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Immune responses in goats to recombinant hemagglutinin-neuraminidase glycoprotein of Peste des petits ruminants virus: identification of a T cell determinant.

Peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus within the family Paramyxoviridae, causes a fatal disease 'peste des petits ruminants' in goats and sheep. This enveloped virus is antigenically closely related to rinderpest virus (RPV), which causes a similar but distinct disease in large ruminants. PPRV harbors two major surface glycoproteins, the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins. The surface glycoproteins of morbilliviruses are highly immunogenic and confer protective immunity. In this study, we investigated the immune responses generated in goats immunized with low doses of purified recombinant extracellular baculovirus carrying a membrane bound form of the HN protein of PPRV without any adjuvant. We report that the immunized goats develop both humoral and cell-mediated immune responses. Antibodies generated in the immunized animals could neutralize both PPRV and RPV in vitro. Further, using a combination of Escherichia coli expressed deletion mutants of PPRV-HN and RPV-H proteins, and synthetic peptides corresponding to the highly conserved N-terminal sequences of MV-H protein, we have mapped an N-terminal T cell determinant (amino acids 123-137) and a C-terminal domain (amino acids 242-609) harboring potential T cell determinant(s) in goats.

Amino Acid Sequence↗

Molecular evolution of growth hormone (GH) in Cetartiodactyla: cloning and characterization of the gene encoding GH from a primitive ruminant, the chevrotain (Tragulus javanicus).

In mammals the sequence of pituitary growth hormone (GH) is generally strongly conserved, indicating a slow basal rate of molecular evolution. However, on two occasions, during the evolution of primates and that of cetartiodactyls, the rate of evolution has increased dramatically (25 to 50-fold) so that the sequences of human and ruminant GHs differ markedly from those of other mammalian GHs. To define further the burst of GH evolution that occurred in cetartiodactyls, the GH gene of the chevrotain (Tragulus javanicus) has been cloned and characterized by use of genomic DNA and a polymerase chain reaction technique. Two very similar gene sequences, which probably reflect allelic variation, were isolated. The deduced sequence for the mature chevrotain GH differs from that of the bovine or red deer GH at only two to three residues, and phylogenetic analysis shows that the burst of rapid evolution of GH that occurred in the Cetartiodactyla must have been completed before the divergence of the Tragulidae and the advanced ruminants (Pecora). The rate of evolution during this burst must therefore have been greater than previously estimated. In other aspects (including signal sequence, 5' upstream sequence, and synonymous substitutions in the coding sequence), the chevrotain GH gene differs considerably from the GH genes of other ruminants and here there is no evidence for the period of accelerated evolution that is seen for GH itself.

Amino Acid Sequence↗

Bioconversion of netobimin pro-drug by gastrointestinal fluids of ruminants.

The biotransformation of netobimin (NTB) pro-drug by ruminal, abomasal and intestinal fluids of sheep and cattle was investigated under anaerobic conditions in vitro. No metabolic conversion of NTB was observed upon incubation with abomasal fluid or boiled samples of different gastrointestinal fluids. NTB pro-drug was reduced and cyclised into albendazole (ABZ) and this further oxidized into ABZ sulphoxide (ABZSO) and ABZ sulphone (ABZSO2) by both sheep and cattle ruminal and ileal fluids. A zwitterion formulation of NTB produced a significantly greater amount of anthelminthically active ABZ metabolites (ABZ and ABZSO) than a trisamine salt formulation of the same compound. Although there was no difference in the total amount of products formed, both cattle ruminal and ileal fluids showed a greater oxidative capacity than sheep derived fluids. This was evidenced by the greater amounts of ABZSO recovered and by the resultant lower ratios of ABZ/ABZSO obtained with cattle fluids.

Animals↗

Genomic insights into Shigella species isolated from small ruminants and manure in the North West Province, South Africa.

This study investigated Shigella species' antibiotic resistance patterns and genomic characteristics from small ruminants and manure collected in Potchefstroom, North West, South Africa. Whole genome sequencing was used to determine resistome profiles of Shigella flexneri isolates from small ruminants' manure and Shigella boydii from sheep faeces. Comparative genomics was employed on the South African 261 S. flexneri strains available from GenBank, including the sequenced strains in this study, by investigating the serovars, antibiotic resistance genes (ARGs), and plasmid replicon types. The S. flexneri strains could not be assigned to known sequence types, suggesting novel or uncharacterized lineages. S. boydii R7-1A was assigned to sequence type 202 (ST202). Serovar 2A was the most common among South African S. flexneri strains, found in 96% of the 250 compared human-derived isolates. The shared mdf(A) was the most prevalent gene, identified in 99% of 261 S. flexneri genomes, including plasmid replicon types ColRNAI_1 (99%) and IncFII_1 (98%). Both species share a core set of resistance determinants mainly involving β-lactams (ampC1, ampC, ampH), macrolides (mphB), polymyxins (eptA, pmrF), multidrug efflux pumps (AcrAB-TolC, Mdt, Emr, Kpn families), and regulatory systems (marA, hns, crp, baeRS, evgAS, cpxA, gadX). However, S. boydii possesses additional resistance genes conferring resistance to tetracyclines (tet(A)), phenicols (floR), sulphonamides (sul2), and aminoglycosides (APH(3'')-Ib, APH(6)-Id), along with the acrEF efflux pump components (acrE, acrF). In contrast, S. flexneri harboured unique genes linked to polymyxin resistance (ugd) and regulatory functions (sdiA, gadW) that were absent in S. boydii. These findings highlight Shigella strains' genomic diversity and antimicrobial resistance potential in livestock-associated environments. Moreover, S. boydii highlights the potential risk of multidrug-resistant bacteria in farming and environmental routes. KEY POINTS: • First whole genome study of Shigella from manure and small ruminants in South Africa. • Shigella boydii strain carried multiple resistance genes to β-lactams and tetracycline. • Multidrug efflux pump gene mdf(A) was detected in 99% of South African Shigella flexneri strains.

Animals↗

Distribution of Cryptosporidium subtypes in humans and domestic and wild ruminants in Portugal.

To investigate the transmission of cryptosporidiosis in Portugal, Cryptosporidium hominis and Cryptosporidium parvum from HIV-infected patients, cattle, and wild ruminants were characterized by sequence analysis of the 60-kDa glycoprotein (GP60) gene. Fourteen subtypes within nine subtype families were identified, and three of the subtype families (If, IIb, and IId) were restricted or largely limited to Portugal. Parasites from cattle from various regions in Portugal and wild ruminants in Lisbon showed limited genetic heterogeneity (only two subtype families). All wild ruminants had the same subtype, which was also the predominant subtype in cattle all over Portugal and was found in nine HIV-infected patients in Lisbon. Two other C. parvum subtypes were only restricted to limited locations. In contrast, human parasites displayed 13 subtypes in nine subtype families, with most of the infections caused by parasites in Ib, IIa, IIc, and IId families. Two of the C. parvum subtype families (IIc and IIb) had only been found in humans. The high overall parasite diversity and high percentage of C. hominis infections attributable to Ib and C. parvum infections to IId represent unique characteristics of Cryptosporidium transmission in humans in Portugal.

Animals↗

Novel approaches to the estimation of intake and bioavailability of radiocaesium in ruminants grazing forested areas.

It is difficult to measure transfer of radiocaesium to the tissues of forest ruminants because they can potentially ingest a wide range of plant types. Measurements on undomesticated forest ruminants incur further difficulties. Existing techniques of estimating radiocaesium intake are imprecise when applied to forest systems. New approaches to measure this parameter are discussed. Two methods of intake estimation are described and evaluated. In the first method, radiocaesium intake is estimated from the radiocaesium activity concentrations of plants, combined with estimates of dry-matter (DM) intake and plant species composition of the diet, using plant and orally-dosed hydrocarbons (n-alkanes) as markers. The second approach estimates the total radiocaesium intake of an animal from the rate of excretion of radiocaesium in the faeces and an assumed value for the apparent absorption coefficient. Estimates of radiocaesium intake, using these approaches, in lactating goats and adult sheep were used to calculate transfer coefficients for milk and muscle; these compared favourably with transfer coefficients previously obtained under controlled experimental conditions. Potential variations in bioavailability of dietary radiocaesium sources to forest ruminants have rarely been considered. Approaches that can be used to describe bioavailability, including the true absorption coefficient and in vitro extractability, are outlined.

Animal Feed↗

Epitopes of the T19 lymphocyte surface antigen are extensively conserved in ruminants.

The reactivity of five monoclonal antibodies (mAbs SBU-T19, 197, IL-A29, CC-15 and CC-39) specific for the T19 molecule on sheep and cattle CD4-CD8- T cells was compared. MAbs SBU-T19 and 197 were shown to recognise separate epitopes on T19. All mAbs reacted with lymphocytes from several different ruminant species and the tissue distribution and frequency of positive cells was similar in each case. None of the mAbs reacted with horse, pig or camel lymphocytes. The extensive conservation of T19 epitopes in ruminants during the mammalian radiation could indicate an important role for this molecule in the ruminant immune system.

Animals↗

Cross-reactivity of workshop antibodies with cells from domestic and wild ruminants.

Reactivities of the monoclonal antibodies (mAbs) from the workshop panel with cells from cattle, sheep, goats, Cape buffalo (Syncerus caffer) and waterbuck (Kobus defassa) were tested. One hundred and sixty-nine mAbs reacted with bovine cells and 111 with sheep cells; 86 were shown to react with goat cells, 71 with buffalo cells and 70 with waterbuck cells. Some mAbs cross-reacted with all five ruminants tested, and are likely to react with epitopes that are conserved in other ruminant species. Such mAbs will obviate the need to produce mAbs panels to leukocyte antigens of other ruminants.

Animals↗

Characterisation of a membrane receptor on ruminants and equine platelets and peripheral blood leukocytes similar to the human integrin receptor glycoprotein IIb/IIIa (CD41/61).

This paper describes two anti-glycoprotein IIb/IIIa or CD41/61 murine monoclonal antibodies (Co.35E4 and Co.2oA1). The cellular distribution and apparent molecular weight of the antigen detected by these antibodies is consistent with their reaction with ruminant and equine glycoprotein IIb/IIIa. Biochemical analysis of the equine molecule using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed bands of 24, 100 and 110 kDa under reducing conditions and 115 and 80 kDa under nonreducing conditions. Biochemical analysis of ruminant antigen revealed that the 24 kDa band did not appear owing to the absence of labelling with sulpho-NHS-biotin. Co.35E4 and Co.20A1 recognised two different Ca2+ complex independent epitopes. The glycoprotein IIb/IIIa was present on ruminant and equine granulocytes, monocytes and platelets. However, binding on granulocytes and monocytes was due to the adsorption of membrane platelet fragments.

Animals↗

Notes on necropsy and herbage processing techniques for gastrointestinal nematodes of ruminants.

Necropsy techniques for the digestive tract of ruminants used at the University of Utrecht differ from those used elsewhere in three respects: (1) the abomasum is opened immediately after slaughter and the "contents" are treated separately from the "washings"; (2) the first 10 m of the small intestine are treated separately from the remainder of the intestine; (3) the aliquots are coloured with iodine before being examined for worms. The majority of the worms are found in the washings of the abomasum and the first part of the small intestine, whereas the contents of the abomasum and the remainder of the small intestine contain the bulk of the digesta. Because inhibited stages of Ostertagia, Haemonchus and, particularly, the very small third stage larvae (L3) of Trichostrongylus can be overlooked easily in digesta, these methods imply a more rapid and accurate enumeration of worms. This is more important in small ruminants than in cattle because a much higher proportion of the inhibited larvae will be washed out of the mucosa and because Trichostrongylus is more important in small ruminants. Herbage sampling methods for monitoring gastrointestinal nematode infections on cattle pastures in northwest Europe should also be suitable for lungworm. The agar-bile technique of Jørgensen is an elegant method, but disadvantages are that many gastrointestinal nematode larvae exsheath, resulting in identification difficulties, and recovery of lungworm larvae decreases as a result of ageing. A simple sucrose flotation method, based on the principle that a sucrose solution does not mix easily with water containing nematodes, has been tested at our laboratory.(ABSTRACT TRUNCATED AT 250 WORDS)

Abomasum↗

Antigenic diversity of ruminant Chlamydia psittaci strains demonstrated by the indirect microimmunofluorescence test with monoclonal antibodies.

Monoclonal antibodies (mAbs) were produced to find strain markers essential to the epidemiological study of chlamydial abortion of ruminants. Their specificity was tested on 53 C. psittaci strains including 35 ruminant strains isolated mainly from abortion, belonging to serotype 1 and which are invasive in our mouse model (Rodolakis et al., 1989), and 14 ruminant strains mostly intestinal, belonging to serotype 2 and non-invasive for mouse. One strain specific mAb was obtained reacting only with the non-invasive strain iB1. Six sub serotype 2 mAbs were found. They reacted only with some non-invasive strains. They allowed the distinction of 9 patterns of response among the 14 non-invasive strains. No serotype 2 specific mAbs reacting with all non-invasive serotype 2 strains were selected. In return all the invasive strains reacted with all the 18 serotype 1 specific mAbs produced. No cross-reactivities between invasive and non-invasive strains were observed, whereas common epitopes were demonstrated between invasive strains and avian or feline strains.

Animals↗

Ruminants genome no longer contains Whey Acidic Protein gene but only a pseudogene.

Whey Acidic Protein (WAP) has been identified in the milk of only a few species, including mouse, rat, rabbit, camel, pig, tammar wallaby, brushtail possum, echidna and platypus. Despite intensive studies, it has not yet been found in the milk of Ruminants. We have isolated and characterized genomic WAP clones from ewe, goat and cow, identified their chromosomal localization and examined the expression of the endogenous WAP sequence in the mammary glands of all three species. The WAP sequences were localized on chromosome 4 (4q26) as expected from comparative mapping data. The three ruminant WAP sequences reveal the same deletion of a nucleotide at the end of the first exon when compared with the pig sequence. Due to this frameshift mutation, the putative proteins encoded by these sequences do not harbor the features of a usual WAP protein with two four-disulfide core domains. Moreover, RT-PCR experiments have shown that these sequences are not transcribed and are, thus, pseudogenes. This loss of functionality of the gene in Ruminants raises the question of the biological role of the WAP. Some putative roles previously suggested for WAP are discussed.

Animals↗

A genetic manipulation tool based on the GP35 recombinase for targeted gene editing in mycoplasmas of ruminants.

Pathogenic ruminant mycoplasmas are major etiological agents in cattle and small ruminants and are responsible for substantial economic losses in the livestock industry. Progress in pathogenesis research and vaccine development has been hampered by a lack of effective genetic tools. The applicability of common genome editing platforms, such as CRISPR, is inherently restricted in these organisms owing to their minimal genomes, the absence of a cell wall, and low homologous recombination efficiency. Although transposon-mediated random mutagenesis and single-base editing are currently used in the editing of bovine mycoplasma, the stochastic nature of transposons, the risk of single-base random deamination, and limitations in editing window selection hinder the genetic manipulation of bovine mycoplasma. Here, we introduce a plasmid-based methodology that employs the GP35 recombinase from bacteriophage SPP1 to mediate long single-stranded DNA (ssDNA) recombineering, thereby enabling precise gene insertions and deletions in Mycoplasma bovis, with a positive-editing rate of 77.78% - 100%. This targeted system eliminates the risk of random deamination. Leveraging this tool, we generated a panel of M. bovis mutants affecting metabolic and virulence genes and obtained key insights into Mb0564, identified as a novel adhesin. The 192 to 287 aa region of GP35 is critical for interaction with SSB. Structural conservation analysis further suggested that this GP35-ssDNA editing system possesses a high potential for translation to other ruminant pathogens. Collectively, our approach expands the existing genetic toolkit for M. bovis, advances synthetic biology and M. bovis pathobiology, facilitates vaccine development, and strengthens the control of high-impact livestock diseases in line with the One Health framework.

Animals↗

Prenatal toxicity of cyanide in goats--a model for teratological studies in ruminants.

Although exposure to cyanogenic plants or cyanide during pregnancy has adverse effects, no teratological study with cyanide has been conducted in goats or any other ruminant. The objective of the present study was to evaluate the effects of the maternal exposure to potassium cyanide (KCN) during pregnancy on both dams and offspring and furthermore, to develop a model for prenatal toxicological studies in ruminants. Twenty-six pregnant goats were allocated into four groups and given 0, 1.0, 2.0, or 3.0mg KCN/kg body weight per day orally (administered via twice-daily gavage) from Day 24 of pregnancy to term. However, one control dam and another from the 3.0mg KCN/kg per day group were sacrificed on Day 120. At birth, the kids were examined carefully for gross abnormalities. Three months after birth, the male kids and one dam from each group were sacrificed for histopathological study. Although clinical signs of poisoning were observed in dams, cyanide treatment did not alter the length of gestation or the number of live kids. Two prognata kids were born in the 3.0mg KCN/kg group, and one dam from the same group aborted two fetuses. There were histological lesions only in the KCN-treated dam (and its fetuses) sacrificed on Day 120; these consisted of an increased number of resorption vacuoles of thyroid follicular colloid, and status spongiosis of nervous white matter. This study proposes a new animal model for teratogenic trials that could be important to evaluate the effects of chemicals throughout pregnancy in goats and potentially other ruminants.

Abnormalities, Drug-Induced↗

Atypical transmissible spongiform encephalopathies (TSEs) in ruminants.

Transmissible spongiform encephalopathies (TSEs) are associated with the accumulation in infected tissues of a disease-associated form of a host-encoded protein, the prion protein (PrP). Contrary to the normal form of the protein, this form of PrP is partially resistant to protease digestion (PrP(res)). Detailed characterisation of PrP(res) has been intensively investigated in recent years to try and decipher the diversity of TSEs in human and animals. This considerably and unexpectedly enlarged our knowledge about such diseases in ruminants. Previously, such a diversity was essentially shown by the demonstration that scrapie from sheep and goats could have different biological behaviours following transmission of the disease in mice, unlike bovine spongiform encephalopathy from cattle (BSE) which showed a distinct and unique behaviour. The properties of the BSE agent were also demonstrated to be very stable, following transmission to a variety of different species. Molecular studies of PrP(res), followed by transmission studies to mice, gave the first evidence for the accidental transmission of the BSE agent to humans where it induced a variant form of the fatal Creutzfeldt-Jakob disease (CJD) and also to different animal species including a goat in France. This last case was found among a few unusual cases of TSEs in small ruminants that showed some molecular similarities with BSE and which are currently under investigation by transmission studies in mice. The application of the molecular methods to characterise PrP(res) has most recently led to the unexpected discovery of deviant BSE forms in a few affected cattle in Europe and in the United States, which raises the question of a possible different origin at least of some cases of BSE in cattle. Finally, considerable numbers of a new TSE form in small ruminants, referred to as "atypical scrapie" or "Nor98", have meanwhile been identified in most European countries by TSE rapid testing using an assay which recognizes also comparatively less PK resistant PrP(res).

Animal Diseases↗

Full genome sequence of peste des petits ruminants virus, a member of the Morbillivirus genus.

Peste des petits ruminants virus (PPRV) causes an acute febrile illness in small ruminant species, mostly sheep and goats. PPRV is a member of the Morbillivirus genus which includes measles, rinderpest (cattle plague), canine distemper, phocine distemper and the morbilliviruses found in whales, porpoises and dolphins. Full length genome sequences for these morbilliviruses are available and reverse genetic rescue systems have been developed for the viruses of terrestrial mammals, with the exception of PPRV. This paper presents the first published full length genome sequence for PPRV. The genome was found to be consistent with the rule-of-six and open reading frames (ORFs) were identified that encoded the eight proteins characteristic of morbilliviruses. At the nucleotide (nt) level, the full length genome of PPRV was most similar to that of rinderpest, the other ruminant morbillivirus. However, at the protein level five of the six structural proteins and the V protein showed a greater similarity to the dolphin morbillivirus (DMV) while only the C and L proteins showed a high relationship to rinderpest.

Base Sequence↗

Ruminant pestiviruses.

The ruminant pestiviruses, bovine virus diarrhoea virus (BVDV) and border disease virus (BDV) are highly successful and important pathogens which infect ruminant species worldwide. Although the serological relationships among ruminant pestiviruses require further clarification, there is growing evidence for two antigenic groups, one of which predominates in cattle and one in sheep. The success of pestiviruses stems from the ability of the non-cytopathic (NCP) biotype of the virus to cross the placenta and establish a persistent infection (PI) in the developing foetus. This biotype should be regarded as the 'normal' biotype with the cytopathic (CP) biotype being an abnormal virus that is usually isolated only from PI animals dying from mucosal disease. Recent molecular evidence points to CP viruses arising from their NCP counterparts by recombination events that include the insertion of host RNA and/or the duplication of viral RNA sequences. However, the biological mechanism through which CP viruses kill cells remains unknown. Virtually all CP and NCP viruses cause only mild, transient clinical symptoms in healthy adult animals and stimulate a protective immune response. Despite the urgent requirement for a safe, effective vaccine, there is still no commercial vaccine that has been shown to immunize dams so that foetal infection is prevented. In the absence of an effective vaccine, reliable diagnostic techniques are essential to implement effective control measures. There is now a range of monoclonal antibody-based enzyme-linked immunosorbent assays for identifying PI or convalescent animals. These tests are specific, rapid, sensitive and reliable but may themselves become redundant as they are superceded by ever-increasing molecular biology-based techniques.

Animals↗

Some relationships between age, immune responsiveness and resistance to parasites in ruminants.

In the Australian livestock industries, susceptibility to infectious diseases is generally greater in young than in mature ruminants. The increased susceptibility is manifest as respiratory and intestinal infections (viral and bacterial) of calves, as well as fleece rot, flystrike and, especially, gastrointestinal parasitic infestations of young sheep. Lower resistance to infectious disease in young ruminants appears to be due largely to immunological hyporesponsiveness, and is not simply a consequence of their not having been exposed sufficiently to pathogens to develop active immunity. Young sheep have significantly lower proportions of CD4+ and CD8+ lymphocytes, but similar proportions of T19+ and B lymphocytes in blood, lymph and skin compared with mature sheep. Blood lymphocytes from young sheep produce less interferon-gamma in culture and young sheep invariably mount smaller antibody responses than do mature animals. Taken together, these findings begin to explain why young ruminants are more susceptible to infectious diseases in general, and to gastrointestinal parasites in particular, when compared to mature animals. Haematological markers of disease resistance, the prevalence of non-selected diseases and immune responses to vaccination were examined in the internal parasite-resistance flocks in Armidale NSW and the fleece rot/flystrike selection flocks at Trangie NSW. Any programme that seeks to improve resistance to parasitic or any other disease should have the capacity to make contemporary measurements of resistance to other diseases which are important in, or threaten, the production system.

Aging↗