parasite-associated lesions in the placenta and fetus. Of the two inoculation routes, the intravenous induced the more acute placental lesions and greater mortality.     

Pathogenesis of bovine neosporosis

The protozoan parasite Neospora caninum is a major pathogen of cattle and dogs, being a significant cause of abortion in cattle in many countries. It is one of the most efficiently transmitted parasites, with up to 90% of cattle infected in some herds. The pathogenesis of abortion due to Neospora is complex and only partially understood. Losses occur after a primary infection during pregnancy but more commonly as the result of recrudescence of a persistent infection during pregnancy. Parasitaemia is followed by invasion of the placenta and fetus. It is suggested that abortion occurs when primary parasite-induced placental damage jeopardises fetal survival directly or causes release of maternal prostaglandins that in turn cause luteolysis and abortion. Fetal damage may also occur due to primary tissue damage caused by the multiplication of N. caninum in the fetus or due to insufficient oxygen/nutrition, secondary to placental damage. In addition, maternal immune expulsion of the fetus may occur associated with maternal placental inflammation and the release of maternal pro-inflammatory cytokines in the placenta. Thus N. caninum is a primary pathogen capable of causing abortion either through maternal placental inflammation, maternal and fetal placental necrosis, fetal damage, or a combination of all three. The question of how N. caninum kills the fetus exposes the complex and finely balanced biological processes that have evolved to permit bovine and other mammalian pregnancies to occur. Defining these immunological mechanisms will shed light on potential methods of control of bovine neosporosis and enrich our understanding of the continuity of mammalian and protozoal survival.     

Absence of human bocavirus from deceased fetuses and their mothers

BackgroundThe human bocavirus (HBoV), a newly discovered parvovirus, is closely related to the bovine parvovirus and the canine minute virus, which are known to cause adverse pregnancy outcomes. Another human parvovirus, B19, can lead to fetal hydrops, miscarriage and intrauterine fetal death (IUFD).ObjectivesTo determine the prevalence of HBoV DNA in aborted fetuses and IUFDs. The HBoV serology of the mothers was also studied.Study designWe retrospectively studied all available fetuses (N = 535) autopsied during 7/1992–12/1995, and 1/2003–12/2005 in Helsinki, Finland. All available formalin-fixed paraffin-embedded fetal tissues – placenta, heart and liver – of 120 miscarriages, 169 IUFDs, and 246 induced abortions were studied by quantitative PCR. We also measured the HBoV IgM and IgG antibodies in the corresponding maternal sera (N = 462) mostly of the first trimester. The IgM-positive sera underwent HBoV PCR.ResultsNone of the fetal tissues harbored HBoV DNA. A total of 97% (448/462) of the mothers were positive for IgG antibodies to HBoV, while only 0.9% (4/462) exhibited HBoV-specific IgM antibodies without viremia or respiratory symptoms. One IgM-positive mother had an unexplained fetal loss.ConclusionsWe did not find HBoV DNA in any of the deceased fetuses. Almost all pregnant women were HBoV-IgG positive.     

DNA extraction by sonication: a comparison of fresh, frozen, and paraffin-embedded tissues extracted for use in polymerase chain reaction assays.

DNA extraction from fixed tissues can be the most laborious and complex step in amplifying DNA by the polymerase chain reaction (PCR). We have previously reported a rapid and efficient method for extracting DNA by the use of sonication and glass beads. We have extended our experiences with this technique using fresh, frozen, and formalin-fixed paraffin-embedded tissues with and without the use of glass beads and report their results. Multiple tissue types were obtained at autopsy or as part of a surgical specimen. DNA was extracted from identical tissue when the sample was fresh, frozen, or formalin-fixed paraffin-embedded. Our results indicate that in most instances the sonication technique, which takes only 30 min from start to finish, can rapidly extract fresh, frozen, or formalin-fixed paraffin-embedded tissue and is superior to other rapid extraction techniques in terms of quality and quantity of DNA. It is much more rapid than those techniques that use long digestion periods. This technique will be of great value to those investigators extracting DNA for polymerase chain reaction assays.     

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.     

Detection of specific human papillomavirus types in paraffin-embedded sections of cervical carcinomas

Human papillomaviruses (HPV) are the causative agents of most cervical carcinomas. A complete understanding of the HPV types that cause cervical carcinoma is needed as vaccines are designed. Fresh tissues are not always available for such studies. We therefore sought to determine the feasibility of HPV studies using formalin-fixed, paraffin-embedded sections of 56 cervical carcinomas, correlating typing information with the pathology and physical state of the HPV sequences within cells. Sections from each specimen were used to extract and purify DNA. Specific HPV types were identified using a PCR/reverse blot strip assay. Tyramide signal-amplified, fluorescent DNA in situ hybridization (FISH) was used to localize HPV within cells. Human beta-globin sequences were amplified in DNA from all specimens. HPV sequences from oncogenic types were identified in 52 of 56 (92.9%) by PCR/reverse blot strip assay, and in one additional case using an HPV 16 multiplex PCR assay. HPV 16 was the most commonly detected type, present in most cases as a solitary isolate. Thirty- five of 42 HPV 16 or HPV 18 PCR-positive specimens were also positive in the FISH assay, in most cases in a pattern consistent with viral integration. We conclude that HPV typing from formalin-fixed, paraffin-embedded sections of cervical carcinomas is possible, with a sensitivity that is similar to that found in studies using fresh tissue.     

Neospora caninum: evaluation of vertical transmission in slaughtered beef cows (Bos indicus)

Neospora caninum is a protozoan parasite that causes the most important reproductive problems in cattle worldwide. The objective of this study was to evaluate the possibility of vertical transmission of N. caninum in zebus breed beef cows (Bos indicus) submitted for slaughter at an abattoir in the northern region of the State of Paraná, southern Brazil. One hundred and fifty-nine cows were evaluated: 83 pregnant (in different stages of gestation) and 76 non-pregnant. Serum determination of N. caninum was evaluated by indirect ELISA (Idexx). Blood (with EDTA) from pregnant cows and tissue samples (brain and heart) from their fetuses were collected and used for PCR analyses. Antibodies against N. caninum were observed in 14.6% (12/83) of pregnant and in 15.8% (12/76) of non-pregnant cows. Antibodies against the parasites were detected in one fetus (1.4%). The PCR analyses revealed that 6.0% (5/83) of cows and 4.8% (4/83) of fetuses evaluated were positive to specific N. caninum primers. These positive fetuses were between 4 and 6 months of age. Thus, considering PCR and serology as an indicative of vertical transmission in fetuses, 4.8% of fetuses were infected by N. caninum during gestation.     

Tissue distribution of bovid herpesvirus-4 in inoculated rabbits and its detection by DNA hybridization and polymerase chain reaction

Summary A DNA hybridization technique, using the polyrepetitiveEcoRI L-fragment of bovid herpesvirus (BHV-4) as a probe, was developed to determine virus distribution in the tissues of BHV-4-infected pregnant rabbits. The cloned fragment did not react with the DNA of rabbits or of other herpesviruses, e.g., infectious bovine rhinotracheitis, bovine herpes virus mammillitis, and pseudorabies viruses. The detection limit was 10^–13g of DNA or approximately 600 genome equivalents of viral DNA, which indicates a level of sensitivity of one viral genome per 500 cells in our assay. Using conventional cell culture techniques, the virus was isolated from only one of fifteen infected rabbits and a few aborted fetuses. However, when organ culture or dot blot hybridization was used, BHV-4 was detected in all rabbits and their fetuses. Viral DNA was detected by DNA hybridization in spleen, ovary, uterus, lung, liver, salivary gland, lymph node, and placentome of adult rabbits and in a composite of fetal tissues. When polymerase chain reaction (PCR) was used, the virus was detected in several organs (including the nervous tissues) that were found negative by other techniques. These results indicate that blot hybridization and PCR are more sensitive than conventional techniques for studying the pathogenesis of BHV-4 in animals. The data obtained by these methods suggest that BHV-4 may be maintained in infected rabbits in a latent state in a variety of tissues including the nervous system.     

Comparative studies on the detection of hepatitis B virus DNA in frozen and paraffin sections by the polymerase chain reaction.

The polymerase chain reaction (PCR) is an extremely sensitive technique that has been used for detection of DNA sequences in formalin-fixed, paraffin-embedded tissues. In order to verify that hepatitis B virus (HBV) DNA sequences are adequately preserved in routinely processed liver tissues, we performed PCR with five different primer pairs for HBV sequences on DNA extracted by two different methods from paraffin and frozen liver sections. The amount of PCR products obtained with DNA templates extracted by the proteinase K-SDS method from frozen sections was significantly larger than that from paraffin sections. However, boiling of deparaffinized sections in water containing Chelex-100 resulted in ample amounts of PCR products irrespective of the primers used. On Southern blots, the location of the bands of amplified DNA obtained by the different methods was consistent with the predicted size, suggesting that the viral sequences had not been altered by processing. Although freezing of fresh tissue yields quantitatively more HBV DNA, formalin fixation qualitatively preserves the viral DNA sequences adequately for detection by PCR. Therefore, formalin-fixed, paraffin-embedded tissues may be used for the detection of viral DNA sequences by PCR. Application of the described procedure to routinely processed tissues significantly broadens the applicability of this powerful diagnostic and investigative method.     

Quantitative detection of Neospora caninum in bovine aborted fetuses and experimentally infected mice by real-time PCR

We report the development of a real-time PCR assay for the quantitative detection of Neospora caninum in infected host tissues. The assay uses the double-stranded DNA-binding dye SYBR Green I to continuously monitor product formation. Oligonucleotide primers were designed to amplify a 76-bp DNA fragment corresponding to the Nc5 sequence of N. caninum. A similar method was developed to quantify the 28S rRNA host gene in order to compare the parasite load of different samples and to correct for the presence of potential PCR-inhibiting compounds in the DNA samples. A linear quantitative detection range of 6 logs with a calculated detection limit of 10(-1) tachyzoite per assay was observed with excellent linearity (R(2) = 0.998). Assay specificity was confirmed by using DNA from the closely related parasite Toxoplasma gondii. The applicability of the technique was successfully tested in a variety of host brain tissues: (i) aborted bovine fetuses classified into negative or positive Neospora-infected animals according to the observation of compatible lesions by histopathological study and (ii) experimentally infected BALB/c mice, divided into three groups, inoculated animals with or without compatible lesions and negative controls. All samples were also tested by ITS1 Neospora nested PCR and a high degree of agreement was shown between both PCR techniques (kappa = 0.86). This technique represents a useful quantitative diagnostic tool to be used in the study of the pathogenicity, immunoprophylaxis, and treatment of Neospora infection.     

Immunohistological detection of human parvovirus B19 in formalin-fixed, paraffin-embedded tissues.

Human parvovirus B19 is a cause of aplastic crises in patients with haemolytic anaemias, prolonged bone marrow failure in the immunosuppressed, and fetal death secondary to non-immune hydrops. The immunohistological detection of parvovirus B19 in formalin-fixed, paraffin-embedded tissues has not previously been reported, and definitive diagnosis of infection in such specimens has relied on the use of specialized DNA hybridization and amplification techniques. A new monoclonal antibody to B19 capsid proteins, R92F6, was found to be capable of labelling infected cells in paraffin-embedded tissues from all 19 cases of parvovirus-related fetal hydrops tested, and in bone marrow from a child with congenital immunodeficiency and chronic parvovirus infection. Viral antigen was detected both in cytoplasmic and in nuclear distributions using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique without preceding proteolytic digestion. The viral epitope recognized appears to be highly conserved, as specimens were obtained over a 13-year period from widely spaced locations in the U.K. Antibody R92F6 should facilitate rapid diagnosis of parvovirus B19 infection in routinely processed and archival specimens.     

Rare detection of cytomegalovirus in severe fetal malformations in China

BackgroundCytomegalovirus (CMV) is a significant cause of fetal abnormalities in developed world. Whether this could be applied in developing world remains unknown.ObjectivesTo investigate CMV infection in severe fetal malformations in China.Study designDuring 2007–2014, 436 fetuses (237 males) with severe malformations and terminated pregnancy at median gestational age of 26⁺¹ weeks were enrolled. CMV DNA was detected in fetal kidneys and other tissues by real-time PCR, and CMV IgG and IgM were measured by ELISA.ResultsCMV DNA was positive in kidneys and other tissues of seven (1.60%) fetuses. Hematoxylin-eosin staining showed intranuclear and intracytoplasmic inclusion bodies in kidneys of three fetuses, which was also positive for CMV antigens in immunohistochemistry. CMV DNA was found in 5 (6.1%) of 82 fetuses with central nervous system anomalies, 1 (11.1%) of 9 fetuses with abdominal anomalies, 1 (0.59%) of 168 fetuses with multiple congenital malformations, and none of fetuses with other anomalies (177). Of 293 pregnant women with plasma available, 279 (95.2%) were CMV IgG positive only and 6 (2.1%) were CMV IgG and IgM positive. Of 5 mothers with infected fetuses 1 (20%) was CMV IgG and IgM positive, while 5 (1.7%) of 288 mothers with uninfected fetuses were positive respectively (P = 0.099).ConclusionsCongenital CMV infection in fetuses with severe congenital malformations is rare, indicating no close association between CMV infection and severe fetal malformations in China. Maternal screening for CMV may have minimal value in identifying fetal malformations in developing world.     

Feasibility of archival non-buffered formalin-fixed and paraffin-embedded tissues for PCR amplification: An analysis of resected gastric carcinoma

Although several factors affecting the sensitivity of potymer-ase chain reaction (PCR) amplification from formalin-fixed tissues have been Investigated mostly by experiments, the feasibility of archival formalln-flxed, paraffin-embedded tissue samples stored in pathology departments for PCR amplification has rarely been examined directly. Thus, the feasibility of 74 archival unbuffered 10% formalin-fixed, paraffin-embedded tissues for PCR amplification with primers producing a 190 b.p. DNA segment of p53 exon 5 was investigated. Fixation time was the critical factor influencing the sensitivity of PCR amplification. All (6/6) of the samples fixed for only 1 day, 44% (7/16) of the samples fixed for 2–3 days and 14% (4/28) of the samples fixed for 4–6 days showed successful amplification, while no amplification was obtained for the samples fixed for 7 days or more. The peak size of DNA extracted from the archival tissues decreased as the fixation time became longer. Experiments using xenografted tumor tissues fixed for various times showed longer permissible fixation time; up to 9 days of fixation, decreasing amounts of PCR products were obtained while no amplification was obtained for the samples fixed for 12 days or more. The time in paraffin seemed to be a minor factor for PCR amplification since all of the 1 day fixation samples, including those that had been embedded for up to 5 years, resulted in efficient amplification. The size of the amplified DNA segments, however, could be another factor influencing the sensitivity of amplification because even the 1 day fixation samples showed less amplification of 345 b.p. DNA compared with those of 167 and 262 b.p. DNA. Additionally, a point mutation was detected in the amplified pS3 products from archival tissues using a non-isotopic method, temperature gradient gel elec-trophoresis. In conclusion, archival tissue samples that had been fixed immediately for only up to 1 day were constantly available for PCR amplification of approximately 200 b.p. DNA segments, suggesting that surgical specimens should be subjected to cutting and paraffin embedding Just after 1 day or less fixation for subsequent use in PCR amplification.