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BackgroundVibrio parahaemolyticus is a marine bacterium causing seafood-associated gastrointestinal illness in humans and acute hepatopancreatic necrosis disease (AHPND) in shrimp. Bacteriophages have emerged as promising biocontrol agents against V. parahaemolyticus. This study characterizes Vibrio phage VPK8, focusing on host specificity, efficiency of plating (EOP) variability across V. parahaemolyticus isolates from diverse sources and other Vibrio species, morphology, genomic features, and bacteriolytic potential.MethodsVibrio phage VPK8 was isolated from blood cockles in Thailand using a mixed-host approach and purified via the double-layer agar method. Host specificity was evaluated using spot assays and EOP measurements against 120 Vibrio strains, including AHPND-associated, clinical, and seafood isolates. Phage morphology was characterized by transmission electron microscopy (TEM), while genomic features were analyzed using next-generation sequencing. Lytic characteristics, including latent period and burst size, were determined through one-step growth curves, and bacterial growth reduction was evaluated over a 24-h.ResultsVibrio phage VPK8 is a lytic phage with a 42,866 bp linear double-stranded genome, G + C content of 49.4%, and 48 coding sequences. Phylogenetic analysis grouped it within the Autographiviridae family, showing 95.96% similarity to Vibrio phage vB_VpaP_MGD1. Viral proteomic analysis placed VPK8 within the Pseudomonadota host group. Spot assays indicated broad lytic activity, but EOP analysis revealed high infectivity in clinical and seafood V. parahaemolyticus isolates, as well as some V. cholerae and V. mimicus strains. TEM revealed an icosahedral head (~ 60 nm) and a short tail. At a multiplicity of infection of 0.01, VPK8 exhibited a latent period of 25 min, a burst size of 115, and effectively inhibited the reference host V. parahaemolyticus PSU5124 within 6 h, maintaining its lytic activity and stability for over 24 h.ConclusionsThis study provides a detailed characterization of Vibrio phage VPK8 which exhibits targeted infectivity with high EOP in clinical and seafood V. parahaemolyticus isolates, as well as selected Vibrio species. Its stable lytic performance, rapid replication, and genomic safety suggest its potential for phage-based applications. Further studies should explore its in vivo efficacy and the genetic features contributing to phage resistance mechanisms, enhancing its potential applicability in managing Vibrio-related diseases.

Bovine mastitis causes severe economic losses to dairy farmers. Staphylococcus aureus, is one of the most important pathogen implicated in etiology of clinical and subclinical mastitis in bovines. In view of increasing antimicrobial resistance alternatives to antibiotic therapy are much needed. The present decade has witnessed a renewed interest in phage based therapeutics and diagnostics. The present study, describes isolation and characterization of two lytic phages SAJK-IND and MSP against Staphylococcus aureus having a potential to be used in therapy against mastitis. SAJK-IND and MSP phages belonged to Myoviridae and Podoviridae families, respectively. TEM imaging of the two phages revealed an iscosahedral head. MSP phage has a short non contractile tail. SAJK-IND and MSP have a burst size of 44 ± 3 and 25 ± 5 PFU/ infected cell, respectively. SAJK-IND and MSP phages revealed ̴ 12 and ̴16 proteins, respectively on SDS-PAGE analysis. The lytic activity of the phages was specific for Staphylococcus aureus. SAJK-IND revealed 100% lytic activity against several strains of Staphylococcus aureus isolated from mastitis milk samples whereas, MSP had only 40% lytic activity. SAJK-IND phage genome was sequenced, assembled and deposited in Genbank under accession no MG010123.

BackgroundThe emergence of antibiotic-resistant bacteria can cause serious clinical and public health problems. This study describes the possibility of using bacteriophages as an alternative agent to control multidrug-resistant Salmonella Typhimurium.MethodsThe potential lytic bacteriophages (P22-B1, P22, PBST10, PBST13, PBST32, and PBST 35) were characterized by morphological property, heat and pH stability, optimum multiplicity of infection (MOI), and lytic activity against S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009.ResultsP22-B1 and P22 belong to Podoviridae family and PBST10, PBST13, PBST32, and PBST 35 show a typical structure with polyhedral head and long tail, belonging to Siphoviridae family. Salmonella bacteriophages were highly stable at the temperatures (< 60 °C) and pHs (5.0–11.0). The reduction rates of host cells were increased at the MOI-dependent manner, showing the highest reduction rate at MOI of 10. The host cells were most effectively reduced by P22, while P22-B1 showed the least lytic activity. The ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 were resistant to ciprofloxacin, levofloxacin, norfloxacin, and tetracycline. P22 showed the highest lytic activity against S. Typhimurium KCCM 40253 (> 5 log reduction), followed by S. Typhimurium ATCC 19585 (4 log reduction) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 (4 log reduction).ConclusionThe results would provide vital insights into the application of lytic bacteriophages as an alternative therapeutics for the control of multidrug-resistant pathogens.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has become a critical concern that necessitates the development of novel antimicrobial approaches. One of the most promising innovative approaches for combating CRAB infections is using effective and lytic bacteriophages (phages), known as phage therapy. Therefore, we recovered and characterized a polyvalent lytic Salmonella_phage_VB_ST-SA173, producing lytic activity against 6 CRAB clinical isolates and 3 multidrug-resistant (MDR) Salmonella serovars. Throughout pH 2–10, and thermal stability at up to 60 °C, the phage maintained its stability and lytic activity against the tested isolates. The presence of a tailed phage with a characteristic prolate head and a contractile tail was detected by the transmission electron microscope (TEM). According to the Oxford nanopore sequencing data, the genome of Salmonella_phage_VB_ST-SA173 was 53,636 bp in size, contained 45.9% G + C, and had 53 opening reading frames (ORFs). According to the TEM, ORFs, and BLASTn analysis findings, it was proved that the Salmonella_phage_VB_ST-SA173 belongs to the Loughboroughvirus genus. The efficacy of the phage-formulated Carbopol 940 hydrogel in wound healing was assessed preclinically in an infected burn wound animal model with a CRABa clinical isolate. The survival rate was enhanced in the phage-treated group compared to the untreated control groups. Histopathological analysis showed improved wound healing in the form of apparently healthy skin with apparently normal epidermal and dermis layers. In conclusion, depending on its in vitro and physicochemical traits, the phage-loaded hydrogel is expected to be a promising candidate for clinical trials against human CRAB-related skin infections.Key points• A polyvalent Loughboroughvirus phage showed lytic activity against CRAB and Salmonella serovars.• The phage showed stability at a wide range of pH and temperature.• The phage hydrogel enhanced healing in the burn-wound animal model infected with CRABa.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-025-13559-2.

Background: Bacteriophages are viruses that specifically target bacteria and offer a promising strategy to control resistant and spoilage-causing strains in dairy products, such as Pseudomonas spp. Their application can improve food safety, extend shelf life, and provide a natural alternative to chemical preservatives and antibiotics. Objectives: In this study, 100 milk and cream samples were aseptically collected and screened for Pseudomonas spp. using biochemical tests and 16S rRNA analysis. Methods: Five strains were isolated, showing high resistance to multiple antibiotics and variable susceptibility to meropenem and amikacin. Three isolates were further analyzed by 16S rRNA sequencing to confirm their phylogenetic relationships with reference Pseudomonas spp. Six lytic bacteriophages were isolated from wastewater and characterized by transmission electron microscopy (TEM). Phages were evaluated for host range, one-step growth, multiplicity of infection (MOI)-dependent efficacy, thermal and pH stability, and lytic activity. Results: Six distinct lytic bacteriophages targeting Pseudomonas spp. were successfully isolated from wastewater and designated Phps1, Phps2, Phps3, Phps4, Phps01, and Phps04. Phages Phps1, Phps3, and Phps4 exhibited broad host ranges and strong lytic activity, while others were more strain-specific. Individual phages and phage cocktails were tested in pasteurized milk at 4°C and 25°C. Phps01 significantly reduced bacterial counts at 4°C, though efficacy declined at 25°C. Phage cocktails, particularly combinations such as Phps1 + Phps4, displayed very strong lytic activity against all tested strains. Conclusions: These results demonstrate that bacteriophages, especially in cocktail formulations, are effective biocontrol agents against Pseudomonas spp. in dairy products. Their stability under various conditions and strong antibacterial activity highlight their potential as natural, safe, and efficient tools to enhance dairy quality, extend shelf life, and mitigate antibiotic-resistant spoilage bacteria.

Background Pseudomonas aeruginosa is a common bacterial species; it can grow in all fields, even in distilled water. One of the obvious characteristics of P. aeruginosa species is its low antibiotic susceptibility (antibiotic-multi-resistant species); this reflects the urgent need for alternative strategies. Unconventionally, we try to use an alternative destructive antibacterial agent, phage. phage application in our environmental fields (engineering relevance) is a promising safe avenue for replacing chemical biocides. Objective To isolate a lytic phage as a bio-control agent for Pseudomonas aeruginosa (ATCC-27853), evaluate the ability of the isolated phage to inhibit or suppress the growth of the target bacteria. We study phage stability while performing biological and physical characterization, optimizing phage action against bacteria. Materials and methods We use an activated, purified young culture of Pseudomonas aeruginosa (ATCC-27853) as the target bacteria to isolate a specific lytic bacteriophage from a mixture of Enriched sewage and marine water through a sequence of experiments: a Broth clearing assay using nutrient broth and a spot assay test by using nutrient agar medium, both for the detection of phage presence and its lytic action. For the purification of phage, the plaque assay test was performed in many successive subcultures by using the serial dilution method on a semisolid medium. The high-titer lysate obtained from the confluent plates was investigated by TEM to determine its morphology and taxonomy. We performed biological characterization of the isolated phage: one-step growth curve, Phage adsorption rate, host range (using nutrient double layer agar), as well as a bacterial challenge test (using nutrient broth, based on the optical densities). Also, we performed physical characterization of the phage to determine phage Stability, pH, thermal stability, and the influence of UV and organic solvents on the lytic action of the phage. Results and conclusion Pyocyanin and pyoverdin pigments (both fluorescent under UV illumination) are secreted by P. aeruginosa (ATCC-27853), also their ability to thrive at 42°C. After spotting P. aeruginosa plates with enriched sewage filtrate, the results indicated lytic area (+ve). Plaques have a pin tip and are translucent. The lytic profile of bacteria after phage infection revealed that the bacteria was reduced to a MOI of 10 in 4 h. However, the multiplicity of infection(MOI) of 1 and 0.01, respectively, was longer (10, 22/h). The electron microscopy revealed that the phage is a member of the podoviridae family, with a noncontractile short tail (18 nm) and a polyhedral (heptagonal) head (45 nm) in diameter and a phage length of 63 nm. The adsorption rate of the phage was greatest at 20 min. Our goal with the phage application is to replace harmful chemical biocides with lytic bacteriophages in various environmental systems (Phage for Biological Control). Phage bio-control treatment thus holds promise for eliminating the requirement for hazardous chemical biocides.

Abstract: The objective of this work was to characterize autochthonous bacteriophages and to determine their lytic activity on Staphylococcus aureus. Six phages were isolated from dairy barn flush water through enrichment cultures with three S. aureus strains. All phages were characterized by DNA digestion by restriction enzymes and sequencing of the DNA fragment encoding endolysin. Each phage was tested against 100 S. aureus strains isolated from bovine mastitis and from dairy products using the lysis-plate method. The sequences of the endolysin gene were highly conserved, with nucleotide similarity higher than 99% among the isolated phages. Three domains involved in the recognition and lysis of the bacterial cell wall were identified. Two bacteriophages isolated from the dairy barns present high lytic activity on S. aureus, on a wide range of host strains, indicating their potential for studies on phage therapy in dairy cattle or as a biological control agent for dairy products.

Comparison of agar-based and checkerboard methods to assess phage-antimicrobial synergy in Pseudomonas aeruginosa.

Aims: To perform the isolation and phenotypic characterization of bacteriophage with lytic activity against Pseudomonas aeruginosa. To demonstrate that this type of viral agent can be isolated from the environment and used for the biocontrol of resistant bacterial types, such as Pseudomonas aeruginosa.&#x0D; Study Design: This study was an experimental study.&#x0D; Place and Duration of Study: The study was conducted at, Bacteriology and Mycology Laboratory in the Veterinary Hospital at the School of Agricultural Sciences, Innovation and Business of the University of Passo Fundo (ESAN/UPF) and Center for Diagnosis and Research in Animal Health of the University of Passo Fundo (CDSA/UPF), between April 2022 and June 2022.&#x0D; Methodology: Samples of untreated water were inoculated with the host bacterium strain Pseudomonas aeruginosa ATCC 27853 in an enriched media After the incubation period in, a phage filtrate was obtained by centrifugation followed by filtration. We verified the presence of bacteriophages using spot test and we carried out its purification by the method of sterile toothpick plate transfer on bacterial overlay semi-solid agar. Amplification was performed using an SM buffer elution procedure to produce a stock of viral material. Through assays in Petri dishes with bacterial overlay, we performed titration and phenotypic characterization regarding the lysis spectrum and efficiency of phage infection in the host.&#x0D; Results: We managed to isolate a morphologically characterized lytic bacteriophage with approximately 1 mm of diameter, high clarity in the inhibition area, the presence of halo and well-demarcated edges. The bacteriophage, named as Pseudomonas aeruginosa Phage UPF_PaBP1, demonstrated the infection capacity of the target bacteria in all tested dilutions and a stock preparation with a titre of 6.5 x 10⁸ PFU/ml was obtained for future use.&#x0D; Conclusion: The isolated phage showed strong lytic activity against the bacterial host, a finding that nourishes our expectations regarding the use of this phage as a biocontrol agent and phage therapy.

Abstract. Narulita E, Utami DAW, Izzaturrohmah L, Arrachmi HI, Febrianti RA, Ludfillah RZ, Widianto AH, Addy HS, Utami BR, Kuswati, Fikri K. 2025. Genomic and evolutionary perspectives on bacteriophage ?Afa-NA1 targeting MDR Gram-negative bacteria in diabetic ulcer. Biodiversitas 26: 6014-6024. The rise of Multidrug-Resistant (MDR) bacterial infections poses a significant challenge in clinical settings, particularly for diabetic ulcer patients who are highly susceptible to persistent infections. This study reports the isolation, characterization, and genomic analysis of a novel lytic bacteriophage, ?Afa-NA1, specifically targeting MDR Gram-negative bacteria associated with diabetic ulcers in Indonesia. The bacteriophage was isolated from wound dressings and tested against four clinical isolates: Alcaligenes faecalis T17, Pseudomonas sp. FP1911, and the Gram-positive control Streptomyces violaceoruber S21. ?Afa-NA1 exhibited strong lytic activity against three Gram-negative isolates but none against the Gram-positive control, confirming host specificity. ?Afa-NA1 produced clear plaques with an average diameter of 3.0±0.5 mm on Alcaligenes faecalis T17, indicative of its potent lytic activity. Stability tests showed the phage retained &gt;90% infective titer after 24 hours at -20°C and 4°C, and exhibited robust stability across a wide pH range (pH 6 to 11). One-step growth analysis revealed a short latent period of 21.7±8.1 minutes and a burst size of 27.7±1.5 PFU per infected cell. Genomic analysis showed ?Afa-NA1 possesses a 78,105 bp double-stranded DNA genome with 45.8% G+C content encoding 83 predicted open reading frames. Comparative genomics and phylogenetic reconstruction confirmed its placement among T7-like Podoviruses, consistent with its obligate lytic lifecycle and genomic architecture. However, the potential for therapeutic application remains preliminary, as host range testing was conducted on a limited panel of single clinical isolates, and all assays were performed in vitro.

Vibrio species are naturally found in estuarine and marine ecosystems, but are also recognized as significant human enteropathogens, often linked to seafood-related illnesses. In aquaculture settings, Vibrio poses a substantial risk of infectious diseases, resulting in considerable stock losses and prompting the use of antimicrobials. However, this practice contributes to the proliferation of antimicrobial-resistant (AMR) bacteria and resistance genes. Our investigation aimed to explore the potential of biological agents such as bacteriophage CH20 and endolysin LysVPp1 in reducing Vibrio bacterial loads in both rotifer and fish larvae. LysVPp1's lytic activity was assessed by measuring absorbance reduction against various pathogenic Vibrio strains. Phage CH20 exhibited a limited host range, affecting only Vibrio alginolyticus GV09, a highly pathogenic strain. Both CH20 and LysVPp1 were evaluated for their effectiveness in reducing Vibrio load in rotifers or fish larvae through short-setting bioassays. Our results demonstrated the significant lytic effect of endolysin LysVPp1 on strains of Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio splendidus. Furthermore, we have showcased the feasibility of reducing the load of pathogenic Vibrio in live feed and fish larvae by using a non-antibiotic-based approach, such as lytic phage and endolysin LysVPp1, thus contributing to the progress of a sustainable aquaculture from a One Health perspective.

Given that it spreads around the world and not just in nations in west and central Africa, monkey pox is a disease of worldwide public health significance. An uncommon condition known as monkey pox is brought on by infection with the monkey pox virus. The virus that causes monkey pox is related to the virus that causes smallpox, or variola virus. Smallpox symptoms are comparable to those of monkey pox, although they are less severe, and monkey pox seldom results in death. Monkey pox has replaced smallpox as the most significant orthopoxvirus for public health since smallpox was eradicated and smallpox vaccinations were subsequently discontinued. Primarily occurring in tropical rainforests in central and west Africa, monkey pox has been steadily spreading into cities. Numerous rodent species and non-human primates serve as hosts for animals. Outside of the African nations where monkey pox is widespread, the United States saw an epidemic. Boston, Massachusetts, recorded the first case. The first monkey pox epidemic outside of Africa occurred in the United States and contact with sick pet prairie dogs was to blame. Fever, chills, swollen lymph nodes, exhaustion, muscle and backaches, headaches, and respiratory problems are some of the signs and symptoms of monkey pox (e.g. sore throat, nasal congestion, or cough). Multiple instances of monkey pox were discovered in numerous non-endemic nations. Studies are being conducted right now to learn more about the epidemiology, sources of illness, and patterns of transmission. Direct contact with the blood, body fluids, cutaneous or mucosal lesions of infected animals can result in animal-to-human (zoonotic) transfer. Numerous animals in Africa, including rope squirrels, tree squirrels, Gambian pouched rats, dormice, several kinds of monkeys, and others, have shown signs of infection with the monkey pox virus. Although rodents are most probable, the natural reservoir of monkey pox has not yet been discovered. Eating undercooked meat and other diseased animal products is a potential risk factor. People who live in or close to forests may be indirectly or minimally exposed to diseased animals. Close contact with respiratory secretions, skin sores on an infected person, or recently contaminated objects can cause human-to-human transmission. Monkey pox typically takes 6 to 13 days to incubate, but it can take anywhere from 5 to 21 days for symptoms to appear. Other rash disorders, such as chickenpox, measles, bacterial skin infections, scabies, syphilis, and medication-associated allergies, must be taken into account when making a clinical differential diagnosis. As a clinical characteristic, lymphadenopathy during the prodromal stage of the illness can help differentiate monkey pox from chickenpox or smallpox. Given its precision and sensitivity, polymerase chain reaction (PCR) is the primary laboratory test. Fluids and food should be provided to patients in order to maintain a healthy nutritional condition. Multiple observational studies have shown that the smallpox vaccine is around 85% effective at preventing monkey pox. The major preventative method for monkey pox is increasing public knowledge of risk factors and teaching individuals about the steps they may take to lessen viral exposure. The feasibility and suitability of vaccination for the prevention and control of monkey pox are now being evaluated through scientific investigations. Some nations have policies in place or are creating them to provide vaccines to those who may be at risk, including laboratory staff, members of quick reaction teams, and healthcare professionals.

In this study, the lytic phage for&nbsp;multidrug-resistant&nbsp;(MDR)&nbsp;Pseudomonas aeruginosa&nbsp;was isolated; this phage belongs to the&nbsp;Myoviridae&nbsp;and&nbsp;Siphoviridae. Analysis of its genome sequences highlighted by different antibiotic resistance gene primers offered the most direct and sensitive method of determining the therapeutic status of this lytic phage. Interestingly, this approach reveals the existence of&nbsp;vanAcassette in the genome of this lytic phage. Whereas van B gene primers highlighted the polymerase chain reaction (PCR) product of 1248 bp, which has shown a relation with ABC transport proteins ofEnterococcus faecalis&nbsp;V583 instead of the ligase meant for the&nbsp;van B&nbsp;resistance trait. BLASTn analysis of the sequenced product has shown the existence of small stretches on Pseudomonas phage PCR product. These predicted signature sequences of phage PCR product are 100% identical with signature sequences of the same size but located at different sites on the genome of&nbsp;E. faecalis&nbsp;V583 corresponding to ABC transporter proteins. Six signature sequences were identified. These signatures are different from Walker A and human ABC signatures. Presumably, these signatures reflected the relation with Walker B sequence. Our data suggested that Pseudomonas lytic phage has some proteins that have partial homologous structure to ABC-transporter with Walker B motifs. The existence of these unique signature sequences once in different ABC transporter in&nbsp;E. faecalis&nbsp;V583 and phage genome has reflected the presence of some functional domains on these proteins that have not yet been identified, and their function need to be elucidated. Key words: Bacteriophage, lytic phage, van B, ABC-transporters, Walker B signature.

Background. The current study focused on the isolation and characterization of two bacteriophages, Psa 12 and Psa 60, which exhibited lytic activity against Pseudomonas aeruginosa. P. aeruginosa is a significant threat due to its widespread distribution, growing antimicrobial resistance, and biofilm formation. The WHO highlights its urgency for new antibiotics. In Ukraine, particularly, war-related injuries are frequently compicated by infections caused by multidrug-resistant P. aeruginosa. Phage therefore emerges as a promising research direction and a potential alternative for managing MDR infections. Methods. Bacteriophages were isolated using the double-layer agar method, and their host range was determined by spot testing against clinical strains. Morphological features were assessed via transmission electron microscopy. Results. Phages Psa 12 and Psa 60 were isolated from wastewater samples and demonstrated strong lytic activity, with titers of 12×107 PFU/mL and 5×106 PFU/mL, respectively. Out of 30 clinical P. aeruginosa strains, phage isolate Psa 12 showed effectiveness against 46 %, while Psa 60 was effective against 53 %. TEM analysis revealed that both phages possess an icosahedral capsid and a contractile tail, consistent with the Caudoviricetes class. Psa 12 had a head diameter of 87±2 nm and a tail length of 137±5 nm. Psa 60 exhibited a head diameter of 100 ± 3 nm and a tail length of 121 ± 3 nm. Сonclusions. The strong lytic activity, broad host range among clinical strains, and structural features characteristic of the Caudoviricetes class highlight the therapeutic potential of phages Psa 12 and Psa 60 against multidrug-resistant P. aeruginosa.

To determine if, as is the case with Trypanosoma brucei and T. congolense, serodemes of T. vivax could be distinguished on the basis of immunity to the metacyclic stages of the parasite, attempts were made to immunize goats by infection with infected tsetse, followed by chemotherapy or eventual 'self-cure'. Thirty goats were infected by tsetse with either clones or stocks of T. vivax from East or West Africa. Twenty-four goats were treated with diminazene aceturate (Berenil, Hoechst A.G.) 2-6 weeks after infection and 6 goats were allowed to self-cure. Infection, followed by treatment, induced immunity to a first homologous challenge by infected tsetse in only 2 of 24 goats (one immune to the East African stock, and the other to a clone of the West African stock). Immunity to a clone of the East African stock was induced in 3 or 4 animals after a second infection and treatment and in the fourth animal of the group following a third infection and treatment. One of 2 goats infected with the clone of the East African stock was immune to challenge at 16 weeks, following self-cure without treatment, and 1 of 4 goats infected with the parent stock was similarly immune when challenged at 40 weeks post-infection. Goats susceptible to infection with East African T. vivax showed evidence of partial immunity by delayed pre-patent periods and depressed parasitaemias after challenge. Goats infected with the relatively more virulent West African T. vivax were, however, completely susceptible to infection after homologous challenge, and showed only a slight delay in pre-patent period. A similar result was obtained in a further 8 goats primed and challenged by large numbers of tsetse (20 or 100 infected tsetse/goat) with the West African T. vivax. In further experiments using a very short treatment interval, infections following challenge were clearly shown to be the result of a lack of immunity rather than relapse following treatment. Lytic antibody activity to cultured metacyclic trypanosomes could not be detected during infection but such activity against bloodstream forms was detected after 2 weeks of infection. It is suggested that the primary reason for the erratic induction of immunity to T. vivax employing this methodology is the low number of metacyclics transmitted by infected tsetse, and thus poor antigenic stimulus encountered by goats upon tsetse challenge.